APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1997, p. 4158–4163 Vol. 63, No.

11
0099-2240/97/$04.0010
Copyright © 1997, American Society for Microbiology

Effects of Lactobacilli on Yeast-Catalyzed

Ethanol Fermentations
N. V. NARENDRANATH, S. H. HYNES, K. C. THOMAS, AND W. M. INGLEDEW*
Department of Applied Microbiology and Food Science, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada S7N 5A8
Received 30 July 1997/Accepted 8 August 1997

Normal-gravity (22 to 24° Plato) wheat mashes were inoculated with five industrially important strains of
lactobacilli at ;105, ;106, ;107, ;108, and ;109 CFU/ml in order to study the effects of the lactobacilli on
yeast growth and ethanol productivity. Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus #3, Lac-

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tobacillus rhamnosus, and Lactobacillus fermentum were used. Controls with yeast cells but no bacterial inocu-
lation and additional treatments with bacteria alone inoculated at ;107 CFU/ml of mash were included.
Decreased ethanol yields were due to the diversion of carbohydrates for bacterial growth and the production
of lactic acid. As higher numbers of the bacteria were produced (depending on the strain), 1 to 1.5% (wt/vol)
lactic acid resulted in the case of homofermentative organisms. L. fermentum, a heterofermentative organism,
produced only 0.5% (wt/vol) lactic acid. When L. plantarum, L. rhamnosus, and L. fermentum were inoculated
at ;106 CFU/ml, an approximately 2% decrease in the final ethanol concentration was observed. Smaller initial
numbers (only 105 CFU/ml) of L. paracasei or Lactobacillus #3 were sufficient to cause more than 2% decreases
in the final ethanol concentrations measured compared to the control. Such effects after an inoculation of only
105 CFU/ml may have been due to the higher tolerance to ethanol of the latter two bacteria, to the more rapid
adaptation (shorter lag phase) of these two industrial organisms to fermentation conditions, and/or to their
more rapid growth and metabolism. When up to 109 CFU of bacteria/ml was present in mash, approximately
3.8 to 7.6% reductions in ethanol concentration occurred depending on the strain. Production of lactic acid and
a suspected competition with yeast cells for essential growth factors in the fermenting medium were the major
reasons for reductions in yeast growth and final ethanol yield when lactic acid bacteria were present.

Bacterial contamination is a major cause of reduction in tions existing in such fermentations (4). In fact, enumeration of
ethanol yield during fermentation of starch-based feedstocks bacteria in many distilleries is often limited to the detection of
by Saccharomyces cerevisiae. Among the bacterial contami- lactic acid bacteria because aerobes and facultative anaerobes
nants encountered, the lactic acid bacteria are the most trou- with little pH tolerance are not considered serious threats to
blesome because of their tolerance to high temperature and product quality or production efficiency.
low pH and their ability to grow rapidly. The genus Lactoba- Yeasts and lactic acid bacteria are often encountered to-
cillus is of major concern to distilleries and fuel alcohol plants, gether in natural ecosystems and may be in competition for the
whereas in breweries, the genus Pediococcus is a persistent same nutrients (1). When both microbes are grown together in
contaminant which often produces off-flavors. As flavor is very a defined medium where yeast growth is restricted through
important in beer, management practices in breweries have provision of suboptimal concentrations of vitamins, a missing
been established to keep these bacteria to a minimum. In substance (nicotinic acid, adenine, guanine, aspartic acid, tryp-
distilleries, cleaning and sanitizing are much less rigorous, and tophan, glycine, alanine, or lysine) essential for the growth of
mashes are subjected to less heat and are not sterile. Contam- Lactobacillus spp. is synthesized in the medium by the yeast
inants can arise from tankage, transfer lines, heat exchangers, cells (5).
raw materials, active dry yeast, poorly stored backset (recycled Uncertainties exist in the literature describing the effect of
thin stillage), or yeast slurry used as the inoculum (23). Micro- lactic acid bacteria on ethanol yield. Chin and Ingledew (6)
bial numbers can be significantly reduced by cleaning and san- reported that Lactobacillus fermentum inoculated at approxi-
itizing the equipment, by maintaining backset at a temperature mately 108 CFU/ml did not seriously affect ethanol productiv-
over 70°C, by pasteurizing or chemically sterilizing the sub- ity in the fermentation of diluted (14° Plato) wheat mash. Only
strates, and by adding antibiotics, such as penicillin (22) or moderate growth (two- to eightfold increases) of the bacteria
virginiamycin (11), to fermentors. In spite of these precautions, occurred. Other workers, however, have reported that when
bacterial contamination still persists in many ethanol produc- bacterial numbers exceeded 108 CFU/ml at 30 h of fermenta-
tion plants. For example, in scotch malt whisky production tion, the spirit loss was approximately 5% (3, 7). According to
plants where wort is not boiled in order to retain the activity of Makanjuola et al. (19), reduced ethanol yields, lower yeast
soluble enzymes of malt, bacterial contamination may compro- crops, reduced carbohydrate utilization, and an increase in
mise the quality of the distilled spirit and reduce the final yield acidity were all caused by the buildup of lactic acid. They found
of this high-value fermentation product (19). Isolates of lactic that a bacterial count of 4.5 3 108 CFU/ml at 30 h resulted in
acid bacteria from distilleries are well-adapted to the condi- a 17% reduction in ethanol yield as a result of stuck fermen-
tation. The concentrations of acetic acid (a minor end product
* Corresponding author. Mailing address: Department of Applied of heterofermentative lactic acid bacteria and wild yeasts or a
Microbiology and Food Science, University of Saskatchewan, 51 Cam- major end product of aerobic bacteria such as Acetobacter spp.)
pus Drive, Saskatoon, Saskatchewan, Canada S7N 5A8. Phone: (306) and lactic acid inhibitory to the growth of S. cerevisiae were 0.5
966-5028. Fax: (306) 966-8898. E-mail: [email protected]. to 9 and 10 to 40 g/liter, respectively, and an 80% reduction in

