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CONTINUOUS AEROBIC F ERM ENTATION
OF SAUERKRAUT BRINE

BY CANDIDA UTILIS

By

Kyu Hang Kyung

A THESIS

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

MASTER OF SCIENCE

Department of Food Science
and Human Nutrition

1978

ABSTRACT

CONTINUOUS AEROBIC FERMENTATION
OF SAUERKRAUT BRINE
BY CANDIDA UTILIS

 

BY

Kyu Hang Kyung

A continuous aerobic fermentation was carried out to convert organic
acids in sauerkraut brine into yeast cells and to reduce biological oxygen
demand (BOD) to a low level. The sauerkraut brine contained about 1.096
titratable acidity as lactic acid and was pasteurized before yeast cultiva-
tion. The culture, Candida utili NRRL Y-900, was adapted to the brine and
inoculated into the 7-liter fermenting vessel containing 5.2 liters of
pasteurized brine. Temperature, agitation and aeration rates were 30 C,
1,200 rpm and l liter/liter/min, respectively. The organic material was
converted into cells which were harvested by centrifugation. A series of
steady-state conditions at different dilution rates was conducted for 5-15 hr
to study the efficiency in BOD reduction and the productivity of yeast cells.
The optimum dilution rate was 0.12/hr, corresponding to a retention time of
about 8 hr and a flow rate of 0.62 liter/hr. Under these conditions, the
adapted culture used about 9796 of total titratable acidity, increased the pH
from 3.2 to.6.2 and reduced the BOD by approximately 8096. The process
produced 11.6 g dried cells/liter of waste, which contained 45.696 crude
' protein. Supplementation of the sauerkraut brine with N and P sources did

not improve the yeast growth appreciably. The continuous cultivation

Kyu Hang Kyung

process was more effective than the batch cultivation process, reducing the

operation time required by about 2596.

ACKNOWLEDGMENTS

I extend my sincere appreciation to Dr. Kenneth E. Stevenson for
guidance and help throughout the course of my research and writing my

thesis.

I would also like to express appreciation to Drs. L.G. Harmon, C.M.

Stine and 5.5. Beneke who helped me as members of my masters committee.

I thank friends and food microbiology colleagues whose advise has been

invaluable.

I am further grateful for the tolerance of all the students and faculty
in the Food Science Building who put up with the unpleasant odor during

some of the fermentation experiments.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . ..................
LIST OF FIGURES . . . . ................ .
1. INTRODUCTION . . ........... . .....
2. LITERATURE REVIEW . . . .............
2.1 Sauerkraut Brine . . . . . . . . . .......
2. 2 Fungi Imperfecti and Their Production . . . .....
2.3 Nutritiononeasts . . . . . . . . . . . .....

2.0 BOD (COD) Reduction by Fungi Imperfecti . . . . . .
3. MATERIALS AND METHODS . .............

3.1 Yeast Culture . . ................
3.2Equipment...... .......
3.3 Continuous Cultivation . ..... . . . . . . . .
3.0 Cultivation Measurements . ............
3.5 Analytical Methods . . . .............

0. RESULTS AND DISCUSSION . . . . . . . . . . . . . .

0.1 Sauerkraut Brine . . . . .......... . . .
0.2 Yeast Cultivation- Batch Process. . . .......
0.3 Yeast Cultivation - Continuous Process .......
5. CONCLUSIONS. ........... . .......
BIBLIOGRAPHY ......................

iii

LIST OF TABLES

Table
1. Characteristics of sauerkraut brine . ..........
2. Effectiveness of Candida utilis in reducing

COD, BOD, K jeldahl nitrogen and titratable
acidity at different dilution rates . . . . ......

iv

29

LIST OF FIGURES

Figure

l.
2.
3.

Changes during batch cultivation . . . . . . . .
Relation between OD 600 and viable cell counts . .

Adjustment of the fermentation from batch process
to the Steady-State o o o o o o o o o o o 0

Protein yield, productivity and cell production
at different dilution rates . . . . . . . . .

pH and titratable acidity changes at different

dilution rates 0 O O O O O I O O O O O O O O O

Page

21
22

25

26

27

1. INTRODUCTION

Sauerkraut packers in the United States annually use about 200,000
tons of cabbage to produce about 13 million cases of 20 No. 303 cans of
sauerkraut, based on data from 1967 to 1976 (USDA, 1977). About 2996 of
the shredded and salted cabbage is discarded as waste brine (Hang 91 31.,
1972a), thus the brine generated each year amounts to about 70,000 tons.
Since waste brine contains extremely high amounts of BOD, lactic acid,
NaCl and phosphorus, simple, conventional disposal of the brine is not only
an environmental problem but also a food and economic loss.

“Elenihough‘some successful breedings of high solid content cabbage
have been reported to reduce the quantity of waste brine (Dickson, 1973,
1977), the waste brine problem will not be solved easily. Recently some
research has been done to utilize sauerkraut brine by cultivating yeasts in it
to produce microbial protein (Hang e_t al_., 1972b, 1975) and the enzyme
"invertase" (Hang get 31., 1973). These research projects have shown the
feasibility, at least on a laboratory scale in shake-flask cultures, of using the
waste, containing high amount of organic acid, to produce single-cell
protein. Much of the organic matter in the waste is converted into yeast
cells, and, hopefully, by selling the yeast cells a portion of the waste treat-
ment cost is returned.

Megiyesgfthis study were to reduce BOD in the waste effluent

and to produce single-cell protein from the waste brine by continuous culti-

vation of yeast. Even though there are_s_orne minor problems, like separating

l

2
yeast cells from the digestion mixture and the highly seasonal operation, the
conversion of waste material into yeast cells containing a high content of
crude protein seems feasible. The practical application of this conversion
lies in ‘the ability to conduct the cultivation as efficiently as possible, with
the final effluent having a low BOD, and to effectively harvest the cells

economically.

