effect of lactobacilli on yeast growth, viability and batch and semi-continuous alcoholic...

Effect of lactobacilli on yeast growth, viability and batchand semi-continuous alcoholic fermentation of corn mash

K.C. Thomas, S.H. Hynes and W.M. IngledewDepartment of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Canada

532/8/00: received 30 August 2000, revised 22 January 2001 and accepted 31 January 2001

K.C. THOMAS, S.H . HYNES AND W.M. INGLEDEW. 2001.

Aims: The aim of this study was to evaluate interactions between Saccharomyces cerevisiae and

selected strains of lactobacilli regarding cell viabilities, and production of organic acids and

ethanol during fermentation.

Methods and Results: Corn mashes were inoculated with yeasts and selected strains of

lactobacilli, and fermented in batch or semi-continuous (cascade) mode. Ethanolic fermentation

rates and viabilities of yeast were not affected by lactobacilli unless the mash was pre-cultured

with lactobacilli. Then, yeast growth was inhibited and the production of ethanol was reduced

by as much as 22%.

Conclusions: Yeasts inhibited the multiplication of lactobacilli and this resulted in reduced

production of acetic and lactic acids. The self-regulating nature of the cascade system allowed

the yeast to recover, even when the lactobacilli had a head start, and reduced the size of the

population of the contaminating Lactobacillus to a level which had an insigni®cant effect on

fermentation rate or ethanol yield.

Signi®cance and Impact of the Study: Contamination during fermentation is normally

taken care of by the large yeast inoculum, although yeast growth and fermentation rates could

be adversely affected by the presence of high numbers of lactobacilli in incoming mash or in

transfer lines.

INTRODUCTION

Fuel alcohol is produced by fermenting sugars derived from

starches of cereal grains (Thomas and Ingledew 1990, 1995;

Thomas et al. 1993, 1995; Wang et al. 1998), from tuber

crops (De Menezes 1978; Win et al. 1996), or by the direct

use of sugars in molasses and sugar cane juice (Lewis 1996).

In North America, corn is the most commonly used source

of starch for the production of fuel alcohol. Starches derived

through wet or dry milling are gelatinized, lique®ed,

sacchari®ed and then fermented with active dry yeast.

Traditionally, fuel alcohol has been produced by batch

fermentation of mashes containing 200±250 g l±1 dissolved

solids. Mashes are usually supplemented with nutrients to

promote yeast growth and thus stimulate fermentation.

Contamination of fermentors with micro-organisms can

result in stuck or sluggish fermentation and in the produc-

tion of undesirable end products (Pampulha and Loureiro-

Dias 1989; Alexandre and Charpentier 1998; Ingledew

1999). The bacteria themselves and their metabolic end

products lead to reduction in ethanol yield (Dolan 1979;

Barbour and Priest 1988; Makanjuola et al. 1992; Narendr-

anath et al. 1997) and considerable economic loss to the

producer.

It is therefore of great advantage to the producer if the

onset of the bacterial contamination can be predicted, and

suitable remedies taken, before problems arise. The presence

of detectable amounts of acetic acid in the fermentor may be

indicative of heavy bacterial contamination. Acetic acid is

known to affect yeast growth and metabolism, and it has

been suggested as one of the causes of stuck and sluggish

fermentation (Rasmussen et al. 1995; Huang et al. 1996;

Edwards et al. 1999). The cause of acetic acid production is

not always bacterial contamination since the yeast itself

produces this acid as a minor end product. If, however,

acetic acid is detected in substantial amounts before the end

of fermentation, it may be an indication that bacterial

contamination has occurred.

Correspondence to: W.M. Ingledew, Department of Applied Microbiology and

Food Science, University of Saskatchewan, 51 Campus Drive, Saskatoon, SK,

Canada S7N 5A8 (e-mail: [email protected]).

ã 2001 The Society for Applied Microbiology

Journal of Applied Microbiology 2001, 90, 819±828

In this study the effects on alcoholic fermentation of

deliberate contamination of corn mashes with different

lactobacilli was investigated. To simulate heavy contamin-

ation with lactobacilli, the bacteria were pre-cultured in corn

mash before initiating alcoholic fermentation with active

dry yeast. In addition, the effects of yeast on growth and

metabolism of lactobacilli and survival of contaminating

bacteria in semi-continuous alcoholic fermentation were

investigated.