4158
VOL. 63, 1997 BACTERIA AND ALCOHOL FERMENTATION 4159

yeast cell mass occurred at concentrations of 7.5 and 38 g/liter, 20 min with periodic shaking. Aliquots (0.25 ml) of this suspension were added
respectively (18). The toxic effect of lactic acid was enhanced to each fermentor to obtain ;106 viable yeast cells/ml.
Mashing of wheat and fermentation. Commercial red spring wheat bought
further by an increase in osmotic pressure (8). A reduction in from a local supplier was ground at setting no. 5 on a model S 500 disk mill (Glen
medium pH due to lactate production may also inhibit the Mills, Inc., Clifton, N.J.). For mashing, 19 liters of distilled water containing 1
saccharification process (12, 19). Despite a significant amount mM CaCl2 z 2H2O was warmed to 60°C in a steam kettle. Seven kilograms of
of research in the area, the effect of lactic acid bacteria on the ground wheat was slowly added, and this was followed by 35 ml of high-temper-
ature a-amylase (Alltech, Inc.). After 5 min, the temperature was raised to 90 to
rate and completion of yeast-catalyzed fermentations remains 95°C by using steam and held at this level for 45 min with stirring to gelatinize
unclear (10). Any correlation between the extent of bacterial starch. The preparation was then cooled to 80°C by passing cold water through
contamination and losses in alcohol yield has yet to be deter- the jacket of the kettle, and a second 35-ml dose of high-temperature a-amylase
mined, although it is obvious that each molecule of sugar was added. The mash was held for 30 min at this temperature to complete
liquefaction of the gelatinized starch. The mash was strained under aseptic
diverted to lactic acid production by the bacteria results in the conditions through a sterile stainless steel food grade sieve (pore diameter, 1.5
loss of two molecules of ethanol that could have been pro- mm), distributed into sterile bottles, and frozen at 240°C. Three days prior to
duced by yeast cells (13). When bacterial contaminants are fermentation, the mash was thawed, and 500-g quantities were aseptically trans-
compared, the problem of the correlation of ethanol yield loss ferred to sterile, jacketed, 1-liter Celstir bioreactors (Wheaton Instruments,
Millville, N.J.). Diethyl pyrocarbonate (Sigma Chemical Co., St. Louis, Mo.) was
with the presence of lactic and/or acetic acid is complicated in then added at a concentration of 0.01% (wt/vol) to sterilize the mash. The
part by the fact that homofermentative lactic acid bacteria

