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This is to certify that the

dissertation entitled

PROPIONIBACTERIA AS INOCULANTS TO

HIGH MOISTURE CORN
presented by

RUBY V. BATO

has been accepted towards fulfillment
of the requirements for

Ph. D. degreein ANIMAL SCIENCE

Q V

Major professor

Date/02"- //~&0£L

MS U i: an Affirmative Action/Equal Opportunity Institution ' 0-12771

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

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DATE DUE

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PROPIONIBACTERIA AS INOCULANTS TO HIGH MOISTURE CORN
By

Ruby V. Bato

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Animal Science

2001

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ABSTRACT
PROPIONIBACTERIA AS INOCULANTS TO HIGH MOISTURE CORN
By

Ruby V. Bato

Two studies were conducted to evaluate the efficacy of prOpionibacteria as
silage inoculants. In the first study, the performance of prOpionibacteria with or
without lactic acid bacteria were evaluated. Results showed that propionibacteria
enhanced the fermentation of reconstituted corn only up d 21 of ensiling. The
combination of P. acidipropionici DH42 with lactic acid bacteria as inoculants
reduced silage pH and butyric acid and increased propionic, acetic and lactic acids.
LAB inoculation did not significantly increase the LAB population in the treated
silages. During aerobic exposure, all the silages appeared well-preserved. Organic
acid levels remained stable throughout the exposure period. Propionibacteria
inoculation did not significantly reduce the yeast and mold population. However,
the silages with P. acidipropionici DH42 + LAB had higher propionic, acetic and
lactic acids and lower pH.

In the second study, the effect of moisture on the efficacy of
prOpionibacteria as silage inoculants was tested. Rolled corn of moisture contents
ranging from 22-35% were used. The 22-28% moisture levels appeared to favor the
growth of the P. acidipropionici DH42 in silage. After 120 d of ensiling, PAB-
inoculated high moisture corn gave higher propionic and acetic acids and lower pH

and butyric acid at 22-28%. P. acidipropionici DH42 inoculated at 1&6ch better

 

gave results as compared to the 105cfu/ g. During aerobic exposure, higher propionic
and acetic acids and lower pH were observed with the FAB-treated silages fiom the
22-28% moisture levels. Propionibacteria inoculation did not significantly reduce
yeast and mold counts.

The vitamin B12 production capability of P. acidipropionici DH42 was also
evaluated in comparison with P. shermam'i. Results showed comparable vitamin B12
production of the two prOpionibacteria strains. After 72 h of incubation, the P.
acidipropionici DH42 and P. shermanii cultures grown at 30°C had vitamin B12
contents of 852.85 and 840.69 ng/ml, respectively. Both strains grew better at 30°C
than at 40°C. P. acidipropionici DH42 cultures tend to have higher propionic and
acetic acids while the P. shermam'i cultures had higher succinic and malic acids.

A PCR-based detection of P. acidipropionici DH42 was developed. Nested
PCR was used with DH42-specific primers dhbl and dhb2 for the secondary
amplification of a 1,267 bp-fragment. Using the established protocols for PCR
amplification, as low as 102 cfu/ml and 103cfu/ml of P. acidzpropionici DH42 in
silage extracts and rumen fluid, respectively, were detected.

The silage studies had shown that moisture level affects the efficacy of P.
acidipropionici DH42. The 22-28% moisture content appears to favor its growth.
An inoculation rate of 106 cfu/ g of ensiling material is recommended. P.
acidipropionici DH42 can produce vitamian that is comparable to P. shermanii’s
producing capability. Moreover, P. acidipropionici DH42 can be detected in silage

and rumen fluid samples using PCR technology.

Copyright by
Ruby V. Bato
2001

3591‘ 311

EA ho'ui

33' that

5.5113" :A

":14.

ACKNOWLEDGMENTS

I would like to thank the members of my committee for their patience and
guidance throughout my graduate study: Dr. Michael Allen, Dr. Paul Coussens, and Dr.
Zeynep Ustunol. My special gratitude to Drs. Melvin Yokoyama and Steven Rust for
serving as co-advisers in my program.

Thanks to Dr. Jeannie Burton and Dr. Paul Coussens for giving me access to their
laboratories, and use of their supplies and facilities. The assistance of Dr. Patty Weber
and Chris Colvin in molecular techniques and Jennifer Wells in DNA sequencing is also
acknowledged. I would like to thank Daniel H. Buckley for designing the primers and
Michael Romanov for his assistance in the phylogenetic analysis of my data I would like
to thank Robert Burnett for helping me with the preparation of the silages and the
laboratory analyses of my samples and to Sunghee, Faith, Sally, Sue, Hugo for their help

My graduate program had not been possible without the financial support from
the Philippine-American Educational Foundation through the Fulbright program and the
office of Dr. Richard Brandenburg, the Graduate School and Department of Animal
Science for financial assistance.

To all the families who had shared their traditions and loving homes with Rob and
me: the Cerdenas, Walters, and the Javiers. To Ines who is like a sister to me and to the
Aviles family for their kindness and support.

To my parents who had always supported me with love and understanding in all
my undertakings, to my sisters and brother whose constant communication had kept me
sane. To Rob, my little lab assistant, this work would not have been possible if not for

his love, extraordinary patience and understanding especially those times I had to work

overtime in the lab. To Papa, whose love, support and prayers had helped immensely in
completing this work, I couldn’t ask for a better partner. Most of all, I thank God for

giving me all that I ever wanted.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................ ix
LIST OF FIGURES ........................................................................ xiii
INTRODUCTION ........................................................................ 1
CHAPTER

1 LITERATURE REVIEW ....................................................... 4

2 PROPIOPIONIBACTERIA AND LACTIC ACID-PRODUCING BACTERIA AS
INOCULANT S FOR ENSILING RECONSTITURED HIGH MOISTURE CORN

Abstract ................................................................ 21
Introduction ................................................................ 22
Materials and methods ................................................................ 23
Results and discussion ................................................................ 26
Implications ................................................................ 46

3 EFFECT OF MOISTURE ON PROPIONIBACTERIA AS INOCULANT S TO
HIGH MOISTURE CORN SILAGE

Abstract ............................................................... 47
Introduction .............................................................. . 48
Materials and methods ............................................................... 48
Results and discussion ............................................................... 52
Implications ............................................................... 84

4 VITAMIN B12 PRODUCTION OF P. ACIDIPROPIONICI DH42 IN BATCH
CULTURE SYSTEM

Abstract ............................................................ 86
Introduction ............................................................ 86
Materials and methods ............................................................ 87
Results and discussion ............................................................. 91
Implications ............................................................. 100

5 PCR-BASED ASSAY FOR THE DETECTION OF P. ACIDIPROPIONICI DH42 IN
SILAGE AND RUMEN FLUID

Abstract ............................................................... 101
Introduction ............................................................... 101

vii

Materials and methods

............................................................... 102
Results and discussion ............................................................... 111
Implications ............................................................... 121

6 SUMMARY AND CONCLUSSIONS .............................................. 126

APPENDICES
Appendix A. Tables presented in chapter 3 ........... - .................................. 131
Appendix B. Data used for analyses in chapter 2 ................................... .154
Appendix C. Data used for analyses in chapter 3 ..................................... 160
Appendix D. Data used for analyses in chapter 4 ....................................... 187

LITERATURE CITED ................................................................ 189

viii

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

LIST OF TABLES

Dry matter content (%) of flesh and ensiled HMC ................ 27
Effect of inoculation on propionic acid content (g/ 100g DM) of flesh and
ensiled HMC ............................................................... 27
Effect of inoculation on acetic acid content (g/ 100g DM) of flesh and
ensiled HMC ............................................................ 29
Effect of inoculation on lactic acid content (g/100g DM) of flesh and
ensiled HMC ............................................................ 29
Effect of inoculation on pH of flesh and ensiled HMC 32

Effect of inoculation on glucose content (g/ 100g DM) of flesh and ensiled
HMC ............................................................ 32

Effect of inoculation on butyric acid content (g/lOOg DM) of flesh and
ensiled HMC ............................................................ 33

Effect of inoculation on ethanol acid content (g/ 100g DM) of flesh and
ensiled HMC ............................................................ 34

Effect of inoculation on citric acid content (g/ 100g DM) of flesh and
ensiled HMC ............................................................ 34

Effect of inoculation on dry matter recovery (%) of ensiled HMC 36

Effect of inoculation on lactic acid bacteria counts (log cfu/ g DM) of

flesh and ensiled HMC ......................................... 36
Effect of inoculation on yeast and mold counts (log cfu/ g DM) of flesh
and ensiled HMC ................................................. 37
Effect of inoculation on propionic acid content (g/ 100 g DM) of exposed
HMC silage ......................................................... 37
Effect of inoculation on lactic acid content (g/ 100 g DM) of exposed HMC
silage ......................................................... 39
Effect of inoculation on pH of exposed HMC silage ............... 39

ix

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Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4—5

Table 4-6

Table 4-7

Effect of inoculation on acetic acid content (g/ 100 g DM) of exposed
HMC silage ............................................................ 40

Effect of inoculation on glucose content (g/100 g DM) of exposed HMC
silage ............................................................ 41

Effect of inoculation on butyric acid content (g/100 g DM) of exposed
HMC silage ............................................................ 41

Effect of inoculation on ethanol content (g/ 100 g DM) of exposed HMC
silage ............................................................ 43

Effect of inoculation on citric acid content (g/ 100 g DM) of exposed HMC
silage ................................. , ............................ 43

Effect of inoculation on lactic acid bacteria counts (log cfu/ 100 g DM) of
exposed HMC silage ................................................... 44

Effect of inoculation on yeast and mold counts (log cfu/ 100 g DM) of
exposed HMC silage ................................................... 44

Effect of inoculation on the dry matter (%) of exposed HMC silage 45
Effect of inoculation on temperature (°C) of exposed HMC silage ...... 45

Effect of incubation temperature on the true vitamin B12(ng/ml) of P.
acidipropionici DH42 and P. shermam'i at different incubation times. . .91

Effect of incubation temperature in the optical density of P.
acidipropionici DH42 and P.3hermanii at different incubation times. . ...95

Effect of incubation temperature on the pH of P. acidipropionici DH42
and P. shermanii at different sampling times ................................. 95

Effect of incubation temperature on the propionic acid of P.
acidipropionici DH42 and P. shermanii at different incubation times... ..97

Effect of incubation temperature on the acetic acid of P. acidipropionici
DH42 and P. shermanii at different incubation times .................... 97

Effect of incubation temperature on the succinic acid of P. acidipropionici
DH42 and P. shermaniz' at different incubation times .................. 98

Effect of incubation temperature on the malic acid of P. acidipropionici
DH42 and P. shermanii at different incubation times .................. 100

 

.a5

 
 

Ana.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table A-1

Table A-2

Table A-3

Table A-4

Table A-5

Table A-6

Table A-7

Table A-8

Table A-9

Table A- l 0

Table A-ll

Bacterial cultures used in the study ................................... 103

Alignment of 16S rDNA of propionibacteria and other bacterial species
with primers dhbl and dhb2 (ambiguous or non-matching positions are

boxed) ............................................................... 106
Synthetic oligonucleotides sequences, positions and calculated melting
temperature(T,,.) ......................................................... 110

Taxonomy BLAST report for 13 propionibacteria species compared

against DH42 using BLAST .............................................. 120

Distribution of top ten Blast hits on the query sequence ............... 125

Effect of inoculation and moisture on the propionic acid content (g/ 100 g)
of flesh and ensiled HMC .................................................. 131

Effect of inoculation and moisture on the pH of flesh and ensiled

HMC .......................................... . ................................. 132

Effect of inoculation and moisture on the acetic acid content (g/lOOg DM)
of flesh and ensiled HMC .................................................. 133

Effect of inoculation and moisture on the lactic acid content (g/lOOg DM)
of flesh and ensiled HMC .................................................. 134

Effect of inoculation and moisture on glucose content (g/ 100 g DM) of
flesh and ensiled HMC. ...................................................... 135

Effect of inoculation and moisture on citric acid content (g/ 100 g DM) of
flesh and ensiled HMC. ..................................................... 136

Effect of inoculation and moisture on ethanol content (g/100 g DM) of
flesh and ensiled HMC. ..................................................... 137

Effect of inoculation and moisture on butyric acid content (g/ 100 g DM)
of flesh and ensiled HMC. .................................................. 138

Effect of moisture and inoculant on the dry matter recovery (%) of ensiled
HMC ........................................................................... 139

Effect of moisture and inoculant on the yeast and molds counts (log cfu/ g
DM) of flesh and ensiled HMC ............................................ 140

Effect of inoculation and moisture on propionibacteria counts (log cfu/ g
DM) of flesh and ensiled HMC ............................................. 141

xi

Table A-12

Table A- l 3

Table A-14

Table A-1 5

Table A-l6

Table A-17

Table A-18

Table A-19

Table A-20

Table A-21

Table A-22

Table B-1

Table B-2

Table C-l

Table C-2

Table D-l

Effect of inoculation and moisture on the propionic acid content (g/ 100g
DM) of exposed HMC silage ................................................ 142

Effect of inoculation and moisture on the acetic acid content (g/ 100g DM)
of exposed HMC silage ...................................................... 143

Effect of inoculation and moisture on the lactic acid content (g/100g DM)
of exposed HMC silage ...................................................... 144

Effect of inoculation and moisture on the pH of exposed HMC silage. 145

Effect of inoculation and moisture on the glucose content (g/ 100g DM) of
exposed HMC silage .......................................................... 146

Effect of inoculation and moisture on the ethanol content (g/ 100g DM) of
exposed HMC silage ............................................................ 147

Effect of inoculation and moisture on the citric acid content (g/lOOg DM)
of exposed HMC silage ........................................................ 148

Effect of inoculation and moisture on the butyric acid content (g/ 100g
DM) of exposed HMC silage ................................................. 149

Effect of inoculation and moisture on the yeast and molds counts (log
cfii/g DM) of exposed HMC silage .......................................... 150

Effect of inoculation and moisture on the dry matter recovery (%) of
exposed HMC silage. ........................................................ 151

Effect of inoculation and moisture on the temperature (°C) of exposed
HMC silage. ..................................................................... 152

Data used for analyses in chapter 2-chemical and microbial analyses... 1 54

Data used for analyses in chapter 2- temperature during aerobic stability
test ................................................................................ 158

Data used for analyses in chapter 3-chemical and microbial analyses... 1 60

Data used for analyses in chapter 3- temperature during aerobic stability
test ................................................................................ 182

Data used for analyses in chapter 4 ....................................... 187

xii

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Figure 3-3

Figure 3-4

Figure 3-5

Figure 3-6

Figure 3-7

Figure 3-8

Figure 3-9

Figure 3-10

Figure 3-11

Figure 3-12

Figure 3-13

Figure 3-14

Figure 3-15

LIST OF FIGURES

Figure 3-1. The effect of inoculation on the propionic acid of flesh and
ensiled high moisture corn at 35% moisture level ....................... 53

' The effect of inoculation on the propionic acid of flesh and ensiled high

moisture corn at 33% moisture level ...................................... 53

The effect of inoculation on the propionic acid of flesh and ensiled high
moisture corn at 28% moisture level ..................................... 54

The effect of inoculation on the propionic acid of flesh and ensiled high
moisture corn at 24% moisture level ..................................... 54

The effect of inoculation on the propionic acid of flesh and ensiled high
moisture corn at 23% moisture level ..................................... 55

The effect of inoculation on the propionic acid of flesh and ensiled high
moisture corn at 22% moisture level .. ............................. 55

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 35% moisture level .................................................. 57

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 33% moisture level ................................................ . 57

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 28% moisture level ................................................. 58

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 24% moisture level ................................................ . 58

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 23% moisture level ................................................ . 59

The effect of inoculation on the pH of flesh and ensiled high moisture
corn at 22 % moisture level ................................................ . 59

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 35% moisture level ...................................... . 62

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 33% moisture level ...................................... . 62

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 28% moisture level ...................................... 63

xiii

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Figure 3-18

Figure 3-19

Figure 3-20

Figure 3-21

Figure 3-22

Figure 3-23

Figure 3-24

Figure 3-25

Figure 3-26

Figure 3-27

Figure 3-28

Figure 3—29

Figure 3-30

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 24% moisture level ...................................... 63

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 23% moisture level ...................................... 64

The effect of inoculation on the acetic acid of flesh and ensiled high
moisture corn at 22% moisture level ........................................ 64

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 35% moisture level ................................... 66

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 33% moisture level .................................. 66

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 28% moisture level ................................ 67

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 24% moisture level ................................. 67

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 23% moisture level .................................. 68

The effect of inoculation on the lactic acid content of flesh and ensiled
high moisture corn at 22% moisture level ................................. 68

The effect of inoculation on the yeast and mold counts of flesh and ensiled .
high moisture corn at 35% moisture level ................................ 71

The effect of inoculation on the yeast and mold counts of flesh and ensiled
high moisture corn at 22% moisture level ................................ 71

The effect of inoculation on the yeast and mold counts of flesh and ensiled
high moisture corn at 28% moisture level ................................ 72

The effect of inoculation on the yeast and mold counts of flesh and ensiled
high moisture corn at 24% moisture level ................................ 72

The effect of inoculation on the yeast and mold counts of flesh and ensiled
high moisture corn at 23% moisture level ................................ 73

The effect of inoculation on the yeast and mold counts of flesh and ensiled
high moisture corn at 22% moisture level ................................ 73

xiv

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Figure 3-32
Figure 3-33
Figure 3-34
Figure 3-35
Figure 3-36
Figure 3-37
Figure 3-38
Figure 3-39
Figure 3-40

Figure 5-1

Figure 5-2

Figure 5-3.

The effect of inoculation and moisture content on the average propionic
acid of exposed high moisture corn silage ................................ 77

The effect of moisture content on the average propionic acid content of
high moisture corn silage during S-day exposure period ................ 77

The effect of inoculation and moisture content on the average acetic acid
content of exposed high moisture corn silage ............................. 78

The effect of moisture content on the acetic acid content of high moisture
corn silage during S-day exposure period ................................. . 78

The effect of inoculation and moisture content on the average lactic acid
content of exposed high moisture corn silage ............................. 80

The effect of moisture content on the lactic acid content of high moisture
corn silage during 5-day exposure period ................................ 80

The effect of inoculation and moisture content on the average pH of
exposed high moisture corn silage ...................................... . 81

The effect of moisture content on the average pH of high moisture corn
silage during 5-day exposure period ....................................... 81

The effect of moisture content and inoculation on the yeast and mold
counts of exposed high moisture corn silage ............................. 83

The effect of moisture content on the yeast and mold counts of high
moisture corn silage during exposure period ........................... 84

DH42 16S rDNA sequence and the target sites of the four primers 107

Effect of annealing temperature (lanes 1-3, 66°C; lanes 4-6, 67°C, lanes 7-
8, 68°C, lanes 9-11, 69°C) and magnesium chloride concentration:1.5 mM
(lanesl,4,7); 1.25 mM (lane 10); 1.0 mM (lanes 2, 5, 8, 9); and 0.5

mM(lanes 3 and 6); lane 12, lkb DNA ladder .......................... 114

Primary PCR showing the 1.5 kb flagment using primers bakll and bak4,
with the following bacterial strains: 1, Propionibacteriumfreudenreichii;
2, P. shermanii CDC3094;3, P. shermanii P92; 4, P. thoenii P15; 5, P.
pentosaceum P11; 6, P. acidipropionici (ATCC 25562); 7, P. shermanii;
8, Propionibacterium sp.; 9, P. acidipropionici DH42; 10, Lactobacillus
sakei; 11, Lactobacillus confusus; 12, Lactobacillus delbruickii; l3,
Bacillus subtilis; 14, Bacillus sp.; 15, Bacillus subtilis (ATCC ); 16,
Eubacterium combesii; 17, 1 kb DNA ladder .................. 114

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Figure 5-5.

Figure 5-6.

Figure 5-7.

Figure 5-8.

Figure 5-9

Figure 5-10

Secondary PCR showing the 1.3 kb flagment amplification using primers
dhbl and dhb2 with the following bacterial strains: 1, Propionibacterium
fieudenreichii; 2, P. shermanii CDC3094; 3, P. shermanii P92; 4, P.
thoenii P15; 5, P. pentosaceum P11; 6, P. acidipropionici (ATCC 25562);
7, P. shermanii; 8, Propionibacterium sp.; 9, P. acidipropionici DH42; 10,
Lactobacillus sakei; ll, Lactobacillus confusus; 12, Lactobacillus
delbruickii; l3, Bacillus subtilis; l4, Bacillus sp.; 15, Bacillus subtilis
(ATCC ); 16, Eubacterium combesii; l7, 1 kb DNA ladder ...... 115

Secondary PCR amplification products showing the 1,267 bp-flagment
size in DH42 (lanes 1-3 and lanes 5-7) and Eubacterium combesii (lanes 4
and 8); lane 9, negative control; lane 10,1-kb DNA ladder. ...... 115

Primary PCR showing the amplification products of primers bak4 and bak
11 using DNA extracts flom rumen fluid (lane 1-5, amplification flom
uninoculated rumen fluid, 102'103, 104, 105 cfu/ml, respectively) and
silage (lanes 6-10, amplification flom uninoculated silage, 102’ 10°, 10°,
105cfu/ml, respectively); lane 9 is negative control; lane 10,1 kb DNA
ladder. ............................................................. 1 17

Secondary PCR showing the amplification products of primers dhbl and
dhb2 using DNA extracts flom rumen fluid (lane 1-5, amplification flom
uninoculated rumen fluid, 102’103, 104, 105 cfu/ml, respectively) and silage
(lanes 6-10, amplification flom uninoculated silage, 102' 10°, 10°, 105
cfu/ml, respectively); lane 9, negative control; lane 10, 1 kb DNA

ladder ...................................................................... 1 18

Secondary PCR of rumen fluid samples: lane 1, uninoculated; lane 2, 102;
lane 3, 103; lane 4, 10‘; lane 5, 105cfu/ml; lane 6, negative control, lane 8,
lkb DNA ladder. .................................................... 119

Phylogenetic tree of the order Actinomycetales. The length of each pair of
branches represents the distance between sequence pairs, while the units at
the bottom of the tree indicate the number of substitution events 123

Partial nucleotide sequence of the 1,267 bp-flagment flom rumen fluid

(RF) sample using primer dhbl as forward primer and dhb2 as reverse
primer. .................................................... 124

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INTRODUCTION

Ensiling of feedstuffs is a means by which the livestock producer is assured of a
year-round feed supply. In the 1990’s, advances in silage technology have made silage
the principal method of forage preservation for dairy and beef cattle producers in North
America (Bolsen, 1998). The main goal of ensiling is to retain as much of the nutritional
value of the original crop as possible. The interaction of various biological and
technological factors during the ensiling process determines silage quality.

Silage is the product formed by the fermentation of grass or other material of
sufficient moisture content (Woolford, 1984). In addition to the oxygen-free
environment, the production of acids (mainly lactic and acetic acids) through the
fermentation of carbohydrates by anaerobic bacteria produces an environment that is
unfavorable for most spoilage microorganisms. The increase in acidity or decline in pH
depends on many factors such as the amount of ferrnentable carbohydrate in the crop to
be ensiled, its buffering capacity and dry matter (DM) content and the type and amount of
microorganisms that are present.

Silage additives had been used to either improve the nutritional value of the silage
or enhance the fermentation thereby reducing dry matter losses. Various silage additives
include fermentation stimulants such as bacterial inoculants and enzymes; fermentation
inhibitors such as organic acids (propionic, formic and sulfuric acids) and substrates or

nutrient sources, such as ammonia, urea, and anhydrous ammonia (Bolsen, 1998).
Propionic acid is one of the acids that are commonly used in silage due to its antifungal

properties. It is normally used at the rate of 1.5% of dry matter (Pitt, 1990). Propionic

 

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acid is used in the storage of grains (Patkar et al., 1995) and in silage preservation
(McDonald et al., 1991) to prevent mold growth. The occurrence of mycotoxins in foods
and feeds due to fungal colonization is a worldwide problem. In the United States, corn
is of a major concern because it is most susceptible to aflatoxin contamination (Wood,
1992). Even silage may also contain aflatoxin if corn is ensiled with the mycotoxin
present. Ground high moisture corn is potentially more hazardous because grinding and
the high moisture content favors the growth of toxigenic molds (Pier et al., 1992).

Propionic acid is commercially produced by petrochemical routes making its use
expensive (Playne, 1986). It is also corrosive. For these reasons, and the increasing
consumer preference for natural products, propionibacteria appear a viable alternative for
propionic acid production in situ. Propionibacteria has many industrial applications
(Boyaval and Corre, 1995) although they are most commonly used as starter cultures in
cheese in which they contribute to the development of the typical flavor and the
characteristic holes in Emmental, Gruyere and other Swiss-type cheeses (Perez-Chaia et
al., 1988). Propionibacteria have been used as natural sources of propionic acid in the
production of bakery products to improve shelf life (Javainen and Linko, 1993; Linko et
al., 1997). Mantere-Alhonen (1995) and Perez-Chaia (1999) reviewed studies in the use
of propionibacteria as probiotics. Their beneficial effects are derived from the
production of propionic acid, bacteriocins, vitamin B12, and ability to grow and survive
gastric digestion (Perez-Chaia et al., 1995a).

The use of propionic acid-producing bacteria (PAB) appears beneficial as an

inoculant for ensiling high moisture feeds. Improved fermentation was Observed in PAB-

treated silages but the effect on the aerobically exposed silages was variable (Florez-

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Galarza et al., 1985; Alio et al., 1994; Dawson, 1994; Weinberg et al., 1995,
Higginbothan et al., 1996 and 1998; Kreikemier et al 1998, Bolsen, 1998; Merry and
Davies, 1999). There is not enough information on the factors affecting the efficacy of
propionibacteria as silage inoculants.

Chapter 1 presents a review of literature on propionibacteria, their metabolism
and properties, and the use of their metabolic products such as propionic and acetic acids
as antimicrobial agents. The use of propionibacteria as silage inoculants is also
discussed. Chapter 2 deals with the use of propionibacteria and lactic acid bacteria as
inoculants for ensiling reconstituted high moisture corn. Chapter 3 evaluates the effect of
moisture on the efficacy of propionibacteria as silage inoculants in high moisture corn.
Chapter 4 presents a study on the vitamin B12 production of P. acidipmpionici DH42 in
a batch culture system. Chapter 5 highlights the development of a PCR-based assay for
the detection of P. acidipropionici DH42 in silage and rumen fluid samples. Chapter 6
gives an overview of the project findings and the recommendations from the results of the
studies.

The overall hypothesis of this project is that the addition of P. acidipropionici
DH42 to silage will increase the production of propionic acid in the ensiled high moisture
com. The increased propionic acid is expected to improve the aerobic stability of the
resulting silage by reducing yeast and mold counts, which is believed to be responsible

for aerobic instability.

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CHAPTER 1

REVIEW OF LITERATURE

Propionibacteria: Properties and Metabolism

Propionibacteria are pleomorphic, non-sporeforming, gram-positive rods that
frequently forms irregular clumps with the “Chinese-character” arrangements. They are
nonmotile, anaerobic to aerotolerant and form small raised colonies that are cream,
yellow, orange or deep red (Glatz, 1992). Their nutritional requirements have been well-
studied (Hettinga and Reinbold, 1972).

Propionibacteria have proteolytic activity. They contain at least two weak
proteinases, one is cell wall-associated and one membrane-bound (Langsrud et al., 1995).
The cell wall-bound enzyme acted preferentially on the B-casein while the second is
released at the stationary phase possibly by autolysis or excretion (Dupuis et al., 1995).
The caseinolytic activity is however low which is not sufficient for effective growth; thus
it is believed the enzyme has other functions. They also have a variety of peptidases and
can degrade many amino acids but large variations in species and strains were noted

(Perez-Chaia, et al., 1990; El-Soda et al., 1992; Langsrud et al., 1995).

Genetics of Propionibacteria
Studies on the genetic systems of propionibacteria are limited. Genetic studies
have been hampered by the lack of sufficient gene transfer systems and convenient
Cloning vector (Gautier et al., 1993). Manipulation of propionibacteria have been limited
'0 Selection of spontaneous mutants and those generated by mutagenesis with N-methyl-

N"?!itro-N-nitrosoguanidine (Glatz and Anderson, 1988). High propionic acid-producing

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strains have also been selected by growing P. acidipmpionici and transferring the strain
into growth medium with increasing levels of propionic acid (Glatz and Anderson, 1988).

Several plasmids in the propionibacteria have been characterized (Rehberger and
Glatz, 1990). Plasmid designated pRGOl is found alone in P. acidipropionici and P.
freudenreichii, and either alone or with other plasmid in P. jensenii. Another plasmid,
pRG02 is also found in P. jensenii. Plasmid pRG03 found in P. freudenreichii has been
linked to lactose fermentation while pRGOS found in P. jensenii, is associated with the
clumping phenomenon.

Bacteriophages have also been found in propionibacteria. In P. freudenreichii
isolated from Swiss cheese, bacteriophage concentration is about 7 x 105 phage forming
units and depends on the sample and strain used for detection (Gautier, et al., 1995a).
Bacteriophages were detected only when the population of propionibacteria reached 108
to 109 colony forming units per gram of cheese.

Many strains of propionibacteria are resistant to lysozyme (Johnson and
Cummins, 1972). Instead of protoplast formation, Gautier et al. (1995b) used
electroporation in which cells are exposed to rapid pulses of a high-strength electrical
field. The electrotransfection of P. freudenreichii with DNA phage produced 7x105
transfectants per pg of DNA under optimal conditions. For lysozyme-sensitive strains,
osmotically fragile cells are produced by exposure to 20 mg/ml of lysozyme for at least
15 min (Baehman and Glatz, 1989). Cells regenerate in 21 days from protoplasts overlaid

With soft agar that had 0.5 M sucrose and 2.5% gelatin.

Molecular '
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Molecular Detection of Propionibacteria

Differentiation of the four classical species of Propionibacteria (P.
acidipropionici, P. freudenreichii, P. jensenii, and P. thoenii) is based on five
biochemical characteristics, which include ability to ferment sucrose or maltose, and
reduce nitrate, and differences in B—hemolysis, color of pigment and isomer of
diaminopimelic acid in cell wall (Cummins and Johnson, 1986). However, distinction
between the species is often difficult since variation in phenotyphic characteristics is not
always reproducible (Grimont and Grimont, 1986; Riedel et al., 1994). Other species
identification techniques include analyses of cellular protein profiles (Baer, 1987; Riedel
and Britz, 1992), and plasmids (Rehberger and Glatz, 1990), immunoblotting (Baer and
Ryba, 1991), and genome analysis (Gautier et al., 1992; Rehberger, 1993). These studies
re-emphasized the problems associated with identification of propionibacteria.

The use of molecular methods in identification and detection of microorganisms
has been widely used in recent years. Ribosomal ribonucleic acid (rRNA) gene
restriction patterns are useful identification tools when biochemical tests are poor or
atypical (Grimont and Grimont, 1986). The rRNAs are ubiquitous and extremely
conserved molecules, which can be sequenced and compared to published or computer-
based data banks for species identification. The polymerase chain reaction (PCR) has
been used to amplify 16S rDN A of the four classical Propionibacteria species (Riedel et
al., 1994) and distinct patterns are observed for each species. Charfleitag and

StEleebrandt (1989) determined the intra-and intergeneric positions of
P r Opionibacterium by comparing l6S rRNA. P. acidipropionici, P. jensenii and P.

”’09?! ii constitute a phylogenetically tight cluster, while P. freudenreichii, P. acnes and

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P. propionicus are loosely related to each other. Likewise, they are not closely related to
the P. acidipropionici-P. jensenii-P. thoem'i cluster either. De Carvalho et al. (1994) and
Riedel and Britz (1996) used rRNA gene restriction patterns (ribotyping) to differentiate
the classical propionibacteria. Their study showed that the four species of
propionibacteria, P. freudenreichii, P. jensenii, P. acidipropionici and P. thoenii, gave
restriction patterns with species-specific fragments. Moreover, de Carvalho (1994) found
that ribotyping also allowed differentiation of P. freudenreichii sub. freudenreichii flom
P. freudenreichii sub. shermanii. Riedel and Britz (1996) on the other hand, found only a
single ribotype profile for the P. freudenreichii group and indicated that there is no need
to separate the group into subspecies. Rossi et al. (1997) reported the PCR-amplification
of the 168-238 spacer region to distinguish 67 strains of dairy propionibacteria. Their
findings validated the four current groupings of classical propionibacteria. They also
indicated that after Hinf I digestion of the 168-238 spacer region, P. freudenreichii, P.
jensenii and P. acidipropionici are easily recognized. Using the Randomly Amplified
Polymorphic DNA (RAPD)-PCR and Conventional Gel Electrophoresis Restriction
Endonuclease Analysis (CGE-REA), Rossi et al. (1998) found a clear distinction of the
current four clusters of propionibacteria. The techniques highlighted the presence of
particular phenotypic characters and allowed intra-specific differentiation. A recent
study developed a method to differentiate the Propionibacterium from other genera using
a modified multiplex-PCR approach (Dasen, et al., 1998). This method detects both
Classical and cutaneous propionibacteria species by the amplification of a
Propionibacterium-genus specific 900-bp. Rossi et al. (1999) had developed a genus-

and Species-specific PCR-based detection of dairy propionibacterium in milk, cheese, soil

and forage samples. They observed that in soil and forage samples, one-step PCR
amplification cannot detect cells lower than 105 and they recommended a double-step

amplification or nested PCR to improve detection.

Antimicrobial Action of Propionic Acid
In the USA, propionic acid and sodium and calcium propionates are considered
GRAS (Generally Recognized as Safe). The sodium and calcium salts are the forms
mainly used in food preservation. In breads and cakes, they are used against molds and
Bacillus mesentericus, the bacterium that causes ropiness in breads (Lueck, 1980).
In cheeses, where propionibacteria are used as starter cultures in combination with lactic
ac id bacteria, propionic acid is produced and provides some protection against molds
(Eklund, 1989).
Propionic acid had been widely used in the storage of grains, nuts, hay, and Silage
‘0 COntrol molding. Ranzani and Fonseca (1995) used ammonium propionate to
determine its effect on the growth of potentially aflatoxigenic fungi in unshelled peanuts
in Comparison with grapefruit seed extract and fungicides such as sodium
01THOphenylphenate and thiabendazole. They observed significant reduction in total
fungal and Aspergillusflavus parasiticus counts when ammonium propionate was used at
SOC)Omg/kg. Patkar et a1. (1995) noted that the incidence of Aspergillusflavus was
greatly reduced when propionic acid was added at Zullg in rice and sorghum and at 3qu
in gr0undnut. Eurotium sp was found in the propionic acid-treated grains and presumed
to be acid-tolerant. The effect Of propionic acid to Gibberella zeae, a common fungi
infeCting wheat and maize residues has also been studied. Khonga and Sutton (1991)

Obser‘ved with a 5% (w/v) solution, propionic acid suppressed production of perithecia

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under laboratory condition. Under field conditions, it reduced inoculum (spore)
production of G. zeae and encouraged its colonization by saprothrops. Toxicity of
propionic acid to fungi depends on pH. Strider and Winstead (1960) observed that
propionic acid is toxic to Cladosporim cucumerinum at 0.01 M at pH 5.0 but not at pH
6.0 indicating that the undissociated acid is the toxicant not its cation. Knapp et a1.
(1976) observed that propionic acid added at 1% to alfalfa hay showed no visible
molding, prevented heating, and reduced dry matter loss. The effect propionic acid has
also been tested in Salmonella. Since feeds are believed to be important source of
infection of food animals, chemical treatments are being explored to reduce the

recontamination' of feeds after heat treatment (Ha et al., 1998).

Several researchers have described the membrane-directed mode of action of
Propionic acid against bacteria (Sheu and Freese, 1972; Sheu et a1. 1972; Freese, et a1.
1973), and yeasts (Moon, 1983). Using whole cells of Bacillus subtilis or membrane
VeSic les, Sheu and Freese (1972) and Sheu et a1. (1972) observed that propionic acid and
Other short chain fatty acids inhibited cell growth which could be due to their inhibitory
effecr on the uptake of amino acids and other compounds necessary for growth or that
ATP generation which depends on the electron transport system is inhibited by fatty
ac ids. Moon (1989) also observed reduction in the cellular growth efficiency YATp

(defined as pg cells/mole glucose consumed) of Saccharomyces ovarum. Salmond et a].
(1984) suggested that the growth inhibition of organic acids to E. coli consists of two
Components, the inhibition of a metabolic function by the undissociated acid (HA), and a

generalized inhibition due to the acidification of the cytoplasm due to the accumulation

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propionic acids against acid-tolerant yeasts. In fungi, Strider and Winstead (1960)
suggested that propionic acid appears to act within the cell with C. cucumerinum, but
with Aspergillusflavus, it appears to be at the cell surface.

In addition to propionic acid, antimicrobial proteins or bacteriocins have also been
detected in propionibacteria. P. jensenii 126 produces a bacteriocin, Jenseniin G that has
inhibitory properties against dairy propionibacteria and several lactic acid bacteria
(Grinstead and Barefoot, 1992). Jenseniin G is stable at 100°C for 15 minutes and to pH
values ranging flom 3 to 12. Activity is detected in 50 to lOO-fold concentrated lO-day
old culture supematants (Grinstead and Barefoot, 1992). Another bacteriocin is
Propionicin PLG-lwhich is produced by P. thoenii 127 (Lyon and Glatz, 1991 and 1993).
Propionicin PLG-l inhibits gram-positive bacteria including P. thoenii, P.
“Cidipropionici, P. jensenii and lactic acid bacteria, gram-negative bacteria
(Pseudomonas, Vibrio, and Campylobacter Spp. and E. coli) and selected yeast and molds
(Lyon and Glatz, 1991). The bacteriocin is stable to temperature 5 85°C and to pH
between 3-9 and is produced at the late stationary phase in agar and broth cultures (Lyon
and Glatz, 1993). A more recent bacteriocin has been identified as being produced by P.
jensenii B1264 (Ratnam et al., 1999). The bacteriocin inhibited propionibacteria and

lactic acid bacteria and was bactericidal to Lactobacillus delbrueckii subs. lactis ATCC
4797. Its maximum activity was detected after 10 days of growth and 10-fold
Com3€=ntration. It is stable at 100°C for 60 minutes and to pH ranging from 2 to 10
(Ratnam et al., 1999). Bacteriocins had been examined as food preservatives (Lyon et al.,

1993).

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Interactions of propionibacteria with lactic acid bacteria
Propionibacteria and Iactobacilli can be found in mixed culture in many
environments such as in cheese, fermented milk, bakery products, and silage. In most
cases, a commensalistic form of interaction between propionibacteria and lactic acid
bacteria had been observed in co-cultures (Lee et al., 1976; Liu and Moon, 1982, Parker
and Moon, 1982; Perez-Chaia et al., 1987;1avainen and Linko, 1993; Perez-Chaia, 1994;
Piveteau et al., 1995; Jimeno et al., 1995). Lactobacilli can ferment glucose into lactic
acid. Propionibacteria can either use glucose or lactate as substrate although would
prefer glucose to the latter (Hettinga and Reinbold, 1972). The stimulatory effect of LAB
on PAB depends on the strains involved. Perez-Chaia et al. (1995b) observed that
Pl'Opionibacteria strains are inhibited in mixed cultures that rapidly reached low pH
Values. When grown in mixed cultures with different propionibacteria strains, only L.
helveticus ATCC 15009 showed the highest pH value allowing the pH sensitive
Propionibacteria to grow (Perez-Chaia et al., 1995b). Piveteau et a1. (1995) observed that
among five LAB Strains, only L. helveticus and Streptococcus thermophilus Stimulated P.
f'eudenreichii and P. acidipropionici. They found that the increase in growth rate and
Cell yield of P. freudenreichii in the presence of L. helveticus RR coincided with an
incil‘eased conversion of lactate to propionate and acetate. Increased cell yields and
gr OWth rates were also observed when P. shermanii and L. acidophilus were grown
together, which may be related to the availability or concentration of lactate (Lui and

M0011, 1982). Thierry et al. (1999) observed that when five strains of P. freudenreichii

were grown in media where different thermophilic lactic acid-producing bacteria had

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been previously grown, stimulatory effect to propionibacteria were observed with a low
proportion of L(+) lactate.

Propionibacteria can use the lactate produced by LAB from the fermentation of
glucose (Lee et al., 1976; Liu and Moon, 1982). LAB produce different isomers of
lactate. L delbrueckii subsp. lactis produces D lactate. S. thermophilus produces L
lactate while L helveticus produces both D and L lactate forms (Piveteau et al., 1995).

Some strains of PAB prefer the L lactate to the D lactate (Crow, 1986; Piveteau et al.,
1995). The consumption of lactate by propionibacteria can slow down the increase in
acidity, which inhibits the growth of the LAB.

Interactions between PAB and LAB are not limited to lactate production and
utilization. Lactobacilli can also benefit from mixed culturing with the added carbon
dioxide produced by propionibacteria (Friedman and Gaden, 1970). Moreover, in
Eminental cheeses, Baer (1995) observed that the growth of propionibacteria was
enhanced by the amino acids released due to the proteolytic activity of starter cultures
such as S. thermophilus and L. delbruickii. In whey, L. helveticus increased the levels of
anlino acids and peptideswhich stimulated the growth of P. freudenreichii (Piveteau et
31-. l 995). However, when the concentration of amino acids is too high, which were
Produced when highly proteolytic lactic bacteria were used, the growth of

Propionibacteria were inhibited (Baer, 1995). Thierry et al. (1999) observed similar
reSults where P. freudenreichii were stimulated by high peptide levels and low free amino
acid levels. This, however, conflicts with the results of Baer and Ryba (1999) who
obSel‘ved that high amino acids stimulated the growth of PAB, regardless of the

lactobacilli strain. Moreover, contrary to earlier observations of Jimeno et al. (1995),

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Baer and Ryba (1999) concluded that neither acetate, formate nor diacetyl, which is
formed when PAB is co-cultured with L. rhamnosus, inhibited the growth of
propionibacteria.

Inhibitory effect of propionibacteria on lactic acid bacteria has also been
observed. When glucose concentration is low, propionic acid has an inhibitory effect on
the growth of LAB (Perez-Chaia et al., 1994 a,b). The researchers suggested that the
propionic acid could diffuse inside the cells, increasing the inward leak of E into the

cells. To compensate for this, extrusion of the excess W by W-ATPase is needed but
when glucose is limiting, metabolism cannot supply the ATP required for the process.

Consequently, growth rate and biomass production of the lactic acid bacteria is reduced.

Production of Vitamin Bu

In addition to propionic acid, vitamin B]; is another major product of
Pr0pionibacterium spp. that has commercial importance. Among the propionibacteria
Strains, the P. freudenreichii and P. shennanii are commonly used. Industrial production
of Vitamin B12 uses Pr0pionibacterium spp. and Pseudomonas denimficans. However,
Since propionibacteria are slow-growers, they have not been used widely for the
CODImercial production of the vitamin.

Vitamin B12 is an essential part of enzyme systems that carry out basic metabolic
furKitions. Humans and animals depend on microbial synthesis for their supply of the
Vitamin. In ruminants, dietary cobalt appears to be the main limiting factor in its
Synthesis by ruminal microflora (Mc Dowell, 1989). However, Sutton and Elliot (1972)

found that on high concentrate diets, vitamin B12 synthesis is decreased and analogues,

whlch have little or no vitamin B12 activity are produced. The ruminal microorganisms

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such as Prevotella ruminicola (Strobel, 1992) and Bacteroides spp (Varel and Bryant,
1974; Chen and Wolin, 1981) require vitamin B12 for growth and propionate production.
Strobe] (1992) found that cell protein yields were reduced by 15 to 25% in the absence of
vitamin B12. In the synthesis of propionic acid, the enzyme methylmalonyl-COA mutase
that catalyzes the conversion of methylmalonyl-COA to succinyl-COA, is a coenzyme
B 1 z-dependent enzyme (Wood, 1981).
P. acidipropionici produces vitamin B12 intracellularly in the form of 5,6
dimethylbenzimidazoylcobamide (Quesada-Chanto et al., 1994a). Propionibacteria strain
arl AKU1251 can excrete the vitamin (mainly in the form of hydroxycobalamin) to the

culture medium (Yongsmith et al., 1982).

Different substrates have been used in the production of vitamin Biz. Using 10%

Whey solids and 1.5% yeast extract, Bullerman and Berry (1966) found vitamin B12
PFOduction of 8.43 pg/ml using P. shermanii. Using molasses, Quesada-Chanto et al.
(1 994a) observed 45 mg/l vitamin BIZ Produced by P. acidipropionici.

Many factors affect the production of vitamin B12, Bullerman and Berry (1966)
obServed increased vitamin B12 levels with the addition of cobalt and the vitamin B12
prficursor 5,6 dimethybenzimidazole (DMBI). Quesada-Chanto et al. (1994b) indicated
that for optimum production of vitamin Biz, cobalt ions, betaine and 5,6 DMBI must be
added to the growth medium at the rates of 5 mg/l, 5 g/l and 2 mg/l , respectively. Cobalt
ions are important in the formation of cobalamin. Betaine (trimethyglycine), on the other
hand. provides the methyl groups of the corrin ring in the synthesis of vitamin B12.

DIVIBI is necessary for the formation of 5,6 dimethylbenzimidazoyl cobamide. Yeast

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extract was found to stimulate growth and product formation of propionibacteria

(Quesada-Chanto et al., 1994b).

Culture conditions such as pH, temperature and aeration also affect vitamin B12
production. P. acidipropionici cultures are highly pH-dependent (Hsu and Yang, 1991).
Quesada-Chanto et al. (1994b) found that the optimal value for the production of
propionic acid and vitamin B12 is pH 6.5-6.8. They also recommended growing cultures
at 40°C for optimal production of vitamin Biz. While propionibacteria can be grown
under anaerobic conditions, they are oxygen-tolerant. With aeration, Bullerman and
Berry (1966) observed increased vitamin B12 yield and that the addition of precursor had
no effect on vitamin production with aeration. Quesada-Chanto et al. (1994b) found
Optimum propionic acid production under completely anaerobic conditions while aeration
Was required for vitamin B12 production. On the other hand, Yongsmith et al. (1982)
“Sing Propionibacterium sp trapped in urethane prepolymers found that vitamin B12

Production is less with aeration compared to static culture.

Silage and Silage Additives

Silage is the product formed by the fermentation of grass or other material of
Sufficient moisture content, generally greater than 50% (Woolford 1984; Bolsen, et al.,
1995). The main objectives of ensiling are to maintain anaerobiosis and to discourage the
proliferation of undesirable microorganisms such clostridia and enterobacteria
(McDonald et al., 1991). The latter is mainly accomplished by production of lactic acid,

which results in the pH reduction and deters the growth of undesirable microorganisms

dUring ensiling.

15

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The ensiling process involves several phases (Pitt, 1990: Bolsen et al., 1995). In
the aerobic phase, two plant activities occur: respiration and proteolysis. In addition,
aerobic microorganisms from the fresh crop predominate using up the oxygen trapped in
the ensiled material. In this phase, excessive heat production can occur reducing the
digestibility of protein and fiber constituents due to browning and Maillard reactions. In

the lag phase, plant cell membranes break down releasing cell juices that provide medium
for microbial growth. Facultative and obligate anaerobes proliferate in the fermentation
phase. The lactic acid bacteria rapidly grow and consume the water-soluble
carbohydrates producing mainly lactic acid. Acetic acid, ethanol, carbon dioxide and
some other minor products are also produced depending on the predominating LAB
strain. The production of lactic acid reduces pH to 3.5-5.0, which limits microbial
activity. In the stable phase, the availability of nutrients can affect the growth of lactic
acid bacteria. Limited water-soluble carbohydrates will slow down the rate of pH
deCline. Oxygen infiltrating the silo through openings in silo walls and plastic covers can
CaUse yeast and molds and other aerobic microorganisms to proliferate causing heating
and substantial dry matter loss. In the feedout phase, aerobic microbial activity
Predominates in the silage that is exposed to air. This is undesirable since significant
10568 in dry matter can occur through the consumption of residual soluble sugars, organic
acids and other fermentation products. Yeast and molds have been implicated in the

aerobic deterioration of silages.