MATERIALS AND METHODS

Micro-organisms

Active dry yeast (ADY) obtained from the Alltech Biotech-

nology Center, Nicholasville, Kentucky, USA was used

throughout this study. This yeast, a polyploid strain of

Saccharomyces cerevisiae, is widely used for industrial-scale

production of fuel alcohol. Four different isolates of

lactobacilli obtained from industrial alcohol plants were

used in this study. These organisms were tentatively

identi®ed by the API 50 CHL testing kits (bioMerieux,

Montreal, PQ, Canada). Lactobacillus collinoides and Lact.fermentum were isolated from separate mixed culture sam-

ples collected from two commercial fermentors (Corn Plus,

Winnebago, MN, USA). Lactobacillus plantarum and Lact.paracasei subsp. paracasei were isolated from mixed cultures

obtained from Copersucar, Centro de Technologia, Pirac-

icaba, SP, Brazil. They were kept at 4 °C on MRS (deMan±

Rogosa±Sharpe) agar slants (Oxoid).

Preparation of corn mash and fermentation

Corn mashes were prepared using feed corn purchased from

a local supplier. Corn was ground by passing it twice

through a plate grinder (Model S500, Glen Mills, Clifton,

NJ, USA). Eighty-six percent of this grind had a particle

size ®ner than 20 mesh. The ground corn (758 g) was

dispersed in 2250 ml of deionized water at 55 °C. To

minimize viscosity development during gelatinization of

starch, a small amount (3á8 ml) of HT-a-amylase (Alltech

Biotechnology Center) was added. Calcium, a stabilizer of

HT-a-amylase, was added in the form of calcium chloride

solution to give a ®nal concentration of 1 mmol l±1. The

temperature of each slurry was raised to 97 °C and held at

that temperature for 60 min. Slurries were continuously

stirred during the cooking stage. The gelatinized starch was

cooled to 80 °C and then lique®ed by adding another 3á8 ml

HT-a-amylase and allowing the enzyme to react for a

further 30 min. During liquefaction, the starch was com-

pletely hydrolysed to dextrins and oligosaccharides as

indicated by negative tests for starch using iodine. The

total yield of each mash was 2500 ml and contained about

230 g l±1 dissolved solids in the supernatant fraction. The

mashes were supplemented with urea (a yeast nutrient) at a

concentration of 16 mmol l±1.

Fermentation of corn mash

Corn mash was distributed in 150 ml quantities into sterile,

250 ml Erlenmeyer ¯asks and sacchari®ed at 30 °C by

adding 0á225 ml glucoamylase (Allcoholase II, Alltech

Biotechnology Center). The mashes were inoculated to

predetermined cell numbers with preconditioned active dry

yeast, a 24-h-old Lactobacillus culture grown in MRS broth,

or with both yeast and a selected bacterial culture. Samples

were withdrawn at regular intervals for analysis.

Enumeration of bacterial and yeast cells

Yeast cells were enumerated either by direct microscopic

count or by determining colony-forming units (cfu) on yeast

extract-peptone-dextrose agar by the membrane ®ltration

technique. Lactobacilli were enumerated by determining the

cfu on MRS agar by the membrane ®ltration technique and

incubating the plates at 30 °C in a CO2 incubator (Model

3630, National Appliance Co., Portland, OR, USA). In some

cases, the percentage viability of yeast cells was determined

by the methylene blue technique reported previously

(Thomas and Ingledew 1990).