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bioreactors were cooled immediately to 4°C by connecting them to a refrigerated
metabolize glucose to lactic acid almost in stoichiometric quan- water bath circulator and then stored at this temperature for 48 h.
tities, whereas heterofermentative lactic acid bacteria produce The bioreactors were connected to a water bath circulator maintained at 30°C
and stirred magnetically (IKA-Labortechnik, Staufen, Germany). One milliliter
lactic acid, CO2, and ethanol and minor amounts of glycerol of filter-sterilized urea was added to each of the bioreactors to give a final
and acetic acid from the supplied glucose. concentration of 8 mM. Saccharification of dextrins to glucose was carried out by
In this paper we describe the effect of five Lactobacillus adding 0.8 ml of glucoamylase (Allcoholase II; Alltech, Inc.) per bioreactor 30
isolates on alcoholic fermentation. The relationships between min before inoculation with yeast. Just prior to yeast inoculation, fermentors
were contaminated (inoculated) with bacteria at the levels mentioned above. The
initial bacterial contamination of wheat mashes and losses in temperature was maintained at 30°C throughout the fermentation. Samples were
alcohol yield were studied. The effects of the bacteria and withdrawn for analysis from each fermentor every 6 h for the first 24 h and then
produced lactic acid on yeast growth and metabolism were also at 36, 48, and 72 h.
investigated. Assay methods. (i) Viable counts of bacteria and yeast cells. Viable cell counts
were monitored by the membrane filtration technique (14). For enumeration of
yeast cells, the membranes were incubated aerobically at 30°C on the surfaces of
YPD plates (10 g of yeast extract per liter, 10 g of peptone per liter, 20 g of
MATERIALS AND METHODS
dextrose per liter, 15 g of agar per liter) supplemented with 0.005% (wt/vol)
Bacteria used. Twelve species of lactobacilli were screened for growth rate in gentamicin and 0.01% (wt/vol) oxytetracycline (Sigma Chemical Co.). The plat-
deMan-Rogosa-Sharpe (MRS) broth (Unipath, Nepean, Ontario, Canada) at ing was done in triplicate for each dilution used.
30°C and for alcohol tolerance. Five species that were capable of extensive Viable counts of bacteria on membrane-filtered samples were obtained by
growth in mash within 36 h and were tolerant to more than 10% (vol/vol) ethanol placing the filters on plates of MRS agar containing 0.001% cycloheximide
were selected for further study so that the information gained would be of use to (Sigma Chemical Co.) and incubating them in a CO2 incubator (National Ap-
the alcohol industry. Two organisms were obtained from Centro de Technologia pliance Co., Portland, Oreg.) at 30°C after two cycles of evacuating and refilling
Copersucar, Piracicaba, São Paulo, Brazil, and were tentatively identified to the with commercial-grade CO2. The results were expressed as CFU per milliliter.
species level and numbered biotype with API 50 CHL test kits (bioMérieux, (ii) Determination of dissolved solids. Portions of samples were centrifuged at
Montreal, Quebec, Canada) as Lactobacillus plantarum biotype 1 and Lactoba- 10,300 3 g for 30 min, and the supernatants were collected and stored in a
cillus paracasei subsp. paracasei biotype 2 (referred to as L. paracasei below). Two freezer (220°C) until they were analyzed. Total dissolved solids in these super-
other strains, Lactobacillus rhamnosus ATCC 15280 and L. fermentum ATCC natants were determined by measuring the specific gravity at 20°C with a model
14931, were obtained from the American Type Culture Collection. The fifth DMA 45 density meter (Anton Parr KG, Graz, Austria). The readings were
strain was an industrial isolate labelled Cargill #3 obtained from Cargill Corn converted to grams of dissolved solids per 100 ml.
Milling (Eddyville, Iowa). An API 50 CHI test kit for Lactobacillus identified this (iii) HPLC analysis. Ethanol and lactic acid were determined by high-perfor-
strain as L. paracasei subsp. paracasei biotype 2, but it differed in microscopic mance liquid chromatography (HPLC) analysis. A 5-ml aliquot from a suitably