The ideal crop for ensiling Should contain sufficient amount of fermentable

Substrate in the form of water-soluble carbohydrates, low buffering capacity and a dry

16

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matter content above 200 g kg". Since many crops used for ensiling do not fulfill these
requirements, material pretreatment and additives are often used (McDonald et al., 1991).
The moisture content of ensiling material affects not only the production of
effluent, which can affect silage quality, but also the availability of nutrients for
microbial growth and the rate of fermentation. If soluble carbohydrates are very high,
lactic acid bacteria rapidly proliferate and rapid pH decline is Observed. Dry matter
affects the production of organic acids in high moisture corn as well. Faber et al. (1989)
observed higher concentrations of lactic acid, acetic acid, ammonia nitrogen and lower
pH in low DM com (68 and 67% shelled com, 71 and 64% car com) compared to high
dry matter com (76% shelled corn and 79% car com). They also observed significant
interaction between dry matter and inoculation. In alfalfa silage, Garcia et al. (1989)
observed that lactic, acetic, and propionic acids are higher with low dry matter (46%
DM) as compared to the high dry matter silages (62%). The observed effects of moisture
on acid production can be attributed to the its effect on microbial metabolic activities.
All chemical reactions of cells require an aqueous environment although bacteria require

relatively higher levels of moisture for their growth than yeast and molds (Jay, 1998).

Microbial Inoculants

In the early studies, additives were used to ensure that lactic acid bacteria
dominate the fermentation process, although more recent developments have focused on
improving the nutritive value of the silage and reducing dry matter losses (McDonald et
al., 1991). Silage additives are classified into fermentation stimulants, fermentation

inhibitors, aerobic deterioration inhibitors, nutrients and absorbents (Woolford, 1985;

McDonald et al. , 1991)-

17

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Among the fermentation stimulants, it is the microbial inoculants that had
received much of the attention in recent years. Commercial microbial inoculants for
silage mainly include different strains of lactic acid-producing bacteria (LAB) such as
Lactobacillus sp., Pediococcus sp. and Enterococcus sp. (Weinberg et al., 1995a). Lower
pH, and higher lactic acid content in LAB-inoculated silages have been observed (Florez-
Galarza et al., 1985; Rust et al., 1989; Faber et al., 1989; Fitzsimons et al., 1992;
Wardynski et al., 1993; Weinberg, et al., 1995a). The increased production of lactic acid
causes rapid decline in pH, which retards the growth of undesirable microorganisms.
However, the stability of the ensiled material once the silos are opened has been
inconsistent. Upon exposure, the LAB-inoculated silages had heating, lower dry matter
recovery, faster deterioration than the uninoculated silages with no effect on the nutritive
value (Rust et al., 1989; Stokes, 1992; Sanderson, 1993; Wardynski et al., 1993).
Inoculation rate and the LAB species also affected silage stability. Lactobacillus
buchneri inoculated at 1x 106 cfu/g gave more stable corn silages compared to those
inoculated at the rate of 1x 105 cfu/g. Lactobacillus plantarum had minimal effects on
silage fermentation and aerobic stability compared to L buchneri—inoculated corn silages
(Ranjit et al., 1998). Reduced aerobic stability was also observed when a mixture of L.
plantarum and Enterococcusfaecium were used to ensile high moisture ear corn
(Sebastian et al., 1995).

The effect of bacterial inoculants in aerobic stability is unclear. Aerobic
instability has been mainly associated with the yeasts and molds. Lower yeast and mold
numbers were observed in the more stable high moisture corn silages (Hara and Ohyama,

1979; Rust and Yokoyama, 1992). Propionic acid has been used to prevent aerobic

18

 

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deterioration in rye grass silage (Hara and Ohyama, 197 8), and high moisture ear corn

silage (Sebastian et al., 1995).

Propionibacteria as inoculants

Recent studies explored the use of propionic acid bacteria to improve the aerobic
stability of silages. Lindgren et al. (1983) evaluated the use of P. shermam'i with LAB
as inoculants to grass silage. No increase in propionic and acetic acids in silages were
observed. Using 27% dry matter high moisture corn, Florez-Galarza et al. (1985)
observed lower pH and yeast population in those inoculated with P. shermanii.
Moreover, mold growth was prevented in all inoculated corn samples. Alio et al. (1994)
on the other hand, observed that PAB inoculation did not influence the aerobic stability
of 24% DM orange pulp silage. Weinberg et al. (1995a) in their study with pear millet
and maize silages observed marginal effect of P. shermanii inoculation with or without
LAB in the aerobic stability of the silages. Kreikeimeier et al. (1997) reported increased
propionate in the propionibacteria inoculated high-moisture corn after 90 d of ensiling.
They also observed increased growth performance of the finishing cattle fed with the
inoculated silage. Higginbotham et al. (1998), using whole plant corn inoculated with P.
acidipropionici alone or in combination with P. cerevisiae noted little effect of
inoculation on pH, concentration of water-soluble carbohydrates, lactic acid,
concentration of volatile fatty acids and on the aerobic stability of the exposed silages.
Dawson et al. (1998) evaluated the effects of P. acidipropionici DH42, a bacterium that
was isolated from high moisture corn (Dawson, 1994) on fermentation characteristics and
aerobic stability of ensiled high moisture corn. After 42 d of ensiling, increased

Propionic acid (0.35 vs. 0.03 g/ 100 g of dry matter), acetic acid, lower pH, lower yeast

19

 

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and mold counts and higher DM recovery were observed in the inoculated silages.
During aerobic exposure, the inoculated silages had higher propionic acid and recovery
of organic matter and the temperature remained unchanged indicating aerobic stability.
The inconsistent results on the use of propionibacteria as a silage inoculant only stress the
need for more studies on factors their efficacy. In a simulation model done by Pitt
(1997) on the growth and fermentation of propionic acid bacteria in silage, factors such

as pH, water activity, temperature, and concentration of organic acids were considered.

Objectives
The current research is aimed at conducting further studies on the use of P.

acidipropionici DH42 as an inoculant for high moisture corn. Specifically, the project

will be conducted to:

1. compare DH42 with commercial propionic acid-producing bacteria with or
without lactic acid-producing bacteria as inoculants to reconstituted high
moisture corn silage;

2. evaluate the effects of moisture content on the fermentation characteristics and
aerobic stability of DH42-inoculated high moisture corn silage;

3. determine vitamin B12 production of DH42 using batch cultures at two incubation
temperatures;

4. develop a PCR-based method of detecting DH42 in silage and rumen fluid

samples.

20

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CHAPTER 2
PROPIONIBACTERIA AND LACTIC ACID-PRODUCING BACTERIA AS
INOCULANTS FOR ENSILING RECONSTITUTED CORN
Abstract

The study was conducted to determine the effects of two propionibacteria
(Propionibacterium sp. P42 and P. acidipropionici DH42) and lactic acid bacteria as
inoculants to reconstituted rolled corn. Treatments were as follows: control (sterile
distilled water), DH42, P42, DH42+LAB, DH42+P42, DH42+P42+LAB, and autoclaved
DH42 culture. Microbial inoculants were added at the rate of 105cf11/g material. Silos
were opened after 7, 21, and 90 d post-ensiling. Samples from the 90-d ensiling period
were used for aerobic stability evaluation. Fresh, fermented and exposed corn samples
were taken for chemical and microbial analyses.

Inoculation enhanced the fermentation of the reconstituted corn up to 21 d of
ensiling. The combination of P. acidipropionici DH42 with lactic acid bacteria as
inoculants reduced silage pH and butyric acid and increased propionic, acetic and lactic
acids. LAB inoculation did not significantly increase the LAB population in the treated
silages.

During aerobic exposure, all the silages appeared well-preserved. Organic acid
levels remained stable throughout the exposure period. However, the silages treated with
P. acidipropionici DH42 + LAB had higher average propionic and lactic acids and lower
pH than the control. Propionibacteria inoculation did not significantly reduce the yeast

and mold counts since the control silages also appeared well-preserved.

21

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Introduction

In recent years, ensiling high moisture corn has gained wide acceptance for
several reasons. Ensiling eliminates the added cost of drying and corn can be harvested 2
to 3 weeks earlier thereby reducing field losses. However, microbial deterioration due to
mold and spoilage organisms can reduce the quality of high moisture corn.

Propionic acid has been used as a preservative to improve storage life of high
moisture corn (Deyoe et al., 1973; Burrell et al., 1973; Jones et al., 1974). In silage, the
benefits of propionic acid are attained with the addition of l-l.5% to the material
(Thomas, 1978). More recently, the use of propionibacteria has been explored as an
alternative to chemical treatments. Lindgren et al. (1983) observed no propionic acid in
the silages inoculated with P. shermanii and lactic acid bacteria. On the other hand,
Flores-Galarza (1985) observed improved reduction in the yeast and mold counts of
ensiled high moisture corn with P. shermanii. Weinberg et al. (1995), Kreikeimer et al.
(1997) and Higginbotham et al. (1998) observed marginal improvement in fermentation
and aerobic stability of propionibacteria-inoculated silages. Dawson et al. (1998)
observed improved fermentation and aerobic stability in high moisture corn inoculated
with P. acidipropionici DH42.

This study was conducted to determine the efficacy of P. acidipropionici DH42 as
a silage inoculant with or without lactic acid bacteria and to detemrine whether the co-
inoculation with another propionibacterium strain, Propionibacterium sp. P42 and LAB
would be advantageous.

The hypothesis of this study is that the addition of propionibacteria is expected to

enhance the fermentation of high moisture corn and reduce the yeast and mold counts in

22

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the silage particularly during aerobic exposure. Moreover, the addition of LAB as a co-

inoculant to PAB is expected to enhance the performance of PAB.

Materials and Methods

Inoculants

The three inoculants used were: P. acidipropionici DH42 (ATCC 55737) which
was taken from cultures maintained at the microbiology laboratory of the Department of
Animal Science, Michigan State University, East Lansing, Michigan. (MSU, East
Lansing, MI), Propionibacterium sp. P42 (Laporte Biochem International, Milwaukee,
WI) and lactic acid-producing bacteria (Laporte Biochem International, Milwaukee, WI).
Propionibacterium sp. P42 and the lactic acid-producing bacteria were received as
freeze-dried cultures and rehydrated with sterile distilled water according to the
manufacturer’s instructions about 30 min before use.

P. acidipropionici DH42 was grown in 0.5X Lactobacilli MRS broth (Difco).
Before use, at least three successive transfers were done to ensure its activation. P.
acidipropionici DH42 was incubated for 18 h at 39°C. The amount of P. acidipropionici
DH42 needed to meet the required inoculation rate of 105 cfu/g material was calculated
from the linear regression formula which had been established beforehand. Based on the
OD value of the culture, estimated cfu/ml was calculated. Serial dilutions of the culture

were also made and plated in Lactobacilli MRS agar (Difco) to verify the calculated

counts.

23

Silage Preparation

Dried corn was rolled and reconstituted by adding distilled water to adjust the
moisture content to 28%. Separate gloves and mixing tubs were used for each treatment
to prevent cross-contamination. Laboratory silos (47.5 x 10.2 cm diameter) were used.
The silos were made of polyvinyl chloride (PVC) pipes with rubber caps fitted with
rubber policeman (Bolsen, 1992). There were seven inoculant treatments: control (sterile
distilled water), DH42, P42, DH42+LAB, DH42+P42, DH42+P42+LAB, and autoclaved
DH42 cultures. All inoculants were added at the rate of 105cfu/g of material. The latter
treatment was used to determine the effect of the culture medium that was added with the
DH42 cultures. It was prepared by autoclaving the DH42 culture for 15 min at 17 psi to
kill all bacteria. The appropriate amounts of inoculants were measured and the volume
adjusted to 100 ml by adding sterile distilled water. The inoculants were added to the
corn and mixed by hand in a tub. For (1 0, two samples were taken from each treatment.
Three silos were prepared for each treatment and ensiling period combination. Silos were
opened after 7, 21 and 90 d of ensiling. Silos were weighed before and after ensiling and
the dry matter recovery was determined. lFresh, fermented and exposed corn samples

were taken for chemical and microbial analyses.

Aerobic Stability

The silage from the 90-d ensiling period was used for the aerobic stability
evaluation. About 1 kg from each silo was measured into a plastic bag, placed in a
Styrofoam container and exposed to air for 5 (1. Samples for chemical and microbial
analyses were taken after 1, 3, and 5 d of exposure. Cooking thermometers were inserted

into the silage to monitor temperature. Temperatures were taken daily.

24

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Chemical Analyses

The dry matter content was determined by drying the samples in a forced air oven
(55°C) for 48h. Aqueous extracts from each sample were obtained by mixing 50 g of
corn in 450 ml of sterile 0.9% saline using a Stomacher (Model 3500, Tekmar) for 5 min
and strained through four layers of cheese cloth into 50-ml centrifuge tubes. The pH of
the extracts was taken. Glucose, ethanol and the organic acids were determined by HPLC
as described by Dawson (1994) with slight modification. Instead of filtering the samples,

they were centrifuged (26,000 x g) for 30 min and the supernatant used for analyses.

Microbial Analyses

Serial dilutions of the extracts were prepared in 1% peptone broth (Difco) and
appropriate dilutions were plated in selective media. Rogosa SL Agar (Difco) was used
to enumerate lactic acid bacteria and Rose Bengal Agar (Difco) with Antimicrobic

Supplement C (Difco) was used to estimate yeast and molds. Culture plates were

incubated at 39°C.

Statistical Analyses

Data for the fermentation phase were analyzed as a one-way completely
randomized design using the General Linear Model subroutine of the Statistical Analysis
System (SAS, 1990). Microbial counts were analyzed using the transformed data (logm
[Y], where Y is the microbial count). Due to the increasing variability of the various
parameters over time, the data were analyzed separately for each collection period (i.e. d
0, 7, 21, 90). At a given collection period, the model used for each parameter (e. g. pH,

Propionic acid, acetic acid) was as follows:

25

Y, = u+oq+ei

Where:
Y = individual variable measured (e.g. pH, propionic acid, glucose)
p. = overall mean
or, = effect of treatment
e, = random residual error

For the aerobic stability phase, data were analyzed by PROC MIXED of the SAS,
using the repeated measures analysis (SAS, 1990) since repeated samplings from the

same sample were done. Treatment means were compared using the Bonferroni (SAS,

1990).

Results and Discussion
Fermentation Phase

The initial dry matter content (Table 2-1) differed (P<0.05) among treatments,
ranging from 69.60-73.57%. This was possibly due to the uneven mixing of water when
the corn was reconstituted. The control had the highest dry matter throughout the ensiling
period. The control had 73.57% dry matter at the start of ensiling and 72.91% after 90 d
of ensiling. The autoclaved DH42, on the other hand, had the lowest dry matter with
slightly below 70% during the fermentation phase.

Propionic acid (Table 2-2) was detected in the silages after 7 d of ensiling.
Propionic acid levels ranged from 0.02-0.04 g/ 100 g DM. The lowest (P<0.05) levels
were observed with DH42 (autoclaved), P42+DH42+LAB and DH42 + LAB. The other
treatments had similar propionic acid levels. After 21 d of ensiling, the combination of

DH42 and P42 gave the highest level with 0.10 g/ 100 g DM followed by DH42 alone

26

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Table 2-1. Dry matter content (%) of fresh and ensiled HMC

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 69.60 d 69.79 c 69.38 d 69.65 d
DH42+P42+LAB 71.90 ° 71.64 b 71.26 b 71.30 b“
DH42+LAB 72.01 c 72.00 b 71.78 b 72.21 3"
DH42+P42 72.45 be 72.16 b 71.76 b 71.99 3"
DH42 70.20 “ 70.44 ° 70.15 ° 70.56 °"
P42 72.80 a” 72.47 a” 71.82 b 71.82 “C
Control 73.57 a 70.32 a 73.03 a 72.91 ‘*
S.E.M." 0.05 0.07 0.05 ' 0.10

 

Elbe“Column means with unlike superscripts differ (P<0.05). .
nStandard error of the mean.

Table 2-2. Effect of inoculation on propionic acid content (g/100 g DM) of fresh and
ensiled HMC

 

 

Treatment Ensiling period ((1)
0 7 21 90
DH42 (autoclaved) 0.00 0.02 b 0.030 'x 0.11
DH42+P42+LAB 0.00 0.02 b 0.007 d 0.12
DH42+LAB 0.00 0.02 b 0.007 d 0.11
DH42+P42 0.00 0.03 a 0.098 a 0.12
DH42 0.00 0.04 a 0.053 b 0.11
P42 0.00 0.04 a 0.029 °“ 0.07
Control 0.00 0.04 a 0.041 ”C 0.07
S.E.M.“ 0.001 0.002 0.004

 

abedColumn means with unlike superscripts differ (P<0.05).
nStandard error of the mean.

27

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(0.05 g/ 100 g DM). After 90 d, differences in the propionic acid levels among
treatments were not significant (P>0.05). The silages treated with the combination of
DH42 + P42 +LAB had 0.12 g/ 100 g DM propionic acid. The data suggest that addition
of propionibacteria did not enhance the production of propionic acid in the silages and
that there was no advantage of adding LAB. In a modeling study done by Pitt (1997),
greater accumulation of propionate was predicted with the co-culture of LAB with
propionibacteria due to the greater production of lactate increasing the substrate for
propionibacteria. Results of this study did not verify or support this hypothesis.
Lindgren et al. (1983) also observed no increase in propionic or acetic acid when a mix
culture of P. shemzanii and lactic acid bacteria were used as silage inoculants. Weinberg
et al. (1995 a,b) observed similar results. Conversely, Kreikemier et al. (1997) observed
increased propionate when LAB and propionibacteria were inoculated into high moisture
corn but their values were much lower than the values observed in this study. Their silos
were however stored at 16°C, which could have affected the growth of the inoculant.

Acetic acid (Table 2-3) was not observed until after (1 7 of ensiling. On (1 7, acetic
acid levels were higher (P<0.05) in the HMC treated with the autoclaved DH42 than the
control and the DH42+LAB. Differences among treatments were not significant after 21
and 90 d of ensiling. Since propionibacteria produce acetic acid in addition to propionic
acid, higher levels of acetic acid are expected when propionibacteria are used as starter
cultures. However, the control had acetic acid levels that were comparable to inoculated
sflages.

LAB inoculation increased lactic acid (Table 24) production in the treated

silages. On (1 7, the combination of DH42+P42+LAB had higher (P<0.05) lactic acid

28

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Table 2-3. Effect of inOculation on acetic acid content (g/ 100 g DM) of fresh and ensiled
HMC

 

 

Treatment Ensilin g period (d)

0 7 21 90
DH42 (autoclaved) 0.00 0.12 a 0.15 0.45
DH42+P42+LAB 0.00 0.10 a” 0.13 0.31
DH42+LAB 0.00 0.07 b 0.15 0.26
DH42+P42 0.00 0.07 a” 0.16 0.41
DH42 0.00 0.08 3" 0.15 0.35
P42 0.00 0.07 a” 0.12 0.32
Control 0.00 0.05 b 0.17 0.34
S.E.M.“ 0.004 0.004 0.014

 

abColumn means with unlike superscripts differ (P<0.05).
nStandard error of the mean.

Table 2-4. Effect of inoculation on lactic acid content (g/ 100 g DM) of fresh and ensiled
HMC .

 

 

Treatment Ensiling period (d)

0 7 21 90
DH42 (autoclaved) 0.00 1.29 “b 1.60 a” 1.06
DH42+P42+LAB 0.00 1.36 a 1.59 3" 1.13
DH42+LAB 0.00 0.94 at” 1.74 a 1.46
DH42+P42 0.00 0.82 a“ 1.48 3" 1.10
DH42 0.00 0.94 a“ 1.84 a 1.15
P42 0.00 0.67 b° 1.17 b 0.80
Control 0.00 0.61 ° 1.20 b 0.79
EM.“ 0.05 0.04 0.05

 

abcColumn means with unlike superscripts differ (P<0.05).

"Standard error of the mean.

29

,ij .

l’l

‘ 1

‘. i;
l‘x‘b ‘
of”: ';
“1.4:“-~

level than P42 and control silages. After 21 d of ensiling, DH42 + LAB and DH42 had
higher (P<0.05) lactic acid levels than P42 and control. However, after 90 d of ensiling,
differences among treatments were not significant. Lactic acid levels observed in this
study are lower than that reported by Wardynski et al. (1993), but higher than that
reported by Phillip and Fellner (1991). Differences in lactic acid production could be due
to differences in grain processing (whether whole or rolled) and dry matter content of the
ensiling material. Both influence the fermentation of lactic acid bacteria by affecting the
substrate available for fermentation (Pitt et al., 1985; Muck, 1990) and water activity.
Goodrich et al. (1975) also observed that com ensiled at harvest had higher lactic acid
content than corn that was dried and reconstituted. Moreover, de Vries et al. (1970) and
Thomas et al. (1979) observed that homofermentative lactic acid bacteria shifts to a
heterofermentative mode of fermentation when glucose is limiting. Instead of only lactic
acid being produced, other metabolites such as acetate, ethanol and formate are formed.
Table 2-6 shows that glucose was rapidly used by d 7 of ensiling.

The effect of lactic acid bacteria inoculation in high moisture corn silage is
variable. While Schaeffer et al. (1989) observed higher lactate in the inoculated silages
(P<0.05), Phillip and Fellner (1992) observed marginal increase (P<0.10) while
Wardynski et a1. (1993) observed higher lactate in the uninoculated silages. This could
be due to differences in the epiphytic lactic acid bacteria numbers in the initial ensiling
material. When there are sufficient bacteria in the material, the added bacterial inoculant
does not appreciably affect the total LAB population. Bolsen et a1. (1996) pointed out
that strain selection is as important as the number of lactic acid bacteria applied per gram

of cr0p.

30

 

as;

-_-..b ..
v

4
w
11-

. .-
4.44;..5‘
-41.». .2

-‘-...c
.“\ \‘
I4-54-

e
’32“;
‘ "bt.>\

.
5"...
h. '3. ‘1‘
""‘ll‘
1
u.
'T'F-o.

"1 «it:

Significant differences in initial pH (Table 2-5) of the corn samples were
observed. The DH42+P42 and DH42 silages had the highest (P<0.05) pH values while
the silages with autoclaved DH42 culture had the lowest pH. After 7 d of ensiling, the
control and the P42 silages had the highest pH with 4.26. On (1 21, all the inoculated
silages had lower (P<0.05) pH values compared to the control. By (1 90, DH42+LAB
silages had lower pH than the silages treated with autoclaved DH42 culture but not
different from the control. The decrease in pH observed in this study is consistent with
results of other studies that used LAB as inoculants (Bolsen et al., 1996).

Glucose content (Table 2-6) of the corn varied initially and ranged from 0.16 to
0.42 g/ 100 g DM. After 7 d of ensiling, more than 90% of the glucose was utilized.
Thereafter, glucose changed little until (1 90 where it was hardly detectable in all
treatments. Decrease in glucose is expected since bacteria use it as a substrate. However,
decrease in pH also promotes hydrolysis of sugars from the cell walls (Jones et al., 1992).
But, it is unlikely that the pH differences were sufficiently large to create a difference in
glucose release from cell walls. All of the glucose was essentially utilized during the
fermentation phase.

Butyric acid (Table 2-7) was detected only after 21 d of ensiling. In silage,
butyrate production is expected at the latter stage of ensiling and is mainly attributed to
clostridial fermentation. However, yeasts and Bacillus sp. can also produce butyrate (Mc
Donald et al., 1991). On (1 21, butyric acid was higher in the silages treated with
autoclaved DH42 culture than the control, DH42+LAB and DH42+P42+LAB. On (1 90,

highest (P<0.05) butyric acid levels were detected in autoclaved DH42-treated silages.

31

9-.

L“. If

.

S‘Lln.

vine l"
‘ -C

H“

Table 2-5. Effect of inoculation on the pH of fresh and ensiled HMC

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 5.28 d 4.10 d 4.16 “d 4.34 "
DH42+P42+LAB 5.44 ° 4.08 d 4.12 d 4.27 “b
DH42+LAB 5.55 b 4.10 d 4.14 d 4.13 "
DH42+P42 5.66 a 4.21 b 4.23 ”c 4.26 3"
DH42 5.69 a 4.16 C 4.14 d 4.25 3"
P42 5.52 b 4.26 a 4.26 b 4.32 a“
Control 5.53 b 4.26 a 4.36 a 4.29 3"
S.E.M.“ 0.01 0.01 0.01 0.01

 

abc°Column means with unlike superscripts differ (P<0.05).
uStandard error of the mean.

Table 2-6. Effect of inoculation on glucose content (g/ 100 g DM) of fresh and ensiled
HMC

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 0.32 b 0.02 0.00 b 0.00
DH42+P42+LAB 0.16 ° 0.01 0.02 a 0.00
DH42+LAB 0.42 a 0.01 0.02 a 0.01
DH42+P42 0.32 b 0.01 0.01 a” 0.00
DH42 0.40 a 0.02 0.01 a” 0.00
P42 0.42 a 0.01 0.00 b 0.00
Control 0.33 b 0.01 0.00 b 0.00
slew 0.002 0.001 0.001 0.001

 

abcColumn means with unlike superscripts differ (P<0.05).
“Standard error of the mean.

32

1’s;

:.

\:D
K

Table 2-7. Effect of inoculation on butyric acid content (g/ 100 g DM) of fresh and
ensiled HMC.

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 0.00 0.00 0.055 a 0.16 a
DH42+P42+LAB 0.00 0.00 0.020 d 0.08 1’
DH42+LAB 0.00 0.00 0.024 °" 0.08 "
DH42+P42 0.00 0.00 0.053 a” 0.10 "
DH42 0.00 0.00 0.050 a” 0.11 3"
P42 0.00 0.00 0.046 a” 0.11 a”
Control 0.00 0.00 0.039 ”c 0.07 b
S.E.M." 0.001 0.004

 

a"""Column means with unlike superscripts differ (P<0.05).
nStandard error of the mean.

Through out the ensiling period, ethanol levels (Table 2-8) were not affected by
inoculation. This is in contrast to results of Dawson et al. (1998) where DH42-inoculated
silages had lower ethanol levels. Bolsen et al. (1996) also reviewed studies on the use of
lactic acid bacteria as silage inoculants and found that over 90% of the inoculated silages
had lower ethanol contents. Ethanol is produced by yeast and heterofermentative lactic
acid bacteria (Mc Donald et al., 1991). However, L. brevis which is the most common
heterofermentative lactic acid bacteria found in high moisture corn does not ferment
glucose anaerobically to ethanol (McDonald et al., 1991).

The initial citric acid levels (Table 2-9) were significantly different among
treatments. P42 had the highest level (0.16 g/ 100 g DM) while the DH42+P42+LAB
had the lowest (0.05 g/ 100 g DM). After 90 d of ensiling, the treatment with the highest

citric acid content was the silages with the autoclaved DH42 culture (0.22 g/ 100 g DM)

33

”.1
‘.9--
‘344
fi..
A.
P?"
v'si‘
”L:
var"
5.":
1.1"“.

0‘

2.: 3

Table 2-8. Effect of inoculation on ethanol content (g/ 100 g DM) of fresh and ensiled
HMC.

 

 

Treatment Ensiling period (d)

0 7 21 90
DH42 (autoclaved) 0.00 0.35 0.38 0.51
DH42+P42+LAB 0.00 0.28 0.32 0.28
DH42+LAB 0.00 0.21 0.37 0.38
DH42+P42 0.00 0.31 0.43 0.49
DH42 0.00 0.28 0.38 0.41
P42 0.00 0.26 0.42 0.46
Control 0.00 0.22 0.35 0.40
S.E.M.° 0.02 0.01 0.02

 

uStandard error of the mean.

Table 2-9. Effect of inoculation on citric acid content (g/ 100 g DM) of fresh and ensiled
HMC.

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 0.11 b 0.00 0.17 a 0.22 "
DH42+P42+LAB 0.05 ° 0.00 0.16 a” 0.17 “b
DH42+LAB 0.11 b 0.00 0.17 a 0.15 b
DH42+P42 0.08 d 0.00 0.17 a 0.15 b
DH42 0.09 C 0.00 0.15 a” 0.15 b
P42 0.16 a 0.00 0.11” 0.13 b
Control 0.11 b 0.00 0.13 “b 0.13 b
LEM.“ 0.001 0.004 0.004

 

abodeColumn mean with unlike superscripts differ (P<0.05).
"Standard error of the mean.

34

which was not different with the DH42+P42+LAB (0.17 g/ 100 g DM). The inoculation
did not affect dry matter recovery (Table 2-10) throughout the ensiling period. On (1 90,
dry matter recovery ranged from 96.14-99.26%. The initial, 01 7 and d 90 LAB
population (Table 2-11) were not affected by inoculation. On d 21, silages with
DH42+LAB and DH42+P42+LAB had lower (P<0.05) LAB counts than control silage.
The LAB counts in the control silages were high and may have limited the effectiveness
of the LAB portion of the inoculant. In a similar study, Wardynski et a1. (1993) did not
observe differences in the LAB population between the control and inoculated silages and
they indicated that the epiphytic bacterial population was much greater than the amount
added by the inoculant.

The initial and d 21 yeast and mold counts (Table 2-12) did not significantly
differ among treatments. On d 90, the control and P42 silages had lower (P<0.05) yeast
and mold counts compared to the DH42+P42+LAB-treated silages. Propionibacteria
inoculation is expected to increase the propionic acid content of the silages. However,
the d 90 propionic acid levels were not significantly different among treatments (Table 2-
2). Dawson (1994) and Wardynski et al. (1993) showed reduced yeast and mold counts
with PAB—inoculated HMC. Levels of propionate in the HMC was much higher than in
their studies, than seen in this study (0.12 g/100g DM). At low propionic acid
concentration, acid-tolerant yeasts are able to metabolize the acid (Lord et al., 1981;

Magan and Lacey, 1986) further reducing the acid available.

Aerobic Phase
Propionic acid contents (Table 2-13) of the exposed silages differed (P<0.01)

among treatments. The DH42, DH42+P42+LAB, and autoclaved DH42-treated silages

35

 

1‘11
713-111

a 4:,
DH42+L
Dinar
Dan

Pl:

Etarot

/.

zt
Column

338410

Table 2-10. Effect of inoculation on dry matter recovery (%) of ensiled HMC.

 

 

Treatment Ensiling period (d)

7 21 90
DH42 (autoclaved) 98.72 99.96 98.86 3"
DH42+P42+LAB 99.84 99.88 96.14 b
DH42+LAB 100.15 101.04 98.91 3‘”
DH42+P42 100.17 101.56 98.08 3"
DH42 98.46 99.46 99.26 3
P42 99.90 102.06 97.97 a”
Control 100.32 101.26 97.94 3"
S.E.M.“ 0.24 0.51 0.26

 

at’Column means with unlike superscripts differ ( P<0.05).
nStandard error of the mean.

Table 2-11. Effect of inoculation on lactic acid bacteria counts (log cfu/ g DM) of fresh
and ensiled HMC.

 

 

Treatment Ensiling period (d)

0 7 21 90
DH42 (autoclaved) 6.37 8.64 8.95 a 8.02
DH42+P42+LAB 6.32 8.62 8.63 °° 8.40
DH42+LAB 6.54 8.64 8.61 c 8.51
DH42+P42 6.33 8.60 8.86 ab 7.57
DH42 6.46 8.59 8.89 a 8.16
P42 6.34 8.58 8.84 a“ 8.09
Control 6.20 8.62 8.93 a 8.45
S.E.M." 0.05 0.02 0.02 0.13

 

°°°Column means with unlike superscripts differ (P<0.05).
"Standard error of the mean.

36

Table 3-1

‘ .
...4 H
5.21le

Ire

N, . .
11:14: 1111
D311}?-

DH4I~L

lit-5's 11

L111 .
«WC srl;

Table 2-12. Effect of inoculation on yeast and mold counts (log cfu/ g DM) of fresh and
ensiled HMC.

 

 

Treatment Ensiling period ((1)

0 7 21 90
DH42 (autoclaved) 7.69 5.59 b 4.00 3.25 3"
DH42+P42+LAB 7.63 6.22 “b 4.11 3.78 “
DH42+LAB 7.76 5.89 8" 4.11 2.35 3"
DH42+P42 7.47 6.27 ab 4.01 2.97 3"
DH42 7.50 5.95 a” 4.15 2.44 3"
P42 7.62 6.18 “b 3.97 2.18 b
Control 7.48 6.69 a 4.18 1.98 "
S.E.M.° 0.05 0.04 0.08 0.11

 

all’Column means with unlike superscripts differ (P<0.05).
11Standard error of the mean.

Table 2-13. Effect of inoculation on propionic acid content (g/100 g DM) of exposed
HMC silage

 

 

Treatment Exposure period ((1) Average
0 l 3 5
DH42 (autoclaved) 0.11 0.12 0.14 0.10 0.12 a
DH42+P42+LAB 0.12 0.1 1 0.12 0.11 0.12 “
DH42+LAB 0.11 0.09 0.12 0.11 0.11 3"
DH42+P42 0.12 0.11 0.09 0.13 0.11 “b
DH42 0.11 0.11 0.13 0.12 0.12“
P42 0.07 0.08 0.09 0.09 0.08 b
Control 0.07 0.08 0.10 0.09 0.08 b
S.E.M.“ 0.01 0.01 0.01 0.01 0.01

 

abColumn means with unlike superscripts differ(P<0.07) except DH42+P42+LAB vs.
control, P<0.09; DH42 vs. control, P<0.10.
“Standard error of the mean.

37

153:6 of da
ctgt‘lsure Vt C

lacti
8 loan
its control a
at: levels c
resection v
at". inocula

V1 , ‘-
trio d1. er

:erterlt of L
28mm stud

,4... ,
l'-‘-’?€T'.r€8 ls

had significantly higher propionic acid than the control and P42-treated silages. The
effects of day and treatment x day interaction on propionic acid level during aerobic
exposure were not significant.

Lactic acid (Table 2-14) levels were also affected (P<0.002) by treatment. The
average lactic acid content of the DH42+LAB silages was higher (P<0.01) compared to
the control and P42 silages with 1.36 vs. 0.83 and 0.86 g/100 g DM, respectively. Lactic
acid levels changed little during exposure period. The effects of day and treatment x day
interaction were not significant. Dawson et al. (1998) reported higher residual lactic acid
with inoculated silages after 5 d of exposure using P. acidipropionici DH42. It could be
due to difference in the ensiling material. They used high moisture corn while this study
used reconstituted corn. Phillip and Fellner (1992) observed a 51% decrease in lactic acid
content of LAB-inoculated high moisture ear corn during aerobic exposure. Unlike the
present study, they did not detect any of the volatile fatty acids whose antimicrobial
properties would have reduced the assimilation of lactic acid by aerobic microorganisms.
The effect of treatment on pH (Table 2-15) was significant (P<0.03). The effect of day
was also significant (P<0.001) but not the day x treatment interaction. The silages
inoculated with DH42+LAB had significantly lower pH than the P42 and autoclaved
DH42-treated silages with 4.15 vs. 4.29 and 4.32, respectively.

Acetic acid (Table 2-16) levels were also relatively stable over time. HMC
treated with autoclaved DH42 culture had higher average acetic acid content compared to
the control, DH42+P42+LAB and DH42+LAB silages. Wardynski et al. (1993) had
higher acetic acid values with at least 3 g/ 100 g DM. Differences in ensiling material

could account for this discrepancy. This study used reconstituted com and had a lower

38

Table 2-14. Effect of inoculation on the lactic acid content (g/ 100 g DM) of exposed
HMC silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5

DH42 (autoclaved) 1.06 0.95 0.95 0.87 0.96 b
DH42+P42+LAB 1.13 1.05 1.14 1.11 1.11 3"
DH42+LAB 1.46 1.22 1.33 1.44 1.36 “
DH42+P42 1.10 0.95 1.06 0.95 1.01 ”
DH42 1.15 1.02 1.08 1.20 1.11 “b
P42 0.80 0.81 0.89 0.92 0.86 "
Control 0.79 0.77 0.87 0.88 0.83 "
S.E.M° 0.11 0.11 0.11 0.11 0.07

 

all’Column means with unlike superscripts differ (P<0.05) except DH42+LAB vs. P42 and
DH42+LAB vs. control, P<0.01 and DH42+LAB vs DH42+P42, P<0.10.
l’Standard error of the mean.

Table 2-15. Effect of inoculation on pH of exposed HMC silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5

DH42 (autoclaved) 4.34 4.32 4.30 4.34 4.32 a
DH42+P42+LAB 4.27 4.26 4.21 4.27 4.25 3"
DH42+LAB 4.13 4.16 4.13 4.18 4.15 b
DH42+P42 4.26 4.27 4.27 4.29 4.27 3"
DH42 4.25 4.25 4.23 4.28 4.25 3"
P42 4.32 4.31 4.29 4.33 4.31 a
Control 4.29 4.28 4.28 4.32 4.29 3"
S.E.M“ 0.03 0.03 0.03 0.03 0.04

 

abColumn means with unlike superscripts differ (P<0.05) except DH42 (autoclaved) vs.
DH42+LAB, P<0.03.
nStandard error of the mean.

39

Table 2-16. Effect of inoculation on acetic acid content (g/100 g DM) of exposed HMC
silage

 

 

Treatment Exposure period ((1) Average
0 1 3 5

DH42 (autoclaved) 0.45 0.51 0.57 0.48 0.50 a
DH42+P42+LAB 0.31 0.30 0.31 0.33 0.31 ”c
DH42+LAB 0.26 0.25 0.30 0.32 0.28 c
DH42+P42 0.41 0.37 0.45 0.40 0.40 3"
DH42 0.35 0.34 0.39 0.44 0.38 3‘”
P42 0.32 0.35 0.40 0.42 0.37 “C
Control 0.34 0.37 0.44 0.43 0.39 ”C
S.E.M° 0.04 0.04 0.04 0.04 0.03

 

abcColumn means with unlike superscripts differ (P<0.07) except DH42 (autoclaved) vs.
DH42+P42+LAB and DH42 (autoclaved) vs. DH42+LAB, P<0.002.
“Standard error of the mean.

moisture content compared to that used by Wardynski et al. (1993). In general, high
moisture enhances the production of organic acids in high moisture corn (Goodrich et al.,
1975; Baron et al., 1986; Faber et al., 1989).

During aerobic exposure, some of the silages had trace amounts of residual
glucose (Table 2-17). The effect of treatment x day interaction was significant
(P<0.0001). From (1 0 to 3, the silages treated with DH42+LAB had generally higher
residual glucose compared to other treatments. This is also the treatment with the lowest
pH. The effect of acid hydrolysis on the release of sugars from cell walls could have
caused the higher glucose level in this treatment. The effects of treatment and day on

butyric acid (Table 2-18) levels were highly significant (P<0.0001). The average butyric

40

 

Table 2-17. Effect of inoculation on the glucose content (g/ 100 g DM) of exposed HMC
silage

 

 

Treatment Exposure period ((1) Average
0 l 3 5
DH42 (autoclaved) 0.00 b 0.00 ° 0.00 c 0.00 0.00
DH42+P42+LAB 0.00 b 0.01 ”C 0.01 l” 0.00 0.00
DH42+LAB 0.01 a 0.02 a 0.02 a 0.01 0.01
DH42+P42 0.00 b 0.01 a” 0.01 a” 0.00 0.01
DH42 0.00 b 0.00 ° 0.00 c 0.00 0.00
P42 0.00 b 0.01 C 0.00 ° 0.00 0.00
Control 0.00 b 0.01 ° 0.01 a” 0.00 0.01
S.E.M“ 0.00 0.00 0.00 0.00 0.00

 

abcColumn means with unlike superscripts differ (P<0.08 for d l and P<0.05 for d 1 and
3).
nStandard error of the mean.

Table 2-18. Effect of inoculation on the butyric acid content (g/ 100 g DM) of exposed
HMC silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5

DH42 (autoclaved) 0.16 0.15 0.17 0.13 0.15 a
DH42+P42+LAB 0.08 0.09 0.08 0.06 0.08 d
DH42+LAB 0.08 0.08 0.09 0.08 0.08 °"
DH42+P42 0.10 0.11 0.12 0.10 0.11 b“
DH42 0.11 0.13 0.13 0.12 012"
P42 0.11 0.14 0.14 0.13 0.13 a”
Control 0.07 0.09 0.09 0.08 0.08 °"
S_.E.M" 0.01 0.01 0.01 0.01 0.01

 

°°°° Column means with unlike superscripts differ (P<0.05).
nStandard error of the mean.

41

acid of the silages treated with autoclaved DH42 culture and the P42 were the highest
with 0.15 and 0.13 g/ 100 g DM, respectively. Ethanol (Table 2-19) levels were high but
were not affected by inoculation. Yeast fermentation could account for the high levels of
ethanol in the silages since the yeast and mold population (Table 2-22) remained
relatively high during the exposure phase. A significant treatment x day interaction
affected the citric acid content (Table 2-20) of the exposed silages. Citric acid content of
the exposed silages with autoclaved DH42 culture was generally higher than other
treatments from d 0 up to d3. LAB population remained high throughout the exposure
period (T able 2-21). Treatment did not affect the LAB population of the silages.
Likewise, inoculation did not affect the yeast and mold counts (Table 2-22) during the
exposure period. Dawson et al. (1998) reported lower yeast and mold counts with DH42-
inoculated high moisture corn silages during aerobic exposure. Their study however
indicated higher propionic acid levels in the treated silages and lower initial yeast and
mold counts. In this study, the initial yeast and mold counts were about 107 cfu/ g DM,
while Dawson et a1. (1998) reported 104cfu/g DM. The differences in inoculation rate and
the quality of the ensiling material could account for the discrepancy in results.
Throughout the exposure period, the dry matter (Table 2-23) of the control silages
remained highest. The inoculated HMC tended to have lower dry matter contents than
the control. Table 2-24 shows the temperature of the silages during the 5—d aerobic
exposure. The effect of treatment on temperature was significant (P<0.01). The control
and P42-treated silages had higher temperature compared to DH42+LAB-treated silages.
However, temperature differences were small that it is likely to have minimal effects on

DM recovery or spoilage. Phillip and Fellner (1991) also observed no significant

42

Table 2-19. Effect of inoculation on ethanol content (g/ 100 g DM) of exposed HMC
silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5
DH42 (autoclaved) 0.51 0.40 0.32 0.20 0.36
DH42+P42+LAB 0.28 0.27 0.28 0.18 0.25
DH42+LAB 0.38 0.35 0.29 0.18 0.30
DH42+P42 0.49 0.42 0.29 0.21 0.35
DH42 0.41 0.36 0.29 0.20 0.32
P42 0.46 0.42 0.36 0.25 0.37
Control 0.40 0.38 0.31 0.20 0.32
S.E.M° 0.04 0.04 0.04 0.04 0.03

 

nStandard error of the mean.

Table 2-20. Effect of inoculation on citric acid content (g/100 g DM) of exposed HMC
silage.

 

 

Treatment Exposure period (d) Average
0 1 3 5
DH42 (autoclaved) 0.22 a 0.27 a 0.26 a 0.17 0.23
DH42+P42+LAB 0.17 a” 0.26 “b 0.17 b 0.14 0.18
DH42+LAB 0.15 b 0.21 ab 0.17 b 0.15 0.17
DH42+P42 0.15 b 0.22 a” 0.19 b 0.13 0.17
DH42 0.15 b 0.22 a” 0.16 b 0.15 0.17
P42 0.13 b 0.213” 0.14 b 0.13 0.15
Control 0.13 b 0.20 b 0.15 b 0.13 0.15
S.E.M“ 0.01 0.01 0.01 0.01 0.01

 

abColumn means with unlike superscripts differ (P<0.03 for d 0, P<0.04 for d 1, P<0.02
for d 3).
nStandard error of the mean.

43

Table 2-21. Effect of inoculation on the lactic acid bacteria counts (log cfu/ g DM) of
exposed HMC silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5
DH42 (autoclaved) 8.02 8.67 8.57 8.69 8.49
DH42+P42+LAB 8.40 8.49 8.46 8.60 8.49
DH42+LAB 8.51 8.44 8.32 8.30 8.39
DH42+P42 7.57 8.47 8.30 8.38 8.18
DH42 8.16 8.44 8.30 8.40 8.33
P42 8.09 8.36 8.20 8.32 8.24
Control 8.45 8.42 8.18 8.23 8.32
S.E.M° 0.18 0.18 0.18 0.18 0.10

 

nStandard error of the mean.

Table 2-22. Effect of inoculation on the yeast and mold counts (log cfu/ g DM) of
exposed HMC silage.

 

 

Treatment Exposure period ((1) Average
0 1 3 5
DH42 (autoclaved) 3.25 1.43 1.63 2.30 2.15
DH42+P42+LAB 3.78 2.75 2.73 2.97 3.06
DH42+LAB 2.35 2.46 2.81 3.08 2.67
DH42+P42 2.97 2.56 1.98 3.01 2.63
DH42 2.44 2.84 2.09 3.24 2.65
P42 2.18 2.90 2.36 3.11 2.51
Control 1.98 2.80 2.27 3.02 2.63
S.E.Mn 0.33 0.33 0.33 0.33 0.19

 

nStandard error of the mean.

Table 2-23. Effect of inoculation on the dry matter (%) of exposed HMC silage.

 

 

Treatment Exposure period (d) Average
0 1 3 5
DH42 (autoclaved) 69.65 d 70.18 C 70.16 ° 71.88 C 70.48
DH42+P42+LAB 71.30 ”C 71.74 b 71.84 b 73.25 l” 72.06
DH42+LAB 72.21 a” 72.42 b 72.42 b 74.20 a” 72.81
DH42+P42 71.99 3‘” 72.63 b 72.60 b 74.30 a” 72.88
DH42 70.56 °“ 71.27 b° 71.27 ”C 73.08 b° 71.54
P42 71.82 a“ 72.64 b 72.64 b 74.28 a” 72.85
Control 72.91 a 74.34 a 74.32 a 75.47 a 74.26
S.E.M“ 0.24 0.24 0.24 0.24 0.20

 

abc"Column means with unlike superscripts differ (P<0.01, except for d 5 with P<0.001).
“Standard error of the mean.

Table 2-24. Effect of inoculation on temperature (°C) of exposed HMC silage.

 

 

Treatment Exposure period (d) Avegge
0 l 2 3 4 5

DH42(autoclaved) 22.41 22.41 21.67 21.67 21.48 21.48 21.85ab
DH42+P42+LAB 22.41 22.50 21.85 21.85 21.67 21.48 21.96ab
DH42+LAB 21.85 21.76 21.21 21.11 21.30 21.11 21.39b
DH42+P42 22.78 22.60 21.57 21.67 21.67 21.48 21.96 3"
DH42 23.15 23.06 22.13 22.22 21.85 22.59 22.50ab
P42 23.33 23.15 22.78 22.78 22.22 22.41 22.78a
Control 23.33 23.33 22.97 23.15 22.78 22.78 23.06a
S.E.M.“ 0.34 0.34 0.34 0.34 0.34 0.34 0.27

 

at’Column means with unlike superscripts differ (DH42+LAB vs. control, P<0.01;
DH42+LAB vs. P42, P<0.06).
“Standard error of the mean.

45

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correlation between the chemical criteria and temperature criteria of aerobic stability in
high moisture corn silage. They concluded that any inferences regarding the effect of

bacterial inoculation on aerobic stability depended on the criterion chosen.

Implications

This study shows that DH42 in co-culture with lactic acid bacteria enhanced the
fermentation of reconstituted corn only up to 21 d of ensiling. P42 inoculation had
marginal effect as an inoculant to the reconstituted corn.