2. LITERATURE REVIEW

2.1 Sauerkraut brine

 

Sauerkraut is produced by an anaerobic lactic acid fermentation of
shredded cabbage which is mixed with 2.25-2.5% NaCl. The fermentation is
completed in 2-3 months at a temperature of about 18 C (65 F) by lactic
acid bacteria which are among the natural microbial flora of cabbage. The
time period varies depending on the temperature employed (Pederson, 1975).
Upon completion of the fermentation, the product has a total titratable
acidity of about 1.7%, expressed as lactic acid, with a pH of 3.3-3.5.

Hang e_t _a_l_. (1972a) gave details on the wastes generated during sauer-
kraut processing. Trim losses were about 35% of the raw cabbage and the
waste brine amounted to about 29% of the salted and shredded cabbage. The
liquid brine produced directly from salted shredded cabbage was around 70
gal. per ton of cabbage processed. Peterson _e_t_ El.- (1925) analyzed sauer-
kraut and its juice and found that the juice contained approximately the
same percentage of nutrients as the sauerkraut itself. Spent brine from
sauerkraut processing plants contained 12,000—29,000 mg/liter BOD, 620-
1,390 mg/liter Kjeldahl nitrogen, 7,000—16,100 mg/liter titratable acidity,
and 81-250 mg/liter phosphorus (Peterson e_t 31., 1925; Hang gt 1L, 1972b,
1973, 1975). The range was rather wide due to variations in individual
batches as well as in factory practices. In their report, Hang gt al. (1972a)

expressed the BOD content in sauerkraut brine on a population equivalent

basis: Total BOD from a 65-ton vat was equal to about 750 pounds, which

4
means that it was equal to that contributed in one day by a population of
about 0,500 persons. If the BOD content of sauerkraut brine is compared
with those for sewage, laundry, dairy and cannery wastes which have values
of 100-300, 300-l,000, 800-l,500 and 200—6,000 mg/liter, respectively
(Eckenfelder and O'connor, 1961), it is apparent why sauerkraut brine
presents a disposal problem.

There have been some efforts to reduce the amounts of waste being
generated and the BOD load in the waste. First, Geisman (1977) suggested a
means to utilize solid wastes, such as cabbage trims, by biological digestion
to get liquid or gaseous fuels and fertilizer. Such solid wastes are generally
returned to the field (Hang gt 91., 1972a). Second, high solid content
cabbage reduces, to some extent, liquid waste from sauerkraut factories.
Dickson (1973, 1977) reported that his research group developed a hybrid
cabbage which contained l.5—2.0% higher sOlid matter. This could reduce
the amount of brine from 29.1% down to 23.7% of the salted shredded
cabbage. Third, biodegradation of liquid waste using Fungi Imperfecti has
been initiated to yield biomass or microbial enzyme (Hang e_t a_l. 1972b,
1973, 1975). If one or more of these techniques become practicable, they

could be economic aids to the industry.

2.2 Fungi Imperfecti and Their Production

 

A large amount of research has been done concerning production of
microbial cells as an important source of protein. There was a considerable
difference in yield and composition of the biomass depending on the
substrate, microbial species and drying conditions employed during proces-
sing (Peppler, 1967). Fungi Imperfecti, including both molds and yeasts,
have been grown on food processing wastes such as whey, molasses, potato

processing waste, pickle brine, brewery wastes, etc.

5

Church and Nash (1970) pointed out some characteristics which should
be considered in selecting proper organisms for use in treating waste water
from food processing plants: 1. The fungi should be capable of reducing
BOD to low levels within a short period of time. 2. They should be able to
maintain themselves as the dominant organism and the cell mass should be
readily separated from the digestion mixture. 3. For the cell material to be
acceptable food or feed, it must be non-toxic, of high yield, of high protein
content, easily digestable and bland in flavor. And they selected two

different filamentous fungi, Trichoderma viride 1-23 for corn waste

 

utilization and Gliocladium deliquescens 1-31 for soy waste utilization. The

 

fungi were cultivated non-aseptically in the wastes and they dominated their
systems. The mycelium was thread-like in nature and was readily filtered
from the effluent by nylon screens. Other filamentous fungi have been

cultivated in waste water from food processing plants. Gliocladium

 

deliquescens, Verticillium sp. (formerly Ayergillus oryzae I-l0), and

 

 

Trichoderma viride QM 9123 were selected as suitable fungi for the

 

utilization of rum distillery waste, coffee waste and mixtures of paunch and
cane mud, respectively (Rolz e_t a_l., 1976). These fungi were tested for
toxicity and proved to be non-toxic when fed to rats (Rolz e_t a_l., 1976).
Even though filamentous fungi have some advantages, such as ease of
harvesting and ability to remain dominant under non-aseptic conditions, they
have a distinct disadvantage due to their slow growth rate. To utilize

organic material, I. viride 1-23 in corn waste and Q. deliquescens in soy

 

waste needed retention times of 17-20 hr and 60 hr, respectively, in
continuous systems (Church and Nash, 1970). Due to the relatively slow
growth rate of filamentous fungi, more research has been focused on the

cultivation of yeasts which have faster growth rates.

6

Sugimoto (1970) screened three different yeasts, Pichia scolyti,

 

 

 

Candida guilliermondii and Debaryomyces kloeckeri, for the purpose of
soybean waste utilization. Among the three, 3. scolyti was eliminated
because it produced an unpleasant aroma during digestion and had a small

cell size. After further studies with the other two species, C. guilliermondii

 

was eliminated because it had relatively small cells and could act as a
secondary pathogen. Debaryomyces kloeckeri produced 10.6 g dried
cells/liter of soy waste and also produced a good fresh aroma.

