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ABSTRACT

EVALUATION OF THE ANAEROBIC MICROORGANISMS
AND THEIR METABOLISM IN AN ANOXIC LAKE BASIN

BY
John Joseph Molongoski

Strict anaerobic culture techniques were employed
to quantitatively and qualitatively evaluate the anaer-
obic heterotrOphic bacteria present at the sediment-
water interface of Wintergreen Lake, a hypereutrOphic
hardwater basin. Anaerobic viable counts remained
constant from March through December, 1973, ranging
from 2.h to 5.7 x 106 organisms per gram dry weight of
sediment. These isolatable bacteria undoubtedly
represented a small percentage of the total microbial
community of these sediments, which was shown by direct
microsc0pic counting techniques to be 2.0 x 1011
organisms per gram dry weight of sediment.

Bacteria of the genus Clostridium dominated the
sedimentary isolates obtained, accounting for 71.8%
of the 960 organisms examined. A single species,
Clostridium bifermentens, comprised 47.7% of the total.
Additional bacterial groups and the percentage in which
they were isolated included: Streptococcus s . (10.8%),
Vibrio s . (9.5%), Gram-positive nonsporing rods (5.6%),
and motile Gram-negative rods (1.9%).

Considered as a whole, the isolates examined were

/‘\
(Igcharacterized by a high percentage of obligate anaerobes
R) (85.7%) as compared to the number of facultative bacteria
to obtained (13.9%), and by the strikingly limited hydro-
lytic capabilities of these facultative organisms. The
obligate anaerobes exhibited strong proteolytic, but
weak saccharolytic capabilities. Temperature growth
studies demonstrated the ability of all the isolates
to grow at 10°C.

Gas-liquid-radiochromatography was employed to
determine the gaseous and soluble metabolic endproducts
produced from lL’C-labeled glucose and an amino acid
mixture by representative sedimentary clostridial
isolates and by a natural sediment microbial community.
At $2.§222 incubation temperatures, the natural sediment
microflora produced soluble fermentative endproducts
characteristic of those elaborated by the clostridial
isolates tested. These results were considered strong
presumptive evidence that clostridia are active in the

decomposition of organic matter in the sediments of

Wintergreen Lake.

EVALUATION OF THE ANAEROBIC MICROORGANISMS
AND THEIR.METABOLISM IN AN ANOXIC LAKE BASIN

By
John Joseph Molongoski

A THESIS

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

MASTER OF SCIENCE

Department of Microbiology and Public Health

1971+

to my parents

ii

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to
Dr. Michael J. Klug for his advice, encouragement, and
understanding patience during the course of this investi-
gation. Appreciation is also due to the members of my
guidance committee, Dr. James M. Tiedje, Dr. Robert L.
Uffen, and Dr. Robert G. Wetzel. I am grateful to Dr.
George H. Lauff, Director, Kellogg Biological Station,
Michigan State University, for financial assistance and
institutional support.

Special thanks are due to Mr. R. E. VanDeusen and
Mr. Joe Johnson for permission to study Wintergreen Lake.
A special note of appreciation is extended to Mr. Art
Wiest for his invaluable aid in the construction of field
equipment used in this study. The generous assistance of
Karen Hogg and the staff of the Microbiology Laboratory
at W. K. Kellogg Biological Station is gratefully acknow—
ledged.

These investigations were supported, in part, by the
National Science Foundation Grant 80-15665 to G. H. Lauff
‘gg.al. (Coherent Areas Program for Investigation of
Freshwater Ecosystem), and by financial assistance pro—
vided by the Department of Microbiology and Public Health,

Michigan State University.

iii

TABLE OF CONTENTS

Pag
INTRODUCTION......................................... 1
MATERIALS AND METHODS................................ 7

Study Site...................................... 7
Temperature..................................... 7
Dissolved Oxygen................................ 8

U)

Bacteriological Sampling Procedures.............
Characterization Of Bacteria.................... 13
Metabolism Of Labeled Organic Compounds By
Selected Bacterial Isolates..................... l8
Metabolism Of Labeled Organic Compounds By A
Natural Sediment Microbial Community............ 21
RESULTS.............................................. 24
Quantitation Of Anaerobic Bacteria At The
Sediment-water Interface........................ 24
Temperature Of The Sediment—Water Interface..... 24
Major Groups Of Anaerobic Heterotrophic Bacteria
Present At The Sediment-Water Interface......... 28
Metabolism Of Labeled Organic Compounds By
Selected Bacterial Isolates..................... 34
Metabolism Of Labeled Organic Compounds By A
Natural Sediment Microbial Community............ 40
DISCUSSION........................................... 45
Organic Inputs To The Lake...................... 45
Density Of Anaerobic Bacteria At The Sediment-

Water Interface...0000000000000.0000000000000000 #6

iv

Page
Anaerobic Bacteria Isolated From The Sediment-
Water Interface............................... 49
Anaerobic Metabolism 0f Labeled Organic Sub-
strates By Clostridial Isolates And By
Sediment Microbial Communities................ 53

LITERATURE CITED.OOOOOOCOOOOOOOOOOOOO00.00.000.000. 58

LIST OF TABLES

Table Page

1 Composition of anaerobic dilution fluid
utilized under different gas atmospheres.... 10

2 Composition of the media utilized in the
isolation of anaerobic bacteria from
Wintergreen Lake sedimentSoooooooooooooooooo ll

3 Columns and conditions for the analysis
of soluble and gaseous metabolic end-
products by gas-liquid and gas-solid
Chromatography.oooooooooooooooooooooooococo. l7

4 Composition of 14C (U) -L- amino acid
mixture utilized in labeling experiments.... 20

5 Presumptive identification features and
distribution of bacterial groups isolated
from Wintergreen Lake sediments............. 29

6 Fermentation and additional physiological
characteristics of isolated sediment

StrainSooooo00000000000000.0.000000000000000

30

7 Hydrolytic characteristics of isolated
Wintergreen Lake sediment strains........... 33

vi

Figure

LIST OF FIGURES
Page

Characterization scheme utilized in the
identification of isolated sedimentary

anaerObeSOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO0... 15

Anaerobic plate counts from the sediment-
water interface of Wintergreen Lake,
February through December, 19730000000000.000 25

Depth-time diagram of the distribution of
dissolved oxygen (mg/l) in Wintergreen
Lake, March through December, l973........... 26

Sediment-water interface temperature at a
depth of 6 meters in Wintergreen Lake,
MarCh through December, 19730000000000.000000 27

Gas-radiochromatogram of 1l”CO produced by
clostridial isolates grown in the presence

of uniformly labeled 4C amino acid mixture
(superimposed upon a chromatographic scan

or a C02 Standard).0.0000000000000000...coco. 35

Gas-iiquid-radiochromatogram of soluble

fatty acids produced by‘Q. bifermentens

grown in 1% FY containing l.n 1

1’0 (U) -1..- aminO aCid mimureooooooooooooooo 36

Gas-liquid-radiochromatogram of soluble

fatty acids produced by C. s oro enes grown
in 1% PY containing 1 uCIVmI Inc (U) -L-
amino acid mixture coo...0.000000000000000... 37
Gas-1iquid-radiochromatogram of soluble
fatty acids produced by.Q. bifermentens
rown in 1% PYG containing .u m
%U)-D— glucose.oo.cocoon...0.000000000000000. 38

Gas-1iquid-radiochromatogram of soluble
fatty acids produced by C. s oro enes grown
in 1% PYG containing I‘MEfi/EI 13% (U) -D-

glucoseoocoono...ocoooooooooooooooooooooooooo 39

vii

10

11

12

Gas-radiochromatogram of thH and 1“002
produced by sediment microflo a

incubated in the presence of uniformly
labeled hC amino acid mixture and 140
glucose respectively (superimposed upon

a chromatographic scan of a standard
mixture or CH]... and 002)....o................