The silages appeared stable based on the lack of changes in residual organic acids
during aerobic exposure. The silages treated with DH42+LAB reconstituted had
relatively higher organic acids than other treatments. However, yeast and mold counts
were not effectively reduced by the inoculation of DH42. Since the control appeared
well-preserved if based on the yeast and mold counts, the effect of propionibacteria
inoculation in reducing yeast and mold counts was not observed in this study. Further
studies need to be conducted to determine the effective inoculation rate and other factors

that affect the growth of DH42 in silage.

46

CHAPTER 3
EFFECT OF MOISTURE ON PROPIONIBACTERIA AS INOCULANTS TO
HIGH MOISTURE CORN SILAGE
Abstract

Rolled corn of different moisture contents (35.2%, 32.9%, 27.9%, 24.1%, 23.1%,
and 21.9%) were inoculated with control (sterile distilled water), P. acidipropionici
DH42 at 105 colony forming units (cfu) per gram of fresh corn, P. acidipropionici DH42
at 10° cfu/g, P. jensenii at 106 cfu/g and uninoculated sterile Reinforced Clostridial
Medium broth. Silos were opened after 10, 21, and 120 days of ensiling. Samples from
the 120-d silos were also taken and exposed to air for 5 days. Fresh, ensiled and exposed
samples were taken for microbial and chemical analyses.

The moisture of the ensiling material affected the efficacy of propionibacteria as
inoculants. PAB-inoculated high moisture corn gave significantly higher propionic and
acetic acid production at 22-28% and 23% moisture levels, respectively after 120 d of
ensiling. Lower pH at 24% and butyric acid at 28% was also observed with the PAB-
treated silages. Inoculation did not affect the yeast and mold counts during ensiling. P.
acidipropionici DH42 inoculated at 10°cfu/g better gave results as compared to the
105cfu/g inoculation rate.

During aerobic exposure, higher propionic and acetic acids were observed with
the PAB-inoculated silages at 22-28% moisture levels. On the other hand, lower pH and
lactic acid were observed with the PAB-inoculated silages at 22-28% and 22%,

respectively. While lower yeast and mold counts were observed in the PAB-treated

47

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silages at 22-23% moisture levels compared to the control, they did not differ with the

RCM-treated silages.

Introduction

Propionic acid has been used considerably in preservation of stored grains (Patkar
et al., 1995) and bakery products (Huitson, 1968; Javainen and Linko, 1993) due to its
antifungal properties. In silage, the benefits of propionic acid treatment had been
demonstrated (Britt et al., 1975; Hara and Ohyama, 1979; Ashbell et al., 1984; Rahnema
and Neal, 1992). One drawback for its widespread use is that it is corrosive. The use of
propionic acid-producing bacteria (PAB) appears advantageous particularly with
increasing consumer preference for natural products. Improved fermentation of PAB-
treated silages were observed but the effects on aerobically exposed silages were variable
(Flores-Galarza et al., 1985; Alio et al., 1994; Weinberg, et al., 1995; Kreikemier et al.,
1997; Higginbotham et al., 1998; Dawson et al., 1998;). More information is needed to
determine the factors affecting efficacy of PAB as silage inoculants. This study evaluated
the effects of the moisture content of corn grain in the efficacy of two Propionibacteria

species as inoculants on the fermentation profile and aerobic stability of silage.

Materials and Methods
Inoculants
Propionibacterium jensenii (ATCC 53962) was purchased from American Type
Culture Collection (ATCC) while P. acidipropionici DH42 (ATCC 55737) was obtained
from cultures maintained in the microbiology laboratory of the Department of Animal

Science, Michigan State University, East Lansing, Michigan. Both inoculants were

48

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grown anaerobically in Reinforced Clostridial Medium (Unipath, England). Before use,
at least three transfers to a fresh medium were made to ensure that the cultures were

active. The cultures were incubated anaerobically at 30°C for about 18 h before use.

Silage preparation

During the fall of 1996, corn was harvested weekly to gather ensiling materials of
various moisture contents. The freshly harvested corn was rolled prior to ensiling.
Laboratory silos made of polyvinyl chloride (PVC) pipes (47 .5x10.2 cm diameter) with
rubber caps fitted with policeman (Fisher) were used. The different moisture contents at
ensiling were: 35.2%, 32.9%, 27.9%, 24.1%, 23.1%, and 21.9%. In each moisture level,
corn was divided and allotted to five inoculant treatments: control (sterile distilled water),
P. acidipropionici DH42 at 105 colony forming units (cfu) per gram of fresh corn, P.
acidipropionici DH42 at 10° cfu/ g, P. jensenii at10° cfu/g and uninoculated sterile
Reinforced Clostridial Medium broth (Unipath, England). The latter treatment was added
to determine the effects of the nutrients in the fermentation broth in the growth of
epiphytic bacteria. The amounts of inoculants needed to meet the inoculation rate were
estimated from optical density (OD) readings of the inocula prior to inoculation. Linear
regression correlating the OD (at 600 nm) with colony forming unit counts had been
prepared separately for each inoculant. Based on the OD value, estimated cfu/ml of the
inoculum was calculated using the regression line. Serial dilutions of the starter cultures
were also made and the appropriate dilutions plated in Reinforced Clostridial Medium.
The plates were incubated anaerobically at 30°C for 5 d. This was done to validate the
calculated estimates of the microbial counts. Prior to inoculation, sterile distilled water

was added to bring the final volume of the inoculant to 100 ml so as not to alter the

49

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moisture level by adding different volumes of liquid. The inoculants were added to the
corn in a plastic tub and mixed by hand. Different tubs and latex gloves were used for
each treatment to prevent any cross-contamination. Four replicate silos were prepared for
each treatment and time combination. However, the 35 and 22% moisture levels had
three replicates since the amount of harvested corn was limited. In addition, 28%
moisture level had eight replicates for the d 0 and 120 sampling periods since the initial
moisture contents of the corn for two successive weeks were not significantly different
from each other. Hence the data were pooled to reflect one moisture level. The silos
were opened after 10, 21 and 120 d of ensiling. Silos were weighed before and after

ensiling and the difference in dry matter weights was expressed as dry matter recovery.

Aerobic Stability Evaluation

Ensiled corn from the 120-d collection was used for aerobic stability evaluation.
About 1 kg from each sample was weighed into a plastic bag, placed in a Styrofoam
container and exposed to air. Cooking thermometers were inserted into each bag to
monitor silage temperature. Morning and afternoon temperatures were taken and the
average used for analyses. Temperatures were monitored for 5 (1. Samples for laboratory

analyses were collected after 1, 3, and 5 d of aerobic exposure.

Chemical and Microbial Analyses
Fresh, fermented and exposed corn samples were taken for chemical and
microbial analyses. Aqueous extracts were prepared by adding 450 ml sterile, 0.9% NaCl

to 50 g silage sample, homogenized in a Stomacher (Tekmar, Model 3500) for 5 min and

50

strained into sterile 50-ml centrifuge tubes. The pH of the extracts was determined using
a pH meter (Cole Palmer, Model 05569-20).

The dry matter was determined by forced-air oven drying at 64°C for 48 h. The
difference in weights before and after drying was expressed as the dry matter. Glucose,
ethanol and organic acids were measured by ion exchange-exclusion high performance
liquid chromatography (HPLC) as described by Dawson (1994) with slight modification.
The extracts were centrifuged (26,000 x g) for 30 min and the supernatant used for the
HPLC analyses.

Serial dilutions of the extracts were prepared in 1% Bacto-peptone broth (Difco)
and appropriate dilutions were plated in various selective media. Rose Bengal Agar
(Difco) with Antimicrobic Supplement C (Difco) was used to estimate yeast and molds
and the plates were incubated aerobically at 39°C for 2 to 3 d. Pal propiobac (Standa
Industrie, France) was used to estimate propionibacteria in 22, 28, 33 and 35% moisture
levels. Plates were incubated anaerobically at 30°C for 5 d. Yellow colonies were
counted and presumptively identified as propionibacteria (Thierry and Madec, 1995).
Yeast and mold counts were done for fresh, ensiled and exposed corn samples while

propionibacteria counts were done for d 0 and 120.

Statistical Analyses

Data for the fermentation phase were analyzed as a one-way completely
randomized design using the General Linear Model subroutine of the Statistical Analysis
System (SAS, 1990). Microbial counts were analyzed using the transformed data (loglo
[Y], where Y is the microbial count). Data were analyzed by moisture level. Due to the

increasing variability of the various parameters over time, the data were analyzed

51

separately for each collection period (i.e. d 0, 10, 21, 120). At a given moisture level and
collection period, the model used for each parameter (e. g. pH. prOpionic acid, acetic acid)

was as follows:

Y, = [L + or, + e
Where:
Y = individual variable measured (e.g. pH, propionic acid, glucose)
1.1 = overall mean
(Xi = effect of treatment
e, = random residual error

For the aerobic stability phase, data were analyzed by PROC MIXED of the SAS, using
the repeated measures analysis (SAS, 1990) since repeated samplings from the same

sample were done. Treatment means were compared using the Bonferroni (SAS, 1990).

Results and Discussion

Fermentation Phase

Initial propionic acid contents (Appendix Table A-1) of the corn were similar
across treatments. This indicates that the amount of propionic acid in the
propionibacteria-containing inoculants was low and had very little impact on the initial
propionic acid content of the corn. Moisture and PAB inoculation (Figures 3-1 to 3-6)
affected the propionic acid content of silages. On d 10, the silage treated with DH42 at
10° cfu had the highest propionic acid at 28 % moisture levels. At 24% moisture level,
the same treatment had similar propionic acid level with the P. jensenii—treated silages but

higher than the other treatments. On d 21, propionic acid of DH42-treated silages

52

inoculated at 10° cfu/g at 24 and 28% moisture levels were the highest (P<0.01) with

0.14% and 0.15%, respectively. At the end of the ensiling period, the same treatment

 

 

 

 

 

 

 

 

 

 

 

E 0.2 _,
g) 0.15 _ — : - ElControl
B, IDH42-A
% 0.1 — ‘ DDH42-B
3 DP. jensem'i
g 0'05 ‘ ' , IRCM
6.9 0 l t .

0 10

Ensiling period ((1)

 

 

Figure 3-1. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 35% moisture level (Standard error of the means: d0=0, d10=0. d21=0,
d120=0.01).

 

 

 

 

 

 

2

Q

N)

8 El Control
33, IDH42-A
% El DH42-B
_; E] P. jensenii
.5 IRCM
a.

o

d:

 

 

 

Ensiling period (d)

 

 

 

Figure 3-2. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 33% moisture level (Standard error of the means: d0=0.01, d10=0,
d21=0, d120=0.01).

53

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2

Q

00

8 13 Control
3, IDH42-A
% El DH42-B
.8 [:1 P. jensenii
,5 IRCM
D.

E

n.

 

 

Ensiling period ((1)

 

 

 

Figure 3-3. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 28% moisture level (Standard error of the means: d0=0, d10=0, d21=0,
d120=0.01).

 

 

 

 

 

2 0.3

a

00

8 El Control

E, IDH42-A
% El DH42-B
; D P. jensenil'
.5. IRCM

8‘

E

 

 

 

Ensiling period (d)

 

 

 

Figure 3-4. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 24% moisture level (Standard error of the means: d0=0, d10=0,
d2l=0.01, d120=0.01).

54

 

 

 

 

 

 

 

 

 

 

E 0.2
00 7
8 0.15 - . EIControl
$0 IDH42-A
E 0" " . . ElDH42-B
g i: 8 i 7 I ,, L'lP.jensenii
:8 005 IRCM
39. o 8' E. l‘l‘l .

0 10

Ensilingperiod(d)

 

 

 

Figure 3-5. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 23% moisture level (Standard error of the means: d0=0.01, d10=0,
d21=0, d120=0.01).

 

 

 

 

 

 

 

 

 

2
Q 0.25 1
DD 1
8 0.2 - DControl
B) 0.15 — = IDH42-A
% DDH42-B
.3 0'1 4 UP. jensenii
.8 0.05 - IRCM
G.
1:12.. 0 t—r—l I . fl T

0 10 21

Ensiling period (d)

 

 

 

Figure 3-6. The effect of inoculation on the propionic acid of fresh and ensiled high
moisture corn at 22% moisture level (Standard error of the means: d0=0, d10=0, d21=0,
d120=0.01).

55

had higher (P<0.05) propionic acid than the control and RCM-inoculated silages at 22-
28% moisture levels. Increasing the inoculation rate of P. acidipropionici DH42 from
105 cfu/g material to 10° cfu/g resulted in numerically greater propionic acid levels in the
resulting silages. Results of other studies that used propionibacteria as silage inoculants,
reported similar (Higginbotham et al., 1996) or lower (Kreikemeier, 1997) propionic
acid levels. Higginbotham et al. (1998) indicated that propionic acid was undetectable in
the corn silage inoculated with P. acidipropionici. Pr0pionic acid levels found in the
present study was lower compared to an earlier study (Dawson et al., 1998) using the
same inoculant. Using high moisture corn with 74% dry matter, Dawson et al. (1998)
reported propionic acid content of 0.35g/ 100 g DM after 42 d of ensiling. In the present
study, the 28% moisture level had about 0.34g/100 g DM propionic acid after 120 d of
ensiling. Differences in the quality of the ensiling material could account for this
discrepancy. It was noted that in the earlier study, the high moisture corn had higher
initial glucose as compared to the amount found in this study (0.34 vs 0.035 g/ 100 g
DM).

The pH (Appendix Table A-2,) was generally lower in the higher moisture level
silages. After 10 d of ensiling, the pH decline (Figures 3-7 to 3-12) is most apparent with
the 33 and 35% moisture levels. The pH at 35% moisture level ranged from 3.89-3.92
while at 22% moisture level, the pH ranged from 5.28-5.30. The effect of inoculation
was observed only after (1 21 of ensiling at 22% moisture level. The DH42-10° inoculated
silages had significantly lower pH compared to the control and RCM-inoculated silages
but similar to the other PAB-inoculated silages. After 120 d of ensiling, the 35%

moisture level had pH ranging from 3.70-3.75 while the 22% moisture level ranged from

56

4.55-4.67. The effect of PAB inoculation was observed at 22 and 24% moisture levels.

The P. jensenii and DH42-10° inoculated silages at 24% moisture levels had lower

(P<0.05) pH than the control and RCM-inoculated silages. At 22% moisture level, the

 

 

 

 

 

Ensiling period ((1)

 

 

E1 Control

I DH42-A
E1 DH42-B
El P. jensenii
I RCM

 

 

 

 

Figure 3—7. The effect of inoculation on the pH of fresh and ensiled high moisture corn at
35% moisture level (Standard error of the means: d0=0.02, d10=0.01, d21=0,

 

 

d120=0.01).
6.00
5.00 7 EIControl
4-00 ‘ 1;; IDH42-A

IQ 3.00 — DDH42-B

2.00 - DP. jensem'i
1.00 . IRCM
0.00 ~

 

 

 

Ensiling period ((1)

 

 

 

 

 

 

 

Figure 3-8. The effect of inoculation on the pH of fresh and ensiled high moisture corn at
33% moisture level(Standard error of the means: d0=0.02, d10=0.01, d21=0.01,

d120=0.02).

57

 

 

 

 

 

El Control

I DH42-A
El DH42-B
[1 P. jensenil'
I RCM

 

 

 

 

 

Ensiling period ((1)

 

 

 

Figure 3-9. The effect of inoculation on the pH of fresh and ensiled high moisture corn at
28% moisture level (Standard error of the means: d0=0.03, d10=0.01, d21=0.01,
d120=0.01)

 

 

 

El Control

I DH42-A
Cl DH42-B
Cl P. jensem'i
I RCM

 

 

 

 

 

Ensiling period ((1)

 

 

Figure 3-10. The effect of inoculation on the pH of fresh and ensiled high moisture corn
at 24% moisture level (Standard error of the means: d0=0.02, d10=0.04, d21=0.03,
d1 20:0. 10)

58

 

 

 

 

 

 

 

4% ElCortrol
ii IDH42-A
g ClDH42-B
1E DPJensenii
: IRCM

a

 

 

 

 

Figure 3-11. The effect of inoculation on the pH of fresh and ensiled high moisture corn
at 23% moisture level (Standard error of the means: d0=0.02, d10=0.03, d21=0.02,
d120=0.01)

 

 

 

I] Cortrol

I DH42-A
E] DH42-B
El P. jenaenl'i
l FEM

 

 

 

 

 

 

 

 

 

Figure 3-12. The effect of inoculation on the pH of fresh and ensiled high moisture corn
at 22 % moisture level (Standard error of the means: d0=0.02, d10=0.02, d21=0.01,
d120=0.01)

59

PAB-inoculated silages had significantly lower pH compared to the uninoculated silages
(control and RCM). The 22% moisture silages had the highest pH ranging from 4.55-
4.67. This indicates that the propionibacteria enhanced silage fermentation. Kreikemeir
et al. (1997) also observed lower pH with PAB-inoculated silages compared to the
uninoculated silages. In the study of Higginbotham et al. (1997), P. acidipropionici
silages inoculated at 10° cfu/g fresh forage had lower pH than the uninoculated silages on
d 30 but differences were not observed after 100 d of ensiling. Weinberg et al. (1995a, b)
and Higginbotham et al. (1998) observed only marginal effect of propionibacteria
inoculation on silage pH. The effect of moisture on propionibacteria is possibly due to
its effect on the pH. The lowest propionic acid production was observed in silages with
the lowest pH level indicating the possible inhibitory effect of acidic conditions to the
grth of propionibacteria. Previous reports using P. shermanii as inoculant to pearl
millet and maize silages (Weinberg et al., 1995a) and wheat and sorghum silages
(Weinberg et al., 1995b), and P. acidipropionici to whole-plant corn silage
(Higginbotham et al., 1998) showed marginal or no effect on pH during ensilement.
Weinberg et al. (1995a) and Higginbotham et al. (1998) observed pH levels ranging from
37-40 after 90 d of ensiling. When used for ensiling high moisture corn, P. shennanii
reduced the yeast population in the inoculated silages (Flores-Galarza et al., 1985). In
this study, the pH of the resulting silages ranged from 4.53-4.73 after 60 d. Higginbotham
et al. (1998) reported that propionic acid was undetectable indicating that the growth of
propionibacteria was not sustained during the ensiling process. In the study of Florez-
Galarza et al. (1985), the higher pH could have favored the growth of propionibacteria

resulting in decreased yeast population. Pahlow and Honig (1994) indicated that the

60

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production of propionic acid by propionibacteria ceases below pH 4.8. Moreover,
Weinberg et al. (1995b) recommended PAB inoculation in slow-fermenting grass silages.
Perez-Chaia et al. (1995) observed that the growth of propionibacteria grown in mixed
cultures with Lactobacilli was inhibited when there is rapid pH reduction, but not when
there is slow pH reduction. In a modeling study done by Pitt (1997), he noted that there is
a wide variation in studies about the optimum pH for growth of PAB which ranged from
pH 3.2-7.0. This is probably due to differences in the sensitivity to pH among
propionibacteria strains. Rehberger and Glatz (1998) indicated that while there are
differences to pH sensitivity, none of the strains they tested was able to initiate growth at
pH below 5. 1n the present study, the rate of pH decline is most apparent at 35% moisture
level. After 10 d of ensiling, silages in this moisture level had pH ranging from 3.89-
3.92. After 120 d of ensiling, most of the silages had pH of about 4.0 while the 35%
moisture level had the lowest (P<0.01) pH. Even at this pH level, propionic acid was
about 0.169 g/100g DM. Since P. acidipropionici DH42 had been isolated from high
moisture corn silage, this strain is presumably more adapted to the acidic silage
environment than other strains used in other studies. When grown in glucose, P.
acidipropionici DH42 had acceptable growth rates between pH 4.9 to 7.8 (Dawson,
1994). Perez-Chaia et al. (1988) observed that among the propionibacteria strains they
studied, P. acidipropionici was the only one that showed activity at pH values that were
inhibitory for growth of propionibacteria indicating that this species could be more
resistant to low pH.

The effect of PAB inoculation on the acetic acid content (Appendix Table A-3,

Figures 3-13 to 3-18) of the silages was observed on d 21 at 24% moisture level. The

61

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Egrre 3-13. ‘
moisture c0n
31:0. dlZO:

 

 

 

 

 

w 025

O

2 1:1 Corirol

El I DH42—A
"-8 El DH42—B
g D P. jensenii
8 I RCM

2

 

 

 

 

 

 

Figure 3-13. The effect of inoculation on the acetic acid of fresh and ensiled high
moisture corn at 35% moisture level (Standard error of the means: d0=0, d10=0.01,
d21=0, d120=0.01)

 

 

 

 

 

 

DD

0

53 1:1 Comol

39 I DH42-A
% 1:1 DH42-B
g [j P. jermu‘i
8 I RCM

C.)

<

 

 

 

 

 

Figure 3-14. The effect of inoculation on the acetic acid of fresh and ensiled high
mOisture corn at 33% moisture level (Standard error of the means: d0=0, d10=0.01,
d21=0, d120=0.01)

62

 

 

 

 

 

 

 

DD

§ EIComol
39 IDH42-A
32 DDH42—B
§ DP.jerm11‘i
8 I RCM

%

 

 

 

 

 

 

Figure 3-15. The effect of inoculation on the acetic acid of fresh and ensiled high
moisture corn at 28% moisture level (Standard error of the means: d0=0, d10=0,
d21=0.01, d120=0.03)

 

 

 

 

 

DD

0

2 [:1 0011101

Eh I DH42-A
g El DH42-B

8 E] P. jenxnl'i
it; I RCM

<

 

 

 

 

 

 

 

 

Figure 3-16. The effect of inoculation on the acetic acid of fresh and ensiled high
moisture corn at 24% moisture level (Standard error of the means: d0=0.02, d10=0.02,
d21=0.01, d120=0.01)

63

 

 

 

 

 

no 0.20 4 —

o

2 2 Como]

E” I DH42-A
E El DH42-B

0

g E] P. jensenii
8 I RCM

<8

 

 

 

Emirgperbdkl)

 

 

 

Figure 3-17. The effect of inoculation on the acetic acid of fresh and ensiled high
moisture corn at 23% moisture level (Standard error of the means: d0=0.02, d10=0.02,
d21=0.01, d120=0.01)

 

 

 

 

 

 

0.35
no
8 0.30-
1 0.25 ~ [10011101
£9 0.20- gm”:
'0 DH42-
"t'l 0.154 _ ..
cu CIRJensam
o 0.104
8 IRCM
% 0154

0.0) ~

 

 

 

 

 

 

Figure 3-18. The effect of inoculation on the acetic acid of fresh and ensiled high
moisture corn at 22% moisture level (Standard error of the means: d0=0, d10=0,
d21=0.01, d120=0.01)

PAB-treated silages had higher acetic acid than the control and the RCM-treated silages.
After 120 d of ensiling and at 23% moisture level, the DH42 with 10° cfu had higher
acetic acid than the control (0.33 vs 0.20 g/ 100 g DM). P. jensenii gave 0.29 g/ 100 g DM
which was similar to the control. In general, numerically higher acetic acid values were
observed with the PAB-inoculated silages at 23-28% moisture levels. Increased acetic
acid with PAB inoculation is expected since acetic acid is also produced in addition to
propionic acid during the fermentation of lactate and glucose by propionic acid bacteria
(Wood, 1981). High propionic acid and acetic acid levels in silage is beneficial because
of their antifungal properties. Moon (1989) observed synergistic effects of mixtures of
acetic, lactic and propionic against acid-tolerant yeasts.

Moisture level affected the lactic acid content of the silages (Appendix-Table A-4,
Figures 3-19 to 3-24). Higher lactic acid production was observed with the high moisture
silages. On (1 120, the 35% moisture level gave lactic acid values ranging from 3.56-3.69
g/ 100 g DM while the 22% moisture level gave values ranging 0.18 to 0.54 g/ 100 g DM.
The lower acetic and lactic acids in the drier silages tend to indicate restricted
fermentation (Jackson and Forbes, 1970; Thomas, 1978; Kung et al., 1984; Luchini et al.,
1997). Garcia et al. (1989) also reported higher acetic and lactic acids with low dry
matter silages. The effect of inoculation was observed with 22% moisture level where
the DH42 at 10° cfu gave significantly lower lactic acid than the control and RCM-treated
silages. This could be due to the increased lactic acid utilization of the DH42 in this
treatment. In addition to glucose, propionibacteria can metabolize lactic acid

(Gottschalk, 1985).

65

 

 

9° P
88

Lactic acid (g/ 100 g
to
8

 

 

10 21
Existitgpm'od (d)

 

120

 

 

I3 0011101

I DH42-A
El DH42-B
E] P. jenmnii
I RCM

 

 

 

 

Figure 3-19. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 35% moisture level (Standard error of the means: d0=0, d10=0.06,

d21=0.03, d120=0. 13).

 

 

Lactic acid (g/ 100 g

 

 

 

 

El Como]
IDH42—A
El DH42—B
El P._jensenii

 

ImM

 

 

 

Figure 3-20. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 33% moisture level (Standard error of the means: d0=0.01, d10=0.04,

d21=0.03, d120=0.06).

66

 

 

 

 

 

 

 

 

 

2

D

on El Control

g I DH42-A
g [:1 DH42-B
E C] P. jensenii
.2

.g I RCM

t—l

 

 

Ensiling period ((1)

 

 

 

Figure 3-21. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 28% moisture level (Standard error of the means: d0=0.01, d10=0.02,
d21=0.02, d120=0.05).

 

 

 

 

 

E

a

00 El Control

§ IDH42-A
g DDH42-B
E? DP. jensenii
.2

g IRCM

t—l

 

 

Ensiling period ((1)

 

 

 

Figure 3-22. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 24% moisture level(Standard error of the means: d0=0, d10=0.l3,
d21=0.05, d120=0.05).

67

 

 

 

 

 

E

on El Control

g I DH42-A
g [1 DH42-B
'g Cl P. jensenl'i
8 IRCM

8

41

 

 

Ensiling period (d)

 

 

 

 

Figure 3—23. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 23% moisture level(Standard error of the means: d0=0.01, d10=0.03,
d21=0.02, d120=0.05).

 

 

 

 

 

E

on Control

§ IDH42-A
@ DDH42-B
g D P. jensenl'i
8 I RCM

63

.1

 

 

 

 

 

Ensiling period (d)

 

 

Figure 3-24. The effect of inoculation on the lactic acid content of fresh and ensiled high
moisture corn at 22% moisture level(Standard error of the means: d0=0.01, d10=0.01,
d21=0.02, d120=0.02).

68

 

The corn from the 35% moisture level had the highest initial glucose content
ranging from 0.75-1.01 g/100g DM (Appendix Table A-5). The high available sugar
may account for the drastic pH decline in this moisture level through the fermentation of
glucose by lactic acid bacteria. On d 21, at 24% moisture level the silages with P.
acidipropionici DH42 at 10°cfu gave glucose values that were higher than P. jensem'i-
treated silages (0.12 vs 0.04 g/ 100 g DM). On (1 120, the 33% moisture level had higher
glucose levels regardless of inoculant. Glucose values ranged from 0.36-0.41 g/ 100 g
DM. Glucose, particularly in the high moisture silages appears to be high even after 120
d of ensiling. This could be due to the lower pH in the wetter silages. Jones et al. (1992)
indicated that decrease in pH promotes hydrolysis of sugars from cell walls.

Citric acid (Appendix Table A-6) was also detected in the silage. McDonald et al.
(1991) indicated that in herbage, citric and malic acids are quantitatively most important.
On (1 0 and 10, the high moisture silages (33 and 35%) tend to have higher citric acid
levels. After 120 d of ensiling, small amounts of citric acid was detected in the silages
with higher values in the drier silages. Bryan-Jones (1969) indicated that citric acid is
fermented by lactic acid bacteria mainly Enterococcusfaecalis.

Ethanol (Appendix Table A-7) tends to be higher with the high moisture silages.
Differences among inoculants were observed only at 28% moisture level at d 120. The
silages with DH42 at 10° cfu had lower (P<0.05) ethanol concentration than the control
silages (0.49 vs 0.75 g/ 100 g DM). Weinberg et al. (1995b) and Kreikemeier et al.
(1997) also observed lower ethanol values in PAB-inoculated silages. However,

Dawson et al. ( 1998) found no difference in ethanol levels between DH42-treated silages

69

and uninoculated high moisture corn silages while Higginbotham et al. (1998) found
higher ethanol concentration in the inoculated silages.

Butyric acid (Appendix Table A-8) was detected after 21 d of ensiling in silages
from the 24% moisture level. The propionibacteria-inoculated silages from the 28%
moisture levels gave lower butyric acid levels compared to the uninoculated silages.
Butyric acid is usually caused by clostridial growth occurring at the latter stages of
ensiling but other organisms such as yeasts and Bacillus spp. also produce small amounts
(Mc Donald et al., 1991). Dry matter recovery (Appendix Table A-9) was affected by
moisture but not by inoculation. The 28-33 % moisture levels gave the lowest dry matter
recovery throughout the ensiling period. On (1 120, 33% moisture level gave the lowest
DM recovery values while the highest was with the 23% moisture level. Dawson et al.
(1998) found higher DM recovery with inoculated high moisture corn silages while
Kreikemeier et al. (1997) and Higginbotham et a1. (1998) found the reverse.
Higginbotham et al. (1998) indicated that the high initial moisture of the corn plant and
bacterial inoculation allowed extensive fermentation of the silages reducing dry matter
recovery in the inoculated silages.

Yeast and mold counts (Appendix Table A-lO, Figures 3-25 to 3-30) decreased as
ensiling progressed. After 10 d of ensiling, reduction in yeast and mold counts were
observed at 24% moisture level. The DH42-10° cfu/g treated silages had lower yeast and
mold counts than the RCM-treated silages but similar to the control. At (1 21, the P.
jensenii-treated silages had significantly lower yeast and mold counts than the control at

22 and 28% moisture levels. On (1 120, regardless of moisture level, the P.

70

 

 

 

      

 

 

 

 

 

S
no 6 _
g 5 ~ , ElControl
g” 4 “r? IDH42-A
§ -- amaze
g ' ‘ DP. jensenii
E Immt
‘5
E

0 10 21 120

Ensiling period ((1)

 

 

 

Figure 3-25. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 35% moisture level (Standard error of the means:d0=0.08,
d10=0.1], d21=0.28, d120=0.22).

 

 

 

 

 

 

 

 

 

Z
0
DD
2
0 Control
DO
:3 I DH42-A
fi DDH42-B
o
E E] P. jensenil'
o
5 I RCM
‘63
8
>‘

 

Ensiling period (d)

 

 

Figure 3-26. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 33% moisture level (Standard error of the means:d0=0.05,
d10=0.07, d21=0.06, d120=0.06).

71

 

 

 

 

 

5 Control

I DH42 -A
D DH42-B
CI P. jensenil'
I RCM

 

 

 

Yeast and molds (log cfu/g DM)

 

 

 

Ensiling period (d)

 

 

 

Figure 3-27. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 28% moisture level (Standard error of the means:d0=0.07,
d10=0.08, d21=0.17, d120=0.20).

 

 

 

 

 

6
Z
D
8”
“6 El Control
00
:0, I DH42—A
fl 1210114213
0
E [I P. jensenii
'0
5 I RCM
>-

 

 

 

 

Ensiling period (d)

 

 

 

Figure 3-28. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 24% moisture level (Standard error of the means:d0=0.06,
d10=0.31, d21=0.22, d120=0.26).

72

 

 

 

 

 

 

 

E

O

§° El Control

:3? I DH42-A
3 1:1 DH42-B
'2 DP. jensenii
; I RCM

8

>-

0 10 21 120
Ensiling period (d)

 

 

 

Figure 3-29. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 23% moisture level (Standard error of the means:d0=0.10,
d10=0.14, d21=0.18, d120=0.29).

 

 

 

 

 

 

 

 

 

E

Q

00

E El Control
3,“ IDH42-A
5 ElDH42-B
E El P. jensem'i
'2 I RCM

N

8

>"

 

Ensiling period ((1)

 

 

 

Figure 3-30. The effect of inoculation on the yeast and mold counts of fresh and ensiled
high moisture corn at 22% moisture level (Standard error of the means:d0=0.07,
d10=0.07, d21=0.04, d120=0.24).

73

acidipropionici DH42 silages with 10° cfu/ g DM generally had numerically lower yeast
and mold counts compared to the uninoculated control silages. The results indicate that
inoculation of P. acidipropionici DH42 reduced the yeast and mold counts. As was
observed in earlier silage trials, microbial counts within treatments are highly variable.
This indicates that a greater number of samples would be needed to detect significant
differences among treatments. Using propionic acid to preserve hay, Magan and Lacey
(1986) indicated that the difficulty of mixing the material with the acid evenly allows
yeasts and fungi to grow in under-treated pockets without competition from other
microorganisms. It had been demonstrated that yeasts could use propionic acid as
substrate when levels are low (Lord et al., 1981; Magan and Lacey, 1986). During the
silage preparation for this study, thorough mixing had been done to ensure that the
inoculants are well distributed, although it is also possible that there were areas or
pockets that were uninoculated allowing the proliferation of yeast and molds. Weinberg
and Muck (1992) pointed out that uneven mixing of the inoculant could be one of the
factors that could lead to an apparent failure of the inoculant to dominate the
fermentation. This may also explain the improved reduction in yeast and mold counts
with increased inoculation rate of the P. acidipropionici DH42. It might be necessary to
have more replicates to get a more representative sample of the microbial flora in the
silage.

The amount propionic acid needed to prevent moulding of hay and silage depends
on the level of pH and moisture content (Lacey et al., 1978; Hara and Ohyama, 1978;
Lord et al., 1981). With a pK of 4.87, more of the propionic acid is in the undissociated

form at lower pH hence, increased antimicrobial action. Rusul et al. (1987) showed that

74

with an initial pH of 5.5 of the medium, a maximum of 1% propionic acid permitted the
growth and aflatoxin production of Aspergillus parasiticus after 3 d of incubation.
However, when the initial pH is 4.5, the maximum concentration was reduced to 0.1%.
The higher the moisture content of the material to be preserved, the more propionic acid
is needed to prevent deterioration due to yeasts and molds. Sauer et a1. (1986) indicated
that 0.4% propionic acid is needed to preserve corn grain. Lacey et al. (1978) also
observed that propionic acid treatment was less effective in preserving hay when baled
with 41% moisture as compared to the drier hays. Lacey et al. (1983) further indicated
that in hay, about 0.12 g propionate/ 100 g water is needed for every 1% moisture content
above 20% to prevent molding. In a more recent findings, Magan and Lacey (1986)
observed the higher tolerance of yeast to propionic acid when water exceeds 30%. This
observation may explain the higher yeast and mold counts at higher moisture levels even
though the propionic acid level were higher compared to the drier silages. Hara and
Ohyama (1978) suggested that the effectiveness of propionic acid depends on the
concentration of the acid in the moisture phase of the silages.

The propionibacteria counts (Appendix Table A-1 1) at d 0 indicate that even the
uninoculated corn had propionibacteria although lower than the inoculated ones. After
120 d of ensiling, propionibacteria counts are high in all the silages at different moisture
levels. The lowest count were observed at 35% moisture level, with the control silage
having propionibacteria counts of 104°ch g DM. It also appears that propionibacteria
counts did not change even after 120 d of ensiling except for the increase in the
uninoculated silages. The low counts could also be due to the sampling technique.

Microbial counts were taken using silage extracts prepared by stomaching the silage

75

samples for 5 rrrin. Sharp et al. (1991) observed that recovery of added microbial cells
from stomaching is only 9 % as compared to 109% using differential centrifugation

technique.

Aerobic Phase

During the exposure period, average daily propionic acid (Appendix Table A-12,
Figures 3-31 to 3-32) decreased only after 3 d of exposure. Significant effect of PAB
inoculation was observed at 22-28% moisture levels. The DH42 10°cfu/g-treated silages
had significantly higher propionic acid levels than the control and RCM-treated silages.
Moreover, this treatment had significantly higher propionic acid than the P. jensenii—
treated silages at 24 and 28% moisture levels. The highest propionic acid was observed at
28% moisture level, with an average of 0.27 g/ 100 g DM for the duration of the exposure
period. While there was no increase in propionic acid concentration during aerobic
exposure as Dawson et al. (1998) observed, the propionic acid levels were higher than
that reported by Kreikemeier et al. (1998). Other studies (Weinberg et al., 1995b;
Higginbotham et al., 1996; 1998) found no propionic acid in the exposed silages. P.
acidipropionici DH42 can be grown aerobically (Dawson, 1994) and thus, can continue
to grow and produce propionic acid even when the silage is exposed to air.
Propionibacteria are considered facultative anaerobes (Cummins and Johnson, 1992).
However, Quesada-Chanto et al. (1997) observed that P. shermanii CDB 10014 can grow
at high volumetric oxygen transfer coefficients (KLa).

The effect of inoculation on the average acetic acid content (Appendix Table A-

13, Figures 3-33 to 3-34) levels in the exposed silages was observed at 22, 23 and 28%

76

 

 

 

 

El Control

I DH42-A
El DH42-B
C] P. jensem'i
I RCM

 

 

 

 

 

Moisture content (%)

 

 

 

Figure 3-31. The effect of inoculation and moisture content on the average propionic
acid of exposed high moisture corn silage (Standard error of the means: 35%=0.0l,
33%=0.0l, 28%:0.01, 24%:0.01, 23%=0.0l, 22%=0.0l).

 

 

0.25

_o
N
1

0.15 -

.0
I—I
1

0.05 ~

Propionic acid (g/ 100 g DM

 

 

O
1

 

   

35

Moisture content (%)

 

 

 

Figure 3-32. The effect of moisture content on the average propionic acid content of high
moisture corn silage during 5-day exposure period.

77

 

 

 

[1 Control

I DH42-A
El DH42-B
D P. jensenii
I RCM

 

 

 

 

 

Moisture contem (%)

 

 

 

 

Figure 3-33. The effect of inoculation and moisture content on the average acetic acid
content of exposed high moisture corn silage (Standard error of the means: 35%:002,
33%:0.02, 28%:0.02, 24%=0.03, 23%:0.02, 22%:0.01).

 

 

M
.0
Ur
o

 

 

Acetic acid (g/ 100

 

   

Moisture content (%)

 

 

 

Figure 3-34. The effect of moisture content on the acetic acid content of high moisture
corn silage during S-day exposure period.

78

moisture levels. At these moisture levels, DH42-10°cfu/g silages had higher acetic acid
level than the control and RCM-treated silages. Moreover, the propionibacteria-
inoculated silages had higher acetic acid than the non-PAB silages. In general, acetic
acid levels were maintained up to d 3 of the exposure and decreased at d 5. Higher acetic
acid levels were also observed at 23-22% moisture levels.

Moisture affected lactic acid levels (Appendix Table A-14, Figures 3-35 to 3-36)
but not inoculation during the exposure period. Lactic acid was highest in the silages
with 33-35% moisture level indicating extensive fermentation in the wetter silages. In
contrast to earlier findings (Dawson et al., 1998), inoculation had no effect on the lactic
acid levels. At 35% moisture level, lactic acid declined at d 5 of aerobic exposure, while
at 24 and 33 % moisture levels, significant decline was noted at d 3. The decline in lactic
acid is due to its metabolism by aerobic microorganisms (Wood et al., 1991).

Except of the silages from the 35% moisture level, the pH of silages (Appendix
Table A-15, Figures 3-37 to 3-38) remained stable during the exposure period. The
increase in pH on d 5 in the 35% moisture level supports the observed decrease in lactic
acid level. The effect of moisture was most apparent with the wetter silages having lower
pH than the drier silages. The effect of inoculation on pH was also significant. The
DH42-10° cfu/g and P. jensenii-treated silages had lower pH compared to the control and
RCM-treated silages at 22, 24, 28% moisture levels. During aerobic exposure, the
increase in pH is attributed to consumption of organic acids by yeast, molds and aerobic
bacteria (Mc Donald et al., 1991). Hence, maintenance of low pH indicates better
preservation of the exposed silage. As discussed earlier, acetic and propionic acid levels

tend to be higher at 22-28% moisture levels with the PAB-treated silages.

79

 

 

 

 

   

 

 

 

   

2 3.50
O 3.00 a '
no 250 - IControl
§ 2'00 g IDH42-A
3’ ' ’ DDH42-B
.12 1.50 g . .-
8 100 fa DP.}ensenzl
o ‘ E:
§ 0.50 i: 'RCM
—‘ 0.00 E?

35 33 28 24 23 22

Moisture content (%)

 

 

 

Figure 3-35. The effect of inoculation and moisture content on the average lactic acid
content of exposed high moisture corn silage (Standard error of the means: 35%=O.42,
33%=O.l 1, 28%:0.08, 24%:0.09, 23%:0.09, 22%:0.02).

 

 

Lactic acid (g/ 100 g DM

 

 

 

 

Moisture content (%)

 

 

 

Figure 3-36. The effect of moisture content on the lactic acid content of high moisture
corn silage during S-day exposure period

80

 

 

 

 

 

. ’ ElControl

:3; ,. g I DH42-A

; P 2; 3; CI DH42-B
f; g E] P. jensenii
if IRCM
g, :

 

 

35 33 28 24 23 22

Moisture content (%)

 

 

 

Figure 3-37. The effect of inoculation and moisture content on the average pH of
exposed high moisture corn silage (Standard error of the means: 35%=O.38, 33%:0.03,

28%:0.04, 24%:0.02, 23%:0.03, 22%:0.02).

 

 

 

 

 

Moisture content (%)

 

 

 

Figure 3-38. The effect of moisture content on the average pH of high moisture corn
silage during 5-day exposure period.

Effect of moisture was also observed in the glucose (Appendix Table A-16) and ethanol
(Appendix Table A-17) levels. The wetter silages tend to have higher glucose and
ethanol than the drier silages. The pH seems to have an effect on the glucose level. The
acidic silages (33-35% moisture levels) had higher glucose content. Higher glucose in the
acidic silages is possibly due to acid hydrolysis of structural components in the silage.
Jones et al. (1992) observed increased solubilization of cell wall sugars as pH decreased.
They further indicated that the effect of inoculation on increased solubilization of cell
wall components is due to its effect in reducing the silage pH. The glucose levels at 33
and 35% moisture levels decreased on d 5 of aerobic exposure. Contrary to the results of
Dawson et al. (1998) inoculation did not increase glucose levels. However, at 22%
moisture level, the P. acidipropionici DH42-105 had higher glucose values than the
control silages. Ethanol values tend to be lower for the inoculated silages particularly at
28% moisture level. Ethanol is a product of fermentation by heterofermentative lactic
acid bacteria and yeast (Mc Donald et al., 1991). Lower ethanol production in the
inoculated silages is possibly due to lower yeast counts in the silages. At (1 3, highest
ethanol production was observed in the control silages from the 35% moisture level with
0.695 g/ 100 g DM. In chemostat cultures, Thomas et al. (1979) observed that the mainly
homofermentative Streptococcus lactis shifts to heterolactic fermentation under low
glucose availability. Instead of producing lactate, there is a shift to formation of ethanol,
formate, and acetate. It would be noted that the drier silages had lower glucose levels.
The 22-24% moisture levels had some amounts of citric acid (Appendix Table A-18),
which were not affected by inoculation and exposure period. Butyric acid (Appendix

Table A-19) production was highest at 33% moisture level ranging from 0.15-0.32 g/ 100

82

g DM. Small amounts were also detected at 23-28% moisture levels. Butyric acid
production is mainly attributed to clostridial fermentation. Inoculation did not affect the
residual butyric acid levels in the silages. The silages with 35% moisture level had the
highest yeast and mold counts (Appendix Table A-20; Figures3-39 to 3-40) after 5 d
with about 108 cfu/g DM. Moreover, at this moisture level, yeast and mold counts
progressively increased over the 5 (1 exposure period. At other moisture levels, the
counts over the 5 d exposure period were variable with slight increases at d 5. While the
control had the highest yeast and mold counts at 22 and 23% moisture levels, the PAB-
treated silages had comparable counts with the RCM-treated silages. It is possible that
the nutrients present in the latter inoculant favored the growth of lactic acid bacteria and
propionibacteria whose growth is beneficial to ensiling. Dry matter recovery (Appendix
Table A-21) was not affected by inoculation. There is no apparent trend in the effect of

moisture in the temperature (Appendix Table A-22) of the silages. After 5 d of exposure,

 

 

 

 

 

 

 

2

Q

00

% Control
E? IDH42-A
% CI DH42-B
S U P. jensem'i
S I RCM

E73

8

>‘

 

Moisture content (%)

 

 

 

Figure 3-39. The effect of moisture content and inoculation on the yeast and mold counts
of exposed high moisture corn silage ((Standard error of the means: 35%=O.40,
33%:0.12, 28%=0.44, 24%=O.36, 23%:042, 22%=O.33).

83

 

 

10.00 '-

 

Yeast and molds (log cfu/ g Div
0‘
8

 

Moisture content (%)

 

 

 

Figure 3-40. The effect of moisture content on the yeast and mold counts of high
moisture corn silage during exposure period.

silages from the 28 and 35% moisture levels tend to have higher temperatures than

silages from other moisture contents (Appendix Table A-23).

Implications

The study shows that both moisture content and inoculation affected the
fermentation profile and aerobic stability of high moisture corn silage. Propionibacteria
enhanced the fermentation of high moisture corn silage. About 22-28% moisture in the
ensiling material is favorable to the growth of propionibacteria when used as silage
inoculants. At these moisture levels, higher propionic and acetic acids and lower pH and
butyric acid were observed with the PAB-treated silages after 120 d of ensiling. At
higher moisture levels (33-35%), the sharp decline in pH appeared to restrict the growth
of propionibacteria. While most of the silages appear stable when judged based on the

temperature of the exposed silages, the propionibacteria-inoculated silages had higher

84

residual organic acids (propionic and acetic) and lower pH. The use of higher inoculation

rate (10° cfu/g material) for DH42 is recommended.

85

CHAPTER 4
VITAMIN B12 PRODUCTION OF P. ACIDIPROPIONICI DH42 IN BATCH
CULTURE SYSTEM
Abstract
P. acidipropionici DH42 and P. shennam'i were grown in batch cultures at two

incubation temperatures (30 and 40°C) using Reinforced Clostridial Medium (RCM)
supplemented with 10 mg/l of CoClz.6H20. Samples were taken after 20, 40 and 72 h of
incubation for analysis. True vitamin B12, pH, optical density and organic acids of the
cultures were determined. Results showed comparable vitamin B12 production of the two
propionibacteria strains. After 72 h of incubation, the P. acidipropionici DH42 and P.
shermanii cultures grown at 30°C had vitamin B12 contents of 852.85 and 840.69 ng/ml,
respectively. Both strains grew better at 30°C than at 40°C. Poor growth of P. shermanir'
was evident at 40°C incubation. Lower pH was observed at lower incubation temperature
but for the P. acidipropionici DH42 cultures, the difference of the pH between the two
incubation temperatures was not significant. P. acidipropionici DH42 cultures tend to
have higher propionic and acetic acids while the P. shermanii cultures had higher

succinic and malic acids.

Introduction
Propionic acid and vitamin B1; are two major products of propionibacteria of
commercial importance. Industrial production of vitamin B12 uses Propionibacterium
spp. and Pseudomonas denim'ficans (Glatz, 1992). Among the propionibacteria strains,
P. shermanii and P. freudenrechiii are commonly used for this purpose. Vitamin Biz is

an essential part of enzyme systems that carry out basic metabolic functions. Humans

86

and animals depend on microbial synthesis for their supply of the vitamin. In ruminants,
dietary cobalt appears to be the main limiting factor in its synthesis by ruminal microflora
(Mc Dowell, 1989). However, Sutton and Elliot (1972) found that in high concentrate
diets, vitamin B12 synthesis is decreased and analogues, which have little or no vitamin
312 activity, are produced. The ruminal microorganisms such as Prevotella rumim'cola
(Strobel, 1992) and Bacteroides spp. (Varel and Bryant, 1974; Chen and Wolin, 1981)
require vitamin B12 for growth and propionate production. Strobel (1992) found that
microbial protein yields were reduced by 15 to 25% in the absence of vitamin Bl;

As a silage inoculant, P. acidipropionici DH42 has been shown to improve the
stability of aerobically exposed silages (Dawson et al., 1998). As a feed additive, it may
provide additional benefits to the animal as a source of vitamin B12. However, the
vitamin B12 production of P. acidipropionici DH42 has not been determined. This study
was conducted to determine the vitamin B12 production of P. acidipropionici DH42 using

two incubation temperatures under anaerobic condition of cultivation.