Hang gt a_l_. (1972b) used three different yeasts for the utilization of

sauerkraut brine and found that Candida utilis gave the best results, with a

 

yield of about 6.3 g dried cells/liter. Shannon and Stevenson (l975a)
reported that Saccharomyces cerevisiae, Saccharomyces uvarum, Candida
M9 and Candida steatolytica yielded 8.9, 9.0, 9.9, and 10.6 g/liter,
respectively, when grown on selected brewery wastes. It was noted that
there was a considerable difference in the cell yield depending on the
substrate and yeast species used. In a subsequent study with nitrogen-
supplemented wastes they obtained much higher yields (Shannon and

Stevenson, l975b). Especially with Q. steatolytica, the cell yield was

 

increased up to 12.7 g/liter. Hadioetomo (1975) reported that Debaryomyces

 

hansenii, among other yeasts isolated from salt-stock pickle brine, utilized
the acid in the brine most rapidly, producing 5 g dried cells/liter.
Saccharomyces fragilis grown on a water suspension containing 2%
lactose prepared from cottage cheese whey produced 12 g dried cells/liter
by batch process (Vananuvat and Kinsella, l975a) and 10 g/liter by
continuous cultivation at a dilution rate of 0.18/hr (Vananuvat and Kinsella,
1975b). Fencl e_t a_l. (1961) reported that they harvested 10.2 g dried

cells/liter of _C_. utilis grown on molasses containing 2% sucrose by batch

7
process, and 12.5 g/liter and 18.5 g/liter on molasses containing 2 and 3%
sucrose each by continuous cultivation at the dilution rate of 0.25/hr. These
examples make it quite obvious that yeasts cultivated continuously have

higher productivity than when cultivated batchwise.

2.3 Nutrition of Yeasts

Peppler (1968) described dried yeasts as nutritional concentrates which
are sources of protein and B vitamins. About 50% of the dry weight of yeast
is crude protein (N x 6.25); the nitrogen is distributed as follows: 80% in
amino acids, 12% in nucleic acids and 8% in ammonia (Rose and Harrison,
1970). Bressani (1968) also reported similar results showing that 80% of the
total nitrogen was due to amino acids, 8-13% to purines, 0% to pyrimidines,
0.5% to choline, and 0.5% to glucosamine and some other nitrogen
compounds.

According to Peppler (1968) the protein content was 50% for _S_.
cerevisiae primarily grown on molasses, 55% for torula yeast grown on
sulfite liquor, 50% for _S_. f_r3gi_li3 grown on whey, and 05% for debittered
brewers' yeast. Shannon and Stevenson (l975a) reported that the protein
content of dried yeasts ranged from 26.7-32.996 for S. cerevisiae and from
27.1-28.7% for 9. % when grown on selected brewery wastes. Candida

steatolytica was reported to contain the lowest percentage of protein

 

despite the production of the highest amount of cell mass. However, the
protein content of the yeast was increased dramatically from 21.3-23.5% to
00.3% by supplementing wastes with nitrogenous material (Shannon and
Stevenson, 1975b). In another study, 9. _l_l_t_I_II_S grown on sauerkraut brine
contained 00.7—06.0% protein, depending on the aeration rates (Hang e_t a_l.
1975). Thus, it is clear that the protein content of the yeast biomass varies

depending on the species and substrates.

8

Cultivation methods, batch or continuous, also affect the protein
content of yeasts. By batch process, S. f_ra_1g111_s grown on 2% crude lactose
was reported to contain 50-60% K jeldahl protein depending on the period of
cultivation (Vananuvat and Kinsella, l975a). In a subsequent investigation,
they (1975b) reported that the yeast cells could be produced with only about
50% Kjeldahl protein when grown continuously on the substrate of the same
composition at a dilution rate of 0.18/hr.

The pattern of amino acids of yeast protein is reasonably good when
compared to high quality proteins such as those in milk and egg (Miller,
1968) and in meat, bread and milk (Dabbah, 1970). Yanez _e__t_ _a1. (1972)
reported that yeast had a high content of lysine when grown on molasses.
Methionine was the only limiting amino acid, and when 0.3-0.5% methionine
was added to the yeast, the protein efficiency ratio (PER) was superior to
that of casein (Bressani, 1968; Yanez £1 a_l., 1972). It was suggested that
since the amino acid pattern of yeast protein was complementary to that of
cereals, the yeast would be valuable as a protein supplement for cereal
products (Dabbah, 1970; Yanez _el £11., 1972).

Yeasts are also excellent sources of vitamins of the B complex
(Peppler, 1967; Bressani, 1968; Dabbah, 1970; Yanez 391.. 1972). The high
content of B vitamins, such as thiamine, riboflavin, pantothenic acid and
niacin is well documented. However, riboflavin and pantothenic acid may be
destroyed under certain conditions. Yeasts also contain small amounts of
vitamin E and D. Irradiation of yeasts by ultraviolet rays for the purpose of
converting ergosterol to vitamin D2 was used in the past, but the practice
has declined (Peppler, 1967). Bakers' yeast usually contains l-2% ergosterol
and extraction of ergosterol from the yeast is practiced in Europe (Peppler,

1967). Being rich in purines, there is a possibility that yeasts can have a

9

detrimental effect on man due to a build-up of uric acid (Bunker, 1968;
Edozien _e_1 g” 1970). Ingestion of large amounts of yeast could result in
high levels of uric acid with the possible accumulation of sodium urate
crystals in urinary tract, joints and soft tissues to give symptoms of kidney
stone formation, gout and tophi (Miller, 1968; Edozien e_t 31., 1970; Stryer,
1975). Uric acid, which has a pKa of 5.0, is the final degradation product of
purines in primates due to a lack of the enzyme "uricase" (Edozien _t al_.,
1970; Stryer, 1975). Considerable research has been done in an attempt to
reduce the nucleic acid levels in yeast cells and thereby reduce the amount
of purines. Methods used to reduce the levels of nucleic acids have included
heat-shock (Maul gt_ a_l., 1970; Ohta e_t a_l., 1971), ribonuclease treatment
(Castro _e_t_ 31., 1971), precipitation (Hedenskog and Ebbinghaus, 1972) and

manipulation of the growth rate and growth conditions (Hedenskog _el a_l.,

1972).