Gas-liquid-radiochromatogram of soluble
fatty acids Eroduced by sediment micro-
flora from 1 C (U) -L- amino acid mixture
(superimposed upon a chromatographic scan
of a 0.1% standard mixture of short-chain
fatty aCidS)ooooooooooooo00.000000000000000.

Gas-1iquid-radiochromatogram of soluble
fatty acids produced by sediment micro-
flora from 1 C (U) -D— glucose (superim-
posed upon a chromatographic scan of a
0.1% standard mixture of short-chain

fatty aCidSoooocoococo-00.000000000000000coo

viii

41

42

43

INTRODUCTION

The importance of anaerobic heterotrophic metabolism
in lacustrine systems is emphasized by its involvement in
all the major cycles of matter in natural waters (Ruttner,
1963; Deevey, 1970). Typically, lake sediments are recog-
nized as having negative oxidation-reduction potentials as
a consequence of the low solubility and diffusion of oxygen
in water and its utilization by aerobic and facultatively
anaerobic microorganisms in the water column and sediments
(Brock, 1966). In the sediments, molecular oxygen is ex-
hausted as an electron acceptor and anaerobic metabolism
commences with the reduction of materials present in a
highly oxidized state such as nitrate, ferric iron, and
sulfate (Wetzel‘gg‘gl., 1972). Among the consequences of
this process are increased solubility and diffusion of
nutrients, including phosphate, ferrous iron, and silicate,
from the sediments to the water often resulting in modified
lake productivity (Mortimer, 1941 and 1942).

The majority of interest has centered on the reduction
of inorganic nitrogen-, sulfur-, and carbon-containing
compounds. The most important and interesting components
of the sediments from a biological standpoint, however, are
the organic substances, the microbial degradation of which
provides the electrons for the above reductive processes to
occur (Ruttner, 1963). The organic materials present in
lake sediments undergo anaerobic decomposition in two or

more distinct stages. In an initial non-methanogenic stage,

1

2

a heterogeneous group of facultative and obligately anaer-
obic bacteria convert proteins, carbohydrates, and lipids
into an array of soluble and gaseous metabolic endproducts,
including volatile short-chain fatty acids, alcohols,
amines, ammonia, hydrogen sulfide, carbon dioxide, and
hydrogen (Toerien and Hattingh, 1969). In a subsequent
methanogenic phase, it was believed that the short-chain
fatty acids and hydrogen resulting from the first stage
were converted to methane and carbon dioxide by a physio-
logically distinct group of strictly anaerobic microorgan-
isms termed the methanogenic bacteria. The latter
observation has recently been challenged by Nelson and
Zeikus (1974) who have demonstrated the conversion of 14092
to thHh in incubated lake sediments, suggesting an auto-
trophic rather than a heterotrophic mode of metabolism for
these bacteria. A third, intermediate group of anaerobic
bacteria links the non-methanogenic and methanogenic stages
by metabolizing volatile fatty acids and additional soluble
organic endproducts to 002 and H2 (Pine, 1971).

Regardless of the preferred mode of metabolism of the
methanogenic bacteria, the production of methane in anoxic
zones of natural waters serves as evidence for the decom-
position of organic matter in these regions (Kuznetsov,
1968; Howard‘gt.gl., 1971), as the production of the
latter gas is dependent upon the initial anaerobic
degradation of complex organic substrates. The quantity

of organic matter carried in natural waters is small

compared to that present in the sediments (Stumm and Morgan,
1970). While aerobic metabolism removes much of the
soluble organic material from the water column, the major-
ity of the insoluble particulate and more refractory
material falls to the bottom (Brock, 1966). This complex
organic matter is metabolized by anaerobic heterotrophic
bacteria, or is lost to the permanent sediments. The fer-
mentation endproducts of these complex substrates in turn
can suffer several fates. As already discussed, short-
chain fatty acids, 002, and H2 are presumptive precursors
of methane formation in lake sediments. Alternatively, in
hardwater lakes, soluble fermentation endproducts may be
removed from the system by sorption to precipitating
calcium carbonate particles (wetzel, 1972; Kerr‘etflgl.,
1973).

Quantitatively, the most significant fate of these
metabolic endproducts may be their diffusion into the
water column (Kerr'gg‘g1., 1973) where they are readily
utilized by a diversity of metabolically distinct bacteria.
Acetate and H28, for example, serve as electron donors for
certain purple and green sulfur bacteria (Pfennig, 1967).
Alternatively, these soluble reduced compounds can be
oxidized by groups of aerobic bacteria which are present
in oxygenated portions of the water column (Kerr‘gg”§1.,
1973). In laboratory microcosm studies, Otsuki and Hanya
(1972a; 1972b) have demonstrated that up to feur times as

much dissolved organic matter is released from decaying

4

green algal cells under anaerobic conditions as compared
to aerobic conditions. In nature, then, a considerable
amount of dissolved organic matter is likely liberated
from lake sediments as a consequence of the anaerobic
decomposition of particulate and complex soluble organic
materials.

Determinations of the bacterial communities of lake
sediments have historically involved the enumeration of
the total number of cells present by direct counting pro-
cedures, or the enumeration of viable cells by plating
techniques, utilizing media previously determined to yield
the greatest number of bacterial colonies under specified
conditions (Rodina, 1972; Sorokin and Kadota, 1972).

More recently, the distribution and density of specific
physiological groups of microorganisms in sediments such
as sulfate reducers, denitrifying bacteria, or anaerobic
heterotrophs have been determined by employing media
presumably designed to selectively enumerate each of these
groups of bacteria (Menon,ggugl., 1971; Vanderpost and
Dutka, 1971; Dutka.e£ug1., 1974). However, few attempts
have been made to isolate and characterize these organisms,
to determine their sensitivities to molecular oxygen, and
to further elucidate their role in sedimentary metabolism.

Collins (1962) and Mah and Sussman (1967) have point-7
ed to the need for the analysis of the bacterial commun-
ities of freshwater habitats under oxygen-limiting

conditions. Nevertheless, workers in aquatic microbiology

5

have been slow to apply the techniques of strict anaerobic
bacteriology originally developed to study other strictly
anaerobic environments (Hungate, 1950; Aranki 22.31,,1969)
to investigations of the unique anaerobic habitats of
natural waters.
Attempts have also been made to study the metabolic

activities of heterotrOphic bacteria in sediments by
measuring the rate at which they convert uniformly labeled

14

organic substrates to 002 (Kadota.e§ugl., 1966; Harrison
3331., 1971; Hall 3331., 1972; Okutani 3321., 1972).
Few of these studies were conducted under anaerobic
conditions, in spite of the anoxic nature of most lake
sediments. Boylen and Brock (1973) compared the incorpor-
ation of th (U)-glucose into cell material and the
conversion of the labeled substrate into 17002 under
aerobic and anaerobic conditions. They obtained similar
values for the incorporation of labeled glucose into cell
material under both conditions, but found that less 1""002
was produced under anaerobic as compared to aerobic con-
ditions. However, as bacterial fermentation is by def-
inition incomplete, it is necessary to employ techniques
which measure the gaseous and soluble fermentative end-
products produced in order to account fully for the fate
of uniformly labeled organic substrates incubated

anaerobically in the presence of natural sediment micro-

bial communities.