Materials and Methods
Cultures and Media
The two propionibacteria species used in this study were Propionibacterium
acidipropionici DH42 (ATCC 55737) and P. shermanii (ATCC 13673). Lactobacillus
leichmannii (ATCC 7830) was used for the microbial assay of vitamin Biz. P. shermanii
(ATCC 13673) and Lactobacillus leichmannii (ATCC 7830) were purchased from the
American Type Culture Collection. Both propionibacteria strains were maintained on

Reinforced Clostridial Medium (Oxoid) agar slants held at 4°C. Before use as inoculants,

87

at least three sub-transfers of each propionibacterium in Reinforced Clostridial Medium
(RCM) broth were done to activate the cultures. The headspace in the culture bottle was
flushed with C02 passed through a hot copper column to eliminate any oxygen.
Lactobacillus leichmannii cultures were maintained in B12 Culture Agar USP
(Difco) slants. Before the culture was used for the assay, sub-transfers using B12
Inoculum Broth USP (Difco) were done twice daily for a period of not less than one
week. The culture was assumed to be active when turbidity was observed within 2 h after

transfer.

Fermentation Media

Separate cultures were prepared for each propionibacterium strain at two
incubation temperatures (30°C and 40°C). The lower temperature setting (30°C) was
used because this is the optimum temperature for the grth of P. acidipropionci DH42.
The higher temperature setting (40°C) was used because this is the optimum temperature
for vitamin 312 Production based on the study of Quesada-Chanto et al. (1994b). After
48 h of growth, the cultures were transferred into 25-ml serum bottles at the rate of 10%
under C02. Reinforced Clostridial Medium (Oxoid) was used and supplemented with 10
mg/l of CoClz.6H20. After 48 h, 5,6 dimethylbenzimidazole (DMBI, Sigma) was added
at the rate of 10 mg/l. Samples were analyzed after 20, 40, and 72 h of incubation. Three
vials were prepared for each strain x incubation temperature x incubation time
combination. The pH of the cultures was measured using a pH meter (Cole Palmer).
Growth was also monitored by the optical density of the cultures at 600 nm using a
spectrophotometer (Spectronic 21D, Milton Roy). The cultures and the uninoculated

medium were analyzed for their contents of true vitamin B12 using a microbial assay and

88

organic acids were analyzed using HPLC. The vitamin B12 and organic acid
concentrations of the uninoculated medium were subtracted from the values determined
in the inoculated cultures to represent that which was produced from the metabolism of

the bacteria.

Vitamin Bu Assay
Vitamin B12 was determined by microbial assay using Lactobacillus leichmannii
(ATCC 7830) as described by Okada et al (1985). This assay is considered to analyze for
true vitamin B12 content. The assay extracts were prepared as follows: 1 ml of the culture
was added to 5 ml of 0.2 M acetate buffer (pH 4.5), 0.2 ml of potassium cyanide
(0.5mg/ml), and 30 ml of distilled water. The mixture was heated to 100°C for 30 min,
cooled and 0.3 ml of 10% metaphosphoric acid solution was added. The solution was put
in ice water for 30 minutes. Distilled water was added to the solution to bring the final
volume to 50 ml. The solution was centrifuged. Two aliquots of 20 ml each were
removed from the supernatant. One portion was adjusted to pH 6.0 and distilled water
added to reach a volume of 40 ml. The solution was recentrifuged to supply test extract
A. The other portion was adjusted to pH ll-12 and heated to 120°C for 30 min.
Thereafter, the pH was adjusted to 6.0 and distilled water added to bring the final volume
to 40 ml. The solution was recentrifuged to obtain test extract B in which the true
vitamin B12 is destroyed.
Using a standard vitamin B12 (Sigma) solution, tubes (16x100 mm) of different
Coneerltrations (0.0 to 0.25 ng/assay tube) of vitamin B12 were prepared as described by
USP ( 1 995). About 2.5 ml of 1312 Assay Medium USP (Difco) was measured into tubes

and . . .
the standard B12 solution or sample extracts were added. Dlstllled water was added

89

to bring the final volume to 5 ml. The tubes were autoclaved at 121°C for 5 min. The
tubes were allowed to cool and one drop of Lactobacillus leichmannii culture
(OD600=0.125) was added aseptically to all tubes except for two blank solutions. The
tubes were covered with sterile rubber stoppers and incubated for 10-15 h at 37°C. After
incubation, the tubes were stuck in the refrigerator for 15-20 minutes to stop the cultures
from growing (Becton Dickenson). The optical density (OD 600nm) of the tubes was
taken. Linear regression analysis was done using the absorbance of the standard vitamin
Br; solutions at a given concentration of vitamin B12. The vitamin Bu content of the

cultures was calculated from the standard curve.

Statistical Analyses
The data were analyzed as a multifactorial design using the general linear model
of the Statistical Analysis System (SAS, 1990). Treatment means were compared using
the Tukey-Kramer test (SAS, 1990). The ANOVA model used was as follows:
Yijkm = Hijk+ai+Bj+Yk+(afiY)ijk+(aB)ij+ (W)ilt+(BY)jlt+€rjltm
Where:

Yijkm = individual response variable measured (e. g. vitamin B12, propionic acid

etc.)
11.in = overall mean
01. = effect of propionibacteria strain (DH42, P. shermanii)
B,- = effect of temperature (30, 40°C)
‘yk = effect of time (20, 40, 72 h)
(011331)in = interaction of strain, temperature and time

(0113);]- : interaction of strain and temperature

90

(aY)11t= interaction of strain and time

(0305;. = interaction of temperature and time

81,-...“ = random residual error (assumed normally distributed)

Results and Discussion
The vitamin B12 concentration of the two propionibacteria cultures at different
incubation times and different incubation temperatures is shown in Table 4-1. After 20 h
of incubation, vitamin B12 production of P. acidipropionici DH42 grown at 30°C was
higher (P<0.01) than the P. shermanii cultures at 30°C but this value was comparable
(412.49 vs 406.13 ng/ml) to that of the vitamin B12 content of the P. shemranii culture at
40°C. Vitamin Biz content of the P. acidipropionici DH42 at 40°C after 20 h of
incubation was the lowest (P<0.01). Significant decline in the vitamin B12 levels of the P.
shennanii was also noted on the 40 h sampling time for the cultures incubated at 40°C.

Table 4-1. Effect of incubation temperature on the true vitamin B12 (ng/ml) of P.
acidipropionici DH42 and P. shennanii at different incubation times.

 

Incubation time (h)

 

 

 

Strains
20 4O 72
Incubation temperature (°C)
30 4O 3O 4O 3O 40
ng/ml

221.44d 406.13bc 327.25c 13.826 852.858 324.31c

P— Shermanii
430.91b 440.97b 840.69a 785.61a

P. acidipropionici 412.49bc 12.89 °

W“ 15.61 (19.11)
. Standard error of mean. Value in parenthesis is the SEM of P. shennanii culture at 20 h

'"Cubation at 40°C.
eails with unlike superscripts differ (P<0.01)

An atIalysis of the uninoculated medium showed that it contained about 132.64 ng/ml of

tru . . . . . .
(2 Vitamin B12. For both strarns, vrtamrn Big contents of the cultures inoculated at

91

30°C increased (P<0.01) over time. For the cultures inoculated at 40°C, there was a
significant decline in the vitamin Biz content of the P. shermanii cultures incubated at
40°C during the 40 h sampling time. Significant increase in vitamin B12 content of the
cultures was observed from 40 to 72 h incubation time. This is possibly due to the
addition of 5,6 DMBI after 48 h. This stimulates the production of vitamin B12 by the
formation of 5,6 dimethylbenzirnidazolyl cobamide (Yongsmith et al., 1982; Marwaha et
al., 1983; Quesada-Chanto et al., 1994b). Marwaha et al. (1983) suggested the addition
of 5,6 DMBI 24 h before the end of the fermentation. Propionibacteria can synthesize
their own DMBI under anaerobic conditions and adding DMBI at the start of the
fermentation inhibits the grth of the organisms (Friedman and Cagen, 1970).
Comparison of the vitamin Biz production of both strains is not easy since they
have different cell densities at a given sampling time. At 30°C incubation temperature
however, it appears that the two propionibacteria strains are comparable when the optical
density and the vitamin B12 contents of their respective cultures are compared. However,
at 40°C after 72 h of incubation P. shennanii appears to make more vitamin B12 than P.
acidipropionici DH42 per unit of cell yield The vitamin Biz content of the P. shermanii
cultures was about 324.31 ng/ml with an OD of 0.265. On the other hand, P.
acidipropionici DH42 had 786.61 ng/ml with an OD of 1.577. These values give an
estimate of vitamin Biz/unit OD of 1223.8 for P. shermanii, and 498.8 for P.
aCidiPropionici. Quesada-Chanto (1994b) observed increased vitamin B12 production as
the temperature is increased with 40°C as the optimum temperature for vitamin B12
p rod“Ction. They used a P. acidipropionici strain. The vitamin B12 contents of the P.

a ° .
Cld’Propionici DH42 cultures at 30 and 40°C incubation temperatures after 72 h

92

incubation were not significantly different although the CD of the 40°C cultures was
lower. At 48 h, the OD of the P. acidipropionicl' DH42 at 30°C incubation temperature
was significantly higher than that incubated at 40°C but their vitamin B12 contents are
comparable. Quesada-Chanto (1994b) noted that product formation might rapidly
increase with increasing temperature even if cell concentration is decreased which was
the case for the propionic acid and vitamin B12 production in this study. The increased
vitamin B12 production at higher incubation temperature could be due to increased cobalt
uptake with increased temperature. Scheneider et a1. (1995) observed increased cobalt-
binding affinity with P. arabinosum exposed to high temperature.

In comparing the vitamin Biz production of the strains, the effect of pH on
vitamin B12 synthesis, particularly for the P. acidipropionici DH42 should be considered.
Propionibacteria are strongly pH-dependent (Hsu and Yang, 1991). Quesada-Chanto
(1994b) indicated that the optimum pH for propionic acid and vitamin B12 production is
between pH 6.5-6.8. The pH of the P. acidipropionici DH42 cultures is significantly
lower than that of P. shemzam'i (Table 4-3). Although P. acidipropionici DH42 appears

to grow well even at low pH, vitamin B12 production could have been affected.

Result of this study is difficult to compare with other published results because of
the differences in culture conditions, substrates and the assay method for vitamin B12.
Unlike most studies which used the continuous culture and methods are geared towards

Conunercial production of vitamin B12, this study used a batch culture method. Hence,
with the accumulation of end products in the media, the resulting decrease in pH may
have affected cell efficiency and vitamin B12 synthesis. Bullerman and Berry (1966) used

th
e Sartre strain P. shermanii (ATCC 13673) to determine its vitamin B12 production

93

using cheese whey and they reported about 8 rig/ml of vitamin B12 incubation of 168 h.
In this study, 852.85 ng/ml was observed after 72 h of incubation. This present study
however accounts for only the true vitamin Biz while their study measured total vitamin
B12. Moreover, their study used continuous cultures with higher cobalt level (20 ppm),
longer incubation time (168 h) and with yeast supplementation. The continuous culturing
method helps the cells in a steady state of growth and prevents the pH decline, which was
a problem in this study. Hatanaka et al. (1988) observed that with pH-controlled batch
cultures, about 2.14 mg/l vitamin B12 was produced. Using a hollow-fiber module that
entrapped the cells and allowed the removal of organic acids produced, the vitamin B12
production rose to 52 mg/l. The growth promoting effect of yeast extract on
propionibacteria had been reported (Quesada-Chanto et al., 1994b, 1997) which was
attributed to vitamins present in the yeast extract (Hettinga and Reinbold, 1972).

Temperature affected the growth of both strains. Based on the OD of the cultures,
it is evident that both strains grew better (P<0.01) at 30°C than at 40°C (Table 4-2).
Moreover, the P. acidipropionici DH42 grew better than P. shermanii up until 72 h of

incubation where P. shermam’i grown at 30°C had comparable OD to the P.
acidipropionici DH42 cultures. P. shermanii grew very poorly at 40°C. On the other
hand, P. acidipropionici DH42 cultures appeared to be more tolerant of the higher
inc ubation temperature. After 72 h of incubation, P. acidipropionici DH42 cultures
grOWn at 30 and 40°C had comparable 01).
As expected, the pH of the cultures decreased with time (Table 4-3). At any

s"“mDIing time, the pH of the P. acidipropionici DH42 was lower (P<0.01) than that of P.

94

Table 4-2. Effect of incubation temperature in the optical density of P. acidipropionici

DH42 and P.shermanii at different incubation times.

 

 

 

 

Strain Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 30 40
P. shermanii 0.803“ 0.114d 1.382° 0.176°° 1.583“ 0.265“
P. acidipropionici 1.7002‘ 1.5073 1.6273 1.497 a 1.61721 1.57721
S.E.M.“ 0.014

 

nStandard error of mean.
Means with unlike superscripts differ (P<0.01)

shermam'i. Since P. acidipropionici DH42 grew faster than P. shermanii, the

accumulation of organic acids (mainly propionic and acetic acids) in the grth medium

could account for the lower pH in the P. acidipropionici DH42 cultures. The pH values

of the P. acidipropionici DH42 cultures in the two incubation temperatures were the

same. For the P. shermam'i cultures, the pH was lower (P<0.01) in the cultures incubated

at 30°C. With the inhibition of propionic acid on cell growth, Quesada-Chanto et al.

( 1994b) developed a two-stage fermentation process to produce vitamin B12 from

Table 4-3. Effect of incubation temperature on the pH of P. acidipropionici DH42 and P.

shermam'i at different sampling times.

*

 

 

 

 

 

\Strain Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 30 40

P- Shemtanii 5.79b 5.99“ 5.25“ 5.89“ 4.95d 5.81°

- aczdipropionlci 4.72“f 4.84““ 4.64f 4.66f 4.61f 4.65f
S.
"3% 0.025

andard error of mean.

earls with unlike superscripts differ (P<0.01).

95

molasses or sugar where the fermentation was switched from anaerobic to aerobic to
reduce the propionic acid production and enhance the production of vitamin B 12.
Propionic and acetic acid are the main products of propionibacteria] fermentation.
Table 4-4 shows the net production of propionic acid in the cultures. Significant
(P<0.01) three-way interaction of inoculant x incubation time x temperature was
observed in the propionic acid levels. The propionic acid content of P. acidipropionici
DH42 cultures was higher (P<0.01) than that of P. shermanii. Regardless of incubation
temperature, the propionic acid levels in the P. acidipropionici DH42 cultures
numerically increased over time but differences were not significant, while the levels in
the P. shennanii cultures increased (P<0.05) during the 40 to 72 h but not from 20 to 40 h
of incubation time. The significant increase could be due to the addition of 5,6
dimethylbenzimidazole at 48 h. Quesada-Chanto et al. (1994b) indicated that 5,6 DMBI
is required for the optimal production of cells and propionic acid.
Unlike the decrease in cell biomass with increased in incubation temperature, the
propionic acid production of P. acidipropionici DH42 was higher (P<0.01) than the P.
shermanii in the 40°C incubation temperatures both at 40 and 72 h sampling times.
Quesada-Chanto et al. (1994b) observed the high temperature dependence of P.
acidipropionici strain. They noted increased product concentration as temperature
inc>I‘eased with the optimum temperature of 37°C for propionic acid production. Unlike
the P - acidipropionici DH42 cultures, the P. shermanii cultures had higher (P<0.01)
p r Opionic acid levels in the 30°C incubation temperature than at 40°C. As noted in Table
4‘ 2’ P. shermanii grew very poorly at 40°C and the propionic acid levels were also the

lOWeSt (P<0.01).

96

Table 44. Effect of incubation temperature in the propionic acid content of P.
acidipropionici DH42 and P. shermanii at different incubation times.

 

 

 

 

 

Strain Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 , 30 40
%
P. shermanii 0048““ 0.009“ 0.154“ 0.015“ 0.280“ 0033““

P. acidipropionici 0.295 a 0.3443 0.329° 0.371a 0.349° 0.388 a

S.E.M.n 0.02
1'Standard error of mean.

Means with unlike superscripts differ (P<0.01).

In general, the acetic acid (Table 4-5) production of P. acidipropionici DH42 was
significantly higher than that of P. shermanii. The acetic acid production of the cultures
after 20 h of incubation did not differ among the cultures. However, after 40 and 78 h of
incubation, the P. acidipropionici DH42 cultures inoculated at 30°C had the highest
acetic acid production. For both propionibacteria strains, the lower incubation
temperature enhanced acetic acid production.

Table 4-5. Effect of incubation temperature in the acetic acid content of P.
acidipropionici DH42 and P. shermanii at different incubation times.

k

 

 

 

 

 

 

\3train Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 30 40
%

P— Shemanti 0.020“b 0.006b 0.051“b 0.010b 0.080“b 0.017b

- acidipropionicr' 0.083“b 0.141“ 0.096“b 0076“" 0.104“b 0.079“b
"88%. 0.024
M ta“(lard error of mean.

eans with unlike superscripts differ (P<0.01)

97

Although levels were low, measurable production of succinic acid (Table 4-6)
was observed particularly for the P. shermanii cultures. Only trace amounts were
detected with the P. acidipropionici DH42 cultures. There was no apparent effect of
incubation temperature although the highest (P<0.01) level (0.034%) was observed in the
P. shermanii cultures incubated at 40°C at 72 h sampling time. Quesada-Chanto et al.
(1997) also observed increased succinate production in P. shermanii cultures with certain
yeast extract brands. The increase in succinate formation correspondingly caused lower
propionic acid production. They speculated that the accumulation of succinic acid could
be due to the presence or absence of a substance interfering with the conversion of
succinic acid to methylmalonyl-CoA in the metabolic pathway to produce propionic acid.
As observed in this study, the P. shermanii cultures had significantly lower propionic
acid levels.

Table 4-6. Effect of incubation temperature in the succinic acid content of P.
acidipropionici DH42 and P. shermanii at different incubation times.

 

 

 

 

 

 

Strain Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 30 40
%
P. shennanii 0.03“b 0.01“d 0.03“b 0.02“b 0.02b 0.03“
P. acidipropionici 0.00(1 0.01“ 0.00“d 0.00d 0.00“d 0.00d
S.E.M°. 0.002
“ Standard error of mean.

Means with unlike superscripts differ (P<0.01).

Malic acid (Table 4-7) was also detected in the cultures. The uninoculated

Reinforced Clostridial Medium did not contain malic acid, so it can be assumed that

98

malic acid was formed from the fermentation of the bacteria. Malic acid is one of the
metabolites in the production of propionic acid via the succinate-propionate pathway
(Gottschalk, 1985). In general, P. shermanii cultures had higher malic acid than the P.
acidipropionici DH42 cultures, which as in the case of succinic acid, could also explain
the lower propionic acid levels in the P. shermanii cultures. Incubation temperature had
no consistent effect on the malic acid production in the cultures. Highest (P<0.01) malic
acid level (0.097%) was observed in the P. shermanii cultures incubated at 40°C sampled
at 40 h. There are no reports as what factors prevent the conversion of malate into
fumarate (Gottschalk, 1985) during the metabolic cycle. It can only be assumed that the
two propionibacteria strains are affected differently by the culture conditions. Using P.
shermanii, Ye et al. (1999) observed increased production of malate and fumarate when
culture condition is switched from anaerobic to aerobic conditions and thereby reducing
propionic acid production. They attributed this to the effect of oxygen on the enzymes.
It should be noted that P. acidipropionici could be grown aerobically (Dawson, 1994)
with substantial production of propionic acid. Although propionibacteria are generally
considered anaerobic microorganisms, some Propionibacterium species have been
observed to possess the components of typical aerobic electron transport chain (Vries et
al., 1972; Quesada-Chanto et al., 1998). The effect of the dissolved oxygen in the
medium to the growth of the two strains could not be discounted. It is possible that the
differences in both the succinic and malic acid contents of the cultures could be due to

differences in the oxygen-sensitivity of the two propionibacteria strains.

99

Table 4-7. Effect of incubation temperature in the malic acid content of P.
acidipropionici DH42 and P. shermanii at different incubation times.

 

 

 

 

 

Strain Incubation time (h)
20 40 72
Incubation temperature (°C)
30 40 30 40 30 40
%
P. shermanii 0092““ 0.031“"““ 0.000“ 0.097 “ 0031““ 0.096“b

P. acidipropionici 0.018 “ 0.052““““ 0.014“ 0.018°°° 0.014 “ 0017““

S.E.M.° 0.012
nStandard error of mean.

Means with unlike superscripts differ (P<0.01).
Implications
Results of the study showed that P. acidipropionici DH42 produce vitamin B12
levels that are comparable with P. shermanii at 30°C. With the P. acidipropionici DH42
cultures, higher cell yield was observed at 30°C but propionic acid production was higher
with the 40°C incubation temperature. Since batch cultures had been used in this study,
vitamin B12 production capacity of the cultures could have been limited by the growth

conditions. Further studies along this line are recommended.

100

CHAPTER 5

PCR-BASED DETECTION OF P. ACIDIPROPIONICI DH42 IN CORN SILAGE
AND RUMEN FLUID

Abstract

A polymerase chain reaction (PCR)-based method of detection for P.
acidipropionici DH42 in silage and rumen fluid samples was developed. Nested PCR
was used with DH42-specific primers dhbl and dhb2 for the secondary amplification of a
1,267 bp-fragment. Using the established protocols for PCR amplification, as low as 102
cfu/ml and 103cfu/ml of P. acidipropionici DH42 in silage extracts and rumen fluid,
respectively, were detected. Moreover, the 16S rDNA of P. acidipropionici DH42 was
sequenced and BLAST search showed its high homology to P. acidipropionici and two
other bacterial species. The results of an earlier study on its metabolic profile and the
168 rDNA sequence confirmed the earlier identification of DH42 as a propionibacterium

of the P. acidipropionici strain.

Introduction

Detection of propionibacteria in the environment is difficult because the media
that are currently used for their isolation are not sufficiently selective and colonies often
appear only after 6 d of incubation (Thierry and Madec, 1995). In addition, many strains
of propionibacteria are resistant to lysozyme (Johnson and Cummins, 1972). Hence,
DNA recovery from a given sample can be limited. Rossi et al. (1999) observed that with
forage and soil samples, cell numbers lower than 105 could not be detected and they
recommended a double-step amplification or semi-nested amplification to improve

sensitivity. Polymerase chain reaction (PCR) techniques have been used to identify and

101

distinguish different propionibacteria species (Riedel et al., 1994; Rossi et al., 1997;
Riedel et al., 1998; Rossi et al., 1999), and to distinguish it from other genera (Dasen et
al., 1998; Mielle et al., 1999).

Nested PCR can eliminate unwanted amplification products while at the same
time dramatically increasing sensitivity (Mullis and Faloona, 1987; Zhang and Ehrlich,
1994). Nested PCR amplifies the DNA in two steps. In the first round of PCR, a pair of
primers is used to generate a long segment that contains the target DNA. An aliquot of
the PCR product is then subjected to another round of amplification using primers
internal to the first set of primers to amplify the target DNA. This approach is often
successful even if the desired product is initially below the level of detection by ethidium
bromide staining and in the presence of visible spurious bands (Roux, 1995). The
efficiency of the second round of PCR is enhanced because of the more rapid and more
complete denaturation of the first reaction product as compared with the total genome
(Porter-Jordan et al., 1990).

With the potential commercial application of P. acidipropionici DH42 as an
inoculant, a system of detection. and monitoring is needed to evaluate its persistence and
efficacy. This study was conducted to develop a sensitive and more rapid detection of P.

acidipropionici DH42 in silage and rumen fluid samples.

Materials and Methods

Bacterial Strains and Culture Conditions
Propionibacteria, Iactobacilli and other bacterial strains as shown in Table 5-1
were used. Eubacten'um combesii (ATCC 25545) was purchased from the American

Type Culture Collection (ATCC). Propionibacteria and E. combesii were cultured using

102

Reinforced Clostridial Medium (Unipath) under C02 atmosphere and incubated at 30°C.

The Iactobacilli strains were cultured in Lactobacilli MRS broth (Difco) and incubated at

37°C. For propionibacteria strains, culture purity was initially checked by plating the

cultures in Purple Broth Base (Difco) agar supplemented with 1% i-erythritol (Sigma).

Some propionibacteria have the ability to ferment erythritol (Hetinga and Reinbold,

1972) and colonies are pigmented yellow in color. Rogosa SL agar (Difco) was used to

check the Iactobacilli strains. Cultures were also gram stained to check purity. Major

products of fermentation such as propionic acid and acetic acid for the propionibacteria

and lactic acid for the Iactobacilli were determined using high performance liquid

chromatography as described by Dawson (1994).

Table 5-1. Bacterial cultures used in the study.

 

Organism

Source

 

Propionibacterium acidipropionici DH42 ATCC 55737

Propionibacterium acidipropionici
Propionibacterium shennanii CDC 3094
Propionibacterium pentosaceum P11
Propionibacterium’ shermanii P92
Propionibacterium shermanii
Propionibacterium freudenreichii
Propionibacterium jensem'i P25
Propionibacterium sp. P42
Lactobacillus sakei

Lactobacillus delbrueckii subs. Iactis
Lactobacillus confusus

Bacillus subtilis

Bacillus sp.

Bacillus subtilis

Eubacterium combesii

ATCC 25562
Laboratory stock
Laboratory stock
Laboratory stock
ATCC 13673
ATCC 1382
Laboratory stock
Laporte Biochem. Intl.
ATCC 15521
ATCC 7830
ATCC 27646
Laboratory stock
Laboratory stock
ATCC 6633
ATCC 25545

 

16S rDNA Sequencing

Pure culture of P. acidipropionici DH42 was sent to Midilabs, Inc.

(http://wwwmidilabs.com) for 168 rDNA sequencing. Partial sequencing of the 1,267

103

base pair fragment was also done in the Molecular Pathogenesis Laboratory of the
Department of Animal Science, MSU. Secondary PCR product amplification from
rumen fluid sample was verified by gel electrophoresis in a 0.8 % agarose gel. The
remaining PCR product was then purified using Wizard® PCR Preps DNA Purification
System (Promega). Sequencing was done using ABI PRISMTM Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin Elmer) with primers dhbl and dhb2 as forward
and reverse primers, respectively. The ABI 373 Automated Sequencer (Perkin Elmer)

was used for in-house sequencing.

Primer Design and Synthesis

Primers dhbl and dhb2 (Table 5-2) were designed using the ARB program
(Strunk and Ludwig, 1996) from the alignment of the 16S rDNA of P. acidipropionici
DH42 with other sequences in the program’s database (as of July 1999). Regions
apparently unique to DH42 were selected and primers complementary to these regions
were designed. The universal primers bakl 1w and bak4 (Dasen et al., 1998)
corresponding to E. coli 16S rRNA positions 8-25 and 1522-1540, respectively, were also
used (Figure 5-1). Primers were synthesized by the Macromolecular Structure Facility of

MSU. Primers were stored at -20°C until use.

Silage and Rumen Fluid Sampling and Inoculation

Corn silage was collected from the Beef Cattle Teaching and Research Center.
Equal amounts (w/v) of silage (250g) and 0.9% saline solution (250 ml) were mixed
using a Stomacher (Tekmar) for about 10 min. The mixture was filtered using four layers

of cheesecloth. The homogenate was measured into five 50-ml centrifuge tubes. Each

104

tube was artificially inoculated with P. acidipmpionici DH42 culture at the rates of
102,103,104, and 10°cfu/ml silage extract. One tube was kept uninoculated. The silage
homogenates were kept in ice until DNA extraction was performed.

Rumen fluid samples were collected from ruminally-fistulated cows fed high
concentrate diets at the MSU Dairy Cattle Research Center. Rumen fluid was strained
using four layers of cheesecloth. Homogenate samples were placed into 50-ml centrifuge
tubes. Each tube was inoculated with DH42 as described above for the silage samples and
one tube was kept uninoculated. The samples were kept in ice until DNA extraction was
performed.

Aliquots of the corn silage and rumen fluid samples were frozen (-20°C). Due to
the sensitivity of the PCR reaction, extra care was taken to prevent cross-contamination
of inoculated and uninoculated samples. Gloves and sterilized glassware were used in

preparation of all the samples.

DNA Extraction

DNA was extracted from pure bacterial cultures and from corn silage and rumen
fluid samples using UltraCleanTM Soil DNA Isolation Kit (Mo Bio Laboratories). About
100 111 of sample was used for extraction. With the pure bacterial cultures, about 0.5 ml
cultures incubated overnight were used in DNA extraction. DNA extracts were stored at
—20°C until use.

DNA was quantified using Beckman DU600 spectrophotometer as described by
Maniatis et al., 1982. DNA integrity and verification of spectrophotometric
determination was checked by gel electrophoresis using a molecular weight marker (Bio-

Rad) in a 0.8 % agarose gel containing 0.5ug/ml ethidium bromide. Approximately 8 111

105

of DNA from each extract was mixed with 2 it] loading dye and electrophoresed at about

80 v for about 2-3 h.

Table 5-2. Alignment of 16S rDNA of propionibacteria and other bacterial species with
primers dhbl and dhb2 (ambiguous or non-matching positions are boxed).

 

Primer dhbl (5’—93’)
P. acidipropionici DH42
E. combesii

P. acidipropionici

P. thoenii

P. jensenii

P. granulosum

P. cyclohexanicum

P. propionicus

A. israelii

F. prausnitzii

CCGGATATGAGCTCCTG
CCGGATATGAGCTCCTG
CCGEhTA
CCGGATA
CCGGATATGAGCTCCTG
CCGGATATGAGCTCEEE
C—‘GATATqECCTCCTG
CjTGATATG GGC
CCGGATA ACATCCEFG
CCGGAT EaAGCTEK
CCGGAT EsAGCch

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Primer dhb2 (3’—>5’)
P. acidipropionici DH42
E. combesii

P. acidipropionici

P. thoem'i

P. jensenii

‘P. avidum

P. propionicus

P. granulosum

P. cyclohexanicum
A. israelii

F. prausnitzii

TTGTGCAAGACGCACCC
AACACGTTCTGCGTGGG
AACACGTTCTGCGTGGG
AACACGTfipTGCGTGmG

m N mmeGGG
E“%§% ..... a...

AAC - ..... T TGT ........ GGG

AACF ..... were 1 ........ GGG
AACACmT T "GTGGG

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4:; ierT :CGG
RC3 EerT EGWG
hcc ; G ..ECmG

 

 

 

 

 

 

 

 

 

 

 

106

Figure 5-1. DH42 168 rDNA sequence and the target sites of the four primers.

TGGAGAGTITGATCCT GGCT CAGGACGAACGCT GGCGGCGTGCTT AACAC AT
bakl l
GCAAGTCGAACGGTAAGGCCC'ITTCGGGGGTACACGAGTGGCGAACGGGTG
AGTAACACGTGAGTAACCTGCCCACI'TCI'I‘CGGGATAACGCTAGGAAACTGG
TGCTAATACCGG ATATGAGCI‘CCTGCCGCATGGTGGGGG'ITGGAAAGTGTTT
dhbl
GTGGTGGTGGATGGACT CGCGGCCT ATCAGCIT GT'I‘GGTGAGGTAGTGGCT C
ACCAAGGCGGTGACGGGTAGCCGGCCTGAGAGGGTGACCGGCCACATTGGG
ACT GAGATACGGCCCAGACT CCT ACGGGAGGCAGCAGTGGGGAATATTGCAC
AATGGGCGGAAGCCI‘ GATGCAGCAACGCCGCGTGCGGGATGACGGCCTT CG
GG'I'I‘GTAAACCGCI'ITCACCAGGGGCGAAGGCATyClTITGGGGTG’lTGACGG
TACCTGGAGAAGAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTG ATACG
TAGGGTGCG AGCGTTGTCCGGAT'I'I‘A'I'I‘GGGCGTAAAGGGCTCGTAGGCGGT
TGATCGCGTCGGAAGTGAAAACTTGGGGCTTAACCCTGAGCGTGCT'ITCGAT
ACGGG'I'I‘GAC’I‘I‘ G AGGAAGGTAGGGG AG AATGG AATTCCT GGTGGAGCGGT
GGAATGCGCAGATATCAGGAGGAAC ACCAGTGGCGAAGGCGGTTCT CT GGA
CCITI‘CCI‘GACGCTGAGGAGCGAAAGCGTGGGGAGCAAACAGGCTI‘AGATAC
CCTGGTAGTCCACGC'I‘GTAAACGGTGGGTACTAGGTGTGGGGTCCA'I’I‘CCAC
GGA’I'I‘CCGTGCCGTAGCI‘AACGC ATTAAGTACCCCGCCTGGGGAGTACGGCC
GC AAGGCT AAAACT C AAAGGAATTGACGGGGCCCCGC ACAAGCGGCGG AGC
ATGCGGATTAATI‘CGATGCAACGCGAAGAACC'I'T ACCT GGGTTTGACATGGA
TTGGTAACGGTC AGAGATGGCCGCCCCCCTTGTGGGCCGGTTCACAGGTGGT
GCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG'I'I‘AAGTCCCGCAACG
AGCGCAACCCTCGTCCACTGTI‘GCCAGCATI'I‘GGTTGGGGACTCAGTGGAGA
CCGCCGGGGTCAACTCGGAGGAAGGTGGGG ATG ACGTCAAGTCATC ATGCCC
CTT ATGTCCAGGGCTT CACGCATGCT AC AATGGCCGGTACAAAGAGTGGCGA
CATCGTGAGGTGGAGCGAATCT CAGAAAGCCGGTCT C AG'I'I‘CGGA'ITGGGGT
CT GCAACI‘ CGACCCCATGAAGTCGGAGTCGCT AGTAATCGCAGATCAGCAAC
GCI‘GCGGTGAATACGTTCCCGGGGCTI‘GTACACACCGCCCGTCAAGTCATGA
AAGTCGGTAACACCCGAAGCCGGTGGCCCAACACGTTCTGCGTGGGGGAGTC
dhb2
GTCGAAGGTGGGACI‘ GGTAATI‘AGG ACT AAGTCGTAACAAGGTAGCCGTACC
GGAAGGTGCGGyTGGATC ACCT CC'IT
bak4

107

Polymerase Chain Reaction

Nested PCR reaction was done as described by Herman et al. (1995) and Rossi et
al. (1999). The first round of PCR was done using 0.2 ml tubes in a 50 pl reaction
mixture containing 5 pl of 10X PCR buffer (GibcoBRL), 2.0 pl of SOmM magnesium
chloride ((GibcoBRL), 1 pl of 1.25 mM dNTPs mixture (GibcoBRL), 2.5 U of Taq
polymerase (GibcoBRL), 20 pmol of each primer (bak4 and bakl l), and 1 pl template
DNA (100 ng). Distilled water (GibcoBRL) was added to make up a volume of 50 pl.
PCR was done using a Perkin-Elmer 9600 thermal cycler as follows: 3 min of
denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for 30 sec, annealing
at 52°C for 30 sec and extension at 72°C for l min, then a final extension step at 72°C for
10 min.

For the second round of PCR, an aliquot of the dilution of the primary PCR
product was used as template. A 50 pl reaction mixture was prepared containing 5 pl of
10X PCR buffer (GibcoBRL), 2.0 pl of SOmM magnesium chloride ((GibcoBRL) for the
control and appropriate amount for-the samples containing DNA, 1 pl of 1.25 mM dNTPs
mixture (GibcoBRL), 2.5 U of Taq polymerase (GibcoBRL), 20 pmol of each primer
(dhbl and dhb2), and 1 pl template DNA. Distilled water (GibcoBRL) was added to
make up a volume of 50 pl. The second amplification was done after an initial
denaturation step at 94°C for 3 min, by 30 cycles of denaturation at 94°C for 30 sec,
annealing at desired temperature for 30 sec, and extension at 72°C for l min then a final
extension step at 72°C for 10 min.

A two-step PCR amplification was also done to compare it with sensitivity of the

nested PCR in detecting the presence of P. acidipropionici DH42 in rumen fluid samples.

108

The first round of PCR was done using 0.2m] tubes in a 50 pl reaction mixture containing
5 pl of 10X PCR buffer (GibcoBRL), 1.50 pl of 50mM magnesium chloride
(GibcoBRL), 1 pl of 1.25 mM dNTPs mixture (GibcoBRL), 2.5 U of Taq polymerase
(GibcoBRL), 20 pmol of each primer (dhbl and dhb2), and 1 pl template DNA (100 ng).
Distilled water (GibcoBRL) was added to make up a volume of 50 pl. PCR was done as
follows: 3 min of denaturation at 94°C, followed by 30 cycles of denaturation at 94°C for
30 sec, annealing at 64°C for 30 sec and extension at 72°C for 1 min then a final
extension step at 72°C for 10 min. Another round of amplification was done using the
same primers and 5 pl of the 1:10 dilution of the primary PCR products. The annealing
temperature was maintained at 64°C but magnesium chloride concentration was reduced

to allow amplification of the target band in the DH42-inoculated samples.

Optimization of PCR Reaction

Primary PCR was done using standard conditions as described previously. For
the secondary PCR, optimum annealing temperature and concentrations of Mg”, primers,
dNTPs, and Taq polymerase were determined. The calculated annealing temperature for
primers dhbl and dhb2 (54°C) was used as the starting temperature for the optimization
(Table 5-3). Different concentrations of Mng (0.5, 0.75, 1.0, 1.25, and 1.5 mM),
primers (7.5, 10, 20 pmols), dNTPs (12.5 pM, 25 pM) and Taq polymerase (2.0 and 2.5
units, U) were tested.

The optimum temperature and concentration were determined as that which
generated the strongest band staining after UV illumination of the gel without the
appearance of other bands. The target DNA in the secondary PCR product is a 1,267-

base pair band when DH42 is present in the sample.

109

Table 5-3. Synthetic oligonucleotides sequences, positions and calculated melting
temperature(T,,.)

 

 

Primer Sequence (5'—)3') E. coli position Tm‘(°C)
dhbl CCGGATATGAGCI‘CCTG 172-188 F 54
dhb2 CCCACGCAGAACGTG’I'I‘ 1429444512 54
bakll AGGAGGTGATCCARCCGCA 8-25 F 50
bak4 AG'I'I‘TGATCMTGGCI‘CAG 1522-1540 R 58

 

iCalculated based on the formula: T," = 4 (G+C) + 2 (A-i-T)

Primer Specificity Evaluation

DNA extracts from pure bacterial cultures and from rumen fluid and corn silage
samples were used as templates. Nested and double-step PCR reactions were done as
described previously using the optimized conditions for the primers. Specificity is

determined by the appearance of the 1,267 bp band in the secondary PCR products.

Phylogenetic Analysis

Using DH42 l6S rRNA sequence as a query sequence, a local alignment search
was done using the Basic Local Alignment Search Tool (BLAST) at the National Center
for Biotechnology Information (NCBI) World Wide Web site
(http://www.ncbi.nlm.nih.gov/BLAST/). BLAST uses a heuristic algorithm, which seeks
local as opposed to global alignments and is therefore able to detect relationships among
sequences that share only isolated regions of similarity (Altschul et al., 1990). Out of 377
BLAST hits on the query sequence, l6S rRNA sequences of 25 Propionibacterineae, 24
Actinobacteria and three environmental samples of unidentified eubacteria were selected
for further analysis (Table 5-4).

Using the computer software package Windows 32 MegAlign© 1993-1999

(DNASTAR Inc.), multiple alignment of 168 rRNA sequences of DH42, 25

110

Propionibacterieae species, 23 other Actinobacteria species and Escherichia coli' (as a
non-related organism) was done and their phylogenetic relationships were estimated. The
alignment of the sequences was performed using the Clustal V method described in
Higgins and Sharp (1989). The Clustal V method groups sequences into clusters by
examining sequence distances between all pairs. Clusters were aligned as pairs then
collectively as sequence groups to produce the overall alignment. After the multiple
alignment was completed, a Neighbor-Joining method was employed to reconstruct
phylogeny for the putative alignment. A phylogenetic tree was generated on which

branch distances (lengths) corresponded to sequence divergence.

Gel Electrophoresis

PCR products were run in 0.8 % agarose gel (GibcoBRL) stained with 0.5 pg/ml
ethidium bromide (GibcoBRL). About 2 pl 10X Blue JuiceTM gel loading buffer
(GibcoBRL) was added to 8 pl of PCR product and electrophoresed at 80V for about 2-3

h. A lkb DNA ladder (GibcoBRL) was used as a marker.

Results and Discussion

PCR Optimization

Figure 5-2 shows the combined effect of magnesium chloride concentration and
annealing temperature on the appearance of the 1,267 bp band with DNA extracted from
P. acidipropionici DH42. It shows that amplification of the target band is possible even
at 69°C using 1.25 mM of Mng. In addition to the magnesium chloride concentration,
the optimum annealing temperature also depends on other factors such as the
concentration of dNTPs, primer and Taq polymerase and amount of DNA. For the primer

concentration, it was found that amplification was possible using 10 pmol of each primer.

1]]

The lowest concentration tested (7.5 pmol) showed no amplification product. Taq
polymerase level was tested at 2.5, 2.0, and 1.5 U. No amplification product was
observed at less than 2.0 U. With pure cultures, stringency was also attained by diluting
the primary PCR products to 1:1,000. At 68°C annealing temperature and 1.5 mM
MgC12, using 1 pl of a 1:1000 dilution of the primary PCR product as template for
secondary PCR prevented the non-specific amplification of other non-propionibacteria
and most propionibacteria] species tested. Among the propionibacteria strains used, only
P. acidipropionici (ATCC 25562) had the 1,267-hp fragment after secondary PCR under
these PCR conditions. Dawson (1994) had used this microorganism as a reference in the
initial identification of DH42 and had observed the similarity its metabolic profile with P.
acidipropionici DH42. This indicates that the two microorganisms are closely related.
The PCR optimization was then aimed at preventing the amplification of the 1,267 bp
band with the P. acidipropionici (ATCC 25562) samples. This was done by adjusting the
primer, dNTP and Taq polymerase concentrations. At 68°C annealing temperature, and
using 10 pmols of each primer, 1.0 mM of MgClz, and 2.0 units of Taq polymerase, the
amplification of Propionibacterium acidipropionici (ATCC 25562) and other bacterial

species was prevented (Figs. 5-3 and 5-4).

Primer Specificity

Based on the alignment of the 168 rDNA of P. acidipropionici DH42 with the
ARB (Strunk and Ludwig, 1996) program’s database, E. combesii was found to have the
closest match differing by only four nucleotides. During the secondary amplification
using 68°C annealing temperature, the estimated 1,267bp fragment was not detected

using E. combesii DNA. If it differed with DH42 by just four nucleotides, a less stringent

112

condition would have allowed amplification of the same size fragment in E. combesii. In
the alignment of the specific-propionibacteria primer gdl, Dasen et al. (1998) observed
that E. combesii showed 100% similarity with the target sequence. Morever, they also
observed it clustered with P. thoenii (92.3%) and had only 78% similarity with other
Eubacterium species. They also found that A. israelii is more similar to P. acnes than to
other species from the genus Actinomyces. They concluded the need for re-evaluation of
these strains or sequencing problems. Based on the HPLC analysis of E. combesii
overnight culture medium, it produced iso-acids but did not produce significant amounts
of either propionic acid or acetic acids, which would indicate that it is not a
propionibacterium. Moreover, it did not form yellow colonies using Purple Base Agar
with i-erythritol while all the propionibacteria species used in this study did. During
PCR, E.combesii DNA did not form the 1,267 bp-fragment with primers dhbl and dhb2.
Under less stringent conditions, amplification of this fragment would have been possible
if it differs with DH42 by only 4 nucleotides. At 68°C annealing temperature, it formed a
fragment size that is about 1.5 kb (Fig. 5-5). A similar fragment size was observed in

other bacteria when the amount of template for secondary amplification was high.

113

Hill

-1636
-1018

—-—-—-——— “' """'

1

Figure 5-2. Effect of annealing temperature (lanes 1-3, 66°C; lanes 4-6, 67°C, lanes 7-8,
68°C, lanes 9-11, 69°C) and magnesium chloride concentration:1.5 mM (lanesl,4,7); 1.25
mM (lane 10); 1.0 mM (lanes 2, 5, 8, 9); and 0.5 mM (lanes 3 and 6); lane 12, lkb DNA
ladder.

1234567891011121314151617_

“
.

“b?

-1636

71018

 

Figure 5-3. Primary PCR showing the 1.5 kb fragment using primers bakll and bak4
with the following bacterial strains: 1, Propionibacterium freudenreichii; 2, P. shemzanii
CDC3094;3, P. shermanii P92; 4, P. thoenii P15; 5, P. pentosaceum P11; 6, P.
acidipropionici (ATCC 25562); 7, P. shemzanii; 8, Propionibacterium sp.; 9, P.
acidipropionici DH42; 10, Lactobacillus sakei; 11, Lactobacillus confusus; 12,
Lactobacillus delbruickii; 13, Bacillus subtilis; 14, Bacillus sp.; 15, Bacillus subtilis
(ATCC 6633); 16, Eubacterium combesii; 17, 1 kb DNA ladder

114

1234567 891011121314151617

-’

 

, ,__.. :. _ _ -. 4 . ;_. , . _

Figure 5-4. Secondary PCR showing the 1.3 kb fragment amplification using primers
dhbl and dhb2 with the following bacterial strains: 1, Propionibacterium freudenreichii;
2, P. shermanii CDC3094; 3, P. shennanii P92; 4, P. thoenii P15; 5, P. pentosaceum
P11; 6, P. acidipropionici (ATCC 25562); 7, P. shermanii; 8, Propionibacterium sp.; 9,
P. acidipropionici DH42; 10, Lactobacillus sakei; 11, L confusus; 12, L delbruickii; 13,
Bacillus subtilis; 14, Bacillus sp.; 15, B. subtilis (ATCC 6633 ); l6, Eubacterium
combesii; 17, 1 kb DNA ladder.

1 2 3 4 5
_'
. bp
2...“ --- -l636
’—

—1018

Figure 5-5. Secondary PCR amplification products showing the 1,267 bpofragment size
in P. acidpropionici DH42, lane 1; P. acidipropionici (ATCC 25563), lane 2;
Eubacten'um combesii, lane 3; negative control, lane 4; l-kb DNA ladder, lane 5.

115

The detection limit of the assay was also determined using rumen fluid and corn silage
extracts. Figure 5-6 shows the primary amplification products in the rumen fluid and
corn silage extracts. The estimated 1.5 kb fragment size was observed in all the samples.
Smearing of the bands in the rumen fluid samples can indicate the abundance of bacterial
DNA in the samples. For the rumen fluid samples, stringency of the secondary
amplification was sufficient at 66°C annealing temperature and 1.5 mM magnesium
chloride. Under these PCR conditions, it was observed the lowest inoculation rate that
can be detected was 103 cfu/ml (Fig. 5-7). Lower annealing temperature for secondary
PCR was used as compared to that found in the PCR optimization protocol (66°C vs
68°C) because it was observed that at higher annealing temperature, the 104 cfu/ml was
the lowest inoculation rate that could be detected. With the silage extracts, the same
annealing temperature was used, but lower magnesium chloride concentration (1.0 mM)
was used to prevent the non-specific amplification of the uninoculated samples. Under
these conditions, as low as 100 cfu/ml of P. acidipropionici DH42 can be detected in the
silage extracts. The difference in the detection limit in rumen fluid and corn silage
samples could be due to their differences in their microbial load and how these affect
amplification efficiency and the presence of contaminants that interfere with PCR. While
it can be assumed that the amplification efficiency is the same for all 16S rDNA in the
rumen fluid samples, the use of universal primers contain degeneracy that may influence
the formation of primer-template hybrids (Wintzingerode et al., 1997). Moreover, varying
molecular percent G + C composition of 16S rRNA genes can also cause differential
amplification. Templates with lower G + C content will have more efficient strand

separation, thus preferential amplification may result (Wintzingerode et al., 1997). With

116

the abundance of DNA in the rumen fluid samples, higher competition with non-DH42
DNA could have reduced the amplification of DH42 16S rDNA during primary PCR.
Consequently, the initial template for secondary amplification may have been less in the
rumen fluid samples as compared to the corn silage extracts. Increasing the amount of
template for secondary PCR or using less stringent condition such as higher magnesium
chloride concentration did not improve the sensitivity of the assay. It is also possible that
certain amounts PCR inhibitors is present in the rumen fluid samples making
amplification less efficient as compared to the silage extracts. While the DNA extraction
kit included a solution to remove PCR inhibitors, it might have not been sufficient to
remove all the inhibitors in the rumen fluid samples. The presence of humic acids and
humic substances in environmental samples and their negative effect in lysis efficiency

PCR amplification had been documented (Wintzingerode et al., 1997).