morphology and colony morphology from the L. paracasei strain obtained from diluted fermentation sample was analyzed by using a FAM-PAK column which
Centro de Technologia Copersucar. Therefore, for the purposes of this study, we analyzes sugars, alcohols, and organic acids (Waters Chromatographic Division,
used the designation Lactobacillus #3. Milford, Mass.) maintained at 65°C. Orthophosphoric acid (1.5 mM) was used as
Preparation of bacterial inocula. Lactobacilli were grown in 250-ml screw-cap the mobile phase at a flow rate of 1 ml/min. The components were detected with
sidearm Erlenmeyer flasks containing 50 ml of MRS broth. Then, 4-ml portions a differential refractometer (model 410; Waters Chromatographic Division).
of late-log-phase cultures were transferred to 1-liter screw-cap flasks containing Methanol was used as the internal standard. The data were processed by using
200 ml of MRS broth. The headspace of each flask was flushed with filter- the Maxima 810 computer program (Waters Chromatographic Division).
sterilized (0.22-mm-pore-size membrane filter) CO2 gas, and the flasks were
incubated in a model G25 Controlled Environmental shaker (New Brunswick
Scientific Co., Inc., Edison, N.J.) at 150 rpm and 30°C. Growth of the organisms RESULTS AND DISCUSSION
was measured with a Klett Summerson colorimeter (Klett Manufacturing Co.,
New York, N.Y.) equipped with a no. 66 red filter (420 to 660 nm), and the time Fermentation rates. The disappearance of dissolved solids in
for growth to the early stationary phase was determined. A relationship between
Klett units and the number of CFU per milliliter in mid-log-phase cultures was
the fermentation supernatant is a measure of glucose conver-
established for each strain. sion to alcohol. Dissolved solids contribute to specific gravity,
Bacterial cells (1,000 ml) were aseptically harvested by centrifugation at and the overall specific gravity decreases as sugar is converted
10,200 3 g for 15 min at 4°C (Sorvall model RC 5C centrifuge; GSA rotor; to ethanol (density, 0.789) and CO2 gas. Only small differences
Sorvall Instruments, Division of Du Pont, Newtown, Conn.). The pellet was
washed twice with sterile 0.1% (wt/vol) peptone water (Difco Laboratories,
in fermentation rates were observed between the treatments
Detroit, Mich.), and the cells were then resuspended in 50 ml of sterile 0.1% containing L. plantarum and the controls with no bacterial
(wt/vol) peptone water and chilled in ice until they were dispensed. Appropriate inoculation (Fig. 1). Although a slight change in the rate of
quantities of 203-concentrated cell suspension were added to 500-g quantities of carbohydrate utilization was observed when the initial bacterial
mash in fermentors to give final viable bacterial cell numbers of ;105, ;106,
;107, ;108, and ;109 cells (CFU)/ml. Volumes of inoculant were always made
numbers were high, all of the fermentations completed (to
equal with sterile 0.1% (wt/vol) peptone water to avoid dilution of nutrients. In constant specific gravity). This suggests that coflocculation (19)
a control mash fermentation, bacteria were not inoculated, but yeasts were added as a reason for incomplete utilization of carbohydrates and loss
at ;106 viable cells/ml. Growth and metabolism of bacteria in the absence of of ethanol yield due to high levels of bacterial contamination
yeast cells were studied by inoculating mash with ;107 CFU of bacteria per ml.
Preparation of yeast inoculum. Eleven grams of S. cerevisiae active dry yeast
was not a factor here. When coflocculation exists, yeasts are
(Allyeast superstart; Alltech, Inc., Nicholasville, Ky.) was dispersed into 99 ml of unable to utilize all of the fermentable carbohydrates in the
prewarmed (38°C), sterile 0.1% (wt/vol) peptone water and incubated at 38°C for mash, resulting in stuck fermentation with residual unfer-
4160 NARENDRANATH ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 1. Concentrations of dissolved solids over time in wheat mash fermen-