6

The present study represents an attempt to quantify
and characterize the predominant groups of strictly
anaerobic heterotrophic bacteria present at the sediment-
water interface of a eutrophic lake, utilizing the strict
anaerobic techniques develOped by Hungate (1950) and Aranki
.EE.2$ (1969). An effort was also made to elucidate the
role of natural sediment microbial communities in the
anaerobic degradation of organic materials and the
production of dissolved organic matter by utilizing gas-
1iquid-radiochromatography to.assess the gaseous and
soluble metabolic endproducts resulting from the catabolism
of uniformly labeled organic substrates by these

communities.

MATERIALS AND METHODS

m are

All investigations were conducted on Wintergreen Lake,
a small hardwater basin located within the W. K. Kellogg
Bird Sanctuary, Kalamazoo County, Michigan, R. 9W., T. 1N.
Sec. 8. The lake has an area of 14.98 ha., a maximum
depth of 6.3 meters, and a mean depth of 3.54 meters
(Manny, 1971). Annual mean primary productivity values
identify Wintergreen Lake as hypereutrophic, the latter
designation based on compression of the trophogenic zone
to a point where light rather than available nutrients
limit productivity (Wetzel, 1966; Manny, 1971). Winter-
green Lake is characterized by a hypolimnion which is
anoxic for nearly 7 months of the year, with anaerobic
conditions extending to 3 meters during midsummer stratifi-
cation. All sediment samples secured during this study
were taken at a depth of 6 meters or greater.
Temperature

The temperature of the sediment-water interface was
measured at approximately two week intervals from.March
through December, 1973. The i}; sit}; temperature was
determined with a Model 46 TUC tele-thermometer (Yellow
Springs Instr. 00., Yellow Springs, Ohio), the probe of
which was weighted to insure its penetration into the

sediment.

Dissolved Oxygen

water samples were collected for dissolved oxygen
analysis on the same dates that sediment temperature was
determined and bacteriological analyses were performed.
Samples were taken at 1 meter intervals with a 3 liter
Van Dorn water sampler and subsamples were siphoned into
300 ml BOD bottles. The samples were immediately fixed in
the field and returned to the laboratory where the dis-
solved oxygen concentration was determined by the Winkler
technique (Standard Methods for The Examination of Water
and Wastewater, 1971). Duplicate titrations were performed
on each subsample.

Bacteriological Sampling Procedures

Sediment samples for bacteriological analysis were

 

collected with a Plexiglass gravity corer (8.5 cm by 110
cm) that preserved the sediment-water interface. Samples
were collected at approximately monthly intervals from
March through December, 1973. Cores were immediately
plugged with airbtight rubber stoppers for transport to
the laboratory where a subsample was taken using a sterile
glass subsampler (3 cm by 23 cm). The subsample was con-
tinually gassed with a mixture of 90% N2-10% H2 or 85%
N2-5% cog-10% H2 before being plugged with rubber stoppers,
clamped into a press, and transferred into an anaerobic ‘
glove box (Aranki‘gg‘gl., 1969). During the initial
period of the study, the anaerobic glove box contained an

atmosphere of 90% N2-10% H2. The gas composition of the

9

chamber was later altered to 85% N2-5% 002-10% H2. No
significant differences were noted in the types or numbers
of bacteria isolated under the two gas atmospheres.

An initial 1:10 dilution (vol/vol) of the surface
sediment (0—2 cm layer) was made in pre-reduced anaerobic
dilution fluid. The composition of the dilution fluid
varied with the gas phase employed (Table l). ,The initial
1:10 dilution was either stirred on a magnetic stirrer for
five minutes, or mixed in a Waring blender for 1 minute.
Appropriate additional dilutions were made in anaerobic
dilution fluid and pre-reduced agar plates containing 0.05%
resazurin as an oxidation-reduction indicator were inoc-
ulated from several different dilutions of the sediment.
Media inoculated included 1% peptone-yeast extract-glucose
(PYG), 0.5% PYG, Brain Heart Infusion (BHI, Difco, Detroit,
Michigan), sediment extract supplemented with trypticase,
yeast extract, and mineral salts, and Medium 10 of Caldwell
and Bryant (1966) modified for use under a predominantly
N2 atmosphere. 1% FIG was pre-determined to yield the
highest counts of bacteria and was routinely used for all
quantitative viable counts. The complete composition of
each medium is listed in Table 2.

Inoculated plates were incubated anaerobically at
15°C for 14 days. In the initial portion of the study,

a stainless steel anaerobic chamber maintained at 15°C
was used to incubate the plates. After August, 1973, a

vinyl glove box (Coy Manufacturing, Ann Arbor, Michigan)

10

Table 1. Composition of anaerobic dilution
fluid utilized under different
gas atmospheres

Atmosphere of 90% N2-10% H2

(NH4)230h 0.05%
MgSOh-7H20 0.005%
NaCl 0.1%

Kzupoh 0.65%
KHZPOA 0.35%
Cysteine-HCl'HZO 0.05%

0.1% Resazurin soln (vol/vol) 0.2%
pH adjusted to 6.9

Atmosphere of 85% N2-5% 002-10% H2

KZHPOh 0.65%
KHZPOQ 0.35%
NaZCO3 0.25%
Cysteine-HGl-H20 0.05%

0.1% Resazurin soln (vol/vol) 0.2%
pH adjusted to 6.9

11

Table 2. Composition of the media utilized in the
isolation of anaerobic bacteria from
Wintergreen Lake sediments

% in medium (wt/vol)

 

 

component 1% PYG 0.5% BHI Sediment *Modified
PYG Extract Medium 10

Peptone 1.0 0.5

Yeast Extract 1.0 0.5 0.4

Glucose 1.0 0.5 .

Cellobiose .

Soluble Starch .

Trypticase 0.2

Hemin (vol/vol)

Volatile Fatty

Acids (vol/vol)

Agar 1.5 1.5 1.5 1.5
Salts Soln 1 90 90 90

(vol/vol)

Salts Soln 2 10 10 10

(vol/vol)

Salts Soln 3 2.0
(vol/vol)

Difco Brain 3.7

Heart Infusion
Cysteine-HCl°H20 0.05 0
0.1% Resazurin 0.4 0
Soln (vol/vol)

Sediment Extract 30*
(vol/vol)

H

OH OHOOOO
O
01 \nCNm\nvum

|—‘
O O0

05 0.05 O O
.4 0.4 0.4

two

*Sterilized separately and added aseptically after
autoclaving

The pH of each media except the sediment extract was
adjusted to 7.8 before autoclaving; the final pH of

the media after eqilibration under 85% N2-5%»CO -lO% H2
was 7.0; the pH of the sediment extract was adj sted to
7.0 before autoclaving and remained at 7.0 after equili-
bration under the anaerobic atmosphere.