2.0 BOD (COD) Reduction by Fungi Imperfecti

Waste treatment has become a heavy burden on the food processing
industry. One of the areas which must receive future emphasis in the food
industry is solving processing waste and pollution problems (Briskey, 1973).
Waste water generated in food processing carries soil particles, chemicals,
microorganisms, product juices, fragments of raw material, and cleaners
(Woodroof, 1975). Estimates of the number of plants expected to be closed
because of pollution control costs, in 1970, varied between 296 and 068
(Olson 91 g” 1970).

There have been several efforts to treat food processing wastes by
growing Fungi Imperfecti. In addition to BOD (COD) reduction the process
can be operated to yield almost no inorganic nitrogen or phosphorus in the

effluent (Woodroof, 1975). The reduction of wastes by Fungi Imperfecti

varies depending on the system used and other factors. When S. fragilis was

10
grown batchwise on whey lactose for 8 hr, and continuously with a dilution
rate of 0.18/hr, the highest BOD reduction by batch process was 70%, and by
continuous cultivation 60% (Vananuvat and Kinsella, 1975a,b). Hang e_t _a_l.
(1972b,‘ 1973) cultivated _C_. gt_i1i_s on sauerkraut brine to produce biomass and
yeast "invertase" and found that BOD was reduced by 87-89% in 08 hr.
Using S. cerevisiae and S. 1r_agi_li_§, they found the BOD was reduced by 68.8
and 83.8%, respectively. With yeasts grown on selected brewery wastes,
Shannon and Stevenson (1975a) reported BOD reductions of 25.9-02.0% by S.
cerevisiae and 20.0-05.5% by Q. U_t_1I_lS_. In subsequent research they reported

BOD reduction of 55% by _C_Z. steatolytica when the substrate was

 

supplemented with nitrogen (Shannon and Stevenson, 1975b). Debaryomyces

 

hansenii grown on salt-stock pickle brine was reported to reduce the BOD by
60-70% when cultivated batchwise for 20-30 hr (Hadioetomo, 1975).
Stevenson e_t_ a. (in press), who studied yeast growth in pickle processing
wastes, reported that 76% of the BOD was reduced by cultivating 9. 551113
for 20 hr. When they supplemented the brine with phosphate, the BOD was
reduced by 91% during the same period of time.

Many investigators (Helmers _e_t_ _a_l., 1952; Sawyer, 1956; Eckenfelder
and O'connor, 1961) studied the quantities of nitrogen and phosphorus
required for effective removal of BOD. Sawyer (1956) suggested both
maximum and minimum ranges of the BOD : N : P ratio; 100 : 5.9 : 1.1 as a
maximum and 100 : 3.1 : 0.67 as a minimum ratio, mentioning that maximum
nutrient concentration was not necessarily required for the biological
stabilization. The ratios reported by Eckenfelder and O'connor (1961) and
Helmers e_t _a_l. (1952) fall into this range. However, the important variables
to be considered before mentioning the ratio are BOD content, availability

of nutrients, temperature, solids and the time of treatment (Eckenfelder and

11

O'connor, 1961). Since sauerkraut brine had a good ratio (Hang e_t 31.,
1972a), BOD reduction from the waste should not be limited by a deficiency

of either P or N.

3. MATERIALS AND METHODS

3.1 Yeast Culture

 

Candida utilis NRRL Y-900 from the culture collection of the

 

department of Food Science and Human Nutrition at Michigan State
University was used in this study. To obtain some information about the
yeast growth in sauerkraut brine prior to continuous cultivation, experiments
were conducted in shake-flasks. In these experiments, 500-ml Erlenmeyer
flasks containing 150 ml of medium were inoculated and held at 30 C on a
gyrotary shaker (New Brunswick Scientific Co., Inc., Model G-25; New
Brunswick, NJ.) operating at 200 rpm.

The stock culture was first inoculated into 10 ml of yeast extract-malt ‘
extract-peptone-glucose (YMPG) broth contained in a 25-ml screw-cap test
tube and incubated at room temperature for approximately 08 hr in a Model
TC-6 rotating drum (New Brunswick Scientific Co.) Operating at 6 rpm.

A 5% v/v inoculum from the 08-hr culture grown in YMPG broth was
inoculated into sterilized brine. Cell growth was monitored by measuring pH
with a Corning Model 7'pH meter (Corning Scientific Instruments; Corning,
N.Y.). The pH rose only slightly from 3.2 to 3.5, in 80 hr. Thus, there was
an obvious delay when compared to the report by Hang e_t_ g. (1972b),
indicating a pH change from 3.0 to 7.0 in 08 hr. Thus, an adaptation process
was necessary.

For adaptation the yeast was inoculated in a medium consisting of

equal amounts of YMPG broth and sterilized brine and shaken under the

12

13

conditions mentioned above for 66 hr. Two more transfers were carried out
in the same medium. Finally a 5% v/v inoculum of 08-hr-old culture was
transferred into sterilized brine and two more transfers were made on the
same Substrate. By this time pH rose significantly, up to 7.1, in 50 hr,
showing a comparable result to the data reported by Hang e_t 31. (1972b).

This culture was streaked on YMPG agar plates and incubated at 32 C
for 08 hr. Isolated colonies were transferred to YMPG agar slants and
incubated under the conditions mentioned above. The culture was stored at

“C.

3.2 Equipment

 

The cultivation was carried out in a 7-liter bench-top fermentor
(Microferm Fermentor, New Brunswick Scientific Co., Inc., New Brunswick,
N.J.) equipped with agitation, aeration and an automatic temperature
controlling device. The structure of the fermentor was theoretically the
same as the one developed by Novick and Szilard (1950).

The continuous cultivation was conducted by continuously pumping
pasteurized brine into the culture vessel through a drip-feed by means of a
Minipuls II peristaltic pump (Gilson Medical Electronics; Middleton, Wisc.)
and the flow rate was controlled by adjusting the pump speed. Treated
brine, which contained yeast cells, overflowed continuously through the
effluent line, thus maintaining a constant volume in the fermenting vessel.
Polyvinyl chloride tubing obtained from the pump manufacturer was used for
the portions of tubing through the pump head.