_
_-1636

-1018

 

Figure 5-6. Primary PCR showing the amplification products of primers bak4 and bakll
using DNA extracts from rumen fluid (lanes 1-5, amplification from uninoculated rumen
fluid, 101103, 104, 10°cfu/ml, respectively) and silage (lanes 6-10, amplification from
uninoculated silage, 102103, 104, 10°cfu/ml, respectively); lane 11, negative control and
lane 12, 1 kb DNA ladder.

117

123456789101112

bp

, ‘. -l636

Figure 5-7. Secondary PCR showing the amplification products of primers dhbl and
dhb2 using DNA extracts from rumen fluid (lane 1-5, amplification from uninoculated
rumen fluid, 102.103, 10“, 10°cfu/ml, respectively) and silage (lanes 640, amplification
from uninoculated silage, 102‘ 103, 104, 105cfu/ml, respectively); lane 9, negative control;
lane 10, 1 kb DNA ladder.

A double-step PCR amplification for the rumen fluid samples was tried to check
if selective amplification of DH42 and better detection could be achieved. Rossi et al.
(1998) recommended this procedure to increase the sensitivity of detection of
propionibacteria in silage or soil samples. In the first round of PCR, slightly lower
annealing temperature (64°C) was used to allow better amplification since at higher
temperature (66-68°C), no primary amplification product was observed in the samples
with lower inoculation rates. In the second step, while the temperature was maintained
at 64°C, magnesium chloride level was reduced 1.25 mM to provide a more stringent
condition and prevent amplification of non-DH42 DNA. In the primary amplification,
the uninoculated sample showed the presence of the 1,267 bp—band (lane 1). This is not
unusual since a less stringent condition would allow amplification of non-specific

hybridization products. The presence of other P. acidipropionici species in the rumen

could have produced this band. In the second amplification, while the decreased amount

118

of magnesium chloride prevented the amplification in the uninoculated samples, the 103

cfu/ml of DH42 was the lowest inoculation rate that could be detected (Figure 5-8).

 

Figure 5-8. Secondary PCR of rumen fluid samples: lane 1, uninoculated; lane 2, 102;
lane 3, 103; lane 4, 10‘; lane 5, 105 cfu/ml; lane 6, negative control, lane 7, lkb DNA
ladder.

Phylogenetic Analysis

Table 5-4 shows the Propionibacteriacea species that was used for the analysis.
The highest BLAST scores (2387-2778) were found for three species: P.
microaerophilus, E. combesii, and P. acidipropionici. This shows that the three
organisms are the ones closely related to DH42 based on their 16S rRNA sequences.
Dawson (1994) observed that the metabolic profile of P. acidipropionici (ATCC 25562)
closely matched that of P. acidipropionici DH42. This confirms the identity of this
species as P. acidipropionici.

Figure 5-9 shows that P. acidipropionici DH42 formed a cluster with the P.

acidipropionici, E. combesii, and P. microaerophilus. P. acidiprapionici DH42 is also

119

within the cluster of P. jensenii and P. thoenii which is in agreement of the findings of

Charfneitag and Stackebrandt (1989).

Table 5-4. Taxonomy BLAST report for 13 propionibacteria species compared against
DH42 using BLAST

Bacteria [eubacteria] BLAST score
. Actinobacteria [ Firmicutes, high GC Gram+] (bits)
. Actinomycetales [Actinobacteridae]

Propionibacterineae [actinomycetes]
Propionibacteriaceae [actinomycetes]

Propionibacterium [actinomycetes]
Propionibacterium microaerophilus --------------- 2778
Propionibacterium acidipropionici ............... 2387
Propionibacterium thoenii ....................... 2240
Propionibacterium jensenii ...................... 2065
Propionibacterium propionicus DSM 43307 ........ 1764
Propionibacterium avidum DSM 4901 ............... 1756
Propionibacterium acnes ......................... 1651
Propionibacterium sp. V07/12348 ................. 978
Propionibacterium cyclohexanicum ................ 821
Propionibacterium sp. LCDC—98AO72 ............... 815
. Propionibacterium propionicus ................... 794
Propioniferax innocua ............................. 786
Eubacterium combesii -------------------------------- 2680

DNA Sequencing

Figure 5-10 shows the alignment of the sequenced fragment with the DH42 16S
rDNA starting from E. coli position 196 to 767. Of the 571 nucleotides in the DH42
DNA sequence, the sequence of the fragment from the rumen fluid sample differed by 6
nucleotides. Differences in the sequences of the DH42 and that of the fragment could be
due to the differences in the primers used for the sequencing. Method of primer synthesis
and approach to primer purification can affect the quality of the sequencing data obtained
in the dye terminator cycle sequencing reactions (Perkin Elmer, 1995). BLAST search
was also done using the partial sequence of the fragment. Table 5 shows the top ten out of

the 200 BLAST hits in the query sequence. The highest BLAST scores (1005-1031)

were found for three species: P. microaerophilus, E. combesii, and P. acidipropionici,

which agree with the earlier BLAST results of the DH42 sequence.

Implications
This study showed that as low as 102 and 103 cfu/ml of P. acidipropionici DH42
can be detected in corn silage and rumen fluid, respectively. While the PCR assay is not
as sensitive in the rumen fluid as compared to the silage samples, it appears sufficient
considering the current suggested inoculation rate of at least 105 cfu/g material in silage.
Improvement in the detection of P. acidipropionici DH42 in rumen fluid samples might

be achieved with a different DNA extraction method or the use of PCR enhancers.

121

Figure 5-9. Phylogenetic tree of the order Actinomycetales. The length of each pair of
branches represents the distance between sequence pairs, while the units at the bottom of
the tree indicate the number of substitution events

122

Myeooactenum cnltae
Propionibacterium acnes

Actinomyces israeli
uncultured Actinomycete sp.

P. sp. V07/12348
P. avidum DSM 4901
P. propionicus DSM 43307
_ DH42

Eubacterium combesii
P. acidipropionici
P. microaerophilus
P. jensenii
P. thoenii
'"E P. cyclohexanicum
P. sp. LCDC-98AO72

Tessaraooocus bendigoensis I
Luteococcus japonicus
Luteococcus sp. CCU638120
Propionilerax innocua
Friedmanniella antarctica
Friedmanniella spumicola
Friedmanniella capsulata
Microlunatus phosphovorus
Nocardioides fulvus
Nocardioides sp. ATCC 39419
'- Hongia koreensis

Marrnoricola aurantiacus
fiocardioides sp. (strain KP7)

 

 
 
 
  
  
 
 

 

 

 

 

 

Nocardioides sp. NSP 41
Noeardioides sp. 084 ______________________________
Actinokineospora globicatena
Actinokineospora diospyrosa
Saccharothrix cryophilis
Kibdelosporangium aridum
Streptoalloteichus hindustanus
Amyoolatopsis sulphurea __ __ ___________
Corynebaeterium sp. 61720
L Corynebaeterium tuberculostearicum
uncultured Corynebaeterium sp. MTcory2P
uncultured Corynebaeterium sp. MTcory20Fl
r- Actinoplanes cyaneus """"""""""""""""
'- Micromonosporaceae, isolate SR 83 IV
Ornithinicoocus hortensis ' """""""""
Streptomyces scabies """"""""""""""""""
Streptomyces sp.
- Streptomyces sp. EF-35
Streptomyces bottropensis
Streptomyces neyagawaensis
- Streptomyces diastatochromogenes
Escherichia coli """""""""""""""
l T l T l

20 15 10 5 0
Figure 5-9

 

 

 

 

 

 

 

 

 

 

 

 

 

20.7

 

1 96
DH42 .......... GGAAAGTG I I I GTGGTGGTGGATGGACTCGCGGCCTATCAGCTT

RF .............. GGAAAGTGTI'I‘GTGGTGGTGGATGGACTCGCGGCCTATCAGCTT
DH42 ........... GTTGGTGAGGTAGTGGCTCACCAAGGCGGTGACGGGTAGCCG
RF .............. G'I'TGGTGAGGTAGTGGCTCACCAAGGCGGTGACGGGTAGCCG
DH42 ........... GCCTGAGAGGGTGACCGGCCACA'I‘TGGGACTGAGATACGGCC
RF .............. GCCTGAGAGGGTGACCGGCCACA'ITGGGACTGAGATACGGCC
DH42 ........... CAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATG
RF .............. CAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATG
DH42 ........... GGCGGAAGCCTGATGCAGCAACGCCGCGTGCGGGATGACGGCC
RF .............. GGCGGAAGCCTGATGCAGCAACGCCGCGTGCGGGATGACGGCC
DH42 ........... TI‘CGGG'I'TGTAAACCGC'I‘ITCACCAGGGGCGAAGGCAT
RF ............... TTCGGGTI‘GTAAACCGC'ITTCACCAGGGGCGAAGGCA
DH42 ........... GGGGTGTTGACGGTACCTGGAGAAGAAGCACCGGCTAACTAC
RF .............. GGGGTG'I'I‘GACGGTACCTGGAGAAGAAGCACCGGCTAACTAC
DH42 ........... GTGCCAGCAGCCGCGGTGATACGTAGGGTGCGAGCGTI‘GTCCG
RF .............. GTGCCAGCAGCCGCGGTGATACGTAEGGTGCGAGCGTTGTCCG
DH42 ........... GATI'TATTGGGCGTAAAGGGCTCGTAGGCGG’T'I‘GATCGCGTCG
RF .............. GATITATTGGGCGTAAAGGGCTCGTAEGCGG’TTGATCGCGTCG
DH42 ........... GAAGTGAAAAC'I'I‘GGGGC’ITAACCCTGAGCGTGC’ITI‘CGATAC
RF .............. GAAGTGAAAAC’ITGGGGCITAACCCI‘GAGCGTGCT'I‘TCGATAC
DH42 ........... GGGTTGACI'I‘GAGGAAGGTAGGGGAGAATGGAATTCCTGGTGG
RF .............. GGGTTGAC'ITGAGGAAGGTAGGGGAGAATGGAATTCCTGGTGG
DH42 ........... AGCGGTGGAATGCGCAGATATCAGGAGGAACACCAGTGGCGA
RF .............. AGCGGTGGAATGCGCAGATATCAGGAGGAACACCAGTGGCGA
DH42 ........... AGGCGGTTCTCTGGACC'ITTCCTGACGCTGAGGAGCGAAAGCG
RF .............. AGGCGGTTCI‘CTGGAECTTTCCTGACGCTGAEGAGCGAAAGCG
767
DH42 ........... TGGGGAGCAAACAGGC’I’I‘AGATAC
RF .............. TGGGGAGCAAACAGGCITAEATAC

Figure 5-10. Partial nucleotide sequence of the 1,327 bp-fragment from rumen fluid (RF)
sample using primers dhbl and dhb2 as forward and reverse primers, respectively.
Ambiguous or non-matching positions are boxed.

124

Table 5-5. Distribution of top ten Blast hits on the query sequence.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Sequences producing significant alignments Score

(bits)

gbbF234623JlAF234623 Propionibacterium microaerophilus 16... 1031
gbIL34614.1[EUBRRDI-I Eubacterium combesii 16S ribosomal RNA 1019
embIX53221.llPACP116S Propionibacterium acidi—propionicija... 1005
emleS3220. llPT H168 Propionibacterium thoenii partial 16S rRNA 862
embIXS3219.l|PJ 16S Propionibacteriufljensenii partial l6S rRNA 813
emblAJOO3058.1|PPAJ3058 Propionibacterium propionicus DSM 4... 549
embIAJ003055. llPAAJ3055 Propionibacterium avidum DSM 4901 1... 549
emblY17821.leRSPl7821 Propionibacterium sp. VO7/ 12348 168 535
gbIAF 154099.11AF 154099 Uncultured hydrocarbon seep bacteriu... 519
gbIAF 1548321 IAF 154832 Propionibacterium acnes l6S ribosoma... 519
blAF145256.1|AF 145256 Propionibacterium acnes 16S ribosoma... 519

 

125

 

CHAPTER 6

SUMMARY AND CONCLUSION

Two studies were conducted to evaluate the efficacy of propionibacteria as silage
inoculants. In the first study, the performance of propionibacteria with or without lactic
acid bacteria were evaluated using reconstituted corn. Results showed that
propionibacteria enhanced the fermentation of reconstituted corn only up (I 21 of ensiling.
The combination of P. acidipropionici DH42 with lactic acid bacteria as inoculants
reduced silage pH and butyric acid and increased propionic, acetic and lactic acids. LAB
inoculation did not significantly increase the LAB population in the treated silages.
During aerobic exposure, all the silages appeared well preserved. Organic acid levels
remained stable throughout the exposure period. Propionibacteria inoculation did not
Significantly reduce the yeast and mold population. However, the silages with P.

acidipropionici DH42 + LAB had higher propionic, acetic and lactic acids and lower pH.
In the second study, the effect of moisture on the efficacy of propionibacteria as
Silage inoculants was tested. Rolled corn of moisture contents ranging from 22-35%
Were used. The moisture of the ensiling material affected the efficacy of propionibacteria
as inOculants. The 22-28% moisture levels appeared to favor the growth of the P.
ac idipropionici DH42 in silage. After 120 d of ensiling, PAB-inoculated high moisture
Com gave significantly higher propionic and acetic acids at 22-28% moisture levels.
LOWer pH at 24 % and butyric acid at 28% moisture levels was also observed with the
PAB~treated silages. Inoculation did not affect the yeast and mold counts during
ensiling. P. acidipropionici DH42 inoculated at 10°cfu/g better gave results as compared

0 the 10°cfu/g inoculatlon rate. During aeroblc exposure, higher propionic and acetic

126

acids and lower pH were observed with the PAB-treated silages from the 22-28%
moisture levels. Propionibacteria inoculation did not significantly reduce yeast and mold
counts.

The vitamin B12 production capability of P. acidipropionici DH42 was also
evaluated in comparison with P. shermanii. Results showed comparable vitamin B12
production of the two propionibacteria strains. After 72 h of incubation, the P.
acidipropionici DH42 and P. shermanii cultures grown at 30°C had vitamin B12 contents
of 852.85 and 840.69 ng/ml, respectively. Both strains grew better at 30°C than at 40°C.
Poor growth of P. shermanii was evident at 40°C incubation. Lower pH was observed at
lower incubation temperature but for the P. acidipropionici DH42 cultures, the
differences of the pH between the two incubation temperatures were not significant. P.
acidipropionici DH42 cultures tend to have higher propionic and acetic acids while the P.
shennanii cultures had higher succinic and malic acids.

A PCR-based detection of P. acidipropionici DH42 was developed. Nested PCR
was used with DH42-specific primers dhbl and dhb2 for the secondary amplification of a
1.267 bp-fragment. Using the established protocols for PCR amplification, as low as 102
Cfll/ml and 10°cfu/ml of P. acidipropionici DH42 in silage extracts and rumen fluid,

respectively, were detected.

The silage studies had shown that moisture level affects the efficacy of P.

“Cidipropionici DH42. The 22-28% moisture contents appear to favor its growth. An
inoCulation rate of 106 cfu/g of ensiling material is recommended. It has been shown that

P‘ “Cidipropionici DH42 can produce vitamian that is comparable to P. shermanii

producing capability. P. acidipropionici DH42 can easily be detected in silage and

rumen fluid using PCR technology.

128

APPENDICES

APPENDIX A

Data Used for Analyses in Chapter 3

130

Table A-1. Effect of inoculation and moisture on the propionic acid content (g/ 100 g) of

 

 

fresh and ensiled HMC.
Moisture level Treatment Ensiling Period ((1)
% 0 10 21 120
Control 0.00 0.00 0.01 a” 0.17
DH42 105 0.00 0.00 0.01” 0.17
35 DH42 106 0.00 0.00 0.01a 0.18
P. jensenii 0.00 0.00 0.01 b 0.18
RCM 0.01 0.00 0.01 b 0.17
S.E.M 0.00 0.00 0.00 0.01
Control 0.00 0.00 0.01 0.34 "
DH42 105 0.00 0.00 0.01 0.30 “b
33 DH42 10° 0.01 0.01 0.02 0.32 a”
P. jensenii 0.05 0.00 0.01 0.29 "
RCM 0.06 0.01 0.01 0.31 "’
S.E.M." 0.01 0.00 0.00 0.01
Control 0.01 0.01 b 0.00 c o. 10 "
DH42 105 0.01 0.01 b 0.06 b 0.26 a
28 DH42 106 0.03 0.04 a 0.15 a 0.34 a
P. jensenii 0.04 0.01 b 0.07 b 0.28 “
RCM 0.02 0.01 b 0.00 ° 0.13 "
S.E.M.“ 0.00 0.00 0.00 0.01
Control 0.01 0.01 c 0.01 b 0.09 "
DH42 105 0.04 0.02 b° 0.03 b 0.15 "
24 DH42 10° 0.02 0.08 a o. 14 a 0.26 a
P. jensenii 0.02 0.05 “b 0.04 b 0.17 3"
RCM 0.02 0.00 ° 0.01 b o. 14 b
S.E.M.“ 0.00 0.00 0.01 0.01
Control 0.00 0.00 0.00 b 0.06 °
DH42 105 0.00 0.00 0.01 a” 0.14 a”
23 DH42 106 0.02 0.02 0.03 a 0.18 a
P. jensenii 0.02 0.02 0.03 a 0.19 a
RCM 0.03 0.00 0.00 b 0.09 b°
S.E.M.“ 0.01 0.00 0.00 0.01
Control 0.00 0.00 0.00 0.03 b
DH42 lo5 0.00 0.00 0.01 o. 15 a
22 DH42 10° 0.01 0.00 0.02 0.20 a
P. jensenii 0.01 0.00 0.01 0.18 a
RCM 0.02 0.00 0.00 0.02 b
S.E.M.“ 0.00 0.00 0.00 0.01

 

“Column means within the same moisture level with unlike superscripts differ (P<0.05).
tahdard error of the mean.

131

Table A-2. Effect of inoculation and moisture on the pH of fresh and ensiled HMC.

 

 

Moisture level Treatment Ensiling period ((1)
% 0 10 21 120
Control 5.78 3.89 3.79 3.70
DH42 105 5.69 3.90 3.80 3.73
35 DH42 10° 5.70 3.89 3.79 3.74
P. jensenii 5.71 3.92 3.79 3.72
RCM 5.71 3.90 3.80 3.75
8.13M.“ 0.02 0.01 0.00 0.01
Control 5.56 4.04 3.90 4.12
DH42 105 5.63 4.06 3.89 4.10
33 DH42 106 5.59 4.01 3.90 4.04
P. jensenii 5.57 4.08 3.92 4.02
RCM 5.64 4.03 3.89 4.05
8.13M.“ 0.02 0.01 0.01 0.02
Control 5.52 4.72 4.51 4.24
DH42 105 5.48 4.62 4.50 4.18
28 DH42 106 5.52 4.61 4.48 4.14
P. jensenii 5.53 4.62 4.55 4.16
RCM 5.49 4.61 4.46 4.22
S.E.M.“ 0.03 0.01 0.01 0.01
Control 5.35 4.55 4.46 4.43 “
DH42 105 5.48 4.53 4.46 4.40 a”
24 DH42 106 5.46 4.60 4.49 4.33 ”C
P. jensenii 5.36 4.36 4.27 4.27 C
RCM 5.41 4.70 4.56 4.40 8"
8.13M." 0.02 0.04 0.03 0.10
Control 5.32 4.79 4.55 4.27
DH42 105 5.22 4.84 4.54 4.33
23 DH42 106 5.26 4.76 4.53 4.30
P. jensenii 5.23 4.88 4.62 4.34
RCM 5.24 4.80 4.51 4.31
8.13M." 0.02 0.03 0.02 0.01
Control 5.26 5.30 5.18 4.67
DH42 105 5.27 5.29 5.06 4.57
22 DH42 106 5.22 5.29 4.97 4.56
P. jensenii 5.24 5.29 5.04 4.55
RCM 5.30 5.28 5.16 4.67
\ 8.13M." 0.02 0.02 0.01 0.01

 

IS2011-1111n means within same moisture level with unlike superscripts differ (P<0.05).
tandard error of the mean.

132

Table A-3. Effect of inoculation and moisture on the acetic acid content (g/100g DM) of

 

 

fresh and ensiled HMC.
Moisture level Treatment Ensilin g period ((1)

% 0 10 21 120
Control 0.00 b 0.18 0.17 0.20
DH42 105 0.00 b 0.19 0.17 0.20

35 DH42 10° 0.00 b 0.20 0.17 0.22
P. jensenii 0.01b 0.19 0.17 0.20
RCM 0.01 a 0.21 0.16 0.22
S.E.M." 0.00 0.01 0.00 0.01
Control 0.03 c 0.15 0.16 0.31
DH42 105 0.04 °° 0.13 0.17 0.30

33 DH42 106 0.07 a 0.17 0.18 0.32
P. jensenii 0.70 a” 0.17 0.16 0.29
RCM 0.08 a 0.15 0.19 0.29
S.E.M." 0.00 0.01 0.00 0.01
Control 0.02 b 0.09 0.16 0.28 °
DH42 105 0.02 a” 0.09 0.20 0.41 a“

28 DH42 106 0.03 a“ 0.12 0.25 0.48 a
P. jensenii 0.04 a 0.11 0.25 0.47 3"
RCM 0.03 a” 0.10 0.16 0.29 3‘”
S.E.M.“ 0.00 0.00 0.01 0.03
Control 0.02 0.07 C 0.08 b 0.24
DH42 105 0.03 0.10 a“ 0.11 a” 0.35

24 DH42 106 0.02 0.15 3" 0.15 a 0.46
P. jensenii 0.03 0.09 ”C 0.15 a 0.40
RCM 0.01 0.15 a 0.07 b 0.26
SEM.“ 0.00 0.01 0.01 0.03
Control 0.00 C 0.07 0.14 a” 0.20 1”
DH42 105 0.02 ”C 0.12 0.19 a” 0.22 b“

23 DH42 10° 0.04 “b 0.14 0.24 a 0.33 a
P. jensenii 0.05 a 0.17 0.14 a” 0.29 “b
RCM 0.03 a” 0.12 0.01 b 0.19 C
SEM." 0.00 0.01 0.02 0.01
Control 0.03 0.00 b 0.01 0.1 l
DH42 105 0.03 0.00 b 0.03 0.17

22 DH42 10° 0.02 0.01b 0.06 0.14
P. jensenii 0.03 0.02 b 0.09 0.16
RCM 0.03 0.05 a 0.08 0.12
S.E.M." 0.00 0.00 0.01 0.01

 

IFolllmn means within the same moisture level with unlike superscripts differ (P<0.05).
tandard error of the mean.

133

Table A-4. Effect of inoculation and moisture on the lactic acid content (g/100g DM) of
fresh and ensiled HMC.

 

 

Moisture level Treatment Ensiling period ((1)
% 0 10 21 120
Control 0.00 2.02 2.28 3.61
DH42 105 0.00 2.08 2.19 3.56
35 DH42 105 0.00 2.19 2.22 3.49
P. jensenii 0.00 1.99 2.19 3.69
RCM 0.01 2.40 2.12 3.62
SEM." 0.00 0.06 0.03 0.13
Control 0.07 1.48 2.07 2.32
DH42 105 0.09 1.61 2.08 2.13
33 DH42 106 0.10 1.64 2.17 2.53
P. jensenii 0.08 1.42 1.95 2.32
RCM 0.10 1.60 2.19 2.53
S.E.M." 0.01 0.04 _ 0.03 0.06
Control 0.08 0.67 1.07 1.25
DH42 105 0.09 0.58 0.83 1.12
28 DH42 106 0.09 0.49 0.78 1.04
P. jensenii 0.11 0.56 0.84 1.31
RCM 0.08 0.63 0.94 1.11
8.13M.“ 0.01 0.02 0.02 0.05
Control 0.01 0.71 0.72 0.63
DH42 105 0.02 0.64 0.59 0.59
24 DH42 10" 0.01 0.58 0.45 0.48
P. jensenii 0.01 0.85 0.79 0.89
RCM 0.00 0.64 0.78 0.88
S.E.M.“ 0.00 0.13 0.05 0.05
Control 0.00 0.60 0.27 0.92
DH42 105 0.01 0.49 0.27 0.65
23 DH42 106 0.09 0.54 0.19 0.56
P. jensenii 0.07 0.40 0.16 0.41
RCM 0.08 0.58 0.15 0.88
S.E.M.“ 0.01 0.03 0.02 0.05
Control 0.05 0.04 b 0.15 b 0.46 3"
DH42 105 0.05 0.04 b 0.16 b 0.31 l”
22 DH42 106 0.05 0.04 b 0.19 b 0.18 b
P. jensenii 0.02 0.07 b 0.32 a” 0.29 b°
RCM 0.04 0.17 a 0.46 a 0.54 a
\ S.E.M.“ 0.01 0.01 0.02 0.02

 

Solumn means within the same moisture content with unlike superscripts differ (P<0.05).
Standard error of the mean

134

Table A-5. Effect of inoculation and moisture on glucose content (g/100 g DM) of fresh

 

 

and ensiled HMC.
Moisture level Treatment Ensiling period ((1)

% 0 10 21 120
Control 1.00 a 0.15 0.14 0.26
DH42 105 0.92 a” 0.18 0.15 0.21

35 DH42 106 1.02 a 0.19 0.15 0.21
P. jensenii 1.01 a 0.14 0.15 0.26
RCM 0.75 b 0.18 0.17 0.29
S.E.M.“ 0.02 0.01 0.00 0.01
Control 0.10 0.17 0.23 0.41
DH42 105 0.10 0.18 0.24 0.36

33 DH42 106 0.11 0.18 0.24 0.40
P. jensenii 0.09 0.16 0.23 0.36
RCM 0.09 0.18 0.28 0.39
8.13M.“ 0.01 0.00 0.00 0.01
Control 0.08 0.10 0.11 0.19
DH42 105 0.10 0.08 0.09 0.20

28 DH42 106 0.06 0.08 0.09 0.17
P. jensenii 0.06 0.07 0.09 0.16
RCM 0.06 0.08 0.09 0.18
S.E.M." 0.01 0.00 0.00 0.01
Control 0.06 0.03 0.08 “b 0.05
DH42 105 0.06 0.04 0.06 a” 0.05

24 DH42 106 0.05 0.05 0.12 a 0.10
P. jensenii 0.05 0.04 0.04 b 0.03
RCM 0.05 0.05 0.07 a” 0.09
8.13M." 0.00 0.00 0.41 0.01
Control 0.27 a 0.05 0.04 0.09
DH42 105 0.25 “b 0.05 0.04 0.08

23 DH42 10° 0.13 ”C 0.04 0.03 0.06
P. jensenii 0.12 c 0.04 0.03 0.10
RCM 0.08 C 0.04 0.03 0.06
8.13M." 0.01 0.00 0.00 0.01
Control 0.05 0.16 a 0.12 a 0.06
DH42 105 0.04 0.17 a 0.08 3" 0.11

22 DH42 106 0.02 0.13 a 0.06 a” 0.09
P. jensenii 0.04 0.10 “b 0.05 b 0.11
RCM 0.05 0.03 b 0.05 b 0.05
8.13M." 0.00 0.01 0.01 0.76

\

 

COlumn means within same moisture content with unlike superscripts differ (P<0.05).

‘1
Standard error of the mean.

135

Table A-6. Effect of inoculation and moisture on the citric acid content (g/ 100 g DM) of

 

 

fresh and ensiled HMC.
Moisture level Treatment Ensiling period ((1)
% 0 10 21 120
Control 0.04 0.04 0.01 0.00
DH42 105 0.05 0.05 0.03 0.00
35 DH42 106 0.05 0.06 0.03 0.00
P. jensenii 0.06 0.02 0.03 0.00
RCM 0.05 0.05 0.05 0.00
S.E.M.“ 0.01 0.01 0.00 0.00
Control 0.12 0.01 0.02 a” 0.00
DH42 105 0.04 0.03 0.01” 0.00
33 DH42 106 0.05 0.02 0.02 a” 0.00
P. jensenii 0.03 0.02 0.02 a” 0.00
RCM 0.03 0.01 0.04 a 0.00
8.13M." 0.01 0.00 0.00 0.00
Control 0.04 0.01 0.01 0.00
DH42 105 0.09 0.01 0.01 0.00
28 DH42 106 0.01 0.01 0.00 0.00
P. jensenii 0.01 0.01 0.01 0.00
RCM 0.01 0.01 0.01 0.00
8.13M.“ 0.01 0.00 0.00 0.00
Control 0.01 0.01 0.01 0.00
DH42 105 0.01 0.01 0.01 0.00
24 DH42 10" 0.01 0.01 0.01 0.01
P. jensenii 0.01 0.01 0.01 0.00
RCM 0.01 0.01 0.01 0.01
S.E.M." 0.00 0.00 0.00 0.00
Control 0.04 0.01 0.01 a 0.01
DH42 105 0.05 0.01 0.012! 0.01
23 DH42 106 0.03 0.01 0.00 b 0.01
P. jensenii 0.01 0.01 0.00 a” 0.02
RCM 0.02 0.01 0.00 b 0.01
S.E.M." 0.00 0.00 0.00 0.01
Control 0.02 0.02 0.02 a 0.02
DH42 105 0.02 0.02 0.02 a” 0.02
22 DH42 10" 0.02 0.02 0.02 a” 0.02
P. jensenii 0.02 0.01 0.01 b 0.02
RCM 0.02 0.01 0.01 b 0.02
S.E.M." 0.00 0.00 0.00 0.00

\

 

(Solumn means within the same moisture level with unlike superscripts differ (P<0.05).
Standard error of the mean.

136

Table A-7. Effect of inoculation and moisture on the ethanol content (g/100g DM) of
fresh and ensiled HMC.

 

 

Moisture level Treatment Ensiling period (d)

% 0 10 21 120
Control 0.00 0.61 0.56 0.48

DH42 105 0.00 0.68 0.55 0.56

35 DH42 106 0.00 0.63 0.54 0.52
P. jensenii 0.00 0.59 0.55 0.49

RCM 0.01 0.75 0.54 0.51

SEM." 0.00 0.02 0.00 0.02

Control 0.13 0.69 0.84 0.85

DH42 105 0.18 0.69 0.85 0.88

33 DH42 106 0.20 0.68 0.83 0.92
P. jensenii 0.16 0.63 0.83 0.86

RCM 0.17 0.69 0.82 0.86

8.13M." 0.00 0.01 0.01 0.02

Control 0.03 0.60 0.66 0.75

DH42 105 0.05 0.53 0.55 0.54

28 DH42 10" 0.07 0.55 0.54 0.49
P. jensenii 0.07 0.53 0.49 0.53

RCM 0.07 0.52 0.50 0.67

8.13M." 0.00 0.02 0.02 0.03

Control 0.04 0.55 0.59 0.42

DH42 105 0.03 0.45 0.52 0.41

24 DH42 106 0.04 0.47 0.59 0.48
P. jensenii 0.03 0.40 0.42 0.32

RCM 0.03 ' 0.50 0.63 0.55

S.E.M.“ 0.00 0.02 0.01 0.03

Control 0.00 0.44 0.15 0.15

DH42 105 0.01 0.40 0.23 0.26

23 DH42 10° 0.03 0.41 0.22 0.34
P. jensenii 0.04 0.37 0.14 0.26

RCM 0.05 0.36 0.00 0.42

S.E.M." 0.01 0.02 0.03 0.02

Control 0.06 0.26 0.63 0.56

DH42 105 0.05 0.32 0.57 0.44

22 DH42 106 0.04 0.31 0.55 0.37
P. jensenii 0.03 0.29 0.59 0.36

RCM 0.04 0.35 0.60 0.59

\ S.E.M." 0.00 0.02 0.02 0.03

 

Solumn means within the same moisture level with unlike superscripts differ (P<0.05).
Standard error of the mean.

137

Table A-8. Effect of inoculation and moisture on the butyric acid content (g/100g DM)

 

 

of fresh and ensiled HMC.
Moisture level Treatment Ensilin g period (d)

% 0 10 21 120
Control 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00

35 DH42 106 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00
S.E.M.“ 0.00 0.00 0.00 0.00
Control 0.00 0.00 0.00 0.32 “
DH42 105 0.00 0.00 0.00 0.23 “b

33 DH42 106 0.00 0.00 0.00 0.27 a”
P. jensenii 0.00 0.00 0.00 0.19 b
RCM 0.00 0.00 0.00 0.26 3"
S.E.M.“ 0.00 0.00 0.00 0.01
Control 0.00 0.00 0.00 0.10 a
DH42 105 0.00 0.00 0.00 0.03 b

28 DH42 106 0.00 0.00 0.00 0.01"
P. jensenii 0.00 0.00 0.00 0.01b
RCM 0.00 0.00 0.00 0.10 “
S.E.M.“ 0.00 0.00 0.00 0.01
Control 0.00 0.00 0.03 0.10 a”
DH42 105 0.00 0.00 0.01 0.07 a”

24 DH42 106 0.00 0.00 0.00 0.03 b
P. jensenii 0.00 0.00 0.00 0.03 "
RCM 0.00 0.00 0.01 0.12 “
S.E.M.“ 0.00 0.00 0.00 0.01
Control 0.00 0.00 0.00 0.00 b
DH42 105 0.00 0.00 0.00 0.00 b

23 DH42 106 0.00 0.00 0.00 0.00 b
P. jensenii 0.00 0.00 0.00 0.00 b
RCM 0.00 0.00 0.00 0.03 a
8.13M." 0.00 0.00 0.00 0.00
Control 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00

22 DH42 106 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00
S.E.M.“ 0.00 0.00 0.00 0.00

\

 

“C01umn means within the same moisture level with unlike superscripts differ (P<0.05).
Standard error of the mean.

138

Table A-9. Effect of moisture and inoculant on the dry matter recovery (%) of ensiled
HMC

 

 

Moisture level Treatment Ensiling period (d)
% 10 21 120
Control 98.65 99.33 98.03
DH42 10s 99.96 99.15 97.49
35 DH42 10° 98.46 98.66 98.21
P. jensenii 99.02 99.27 97.13
RCM 97.22 98.85 96.62
S.E.M." 0.41 0.17 0.21
Control 97.69 96.88 a” 95.93
DH42 105 96.27 97.61ab 95.53
33 DH42 106 96.74 96.99 3" 95.69
P. jensenii 96.45 98.09 a 96.38
RCM 96.39 95.95 b 94.52
S.E.M.“ 0.61 0.18 0.29
Control 92.95 93.07 96.77
DH42 105 95.09 93.76 97.17
28 DH42 10" 93.68 95.46 97.23
P. jensenii 93.36 92.70 97.58
RCM 90.03 95.69 97.34
S.E.M.” 0.55 0.00 0.12
Control 98.67 97.91 97.59
DH42 105 97.17 97.95 97.24
24 DH42 106 97.96 97.45 97.34
P. jensenii 97.78 97.81 97.70
RCM 97.65 97.42 97.41
8.15M." 0.23 0.13 0.09
Control 99.53 99.47 a 99.11
DH42 105 99.02 98.95 “b 98.91
23 DH42 10° 99.19 99.12 a 98.33
P. jensenii 99.05 99.16 a 98.78
RCM 98.47 97.46 b 98.22
S.E.M.“ 0.13 0.15 0.13
Control 99.00 97.93 97.84
DH42 105 99.12 97.85 98.05
22 DH42 10" 99.47 99.21 98.54
P. jensenii 99.16 98.15 97.92
RCM 99.49 99.52 98.30
S.E.M." 0.16 0.40 0.16

\

 

COlumn means within the same moisture level with unlike superscripts differ (P<0.05).

n
Standard error of the mean.

139

Table A-lO. Effect of moisture and inoculant on the yeast and molds counts (log cfu/g
DM) of fresh and ensiled HMC.

 

 

Moisture level Treatment Ensiling period ((1)

% 0 10 21 120
Control 4.87 4.56 4.52 3.20

DH42 105 5.17 4.84 4.51 2.11

35 DH42 106 5.23 5.17 4.40 2.02
P. jensenii 5.31 4.72 5.05 2.98

RCM 4.60 4.25 4.99 3.13

S.E.M.“ 0.08 0.11 0.28 0.22

Control 5.44 4.64 4.13 0.18

DH42 105 5.31 4.58 4.37 0.25

33 DH42 106 5.36 4.23 3.82 0.13
P. jensenii 5.35 4.71 4.35 0.00

RCM 5.44 5.05 4.33 0.00

8.13M." 0.05 0.07 . 0.06 0.06

Control 4.94 5.03 5.59 2.22

DH42 105 4.79 4.45 4.92 1.65

28 DH42 106 4.89 4.81 3.70 0.91
P. jensenii 4.72 4.45 2.93 1.05

RCM 4.80 4.51 5.49 1.73

S.E.M.“ 0.07 0.08 0.17 0.20

Control 4.71 1.89 5.02 1.21

DH42 105 4.66 3.80 4.21 1.39

24 DH42 10° 4.67 0.00 3.26 0.19
P. jensenii 5.04 1.82 4.06 0.56

RCM 4.58 3.92 4.97 0.26

S.E.M.“ 0.06 0.31 0.22 0.26

Control 4.54 4.95 5.62 2.46

DH42 105 4.76 5.05 5.34 1.73

23 DH42 106 4.48 4.24 4.48 0.53
P. jensenii 4.38 4.94 4.72 0.85

RCM 4.62 5.27 6.22 1.45

S.E.M." 0.10 0.14 0.18 0.29

Control 5.78 5.98 5.89 2.91

DH42 105 5.63 6.32 5.79 1.19

22 DH42 10" 5.64 6.32 5.48 0.00
P. jensenii 5.97 6.18 5.44 0.94

RCM 5.59 6.34 5.73 1.72

\ SEM." 0.07 0.07 0.04 0.24

 

Solumn means within the same moisture level with unlike superscripts differ (P<0.05).
Standard error of the mean.

140

Table A-1 1. Effect of inoculation and moisture on propionibacteria counts (log cfu/g
DM) of fresh and ensiled HMC.

 

 

Moisture level Treatment Ensiling period (d)
% 0 120
Control 1.84 °° 4.80
DH42 105 3.52 “C 5.56
35 DH42 106 5.90 a” 6.15
P. jensenii 6.51 a 6.20
RCM 0.00 C 5.28
SEM.“ 0.41 0.16
Control 1.68 5.53
DH42 105 5.10 5.79
33 DH42 10° 5.74 5.88
P. jensenii 6.25 5.80
RCM 1.68 5.40
8.15M." 0.29 0.04
Control 1.96 b 4.88 b
DH42 105 5.62 a 5.19 a”
28 DH42 10° 6.32 a 6.09 a
P. jensenii 6.55 a 6.16 a
RCM 4.32 a” 5.47 3"
8.13M.“ 0.16 0.07
Control 0.00 d 5.88
DH42 105 5.51 b 6.12
'22 DH42 106 6.11 “b 6.12
P. jensenii 6.35 a” 5.89
RCM 3.67 C 6.20
SEM.“ 0.07 0.07

 

Solumn means within the same moisture level with unlike superscripts differ (P<0.05).
Standard error of the mean.

141

Table A-l2. Effect of inoculation and moisture on the propionic acid content (g/100g
DM) of exposed HMC silage

 

 

Moisture level Treatment Exposure period (d)
% 0 1 3 5 Averagen
Control 0.17 0.19 0.17 0.11 0.10
DH42 105 0.17 0.19 0.17 0.13 0.11
35 DH42 106 0.18 0.18 0.16 0.14 0.11
P. jensenii 0.18 0.18 0.16 0.14 0.11
RCM 0.18 0.17 0.15 0.09 0.10
Average 0.11 a 0.12 a 0.11 a 0.08 b (0.01)
Control 0.34 0.32 0.32 0.25 0.20
DH42 105 0.30 0.33 0.31 0.26 0.20
33 DH42 106 0.32 0.34 0.32 0.26 0.20
P. jensenii 0.29 0.30 0.30 0.24 0.19
RCM 0.31 0.32 0.31 0.27 0.20
Average 0.20 0.21 0.21 0.17 (0.01)
Control 0.10 0.11 0.11 0.10 0.10 c
DH42 105 0.26 0.23 0.24 0.20 0.21b
28 DH42 106 0.34 0.30 0.28 0.25 0.27 a
P. jensenii 0.28 0.23 0.24 0.21 0.20 b
RCM 0.13 0.12 0.12 0.11 0.11c
Average 0.18 0.19 0.19 0.15 (0.01)
Control 0.09 0.08 0.10 0.09 0.07 c
DH42 105 0.15 0.16 0.17 0.14 0.12 b
24 DH42 106 0.26 0.24 0.25 0.20 0.18 a
P. jensenii 0.17 0.15 0.17 0.14 0.12 b
RCM 0.14 0.11 0.11 0.08 0.08 be
Average 0.12 0.1 1 0.12 0.01 (0.01)
Control 0.06 0.05 0.06 0.05 0.06 C
DH42 105 0.14 0.14 0.14 0.13 0.14 b
23 DH42 106 0.19 0.18 0.17 0.18 0.18 a
P. jensenii 0.19 0.17 0.20 0.15 0.18 3'
RCM 0.09 0.09 0.07 0.11 0.09 °
Average 0.12 0.11 0.12 0.01 (0.01)
Control 0.03 0.02 0.03 0.02 0.03 °
DH42 105 0.15 0.15 0.16 0.11 0.14 b
22 DH42 106 0.20 0.21 0.19 0.15 0.19 a
P. jensenii 0.18 0.17 0.18 0.14 0.17 a
RCM 0.02 0.02 0.03 0.01 0.02 c

Average 0.123 0.11ab 0.13a 0.09b (0.01)

COlumn or row means within the same moisture level with unlike superscripts differ
(P<0.05).
umber in parenthesis denotes the standard error of the mean of the column.

142

Table A-l3. Effect of inoculation and moisture on the acetic acid content (g/100g DM)

 

 

of exposed HMC silage.
Moisture level Treatment Exposure period ((1)

% 0 1 3 5 Averagen
Control 0.20 0.22 0.14 0.00 0.14
DH42 105 0.20 0.22 0.20 0.05 0.17

35 DH42 10° 0.22 0.21 0.21 0.03 0.17
P. jensenii 0.20 0.21 0.18 0.04 0.16
RCM 0.22 0.16 0.16 0.03 0.14
Average 0.21a 0.20 a 0.18 a 0.03 b (002)
Control 0.31 0.29 0.31 0.25 0.29
DH42 105 0.30 0.34 0.32 0.27 0.31

33 DH42 106 0.32 0.35 0.35 0.28 0.32
P. jensenii 0.29 0.30 0.31 0.24 0.28
RCM 0.29 0.31 0.30 0.26 0.29
Average 0.30 a 0.32 ‘ 0.31a 0.26 b (0.02)
Control 0.28 0.13 0.18 0.11 0.17 ‘
DH42 105 0.41 0.25 0.33 0.21 0.30 3"

28 DH42 106 0.48 0.26 0.32 0.22 0.32 a
P. jensenii 0.48 0.27 0.39 0.22 0.34 a
RCM 0.29 0.16 0.31 0.09 0.21 b°
Average 0.39 a 0.21 c 0.30 b 0.17 ° (0.02)
Control 0.24 0.23 0.27 0.24 0.24 a”
DH42 105 0.35 0.34 0.35 0.29 0.33 “b

24 DH42 106 0.46 0.39 0.44 0.36 0.412'
P. jensenii 0.40 0.35 0.40 0.33 0.37 a
RCM 0.26 0.21 0.21 0.16 0.21 b
Average 0.34 a 0.30 “b 0.33 a 0.28 b (0.03)
Control 0.20 0.16 0.17 0.15 0.17 °
DH42 105 0.22 0.21 0.20 0.20 0.21bc

23 DH42 106 0.33 0.30 0.30 0.28 0.30 a
P. jensenii 0.29 0.24 0.27 0.23 0.26 3"
RCM 0.19 0.16 0.12 0.18 0.16 “
Average 0.24 0.21 0.21 0.21 (0.02)
Control 0.11 0.11 0.10 0.07 0.10 °
DH42 105 0.17 0.15 0.16 0.12 0.15 a”

22 DH42 106 0.14 0.17 0.15 0.12 0.14 3'”
P. jensenii 0.16 0.20 0.18 0.13 0.16 a
RCM 0.12 0.15 0.12 0.07 0.11 "
Average 0.14 a 0.15 a 0.13 a 0.10 b (0.01)

 

Column or row means within the same moisture level with unlike superscripts differ

(P<0.05).

"Number in parenthesis denotes the standard error of the mean of the column.

143

Table A-14. Effect of inoculation and moisture on the lactic acid content (g/100g DM) of
exposed HMC silage

 

 

Moisture level Treatment Exposure period ((1)
% 0 1 3 5 Averagen
Control 3.61 3.79 3.51 1.14 3.01
DH42 105 3.56 3.68 3.45 1.57 3.07
35 DH42 106 3.49 3.38 3.39 2.32 3.14
P. jensenii 3.69 3.63 3.38 1.79 3.12
RCM 3.62 3.25 2.29 1.06 2.56
Average 3.59 a 3.55 a 3.20 a 1.58 b (0.42)
Control 2.32 2.16 2.14 1.73 2.09
DH42 105 2.13 2.21 2.04 1.71 2.02
33 DH42 106 2.53 2.53 2.38 1.93 2.34
P. jensenii 2.32 2.35 2.36 1.92 2.23
RCM 2.53 2.44 2.34 2.08 2.35
Average 2.36 a 2.34 a 2.25 b 1.87 c (0.11)
Control 1.25 1.28 1.14 0.94 1.15
DH42 105 1.12 1.12 1.15 0.92 1.08
28 DH42 106 1.04 1.08 1.10 0.87 1.02
P. jensenii 1.31 1.18 1.12 0.89 1.13
RCM 1.11 1.24 0.88 0.96 1.05
Average 1.17 1.18 1.08 0.91 (0.08)
Control 0.63 0.56 0.68 0.60 0.62
DH42 105 0.59 0.58 0.60 0.48 0.56
24 DH42 10° 0.48 0.48 0.52 0.41 0.47
P. jensenii 0.89 0.80 0.88 0.75 0.83
RCM 0.88 0.70 0.73 0.55 0.72
Average 0.69 a 0.62 b 0.68 b 0.56 b (0.09)
Control 0.92 0.73 0.79 0.76 0.80
DH42 105 0.65 0.64 0.61 0.63 0.63
23 DH42 10° 0.56 0.53 0.46 0.57 0.53
P. jensenii 0.41 0.38 0.51 0.47 0.44
RCM 0.88 0.82 0.61 0.76 0.77
Average 0.68 0.62 0.60 0.64 (0.09)
Control 0.46 0.46 0.44 0.44 0.45 “
DH42 105 0.31 0.35 0.36 0.27 0.32 b
22 DH42 10" 0.18 0.43 0.20 0.15 0.24 °
P. jensenii 0.29 0.60 0.32 0.25 0.36 b
RCM 0.54 0.61 0.54 0.34 0.51 3

Average 0.36b 0.49“ 0.36b 0.29b (0.02)

Column or row means within the same moisture level with unlike superscripts differ
(P<0.05).
nNumber in parenthesis denotes the standard error of the mean of the column.