12

Table 2 (continued)

Salts Solution 1 (per 900 m1)

KZHPOI+
KHZPOA
Na2003
MgSOh'7H20
(NHA)2804
NaCl

FeCl3

520
280
200
100
200
480

5

Salts Solution 2 (per 100 m1)

CaC12°2H20

60

Salts Solution 3 (per 1000 ml)

(NHh)2804
MgSOh'7H20
NaCl
K2HPOh
KHZPOA

Volatile Fatty Acid Mix

Acetic acid
Propionic acid
Butyric acid
Valerie acid
Iso-valeric acid
Iso-butyric acid

l.O
0.1
2.0
13.0
7.0

FJFWdF‘OVQ

mg
mg
mg
mg
mg
mg
mg

mg

0909090909

m1
m1
m1
m1

m1

13

was used for all bacteriological procedures. Inoculated
plates were placed in anaerobic gas-pak jars (BBL,
Cockeysville, Maryland) containing a palladium catalyst.
The jars were sealed and removed from the glove box to a
15°C incubator. After 14 days, the jars were returned to
the glove box, opened, and the plates were examined
qualitatively and quantitatively.

Characterization‘gf Bacteria

All plates were carefully examined for colony
diversity. Fifty to 60 colonies were randomly picked
from each medium and transferred to 1%TPYG plates.
Although colonies were selected at random, an effort was
made to insure that representatives of all distinct
colony types present on the plates were included in the
organisms transferred. The PYG plates were incubated at
room temperature within the glove box. Greater than 95%
of the isolates grew within three days. The colonies
were then streaked onto 1%»PYG plates and isolated
colonies obtained were transferred to anaerobic PYG
slants and refrigerated at 4°C until needed for further
characterization.

The isolates were characterized primarily by the
methods described in the Virginia Polytechnic Institute.
(VPI) Anaerobe Laboratory Manual (Holdeman and Moore,
1972). All characterization tests were performed using
pre-reduced anaerobically sterilized (PEAS) media,

except for the determination of oxygen sensitivity when

14

incubation was aerobic. In addition, the nitrate reduction
broth described by Holdeman and.Moore (1972) lacked
cysteine in order to avoid chemical reduction of the
nitrate in the medium. All characterization tests were
performed at room temperature, except for the temperature
growth studies.

In the characterization scheme utilized (Figure l),
1% PYG was used as the basal medium with the various test
substrates being added at a concentration of 1%.(wt/vol).
Morphology, motility, and the presence of spores were
determined by examination of 18-24 hour PYG broth cultures
by phase-contrast microscopy. In cultures in which no
spores were apparent, spore production was determined by
inoculating 1% PY-starch with 2-3 dr0ps of the suspected
culture, pasteurizing the stoppered tube for 10 minutes at
80°C in a water bath, and incubating the tubes at room
temperature. Outgrowth within four days was scored as
indicating positive spore formation (Holdeman and.Moore,
1972).

Hydrogen sulfide production was determined by suspend-
ing a strip of Whatman # 1 filter paper previously soaked
in 5% lead acetate within the headspace of each PYG broth.
Black discoloration of the strip caused by the formation
of lead sulfide indicated positive H25 production.

In order to detect H2 and CO2 production by the in-
dividual isolates, tubes of 1% PYG broth were prepared

under 100% N2 utilizing the Hungate modification of the

15

Figure 1. Characterization scheme utilized in
the Identification of isolated
sedimentary anaerobes

Isolate on 1% PYG
anaerobic slant

1% PYG broth 7 ml 1% PYG broth Triplicate sets

under 100% N of 1% PYG broth
for detection of 2 dr0p incubated at 5°,
H2 + 002 production inoculm ““.l ,150 C

   
   

2 drop inoculum

1% FY broth Examination after
(Indole production) 24 hours for:
(pH) Morphology
Motility
Gram reaction
2 drop Presence of spores
inoculum Growth in the presence of 02
H S production
Final pH in: Metabolic endproducts
1% PY-cellobiose pH

1% PY-galactose
1% PY-lactose
1% PY-maltose
1% PY-sucrose

2 drop
inoculum

Nitrate reduction broth
Catalase production
Tributyrin hydrolysis
Gelatin liquefaCtion
Casein hydrolysis
Starch hydrolysis
Cellulose degradation
Carboxymethylcellulose digestion

16

anaerobic r011 tube as described by Macy'et.al,, (1972).

H2 and CO2 were analyzed by injecting 0.2 m1 of the head-
space of each culture into a Packard Model 409 gas
chromatograph (Packard Instrument Co., Downers Grove, Ill.)
equipped with a thermal conductivity detector. The

column and operating conditions employed are listed in
Table 3. Part A.

The ability of each isolate to grow in the presence
of molecular oxygen was determined by inoculating 1% PYG
agar plates lacking cysteine with 1 drop of an actively
growing broth culture of each organism, and incubating the
plates aerobically at room temperature. Aerobic PYG
plates were also streaked with each isolate. Both methods
yielded identical results in nearly all cases.

The cellulolytic capabilities of the isolates were
tested by incorporating 0.1%iball-milled cellulose (made
from Whatman #1 filter paper and kindly supplied by Dr.

M. P. Bryant) into pre-reduced PY plates. Zones of
clearing around inoculated colonies indicated positive
cellulolytic activity. Casein digestion and tributyrin
hydrolysis were determined in a similar manner.

All additional tests, including indole production,
nitrate reduction, Gram reaction, sugar fenmentations,
gelatin liquefaction, starch hydrolysis, catalase pro-
duction, and endproduct analysis, were performed as
outlined in the VPI Anaerobe Laboratory Manual (Holdeman

and Moore, 1972). Volatile short-chain fatty acids

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18

produced as metabolic endproducts by the isolates were
analyzed on a Packard Model 409 or a Varian Model 600
(Varian Aerograph, Walnut Creek, Cal.) gas chromatograph
equipped with hydrogen flame ionization detectors.
Columns and operating conditions utilized are given in
Table 3, parts B and C. When the fatty acids were analyzed
on a stainless steel column, a glass liner was inserted
into the injection port of the gas chromatograph in order
to insure that the samples were volatilized on glass and
not lost by adsorption to the column. Lactic acid was
determined by converting it to its propyl ester (boron
trifluoride-propanol esterification reagent obtained from
Anspec 00., Ann Arbor, Michigan) and analyzing it by gas
chromatography as described in Table 3, part 0.
Metabolism of Labeled Organic Compounds 21 Selected
Bacterial Isolates

Two representative isolates obtained from the
sediments of Wintergreen Lake, and tentatively identified
on the basis of fermentation endproducts and additional
biochemical tests as Clostridium bifermentens and.Q.
sporogenes, were selected for additional metabolic
experiments. Each organism.was grown in pre-reduced PYG
broth. Two drops of a 24-hour culture of each isolate
were added to 2 ml of 1% PYG broth containing 1,uCi/m1
of th (U)-D-glucose (Amersham/Searle, Arlington Heights,
I11.) and to 2 ml of 1%.PY broth containing l‘pCi/ml of

14C (U) -L— amino acid mixture (New England Nuclear,

19

Boston, Mass.) respectively. The composition of the la-
beled amino acid mixture is given in Table 4.