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tations at 30°C inoculated with various concentrations of L. plantarum. Most
mashes were inoculated with yeast cells at approximately 106 CFU/ml. Symbols:
h, control (no bacterial inoculation); ■, ;105 CFU of bacteria/ml; E, ;106 CFU
of bacteria/ml; F, ;107 CFU of bacteria/ml; Ç, ;108 CFU of bacteria/ml; å,
;109 CFU of bacteria/ml; j, ;107 CFU of bacteria/ml (no yeast inoculation).

mented sugar and a concomitant loss (up to 17%) of ethanol

(19). Less than 0.1% fermentable sugars remained at the end
of our fermentations, ruling out coflocculation.
When mash was inoculated with L. plantarum alone at ap-
proximately 107 CFU/ml, the bacteria did not consume more
than 1 g per 100 ml of mash dissolved solids (fermentable
carbohydrates) for growth and metabolism (Fig. 1). Similar
trends were observed in the experiments done with L. paraca-
sei, Lactobacillus #3, L. rhamnosus, and L. fermentum (data
not shown). This implies that most glucose remained to be
converted to ethanol by yeast cells, as suggested by Chin and FIG. 2. Growth of L. plantarum (A), L. paracasei (B), Lactobacillus #3 (C),
Ingledew (6). However, when two organisms grow together in L. rhamnosus (D), and L. fermentum (E) inoculated at various levels into wheat
mash fermented at 30°C. The mashes contained yeast cells at approximately 106
the same medium, there is always a competition between them CFU/ml. Bacteria were inoculated at ;105 CFU/ml (■), ;106 CFU/ml (E),
for certain nutrients. The lactic acid bacteria anaerobically ;107 CFU/ml (F), ;108 CFU/ml (Ç), ;109 CFU/ml (å), and ;107 CFU/ml (no
metabolize glucose to lactic acid to derive energy for growth yeast inoculation) (j).
and cell maintenance, and it is the production of lactic acid
which leads to a reduction in ethanol yield (since every gram of
lactic acid formed is at the expense of 0.51 g of ethanol).
Growth of lactobacilli in yeast-catalyzed fermentations. The cells, their growth rates were initially slightly lower than those
growth of the five different lactobacilli inoculated at various observed when they were grown in the presence of yeast cells.
levels into fermenting wheat mash is shown in Fig. 2. When This might be attributed to the fact that these bacteria benefit
these bacteria were grown in the absence of yeast cells, they from undetermined growth factors excreted by the yeast cells
remained viable, in contrast to the treatments with yeast cells, during growth (15, 17, 24). In contaminated yeast fermenta-
where they died off toward the end of fermentation. Moreover, tions, the final lactic acid concentration measured at 72 h is
the death rate of the bacteria increased with increases in the proportional to the number of new bacterial cells produced.
final concentration of lactic acid when lactic acid was present Once the maximum population is achieved, it is followed by the
with ethanol. This suggests that ethanol acts synergistically death of a proportion of the cells, while the survivors still
with lactic acid to kill these bacteria and that the toxicity of metabolize glucose for cell maintenance. Therefore, when high
ethanol is enhanced by the decrease in pH caused by the lactic numbers of cells are produced, higher rates of lactic acid pro-
acid in the medium. Based on the results obtained (Fig. 2), the duction occur. Under anaerobic conditions, the bacteria derive
homofermentative organisms L. paracasei and Lactobacillus energy by fermenting carbohydrates to lactic acid (homofer-
#3 (since they did not die off as fast as the other organisms) mentative strains) or to a mixture of end products, such as
appear to be more tolerant to ethanol than L. rhamnosus and ethanol, CO2, and lactic acid, and minor end products, like
L. plantarum. The former two organisms may be well-adapted acetic acid (heterofermentative strains).
to the prevailing conditions, as they are recent industrial iso- Effect of lactobacilli on growth and metabolism of yeast
lates (4). In case of L. fermentum, a heterofermentative organ- cells. As the inoculum size of L. plantarum was increased, there
ism, the amount of lactic acid produced was only 0.5% (wt/vol), were corresponding decreases in yeast growth rates. The
compared to Lactobacillus #3, which produced 1.59% (wt/vol) growth rate of yeast cells in the fermentations with the smallest
(Fig. 3). As the effect of ethanol is accentuated by the low pH bacterial inoculum (105 CFU/ml) was 0.42 h21, while the
of the medium due to lactic acid, Lactobacillus #3 died faster growth rate with the largest bacterial inoculum (109 CFU/ml)
than L. fermentum, while L. paracasei was more tolerant to was 0.36 h21. Decreases in the maximum yeast growth (Fig. 4),
ethanol even at higher lactic acid concentrations in the me- in the final ethanol concentration (Table 1), and in the final pH
dium. of the medium also were seen. This is in agreement with the
When the bacteria were grown alone in the absence of yeast results obtained by Makanjuola et al. (19). During fermenta-
VOL. 63, 1997 BACTERIA AND ALCOHOL FERMENTATION 4161