Culture vessel, air filter and tubings were autoclaved at 121 C for 15
min. Adaptors used to connect lines were treated with 75% ethyl alcohol at
room temperature for 15 min. The feed reservoir was a 13-liter pyrex glass

jar. A magnetic stirrer (Arther H. Thomas Co., Philadelphia, PA) was used

10
to prevent settling of solid material.

Several types of antifoam were tried to effectively depress foam
formation. Polypropylene glycol 2025 (BDH Chemicals LTD; Poole, England)
was the antifoam chosen since a relatively small amount was required and it
did not significantly interfere with optical density measurements.

The cell concentration during batch process was determined by
measuring optical density at 600 nm with a Spectronic 20 spectrophotometer
(Bausch 6c Lomb Inc., Rochester, N.Y.). Due to high cell concentrations, the

dilution method proposed by Lawrence and Maier (1977) was used.

3.3 Continuous Cultivation

 

To begin the cultivation, the fermentor was filled with 5.2 liters of the
pasteurized brine. Initially, 5 ml of antifoam was added before aeration and
agitation began.

The inoculum was prepared by seeding 5 ml of adapted culture into 150
ml of pasteurized brine contained in a 500-ml flask and by incubating at 30
C for 36 hr. The entire contents of the flask were used as an inoculum
following the adjustment of the temperature of the medium in the
fermentor.

The temperature, agitation and air supply were set at 30 C, 1,200 rpm
and l liter/liter medium/min, respectively, and kept constant.

The process was allowed to proceed batchwise for approximately 10 hr.
When the total titratable acidity as~lactic acid decreased to ca. 0.02%, the
fermentor was connected to a feed reservoir tank to start continuous
cultivation. Pasteurized brine was supplied constantly at the designated
dilution rates (0.08, 0.12, or 0.16/hr). The dilution rate (D) is defined as f/ V,
where f is medium flow rate (liters/hr) and V is culture volume in liters, i.e.

D=f/ V.

15
Treated brine with yeast cells was withdrawn continuously to keep a
constant volume in the culture vessel. At hourly intervals samples were
taken through the effluent line and analyzed for titratable acidity to
confirm that steady-state conditions existed. Final samples were always
taken only after the culture had maintained a steady-state for more than 6
hr. Foaming during the continuous cultivation was controlled by the addition

of Z-ml portions of antifoam when necessary.

3.0 Cultivation Measurements

 

The optical densities (00600) of the culture medium during batch
cultivation were regularly checked with a spectrophotometer using pasteur-
ized brine as a standard. In the case of continuous cultivation, OD600
measurement was not performed because of the interference by antifoam
added irregularly. The pH of each of the samples was also measured using a
pH meter which was calibrated against buffers at pH 0.0 or 7.0.

The volume of the effluent of the continuous system was measured
from time to time by use of a graduated cylinder to determine the flow rate
(f). Samples were placed on ice during sample collection and two separate
samples were gathered for each set of steady-state conditions. The samples
were centrifuged in a RC-IIB refrigerated centrifuge at 5,000 x G (Ivan
Sorvall Inc., Norwalk, Conn.). To determine dry cell weight, the cell pellet
was washed 0 times with distilled water to remove antifoam. The washed
cells were transferred from centrifuge bottles to tared aluminum weighing
dishes using ca. 20 ml of deionized distilled water and were dried in an oven
at 105 C for 20 hr. Dried cells were transferred to a dessicator before
weighing. About 100 ml of the sample were centrifuged at 5,000 x G for 15
min to obtain cell-free supernatant liquid which was decanted into a screw-

cap flask. Cell-free liquid and dried cells were stored at -20 C for future

analyses.

16

 

3.5 Analytical Methods

- Waste samples were analyzed before and after yeast cultivation to
determine the effects of yeast growth. Both noncentrifuged and centrifuged
brine were examined because residual solid material in the brine could settle
and be removed with yeast cells during centrifugation of cells.

Samples were analyzed for BOD, COD, titratable acidity and total
nitrogen. BOD and COD were determined according to the methods
described in Standard Methods For Examination of Water and Waste-water
(APHA, 1971). For the determination of dissolved oxygen for BOD
measurements, the modified azide method was used. Treated sewage
effluent in the final step from the East Lansing Sewage Treatment Plant was
used as a seeding material at a concentration of 2 ml/liter.

Total titratable acidity as lactic acid was determined by titration with
0.05 N NaOH. During titration the pH was monitored with a pH meter and a
pH of 8.3 was chosen as the titration endpoint.

Total nitrogen was determined by the modified micro-Kjeldahl method
(AOAC, 1970). Approximately 15 ml of liquid sample and about 0.1 g of
dried yeast were used for the analysis. The nitrogen content was determined
by titration with 0.1 N H2504. Crude protein contents (6.25 X N) of the

dried yeast cells were also calculated.

0. RESULTS AND DISCUSSION

0.1 Sauerkraut Brine

 

A group of investigators (Peterson _e_t__3_1_., 1925; Hang gt 31:, 1972a,
1972b, 1973, 1975) studied the composition of sauerkraut brine and
concluded that the brine was unfavorable for conventional waste treatment
because of the extremely high BOD and NaCl content and the low pH. The
strength of the waste brine varied depending on different batches and
individual factory practices. Preliminary analysis of the waste brine used in
this study showed that BOD, Kjeldahl nitrogen and titratable acidity were
very high, yet still within the ranges reported by previous investigators. The
characteristics of sauerkraut brine reported previously ranged widely,
presumably due to the degree of dilution of original brine with vat wash, vat
soak or can cooling water. There was a big difference between the values
for BOD and COD of the brine used in this study, indicating that a large
portion of the organic matter was not biologically degradable under the
conditions used for the 5-day BOD. Table 1 shows that, when the brine was
centrifuged, the K jeldahl nitrogen decreased by 08%, whereas BOD and COD
contents decreased by only 21% and 25%, respectively. Thus, the insoluble
constituents in the brine contributed around 20-25% of the total BOD and
COD, which means that most of the BOD and COD was due to substances
which were dissolved in the brine. The soluble matter would be primarily
lactic acid and some acetic acid, and ethyl alcohol. Almost one half of the

nitrogenous substances were not soluble. Peterson gt 31. (1925) in their

17

18
study about the chemical composition of the cabbage and sauerkraut
reported that sauerkraut and its juice contained approximately the same
percentage of nutrients and that sauerkraut contained 12-76% insoluble
nitrogen based on total nitrogen in sauerkraut, depending on the variety,
growing area and degree of maturity. The percentage of insoluble nitrogen
of the brine used in this study was comparable to their average value (00%).