Immediately after inoculation, the culture tubes were
sealed with butyl rubber septa and incubated for 24 hours
at room temperature. The gaseous phase of each tube was
analyzed for labeled C02 by injecting 0.2 ml of the culture
headspace into the Packard M0del 409 gas chromatograph
equipped with a thermal conductivity detector. Column
and Operating conditions are given in Table 3, part D. A
one eighth inch (0D) section of stainless steel tubing was
swaged (Swagelok Fittings, Grand Rapids Valve and Fitting
00., Grand Rapids, Mi.) onto the thermal conductivity
effluent port of the gas chromatograph such that the
radioactive effluent passed directly into a Packard Model
325 Tri-carb combustion furnace (Packard Instru. Co.).

The latter contained a quartz glass tube filled with
copper oxide which was maintained at a temperature of
750°C. Organic components of the gas chromatographic
effluent gas were thereby combusted to 002, passed through
a magnesium perchlorate water trap, and the radioactive
effluent collected at selected time intervals by bubbling
it into a scintillation vial containing 9 m1 of methanol,
6 ml of scintillator (15g PPO, 1 g bis-MSB, made up to 1
liter with toluene), and 4 ml of purified monoethanolamine
(Packard Instrument Co.). The scintillation vials were
counted in a Beckman Model LS-150 liquid scintillation

spectrometer.

20

Table 4. Composition of lac (U) -L- amino acid mixture
utilized in labeling experiments

 

14C (U) -L- amino millicuries per microcuries
acid millimole amino acid per
millicurie mix-
ture
Alanine 120 80
Arginine 256 70
Aspartic Acid 172 80
Glutamic Acid 215 125
Glycine 84 40
Histidine 282 15
Isoleucine 258 50
Leucine 258 140
Lysine 252 60
Phenylalanine 423 80
Proline 215 50
Serine 140 40
Threonine 188 50
Tyrosine 387 40

Valine 215 80

21

The clostridial cultures were centrifuged for 10
minutes in a clinical centrifuge. The supernatant from
each was acidified with 3 drops of 50% ”2804' extracted
twice with 2 m1 of redistilled ethyl ether, and concentrat-
ed under N2 to a volume of 0.1 ml. A 10 p1 sample of each
extract was injected into the Packard Model 409 gas
chromatograph. Operating conditions are given in Table 3,
part E. Radioactive endproducts were chromatographed,
combusted to 002, trapped in ethanolamine, and counted as
described above for the gaseous phase, except that for the
analysis of the soluble endproducts, the stainless steel
tubing connecting the gas chromatograph to the combustion
furnace was wrapped in asbestos heating tape (Cole-Farmer
Co., Chicago, Ill.) and maintained at a temperature of
210°C to insure that sample condensation did not occur
during passage from the gas chromatograph to the combustion
furnace. An efficiency of approximately 50%, measured by
extracting a sample of lL’C—acetate of known activity with
ether and injecting it into the chromatograph, was deter-
mined for the system.

Metabolism <_>_f_ Labeled Organic Compounds By _A_ Natural
Sediment Microbial Community»

The anaerobic metabolism of uniformly ll"C-labeled .
amino acid mixture and uniformly ll’C--labe1ed glucose by a
natural sediment microbial community was also examined.
Sediment samples were collected in the summer of 1974

using the same anaerobic collection techniques as

22

described previously. A 1:10 (vol/vol) dilution of
sediment (0-2 cm layer) was made in the anaerobic glove
box with pre-reduced filterbsterilized interstitial water
(pH 7.2) which had previously been extracted from Winter-
green Lake sediments by centrifugation at 15,000 rpm for
30 minutes. Ten ml of the 1:10 sediment dilution were
added to duplicate 50-m1 flasks containing 4upCi/m1 of
pre-reduced 140 (U) -D— glucose and 3.4 pCi/ml of pre-
reduced lac (U) -L- amino acid mixture respectively. Cold
glucose (0.115 mg/ml) and cold amino acid mixture (Sigma
Chemical Co., St. Louis, M0.) were also added to the
respective flasks as carrier substrate. Control flasks
were killed by the addition of 1 m1 of 2% HgCl2 or 1 m1

of 6N H280“ before addition of sediment. The flasks were
sealed with rubber serum bottle stoppers, removed from the
glove box, and incubated with shaking for 72 hours at 10°C.
The latter temperature closely approximated the actual
temperature of the sediments at the time of sampling.

The reactions were terminated after 72 hours by in-
jecting 1 ml of 6N H2804 into each flask. The flasks were
allowed to shake an additional hour at 10°C to allow CO2
release from the sediment to occur. After equilibration
to room temperature, the gas phase of each flask was
analyzed for radioactive components as described previous-
ly. The contents of each flask were extracted with

successive 5-m1 portions of ethyl ether. The ether

23

fraction was collected, concentrated to a volume of 0.1
m1 under N2, and a 10 ul sample was analyzed by gas-radio-

liquid chromatography.

RESULTS

Qiiantitation 23 Anaerobic Bacteria at 222 Sediment- Water
Interface

Figures 2 and 3 illustrate the density of anaerobic
heterotrophic bacteria at the sediment-water interface
and the concentration of dissolved oxygen in the water
column of Wintergreen Lake from February through December,
1973. The density of anaerobic bacteria remained nearly
constant throughout this period, with a moderate increase
in June corresponding to the onset of intense anoxic
conditions in the hypolimnion (Figure 3). The density of
anaerobes at the sediment-water interface did not decline
appreciably in November after lake turnover had occurred,
a reflection of the near permanent anaerobiosis of these
sediments and of the ability of the sediment microflora
to withstand brief exposure to oxygenated conditions.
Temperature 9; 2113 Sediment-M Interface

The temperature at the sediment-water interface of
Wintergreen Lake from March through December, 1973
(Figure 4) varied considerably, ranging from a low of
3.4°C in December to a high of 13.3°C in October. Despite
these seasonal flucuations, the measured sediment tempera-
ture remained 10°C or greater for a minimum of 6 months

of the year.

24

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28

‘nggg Groups.g£ Anaerobic Heterotrophic Bacteria Present
At The Sediment-Water Interface

Table 5 summarizes the major groups of facultative
and strictly anaerobic bacteria isolated from the sediment-
water interface of Wintergreen Lake. Fermentation and
additional physiological characteristics of each group are
found in Table 6. Group I, tentatively identified as a
Streptococcus gp,, was composed of Gram-positive faculta-
tively anaerobic cocci, l-1.5 pm.in diameter. The cells
were either spherical or dumbbell shaped and occurred
singly, in pairs, or in short chains. No H23 or indole
were produced, and fermentation endproducts in PYG included
major amounts of lactic and acetic acids with trace
quantities of propionic, butyric, and iso-butyric acids.
Strains of Group I fermented all sugars tested, produced
neither H2 or CO2 from PYG, and exhibited the ability to
grow at 5°C. These isolates comprised 10.8% of the total
organisms examined.