144

Table A-15. Effect of inoculation and moisture on the pH of exposed HMC silage.

 

 

Moisture level Treatment Exposure period (d)
% 0 1 3 5 Averagen
Control 3.70 3.69 4.82 5.15 4.06
DH42 105 3.73 3.72 3.72 5.00 4.05
35 DH42 10C 3.74 3.72 3.74 3.91 3.78
P. jensenii 3.72 3.71 3.75 4.82 3.99
RCM 3.75 3.76 3.73 6.26 4.65
Average 3.73 C 3.72 C 3.95 C 5.03 C (0.38)
Control 4.12 4.14 4.13 4.10 4.12
DH42 105 4.10 4.11 4.10 4.07 4.09
33 DH42 10C 4.04 4.08 4.05 4.02 4.05
P. jensenii 4.02 4.03 4.02 4.01 4.02
RCM 4.05 4.07 4.06 4.01 4.05
Average 4.06 C 4.09 C 4.07 CC 4.04 C (0.03)
Control 4.24 4.24 4.27 4.56 4.33 a
DH42 105 4.18 4.17 4.20 4.20 4.19 C
28 DH42 10" 4.14 4.13 4.16 4.16 4.15 C
P. jensenii 4.16 4.17 4.19 4.20 4.18 c
RCM 4.22 4.23 4.24 4.37 4.26 CC
Average 4.19 C 4.19 C 4.21 C 4.30 C (0.04)
Control 4.43 4.42 4.42 4.44 4.43 a
DH42 105 4.40 4.40 4.39 4.42 4.40 C
24 DH42 10C 4.33 4.33 4.32 4.35 4.33 C
P. jensenii 4.27 4.26 4.25 4.29 4.27 c
RCM 4.40 4.38 4.39 4.41 4.39 C
Average 4.37 C 4.36 C 4.36 C 4.38 C (0.02)
Control 4.27 4.26 4.28 4.33 4.28
DH42 105 4.33 4.31 4.34 4.39 4.34
23 DH42 10C 4.30 4.30 4.32 4.38 4.32
P. jensenii 4.34 4.33 4.34 4.39 4.35
RCM 4.31 4.32 4.33 4.39 4.33
Average 4.31 C 4.30 C 4.32 C 4.38 C (0.03)
Control 4.67 4.66 4.66 4.72 4.68 C
DH42 105 4.57 4.55 4.54 4.56 4.55 C
22 DH42 10C 4.56 4.57 4.57 4.59 4.57 C
P. jensenii 4.55 4.52 4.53 4.55 4.54 C
RCM 4.67 4.65 4.63 4.70 4.66 C
Average 4.60 CC 4.59 C 4.58 C 4.62 C (0.02)

 

Column or row means within the same moisture level with unlike superscripts differ

(P<0.05).

nNumber in parenthesis denotes the standard error of the mean of the column.

145

Table A-16. Effect of inoculation and moisture on the glucose content (g/100g DM) of
exposed HMC silage

 

 

Moisture level Treatment Exposure mriod (d)
% 0 1 3 5 Averagen
Control 0.26 0.27 0.26 0.15 0.23
DH42 105 0.21 0.22 0.21 0.18 0.20
35 DH42 10C 0.21 0.21 0.21 0.19 0.21
P. jensenii 0.26 0.25 0.24 0.18 0.23
RCM 0.29 0.25 0.22 0.16 0.23
Average 0.25 C 0.24 C 0.23 C 0.17 C (0.02)
Control 0.41 0.39 0.39 0.31 0.38
DH42 105 0.36 0.38 0.36 0.30 0.35
33 DH42 10" 0.40 0.40 0.39 0.32 0.38
P. jensenii 0.36 0.37 0.37 0.30 0.35
RCM 0.39 0.39 0.38 0.34 0.38
Average 0.38 C 0.39 C 0.38 C 0.32 C (0.01)
Control 0.19 0.17 0.16 0.12 0.16
DH42 105 0.20 0.19 0.15 0.13 0.17
28 DH42 10C 0.17 0.15 0.13 0.10 0.14
P. jensenii 0.16 0.17 0.14 0.15 0.16
RCM 0.18 0.16 0.15 0.14 0.16
Average 0.18 C 0.17 C 0.15 C 0.13 C (0.02)
Control 0.05 0.04 0.05 0.05 0.05
DH42 105 0.05 0.05 0.07 0.05 0.06
24 DH42 10C 0.10 0.08 0.10 0.07 0.09
P. jensenii 0.03 0.02 0.02 0.02 0.02
RCM 0.09 0.06 0.07 0.05 0.07
Average 0.06 C 0.05 C 0.06 C 0.05 C (0.02)
Control 0.09 0.07 0.08 0.06 0.08
DH42 105 0.08 0.08 0.08 0.07 0.08
23 DH42 10° 0.06 0.06 0.07 0.07 0.06
P. jensenii 0.10 0.08 0.10 0.07 0.09
RCM 0.06 0.05 0.05 0.06 0.05
Average 0.08 C 0.07 C 0.07 CC 0.06 C (0.01)
Control 0.06 0.05 0.06 0.07 0.06 C
DH42 105 0.11 0.10 0.14 0.09 0.11 C
22 DH42 10" 0.09 0.05 0.08 0.08 0.07 C
P. jensenii 0.11 0.00 0.12 0.10 0.08 C
RCM 0.05 0.00 0.00 0.00 0.01 C
Average 0.08 C 0.04 C 0.08 C 0.07 C (0.01)

 

Column or row means within the same moisture level with unlike superscripts differ

(P<0.05).

nNumber in parenthesis denotes the standard error of the mean of the column.

146

Table A-17. Effect of inoculation and moisture on the ethanol content (g/100g DM) of
exposed HMC silage.

 

 

Moisture level Treatment Exposure period (d)
% 0 1 3 5 Average"
Control 0.48 0.46 0.70 0.00 0.30
DH42 105 0.56 0.55 0.28 0.06 0.38
35 DH42 10C 0.52 0.48 0.35 0.02 0.34
P. jensenii 0.49 0.48 0.33 0.02 0.33
RCM 0.51 0.36 0.32 0.01 0.27
Average 0.51 C 0.46 C 0.29 C 0.02 C (0.03)
Control 0.85 0.81 0.20 0.35 0.66
DH42 105 0.88 0.86 0.65 0.37 0.69
33 DH42 10° 0.92 0.79 0.67 0.33 0.68
P. jensenii 0.86 0.79 0.68 0.32 0.67
RCM 0.86 0.77 0.68 0.33 0.66
Average 0.87 C 0.80 C 0.67 C 0.34 C (0.03)
Control 0.75 C 0.55 C 0.38 CC 0.14 0.45
DH42 105 0.54 C 0.39 C 0.37 CC 0.14 0.36
28 DH42 106 0.49 C 0.36 C 0.28 C 0.11 0.31
P. jensenii 0.53 C 0.35 C 0.34 CC 0.13 0.34
RCM 0.67 C 0.55 C 0.42 C 0.13 0.45
Average 0.60 0.44 0.36 0.13 (0.04)
Control 0.42 0.34 0.29 0.13 0.29
DH42 105 0.41 0.38 0.32 0.16 0.32
24 DH42 106 0.48 0.39 0.34 0.18 0.35
P. jensenii 0.32 0.26 0.24 0.13 0.24
RCM 0.55 0.39 0.33 0.15 0.35
Average 0.44 C 0.35 C 0.30 C 0.15 C (0.04)
Control 0.15 C 0.26 CC 0.21 0.09 0.18
DH42 105 0.26 C 0.16 C 0.15 0.05 0.15
23 DH42 10° 0.34 CC 0.24 CC 0.17 0.06 0.20
P. jensenii 0.26 ° 0.18 b 0.17 0.05 0.16
RCM 0.42 C 0.30 C 0.16 0.11 0.25
Average 0.29 0.23 0.17 0.07 (0.02)
Control 0.56 0.33 0.22 0.07 0.29
DH42 105 0.44 0.31 0.25 0.12 0.28
22 DH42 106 0.37 0.26 0.15 0.08 0.22
P. jensenii 0.36 0.27 0.22 0.10 0.24
RCM 0.59 0.37 0.23 0.06 0.31
Average 0.46 C 0.31 C 0.21 C 0.08 C (0.03)

 

Row means within the same moisture level with unlike superscripts differ (P<0.05).

nNumber in parenthesis denotes the standard error of the mean of the column.

147

Table A-18. Effect of inoculation and moisture on the citric acid content (g/100g DM) of
exposed HMC silage

 

 

Moisture level Treatment Exposure period (d)
% 0 1 3 5 Averagen
Control 0.00 0.01 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00 0.00
35 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00 0.00
Average 0.00 0.00 0.00 0.00 (0.00)
Control 0.00 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00 0.00
33 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00 0.00
Average 0.00 0.00 0.00 0.00 (0.00)
Control 0.00 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00 0.00
28 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 I 0.01 0.00
RCM 0.00 0.00 0.00 0.00 0.00
Average 0.00 0.00 0.00 0.00 (0.00)
Control 0.00 0.00 0.00 0.00 0.00
DH42 105 0.00 0.01 0.01 0.00 0.00
24 DH42 10C 0.01 0.01 0.01 0.01 0.01
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.01 0.01 0.01 0.00 0.01
Average 0.01 0.01 0.01 0.00 (0.00)
Control 0.01 0.01 0.01 0.01 0.01
DH42 105 0.01 0.01 0.01 0.01 0.01
23 DH42 10C 0.01 0.01 0.01 0.01 0.01
P. jensenii 0.02 0.01 0.01 0.01 0.01
RCM 0.01 0.01 0.01 0.01 0.01
Average 0.01 0.01 0.01 0.01 (0.00)
Control 0.02 0.02 0.02 0.03 0.02
DH42 10C 0.02 0.02 0.02 0.02 0.02
22 DH42 10C 0.02 0.02 0.02 0.01 0.02
P. jensenii 0.02 0.01 0.02 0.02 0.02
RCM 0.02 0.01 0.01 0.02 0.02

Average 0.02 0.02 0.02 0.02 (0.00)
"Number in parenthesis denotes the standard error of the mean of the column.

 

148

Table A-19. Effect of inoculation and moisture on the butyric acid content (g/100g DM)
of exposed HMC silage.

 

 

Moisture level Treatment Exposure period ((1)
% 0 l 3 5 Averagen
Control 0.00 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00 0.00
35 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00 0.00
Average 0.00 0.00 0.00 0.00 (0.00)
Control 0.32 0.29 0.29 0.23 0.28
DH42 105 0.23 0.24 0.22 0.18 0.22
33 DH42 10C 0.27 0.27 0.25 0.20 0.25
P. jensenii 0.19 0.19 0.18 0.15 0.17
RCM 0.26 0.26 0.24 0.22 0.24
Average 0.25 0.25 0.24 0.19 (0.02)
Control 0.10 0.07 0.10 0.10 0.09
DH42 105 0.03 0.01 0.03 0.04 0.03
28 DH42 10C 0.01 0.00 0.01 0.02 0.01
P. jensenii 0.01 0.00 0.01 0.02 0.01
RCM 0.10 0.08 0.09 0.09 0.09
Average 0.05 0.03 0.05 0.05 (0.01)
Control 0.10 0.10 0.12 0.11 0.01
DH42 105 0.07 0.07 0.08 0.06 0.07
24 DH42 10° 0.03 0.03 0.04 0.03 0.03
P. jensenii 0.03 0.03 0.04 0.03 0.03
RCM 0.12 0.11 0.11 0.08 0.11
Average 0.07 0.07 0.08 0.06 (0.02)
Control 0.00 0.01 0.00 0.01 0.01
DH42 105 0.00 0.00 0.00 0.00 0.00
23 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.03 0.03 0.01 0.02 0.02
Average 0.01 0.01 0.00 0.01 (0.00)
Control 0.00 0.00 0.00 0.00 0.00
DH42 105 0.00 0.00 0.00 0.00 0.00
22 DH42 10C 0.00 0.00 0.00 0.00 0.00
P. jensenii 0.00 0.00 0.00 0.00 0.00
RCM 0.00 0.00 0.00 0.00 0.00

Average 0.00 0.00 0.00 0.00 (0.00)
l"Number in parenthesis denotes the standard error of the mean of the column.

149

Table A-20. Effect of inoculation and moisture on the yeast and molds counts (log cfu/g
DM) of exposed HMC silage.

 

 

Moisture level Treatment E&osure period ((1)
% 0 l 3 5 Averagen
Control 3.20 3.67 6.40 7.67 5.24
DH42 105 2.11 2.10 5.63 8.04 4.58
35 DH42 10" 2.02 3.16 5.85 7.68 4.68
P. jensenii 2.98 3.35 5.74 7.60 4.92
RCM 3.13 4.21 6.50 7.89 5.43
Average 2.68 C 3.39 C 6.02 C 7.78 C (0.40)
Control 0.16 0.16 0.73 0.88 0.48
DH42 105 0.24 0.40 0.88 0.00 0.38
33 DH42 10C 0.09 0.00 0.49 0.00 0.14
P. jensenii 0.00 0.09 0.32 0.00 0.10
RCM 0.00 0.00 0.55 0.68 0.31
Average 0.10 C 0.13 C 0.59 C 0.31 CC (0.12)
Control 2.29 2.09 2.29 1.81 2.12
DH42 105 1.38 0.52 0.37 1.58 0.96
28 DH42 10C 0.93 0.71 0.65 0.72 0.75
P. jensenii 0.91 0.74 0.74 1.03 0.86
RCM 0.44 0.48 0.49 0.86 0.57
Average 1.19 0.91 0.91 1.20 (0.44)
Control 1.14 0.88 0.58 3.55 1.53
DH42 105 1.39 1.27 1.11 1.91 1.42
24 DH42 10C 0.17 0.00 0.34 0.86 0.34
P. jensenii 0.53 0.53 1.01 1.42 0.87
RCM 0.25 0.35 0.39 0.42 0.35
Average 0.70 0.60 0.69 1.63 (0.36)
Control 2.43 2.68 3.25 3.26 2.91 C
DH42 105 1.73 1.33 1.87 1.48 1.60 CC
23 DH42 10° 0.54 0.37 1.50 1.45 0.96 C
P. jensenii 0.85 0.27 0.87 0.84 0.71 C
RCM 1.45 0.52 0.49 0.45 0.72 C
Average 1.40 CC 1.03 C 1.59 C 1.50 CC (0.42)
Control 2.90 2.97 3.41 3.95 3.31 a
DH42 105 1.19 0.91 1.96 1.78 1.46 C
22 DH42 10“ 0.00 0.00 1.72 1.05 0.69 C
P. jensenii 0.92 0.30 1.20 1.56 1.00 C
RCM 1.72 0.70 1.06 0.91 1.10 C

Average 1.35CC 0.97C 1.87C 1.85 CC (0.33)

Column or row means within the same moisture level with unlike superscripts differ
(P<0.05).
nNumber in parenthesis denotes the standard error of the mean of the column.

 

150

Table A-21. Effect of inoculation and moisture on the dry matter recovery (%) of
exposed HMC silage.

 

 

Moisture level Treatment Exposure period (d)
% 0 1 3 5 Averagen
Control 98.03 101.43 99.56 99.10 99.53
DH42 105 97.49 101.31 101.49 98.94 99.81
35 DH42 106 98.21 101.56 100.36 101.11 100.31
P. jensenii 97.13 100.47 99.90 100.04 99.38
RCM 96.62 99.08 100.87 98.24 98.71
Average 97.49C 100.77C 100.43 CC 99.49C (0.35)
Control 95.93 99.61 99.16 99.75 98.61
DH42 105 95.53 100.43 99.22 100.11 98.82
33 DH42 106 95.69 100.84 98.75 100.62 98.98
P. jensenii 96.38 100.25 99.33 100.93 99.22
RCM 94.52 101.10 98.79 100.57 98.74
Average 95.61C 100.45C 99.05C 100.39C (0.20)
Control 96.77 101.43 97.54 101.41 99.29
DH42 10s 97.17 101.01 96.69 99.81 98.67
28 DH42 10C 97.23 101.61 97.77 101.51 99.53
P. jensenii 97.58 101.82 96.95 101.33 99.42
RCM 97.34 101.34 97.34 101.30 99.33
Average 97.22C 101.44C 97.26C 101.07C (0.23)
Control 97.59 100.81 99.21 99.89 99.37
DH42 105 97.24 100.93 98.99 100.30 99.36
24 DH42 10C 97.38 95.63 99.17 99.27 97.85
P. jensenii 97.70 100.98 97.68 99.59 99.71
RCM 97.41 100.91 98.92 100.09 99.38
Average 98.67C 100.37C 98.79b 99.20C (0.21)
Control 99.1 1 100.00 100.00 99.52 99.65
DH42 105 98.91 100.79 100.79 98.96 99.86
23 DH42 10" 98.33 100.46 100.47 99.35 99.65
P. jensenii 98.78 100.29 100.27 99.47 99.71
RCM 98.22 100.31 100.33 98.68 99.38
Average 98.67C 100.37C 100.37C 99.20C (0.21)
Control 97.83 99.26 102.52 101.53 100.29
DH42 105 98.05 100.61 102.00 100.41 100.27
22 DH42 10C 98.54 100.67 100.20 99.29 99.68
P. jensenii 97.92 102.43 100.51 99.04 102.48
RCM 98.29 100.50 99.67 99.84 99.58
Average 98.13C 102.69C 100.98C 100.02"C (1.21)

 

Row means within the same moisture level with unlike superscripts differ (P<0.05).

“ Number in parenthesis denotes the standard error of the mean of the column.

151

Table A-22. Effect of inoculation and moisture on the temperature (°C) of exposed HMC

sflage.

 

Moisture Treatment

level (%)

35

33

28

24

23

22

Control
DH42 105
DH4210°
P. jensenii
RCM
Average
Control
DH42 105
DH4210°
P. jensenii
RCM
Average
Control
DH42105
DH42 106
P. jensenii
RCM
Average
Control
DH42 105
DH42 10"
P. jensenii
RCM
Average
Control
DH42 10C
DH42 106
P. jensenii
RCM
Average
Control
DH42 105
DH42 10C
P. jensenii
RCM
Average

0

1

18.52 CC 19.54

17.78
18.89
18.70
19.07

18.59 c

21.81
21.53
21.94
21.67
21.11

21.61 a

24.44
24.03
24.51
24.65
24.58

24.44 C 24.77 C

23.89
23.06
23.19
23.47
23.19

23.36 a

21.25
21.11
21.25
21.67
21.53
21.36
21.67
21.67
21.48
21.85
22.22

19.35
19.63
19.44
22.87

20.17 C

20.21
20.00
20.42
20.14
19.44

20.04 C

24.83
24.34
24.86
24.86
24.97

20.76
20.83
20.56
20.83
20.90

20.78 C

20.76
20.42
21.11
21.53
21.18
21.00
21.48
21.67
21.02
21.94
21.85

Exposure period ((1)

2

18.89
18.52
19.26
19.54

21.57
19.56 c

19.51
19.31
19.72
19.51
18.82

19.36 C

22.99
22.64
22.85
22.85
22.92

22.85 C

20.56
20.69
20.42
20.69
20.42

20.56 c

20.49
20.63
20.69
21.60
21.32
20.94
22.04
22.22
21.48
21.85
22.22

3

19.63
17.78
18.33
18.70
19.63

18.81 c

19.65
19.44
19.79
17.99
18.68

19.11C

24.38
23.33
23.85
24.03
24.06

23.93 c

20.76
20.56
20.69
20.69
20.83

20.71 c

19.72
19.72
20.42
20.56
20.56
20.19
21.02
21.20
20.74
21.57
20.83

4

21.67
20.74
19.26
23.89
26.48

22.41 C

19.79
19.51
19.86
19.44
19.10

19.54 c

26.25
25.69
25.83
25.97
26.11

25.97 a

20.56
20.14
20.56
20.42
20.56

20.44 C

19.72
19.44
20.00
20.97
20.56
20.14
21 .67
21.48
21.30
21.48
21.48

5

27.96
25.00
25.28
23.98
25.56

25.56 a

20.28
20.00
20.14
20.14
19.58

20.03 C

27.01
25.69
26.04
26.18
26.60

26.31 a

21.39
20.97
21.39
21.11
21.39

21.25 C

20.42
19.58
20.28
21.11
20.42
20.36
21.30
21 .48
20.56
21 .11
20.93

Avg.

21.03
19.86
20.11
20.71
22.53
(0.98)
20.21
19.97
20.31
19.81
19.46
(0.25)
24.98
24.29
24.66
24.76
24.87
(0.33)
21.32
21.04
21.13
21.20
21.22
(0.34)
20.39
20.15
20.63
21.24
20.93
(0.22)
21.53
21.62
21.10
21.64
21.59

21.78 CC21.59C 21.96C 21.07C 21.48C 21.07C (0.13)

n

 

Row means within the same moisture level with unlike superscripts differ (P<0.05).

nNumber in parenthesis denotes the standard error of the mean of the column.

152

APPENDIX B

Data Used for Analyses in Chapter 2

153

Table B-1. Data used for analyses for chapter 2-chemical and microbial analyses’.

Trt Rep Time Pa

1

t‘r‘r‘WWWC—HC—HHHIEQOQZZKZI‘P‘WNHHHNEEQQ

wN—th—wNu—wwwNwwN—N—NHN—tN—N—N—iN—‘N

0

\lflflflflflflflflflflflflflfl\IQOOOOOOCOOOCOOOO

0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.018
0.017
0.017
0.039
0.036
0.009
0.018
0.022
0.019
0.007
0.021
0.033
0.027
0.037
0.039
0.040
0.039

La GI

0.000 0.321
0.000 0.310
0.000 0.249
0.000 0.251
0.000 0.160
0.000 0.161
0.000 0.419
0.000 0.419
0.000 0.319
0.000 0.321
0.000 0.406
0.000 0.389
0.000 0.335
0.000 0.333
0.000 0.432
0.000 0.416
1.035 0.012
1.418 0.016
1.410 0.019
1.205 0.011
1.201 0.006
1.230 0.011
1.801 0.012
1.053 0.011
0.861 0.009
0.821 0.010
1.123 0.008
0.938 0.009
0.588 0.010
0.938 0.017
0.916 0.010
1.183 0.021
0.708 0.021

Ac

0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.099
0.127
0.133
0.099
0.073
0.089
0.133
0.077
0.068
0.060
0.067
0.077
0.053
0.090
0.083
0.102
0.057

Et
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.307
0.351
0.395
0.392
0.275

0.215 '

0.407
0.229
0.195
0.205
0.226
0.324
0.255
0.361
0.264
0.384
0.185

Bu
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.106
0.106
0.079
0.079
0.055
0.055
0.1 13
0.1 12
0.082
0.081
0.092
0.094
0.1 1 1
0.1 l 1
0.155
0.160
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002
0.002

Lab an

6.29
6.30
6.01
6.05
6.30
6.28
6.76
6.75
6.20
6.20
6.59
6.60
6.05
6.05
6.22
6.20
8.73
8.64
8.55
8.80
8.82
8.49
8.71
8.66
8.60
8.63
8.68
8.54
8.63
8.63
8.51
8.58
8.67

7.54
7.52
7.25
7.30
7.51
7.50
7.61
7.60
7.29
7.30
7.44
7.41
7.61
7.62
7.53
7.54
6.10
5.37
5.30
6.52
6.44
6.47
6.40
5.79
6.21
5.51
5.95
5.88
6.62
6.31
5.82
5.65
6.38

pH Drn Dmr
5.29 69.59 ND
5.27 69.60 ND
5.74 70.52 ND
5.70 70.47 ND
5.45 71.80 ND
5.42 72.00 ND
5.54 72.22 ND
5.55 71.80 ND
5.66 72.39 ND
5.65 72.50 ND
5.68 70.19 ND
5.69 70.20 ND
5.53 73.54 ND
5.53 73.60 ND
5.52 72.60 ND
5.51 73.00 ND
4.11 70.04 97.92
4.10 69.66 99.07
4.10 69.66 99.17
4.04 70.54 100.66
4.01 70.08 100.58
4.09 71.61 100.16
4.08 71.68 99.89
4.06 71.62 99.46
4.10 72.01 100.13
4.10 72.42 98.67
4.10 71.56 101.64
4.21 72.02 100.75
4.21 72.16 100.07
4.21 72.30 99.69
4.17 70.13 99.57
4.15 70.47 98.32
4.17 70.72 97.50

.Abbreviations used: Trt-treatment, Rep-replication, Pa—propionic acid, La-lactic acid, Gl-

glucose, Ac-acetic acid, Et-ethanol, Bu-butyric acid, Ca-citric acid, Lab-lactic acid

bacteria, Ym—yeast and molds, Dm-dry matter, Dmr-dry matter recovery, G-autoclaved
DH42, H-DH42+LAB(reconstituted with tap water), I-DH42+P42+LAB, J- DH42+LAB
(reconstituted with distilled water), K-P42+DH42, L-DH42, M- control, N-P42, ND-not

determined.

Table B-1 (contd).

154

Trt Rep Time Pa

1

t—ri—t—r—«t—r—immaoozzzzzzt—rtrwxwuuH—i—t—tmmooozzzzzg

tutu-—th—Nt—ww~u-w-w-uN—wNu—sz—N—an—wN—wn

0.038
0.038
0.039
0.043
0.040
0.043
0.025
0.030
0.035
0.010
0.006
0.008
0.012
0.000
0.000
0.010
0.011
0.107
0.084
0.104
0.045
0.061
0.051
0.040
0.042
0.040
0.038
0.023
0.026
0.091
0.107
0.123
0.080
0.068
0.118
0.094
0.160
0.117
0.121
0.081

Table B-1 (contd).

La
0.521 0.010

0.660 0.016
0.637 0.016
0.763 0.017
0.671 0.012
0.583 0.010
1.280 0.000
1.761 0.000
1.771 0.000
1.379 0.023
1.374 0.022
1.738 0.017
1.562 0.021
1.478 0.013
1.646 0.023
1.916 0.018
1.664 0.007
1.514 0.014
1.467 0.010
1.453 0.014
2.066 0.006
1.937 0.007
1.517 0.000
1.129 0.000
1.242 0.007
1.241 0.000
1.269 0.000
1.141 0.000
1.101 0.000
0.868 0.000
1.103 0.000
1.199 0.000
0.953 0.008
0.854 0.007
1.579 0.000
1.134 0.000
0.667 0.000
1.636 0.008
1.628 0.009
1.125 0.010

G1

Ac

0.043
0.060
0.052
0.076
0.064
0.062
0.118
0.155
0.179
0.076
0.075
0.142
0.118
0.124
0.140
0.157
0.158
0.176
0.170
0.141
0.154
0.144
0.137
0.203
0.135
0.159
0.129
0.122
0.113
0.389
0.426
0.519
0.209
0.151
0.287
0.226
0.416
0.276
0.300
0.198

Et
0.190

0.224
0.261
0.314
0.249
0.217
0.390
0.376
0.371
0.398
0.404
0.380
0.233
0.343
0.325
0.414
0.378
0.437
0.390
0.465
0.318
0.396
0.436
0.246
0.437
0.3 76
0.480
0.347
0.423
0.456
0.530
0.556
0.477
0.413
0.394
0.391
0.051
0.473
0.360
0.298

Bu

0.000
0.000
0.000
0.000
0.000
0.000
0.049
0.059
0.058
0.074
0.074
0.017
0.020
0.024
0.024
0.024
0.025
0.054
0.050
0.054
0.054
0.052
0.044
0.033
0.045
0.038
0.057
0.040
0.042
0.130
0.168
0.184
0.236
0.238
0.088
0.078
0.072
0.108
0.077
0.049

155

Ca

0.002
0.002
0.002
0.002
0.002
0.002
0.132
0.187
0.196
0.159
0.153
0.162
0.162
0.143
0.173
0.184
0.161
0.188
0.158
0.154
0.155
0.152
0.132
0.127
0.132
0.136
0.122
0.106
0.110
0.202
0.216
0.248
0.191
0.161
0.162
0.137
0.199
0.173
0.156
0.134

Lab Ym pH Dm Dmr

8.57
8.63
8.67
8.69
8.53
8.52
8.87
8.98
9.00
8.67
8.50
8.63
8.62
8.63
8.65
8.61
8.57
8.89
8.92
8.77
8.99
8.78
8.89
8.83
9.04
8.90
8.94
8.82
8.75
8.47
7.68
7.91
8.36
8.27
8.32
8.36
8.51
8.66
8.47
8.41

6.66
6.86
6.56
5.92
6.03
6.60
3.85
4.09
4.07
3.92
3.80
4.14
4.13
4.05
4.19
4.23
3.90
4.12
4.12
3.79
3.87
4.29
4.38
4.24
3.96
4.26
4.01
3.90
4.00
3.57
3.04
3.13
4.15
2.73
3.41
3.35
4.58
2.58
2.58
1.88

4.27
4.26
4.26
4.25
4.26
4.28
4.18
4.17
4.14
4.12
4.12
4.11
4.13
4.13
4.15
4.15
4.11
4.25
4.25
4.18
4.13
4.15
4.14
4.38
4.35
4.35
4.22
4.28
4.27
4.36
4.33
4.33
4.36
4.40
4.16
4.18
4.47
4.14
4.13
4.13

73.03
73.62
73.31
72.46
72.04
72.90
69.08
69.36
69.70
70.20
70.30
71.20
71.16
71.41
71.94
71.61
71.78
71.64
71.94
71.70
70.58
70.02
69.84
72.94
73.04
73.12
71.66
72.14
71.67
69.79
69.52
69.65
70.62
70.51
71.96
71.83
70.10
72.18
71.92
72.52

101.38
99.20
100.38
100.01
101.39
98.30
100.94
100.02
98.91
99.20
99.15
104.52
101.49
93.64
100.37
101.68
101.08
101.94
100.87
101.87
98.08
99.90
100.40
101.57
101.23
100.97
102.68
100.93
102.58
98.90
98.71
98.97
99.41
99.08
93.29
99.31
95.83
98.83
98.58
99.31

Table B-1 (contd).

Trt Rep Time Pa

EEQQQZZZZZZFF‘P‘WWWC—C—HHHH‘LGEOQOZZZZZZF‘FCI'CKWN

NHwNHwNu—wN—th—IwN—MNHwNHNHWN—‘wwwwN—rWN-HDJNH

90
90
90
90
90
90
90
90
90
90
90
90
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
91
93
93
93
93
93

0.122
0.130
0.1 12
0.1 10
0.107
0.1 13
0.080
0.047
0.080
0.076
0.073
0.061
0.1 15
0.126

0.124

0.095
0.095

La
1.109
1.177
1.018
1.120
1.144
1.190
0.901
0.555
0.918
0.847
0.780
0.768
0.838
1.002
1.005
1.065
1.117

0.066 0.889
0.106 1.324
0.170 0.933
0.071 1.010
0.132 1.640
0.070 1.005
0.108 0.907
0.112 0.941
0.115 0.991
0.125 1.119
0.117 1.179
0.076 0.774
0.084 0.811
0.048 0.521
0.097 0.985
0.074 0.843
0.070 0.680
0.081 0.908
0.124 0.788
0.131 0.886
0.153 1.184
0.095 0.881
0.103 0.882

G1 Ac Et Bu

0.000 0.402 0.502 0.100
0.008 0.439 0.518 0.105
0.000 0.378 0.438 0.088
0.000 0.345 0.393 0.106
0.000 0.332 0.333 0.097
0.000 0.377 0.509 0.121
0.000 0.399 0.432 0.077
0.008 0.266 0.336 0.053
0.000 0.364 0.416 0.076
0.000 0.295 0.487 0.109
0.006 0.401 0.492 0.114
0.000 0.256 0.415 0.105
0.000 0.491 0.457 0.151
0.007 0.503 0.363 0.153
0.000 0.529 0.365 0.139
0.014 0.258 0.430 0.278
0.016 0.233 0.398 0.283
0.011 0.160 0.341 0.096

0.013 0.281 0.401 0.090'

0.000 0.468 0.075 0.075
0.018 0.193 0.373 0.098
0.016 0.335 0.313 0.080
0.014 0.206 0.360 0.072
0.011 0.362 0.412 0.099
0.016 0.366 0.430 0.114
0.012 0.375 0.407 0.118
0.000 0.393 0.343 0.124
0.000 0.351 0.351 0.137
0.000 0.275 0.382 0.128
0.014 0.403 0.383 0.089
0.016 0.261 0.391 0.080
0.012 0.440 0.352 0.087
0.012 0.318 0.413 0.140
0.011 0.398 0.425 0.136
0.011 0.333 0.435 0.144
0.000 0.525 0.351 0.152
0.000 0.540 0.330 0.165
0.000 0.641 0.285 0.186
0.018 0.241 0.280 0.293
0.011 0.203 0.305 0.308

156

Ca Lab

0.136
0.160
0.148
0.152
0.135
0.158
0.134
0.1 10
0.137
0.125
0.135
0.1 14
0.289
0.285
0.241
0.254
0.224
0.238
0.247
0.293
0.223
0.199
0.212
0.193
0.231
0.244
0.217
0.231
0.206
0.204
0.196
0.21 1
0.208
0.205
0.202
0.246
0.249
0.272
0.251
0.209

8.57
6.88
7.25
8.77
8.78
6.93
8.47
8.50
8.37
7.81
8.47
7.99
8.74
8.76
8.52
8.42
8.45
8.30
8.22
8.95
8.46
8.48
8.38
8.39
8.57
8.45
8.44
8.44
8.43
8.46
8.37
8.44
8.29
8.42
8.38
8.61
8.51
8.59
8.44
8.37

Ym
2.18
3.24
3.47
3.07
2.37
1.89
2.18
1.88
1.88
2.18
2.18
2.19
2.19
0.19
1.89
2.67
2.37
2.18
2.58
3.49
2.65
1.88
2.83
2.36
1.88
3.46
3.47
3.17
1.89
2.17
3.65
2.57
3.28
2.18
3.26
2.80
1.89
0.20
2.67
2.37

pH

4.27
4.24
4.27
4.27
4.24
4.25
4.29
4.31
4.28
4.33
4.32
4.31
4.35
4.30
4.31
4.36
4.39
4.16
4.21
4.40
4.16
4.16
4.15
4.27
4.25
4.28
4.27
4.23
4.24
4.28
4.30
4.27
4.33
4.30
4.30
4.34
4.30
4.27
4.34
4.35

Dm

72.08
72.07
71.82
70.59
70.56
70.54
73.03
72.66
73.04
72.13
71.89
71.45
70.17
70.20
70.18
71.25
71.03
72.08
72.57
70.88
72.82
72.04
72.40
72.50
72.50
72.90
71.50
71.27
71.03
74.20
74.28
74.53
72.97
72.51
72.45
70.17
70.21
70.18
70.97
71 .03

98.22
98.12
97.91
99.20
99.21
99.38
98.07
97.64
98.12
98.09
97.96
97.85

866%666666666666666668666666

Table B-1 (cont’d).

Trt Rep Time Pa

ZZZZZZt-‘t-‘t-Cz‘xra‘t-t—av—HHEEQQOZZZZZZt—r‘t—xth—at—r—«m—t

l

MN—WNt-‘UJNF—WNh—UJNh-UJNi—N—wN—‘mNr—MNHWNHWNHWNHWN

93
93
93
93
93
93
93
93
93
93
93
93
93
93
93
93
93
93
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95
95

La Gl Ac Et Bu Ca

0.130 1.569 0.011 0.326 0.308 0.111 0.204
0.098 1.179 0.007 0.239 0.265 0.082 0.146
0.135 0.682 0.000 0.366 0.261 0.043 0.170
0.118 1.289 0.014 0.275 0.315 0.100 0.164
0.132 1.493 0.019 0.325 0.287 0.089 0.190
0.112 1.195 0.018 0.289 0.268 0.069 0.169
0.129 0.953 0.007 0.397 0.284 0.107 0.171
0.130 0.910 0.008 0.413 0.269 0.110 0.173
0.000 1.306 0.015 0.526 0.307 0.145 0.217
0.116 0.856 0.000 0.359 0.258 0.115 0.151
0.135 1.229 0.000 0.384 0.258 0.129 0.173
0.134 1.155 0.000 0.437 0.364 0.131 0.158
0.103 0.931 0.009 0.444 0.289 0.091 0.171
0.091 0.715 0.012 0.426 0.337 0.079 0.125
0.106 0.971 0.009 0.436 0.315 0.092 0.145
0.092 0.948 0.000 0.359 0.349 0.132 0.117
0.090 0.722 0.000 0.463 0.354 0.130 0.150
0.094 0.999 0.000 0.388 0.389 0.148 0.160
0.106 0.846 0.000 0.502 0.228 0.126 0.179
0.099 0.853 0.000 0.449 0.170 0.124 0.158
0.102 0.905 0.000 0.494 0.195 0.129 0.180
0.070 0.900 0.000 0.207 0.141 0.223 0.155
0.067 0.872 0.000 0.169 0.168 0.234 0.131
0.087 1.298 0.000 0.257 0.153 0.073 0.117
0.100 1.161 0.000 0.277 0.186 0.072 0.121
0.144 0.870 0.000 0.468 0.187 0.047 0.167
0.106 1.396 0.008 0.300 0.214 0.093 0.156
0.108 1.462 0.009 0.314 0.154 0.074 0.148
0.108 1.463 0.008 0.334 0.183 0.063 0.141
0.115 0.983 0.000 0.408 0.221 0.101 0.132
0.114 0.935 0.000 0.393 0.212 0.103 0.128
0.157 0.927 0.000 0.387 0.207 0.095 0.130
0.139 1.317 0.000 0.477 0.218 0.135 0.179
0.118 1.173 0.000 0.399 0.191 0.118 0.136
0.114 1.101 0.000 0.431 0.191 0.116 0.138
0.085 0.862 0.000 0.426 0.168 0.080 0.135
0.082 0.883 0.000 0.445 0.228 0.079 0.139
0.087 0.895 0.000 0.421 0.189 0.074 0.126
0.073 0.928 0.000 0.363 0.235 0.125 0.124
0.089 0.809 0.000 0.494 0.257 0.126 0.132
0.092 1.027 0.000 0.397 0.255 0.138 0.141

157

Lab

8.46
8.34
8.58
8.37
8.34
8.24
8.30
8.34
8.25
8.33
8.31
8.27
8.19
8.22
8.14
8.24
8.20
8.15
8.65
8.72
8.69
8.60
8.36
8.38
8.39
9.02
8.27
8.23
8.41
8.28
8.37
8.49
8.49
8.38
8.33
8.27
8.23
8.17
8.26
8.36
8.34

an pH Dm Dmr

2.36
2.36
3.48
2.72
2.58
3.14
2.18
1.88
1.88
1.89
2.49
1.89
2.17
2.47
2.17
2.35
1.88
2.83
1.89
2.18
2.83
2.57
2.35
2.47
3.08
3.36
3.02
3.39
2.82
2.65
3.28
3.10
3.35
3.29
3.08
3.12
3.14
2.82
2.82
3.27
3.23

4.13 72.07
4.16 72.57
4.35 70.88
4.14 72.83
4.14 72.04
4.12 72.40
4.24 72.50
4.25 72.50
4.32 72.79
4.26 71.50
4.20 71.27
4.22 71.03
4.29 74.20
4.28 74.27
4.26 74.48
4.32 72.97
4.28 72.51
4.28 72.45
4.35 71.36
4.34 72.08
4.33 72.21
4.39 73.90
4.39 72.87
4.19 73.54
4.22 73.66
4.40 72.54
4.18 73.86
4.18 74.25
4.17 74.49
4.29 74.44
4.28 74.29
4.29 74.18
4.30 73.03
4.24 72.55
4.29 73.65
4.31 75.82
4.34 75.35
4.30 75.25
4.34 74.62
4.33 74.16
4.33 74.06

66666666666666666666%66666666666666866666

Table B—2. Data used for analyses in chapter 2- temperature during aerobic stability test.

Trt Rep temp90

1

ZZZZZZK‘F‘F‘WKWHHHH—‘HIEQOQ
wN—th—th—wN—wwu—wwt—Nt—wm

22.78
21.67
22.78
22.78
21.67
22.22
22.22
22.78
22.22
22.22
21.11
23.33
21 .67
23.33
22.22
23.89
23.33
23.89
23.33
22.78
22.78
23.89
23.33

temp91

22.78
21.67
22.78
22.78
21.67
22.22
22.50
22.78
21.94
22.22
21.11
23.06
21.67
23.06
22.22
23.89
23.06
23.89
23.33
22.78
22.78
23.61
23.06

temp92

22.22
21.67
21.11
21.67
21.11
21.67
21.67
22.22
21.39
21.67
20.56
21.94
21.11
21.67
21.11
23.33
21 .94
23.06
22.78
23.06
22.78
22.78
22.78

ternp93

22.22
21.11
21.67
21.67
21.11
21.67
21.67
22.22
21.11
21.67
20.56
22.22
21.11
21.67
21.11
23.33
22.22
23.33
22.78
23.33
22.78
22.78
22.78

temp94

21.11
21.11
22.22
20.56
21.67
21.67
21.67
21.67
21.11
21.67
21.11
21.11
22.22
21.67
21.11
22.78
21.67
22.22
23.33
22.78
22.22
22.22
22.22

temp95

21.11
21.11
22.22
20.56
21.67
21.67
21.67
21.11
21.11
21.11
21.11
21.11
22.22
21.11
22.22
23.33
22.22
22.22
23.33
22.78
22.78
22.78

_ 21.67

‘Abbreviations used: Trt-treatment, Rep-replication, temp-temperature

158

APPENDIX C

Data Used for Analyses in Chapter 3

159

Table

C
(n.1-
N

.AAAA

AAAAAAAAAAABBBBBBBB

‘ . .

Table 01. Data used for analyses in chapter 3-chemical analyses.

Mc Inoc Rep Time

1

nwwwwwwwwwwwwwwwwwwww>>>>>>>>>>>>>>>
wxxxxgssgccccaeaewwwwxxxg22cccaeewmm
C—AWN—CbWN—‘hLaJNi-ehMN—AWNHMNHWNI—WNHWNHWN

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOCOOOOO

pH Dmr Ym Ac

5.80
5.76
5.77
5.63
5.75
5.69
5.73
5.81
5.57
5.73
5.75
5.66
5.65
5.69
5.78
5.56
5.55
5.61
5.53
5.63
5.67
5.54
5.67
5.63
5.62
5.46
5.66
5.68
5.59
5.55
5.46
5.55
5.63
5.68
5.68
5.61

666666666666666%66666666666666666666

4.33
5.18
5.11
5.34
5.24
4.93
5.18
5.41
5.11
5.48
5.11
5.33
4.34
5.12
4.34
5.19
5.92
5.40
5.25
5.35
5.22
5.28
5.38
5.30
5.42
5.34
5.38
5.57
5.45
5.16
5.21
5.05
5.60
5.54
5.57
5.32

0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.003
0.002
0.009
0.007
0.022
0.009
0.01 1
0.037
0.022
0.056
0.033
0.033
0.044
0.054
0.070
0.080
0.083
0.063
0.069
0.081
0.062
0.063
0.054
0.069
0.084
0.097
0.021

Bu
0.001
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.005
0.000
0.000
0.000
0.002
0.000
0.009
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.000
0.056
0.058
0.033
0.055
0.055
0.059
0.051
0.052
0.065
0.059
0.055
0.050
0.062
0.049
0.132
0.195
0.102
0.048
0.039
0.044
0.048
0.029
0.037
0.052
0.038
0.052
0.036
0.031
0.023
0.042
0.025
0.030
0.045
0.031
0.074

Et
0.000
0.000
0.000
0.000
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.025
0.073
0.138
0.142
0.146
0.175
0.172
0.191
0.184
0.205
0.200
0.204
0.190
0.170
0.143
0.168
0.162
0.163
0.183
0.156
0.158
0.007

Gl
1.036
0.981
0.980
0.820
0.970
0.970
1.052
1.019
0.973
0.978
1.031
1.023
0.900
0.719
0.622
0.108
0.098
0.130
0.079
0.048
0.108
0.145
0.090
0.135
0.1 12
0.103
0.103
0.093
0.089
0.079
0.099
0.074
0.094
0.106
0.094
0.181

La
0.000
0.000
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.002
0.029
0.039
0.041
0.052
0.141
0.1 13
0.066
0.156
0.040
0.145
0.053
0.098
0.105
0.056
0.063
0.048
0.152
0.033
0.190
0.091
0.089
0.010

Pa
0.000
0.002
0.000
0.001
0.000
0.001
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.035
0.003
0.000
0.004
0.002
0.000
0.000
0.000
0.000
0.044
0.001
0.000
0.000
0.000
0.052
0.000
0.138
0.000
0.228
0.000
0.000
0.004

‘Abbreviations used: Mc-moisture content, Inoc-inoculant, Rep-Replication, Dmr-dry
matter recovery, Ym-yeast and molds, Ac-acetic acid, Bu-butyric acid, Ca-citric acid, Et-
ethanol, Gl-glucose, La-lactic acid, Pa-propionic acid, ND- not determined.

160

Table

110 l

‘I

C

CCCCCCCCCC

CCCCCCCCCCCCCCCCCCCrKr\r\r\r\r\l\l\

Table C-l (cont’d).

Mc Inoc Rep Time

1T11'11OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

wwxxxxggggccccaaaammmwxxxxgggsccccaaaawmm

Nt—MQONmeQMOOQGMOOQGMOOQONM-RWNF—AWNC—AUJNi-ChMNH-fibWN

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

pH Dmr Ym Ac

5.63
5.78
5.50
5.71
5.60
5.42
5.64
5.60
5.74
5.81
5.45
5.58
5.74
5.81
5.45
5.58
5.47
5.62
5.75
5.35
5.43
5.45
5.39
5.39
5.46
5.24
5.35
5.50
5.44
5.28
5.31
5.40
5.49
5.42
5.38
5.37
5.48
5.36
5.26
5.43
5.33

66%666666666666666%6666666666666666666666

4.76
5.76
4.97
4.86
5.10
4.96
5.14
5.61
5.27
5.28
5.12
4.78
4.78
4.81
4.49
5.34
5.32
4.79
4.74
4.84
4.81
4.46
4.63
4.23
4.46
5.17
4.38
3.86
4.83
4.49
4.66
4.94
4.99
4.66
4.33
4.67
4.99
3.68
4.90
4.74
4.73

0.020
0.021
0.014
0.020
0.015
0.016
0.021
0.018
0.042
0.029
0.035
0.024
0.036
0.032
0.037
0.033
0.027
0.014
0.018
0.020
0.010
0.01 1
0.015
0.030
0.027
0.010
0.027
0.045
0.023
0.009
0.010
0.027
0.052
0.059
0.009
0.012
0.049
0.035
0.026
0.008
0.017

161

Bu
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0. 000
0. 000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.065
0.082
0.086
0.076
0.044
0.062
0.475
0.027
0.01 1
0.012
0.016
0.014
0.012
0.006
0.018
0.007
0.01 1
0.006
0.005
0.004
0.006
0.007
0.008
0.010
0.010
0.008
0.008
0.009
0.01 1
0.008
0.009
0.007
0.01 1
0.010
0.012
0.008
0.013
0.013
0.008
0.010
0.017

Et
0.015
0.018
0.023
0.046
0.059
0.055
0.074
0.075
0.077
0.091
0.104
0.061
0.093
0.085
0.069
0.076
0.053
0.051
0.078
0.058
0.022
0.015
0.044
0.103
0.053
0.015
0.022
0.054
0.098
0.015
0.020
0.020
0.070
0.029
0.091
0.081
0.102
0.101
0.040
0.033
0.043

G1
0.084
0.086
0.103
0.058
0.085
0.065
0.381
0.057
0.047
0.051
0.071
0.056
0.062
0.057
0.068
0.059
0.066
0.066
0.066
0.042
0.048
0.048
0.046
0.062
0. 066
0. 044
0. 050
0.055
0.059
0.047
0.053
0.046
0.071
0.055
0.052
0.050
0.075
0.074
0.049
0.070
0.064

La
0.005
0.026
0.010
0.010
0.010
0.008
0.018
0.010
0.034
0.021
0.033
0.015
0.020
0.007
0.027
0.000
0. 007
0. 000
0. 016
0.170
0.130
0.117
0.162
0.183
0.156
0.116
0.181
0.163
0.178
0.159
0.137
0.168
0.180
0.306
0.161
0.175
0.177
0.193
0.082
0.010
0.023

Pa
0.005
0.009
0.000
0. 000
0. 002

0000
0. 000
0. 001
0.024
0.046
0.034
0.000
0.027
0.024
0.097
0.124
0.000
0.000
0. 000
0. 005
0. 010
0. 004
0. 005
0.044
0. 004
0. 008
0.046
0.025
0.053
0.042
0.052
0.031
0.063
0.033
0.053
0.000
0. 000
0. 003
0. 000
0. 007
0.007 ‘

Table

C
1
\IV

iEEEEEEEEEEEEEEEEEE

FFFFFFFFFPII..