Determination of total dissolved solidsand fermentation products

Total dissolved solids were estimated indirectly by measur-

ing, at 20 °C, the speci®c gravity of the supernatant portion

of the mash as described previously (Thomas and Ingledew

1990). Sugars, ethanol, glycerol, lactic acid and acetic acid

were determined by high performance liquid chromatogra-

phy (HPLC) using a Waters Chromatographic System

(Waters Corporation, Milford, MA, USA). Supernatant

portions of mash obtained by centrifugation (10 300 g,

15 min) were ®ltered through a Millipore membrane

(0á22 lm pore size), diluted with distilled water, and 5 ll

of the diluted sample were injected into an Aminex HPX-

87H column (Bio-Rad Laboratries, Hercules, CA, USA)

maintained at 40 °C. Deionized water (Milli-Q) containing

5 mmol l±1 sulphuric acid was used as the eluant. The

elution rate was 0á7 ml min±1 and boric acid was used as the

internal standard. The separated components were detected

with a differential refractometer (Model 410) and quanti®ed

using the Millennium32 Chromatography Manager compu-

ter program both supplied by Waters Corporation.

820 K.C. THOMAS ET AL .

ã 2001 The Society for Applied Microbiology, Journal of Applied Microbiology, 90, 819±828

RESULTS

Fermentation of corn mash

Fully sacchari®ed corn mash was inoculated with bacteria

alone, with yeast alone or with bacteria and yeast together.

Both organisms were inoculated into mash at a level of

1á0 ´ 107 ml±1 cells. Mashes inoculated with bacteria at 0 h

were incubated for 24 h and then inoculated with yeast

to give 1á0 ´ 107 ml±1 cells. The amounts of carbohydrate

consumed by lactobacilli in the absence of yeast varied from

1á2 g l±1 (Lact. collinoides) to 6á6 g l±1 (Lact. paracasei). The

rates of consumption of carbohydrate after inoculation

with the yeast were about the same whether the yeast was

growing alone, growing in combination with the added

bacteria, or growing after the 24 h delayed inoculation

(Fig. 1). Fermentation proceeded at about the same rate and

all of the sugars were consumed in less than 36 h.

Although all of the sugars were used up, the presence of

lactobacilli in mash resulted in decreased ethanol yield and

an increased production of both glycerol (Table 1) and lactic

acid (Table 2). The loss of ethanol yield was as high as 22%

if Lact. collinoides or Lact. fermentum was grown in the mash

for 24 h prior to inoculation with the yeast. If the yeast and

the bacteria were inoculated at the same time, ethanol loss

amounted to 4á6±12%. Decreased growth of the lactobacilli

corresponded to reduced loss of ethanol yield. No glycerol

was produced by any of the Lactobacillus strains growing

alone in the corn mash, while the yeast always produced

small amounts of glycerol. It appears that the presence of

lactobacilli in the mash stimulated the production of glycerol

by the yeast. In the case where Lact. fermentum was

inoculated 24 h prior to the yeast, glycerol production

increased by 46%.

Fig. 1 Changes in total carbohydrate during fermentation of corn

mash at 30 °C by yeast, or by a combination of yeast and a chosen

Lactobacillus. Open symbols: yeast and Lactobacillus culture inoculated

at 0 h. Filled symbols: yeast inoculated 24 h after the bacterial culture.

Yeast alone (´); Lact. collinoides (s, d); Lact. fermentum (h, j); Lact.

plantarum (n, m); Lact. paracasei (e, r). The inoculation level of each

organism was 1á0 ´ 107 cells ml)1 of the mash. The results are average

of duplicate determinations

Table 1 Amounts of ethanol and glycerol produced by fermentation

of corn mash by Saccharomyces cerevisiae in the presence or absence of

four different strains of lactobacilli. The mashes were inoculated with

the chosen Lactobacillus strain, and yeast was added at 0 or 24 h

Time of yeast Ethanol Glycerol

Treatment inoculation (h) (g l)1) (g l)1)

Yeast alone 0 127á7 (100) 6á53 (100)

Lact. collinoides 0 119á5 (94) 7á05 (108)

24 99á6 (78) 6á93 (106)

Lact. fermentum 0 124á4 (97) 8á03 (123)

24 99á5 (78) 9á55 (146)

Lact. plantarum 0 121á8 (95) 6á80 (104)

24 117á6 (92) 8á60 (132)

Lact. paracasei 0 112á4 (88) 6á70 (103)

24 105á9 (83) 8á09 (124)

Values are average of duplicate determinations. Figures in the

parentheses indicate production of ethanol or glycerol as a percentage

of that observed in the control.