Group II, comprising 9.5% of the total isolates,
consisted of motile Gram-negative curved rods measuring
0.5 by 3‘um. These organisms were obligately anaerobic,
produced H28, and were indole-negative. Fermentation
endproducts included major amounts of acetic and propionic
acids with lesser quantities of butyric, iso-butyric, and
iso-valeric acids. Presumptively classified as Vibrio
species, these organisms fermented only maltose among the

six sugars tested. These strains grew at 10° and 15°C,

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30

Table 6. Fermentation and additional physiological
characteristics of isolated sediment strains

Group Number*

 

 

Characteristic 1 2’ 3a 3b 3c
Glucose a n w w a
Cellobiose a n n n a
Galactose a n n n a
Lactose a n n n a
Maltose a a w w a
Sucrose a n n n a
Indole Production - - + _ _
H S Production - + + + +
Catalase - - — - _
Nitrate Reduction - - - - _
H Production - ND + + +
CO Production - ND + + +
Grgwth at:
SC + - - + -.
100 + + + + +
15C + + + +

* Fermentation and additional physiological tests were
performed on the first three groups of isolates only.

Symbols: a, acid reaction, terminal pH 5.5 or less
w, weak reaction, terminal pH 5.5-6.0
n, no fermentation, terminal pH greater than 6.0
ND, not determined

31

but not at 5°C.

By far the largest group of isolates obtained, repre-
senting 71.8% of the total, Group III consisted of
anaerobic sporeforming rods of the genus Clostridium.

This group could be further subdivided into three subgroups
identified on the basis of fermentation endproducts and
additional biochemical reactions as'g. bifermentens,lg.
sporogenes, and'g. butyricum. ‘Q. bifermentens represented
the most predominant species obtained, comprising 47.7%

of the total isolates.

The predominantly proteolytic nature of these
clostridial isolates (Barker, 1961) was substantiated by
their inability to ferment a variety of sugars, the sole
exception being‘g. butyricum which was able to ferment all
of the sugars tested. Also of note was the ability of the
clostridial isolates to produce H2 in PYG broth and their
capability to grow at environmental temperatures. All
three clostridial isolates grew at 10°C;'Q. sporogenes
was able to grow at 5°C as well (Table 6).

Groups IV and V represented a minor portion of the
isolates, together comprising only 7.5% of the total.
Group IV consisted of Gram-positive nonsporing rods and
included two subgroups, the first an obligately anaerobic
rod which produced acetic and butyric acids as major
fermentation endproducts and which may be related to a

species of Eubacterium, and the second, as unidentified

32

facultatively anaerobic rod. Group V consisted of
unidentified motile Graménegative rods which comprised
less than 2% of the total isolates. (Table 5).

Table 7 illustrates the hydrolytic capabilities of
each group of isolates towards a number of complex sub-
strates. No cellulolytic activity was demonstrable as
shown by the inability of all of the groups to hydrolyze
cellulose or carboxymethyl-cellulose. Moreover, only the
presumptive Eubacterium, the group V isolates, and one
subgroup of clostridia,.Q. butyricum, were able to ferment
starch. Similarly, only two groups, the group V isolates,
and the presumptive Eubacterium exhibited lipase activity
as demonstrated by the ability to hydrolyze tributyrin.

The majority of the isolates, on the other hand,
exhibited proteolytic capabilities. The proteolytic
nature of the clostridia was evident from the ability of
.Q. bifermentens and‘g. sporogenes to hydrolyze both
casein and gelatin. OnlyIQ. butyricum failed to attack
these compounds (Table 7). The remaining strictly
anaerobic isolates, the group II Vibrio and the group IV
Eubacterium, also demonstrated proteolytic capabilities,
while among the facultative isolates, only the motile
Gram-negative rods of group V were able to liquify gelatin

(Table 7).

33

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34

Metabolism‘gf Labeled Organic Compounds‘gy Selected
Bacterial Isolates

Figures 5 through 9 illustrate the results of the
analysis of the gaseous and soluble metabolic endproducts
produced by.g. bifermentens and.Q.A§porogenes when grown
in the presence of 1[IO labeled amino acid mixture or
glucose reSpectively. Neither organism produced great
amounts of lhco2 from the amino acid mixture (Figure 5),
or from glucose (results not shown), even though both
isolates had been previously demonstrated to produce CO2
in PYG broth (Table 6). Both isolates incorporated label
into an array of short-chain fatty acids when grown in the
presence of the lhC-amino acid mixture (Figures 6 and 7).
The labeled compounds represented by the histograms in the
latter two figures, and in Figures 8 and 9 as well, were
separated chromatographically, using a thermal conductivity
detector and the chromatographic conditions listed in
Table 3, part E. The fatty acid peaks in Figures 6 through
9 are authentic endproducts produced by the two isolates
as determined by the more sensitive hydrogen flame ioniza-
tion detector (Table 3, part C). By comparison, both'g.
bifermentens and.Q. sporogenes incorporated far lesser
amounts of label into the soluble fatty acid fraction when
grown in the presence of ll’C—glucose (Figures 8 and 9).
These observations appear to substantiate the proteolytic
nature of these organisms, but as the specific activities

of the individual fatty acid peaks were not determined,

35

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this type.

Metabolism‘gg’Labeled Organic Compounds‘gy‘A.Natural

 

Sediment Microbial Community

Figures 10 through 12 illustrate the ability of a
natural sediment microbial community to convert ll’C--amino
acids and ll‘C-glucose to lI’D-labeled soluble fatty acids,
C02, and CH1+ when incubated anaerobically at 10°C. When
absolute counts of radioactivity (cpm) alone are considered,
more label appears to be incorporated into both the soluble
fatty acid fraction and the gaseous fraction when labeled
glucose is supplied as substrate as compared to the
labeled amino acids. However, as the small quantities of
gaseous and soluble metabolic endproducts produced by the
sediment microflora were below the sensitivity of both the
thermal conductivity and flame ionization detectors under
the chromatographic conditions employed, the specific
activity of each endproduct cannot be determined. More-
over, the specific activities of the two initial substrates
were not the same. Nonetheless, when superimposed upon
chromatographic scans of standard mixtures of 002 and CHh’
and of a 0.1% standard mixture of fatty acids which were
analyzed under the same conditions as the sediment samples,
the histograms of radioactivity depicted in Figures 10.
through 12 correspond to authentic metabolic endproducts,
illustrating the ability of the sediment microbial

community to convert labeled organic substrates to

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gaseous and soluble reduced metabolites under the exper-

imental conditions described.

DISCUSSION
Organic Inputs lg [he 9.23.2.

Provided that the rates of benthic production and
permanent loss of carbon to the sediments are known, the
rate of flux of carbon from freshwater lake sediments
provides a good estimate of overall lake productivity,
since benthic metabolism tends to integrate all sources
of carbon production (Wetzel and Rich, 1972). Originally
applied to studies concerning CO2 release from the
sediments of an oligotrophic marl lake (Rich and Wetzel,
1972), the above generalization can be applied to a
hypereutrophic basin such as Wintergreen Lake as well if
the flux of carbon from the sediments is expanded to in-
clude not only 002, but also CH4, soluble fatty acids and
alcohols, and additional forms of dissolved organic carbon
released from these sediments. The sediments of Winter—
green Lake are particularly important as a focal point
for the accumulation of particulate organic materials.
During the study period, the lake received large inputs
of allochthonous dissolved and particulate carbon,
nitrogen, and phosphorus from migrating and resident
waterfowl, as well as from a dairy feedlot effluent. The
surface area of the watershed is 7-10 times that of the
lake (Manny, 1971). Surface runoff from this large area,
which includes a considerable amount of fertilized and

cultivated farmland, provides additional inputs of

45

A6

nutrients to the lake, further fueling the massive algal
blooms characteristic of Wintergreen Lake.