Table C-l (cont’d).

Mc Inoc Rep Time

Cigar-urnC-rl-nC-nC-riC-i-lC-nC-n-nurinal-11mmmmmmmmmmmmmmmmmmmmmmmm

wwwxxxxggssccccaaeawwwwxxxxggggccccaaaaww

WNHAwN—thH-BUJNi—AwNflhWNH-RMNHPLWNH-hWNthJNH-hw

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

pH Dmr an Ac

5.29
5.36
5.55
5.47
5.49
5.42
5.52
5.39
5.53
5.40
5.33
5.29
5.37
5.45
5.48
5.45
5.30
5.42
5.37
5.44
5.32
5.14
5.13
5.39
5.17
5.18
5.24
5.27
5.32
5.21
5.19
5.24
5.24
5.24
5.29
5.28
5.13
5.24
5.28
5.21
5.29

6%6666666666666666%6666666666668666666666

4.92
4.44
4.80
4.66
4.28
4.88
4.40
4.80
4.68
4.82
5.20
5.24
4.80
4.93
4.58
5.01
4.55
4.18
4.41
5.05
5.00
3.71
4.41
4.89
5.26
4.49
4.18
4.52
4.84
4.36
3.89
4.90
4.19
4.52
5.05
4.69
4.01
4.73
5.78
5.92
5.63

0.020
0.021
0.051
0.018
0.043
0.013
0.020
0.030
0.013
0.010
0.065
0.019
0.016
0.016
0.000
0.019
0.029
0.002
0.000
0.000
0.007
0.006
0.034
0.015
0.001
0.015
0.031
0.042
0.040
0.033
0.035
0.029
0.061
0.058
0.046
0.028
0.025
0.018
0.010
0.047
0.022

162

Bu
0.006
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.010
0.008
0.018
0.013
0.014
0.01 1
0.010
0.011
0.012
0.003
0.013
0.006
0.008
0.008
0.014
0.010
0.014
0.009
0.028
0.048
0.046
0.034
0.033
0.084
0.044
0.040
0.057
0.038
0.007
0.01 1
0.009
0.012
0.013
0.018
0.040
0.013
0.015
0.012
0.024
0.022
0.018

Et
0.023
0.056
0.053
0.025
0.017
0.020
0.043
0.037
0.052
0.028
0.034
0.026
0.029
0.029
0.032
0.022
0.061
0.021
0.000
0.000
0.000
0.000
0.022
0.024
0.000
0.005
0.008
0.072
0.017
0.014
0.025
0.012
0.059
0.079
0.1 14
0.006
0.002
0.057
0.066
0.065
0.050

G1
0.067
0.040
0.058
0.054
0.065
0.041
0.058
0.048
0.053
0.042
0.050
0.049
0.045
0.048
0.046
0.047
0.056
0.046
0.295
0.256
0.267
0.264
0.207
0.286
0.288
0.210
0.238
0.142
0.035
0.1 16
0.141
0.155
0.091
0.071
0.081
0.026
0.076
0.135
0.047
0.045
0.048

La
0.01 1
0.008
0.008
0.005
0.028
0.037
0.003
0.006
0.012
0.000
0.000
0.003
0.015
0.020
0.003
0.002
0.000
0.003
0.005
0.000
0.001
0.001
0.001
0.043
0.000
0.008
0.063
0.177
0.067
0.058
0.025
0.016
0.079
0.146
0.245
0.024
0.01 1
0.055
0.033
0.021
0.107

Pa
0.005
0.007
0.007
0.048
0. 107
0.007
0.021
0.023
0.014
0.020
0.031
0.033
0.018
0.016
0.013
0.020
0.024
0.010
0.000
0.000
0.000
0.000
0.004
0.003
0.000
0.004
0.005
0.054
0.007
0.005
0.000
0.008
0.003
0.065
0.109
0.003
0.005
0.004
0.005
0.003
0.006 ’

Table

MC l

0666

Table C-I (cont’d).

Me Inoc Rep Time
1

OOOOOOOOOOOO

wmwwwwwwwwwwww>>>>>>>>>>>>>>>Qooooooooooo

2ficcccfifififimmmmxxxfifificcc-ifi—lmmmxxxsggcccqqq

Nt—Aw-¢w-4>wm~ww~wm—wN—wman—uN—wN—wN—um
'5

pH Dmr

5.15
5.41
5.24
5.28
5.20
5.19
5.31
5.24
5.17
5.44
5.23
5.22
3.88
3.91
3.89
3.91
3.90
3.88
3.89
3.87
3.92
3.89
3.94
3.92
3.90
3.87
3.93
4.04
4.01
4.07
4.03
4.02
4.11
4.06
4.06
4.02
3.96
4.05
4.01
4.12
4.09

666666666666

Ym

5.39
5.69
5.81
6.17
5.33
5.41
6.07
5.93
5.91
5.94
5.37
5.46
4.54
4.66
4.48
4.85
4.26
5.41
5.47
5.41
4.64
4.70
5.26
4.21
4.57
4.01
4.16
4.80
4.65
4.65
4.47
4.84
4.48
4.55
4.45
4.52
4.56
3.41
4.42
4.77
4.69

Ac
0.029
0.016
0.057
0.014
0.015
0.035
0.010
0.025
0.039
0.047
0.023
0.029
0.187
0.189
0.165
0.169
0.194
0.215
0.202
0.193
0.216
0.165
0.183
0.215
0.242
0.194
0.203
0.136
0.145
0.159
0.155
0.1 18
0.117
0.098
0.168
0.163
0.178
0.164
0.186
0.136
0.215

163

Bu
0.000
0.000
0.001
0.000
0.000
0.001
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.002
0.001
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.002
0.000
0.000
0.002
0.000
0.001
0.002
0.000
0.001
0.000
0.000
0.000
0.000

Ca
0.025
0.019
0.016
0.020
0.01 1
0.017
0.028
0.020
0.014
0.016
0.012
0.021
0.017
0.102
0.014
0.032
0.068
0.036
0.039
0.040
0.099
0.013
0.012
0.034
0.070
0.036
0.033
0.009
0.01 1
0.014
0.01 1
0.042
0.015
0.061
0.014
0.012
0.013
0.051
0.014
0.010
0.012

Et
0.023
0.048
0.070
0.063
0.012
0.044
0.046
0.034
0.021
0.054
0.037
0.029
0.618
0.658
0.556
0.657
0.681
0.691
0.630
0.575
0.676
0.553
0.604
0.617
0.892
0.683
0.679
0.635
0.673
0.759
0.703
0.624
0.700
0.671
0.769
0.691
0.647
0.650
0.736
0.515
0.624

01
0.047
0.033
0.049
0.037
0.009
0.023
0.035
0.04-4
0.039
0.048
0.050
0.037
0.139
0.190
0.116
0.148
0.209
0.174
0.160
0.159
0.247
0.116
0.127
0.161
0.230
0.155
0.160
0.142
0.163
0.183
0.178
0.171
0.171
0.172
0.192
0.184
0.179
0.166
0.198
0.11 1
0.153

La
0.042
0.055
0.041
0.109
0.034
0.011
0.039
0.019
0.003
0.022
0.071
0.011
2.061
2.210
1.800
1.780
2.211
2.243
2.146
2.105
2.317
1.768
1.962
2.243
2.766
2.214
2.227
1.349
1.424
1.514
1.617
1.495
1.675
1.634
1.620
1.601
1.787
1.412
1.753
1.087
1.397

Pa
0.004
0.008
0.002
0.002
0.002
0.035
0.013
0.003
0.003
0.031
0.038
0.002
0.004
0.003
0.001
0.000
0.001
0.004
0.006
0.000
0.006
0.002
0.003
0.003
0.007
0.001
0.002
0.001
0.000
0.003
0.004
0.004
0.002
0.002
0.004
0.002
0.006
0.008
0.007
0.004
0.003 ‘

Tabl

Table 01 (cont’d).

Mc Inoc Rep Time

1111111111T1mmmml'I'JlTllTllTl1T111111100OOOOOOOOOOOOOOOOOOUwawww

22ecccceaaewmmwxxxxggzgccccaaaammwwxxxxas

3

(AN—CAMN—AL‘JNi—ADJN—ADJNr-CRWNF—RMNH-fi-WNH-DLMNHRWNH-h

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

pH

4.05
4.05
4.00
3.97
4.07
4.06
4.69
4.66
4.93
4.61
4.62
4.63
4.60
4.61
4.68
4.65
4.57
4.52
4.66
4.67
4.64
4.51
4.63
4.62
4.66
4.52
4.95
4.41
4.48
4.36
4.83
4.38
4.65
4.24
4.67
4.48
4.65
4.59
4.33
4.27
4.48

Dmr

98.26
98.82
96.92
98.43
97.52
92.69
91.14
87.52
94.81
98.34
95.47
92.07
94.87
97 .96
92.02
93.29
91.53
97.89
95.75
87.49
92.20
98.00
89.34
89.64
88.65
92.50
99.07
97.79
98.09
99.72
94.66
98.39
98.08
97.56
98.24
98.49
97.39
97.74
96.48
99.03
98.17

Ym

5.20
4.16
5.01
5.10
4.89
5.20
4.90
5.11
5.63
4.48
5.01
4.27
4.89
3.63
4.80
5.33
5.26
3.87
4.85
4.59
4.20
4.14
4.84
4.36
4.42
4.43
3.87
0.00
3.70
0.00
3.40
4.30
3.40
4.10
0.00
0.00
0.00
0.00
3.88
3.40
0.00

Ac
0.168
0.166
0.158
0.176
0.129
0.154
0.078
0.072
0.132
0.087
0.084
0.090
0.110
0.088
0.094
0.115
0.138
0.138
0.101
0.092
0.121
0.118
0.090
0.084
0.101
0.112
0.089
0.079
0.062
0.061
0.149
0.096
0.080
0.074
0.172
0.113
0.181
0.118
0.092
0.083
0.103

164

Bu
0.001
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.015
0.045
0.013
0.014
0.016
0.013
0.010
0.009
0.021
0.014
0.009
0.009
0.014
0.010
0.008
Mm
0.010
0.014
0.01 1
0.009
0.012
0.015
0.012
0.010
0.014
0.016
0.01 1
0.007
0.007
0.006
0.009
0.007
0.005
0.005
0.008
0.008
0.005
0.005
0.006
0.006
0.008

Et
0.674
0.716
0.681
0.645
0.764
0.653
0.485
0.435
0.861
0.636
0.467
0.525
0.540
0.575
0.494
0.486
0.621
0.597
0.521
0.471
0.544
0.575
0.467
0.490
0.509
0.614
0.601
0.518
0.539
0.520
0.596
0.474
0.458
0.289
0.566
0.483
0.469
0.340
0.416
0.370
0.430

G1
0.185
0.196
0.178
0.192
0.187
0.166
0.070
0.073
0.160
0.089
0.089
0.076
0.087
0.076
0.062
0.073
0.091
0.090
0.038
0.072
0.076
0.081
0.067
0.068
0.076
0.092
0.049
0.025
0.035
0.022
0.049
0.055
0.039
0.018
0.053
0.052
0.044
0.037
0.050
0.047
0.041

La
1.623
1.552
1.697
1.754
1.466
1.497
0.536
0.453
1.007
0.682
0.610
0.496
0.598
0.596
0.336
0.383
0.566
0.665
0.440
0.458
0.641
0.685
0.558
0.540
0.674
0.765
0.510
0.743
0.793
0.792
0.445
0.804
0.559
0.730
0.647
0.821
0.400
0.469
0.985
1.050
0.678

Pa
0.003
0.005
0.003
0.009
0.004
0.007
0.008
0.004
0.009
0.002
0.003
0.007
0.009
0.002
0.028
0.022
0.053
0.040
0.012
0.004
0.028
0.014
0.013
0.002
0.007
0.002
0.004
0.010
0.006
0.005
0.043
0.015
0.029
0.001
0.100
0.075
0.092
0.034
0.041
0.033
0.069

Table C-1 (cont’d).

Mc Inoc Rep Time

omooooooooooooommmmmmmmmmmmmmmmmmmmmmmmm

xxxgggcccaaawwwxxxxggggccccaaaammmmxxxxg

4

wN—th—wN—wmr—wm—Auw~bw-hm-hw-hwm~bwmw

10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10

pH

4.36
4.79
4.53
4.87
4.59
4.59
4.84
4.91
4.81
4.66
4.93
4.97
4.81
4.61
4.75
4.96
4.72
4.85
4.91
4.88
4.89
4.72
4.83
4.81
4.85
5.37
5.29
5.23
5.39
5.24
5.25
5.37
5.30
5.20
5.40
5.21
5.26
5.38
5.26
5.21

Dmr Ym Ac

97.43 0.00 0.075
97.37 4.24 0.148
97.99 3.70 0.138
97.40 3.40 0.174
97.82 4.35 0.131
99.59 4.44 0.010
99.20 5.21 0.029
99.94 5.29 0.143
99.38 4.87 0.108
98.78 4.69 0.024
99.79 5.06 0.149
98.85 6.18 0.178
98.65 4.27 0.131
99.15 5.13 0.044
99.26 3.81 0.181
99.28 3.77 0.199
99.07 4.24 0.149
98.72 5.27 0.110
99.51 4.81 0.188
98.76 5.75 0.176
99.21 3.95 0.191
99.17 4.90 0.139
98.74 5.20 0.100
96.86 5.72 0.059
99.12 5.26 0.166
98.27 5.82 0.002
99.07 6.12 0.005
99.66 6.01 0.002
99.32 5.99 0.006
99.79 6.05 0.002
98.26 6.90 0.001
99.94 6.17 0.005
99.45 6.63 0.011
99.01 6.16 0.009
99.08 6.36 0.010
99.95 6.07 0.013
98.45 6.11 0.023
99.16 6.36 0.043
99.38 6.46 0.041
99.94 6.20 0.057

165

Bu
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000

Ca
0.006
0.004
0.009
0.008
0.007
0.010
0.014
0.012
0.012
0.006
0.011
0.011
0.010
0.008
0.010
0.012
0.009
0.010
0.010
0.010
0.007
0.009
0.010
0.008
0.009
0.015
0.017
0.012
0.020
0.014
0.020
0.014
0.021
0.012
0.014
0.013
0.011
0.007
0.01 1
0.006

Et
0.367
0.386
0.532
0.497
0.573
0.443
0.460
0.399
0.475
0.453
0.350
0.363
0.423
0.350
0.427
0.380
0.472
0.409
0.368
0.268
0.426
0.421
0.393
0.1 18
0.513
0.269
0.298
0.214
0.263
0.303
0.384
0.224
0.375
0.318
0.271
0.240
0.355
0.336
0.362
0.348

G1
0.037
0.035
0.052
0.044
0.050
0.044
0.051
0.047
0.041
0.040
0.044
0.052
0.042
0.031
0.044
0.046
0.046
0.045
0.042
0.044
0.041
0.044
0.043
0.031
0.046
0.155
0.155
0.166
0.226
0.164
0.1 10
0.166
0.1 13
0.1 16
0.092
0.124
0.071
0.032
0.033
0.030

La
0.684
0.502
0.789
0.438
0.829
0.709
0.648
0.486
0.567
0.676
0.352
0.351
0.578
0.536
0.456
0.425
0.737
0.408
0.391
0.320
0.483
0.631
0.533
0.437
0.708
0.029
0.036
0.042
0.040
0.048
0.026
0.028
0.043
0.056
0.041
0.069
0.108
0.157
0.154

Pa
0.046
0.000
0.003
0.007
0.001
0.000
0.002
0.004
0.004
0.000
0.003
0.006
0.003
0.004
0.047
0.013
0.035
0.020
0.024
0.003
0.018
0.004
0.002
0.009
0.004
0.000
0.000
0.001
0.000
0.000
0.001
0.000
0.002
0.003
0.000
0.002
0.001
0.001
0.001

0.188 0.002

Table C

Table 01 (cont’d).

Mc Inoc Rep Time

1

n000000wwaswwwwwwwwwwwwwwww>>>>>>>>>>>>>>>
wawwxxxxgggeccccaaaamwwwxxxgggccceaawmw
v—kWNr—ALAN—tburgu—Ammu—buoNh—AUJNHWNr—WNv—MNh-wN—wtx)

21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21

pH Dmr Ym Ac

3.79
3.80
3.79
3.79
3.80
3.80
3.79
3.79
3.79
3.80
3.80
3.77
3.82
3.81
3.76
3.90
3.90
3.93
3.88
3.86
3.93
3.88
3.90
3.91
3.83
3.94
3.91
3.92
3.92
3.89
3.93
3.88
3.83
3.93
3.91
4.51
4.55
4.46
4.50
4.44

99.06
98.89
98.32
99.88
99.27
98.88
99.30
97.79
98.69
99.57
99.54
98.35
98.78
99.43
96.86
97.90
96.84
95.91
98.19
97.05
97.08
98.13
97.11
97.30
96.69
96.89
97.94
98.83
96.90
98.69
94.42
96.70
96.82
95.87
94.76
93.26
92.07
92.18
92.14

100.00 4.25

5.65
3.68
4.10
5.96
3.48
4.08
4.97
4.15
4.54
6.21
4.40
6.38
4.94
3.65
4.04
4.29
4.26
3.91
4.16
4.57
4.26
4.48
3.45
3.42
4.38
4.04
4.28
4.65
4.24
4.22
4.17
4.48
4.35
4.30
5.55
5.85
5.43
5.51
4.98

0.176
0.155
0.176
0.150
0.165
0.182
0.181
0.150
0.190
0.165
0.152
0.183
0.163
0.145
0.168
0.154
0.167
0.165
0.159
0.192
0.142
0.168
0.167
0.175
0.199
0.159
0.176
0.151
0.165
0.163
0.159
0.180
0.225
0.163
0.170
0.164
0.185
0.106
0.170
0.165

166

Bu
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.009
0.007
0.025
0.027
0.008
0.043
0.032
0.008
0.060
0.039
0.026
0.008
0.040
0.049
0.052
0.009
0.012
0.013
0.038
0.009
0.010
0.013
0.012
0.01 1
0.019
0.010
0.037
0.012
0.026
0.01 1
0.01 1
0.034
0.069
0.033
0.034
0.010
0.009
0.014
0.006
0.006

Et
0.546
0.563
0.568
0.554
0.543
0.555
0.538
0.523
0.572
0.555
0.552
0.535
0.533
0.549
0.532
0.904
0.797
0.844
0.821
0.803
0.851
0.810
0.928
0.791
0.747
0.854
0.917
0.841
0.793
0.778
0.893
0.839
0.773
0.792
0.870
0.683
0.627
0.699
0.643
0.482

GI
0.139
0.126
0.156
0.137
0.134
0.182
0.146
0.123
0.169
0.169
0.141
0.139
0.160
0.169
0.178
0.210
0.231
0.256
0.228
0.224
0.226
0.264
0.247
0.229
0.236
0.233
0.254
0.207
0.251
0.238
0.220
0.259
0.322
0.255
0.268
0.098
0.122
0.1 12
0.107
0.080

La
2.308
2.218
2.304
2.078
2.218
2.285
2.283
2.035
2.341
2.193
2.087
2.276
2.136
2.056
2.161
1.986
2.120
2.093
2.067
2.205
1.830
2.166
2.102
2.193
2.269
1.998
2.201
1.845
1.986
2.079
1.906
2.188
2.436
2.028
2.091
0.924
1.047
1.237
1.050
0.721

Pa
0.006
0.007
0.010
0.006
0.006
0.005
0.01 1
0.009
0.010
0.005
0.006
0.006
0.006
0.006
0.005
0.008
0.015
0.008
0.028
0.014
0.005
0.014
0.008
0.009
0.020
0.013
0.027
0.006
0.012
0.010
0.01 1
0.008
0.022
0.005
0.019
0.004
0.001
0.002
0.002
0.058

Table

W.

CCCCCCCC

‘Ili . .

Table C-l (cont’d).

Me Inoc Rep Time

~r1'11mmmmmmmmmmmmmmmmmmmmmmmnnnnnnonnnnnonn

awmwwxxxxggggccccaaaquwmxxxxggggccccaaa

2

l—4300191—AuNH-thr—Aww—AWNthN—wa—AwNv—thv—Aw

21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21

pH

4.58
4.47
4.49
4.49
4.50
4.47
4.44
4.59
4.63
4.49
4.48
4.46
4.51
4.41
4.47
4.70
4.39
4.38
4.37
4.69
4.37
4.51
4.25
4.57
4.37
4.60
4.43
4.25
4.14
4.37
4.31
4.57
4.48
4.66
4.53
4.39
4.57
4.70
4.54
4.43

Dmr Ym Ac

92.93
95.11
94.88
96.26
96.94
94.94
93.69
92.69
92.78
90.42
94.93
95.39
91.88
97.38
98.11
98.42
97.04
97.93
98.26
97.92
97.98
97.69
98.23
98.12
97.58
96.59
97.51
98.09
98.58
97.86
96.71
96.70
97.83
97.59
97.57
99.86
99.42
99.24
99.35
99.06

5.09
4.46
5.16
5.17
3.46
3.33
2.85
3.15
0.00
3.75
4.82
5.49
5.73
5.08
5.65
4.99
4.74
4.79
5.56
3.00
6.31
3.84
3.68
2.87
2.87
2.87
4.45
5.52
3.78
2.87
4.09
5.38
3.68
5.41
5.39
5.36
5.54
5.74
5.85
5.11

0.209
0.213
0.214
0.199
0.304
0.253
0.257
0.137
0.269
0.308
0.302
0.087
0.178
0.237
0.124
0.035
0.1 17
0.085
0.092
0.093
0.134
0.107
0.103
0.157
0.155
0.146
0.153
0.146
0.119
0.135
0.185
0.065
0.050
0.084
0.074
0.061
0.201
0.246
0.047
0.176

167

Bu
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.002
0.087
0.043
0.000
0.000
0.022
0.030
0. 005
0. 000
0. 019
0. 000
0. 000
0. 002
0.000
0. 000
0. 003
0.008
0. 036
0. 000
0. 012
0.001
0.000
0.000
0.000
0.002

Ca
0. 009
0. 004
0. 007
0.001
0. 005
0. 004
0. 007
0.005
0.013
0.007
0.008
0.007
0. 005
0. 006
0. 008
0.009
0.005
0.006
0.007
0.009
0.006
0. 006
0. 005
0.008
0.007
0.012
0.007
0.005
0.006
0.006
0. 006
0. 008
0.007
0.007
0.010
0.007
0.006
0.003
0.008
0.005

Et
0.540
0.555
0.624
0.442
0.630
0.537
0.549
0.219
0.503
0.657
0.598
0.290
0.573
0.679
0.463
0.648
0.551
0.565
0.587
0.571
0.505
0.529
0.484
0.551
0.533
0.623
0.639
0.390
0.393
0.431
0.447
0.649
0.657
0.532
0.670
0.092
0.244
0.253
0.026
0.393

G]
0. 092
0. 090
0. 101
0.089
0.099
0.071
0.092
0.067
0.076
0.098
0.098
0.076
0.100
0.1 10
0.091
0.124
0.057
0.067
0.074
0.089
0.039
0.055
0.045
0.174
0.085
0.152
0.057
0.041
0.044
0.042
0.045
0.066
0.082
0.052
0.066
0.036
0.038
0.043
0.025
0.045

0.688
0.908
1.016
0.722
0.883
0.634
0.872
0.657
0.693
1.028
0.964
0.770
0.919
1.195
0.882
0.507
0.753
0.783
0.827
0.212
0.765
0.448
0.927
0.264
0.682
0.290
0.579
0.827
1.099
0.551
0.669
0.719
0.826
0.639
0.928
0.361
0.319
0.236
0.149
0.470

Pa
0.046
0.089
0.041
0.164
0.163
0.151
0.120
0.066
0.070
0.089
0.068
0.003
0.007
0.002
0.005
0.005
0.01 1
0.01 1
0.008
0.076
0.01 1
0.015
0.007
0.144
0.116
0.181
0.122
0.021
0.050
0.014
0. 067
0. 006
0. 015
0.005
0.006
0.002
0. 001
0. 004
0. 002
0.007

Table C-l (cont’d).

Mc Inoc Rep Time

>>>>>>>>>>ooomooooooooamommmmmmmmmmmmmwm

fiddddfiflwwwxxx2g2cccaaammmxxxxg2ggccccaaa

2

-—-U)Nt—UJNv—wN—L»NHwN—WN—ri—wmv—AwN—Ale—«bwtut—45w

21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
21
120
120
120
120
120
120
120
120
120
120

pH

4.57
4.62
4.55
4.51
4.48
4.66
4.46
4.65
4.61
4.64
4.57
4.51
4.54
4.50
4.50
5.22
5.20
5.13
5.09
5.09
5.01
5.00
4.98
4.92
5.00
5.10
5.01
5.22
5.17
5.08
3.70
3.70
3.70
3.80
3.73
3.67
3.69
3.74
3.79
3.72

Dmr Ym Ac

99.16
99. 19
98.36
99.63
98.64
99.79
98.44
99.17
99.82
98.80
98.83
95.79
97.36
98.23
98.46
96.14
98.68
98.97
95.08

100.18

98.31
99.81
99.19
98.64
96.49
99.50
98.45
99.14
99.87
99.54
98.36
98.14
97.59
96.86
98.58
97.02
97.70
98.51
98.41
97.54

5.07
5.43
5.74
5.16
4.55
4.91
3.31
5.90
3.45
3.45
6.07
5.96
5.90
6.41
6.63
6.02
5.92
5.73
5.89
5.74
5.75
5.24
5.59
5.61
5.41
5.55
5.36
5.77
5.87
5.54
2.47
4.73
2.41
3.16
1.81
1.33
1.63
1.81
2.61
2.28

0.258
0.288
0.044
0.159
0.257
0.279
0.278
0.000
0.259
0.277
0.005
0.009
0.016
0.000
0.000
0.007
0.006
0.028
0.008
0.015
0.074
0.040
0.050
0.095
0.059
0.100
0.11 1
0.078
0.052
0.117
0.224
0.189
0.171
0.234
0.183
0.196
0.171
0.223
0.259
0.191

168

Bu
0.001
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.001
0.001
0.001
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.006
0.006
0.005
0.001
0.001
0.004
0.000
0.003
0.003
0.006
0.002
0.001
0.002
0.003
0.002
0.016
0.021
0.024
0.014
0.021
0.020
0.016
0.018
0.013
0.010
0.012
0.012
0.008
0.010
0.013
0.006
0.004
0.002
0.004
0.002
0.003
0.004
0.002
0.002
0.003

Et
0.278
0.221
0.017
0.164
0.233
0.128
0.333
0.000
0.272
0.282
0.002
0.000
0.004
0.002
0.002
0.580
0.641
0.664
0.509
0.623
0.590
0.449
0.588
0.602
0.446
0.659
0.678
0.607
0.630
0.556
0.599
0.413
0.422
0.622
0.546
0.521
0.499
0.459
0.591
0.491

G1
0.042
0.040
0.029
0.032
0.035
0.025
0.043
0.024
0.034
0.037
0.020
0.031
0.027
0.026
0.026
0.096
0.120
0.131
0.072
0.127
0.054
0.080
0.043
0.053
0.037
0.048
0.052
0.043
0.052
0.053
0.307
0.241
0.231
0.106
0.249
0.267
0.220
0.270
0.153
0.244

La
0.226
0.203
0.168
0.239
0.161
0.085
0.276
0.131
0.203
0.141
0.154
0.126
0.141
0.152
0.168
0.115
0.143
0.193
0.110
0.193
0.173
0.167
0.143
0.263
0.226
0.344
0.389
0.311
0.516
0.540
4.112
3.563
3.140
3.260
3.233
4.191
3.173
3.860
3.430
3.265

Pa
0.016
0.018
0.01 1
0.033
0.026
0.018
0.052
0.009
0.050
0.042
0.024
0.001
0.000
0.002
0.004
0.000
0.000
0.000
0.001
0.005
0.009
0.008
0.012
0.034
0.004
0.007
0.016
0.002
0.003
0.000
0.200
0.165
0.141
0.163
0.153
0.206
0.151
0.195
0.181
0.157

Table C-

BBCCCCCCCCCCCCCC

Table C-l (cont’d).

Mc Inoc Rep Time

Oflnnnnonnnnnnnwwwwwwwwwwwwwwwwwwww>>>>>

ssccccaaaawwwwxxxxgzggccccaaaawmwwxxxss

2

N'—#UJNt-Cbulgh—AWN—thF-‘AWN—RNN—kWN—hWN—WNi—W

120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120

pH

3.77
3.66
3.7 8
3.78
3.69
4.11
4.06
4.17
4.12
4.07
4.05
4.05
4.22
4.04
3.94
4.06
4.13
3.99
4.13
4.00
3.96
4.04
3.97
4.11
4.07
4.18
4.36
4.19
4.23
4.08
4.22
4.11
4.24
4.06
4.04
4.08
4.16
3.97
4.33

Dmr Ym Ac

98.08 0.212
95.75 0.192
96.65 0.220
97.35 0.249
95.85 0.186
95.78 0.333
97.56 0.278
94.70 0.292
95.66 0.339
95.76 0.346
95.15 0.241
95.65 0.295
95.57 0.315
96.00 0.330
96.18 0.354
95.30 0.00 0.246
95.28 0.00 0.364
96.84 0.00 0.182
96.63 0.00 0.322
95.90 0.00 0.338
96.14 0.00 0.326
95.70 0.00 0.293
96.54 0.00 0.317
94.95 0.00 0.267
90.90 0.00 0.262
96.09 0.123
96.21 0.087
95.83 0.00 0.127
96.75 0.168
97.41 0.298
97.18 0.292
96.60 0.256
96.32 0.270
96.85 0.350
96.36 0.268
96.94 0.307
96.45 0.247
96.32 0.281
97.35 0.380

169

Bu
0.000
0.002
0.000
0.002
0.000
0.306
0.301
0.328
0.348
0.187
0.211
0.237
0.288
0.268
0.210
0.281
0.316
0.136
0.266
0.192
0.145
0.320
0.224
0.241
0.263
0.162
0.072
0.184
0.090
0.023
0.000
0.018
0.015
0.000
0.000
0.000
0.004
0.000
0.000

Ca
0.005
0.002
0.001
0.005
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.002
0.000
0.005
0.000
0.007
0.000
0.006
0.000
0.002
0.000
0.002
0.002
0.000

Et
0.444
0.530
0.462
0.540
0.512
0.734
0.796
0.887
0.984
0.713
0.833
1.012
0.948
0.883
0.908
0.916
0.961
0.744
0.905
0.906
0.890
0.929
0.873
0.631
1.000
0.849
0.773
0.844
0.745
0.787
0.607
0.635
0.665
0.670
0.516
0.628
0.380
0.523
0.603

O]
0.284
0.256
0.298
0.310
0.247
0.396
0.393
0.434
0.412
0.306
0.381
0.397
0.363
0.388
0.393
0.398
0.407
0.315
0.379
0.373
0.354
0.413
0.422
0.364
0.377
0.287
0.122
0.264
0.324
0.251
0.373
0.255
0.160
0.235
0.191
0.21 1
0.162
0.284
0. 187

La
3.368
4.425
3.400
3.410
4.041
2.401
2.484
2.244
2.130
2.136
2.271
2.339
1.776
2.510
2.951
2.425
2.233
2.181
2.076
2.381
2.624
2.724
2.966
2.143
2.288
1.640
1.051
1.930
1.516
1.658
1.106
1.318
1.396
1.401
1.378
1.195
1.143
1.816
1.591

Pa
0.159
0.217
0.169
0.158
0.197
0.354
0.323
0.345
0.326
0.307
0.303
0.303
0.297
0.341
0.300
0.315
0.312
0.245
0.298
0.304
0.297
0.335
0.312
0.302
0.288
0.230
0.030
0.158
0.024
0.370
0.314
0.315
0.270
0.445
0.456
0.416
0.315
0.390
0.290

Table C

MC 1111

WWVAXXXSSQC‘C.

CCCCCCCCCCCCCCCCC

CCCCCCCCCEEEEEEEEEEEEEE

Table C-1 (cont’d).

Mc Inoc Rep Time

C

mmmmmmmmmmmmrrlmnononononnoonnonnnnonnooo

W

ssccccaaaammmwxxxxagggccccaaaawmwmxxxxg

N'—$0310!—DWNC—«rP-UJNF—OOQC‘UIOOQO‘UIOOQO'JIOOQONUIOOQOLII4503N—C-AUJ

120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120

pH Dmr

4.03
4.22
4.17
4.27
4.19
4.18
4.29
4.23
4.22
4.18
4.17
4.23
4.18
4.18
4.17
4.18
4.19
4.20
4.22
4.17
4.17
4.20
4.27
4.26
4.18
4.22
4.38
4.48
4.46
4.39
4.42
4.41
4.40
4.38
4.33
4.32
4.38
4.30
4.25
4.24

96.90
97.21
96.84
97.99
96.52
95.67
96.72
98.16
97.06
97.37
97.12
97.81
97.15
97.81
97.48
98.26
97. 13
98.39
97.87
97.66
98.20
99.13
98.13
98.68
97.35
97.58
98.03
96.68
97.54
98.11
97.34
96.91
96.95
97.75
97.84
97.62
96.98
96.91
97.66
97.53

Ym

0.00
0.00
0.00
0.38
0.00
1.36
1.07
3.07
0.68
3.61
1.82
0.98
2.74
1.94
0.00
1.49
0.00
1.75
0.38
3.13
0.00
1.07
2.09
2.89
4.95
2.00
0.39
3.76
0.40
0.00
2.73
0.00
0.00
2.84
0.00
0.00
0.00
0.69
0.00
0.00

Ac
0.226
0.393
0.192
0.128
0.215
0.216
0.448
0.356
0.544
0.356
0.446
0.623
0.589
0.535
0.518
0.675
0.706
0.786
0.731
0.448
0.609
0.737
0.515
0.218
0.105
0.735
0.155
0.266
0.202
0.325
0.240
0.403
0.319
0.424
0.554
0.697
0.293
0.306
0.315
0.439

170

Bu
0.000
0.000
0.137
0.193
0.122
0.095
0.150
0.079
0.076
0.012
0.102
0.007
0.065
0.014
0.024
0.019
0.013
0.013
0.012
0.038
0.011
0.013
0.1 16
0.103
0.032
0.007
0.061
0.1 16
0.123
0.101
0.008
0.109
0.078
0.068
0.032
0.067
0.01 1
0.014
0.028
0.035

Ca
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.009
0.000
0.000
0.004
0.007
0.005
0.001
0.002
0.011
0.005
0.017
0.003
0.001
0.001

Et
0.567
0.505
0.821
0.887
0.807
0.857
0.694
0.554
0.717
0.846
0.444
0.522
0.297
0.384
0.402
0.393
0.393
0.539
0.651
0.210
0.563
0.595
0.764
0.550
0.092
0.606
0.444
0.225
0.371
0.618
0.482
0.474
0.419
0.270
0.545
0.497
0.596
0.287
0.215
0.344

G]
0.138
0.191
0.206
0.210
0.210
0.202
0.144
0.112
0.150
0.124
0.118
0.134
0.136
0.146
0.114
0.153
0.151
0.123
0.132
0.123
0.122
0.128
0.167
0.117
0.108
0.178
0.117
0.034
0.022
0.044
0.099
0.037
0.032
0.030
0.125
0.056
0.188
0.034
0.020
0.021

La
1.367
1.602
1.465
1.365
1.373
1.435
0.736
0.751
1.055
1.279
0.666
0.713
0.960
1.146
0.680
0.895
0.860
0.781
1.120
0.963
1.024
1.024
0.993
0.853
0.506
0.925
0.584
0.510
0.476
0.934
0.234
0.810
0.330
0.970
0.433
0.559
0.463
0.481
0.876
1.214

Pa
0.285
0.231
0.183
0.156
0.164
0.158
0.1 11
0.069
0.109
0.088
0.183
0.232
0.212
0.210
0.223
0.369
0.271
0.253
0.305
0.223
0.244
0.295
0.1 11
0.088
0.062
0.106
0.114
0.083
0.060
0.109
0.177
0.150
0.125
0.157
0.294
0.240
0.362
0.149
0.144
0.158

f

b C
a [Ill

Table C-l (cont’d).

Mc Inoc Rep Time

oooooooooooooaommmmmmmmmmmmmmmmmmmmmmmmmm

><><><ssgccc—l—l—lmmmxxxxggggcccc—l—l—l—lmmmmxxxx22

WN—WNFUJN—CWNI—WNI—AMNHAMN~&WN—&MN—-§WN~4§WNHJ§W

120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120
120

pH

4.28
4.32
4.36
4.41
4.40
4.41
4.17
4.27
4.32
4.31
4.20
4.43
4.36
4.31
4.25
4.30
4.36
4.29
4.35
4.34
4.36
4.29
4.31
4.29
4.34
4.29
4.71
4.62
4.69
4.56
4.61
4.54
4.57
4.53
4.57
4.55
4.54
4.55
4.69
4.68
4.64

Dmr

97.69
97.92
97.39
97.40
97.07
97.49
99.03
99.51
99.25
98.64
99.57
98.77
99. 12
98.18
98.83
97.28
98.79
98.43
98.59
98.91
99.09
98.53
97.04
98.87
98.67
98.30
98.20
97.38
97.93
98.19
98.62
97 .35
98.94
98.58
98.10
98.29
97 .98
97 .48
97.14
99.13
98.62

Ym

1.70
0.40
0.00
0.00
0.00
1.00
2.68
2.90
3.58
0.58
1.66
2.47
0.00
2.77
0.00
0.00
0.00
2.14
0.00
1.58
1.82
0.00
0.00
3.26
2.54
0.00
2.59
2.76
3.36
1.37
2.19
0.00
0.00
0.00
0.00
0.00
1.28
1.49
0.00
2.97
2.19

Ac
0.398
0.433
0.366
0.297
0.165
0.195
0.177
0.197
0.209
0.213
0.219
0.246
0.255
0.168
0.347
0.342
0.343
0.266
0.356
0.274
0.255
0.276
0.214
0.239
0.134
0.151
0.093
0.112
0.138
0.1 14
0.166
0.219
0.114
0.120
0.182
0.108
0.168
0.205
0.106
0.109
0.138

171

Bu
0.028
0.025
0.136
0.176
0.077
0.107
0.000
0.010
0.005
0.000
0.002
0.002
0.002
0.012
0.000
0.007
0.000
0.002
0.000
0.002
0.002
0.002
0.057
0.017
0.013
0.043
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001

Ca
0.005
0.002
0.009
0.004
0.009
0.012
0.006
0.010
0.016
0.012
0.010
0.013
0.013
0.006
0.008
0.009
0.01 1
0.005
0.012
0.026
0.016
0.01 1
0.01 1
0.006
0.005
0.010
0.016
0.019
0.018
0.015
0.020
0.023
0.012
0.012
0.019
0.010
0.018
0.017
0.018
0.024
0.027

Et
0.349
0.387
0.706
0.548
0.539
0.394
0.194
0.251
0.166
0.000
0.327
0.194
0.289
0.221
0.436
0.320
0.256
0.360
0.223
0.320
0.195
0.303
0.443
0.340
0.348
0.562
0.536
0.602
0.529
0.225
0.589
0.502
0.379
0.385
0.342
0.302
0.509
0.261
0.546
0.689
0.530

Gl
0.042
0.031
0.101
0.049
0.121
0.077
0.059
0.091
0.123
0.074
0.067
0.103
0.1 10
0.042
0.058
0.058
0.086
0.038
0.104
0.122
0.1 10
0.075
0.076
0.057
0.031
0.055
0.044
0.068
0.062
0.081
0.092
0.147
0.062
0.090
0.102
0.066
0.162
0.104
0.045
0.048
0.070

La
0.866
0.596
1.053
0.814
0.795
0.864
1.142
0.950
0.706
0.875
1.213
0.453
0.405
0.531
0.838
0.732
0.178
0.508
0.402
0.456
0.405
0.383
0.966
0.848
0.737
0.971
0.372
0.514
0.492
0.285
0.306
0.330
0.140
0.205
0.194
0.200
0.341
0.340
0.415
0.656
0.560

Pa
0.210
0.171
0.160
0.124
0.139
0.129
0.057
0.082
0.030
0.084
0.142
0.164
0.142
0.108
0.197
0.241
0.139
0.162
0.151
0.206
0.228
0.158
0.085
0.109
0.067
0.096
0.027
0.024
0.036
0.108
0.150
0.203
0.167
0.157
0.267
0.149
0.198
0.186
0.008
0.018
0.027 '

Table C-1 (cont’d).

Mc Inoc Rep Time
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121

n000000wwwwwwwwwwwwwwwwwww>>>>>>>>>>>>>>>
amwmmxxxxgggeccccaaaawwwmxxxg22cccaaawmw
v—bulgi—brawl—ALAN—huts)—thHAwN—uN—MNHwN—wNv—MNH

pH Dmr Ym Ac

3.66
3.68
3.72
3.77
3.73
3.66
3.68
3.69
3.78
3.70
3.75
3.67
3.81
3.78
3.68
4.11
4.09
4.16
4.18
4.11
4.05
4.04
4.23
4.09
3.97
4.12
4.12
4.00
4.12
4.02
3.99
4.06
4.01
4.12
4.10
4.22
4.35
4.21
4.24
4.07

103.31
100.59
100.39
102.48

99.63
101.82
102.75
102.35

99.58

99.95

100.96 3.96

100.51
99.45
94.88

102.91
99.57

0.226

0.216

0.206
0.260

3.60 0.214
0.00 0.197

0.194
0.180
0.250

3.19 0.221

0.203
0.191
0.059
0.234
0.174

0.00 0.251

99. 16 0.00 0.289

100.20 0.00 0.290 0.304 0.000
0.64 0.334
0.00 0.432

99.52
100.77

101.10 1.60 0.239
100.16 0.00 0.335

99.68

0.00 0.338

100.80 0.00 0.401
100.79 0.00 0.318
101.30 0.00 0.296
100.49 0.00 0.379

100.82
100.15

0.00 0.161
0.34 0.335

101.00 0.00 0.339

99.04
102.11
100.88
100.61
100.79
100.17
101.95
103.52
101.48
101.00

0.00 0.346
0.00 0.312
0.00 0.379
0.00 0.259

0.277
0.129
0.141
0.093
0.139

0.269

172

0.000 0.006
0.000 0.005
0.000 0.005
0.000 0.002

0.000 0.003

0.000 0.002
0.000 0.002

Et
0.487
0.397
0.489
0.544
0.537
0.553
0.477
0.452
0.501
0.489
0.460
0.490
0.045
0.501
0.518
0.657
0.854
0.834
0.890
0.752
0.809
1.028
0.834
0.702
0.830
0.767
0.875
0.639
0.853
0.798
0.871
0.709
0.720
0.691
0.951
0.627
0.463
0.466
0.565
0.537

G1 La Pa

0.301 3.979 0.200
0.260 3.835 0.185
0.255 3.564 0.176
0.105 3.295 0.170
0.273 3.521 0.176
0.267 4.236 0.215
0.246 3.566 0.183
0.243 3.382 0.175
0.132 3.179 0.168
0.269 3.741 0.183
0.254 3.100 0.151
0.231 4.049 0.193
0.248 2.513 0.168
0.272 3.321 0.157
0.237 3.913 0.189
0.327 1.946 0.289
0.403 2.441 0.335
0.424 2.174 0.339
0.405 2.061 0.317
0.362 2.359 0.377
0.393 2.365 0.307
0.424 2.494 0.334
0.347 1.609 0.291
0.391 2.414 0.359
0.390 2.894 0.329
0.427 2.608 0.344
0.406 2.205 0.316
0.297 2.124 0.232
0.406 2.208 0.327
0.374 2.309 0.302
0.388 2.752 0.343
0.400 2.565 0.335
0.408 2.755 0.323
0.351 2.029 0.300
0.409 2.396 0.318
0.272 1.545 0.229
0.150 1.258 0.071
0.235 1.727 0.155
0.218 1.444 0.147
0.224 1.550 0.335

Table

l
C
M

CCCCCCCC

CCCCCCCCCCCCCCCC(

II‘ ( ll 1

Table C-l (cont’d).

Mc Inoc Rep Time
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121

mmtnmmoonnoonnonnnonooonnnnnonononnnnonon
amwmwxxxxgg2acct:caaaawwmwxxxxggggcccc—l—i-d
C-CDWNt—OO\lO‘UIOOQOUIOOQO‘UIOOQOMOOQO‘UI-RUJNH-RWNV—«hbJNI-CAWN

pH Dmr Ym Ac

4.22
4.12
4.25
4.07
4.06
4.09
4.16
3.98
4.35
4.06
4.24
4.20
4.29
4.19
4.21
4.28
4.23
4.20
4.17
4.20
4.21
4.15
4.16
4.16
4.17
4.17
4.18
4.16
4.22
4.16
4.19
4.28
4.27
4.17
4.20
4.39
4.48
4.44
4.38
4.42

100.81
101.81
102.25
101.80
102.01
100.61
102.28
104.30
100.10
101.38
102.00
101.61
100.57
101.89
102.52
100.58
101.65
100.47
101.62
100.76
100.99
100.03
100.41
101.08
101.49
102.58
101.04
101.65
102.69
100.81
101.67
100.46
100.40
101.05
102.21
100.33
100.82
101.13
100.96
100.82

0.38
0.00
0.34
0.99
0.86
0.98
0.00
1.59
0.68
0.68
0.00
0.68
0.38
0.00
0.86
0.00
0.00
0.38
3.59
0.38
0.86
1.64
1.49
0.00
0.00
0.00
2.19
1.59
0.00
0.38
2.21
0.00
2.23
4.38
2.23
0.00
2.62
0.88
0.00
3.34

0.263
0.234
0.261
0.292
0.242
0.308
0.239
0.247
0.388
0.257
0.312
0.153
0.109
0.129
0.217
0.131
0.157
0.181
0.103
0.227
0.261
0.263
0.21 1
0.250
0.210
0.231
0.297
0.257
0.252
0.192
0.255
0.133
0.1 10
0.176
0.240
0.122
0.263
0.296
0.233
0.258

173

Bu
0.015
0.000
0.015
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.137
0.193
0.109
0.047
0.1 14
0.001
0.000
0.000
0.039
0.000
0.000
0.000
0.000
0.000
0.000
0.005
0.015
0.000
0.000
0.002
0.076
0.079
0.015
0.013
0.043
0.1 16
0.178
0.071
0.013

Ca
0.003
0.000
0.006
0.000
0.002
0.000
0.002
0.000
0.000
0.001
0.000
0.000
0.000
0.000
0.000
0.001
0.001
0.000
0.000
0.000
0.003
0.001
0.001
0.002
0.003
0.000
0.001
0.001
0.005
0.001
0.001
0.001
0.002
0.000
0.001
0.003
0.000
0.000
0.003
0.012

Et
0.439
0.427
0.413
0.479
0.320
0.457
0.301
0.308
0.375
0.381
0.345
0.647
0.629
0.497
0.732
0.588
0.590
0.563
0.552
0.239
0.340
0.430
0.267
0.314
0.348
0.265
0.41 1
0.231
0.396
0.359
0.366
0.560
0.600
0.368
0.399
0.308
0.212
0.427
0.424
0.420

G1
0.343
0.239
0.271
0.215
0.180
0.21 1
0.262
0.273
0.192
0.209
0.183
0.206
0.199
0.174
0.223
0.183
0.092
0.142
0.061
0.1 16
0.131
0.078
0.086
0.082
0.139
0.066
0.072
0.090
0.211
0.062
0.161
0.086
0.062
0.130
0.173
0.095
0.019
0.024
0.027
0.131

La
0.981
1.282
1.247
1.273
1.229
1.123
1.155
1.596
1.458
1.293
1.631
1.488
1.246
1.217
1.622
0.978
1.108
1.071
1.110
0.897
0.859
1.144
1.017
0.913
1.038
0.986
0.911
0.982
0.708
0.870
0.918
1.080
0.963
1.068
1.223
0.493
0.498
0.601
0.642
0.254

Pa
0.296
0.310
0.250
0.422
0.399
0.400
0.363
0.329
0.257
0.265
0.284
0.185
0.138
0.146
0.173
0.085
0.077
0.071
0.053
0.162
0.190
0.144
0.150
0.208
0.253
0.179
0.191
0.171
0.206
0.143
0.199
0.089
0.067
0.066
0.071
0.089
0.069
0.096
0.066
0.201

Table 0 l (c

Me 11100 R1

TTTUUUUWWWWVAXXVASSSSTTTTU

EEEEEEEEEEEEEE

EFFFFFFFFFFFFFFFFF

FFFGGGGGG

Table C- 1 (cont’d).