Table 2 Production of lactic acid by active dry yeast (Saccharomyces

cerevisiae) and by combination of active dry yeast and selected

lactobacilli

Time of

yeast

inoculation

Maximum

lactic acid

produced

Lactic acid at

the end of

fermentation

Treatment (h) (g l)1) (g l)1)

Yeast alone 0 0á3 (36) 0 (48)

Lact. collinoides 0 2á0 (24) 0á9 (48)

24 8á5 (48) 4á1 (72)

Lact. fermentum 0 1á6 (24) 0á4 (48)

24 8á8 (24) 4á3 (72)

Lact. plantarum 0 5á2 (24) 2á4 (48)

24 17á2 (36) 5á1 (72)

Lact. paracasei 0 6á3 (24) 5á1 (48)

24 11á9 (36) 6á9 (72)

Values are average of duplicate determinations. Numbers in

parentheses are the time in hours when the above lactic acid

concentrations were observed.

LACTOBACILLI AND YEAST VIABIL ITY 821


Yeast growth

Fewer yeast cells were produced if bacteria had a head start

of 24 h before yeast was introduced into the fermentor

(Fig. 2). A maximum of 3á2 ´ 108 ml±1 yeast cells was

produced in the absence of bacterial inoculation, while

the maximum cell number declined to 2á2 ´ 108 ml±1 if the

heterofermentative bacterium, Lact. fermentum, was allowed

to grow for 24 h before the yeast was inoculated into the

mash. The effect of the homofermentative Lact. plantarumon yeast growth was less severe, but its inhibitory effect was

evident whether the yeast was inoculated at the same time as

the bacterium or later.

Viability of yeast in the presence of lactobacilli

Bacterial growth and metabolism affected yeast growth. The

yeast cells lost viability at a rapid rate, and to a greater

extent, if bacteria had a chance to grow in the mash prior to

the yeast inoculation (Fig. 3). In the absence of lactobacilli,

viability of the yeast cells remained above 90% for a longer

time after the completion of fermentation (exhaustion of

sugars) than in their presence. Pre-culturing lactobacilli in

the mash did cause loss of viability of yeast cells to varying

degrees. With the heterofermentative Lact. fermentum pre-

cultured in the mash for 24 h prior to the yeast inoculation,

the viability of the yeast cells declined to 45% within 12 h

after the exhaustion of sugars.

Bacterial growth

The loss of yeast viability after completion of fermentation

was related to the extent of bacterial growth. For example,

Lact. fermentum grew to a maximum of 1á8 ´ 109 cfu ml±1

Fig. 2 Growth of yeast during fermentation of corn mash. The

mash was inoculated with 1á0 ´ 107 cells ml)1 of (a) Lactobacillus

fermentum or (b) Lact. plantarum at 0 h. Yeast inoculation was done

(s, j) at 0 h or (h) at 24 h. The open circle represents the control

that did not receive bacterial culture

Fig. 3 Changes in the viability of yeast cells during fermentation

of corn mash, with or without various lactobacilli in the mash. The

control treatment (s) received at 0 h yeast as the only inoculum. In

other treatments, the mashes were inoculated at 0 h with Lact.

collinoides (d), Lact. fermentum (j), Lact. plantarum (m), or Lact.

paracasei (r), and with yeast 24 h after the bacterial inoculation. The

results are average of duplicate determinations



within 24 h if the mash was not inoculated with the yeast.

Even when the mash was inoculated with the yeast and the

bacterium at the same time, bacterial growth still occurred

but was reduced by 94% (1á2 ´ 108 cfu ml±1). Yeast growth,

however, was not affected (Fig. 2a). Starting with 1á0 ´ 107

cells ml±1, the fast growing homofermentative Lact. paracaseimultiplied to 4á6 ´ 109 cfu ml±1 within 24 h in the absence of

added yeast, while it grew to only 2á1 ´ 109 cfu ml±1 in the

presence of the yeast. The yeast inoculated into the mashes

in which the lactobacilli were grown for 24 h (by which

time the bacteria had grown to the maximum) grew and

fermented the sugars completely. However, up to 55% of

the yeast cells lost viability within 12 h after the completion

of fermentation (Fig. 3). When growth of the lactobacilli

was poor, as when the yeast and the bacteria were

inoculated together, the yeast did not lose viability (data

not shown). Nevertheless, 98% of both bacterial popula-

tions (Lact. fermentum and Lact. paracasei) died within 48 h

after the addition of the yeast to mashes in which the

lactobacilli were pre-cultured before inoculating with the

yeast.