The quantity of nitrogen and phosphorus required for effective BOD
removal and microbial synthesis was recommended as BOD : N : P = 100:
5.9 : 1.1 to 100 : 3.1 : 0.67 (Sawyer, 1956). In this study total phosphorus
content was not determined, so the ratio could not be calculated directly.
However, since all the other values were close to the highest figure of the
reported ranges, from the Table l the phosphorus content was probably
around 200 mg/liter. By using this value, BOD : N : P ratio could be
calculated as 100 : 0.2 : 0.67, which falls within the recommended range.

Hang gt g1. (1972a) in their investigation on the composition of sauer-
kraut waste brine mentioned that neither nitrogen nor phosphorus was
required as a nutrient supplement to achieve optimal biological stabilization.
Hang _e_t a_l. (1972b) worked with a brine having a BOD : N : P ratio of 100:
5.5: 0.85 and reported that the brine was a more favorable medium for
cultivating yeasts than malt extract broth and was as good or better than
peptone dextrose broth. In a separate paper (Hang _el 31., 1973), they also
reported that sauerkraut brine with a BOD : N : P ratio of 100 : 5.0: 0.65
was favorable for the production of the enzyme "invertase".

Although brine used in this work had a somewhat lower nitrogen
content than the brines used by others (Hang _e_t_ a_l., 1972b, 1973, 1975), the
brine was a good medium for cultivation of yeasts and had a relatively good
nutrient ratio. N- and P- supplementation did not increase yeast growth

appreciably.

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20

0.2 Yeast Cultivation -Batch Process-

 

As a basis for reference, batch cultivation was carried out under the
conditions (except for nutrient feed) used for continuous cultivation.
Samples were taken at one-hour intervals during incubation of a culture in
the brine at 30 C in a fermentor operating at 1,200 rpm and 1 liter/liter
fermentor volume/min of air supply. Portions of these samples were used
for measuring titratable acidity, OD 600 and viable cell counts. Experiments
showed that OD 600 was a linear function of population density within the
ranges of OD 600 of 0.0-0.25. Continuous two—fold dilutions were made until
OD 600 fell into this range and the resultant readings were multiplied by the
dilution factor to give the correct OD 600' The typical growth curve of the
culture and the utilization of lactic acid are illustrated in Figure 1. It took
approximately 12 hr for the yeast culture to use up virtually all of the lactic
acid.

When growth was measured by OD 600’ the duration of the lag phase, as
defined by Lodge and Hinshelwood (1903), was about 2.5 hr. However, when
the lag phase was measured by viable cell counts on plate count agar (PCA),
it was around 5 hr. This discrepancy probably was due to the fact that, even
though cell mass was being synthesized during the latter portion of this
period, propagation of the cells did not actually occur until somewhat later.

Specific growth rates during the exponential growth phase were
calculated using curves based on two growth parameters, OD 600 and viable
cell counts. Based on the OD 600 curve, the specific growth rate was
0.09/hr, corresponding to a generation time of 1.0 hr. The specific growth
rate calculated from the curve based on viable cell counts was 0.53/hr,
corresponding to a generation time of 1.3 hr. The specific growth rates,
0.09/hr and 0.53/hr, calculated from the two curves were in close agreement

with each other.

 

21

 

 

  
  
  

 

% titratable acidity and viable cell counts on PCA

 

 

10 _ —.—.— : 00600 curve
' -C)—<)- : viable cell counts curve
“D—i}—' : titratable acidity curve
p-
p
p
8 3
no -'
o 9 : i
9
1.0L.
P
p-
l- V
0
P
b I o
I
0
I
" I
I I
I I
I f D
’l ’I
--.. --_.-:,1......- O [I
Lag ’l 'I »
1- ------- s-—---——----;’------
I
Lag II \
I l l g 1 a 1 l l l
0.1 5 10

Figure 1.

Changes during batch cultivation.

(107 cells/m1)

22

 

10..

1.0 _

OD600

 

_Llllj l

 

 

l
0.1

Figure 2.

1.0

8
viable cell counts (x 10 cells/ml)

Relation between OD

600

and viable cell counts.

23

Microscopic observation revealed that budding cells in the exponential
growth phase (OD 600 curve) were not separated immediately and formed
small pseudomycelium-like cell clusters. As the growth rate declined, the
cell clUsters began to disappear until no such clusters were found in the
maximum stationary phase. Based on the OD 600 curve as shown in Figure 1,
the exponential growth phase ended after ca. 9 hr. of cultivation. In
contrast, the exponential growth phase based on viable cell counts was
extended for about two more hours. Other investigators have also reported
that cell division continued even after there were no increases in cell mass
(Stanier _et Q... 1976). The presence of cell clusters and their subsequent
disintegration might explain the difference in the slopes of the two curves
and the extension of the exponential growth phase based on viable cell
counts.

The correlation between OD 600 and viable cell counts of the culture is
shown in Figure 2. At the time the residual titratable acidity was less than

0.01%, viable cell count was about 5.0 x 108

cells/ml. Comparisons of
OD 600 with viable cell counts showed that the two properties were
proportional only during the exponential growth phase and that an OD 600 of
0.0 corresponded to a viable cell count of about 1.0 x 108 cells/ml. But, for

viable cell counts of (3.0 x 107 cells/ml and >2.0 x 108

cells/ml, the
proportionality did not hold. For low counts, this could be due to continuous
synthesis of cell material without propagation. And for the high counts, this
might be due to smaller cell size and/or continuing division of cells even
after increases in cell mass had stopped (Stanier gt 31., 1976). Endogenous
metabolism (maintenance metabolism) or autolysis (Pirt, 1975; Herbert,

1976) could also be responsible for the latter phenomenon. This would

confirm that cells in stationary phase (OD600 curve) were smaller than those

20
of exponential growth phase due to the reason mentioned above. Obvious
differences in cell size between the two phases were noted by microscopic
observation.