TABLE 1. Concentration of ethanol produced after fermentation of

normal-gravity (22 to 24° Plato) wheat mash at 30°C for 72 h by
yeast cells coinoculated with lactobacilli at various levelsa
Approx no. of Maximum concn of ethanol produced (%, vol/vol)b
bacteria
inoculated L. L. Lactobacillus L. L.
(CFU/ml) plantarum paracasei #3 rhamnosus fermentum

None (control) 12.71 12.46 12.24 12.71 13.14

105 12.55 12.20 11.99 12.50 13.04
106 12.40 11.99 11.77 12.32 12.90
107 12.31 11.80 11.60 12.20 12.82
108 12.19 11.65 11.44 12.07 12.75
109 11.99 11.51 11.30 11.86 12.63
a
Mashes were inoculated with approximately 106 CFU of yeast cells/ml.
b
All assays were done in duplicate with HPLC analysis. The variations in
ethanol concentrations (in duplicate assays) were in all cases less than 0.04%

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(vol/vol).

biomass quickly in order to create enough metabolic potential

to compete with yeast cells for sugar and create ethanol yield-
FIG. 3. Effect of the initial numbers of lactobacilli on the final lactic acid reducing levels of lactic acid prior to termination of fermenta-
concentration. The mashes contained yeast cells at approximately 106 CFU/ml.
Symbols: h, L. plantarum; ■, L. paracasei; E, Lactobacillus #3; F, L. rhamnosus;
tion.
Ç, L. fermentum. The increases in lactic acid with the five bacterial strains
correlated with the decreases in the final ethanol concentra-
tions (correlation coefficients, 0.92 to 0.99) and the viable yeast
numbers. The production of the metabolic end product of
tion, the concentrations of lactic acid achieved increased as the these bacteria, lactic acid, inhibits yeast growth and metabo-
levels of bacteria inoculated increased (Fig. 3). This was also lism (18) and was the major cause of the decrease in ethanol
observed by other authors (3, 19). In all experiments, the total yield in this study. In the case of L. fermentum, a heterofer-
yeast cell numbers reached a maximum at 24 h of fermentation mentative organism, the lactic acid concentrations finally
and then started to decrease (Fig. 4). Only data pertaining to achieved were not as high as those achieved with the homof-
the L. plantarum coinoculation experiment are shown. The ermentative strains used in this study (Fig. 3), yet the percent
trend was similar with the other four strains (data not shown). reduction in ethanol production by yeast cells was comparable
The effects of each Lactobacillus sp. at the five different inoc- to that of the control (about 2%) when the preparations were
ulation levels on the maximum amount of ethanol produced by inoculated at approximately 106 CFU/ml with the homofer-
yeast cells were studied in separate sets of experiments (Table mentative organisms (L. plantarum and L. rhamnosus). This
1). Single mashes were used for each experiment because be- may be partly due to the production of 0.03 to 0.05% (wt/vol)
tween experiments there were minor changes in the dissolved acetic acid by L. fermentum toward the end of fermentation
solid contents of the mashes that would have led to variations (the actual amount depended on the inoculation level). Acetic
in the final ethanol concentrations produced by yeast cells in acid is more toxic than lactic acid since it has a higher pKa than
the absence of bacteria. Yeast-mediated fermentations, as de- lactic acid. It is the undissociated form of the organic acid that
scribed above, are over within 48 h. Therefore, even when is responsible for antimicrobial activity (2), and therefore a
bacteria are present in high numbers, they must increase in higher concentration of the undissociated form of acetic acid
exists at the pH values of mash fermentation. The two acids
together have been shown to have a synergistic and negative
effect on yeast growth and metabolism (20). It has also been
found that ethanol accentuates the effect of acetic acid in the
inhibition of fermentation by yeast cells (21). Lactic acid and
acetic acid along with ethanol should have acted synergistically
on yeast growth and metabolism, resulting in a decrease in
ethanol yield. It has been reported that acetic acid, in its
undissociated form, causes an increased expenditure of energy
(ATP) for cell maintenance (18). However, data for the action
of acetic acid and lactic acid on yeasts show growth inhibition
different from that predicted on the basis of dissociation con-
stants, indicating that these acids may not act in the same
manner on yeast cells (18, 20).
Lactobacilli are extremely fastidious. They require a variety
of growth factors, like nucleotides, amino acids, and vitamins
(15). Biotin and vitamin B12 are required by a few strains (15,
FIG. 4. Growth of yeast cells in fermenting wheat mash at 30°C coinoculated 16). Biotin is also an essential growth factor for S. cerevisiae
with L. plantarum at various levels. The mashes contained yeast cells at approx- (16). Therefore, lactobacilli, when they are present in high
imately 106 CFU/ml. Symbols: h, control (no bacterial inoculation); ■, ;105
CFU of bacteria/ml; E, ;106 CFU of bacteria/ml; F, ;107 CFU of bacteria/ml;
numbers, can quickly scavenge from the medium large
Ç, ;108 CFU of bacteria/ml; å, ;109 CFU of bacteria/ml; h, ;107 CFU of amounts of the essential growth factors required by the yeast
bacteria/ml (no yeast inoculation). cells. Since the growth rates of the selected lactobacilli are
4162 NARENDRANATH ET AL. APPL. ENVIRON. MICROBIOL.