Production of lactic and acetic acids

The yeast by itself produced small amounts of lactic acid but

not enough to make any signi®cant impact on alcohol

productivity or yeast metabolism (Table 2). The homofer-

mentative Lact. plantarum and Lact. paracasei produced

17á2 g l±1 and 11á9 g l±1 of lactic acid, respectively. The

amounts of lactic acid produced by the facultatively

heterofermentative Lact. collonoides and Lact. fermentumwere much less than those observed for the homofermen-

tative organisms. If the mash was inoculated with bacteria

and yeast simultaneously, lactic acid production declined

considerably. This was to be expected since yeast inhibited

growth of these lactobacilli. The percentage inhibition of

lactic acid production was greater for the heterofermentative

Lact. collonoides and Lact. fermentum than for the homofer-

mentative Lact. plantarum or Lact. paracasei.After reaching a maximum, the lactic acid contents of the

mashes declined in all cases where the yeast was present,

indicating probable uptake and utilization of lactic acid

by the yeast. The beginning of the decline of lactic acid

coincided with the exhaustion of sugars from the medium.

Effect of acetic acid on yeast growthand viability

Saccharomyces cerevisiae under certain conditions can pro-

duce trace amounts of acetic acid, and it can also use acetic

acid as the sole source of carbon if the pH of the medium is

kept at about 5á0 or higher (Casal et al. 1996). At lower pH

values, acetic acid is a potent inhibitor of yeast growth. It has

been reported that acetic acid at a concentration as low as

0á5 g l±1 can inhibit yeast growth (Maiorella et al. 19831 ). In

the absence of any bacterial cells, the Saccharomyces yeast

produced 0á69 g l±1 acetic acid by the end of fermentation.

In spite of this concentration of acetic acid in the mash, the

yeast did not lose viability as rapidly as when lactobacilli

were inoculated into the mash (Fig. 3). No acetic acid was

detected until after 36 h, by which time no detectable

fermentable sugars remained in the mash. Acetic acid

production by the yeast seemed to be triggered by the

depletion of glucose, and it may also be related to the

availability of oxygen.

The amounts of acetic acid produced in the treatment

where the heterofermentative lactobacilli (Lact. collonoidesand Lact. fermentum) and yeast were co-inoculated were

similar to those produced by the yeast alone (Fig. 4). If these

lactobacilli were added 24 h prior to the yeast inoculation,

Fig. 4 Production of acetic acid by (a) Lactobacillus collonoides, (b) Lact. fermentum and (c) Lact. plantarum during fermentation of corn mash with

active dry yeast. The mash was inoculated at 0 h with the yeast alone (s), with the bacterial culture and yeast at 0 h (d), or with bacterial culture at

0 h and yeast at 24 h (h). The results are an average of duplicate determinations



acetic acid production started early and reached much

higher values. At the time of yeast inoculation of the mash in

which Lact. fermentum was pre-grown for 24 h, the acetic

acid concentration was 1á1 g l±1, and it increased to 2á0 g l±1

towards the end of fermentation. This may have contributed

to the decreased proliferation of yeast cells and to the

increased loss of yeast cell viability (Figs 2 and 3).

Only small amounts of acetic acid were produced before

the exhaustion of fermentable sugars (the end of fermenta-

tion) in experiments where homofermentative lactobacilli

(Lact. plantarum and Lact. paracasei) were included. After

this point, the levels of acetic acid in the treatments where

the yeast was added 24 h after the bacteria, increased to

approximately twice the amount produced by yeast alone.