These explosive phytoplankton blooms, along with
decaying vegetation derived from the extensive littoral
macrophyte beds, constitute the primary autochthonous
source of particulate organic matter in the lake. Because
of the shallow nature of Wintergreen Lake, and the fact
that during summer stratification the hypolimnetic oxygen
concentration (Figure 3) is insufficient to oxidize the
steady influx of organic matter into the basin, much of
this material likely reaches the pelagic sediments in a
primarily undegraded form. The immediate consequence of
this increased accumulation of sedimentary organic matter
is a greatly expanded role for the sedimentary anaerobic
heterotrophic microflora in the re-cycling of organic
compounds in the lake.

Densitylgf Anaerobic BacteriaIAEHZQE Sedimentfiflgggr
Interface

The anaerobic heterotrOphic bacterial population
present at the sediment-water interface of Wintergreen
Lake (Figure 2) showed little variation from March through
December, 1973. The mean viable count obtained on pre-
reduced PYG plates during this period was h.24 x 106
organisms/gram dry wt. of sediment. The sediment viable
counts reached a maximum in June (Figure 2) corresponding
to the onset of significant oxygen depletion in the

hypolimnion (Figure 3). However, the counts did not

#7

decline significantly in November and December when the
entire water column had become reoxygenated as a conse-
quence of lake turnover. These results likely reflect
the near permanent anoxia of these sediments as well as
the ability of the large numbers of clostridia present
to withstand periodic exposure to oxygenated conditions.
The anaerobic heterotrophic bacterial populations
reported in this study are similar to the typical distri-
bution patterns encountered in other lakes of similar
trophic level as Wintergreen Lake. Boylen and Brock
(1973) reported anaerobic heterotrophic bacterial
populations of h.h x 105 to 2.8 x 106 organisms/gram wet
wt. of sediment in Lake Wingra, Wisconsin during the
winter. Surface sediment heterotrophic bacterial
densities in the eutrophic lower Great Lakes have consist-
ently ranged between 106 and 107 organisms/gram dry wt.
of sediment regardless of the medium used or the time of
sampling (Menon g£_al,, 1971; Vanderpost and Dutka, 1971;
Bell and Dutka, 1972a, 1972b; Dutka‘ggigl., 1974). Such
viable counts represent a very small percentage of the
bacterial populations of these sediments. Periodic
direct microscopic counts were performed on the sediment-
water interface of Wintergreen Lake during the summer of
l97h. Organisms were dispersed from the sediment
particles by the method of Bohlool and Schmidt (1973) and
samples were examined with a fluorescence microscope

utilizing acridine orange staining techniques

#8

(Francisco‘gg‘gl., 1973). A mean direct microsc0pic

count of 2.03 x 1011

organisms/gram dry wt. of sediment
was obtained, a value not unrealistic for organically
enriched habitats.

Heterotrophic bacteria are present wherever suffi-
cient organic matter is present, in numbers that are
usually proportional to the quantity of organic matter
available (Dutka 22.31., 1974). However, as organic
nutrients are generally present in greater than optimal
quantities in eutrophic lake sediments, heterotrophic
bacterial communities are limited by the ability of
their members to survive and metabolize in a deoxygenated
environment rather than by the organic content of the
habitat. Consequently, once a stable bacterial community
is established in these sediments, the continued addition
of organic material to the system such as occurs in
Wintergreen Lake may not necessarily result in an in-
creased bacterial biomass (Dutka gt_§1,, 1974). Rather,
the sudden and dramatic crashes of phytoplankton blooms
observed in Wintergreen Lake, similar to the massive
algal rains documented in Lake Erie (Burn and Boss, 1972),
may, while providing additional nutrients to the sediments,
also tend to bury the endemic population. Only the sur~
face bacterial population would be actively involved in
the degradation of this added nutritive material, thus
explaining the relatively stable bacterial community and

steady build-up of organic materials in the sediments of

49

Wintergreen Lake and other eutrophic basins (Dutka‘gg.§1.,
1974).
Anaerobic gacteria Isolated.§zgmb2hgiSedimentfiflgggr
Interface

The most striking feature of the bacterial groups
isolated from Wintergreen Lake sediments was the high
predominance of clostridia among the organisms examined.
The genus Clostridium accounted for 71.8% of the total
isolates (Table 5). Greater than 50% of the clostridial
strains in turn were identified as Clostridium bifgg—

mentens and'g. sporogenes (47.7% and 17.8% respectively).

 

Although clostridia are generally held to be widely
distributed in nature (Stanier'gg.gl., 1970; Slepecky,
1972; Doetsch and Cook, 1973), studies of their occur—
rence in freshwater and marine sediments have, for the
most part, been limited to considerations of these
habitats as reservoirs of infection for spores of
clostridia pathogenic to humans and animals (Laycock and
Longard, 1972; Laycock and Loring, 1972; Sugiyama‘gg.gl.,
1972). Smith (1968), working with marine sediments,
compared the clostridial flora of a productive sediment
and an organically deficient sediment. On the basis of
preliminary data, he suggested that (l) proteolytic
clostridia such as.g. sporogenes,.Q. bifermentens, and.g.
sticklandii may be essential members of most marine
sediment microbial communities regardless of their organic

content; (2) that primarily saccharolytic clostridia can

50

sustain themselves in a sedimentary habitat only when the
latter is organically enriched and constantly replenished
with nutrients, and (3) that more species of clostridia
may be present in environments with a high organic
content.

Matches and Liston (197k) examined the sediments of
Puget Sound for the presence of clostridia. They reported
anaerobic plate counts ranging from 0.73 to 23.5 x 10’+
cells/ml of sediment-water slurry. Approximately 30% of
the organisms obtained were determined to be clostridia,
the three species isolated in greatest numbers being'g.
perfringens, _g. bifermentens, and _g. 321. As the
sediment temperature of Puget Sound rarely rises above
10°C, which has been reported to be the minimum temper-
ature for the germination and growth of most clostridia
(Matches and Liston, 197A), they concluded that the
resident sedimentary population of these organisms was
derived from terrestrial sources rather than from active
growth within the sediments.

The highly proteolytic nature of most of the
clostridial isolates was particularly striking (Table 7).
Although the sediments of Wintergreen Lake are organically
enriched, very few saccharolytic clostridial strains
were found, the single saccharolytic representative,'ga
butyricum comprising only 7% of the total isolates. The
diversity of the clostridial isolates was particularly

low as well (Table 5). As clostridial taxonomy has

51

traditionally been problematical, the performance of
additional definitive biochemical tests might demonstrate
the presence of other species of clostridia among the
isolates examined. Enrichments for specific physiological
types of clostridia such as cellulose degraders, while

not performed in the present study, might also reveal the
presence of additional members of this genus in Winterb
green sediments.