production did not affect the saccharification process. When

the activity of the glucoamylase used was assayed at various pH
levels, it was found that the enzyme retained about 91% of its
original activity at pH 4.0 and 70% of its original activity at pH
3.0 (data not shown). It has been reported that there is a
significant loss in glucoamylase activity when the pH falls be-
low 3.5 (6), but the lowest pH observed in the present study
was only 3.9.
As mentioned by Makanjuola et al. (19), studies on the
direct effects of contamination are not easily carried out. The
differences between and within batches of raw materials pro-
duce differences in chemical composition and measured fer-
mentation parameters. Raw materials themselves may harbor
contaminating bacteria which compete with yeasts for growth-
promoting nutrients. In this study, the effects of five contami-
nating lactobacilli were studied under conditions where all

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factors other than initial numbers of bacteria present in the
mash prior to yeast inoculation were controlled.
The results indicate that apart from the diversion of small
amounts (less than 1%, wt/vol) of fermentable sugar for growth
FIG. 5. Effect of the initial numbers of lactobacilli on the reduction in final of the bacteria, the production of lactic acid and a suspected
ethanol concentration compared to the control with no bacterial inoculation. The
final ethanol concentrations (Table 1) were approximately 10% (wt/vol) (12.7%, competition by the bacteria for nutrients that promote yeast
vol/vol), so the losses in produced ethanol ranged from 0 to 7.5%. The mashes growth are the important reasons for the reductions in yeast
contained yeast cells at approximately 106 CFU/ml. Symbols: h, L. plantarum; ■, growth and metabolism and, ultimately, the final ethanol yield.
L. paracasei; E, Lactobacillus #3; F, L. rhamnosus; Ç, L. fermentum. Little research has been done to determine the exact mecha-
nism of action of lactic acid on yeast growth and metabolism.
Such work is in progress.
higher than the growth rates of S. cerevisiae and lactic acid
bacteria found in breweries and food products, removal of ACKNOWLEDGMENTS
essential growth factors could result in reductions in the yeast
We thank Jaime Finguerut, Centro de Technologia Copersucar,
growth rate and catalytic activity. This would reduce the final
Piracicaba, São Paulo, Brazil, and Cargill Corn Milling, Eddyville,
ethanol yield. Iowa (David Schisler) for strains which they provided.
Results obtained in this study show that initial bacterial N.V.N. thanks the University of Saskatchewan for providing schol-
contamination of mash with approximately 106 CFU/ml led to arship support. The Western Grains Research Foundation and Natural
as much as a 2% reduction in ethanol yield compared to the Sciences and Engineering Research Council are thanked for grant
control with no bacteria (Table 1). Higher levels of bacteria support (to W.M.I.).
(109 CFU/ml) led to greater than 7% losses in produced eth-
anol. In fact, both final lactic acid concentrations and decreases REFERENCES
in ethanol yields at the end of fermentation were directly 1. Alexander, M. 1971. Microbial ecology. John Wiley & Sons, Inc., London,
United Kingdom.
correlated with initial numbers of viable bacteria in the mash 2. Baird-Parker, A. C. 1980. Organic acids, p. 126–134. In J. H. Silliker, R. P.
(Fig. 3 and 5). This is the first report which shows that there is Elliott, A. C. Baird-Parker, F. L. Bryan, J. H. B. Christian, D. S. Clark, J. C.
a linear relationship among these parameters. Likewise, there Olson, Jr., and T. A. Roberts (ed.), Microbial ecology of foods, vol. I.
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