It is not clear whether this increase was due to yeast, or

bacterial metabolism, or both. No acetic acid, however, was

produced by these two lactobacilli growing alone (data not

shown), although the bacteria had multiplied several fold.

This indicates that these lactobacilli do not produce acetic

acid under normal fermentation conditions, but it does not

rule out the possibility that they may produce small amounts

under more aerobic conditions when the carbohydrates have

been depleted and CO2 evolution has ceased. It has been

reported that `homofermentative' lactobacilli in the absence

of hexoses may catabolize pentoses aerobically to produce

acetic acid (Kandler 1983).

The viability of S. cerevisiae declined rapidly after the

completion of fermentation if lactobacilli had become

established in the fermentor before the yeast had a chance

to grow and multiply. It can therefore be stated that

lactobacilli and yeast are antagonistic to each other. It is

apparent that other factors, in addition to lactic and acetic

acids, may be involved in the inhibition of yeast growth and

loss of viability (Huang et al. 1996; Alexandre and Char-

pentier 1998; Edwards et al. 1999). These may include

nutritional de®ciencies and/or inhibitory substances such

as toxic fatty acids (Alexandre and Charpentier 1998).

Ethanol produced by the yeast plays an important role in

the inhibition of lactobacilli (Narendranath et al. 1997).

However, since the studies reported above simulated batch

operation, the results obtained and the conclusions drawn

from them may not truly re¯ect the current continuous

or semi-continuous fermentation beginning to be adopted

by industry. For this reason, a set of experiments, using

cascade fermentation, was conducted. The aim of the

experiments was to give a bacterial culture, which showed

maximal inhibitory effect on yeast growth and viability, a

head start and to see how it performed and survived during

repeated fermentations. For this, 200 ml of a fully-saccha-

ri®ed corn mash was inoculated with 2á67 ml of a 24-h-old

MRS broth culture of heterofermentative Lact. fermentumand incubated for 24 h at 30 °C. The mash was then

inoculated with active dry yeast and incubated for another

24 h. At this time, half of the fermented mash was removed

and replaced with an equal amount of fresh, fully-saccha-

ri®ed mash. The cycle was repeated several times. The

fermented mash removed each time was analysed for yeast

cell number, viability, total dissolved solids, and metabolites

such as lactic acid, acetic acid, glycerol and ethanol. A

control set of experiments without the bacteria was also

conducted.

The results showed that in the absence of Lact. fermentum,

the yeast multiplied and reached a maximum of 3á2 ´ 108

cells g±1 mash in the ®rst cycle. If Lact. fermentum was grown

for a period of 24 h before inoculating with the yeast, the

maximum yeast cell number attained was only 2á2 ´ 108

cells g±1 mash (a reduction of 31%). The decreased

proliferation of the yeast may be related to the production

by the bacteria of metabolites that are toxic to the yeast

and/or to the depletion of some nutrients essential for yeast

growth (Fig. 5a). In subsequent fermentation cycles, the

inhibitory effects of metabolites (for example, ethanol) in the

control (with no bacteria) started to in¯uence the multipli-

cation of yeast cells, leading to an initial decline in cell

number followed by a gradual stabilization. It is apparent

that by the fourth fermentation cycle, the maximum number

of yeast cells produced was about the same in the control and

in the mash inoculated with yeast and bacteria.

The viability of the yeast cells in the control remained

above 95% throughout all fermentation cycles, while in the

experiments that had received Lact. fermentum at the start,

the yeast viability declined in the early fermentation cycles.

The viability then gradually increased and by the sixth

fermentation cycle, it was equal to that observed in the

control. The changes in viability determined by the

methylene blue technique (Fig. 5b) correlated with changes

in the total cell number of yeasts. These changes seemed to

be related to the number of bacterial cells surviving in the

mash (see the results below).

Surprisingly, the yeast cell number in mashes which were

inoculated with Lact. fermentum also stabilized by the fourth

fermentation cycle to a value similar to that in the control.