The ability of most clostridia to grow at 10°C
(Matches and Liston, 197A) should impart these organisms
with a favorable selective advantage over other species
of strictly anaerobic bacteria by allowing them to grow
and metabolize in cold environments. Such appears to be
the case in Wintergreen Lake. The temperature of the
sediment-water interface remained at 10°C or greater for
nearly 6 months of 1973, reaching a yearly high of
13.3°C in October (Figure A). This upper range of
sediment temperature correlates well with the period of
most extensive oxygen depletion in the hypolimnion (Figure
3) and with the peak in anaerobic heterotrophic bacteria
(Figure 2), indicating that conditions during the summer
months are optimal for the growth of the clostridia.

The three clostridial isolates obtained from
Wintergreen Lake were able to grow at 10°C (Table 6).
Thus, while additional clostridial inocula were supplied
to Wintergreen sediments frmm terrestrial sources and

from fecal contamination from aquatic waterfowl, a

52

resident sedimentary community was metabolizing and
growing'ig_§itu, particularly during periods in the

summer and early fall when sediment temperature and

anoxic conditions in the hypolimnion were most favorable
for growth. The ability of these organisms to form

spores would then presumably allow them to withstand
periods unfavorable for growth, as during lake turnover,
or during periods of reduced sediment temperature (Doetsch
and Cook, 1973).

Although isolated in far fewer numbers than the
clostridia, the facultative cocci of group I and the
anaerobic Vibrio of group II (Table 5) are probably
active members of the natural sediment community. Each
demonstrated the ability to grow at environmental temper-
atures (Table 6). The group I Streptococcus failed to
hydrolyze any of the complex substrates tested (Table 7),
but vigorously fermented all of the sugars tested
(Table 6). Presumably, this organism thrives in the
sediments by metabolizing the soluble carbohydrates
present or the breakdown products resulting from the
attack of other bacteria on more complex substrates.
Unlike the Streptococcus, the anaerobic Vibrio may
actively be involved in the degradation of particulate
material in the sediments. This organism, like the
clostridia, was primarily proteolytic (Table 7) rather

than saccharolytic (Table 6).

53

Considered as a whole, the Wintergreen sedimentary
isolates are characterized by a high percentage of
obligate anaerobes as compared to the number of faculta-
tive bacteria obtained and by the strikingly limited
hydrolytic capabilities of these facultative organisms.
The greater hydrolytic abilities of the strictly
anaerobic isolates, particularly their proteolytic
capabilities, could provide these organisms with a
selective advantage in an organically enriched environ-
ment such as the sediments of Wintergreen Lake, where a
predominant portion of the organic matter present is in
a particulate or complex form.

Anaerobic Metabolism g£_Labeled Organic Substrates‘gy
Clostridial Isolates aggugy Sediment Microbial Communities

The genus Clostridium includes a large number of
species capable of utilizing widely differing catabolic
pathways for decomposition of proteins, carbohydrates,
and additional complex substrates (Barker, 1961; Wood,
1961; Stanier‘gg.gl., 1970; Mead, 1971). Moreover,
these bacteria have been demonstrated to produce a wide
array of metabolic endproducts depending upon the medium,
cultural conditions, and analytical techniques employed
(Stanier, 1970; Farshy and Moss, 1970; Anema‘ggugl.,
1973). Due to these complexities, many of the details
of the diverse catabolic capabilities of these organisms

remain unresolved (Barker, 1961).

5h

The catabolism of amino acids is carried out by a
great number of obligate and facultative anaerobic
bacteria, including numerous clostridia (Doelle, 1969).
Although many clostridia ferment single amino acids
(Barker, 1961; Doelle, 1969), the two isolates dealt
with in the present study, 9. bifermentens and‘Q.
sporogenes, obtain most of their energy by a coupled
oxidation-reduction reaction between suitable amino acids
or between amino acids and a non-nitrogenous compound.
The coupled decomposition of amino acids is referred to
as the Strickland reaction (Barker, 1961). The
characteristic feature of this reaction is that while
single amino acids are not decomposed appreciably,
appropriate pairs are degraded rapidly. One member of
the pair is oxidized while the other is reduced (Barker,
1961).

The volatile short-chain fatty acids produced by.Q.
bifermentens and Q. sporogenes in PYG broth (Table 5) are
characteristic for these organisms (Moore'gt'gl., 1966;
Holdeman and Moore, 1972). With the exception of a few
species of anaerobic cocci (Peptostreptoccus s .) and a
small number of Bacteroides g2, and Eubacterium‘gp., the
production of valeric and iso-valeric acids as fermenta-
tion endproducts is generally limited to the genus

Clostridium (Moore‘gtlgl., 1966). Furthermore, production

 

of major quantities of caproic and iso-caproic acids as

fermentative endproducts is almost exclusively restricted

55

to the clostridia (Barker, 1961; Wood, 1961; Holdeman
and Moore, 1972). A pertinent consequence of these
observations is that the production of significant
amounts of these fatty acids by a natural sediment
microbial community would provide strong presumptive
evidence for the active role of clostridia in the de-
composition of organic substrates in these sediments.
Gas-liquid-radiochromatographic experiments were
performed to test this hypothesis in the sediments of
Wintergreen Lake (Figures 10 through 12). Although the
specific activity of each individual labeled metabolite
could not be determined, it is nonetheless clear that
significant quantities of radioactivity were detected
at time intervals corresponding to the chromatographic
retention times of a series of soluble fatty acids
(acetic through caproic) after incubation of a sediment
microbial community at 10°C in the presence of labeled
organic substrates (Figures 11 and 12). Of particular
significance is the incorporation of radioactivity in
those regions corresponding to the valeric and caproic
series of fatty acids respectively, when either 140-
1abeled glucose or amino acids were utilized as the ini-
tial substrate. The incorporation of radioactivity into
these "marker" fatty acids provides strong presumptive
evidence that clostridia, and possibly'g. bifermentens
and'Q. sporogenes in particular (Figures 6 and 7), are

active in the degradation of organic substrates in

56

Wintergreen Lake sediments.

Similarly, an analysis of the gaseous metabolites
produced by the natural sediment microflora after
anaerobic incubation at 10°C (Figure 10) demonstrated
the production of thHh and thO2’ the collected radio-
active effluents corresponding well with the retention
times of CHA and CO2 standards analyzed under the same
conditions. The significance of this finding is that it
points out the role played by anaerobic heterotrophic
bacteria, such as the clostridia in the production of
soluble and gaseous metabolites which can serve as
precursors for further metabolism by additional groups
of lake bacteria, such as the methanogenic bacteria.
For instance, the vigorous production of H2 by the
clostridia (Table 6; Barker, 1961; Gray and Gest, 1965)
assumes added importance in light of the recent assertion
that the methanogenic bacteria of lake sediments are
autotrophs and utilize 002 and H2 in the formation of
methane (Nelson and Zeikus, 197A).

The decomposition and recycling of organic carbon
in the pelagic zone of Wintergreen Lake is undoubtedly
controlled by primarily anaerobic processes in the
sediments which result in the production and release of
significant quantities of reduced dissolved organic
matter and gaseous metabolites. Although not specifi-
cally considered in the present study, decomposition in

the littoral zone of the lake is likely controlled by

57

similar processes as well. This investigation has
demonstrated the predominance of clostridia among the
isolatable strictly anaerobic bacteria of the pelagic
sediments of a eutrOphic hardwater lake, and has pre-
sented strong presumptive evidence that these clostridial
species are active in the decomposition of organic

matter present in these sediments.

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