Mc Inoc Rep Time
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121
121

oooomo-n-nm-n-nmm~n~r1~r1~r1Cum-nm-nm-nmrnmmmmmmmmmmmmmmm
r-l—l—lwwwxxxxggggcccc—l—l—l—lmmmwxxxxfigsscccc—i—i—l
Lawn—WN—«thh—JiLDNU—kri-‘bUJNh-bUJN~§mN—&WN—AUJNH«§UJN

pH

4.39
4.41
4.37
4.33
4.31
4.39
4.30
4.25
4.20
4.29
4.31
4.36
4.41
4.38
4.37
4.15
4.27
4.30
4.30
4.20
4.39
4.35
4.30
4.23
4.30
4.36
4.29
4.33
4.34
4.38
4.28
4.32
4.31
4.32
4.32
4.72
4.60
4.67
4.59
4.57
4.48

Dmr
100.74
100.71
101.43
100.77
100.22

79.79
101.74

99.53
101.67
100.34
102.37
101.31
101.26
100.48
100.58

99.61

99.02
100.50
100.86
101.77
100.11
100.79
100.48
100.81
101.34

99.72

99.99
100.36
100.14
100.16
100.49
101.53

99.54
100.18

99.98

98.84
100.11

98.81
101.00
100.86

99.96

Ym

0.00
0.00
1.73
0.00
0.00
0.00
0.00
0.40
0.00
1.00
0.71
0.00
0.00
0.70
0.70
3.37
2.40
3.89
1.06
0.00
3.01
0.00
2.32
0.00
0.00
0.00
1.48
0.00
0.00
1.06
0.00
0.00
0.88
1.19
0.00
3.07
2.08
3.76
0.00
2.72
0.00

Ac
0.365
0.407
0.326
0.440
0.388
0.267
0.474
0.293
0.375
0.391
0.337
0.236
0.267
0.139
0.189
0.115
0.132
0.189
0.197
0.165
0.203
0.218
0.233
0.302
0.212
0.346
0.334
0.202
0.218
0.213
0.344
0.126
0.178
0.169
0.171
0.132
0.086
0.102
0.170

Bu
0.099
0.117
0.066
0.031
0.044
0.016
0.031
0.029
0.043
0.025
0.022
0.098
0.161
0.057
0.117
0.000
0.014
0.000
0.019
0.000
0.000
0.000
0.016
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.027
0.020
0.024
0.054
0.000
0.001
0.000
0.000

0.130 0.000
0.160 0.000

174

Ca
0.003
0.004
0.000
0.010
0.003
0.010
0.007
0.000
0.004
0.004
0.000
0.006
0.004
0.009
0.013
0.004
0.006
0.014
0.009
0.005
0.009
0.013
0.009
0.005
0.005
0.01 1
0.007
0.006
0.008
0.010
0.010
0.005
0.005
0.008
0.007
0.023
0.014
0.018
0.023
0.017
0.01 1

Et
0.393
0.486
0.236
0.400
0.372
0.413
0.359
0.198
0.323
0.296
0.236
0.440
0.428
0.345
0.325
0.217
0.290
0.229
0.283
0.158
0.088
0.189
0.215
0.226
0.140
0.234
0.358
0.128
0.209
0.143
0.219
0.199
0.241
0.328
0.447
0.484
0.165
0.342
0.337
0.353
0.243

O]
0.024
0.039
0.021
0.082
0.022
0.145
0.057
0.016
0.014
0.035
0.023
0.065
0.038
0.077
0.077
0.033
0.055
0.124
0.071
0.049
0.077
0.1 15
0.065
0.039
0.040
0.105
0.052
0.063
0.079
0.091
0.080
0.023
0.045
0.063
0.057
0.089
0.039
0.018
0.104
0.086
0. 102

La
0.705
0.469
0.877
0.337
0.443
0.409
0.748
0.825
1.131
0.816
0.437
0.674
0.698
0.637
0.793
0.787
0.644
0.641
0.864
1.001
0.345
0.451
0.769
0.733
0.458
0.216
0.708
0.315
0.364
0.357
0.496
0.626
0.690
0.902
1.073
0.549
0.424
0.404
0.400
0.362
0.274

Pa
0.124
0.168
0.126
0.241
0.164
0.319
0.230
0.134
0.147
0.189
0.125
0.115
0.101
0.098
0.130
0.038
0.053
0.035
0.085
0.112
0.127
0.173
0.164
0.166
0.148
0.183
0.220
0.135
0.154
0.198
0.181
0.055
0.088
0.089
0.110
0.040
0.009
0.017
0.156
0.133
0.147

Table C-1 (cont’d).

Mc Inoc Rep Time

03070000600000wwwwwwwwmw>>>>>>>>>>>>>>>ooooooaoa

xééééccccdfifiawwmwxxxsegcccaeawwmxxxgggccc

1

i—hum—Ammu—war—Awmt—WN—wN—WN—uNHwN—WN—th—WN

121
121
121
121
121
121
121
121
121
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123

pH Dmr Ym Ac

4.63
4.53
4.55
4.53
4.55
4.49
4.70
4.64
4.62
3.69
3.76
3.72
3.80
3.74
3.68
3.71
3.72
3.82
3.72
3.78
3.70
6.95
3.80
3.72
4.10
4.07
4.16
4.18
4.08
4.05
4.04
4.23
4.07
3.97
4.04
4.12
3.96
4.10
4.01
3.99
4.04

100.60
101.82
99.60
101.85
101.50
133.93
100.26
100.37
100.86
99.37
100.28
99.03
101.68
102.35
100.43
100.69
99.87
100.52
99.79
100.00
99.90
101.44
101.91
99.26
97.91
98.67
100.67
99.41
98.03
99.35
99.34
100.17
98.11
98.53
98.77
99.61
97.67
99.44
98.73
101.48
97.04

0.00
0.00
0.00
0.00
0.00
0.89
0.00
0.89
1.20
5.89
7.47
5.84
6.39
6.07
4.42
5.84
5.58
6.14
5.75
6.38
5.08
8.08
6.43
5.00
0.34
0.82
0.95
0.82
0.94
0.35
2.23
0.00
0.34
0.64
0.64
0.34
0.00
0.95
0.34
0.00
0.00

0.224
0.145
0.141
0.179
0.244
0.161
0.187
0.135
0.126
0.21 1
0.028
0.182
0.213
0.207
0.176
0.213
0.200
0.210
0.212
0.154
0.168
0.000
0.195
0.290
0.299
0.271
0.284
0.364
0.388
0.229
0.308
0.358
0.401
0.301
0.286
0.390
0.207
0.344
0.384
0.290
0.297

175

Bu
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.004
0.000
0.000
0.000
0.000
0.002
0.002
0.001
0.002
0.000
0.000
0.002
0.000
0.001
0.002
0.000
0.003
0.242
0.273
0.302
0.321
0.185
0.193
0.226
0.271
0.253
0.189
0.254
0.303
0.147
0.270
0.189
0.1 18
0.260

Ca
0.024
0.017
0.018
0.013
0.012
0.014
0.012
0.01 1
0.006
0.006
0.004
0.002
0.005
0.002
0.003
0.005
0.002
0.002
0.002
0.002
0.002
0.002
0.003
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Et
0.335
0.265
0.189
0.248
0.322
0.253
0.443
0.443
0.217
0.364
0.135
0.343
0.326
0.359
0.356
0.433
0.330
0.225
0.343
0.310
0.309
0.000
0.285
0.301
0.559
0.752
0.572
0.698
0.565
0.649
0.739
0.715
0.618
0.695
0.677
0.733
0.671
0.761
0.759
0.589
0.733

01
0.135
0.019
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.305
0.218
0.241
0.108
0.263
0.251
0.267
0.257
0.1 18
0.288
0.232
0.207
0.190
0.261
0.21 1
0.355
0.384
0.416
0.410
0.321
0.356
0.395
0.356
0.394
0.341
0.401
0.406
0.369
0.404
0.402
0.315
0.362

La
0.506
0.426
0.369
0.598
0.810
0.381
0.725
0.652
0.463
3.937
3.216
3.381
3.239
3.307
3.806
3.705
3.603
2.854
3.771
2.813
3.560
0.272
3.131
3.478
2.029
2.353
2.113
2.082
2.119
2.149
2.273
1.630
2.411
2.544
2.395
2.180
2.579
2.202
2.448
2.200
2.326

Pa
0.231
0.184
0.199
0.195
0.192
0.130
0.036
0.013
0.019
0.191
0.166
0.159
0.157
0.155
0.184
0.176
0.168
0.134
0.185
0.121
0.166
0.147
0.143
0.164
0.313
0.324
0.328
0.315
0.332
0.288
0.313
0.299
0.357
0.293
0.320
0.314
0.287
0.322
0.332
0.273
0.31 l

kaC

CCCCCCCCCCCCCCCCCCCCC

Table 01 (cont’d).

Mc Inoc Rep Time

nononnnnnnnnnnnnnonnnnnnonnnnnnonononnwww

xxgzggccccaaaawmmwxxxxggsgCCCCHHHHmwwwxxx

O‘UIOOQQLIIOOQO‘MOOQOMOOQOUI-P-WNF—#WNl-‘AUJNH-RUJNF—#UJNC—AWN

123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123

pH

4.00
4.11
4.09
4.24
4.36
4.22
4.27
4.12
4.23
4.12
4.25
4.08
4.07
4.09
4.18
4.00
4.35
4.05
4.26
4.20
4.28
4.21
4.22
4.30
4.28
4.27
4.25
4.23
4.27
4.19
4.22
4.20
4.21
4.21
4.20
4.20
4.22
4.20
4.22
4.30
4.31

Dmr

99.16
99.42
99.53
98.74
96.84
95.65
97 .51
97 .74
97.14
96.43
98.27
96.42
96.49
99.07
97 .42
95.79
95.62
96.90
96.89
98.71
96.39
96.92
96.74
97 .43
97 .13
98.03
99.02
98.57
99.70
99.71
85.98
98.71
98.23
98.1 1
97.73
97.69
98.03
98.81
95.88
96.82
98.04

Ym

1.85
0.34
0.00
0.38
3.85
0.98
3.93
1.16
0.00
0.32
0.00
0.85
0.98
0.38
0.38
1.22
0.68
0.37
0.68
1.23
0.37
0.00
0.37
1.72
3.93
1.50
3.63
0.00
1.47
1.16
3.58
0.99
0.00
1.29
3.24
1.16
0.38
1.96
1.55
2.73
4.21

Ac
0.368
0.270
0.251
0.247
0.105
0.199
0.360
0.314
0.489
0.394
0.459
0.325
0.278
0.450
0.503
0.354
0.621
0.472
0.608
0.397
0.402
0.365
0.484
0.125
0.140
0.179
0.060
0.201
0.253
0.233
0.290
0.249
0.236
0.247
0.233
0.250
0.353
0.216
0.265
0.219
0.213

176

Bu
0.217
0.253
0.224
0.157
0.101
0.208
0.1 15
0.030
0.040
0.029
0.024
0.008
0.008
0.016
0.019
0.023
0.010
0.024
0.014
0.150
0.121
0.156
0.115
0.109
0.055
0.022
0.013
0.061
0.000
0.014
0.004
0.010
0.017
0.01 1
0.006
0.015
0.007
0.000
0.003
0.062
0.078

Ca
0.000
0.000
0.000
0.001
0.003
0.002
0.003
0.000
0.003
0.000
0.003

Et
0.705
0.633
0.649
0.478
0.176
0.546
0.528
0.478
0.395
0.555
0.430

0.000 0.388

0.003

0.352

0.000 0.460

0m2
0M1
0m3
owl
omz
0W1
omz
owl
omz
0M1
mm
mm
0m1
0m1
omz
owl
mm
mm
0m3
mm
oml
mm
owl
0m1
owl
0W1
mm

0.330
0.297
0.471
0.468
0.476
0.433
0.642
0.536
0.620
0.323
0.425
0.332
0.205
0.258
0.271
0.250
0.291
0.179
0.235
0.138
0.143
0.131
0.323
0.293
0.280
0.383
0.389

G1
0.412
0.395
0.353
0.185
0.176
0.237
0.261
0.158
0.213
0.195
0.194
0.126
0.119
0.173
0.204
0.145
0.208
0.190
0.195
0.171
0.209
0.186
0.215
0.120
0.133
0.117
0.081
0.120
0.158
0.146
0.042
0.103
0.114
0.104
0.115
0.090
0.108
0.114
0.090
0.101
0.117

2.769
2.247
2.013
1.094
0.698
1.890
1.548
1.253
1.389
1.469
1.301
0.922
0.968
1.206
1.408
1.048
1.130
1.617
1.264
0.685
0.809
0.903
1.039
0.841
1.032
1.028
0.987
0.773
0.975
1.081
0.936
1.002
1.1 13
1.082
1.102
0.882
0.960
1.017
1.026
0.758
1.009

Pa
0.322
0.328
0.276
0.191
0.090
0.190
0.132
0.315
0.327
0.390
0.269
0.343
0.299
0.455
0.401
0.261
0.291
0.352
0.324
0.157
0.150
0.170
0.187
0.065
0.068
0.064
0.053
0.134
0.186
0.133
0.187
0.205
0.221
0.178
0.146
0.148
0.211
0.159
0.184
0.070
0.084 '

Table 1

VA ) ‘1‘ ‘0

C

M

CCEEEEEEEEEEE

Table C-1 (cont’d).

Mc Inoc Rep Time

mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmoo

xxfifisficccc“3"3—3”3mmV’V’xxxxgfififiCCCCH—JH—lmmmmxx

7

NH-BUJND—AMNHDMNh—AUNh-hUJNh-ANNF—hUJNl—hWNU-‘hUJNi-‘m

123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123
123

pH

4.20
4.22
4.39
4.49
4.43
4.37
4.41
4.40
4.39
4.36
4.31
4.30
4.38
4.30
4.24
4.20
4.27
4.30
4.36
4.41
4.38
4.41
4.18
4.27
4.35
4.31
4.21
4.43
4.38
4.35
4.25
4.32
4.38
4.33
4.31
4.34
4.38
4.31
4.33
4.31

Dmr Ym Ac

97.13
97.99
99.25
99.91
97.99
99.69
97.84
101.30
99.36
97.44
98.74
99.01
98.88
100.05
98.34
98.13
98.03
96.23
98.42
98.40
99.58
99.26
100.66
100.43
99.69
99.87
98.67
100.21
99.09
100.95
99.95
99.88
101.00
100.86
99.38
99.01
101.20
98.76
99.84
100.99

5.74
4.41
0.00
1.92
0.40
0.00
2.85
0.00
0.88
0.70
0.00
0.00
0.00
1.36
0.70
1.76
0.70
0.88
0.00
0.00
0.00
1.55
3.54
3.13
4.82
1.49
1.63
3.23
0.59
2.01
1.37
1.59
3.03
0.00
1.29
0.59
1.59
0.00
1.37
0.00

0.137
0.236
0.160
0.310
0.300
0.306
0.300
0.348
0.402
0.360
0.467
0.539
0.293
0.439
0.296
0.421
0.414
0.467
0.205
0.278
0.173
0.185
0.139
0.126
0.189
0.216
0.152
0.174
0.237
0.244
0.256
0.180
0.368
0.386
0.223
0.269
0.222
0.357
0.052
0.180

177

Bu
0.038
0.024
0.067
0.137
0.166
0.094
0.024
0.094
0.121
0.070
0.034
0.071
0.027
0.030
0.039
0.048
0.019
0.035
0.083
0.172
0.078
0.099
0.000
0.000
0.003
0.007
0.000
0.000
0.000
0.006
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.002
0.003
0.018

Ca
0.000
0.000
0.008
0.000
0.002
0.005
0.01 1
0.002
0.004
0.003
0.010
0.004
0.012
0.006
0.000
0.003
0.005
0.000
0.005
0.004
0.008
0.007
0.005
0.005
0.016
0.010
0.005
0.010
0.014
0.010
0.005
0.005
0.015
0.012
0.008
0.017
0.013
0.009
0.014
0.006

Et
0.173
0.217
0.280
0.155
0.305
0.408
0.396
0.291
0.373
0.225
0.344
0.354
0.378
0.262
0.145
0.263
0.217
0.342
0.329
0.388
0.364
0.249
0.186
0.241
0.172
0.239
0.146
0.083
0.194
0.156
0.154
0.101
0.196
0.216
0.1 15
0.190
0.104
0.265
0.046
0.165

G]
0.090
0.102
0.1 16
0.021
0.030
0.045
0.169
0.023
0.044
0.026
0.096
0.037
0.198
0.055
0.016
0.015
0.035
0.031
0.059
0.048
0.105
0.066
0.045
0.053
0.133
0.085
0.049
0.077
0.1 17
0.067
0.040
0.039
0.1 17
0.090
0.077
0.118
0.126
0.064
0.014
0.051

La
0.958
0.897
0.678
0.606
0.586
0.848
0.321
0.663
0.505
0.926
0.370
0.592
0.447
0.668
0.881
1.196
0.829
0.612
0.555
0.757
0.807
0.805
0.917
0.612
0.649
0.999
0.848
0.284
0.473
0.827
0.617
0.386
0.254
0.571
0.339
0.473
0.385
0.839
0.246
0.710

Pa
0.065
0.062
0.127
0.089
0.091
0.103
0.220
0.125
0.177
0.140
0.247
0.222
0.346
0.200
0.144
0.155
0.191
0.191
0.081
0.1 13
0.134
0.108
0.046
0.050
0.034
0.097
0.096
0.1 12
0.176
0.166
0.143
0.129
0.209
0.189
0.148
0.198
0.219
0.236
0.018
0.091

Table 01 (cont’d).

Mc Inoc Rep Time

wwwwwwww>>>>>>>>>>>>>>>OQQQQOQQQOQQOQQWW

aaaawwwmxxxgzgcccaaawwmxxxgggcccaaawwwxx

Autoi—AwN—wN—uN—ww~w-w-w-um~w-w-ww~¢w

123
123
123
123
123
123
123
123
123
123
123

~123

123
123
123
123
123
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125

w

4%
4n
“1
4m
4m
m5
4m
4m
4m
4%
4%
4m
4%
M1
4m
4M
4M
4%
M1
33
4M
6m
3w
3%
M8
4n
4%
6M
3%
8m
6%
3m
4m
4m
4m
4m
4M
4m
3m
4m

Dmr
99.49
100.04
102.84
101.79
102.93
102.05
102.37
101.59
101.19
101.38
98.04
100.99
100.33
100.22
100.00
99.46
99.56
100.12
96.04
101.13
98.08
98.66
100.07
102.16
100.46
100.70
101.02
97 .79
101.31
94.88
98.02
101.96
99.96
99.88
97.74
101.42
101.43
100.95
99.70
98.34

Ym

0.00
0.59
3.45
2.90
3.88
1.60
2.38
1.90
1.20
1.38
2.59
1.20
0.90
1.50
1.38
0.89
0.90
7.95
7.54
7.54
8.21
8.36
7.54
7.59
7.55
7.91
7.90
8.26
6.64
8.35
8.26
7.07
0.00
1.83
0.00
1.67
0.00
0.00
0.00
0.00

Ac
0.198
0.062
0.103
0.088
0.107
0.123
0.141
0.222
0.129
0.161
0.145
0.125
0.185
0.215
0.074
0.121
0.149
0.002
0.000
0.000
0.005
0.005
0.132
0.103
0.000
0.000
0.007
0.016
0.106
0.028
0.002
0.060
0.243
0.233
0.232
0.270
0.336
0.179
0.270
0.301

178

Bu
0.015
0.017
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.001
0.000
0.004
0.000
0.004
0.000
0.000
0.000
0.000
0.000
0.005
0.002
0.000
0.206
0.230
0.222
0.245
0.154
0.150
0.206
0.212

Ca
0.010
0.022
0.015
0.015
0.022
0.017
0.021
0.021
0.014
0.016
0.014
0.013
0.021
0.025
0.008
0.022
0.008
0.000
0.002
0.002
0.002

0.002
0.002
0.002
0.003
0.005
0.000
0.001
0.002
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Et
0.331
0.11 1
0.194
0.254
0.199
0.219
0.282
0.258
0.143
0.193
0.119
0.142
0.253
0.275
0.143
0.342
0.196
0.002
0.000

G]
0.092
0.027
0.059
0.056
0.066
0.1 17
0.107
0.181
0.061
0.105
0.059
0.084
0.138
0.125
0.002
0.000
0.000
0.1 14
0.144

0.000 0.192
0.000 0.161
0.002 0.000 0.158

0.175
0.055
0.000
0.007
0.000
0.000
0.071
0.000
0.000
0.039
0.279
0.385
0.383
0.334
0.302
0.334
0.453
0.399

0.224
0.209
0.192
0.175
0.172
0.164
0.197
0.153
0.144
0.179
0.307
0.320
0.315
0.311
0.282
0.284
0.344
0.287

La
1.066
0.433
0.393
0.491
0.439
0.298
0.371
0.398
0.127
0.286
0.171
0.241
0.352
0.358
0.344
0.615
0.656
0.808
0.058
2.566
0.956
0.289
3.461
3.01 1
2.473
1.482
1.783
0.260
3.317
0.025
0.169
2.998
1.793
1.926
1.620
1.580
1.855
1.722
1.990
1.287

Pa
0.1 10
0.040
0.025
0.027
0.030
0.115
0.142
0.213
0.163
0.193
0.203
0.152
0.200
0.200
0.015
0.024
0.035
0.062
0.105
0.152
0.109
0.1 13
0.162
0.152
0.135
0.130
0.137
0.132
0.151
0.015
0.108
0.145
0.264
0.266
0.242
0.245
0.288
0.225
0.265
0.241

Tabl

tote.

BBBBBBBBBBB

BCCCCrKth1ill

Table C-l (cont’d).

Mc Inoc Rep Time

nonnnnonnnonnnnnnnonnnnnnonnnwwwwwwwwwwww

caaaawwmwxxxxggggcccc—l—i—l—immwwxxxxg22scat-Jr:

MOOQONMOOQOKUI-b-UJN—Awwt—AwN~thHAUJN~hUJNHAUJN—#wwr—

125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125

pH Dmr Ym Ac

4.04
3.96
3.98
4.09
3.93
4.11
3.96
4.04
4.00
4.02
4.07
3.95
4.25
4.60
4.28
4.23
4.12
4.19
4.16
4.27
4.11
4.08
4.14
4.20
4.03
4.37
4.08
4.28
4.22
4.30
4.25
4.24
4.31
4.26
4.24
6.28
4.23
4.26
4.20
4.18
4.19

100.16
101.13

99.24
101.94
102.56
101.50

99.97

99.70
102.37
100.55
100.19

99.16
102.52
103.55
100.35
102.12

99.97
101.21
101.55

99.78
102.02
101.89
101.84

99.83
101.05
100.93
102.27
101.61

99.31
102.09
100.81
103.05
102.97
100.08
102.71

96.99

99.55

97.87

98.30
100.24
104.23

0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
0.00
1.36
1.36
0.00
0.40
4.66
0.00
2.18
0.69
3.27
0.69
1.67
0.00
1.99
0.00
0.87
0.98
1.88
0.87
0.40
1.23
1.81
0.39
0.00
0.00
1.36
0.42
1.19
2.91
0.41
0.42
1.37
0.70

0.338
0.250
0.217
0.296
0.155
0.256
0.294
0.250
0.254
0.313
0.237
0.233
0.114
0.010
0.120
0.178
0.229
0.170
0.202
0.153
0.219
0.174
0.246
0.179
0.160
0.188
0.222
0.213
0.104
0.055
0.126
0.102
0.136
0.144
0.156
0.002
0.292
0.256
0.229
0.171
0.264

179

Bu
0.212
0.166
0.201
0.218
0.1 16
0.213
0.163
0.1 15
0.239
0.179
0.228
0.212
0.131
0.073
0.141
0.148
0.051
0.018
0.025
0.027
0.029
0.019
0.010
0.009
0.013
0.009
0.035
0.01 1
0.107
0.177
0.143
0.033
0.136
0.090
0.039
0.015
0.1 13
0.034
0.035
0.004
0.015

Ca
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.007
0.000
0.011
0.001
0.005
0.004
0.005
0.002
0.003
0.002
0.005
0.002
0.006
0.001
0.008
0.000
0.001
0.001
0.000
0.003
0.004
0.000
0.005
0.004
0.004
0.000
0.000
0.003

Et
0.320
0.347
0.307
0.340
0.268
0.349
0.352
0.323
0.328
0.317
0.290
0.389
0.197
0.000
0.200
0.366
0.256
0.124
0.241
0.192
0.126
0.179
0.222
0.114
0.085
0.175
0.175
0.196
0.186
0.326
0.202
0.194
0.116
0.202
0.006
0.000
0.089
0.141
0.086
0.003
0.009

G]
0.326
0.316
0.319
0.309
0.299
0.305
0.321
0.286
0.347
0.349
0.344
0.326
0.198
0.062
0.194
0.279
0.179
0.231
0.081
0.230
0.063
0.1 13
0.067
0.207
0.090
0.285
0.073
0.213
0.172
0.212
0.239
0.185
0.057
0.080
0.041
0.023
0.079
0.109
0.058
0.062
0.074

1.958
2.263
1.893
1.611
2.080
1.660
1.964
1.969
2.213
2.311
1.936
1.855
0.885
0.596
1.313
1.516
1.172
0.647
1.103
0.745
0.968
0.864
0.881
0.845
1.029
0.540
1.125
0.951
0.792
0.942
1.255
0.939
0.886
1.015
0.979
0.289
1.050
0.831
0.982
0.806
0.831

Pa
0.291
0.250
0.246
0.233
0.235
0.238
0.255
0.245
0.282
0.264
0.280
0.253
0.165
0.076
0.131
0.178
0.285 '
0.195
0.275
0.186
0.307
0.284
0.317
0.277
0.224
0.193
0.245
0.229
0.127
0.1 17
0.174
0.121
0.082
0.069
0.059
0.038
0.210
0.194
0.132
0.1 15
0.210 ‘

Table

11c 1.

CCCCCC

Table C-l (cont’d).

Mc Inoc Rep Time
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125

~11~rl~rl~rl~11Cn-nrrlwlrnmmmmmmmmmmmmmmmmmmmmnonnonnoono

cc~1—1—14mmmm><><><><gaggcccc—l—i-i—lmmmmxxxxgagged:
Nr—Awwv—ALAN—ALAND-AwNi—AUJNr-Ari—AUJNr—OOQGUIOOQOUIOOQG

pH Dmr Ym Ac

4.18
4.19
4.20
4.18
4.24
4.18
4.22
4.29
4.36
5.05
4.25
4.40
4.50
4.47
4.39
4.45
4.42
4.41
4.39
4.35
4.35
4.40
4.29
4.26
4.23
4.32
4.34
4.37
4.44
4.41
4.43
4.23
4.31
4.40
4.38
4.28
4.47
4.42
4.40
4.33
4.36

99.46
101.30
101.51
100.27

99.64
101.24
103.60
102.68
101. 13
100.80
100.51
100.20

99.83
100.84

98.68
101.08

99.15
100.40
100.57

99.56

99.65
100.84

97.03
101.21
100.40
102.35
100.54
100.04
100.14
101.01

99.54
100.02

99.41

99.13

99.53

99.88

99.69

98.56

97.71

97.89
100.32

1.59
0.00
0.00
0.71
1.73
0.00
0.71
1.52
0.00
0.71
0.41
4.15
3.97
4.26
1.80
1.20
1.68
2.69
2.06
0.89
0.60
1.37
0.60
3.19
0.59
1.29
0.59
0.77
0.00
0.89
0.00
4.04
3.72
4.08
1.21
1.60
2.50
0.91
0.90
1.21
2.78

0.220
0.231
0.246
0.275
0.305
0.209
0.218
0.130
0.051
0.009
0.135
0.146
0.305
0.281
0.210
0.257
0.315
0.348
0.243
0.513
0.415
0.213
0.295
0.303
0.372
0.297
0.343
0.210
0.206
0.127
0.098
0.132
0.149
0.144
0.171
0.158
0.192
0.204
0.229
0.313
0.235

180

Bu
0.020
0.009
0.005
0.033
0.014
0.021
0.001
0.088
0.104
0.037
0.007
0.073
0.138
0.157
0.058
0.023
0.079
0.101
0.051
0.044
0.052
0.015
0.018
0.037
0.039
0.006
0.022
0.076
0.127
0.063
0.057
0.000
0.009
0.003
0.017
0.004
0.001
0.001
0.005
0.003

Ca
0.004
0.002
0.000
0.002
0.012
0.006
0.002
0.003
0.009
0.004
0.002
0.007
0.003
0.002
0.000
0.012
0.000
0.003
0.001
0.008
0.003
0.01 1

Et
0.094
0.030
0.090
0.037
0.216
0.100
0.065
0.096
0.000
0.000
0.016
0.075
0.132
0.136
0.164
0.270
0.165
0.1 16
0.075
0.224
0.147
0.202

0.000 0.148

0.001
0.003
0.004
0.007
0.007
0.001
0.006
0.003
0.007
0.006
0.009
0.006
0.001
0.010
0.020
0.010
0.006

0.086
0.179
0.088
0.148
0.134
0.240
0.184
0.035
0.098
0.142
0.056
0.054
0.089
0.017
0.103
0.000
0.063

0.004 0.011 0.077

01
0.121
0.082
0.054
0.083
0.234
0.070
0.137
0.068
0.091
0.127
0.039
0.1 19
0.023
0.024
0.029
0.132
0.017
0.040
0.014
0.104
0.026
0.1 19
0.035
0.018
0.012
0.024
0.022
0.049
0.026
0.078
0.033
0.047
0.054
0.098
0.055
0.041
0.072
0.096
0.052
0.046
0.061

La
0.950
0.908
0.724
0.973
0.826
0.895
0.802
0.975
1.140
0.646
0.966
0.712
0.564
0.563
0.576
0.265
0.580
0.455
0.624
0.412
0.458
0.317
0.445
0.887
1.082
0.591
0.440
0.619
0.540
0.569
0.481
0.972
0.771
0.507
0.777
0.950
0.341
0.430
0.778
0.817
0.549

Pa
0.251
0.181
0.159
0.179
0.258
0.166
0.175
0.083
0.095
0.073
0.062
0.128
0.100
0.078
0.060
0.199
0.102
0.156
0.101
0.270
0.171
0.240
0.127
0.146
0.137
0.140
0.129
0.087
0.067
0.093
0.070
0.055
0.060
0.011
0.081
0.104
0.116
0.149
0.148
0.160
0.174 '

Table C-1 (cont’d).

Mc Inoc Rep Time

QQQOOQQQQQQQOQOWWWWWW'UW'U'H

xxxgggcccaaemmmxxxxgggscc

3

WNHMNF—WNHUNHMNH-BWNH-BWN—C-k

125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125
125

pH Dmr Ym Ac

4.43
4.39
4.39
4.38
4.43
4.37
4.41
4.38
4.38
4.37
4.73
4.67
4.75
4.58
4.58
4.52
4.60
4.55
4.62
4.58
4.55
4.52
4.71
4.70
4.68

99.26 0.60
99.94 1.21
98.91 1.78
100.62 0.00
98.44 0.91
99.89 0.69
98.25 0.84
98.98 0.00
98.18 0.94
99.30 0.00
101.32 4.04
101.75 3.72
101.52 4.08
103.27 1.24
99.36 1.61
98.61 2.51
100.26 0.96
99.08 0.95
98.53 1.24
99.50 2.78
99.21 0.70
98.42 1.23
99.28 1.79
100.98 0.00
99.27 0.96

0.307
0.281
0.219
0.152
0.221
0.335
0.175
0.211
0.165
0.166
0.068
0.091
0.053
0.094
0.107
0.154
0.083
0.158
0.121
0.121
0.134
0.138
0.076
0.064
0.078

181

Bu
0.000
0.000
0.000
0.003
0.002
0.002
0.034
0.000
0.003
0.049
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000

Ca
0.012
0.008
0.008
0.004
0.012
0.008
0.006
0.012
0.010
0.024
0.021
0.028
0.042
0.011
0.016
0.017
0.01 1
0.016
0.015
0.019
0.015
0.012
0.016
0.022
0.021

Et
0.087
0.000
0.044
0.024
0.044
0.075
0.045
0.095
0.130
0.157
0.058
0.138
0.000
0.074
0.182
0.1 16
0.038
0.180
0.012
0.096
0.109
0.083
0.060
0.070
0.038

GI
0.102
0.049
0.069
0.033
0.094
0.070
0.038

La
0.234
0.673
0.332
0.646
0.420
0.484
0.777

Pa
0.183
0.201
0.152
0.075
0.21 1
0.156
0.073

0.079 0.396 0.152

0.046
0.059
0.055
0.105
0.044
0.083
0.081
0.116
0.048
0.117
0.084
0.101
0.131
0.072
0.006
0.000
0.000

0.842
1.039
0.326
0.602
0.386
0.237
0.297
0.279
0.088
0.236
0.138
0.260
0.265
0.234
0.320
0.340
0.357

0.094
0.1 14
0.017
0.036
0.020
0.082
0.101
0.135
0.104
0.186
0.168
0.151
0.136
0.133
0.012
0.013
0.016

Tab

Adi-I‘ll,-

Table 02. Data used for analyses in chapter 3- temperature during aerobic stability test.

Mc Inoc temp0 templ

meowcommunalwwwwwwwwwwww>>>>>>>>>>>>>>>

><><><><€€€SCCCCH—i—l—ammmmxxxéééccc—l—l—immm

Rep

p...

hWNh—hb-IN—bWNW-bulb)!—hWNHWNi—WNHWN—MNHDJN

18.33
17.78
19.44
18.33
17.22
17.78
17.78
20.00
18.89
17.78
19.44
18.89
19.44
18.89
18.89
22.22
20.56
22.78
21.67
21.67
21.67
22.22
20.56
21.67
21.67
21.11
23.33
21.67
21.67
21.67
21.67
21.67
21.11
20.00
21.67

19.44
18.61
20.56
20.00
19.17
18.89
18.89
20.56
19.44
19.44
19.72
19.17
29.44
19.44
19.72
20.83
19.44
20.83
19.72
20.00
20.28
20.56
19. 17
20.28
20.28
19.44
21.67
20.28
20.28
19.72
20.28
19.44
19.72
18.61
20.00

18.61
18.61
19.44
19.44
17.78
18.33
18.33
20.56
18.89
19.44
19.72
19.44
26.67
18.89
19.17
20.00
18.89
20.00
19.17
19.44
19.44
19.72
18.61
19.44
19.44
19. 17
20.83
19.44
19.44
19.17
20.00
18.89
18.89
18.06
19.44

temp2 temp3

18.33
21.11
19.44
18.33
17.78
17.22
17.78
19.44
17.78
18.33
19.44
18.33
21.67
18.89
18.33
20.00
18.89
20.28
19.44
19.44
19.72
20.00
18.61
19.44
19.72
19.17
20.83
13.89
19.17
19.17
19.72
18.89
18.61
18.06
19.17

18.89
26.67
19.44
21.11
23.33
17.78
17.78
21.11
18.89
18.33
35.00
18.33
23.89
37.78
17.78
20.28
19.17
20.00
19.72
19.44
19.44
20.28
18.89
19.44
19.44
19.44
21.11
19.44
19.44
18.89
20.00
19.44
18.89
18.33
19.72

temp4 temp5

29.44
25.56
28.89
24.72
29.44
20.83
22.22
28.06
25.56
23.61
26.39
21 .94
27.22
26.39
23.06
20.56
19.44
20.56
20.56
20.00
20.00
20.56
19.44
20.00
19.44
19.44
21 .67
20.56
20.00
19.44
20.56
20.00
19.44
18.89
20.00

Abbreviations used: Mc-moisture content, Inoc-inoculant, Rep-replication, temp0-
temperature at d0, templ- temperature at d1, temp2- temperature at d2, temp3-‘
temperature at d3, temp4- temperature at d4, d5- temperature at d5.

182

 

Mc Inoc

Mc Inoc

O00OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

><><><><€2€2cccc1—l-4—l—lmwmmxxxx2sszcccc—lr-i—l—lmmmm

Rep

fl

OO\)O\UIOO\IO\UIOO\IO\UIOO\IO\LAOO\IO\LIIJ>UJNI—hWN—C$WNC-‘4BWNi-C-RUJN

26.11
24.44
24.44
23.33
23.89
25.00
23.89
25.00
24.44
25.00
25.00
25.00
25.00
25.00
26.67
26.11
24.44
25.00
26.67
26.11
24.44
23.89
23.89
25.00
23.89
23.89
23.89
22.78
24.44
23.89
23.33
25.00
23.89
23.89
22.78
23.89
23.89
22.78
22.78
25.00

tempO templ

23.89
23.89
22.78
22.78
22.78
23.89
22.78
23.89
23.33
23.89
23.89
23.33
23.33
23.89
25.00
24.44
23.33
23.33
25.00
25.00
26.39
25.56
26.11
27.22
25.83
25.56
25.83
24.17
26.11
25.56
25.28
27.50
25.56
25.83
24.44
26.39
25.56
25.00
25.56
26.94

temp2

20.56
21.11
20.56
19.44
20.00
21 .11
19.44
21.11
20.00
20.00
21.11
20.00
20.00
20.56
21 .67
21 .11
20.56
20.56
21.11
21.11
25.56
25.00
25.56
26.11
25.56
25.00
25.00
23.89
25.56
25.00
24.44
26.67
25.00
25.00
23.89
25.56
25.00
24.44
25.00
25.56

183

temp3

23.89
23.89
23.06
22.78
23.06
23.61
22.50
23.89
24.44
23.33
24.17
23.33
23.33
23.61
24.72
23.89
23.06
23.89
24.72
24.44
24.44
24.17
24.44
28.33
23.61
23.89
24.44
21 .67
23.33
23.61
23.33
25.28
23.61
24.44
23.89
24.72
23.61
23.06
25.00
24.72

25.56
25.00
23.89
23.89
25.00
25.00
23.89
25.00
23.33
24.44
25.00
25.00
23.89
24.44
25.00
26.1 1
23.89
24.44
25.00
26.1 1
27.22
26.67
27.22
30.56
27.22
26.67
27.22
25.56
27.22
26.67
26.67
28.33
26.67
26.67
26.67
28.33
26.67
26.67
27.78
28.33

temp4 temp5

25.56
26.67
27.78
25.00
25.00
25.56
23.89
26.11
24.44
24.44
26.11
25.56
25.00
25.00
26.11
26.67
24.44
24.44
26.11
26.67
27.22
27.78
26.67
29.44
26.67
26.67
27.22
24.44
26.67
26.67
26.11
28.33
27.22
27.22
25.56
26.67
27.22
26.67
28.33
28.89

E

CLEEtll...

Mc Inoc Rep

~n~n~n~n~nCnCnCnCnCnCn-nm~11~11Corn-r1mmmmmmmmmmmmmmmmmmmmmm

><><><><2ééficccc—l—i—i—lmmmmxxxxfiéfiéccccafiflammmm

1

AWN-d-bWNH-hLANt-iAli-INF—AMNHAMNH-kLRNH-D-WNt—QWNHJBWN

temp0

23.33
24.44
24.44
23.33
21.67
23.89
22.78
23.89
22.78
22.78
24.44
22.78
23.33
23.89
23.33
23.33
21.67
23.89
22.22
25.00
21.11
21.11
20.56
22.22
21.11
20.56
21.67
21.11
21.11
21 .67
21.11
21.11
21.67
21.67
22.22
21.11
21.67
21.67
21.67
21.1 1

templ

20.56
20.83
20.28
21.39
20.00
20.56
21.11
21.67
20.83
21.11
20.28
20.00
20.00
21.39
20.56
21.39
20.00
21.11
20.83
21.67
20.28
20.00
20.56
22.22
20.28
20.00
20.83
20.56
21.11
21.39
21 .11
20.83
21.67
21.11
22.22
21.11
21.39
21.39
20.83
21.11

20.00
20.56
20.00
21.67
20.56
20.00
20.56
21.67
21.11
21.11
18.89
20.56
18.89
21 . 11
21.11
21.67
20.00
20.00
21.11
20.56
19.72
20.28
20.56
21.39
21.11
19.72
21.11
20.56
20.83
21.39
20.83
19.72
21.67
21.67
22.22
20.83
21.39
21 .67
20.56
21 .67

184

temp2 temp3

19.17
20.56
21 .11
22.22
20.56
19.72
20.00
21.94
20.83
20.28
21.11
20.56
20.00
21.11
19.44
22.22
20.28
21 .94
21.11
20.00
19.44
19.44
19.44
20.56
20.00
19.44
20.00
19.44
21.11
21.1 1
20.00
19.44
20.00
21.11
21.67
19.44
20.56
20.56
20.00
21.1 1

18.89
20.56
20.56
22.22
20.56
18.89
20.00
21.11
20.56
20.56
20.56
20.56
20.00
21.11
18.33
22.22
20.00
22.22
20.56
19.44
19.44
19.44
19.44
20.56
20.00
18.89
19.44
19.44
20.00
21.11
19.44
19.44
20.00
21.11
21 .67
21.11
21.11
20.56
20.00
20.56

temp4 temp5

20.00
21 .11
22.22
22.22
21.11
20.00
20.56
22.22
21.11
21.11
21.67
21.67
20.56
21.11
20.56
22.22
20.56
22.78
21.67
20.56
20.56
20.00
20.00
21.11
20.00
18.89
20.00
19.44
20.56
21.11
20.00
19.44
20.56
21 .11
21 .67
21.11
20.56
20.56
20.00
20.56

Mc 1110c

SSSTTTIIIIlll

GGGGGGGGGGGGGGG

Mc Inoc

OOQQOOOQOQQQQOQ

XXXéfiéCCCHHHmmr/a

Rep

)—

MN—CMNHLfiN—MNHWN

22.22
21.67
21.11
22.22
21.11
21.67
21.67
21.11
21.67
21.67
22.22
21.67
22.22
22.22
22.22

tempO templ

21.94
21.39
21.11
21.94
21.11
21.94
20.56
21.11
21.39
22.22
22.22
21.39
22.22
21.39
21.94

temp2

22.78
21.67
21.67
22.78
21.67
22.22
21.67
21.67
21.11
22.22
21.67
21.67
22.22
22.22
22.22

185

temp3

20.83
21.11
21.11
20.56
21.11
21.94
20.28
20.56
21.39
21.39
22.22
21.11
20.83
20.56
21.11

temp4

22.22
21.67
21.11
21.67
21.11
21.67
21.67
21.11
21.11
21.11
22.22
21.11
21.67
21.11
21.67

temp5

20.56
22.22
21.11
21.67
21.11
21.67
20.00
21.11
20.56
20.56
21.67
21.11
20.56
21.11
21.11

APPENDIX D

Data Used for Analyses in Chapter 4

186

[I‘ll

Table D-l. Data used for analyses in chapter 4'.

Inoc Temp Time Rep Ace Mal Prop Suc OD pH
DH42 30 20 1 0.082 0.019 0.294 0.000 1.70 4.72
DH42 30 20 2 0.086 0.016 0.298 0.000 1.70 4.72
DH42 30 20 3 0.081 0.020 0.292 0.000 1.70 4.72
DH42 40 20 1 0.308 0.062 0.485 0.021 1.43 4.95
DH42 40 20 2 0.061 0.047 0.279 0.006 1.54 4.78
DH42 40 20 3 0.055 0.048 0.270 0.005 1.55 4.78
SHER 30 20 1 0.023 0.092 0.052 0.028 0.82 5.81
SHER 30 20 2 0.021 0.094 0.051 0.028 0.80 5.73
SHER 3O 20 3 0.015 0.090 0.043 0.027 0.79 5.84
SHER 40 20 1 0.002 0.093 0.008 0.009 0.11 6.09
SHER 40 20 2 0.007 0.000 0.011 0.011 0.11 5.96
SHER 40 20 3 0.007 0.000 0.009 0.010 0.13 5.91
DH42 30 40 1 0.092 0.014 0.325 0.007 1.63 4.64
DH42 30 40 2 0.096 0.014 0.330 0.002 1.62 4.64
DH42 30 40 3 0.101 0.014 0.332 0.002 1.63 4.64
DH42 40 40 1 0.077 0.019 0.374 0.000 1.50 4.66
DH42 40 40 2 0.072 0.018 0.364 0.000 1.49 4.66
DH42 40 40 3 0.079 0.019 0.376 0.000 1.50 4.66
SHER 30 40 1 0.054 0.000 0.156 0.026 1.38 5.25
SHER 30 40 2 0.047 0.000 0.152 0.025 1.39 5.24
SHER 30 40 3 0.051 0.000 0.154 0.026 1.38 5.26
SHER 40 40 1 0.009 0.096 0.016 0.020 0.16 5.91
SHER 40 40 2 0.009 0.096 0.014 0.026 0.19 5.87
SHER 40 40 3 0.012 0.099 0.014 0.024 0.18 5.89
DH42 30 72 1 0.104 0.014 0.349 0.005 1.61 4.60
DH42 30 72 2 0.102 0.014 0.347 0.005 1.62 4.64
DH42 30 72 3 0.106 0.014 0.351 0.002 1.62 4.60
DH42 40 72 1 0.078 0.018 0.385 0.000 1.57 4.65
DH42 40 72 2 0.079 0.017 0.388 0.000 1.57 4.65
DH42 40 72 3 0.081 0.018 0.392 0.000 1.59 4.64
SHER 30 72 1 0.081 0.000 0.280 0.021 1.58 4.95
SHER 30 72 2 0.076 0.000 0.276 0.024 1.59 4.95
SHER 30 72 3 0.085 0.093 0.283 0.021 1.58 4.95
SHER 40 72 1 0.016 0.096 0.039 0.034 0.23 5.83
SHER 40 72 2 0.017 0.098 0.027 0.033 0.30 5.81
SHER 40 72 3 0.016 0.096 0.034 0.034 0.26 5.79

'Abbreviations used: Inoc-inoculant, temp-temperature, rep-replication, ace-acetic
acid, mal-malate, prop-propionic acid, sue-succinic acid, OD-optical density,
SHER-P. shermanii

187

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