It is apparent that the bacteria, which had a head start of

24 h and had multiplied to a high number (1á9 ´ 109 cfu

ml±1), did not seem to have multiplied since2 the mash was

inoculated with the yeast (Fig. 6). By the seventh fermen-

tation cycle, the bacterial number had declined to 2á4 ´ 104

cfu ml±1. The yeast counts, on the other hand, stabilized to

a value of 1á3 ´ 108 cfu ml±1 after an initial decline. Total

visual count by the microscope method (Fig. 5) always gave

a greater estimate of the cell population than the cfu

determined by the plate count method (Fig. 6). In these

cascade fermentations, the decline in the production of

lactic and acetic acids (Fig. 7) corresponded to the decrease

in bacterial cell number (Fig. 6). The steady state concen-

trations of acids attained after repeated cycles were low, and



it appeared that they were not high enough to cause

inhibition of yeast growth or to affect the rate of

fermentation.

DISCUSSION

It is clear from the results presented here that contamination

of corn mashes with different lactobacilli during the course

of alcoholic fermentation by batch fermentation may result

in (a) decreased ethanol yield, (b) increased channeling of

carbohydrates for the production of glycerol and lactic acid,

(c) a rapid loss of the yeast viability after exhaustion of

fermentable sugars, (d) decreased proliferation of the yeast

in the mash in which the contaminating lactobacilli had

already grown to a high number, and (e) inhibition of

growth of lactobacilli in mash in which the yeast was present

in high numbers or in mashes which were inoculated with

the lactobacilli and then yeast in equal numbers.

Under the semi-continuous system (simulated cascade

fermentation), the contaminating bacterial population, even

when the initial cell number was high, declined in number

over successive cycles and reached a level low enough to

cause no major problem in terms of ethanol yield or yeast

viability. Following an initial decline, the yeast population

increased and recovered to the level of the control. The

concentrations of lactic acid and acetic acid declined

proportionally with the decline of the bacterial population.

In general, it can be stated that unless the incoming mash or

transfer lines themselves are contaminated, the self-regula-

ting feature of a semi-continuous system (and most likely a

continuous system as well) would restore the yeast popu-

lation and maintain the fermentation without appreciable

loss in ethanol yield or yeast viability. Published work on

Fig. 5 Maximum cell number (a) and percentage viability (b) of yeast

populations at the end of successive fermentation cycles in a semi-

continuous (cascade) fermentation system with (j) and without (h)

the heterofermentative Lactobacillus fermentum in the mash. In the

experimental set up, the mash was inoculated ®rst with the Lactoba-

cillus culture grown for 24 h, at which time the yeast was added at the

beginning of the ®rst cycle. The results are an average of duplicate

determinations3

Fig. 6 Viable counts of Saccharomyces cerevisiae (d) and Lactobacillus

fermentum (h) during successive (cascade) fermentation cycles. The

mash was inoculated with the Lactobacillus culture 24 h prior to the

addition of the yeast. The number of cells of each organism added

initially to the mash was 1á0 ´ 107 ml)1



Lactobacillus contamination of alcohol plants deals mainly

with effects of products of bacterial metabolism on yeast

growth and metabolism. Not only can the lactobacilli

tolerate low pH, high acidity, and relatively high concen-

trations of ethanol, but they also multiply under the

conditions of alcoholic fermentations. It is fairly well

established that the products of metabolism of lactobacilli

affect yeast growth and viability (Rasmussen et al. 1995;

Huang et al. 1996; Alexandre and Charpentier 1998;

Edwards et al. 1999) and a number of mechanisms of

inhibition have been proposed (Pons et al. 1986; Rasmussen

et al. 1995; Pampulha and Loureiro-Dias 1989, 1990, 2000),

but not much attention has been paid as to the nature of

inhibition of lactobacilli during the course of alcoholic

fermentation by yeast. This study has shown that if the yeast

cell number is high or at least equal to the number of cells

of lactobacilli, growth of lactobacilli is inhibited. At least

two reasons may be suggested for the reduced growth of the

bacteria in the mash. First, there is a reduced availability of

essential nutrients because of the competition from the

yeast, which leads to decreased bacterial growth and

multiplication. Since the yeast cell size is approximately

20±50 times greater than that of bacterial cells, on a cell basis

a greater proportion of nutrients would be taken up by yeast

cells. Second, the alcohol produced by the yeast can exert

inhibitory effects on the multiplication of lactobacilli.