As mentioned earlier, abundant cell clusters were observed during the
period of exponential growth. Fencl and Burger (1958) mentioned that the
formation of pseudomycelium-like cell clusters was due to the effect of
nutrients as well as a lack of oxygen. They mentioned that the formation of
pseudomycelium occurs frequently in the fermentor when there is some
nutrient deficiency. In this study it was not possible to determine if pseudo-
mycelium formation was possibly due to oxygen deficiency, nutrient
deficiency or some other factor. However, foam formation limited the
amount of aeration and agitation which could be employed with the

instrument used in this study.

0.3 Yeast Cultivation -Continuous Process-

Continuous cultivation of the yeast Candida utilis in sauerkraut brine

 

was studied from the viewpoints of biomass production and BOD reduction of
the waste. Figure 3 shows the transition of the culture from batch process
to continuous process and steady state during continuous cultivation. Figure
0 shows the effects of dilution rate on the crude protein yield, productivity
and biomass in the effluent. The values plotted are averages of at least
three measurements during steady-state continuous cultivation. The highest
productivity was observed at a dilLItion rate of 0.16/hr. However, at this
dilution rate the cell concentration in the effluent was much lower than at
the lower dilutions, implying some nutrients were not utilized.

As shown in Figure 5, there are significant differences in the residual
titratable acidity and pH at different dilution rates. Although no obvious

difference was found at dilution rates of 0.08 and 0.12/hr, with a dilution

% titratable acidity

(as lactic acid)

25

 

A
Ul

0.1

0.0

 

   

----~‘----

flow rate.
=14.6O ml/hin

flow rate = 13.75 ml/min

 

 

10

15 20 25 30 35

Time in hours

Figure 3. Adjustment of the fermentation from
batch process to the steady-state.

4O

26

 

 

 

 

Dilutioanate
(hr' )

.40 . €106
l T productivity
.5 C 1?
w ~ e
.0 ‘5 1.20- 1.4 H
a a. ' l a
';,M crude protein a
yield C‘ 0 I12
c m o
r, a >~
p 'H 1.00. . 11.2 0"
O (D I v
8.8 >.
H cell 4-1 l-ll
0) Q; ' "'1
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512 '3
0.80 .
0 g l 1.0 g
H "O
o ' o
'r'\ A H
:4 ° 9* L10
3‘
0.600 10.8
I J l
0.08 0.12 0.16

Figure 4. Protein yield, productivity and cell production

at different dilution rates.

—@D—C)~; calculated for centrifuged brine.

Yeast cell production (g/l)

27

 

    
 

 

7.0 _
O__pI-I
6.0 _
m
a. 3.0!-
4-0'- titratable
acidity
3.0 -
l | n

0.18

0.10

0.06

 

 

0.08 0.12 0.16

Dilution rate
(hr-l)

Figure 5. pH and titratable acidity changes depending

on different dilution rates.

% titratable acidity as lactic acid.

28

rate of 0.16/hr, the pH was close to that of untreated brine and the
titratable acidity remained high. A reasonable goal for titratable acidity of
the brine after fermentation would be a residual acidity of less than 0.05%
in the final effluent. This was achieved in the continuous cultivation at
dilution rates of 0.08 and 0.12/hr. However, productivity is important and
dilution rates should not be lowered only to achieve the maximum utilization
of nutrients in the substrate. Herbert (1976) reported that as the dilution
rate decreased, the portion of substrate converted to cells decreased and
more substrate was converted to C02, thus the 02 used per molecule of
substrate increased. This is due primarily to endogenous (maintenance)
metabolism (Pirt, 1975; Herbert, 1976) and may be influenced by autolysis
(Pirt, 1972). Therefore, cells should be grown at the highest practicable
dilution rate in order to obtain maximum cell yields for the smallest
expenditure of substrate and oxygen. With respect to cell production,
0.12/hr was the optimum dilution rate of those tested in this investigation.

The values for crude protein (Figure 0) are very high because protein in
the biomass was determined by Kjeldahl analysis (N x 6.25); thus, the values
include nitrogen contained in nucleic acids, cell walls, and other organic
nitrogenous compounds in addition to that in protein (Bressani, 1968; Rose
and Harrison, 1970). The highest protein yield was obtained at a dilution
rate of 0.12/hr.

For the purpose of comparison, protein yield (Figure 0), and per cent
reductions of BOD, COD and Kjeldahl nitrogen (Table 2) were calculated in
two ways; one against noncentrifuged natural brine and the other against
centrifuged brine. The latter calculation was carried out to take into
account the solids which were removed during the harvesting of cells.

Otherwise, the solids would be mechanically removed with yeast cells during

29

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comouuwc anmcaonx .00m .900 mcwoscou ca mwawus unaccmo mo mmoco>wuoommm .N manna

 

 

30

centrifugation and would give higher per cent reductions attributed to yeast
growth. Hence, the actual per cent reductions would be between two sets of
data; higher than the plot based on centrifuged brine, and lower than the one
calculated for noncentrifuged brine. There is only a small difference (5-
10%) between the values for BOD and COD reduction using the noncentri-
fuged and centrifuged brines. Values for reduction of Kjeldahl nitrogen show
an interesting difference between the two brines. This difference might be
interpreted to mean that the suspended solids (precipitated during centri-
fugation) in the brine contained much more nitrogenous and biologically
stable material than the substances dissolved in the brine. Since the per
cent reductions of Kjeldahl nitrogen at all the dilution rates tested were
between 39 and 70%, these figures agree with those of Helmers e_t a_l. (1952)
who reported 30 to 70% of the nitrogenous substances were removed,
depending on the source, the temperature, and other variables. From data in
Figuresll and 5 it is apparent that part of the suspended solids is utilized by
the yeasts. The biologically degradable material, however, is mostly in the
liquid portion and is primarily lactic acid. Therefore, depending on the
method used, the per cent reductions of COD and Kjeldahl nitrogen could
differ greatly and could be misleading.

Hang gt 3. (1972b) studied three different yeasts to evaluate the
suitability of sauerkraut brine as a substrate for yeast cultivation. Using a
shake-flask culture method, the yeasts 9. M, S. tt3gi_lit and S. cerevisiae,
reduced the BOD in the waste by 87, 80 and 69%, respectively, in 08 hr. In a
subsequent study, they (Hang e_t Q” 1973) reported that 9. M reduced the
BOD and Kjeldahl nitrogen by 89 and 73%, respectively, in 08 hr of batch
cultivation. As shown in Figure 0, the per cent reductions (calculated for

noncentrifuged brine) of BOD and Kjeldahl nitrogen at the dilution rates of

31
0.08 and 0.12/hr were close to those results reported previously (Hang e_t 31.,
1973). The actual per cent reductions of BOD were between 79 and 83% at
the dilution rate of 0.08/hr and between 76 and 81% at the dilution rate of
0.12/hr, regardless of the basis for calculations.

Calculated for noncentrifuged brine, the Kjeldahl nitrogen was reduced
by 70% and 70% at the dilution rates of 0.08 and 0.12/hr, respectively, which
exceeds the results reported by Hang gt 31. (1973) after a 08—hr batch
process. However, the results for Kjeldahl nitrogen were somewhat lower
based on centrifuged brine. As stated previously these results reveal that
the waste brine contains a significant amount of soluble, biologically stable
nitrogenous material. Sugimoto (1970) in his investigation of yeast
cultivation on soybean spent solubles mentioned that a large part of residual
BOD or COD might consist of higher molecular nitrogenous substances.
Considering the results shown in Table 2, his conclusion could also be true
for sauerkraut brine.

In this investigation reduction of COD was much lower than reduction
of BOD at the same dilution rate. The data indicates that about 70% of the
total organic matter was utilized by the yeasts. However, approximately
80% of the biodegradable matter was consumed at the dilution rates of 0.08
and 0.12/hr. 1

Considering factors such as protein yield, productivity, BOD and COD
reductions, a dilution rate of 0.12/hr was most suitable for treatment of the
sauerkraut brine. At the. same time, continuous cultivation itself had a
distinct advantage. About 10 hr was required to reduce titratable acidity
below the level of 0.05% by batch process. By continuous cultivation,
however, the mean residence time (l/D; the average time a particle spends

in the culture vessel) was about 8 hr at the dilution rate of 0.12/hr, which

32
means that continuous operation would reduce the time required for one
cycle by 25%. Actually, it is generally accepted that any organism which
can grow batchwise can almost certainly be grown more efficiently in a
continuous culture (Herbert, 1976; Pirt, 1972). Pirt (1972) stated that,
because of this reason, continuous cultivation should be required for the
biodegradation of wastes such as effluents.

In spite of all the potential of continuous cultivation of sauerkraut
brine, there are some factors which require further study. Like most wastes
and by—products (except molasses), sauerkraut brine is not suitable strictly
for single-cell protein production because it can not be used on a large scale
and it can not be a capital-intensive process (MacLennan, 1976). Insufficient
amounts of brine are produced, and the sauerkraut fermentation is highly
seasonal from September to February.

The yeast 9. u_tili_s used in this study has the necessary characteristics
described by Church and Nash (1972) for microorganisms to be used for
waste treatment, although it is not very easy to separate yeast cells from
the digestion mixture. Yeast cells can be harvested by centrifugation only

or by centrifugation after alkali treatment.

33

5. CONCLUSION

This study was carried out to investigate the utilization of sauerkraut
brine for the production of yeast cells and at the same time for the
reduction of BOD in the waste. Reductions of BOD, COD, K jeldahl nitrogen,
titratable acidity and productivity of yeast cells were chosen as experimen-
tal parameters.

The culture Candida utilis NRRL Y-900 was adapted to the brine by a

 

continuous transfer technique. Continuous aerobic cultivation of the yeast
in the brine is very promising since 76—81% of biologically degradable matter
were utilized in 8 hr, increasing the pH from 3.2 to 6.2. For comparison, the
per cent reductions were calculated for both noncentrifuged and centrifuged
brine. This could eliminate some possible misinformation about per cent
reductions caused by those suspended solids removed during harvesting of
the yeast cells.

The brine had a well-balanced nutrient ratio (BOD : N : P = 100 : 0.2:
0.67) which supported effective BOD removal and microbial synthesis.
Supplementation tests with N and P sources showed no appreciable
enhancement of yeast growth. A previous report mentioned that neither N
nor P was necessary for proper biological stabilization of sauerkraut brine
(Hang _et 31., 1972a). As noted earlier, sauerkraut brine had an extremely
high BOD, ranging from 12,000 to 30,000 mg/liter depending on the
individual factory practices. Since yeast cultivation can reduce BOD to a

very low level and high protein yeast cells are produced, the process would

30

alleviate the pollution problem and would be an economic aid to the
industry. In the United States, approximately 200,000 tons of cabbage is
processed to produce sauerkraut every year. This would generate about
70,000 "tons of high BOD waste which would be used to produce about 800
tons of yeast cells.

Under the same conditions, a continuous process for utilization of
sauerkraut waste by Q. 3ti1i3 could reduce the operation time by about 25%
compared to a batch process. Moreover, since batch process involves
certain operations between batches, e.g. time for sterilization of the tanks
etc., more time will be saved with continuous cultivation. Continuous
cultivation required a retention time of about 8 hr (dilution rate = 0.12/hr),
whereas a batch process took about 10 hr to achieve the same level of

certain parameters.

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