Unlike in wine making where acetic acid produced by

contaminating lactobacilli can result in `stuck' fermentation

(Rasmussen et al. 1995; Alexandre and Charpentier 1998),

no `stuck' fermentation was observed during alcoholic

fermentation of grain mashes in the presence of various

lactobacilli. Similar observations were made by Chin and

Ingledew (1994) and by Narendranath et al. (1997) who

studied the effect of lactobacilli on wheat mash fermenta-

tions. Growth of the yeast and rates of fermentation were,

however, affected by the presence of lactobacilli in mash.

These effects were pronounced if the contamination by

Lactobacillus was heavy, or if the bacteria had a chance to

grow for a period of time in the mash in the absence of yeast.

The ethanol yield was reduced signi®cantly, in some cases

by as much as 22%. Such high losses were associated with

heavy contamination of mash with heterofermentative

lactobacilli. In general, it can be concluded from the results

reported here and those published previously (Narendranath

et al. 1997) that the loss in ethanol yield is related to the

level of bacterial contamination. Makanjuola et al. (1992)

also observed that increasing additions of bacteria produced

progressive decreases in ethanol, ranging from 6 to 22%

with the highest loss occurring in the presence of a massive

bacterial inoculum. These workers suggested that reduction

in ethanol yield was caused, at least in part, by the

incomplete utilization of the carbohydrates. In the study

reported here, the utilization of fermentable sugars was

Fig. 7 Concentrations of lactic acid (a) and acetic acid (b) at the end of

successive fermentation cycles in a semi-continuous (cascade)

fermentation system with (j) and without (h) the heterofermentative

Lactobacillus fermentum in the mash. In the control experiment, the

mash was inoculated at the beginning of the ®rst cycle with active dry

yeast. In the experimental set up where both organisms were used, the

mash was inoculated ®rst with the Lactobacillus culture and grown for

24 h and then the yeast was added at the beginning of the ®rst cycle.

The results are average of duplicate determinations4



complete, and yet there was a substantial decrease in ethanol

yield. The loss of this magnitude could not be explained in

terms of sugars being diverted for bacterial growth,

production of acetic acid or lactic acid, or by increased

synthesis of glycerol alone. It is not clear whether the

observed gradual decrease of lactic acid from the medium

(Table 2) was the result of its uptake by the yeast and

lactobacilli or its conversion to ethyl lactate. Small amounts

of ethyl lactate were detected in samples at the end of

fermentation (results not shown). As expected, formation of

ethyl lactate would result in decreased ethanol yield.

Although none of the lactobacilli growing in the absence

of yeast produced any glycerol, production of glycerol by

yeast increased in the presence of lactobacilli. The reason for

this enhanced production of glycerol is not known, although

increased synthesis of glycerol as a compatible solute by

yeast in response to osmotic stress has been reported (Meikle

et al. 1988). There appears to be a relationship between

acetic acid production and the amount of glycerol produced

by yeast. It has been reported that selective breeding of yeast

strains to enhance glycerol production resulted in a threefold

increase in acetic acid-producing capacity (Michnick et al.1997). Similarly, Prior et al. (1999) also observed increased

production of glycerol by yeast when there were elevated

levels of acetic acid synthesis. However, it must be pointed

out that relatively large amounts of glycerol could be

produced by some strains of yeast without a parallel increase

in acetic acid production. It is clear from these reports that

both metabolites (glycerol and acetic acid) were produced by

yeast. As far as is known, no information is available about

the effects of acetic acid produced by bacteria on glycerol

production by yeast. Observation suggests that there may be

such a relationship. It is not clear whether altered nutritional

conditions due to the presence of lactobacilli in the medium

caused the increased production of glycerol, or whether the

yeast increased its production to combat the `stress' caused

by the products of bacterial metabolism such as lactic and

acetic acids. This aspect needs further study.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the Natural Sciences

and Engineering Research Council of Canada, and Chip-

pewa Valley Ethanol Company, Delta T Corporation and

Corn Plus Co-operative for grants which supported this

project.

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