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Applied and Environmental Microbiology, July 1999, p. 3134-3141, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Nisin Production by a Mixed-Culture System
Consisting of Lactococcus lactis and
Kluyveromyces marxianus
Hiroshi
Shimizu,
Taiji
Mizuguchi,
Eiji
Tanaka, and
Suteaki
Shioya*
Department of Biotechnology, Graduate School
of Engineering, Osaka University, Suita, Osaka 565-0871, Japan
Received 24 August 1998/Accepted 23 April 1999
 |
ABSTRACT |
To control the pH during antimicrobial peptide (nisin) production
by a lactic acid bacterium, Lactococcus lactis subsp.
lactis (ATCC11454), a novel method involving neither
addition of alkali nor a separation system such as a ceramic membrane
filter and electrodialyzer was developed. A mixed culture of L. lactis and Kluyveromyces marxianus, which was
isolated from kefir grains, was utilized in the developed system. The
interaction between lactate production by L. lactis and its
assimilation by K. marxianus was used to control the pH. To
utilize the interaction of these microorganisms to maintain high-level
production of nisin, the kinetics of growth of, and production of
lactate, acetate, and nisin by, L. lactis were
investigated. The kinetics of growth of and lactic acid consumption by
K. marxianus were also investigated. Because the pH of the
medium could be controlled by the lactate consumption of K. marxianus and the specific lactate consumption rate of K. marxianus could be controlled by changing the dissolved oxygen
(DO) concentration, a cascade pH controller coupled with DO control was
developed. As a result, the pH was kept constant because the lactate
level was kept low and nisin accumulated in the medium to a high level
compared with that attained using other pH control strategies, such as
with processes lacking pH control and those in which pH is controlled
by addition of alkali.
| |
INTRODUCTION |
Nisin is an antimicrobial peptide
produced by certain Lactococcus species. Nisin has been
accepted as a safe and natural preservative in more than 50 countries
(4, 10, 12). This peptide inhibits the vegetative growth of
a range of gram-positive bacteria. Since, in particular, nisin inhibits
the food-borne pathogens Listeria monocytogenes,
Staphylococcus aureus, and psychrotrophic enterotoxigenic Bacillus cereus, the effectiveness of nisin as a food
preservative against these organisms under various preservation
conditions has been investigated in detail (2, 20, 25).
Not only the use of nisin-producing lactic acid bacteria (LAB) as a
fermentation starter culture but also the direct addition of nisin to
various kinds of foods, such as cheese, margarine, flavored milk,
canned foods, and so on, is permitted (4). The development
of effective nisin production systems using LAB is a new field of
interest. Nisin Z is a lantibiotic bacteriocin similar to nisin A which
is produced by Lactococcus lactis IO-1 (15).
Although nisin Z has not yet been accepted as a food additive, the
effects of culture conditions such as pH, temperature, and calcium ion
concentration on nisin production were investigated. It was suggested
that an increased calcium ion concentration stimulated the production
of nisin Z by L. lactis IO-1. However, the most important
problem in nisin production is the inhibition of growth due to the
increase in lactate concentration and the decrease in pH
(24).
To avoid growth inhibition by the decrease in pH caused by the
accumulation of lactate in LAB fermentation processes, pH control methods involving the addition of alkali or the extraction of lactate
by using organic solvents have been reported (9, 27). However, the methods of extraction with organic solvents could not be
used for food additive production. Although the addition of alkali as a
means of pH adjustment was sometimes adopted for production of
fermented foods, more attention has been paid to purely natural foods,
without addition of artificial ingredients, as described in a paper
forecasting the food industry in the year 2020 (22), and
consumers who are sensitive to environmental issues are increasing in
number. In this regard, removal of lactate from the reactor in an
appropriate way will be preferable for pH control. Continuous-culture
methods for lactate fermentation processes, using free or immobilized
cells, have been reported (1, 7). Continuous culture with
separation systems such as membranes (8, 18, 21) or
electrodialyzers (11, 16, 26) also has been reported.
Immobilization of cells in an L. lactis IO-1 cultivation
process (6) and microfiltration in an L. lactis
IFO12007 cultivation process (24) enabled continuous production of nisins Z and A, respectively. In these studies, nisin was
produced continuously by keeping the cells in the fermentors. Removal
of lactate from the cultivation medium with these separation systems
precluded growth inhibition and achieved an extension of the
fermentation period. However, the total fermentation processes including these separation systems were mechanically complex and costly.
In this study, L. lactis subsp. lactis (ATCC
11454) was used as a nisin-producing microorganism, and lactate was
assimilated by a yeast, Kluyveromyces marxianus, which was
isolated from kefir grains. The developed pH control strategy, without
addition of alkali, will meet the demand of consumers who are sensitive
to environmental issues. Moreover, the mixed-culture system gives us an
example of the microorganisms' interaction and provides useful
information for future study of ecosystems. To exploit the interaction
of these microorganisms in order to maintain a high level of nisin
production, the kinetic parameters of both microorganisms, including
specific growth rates and specific rates of production of nisin and
lactate by L. lactis and the specific consumption rate of
lactate by K. marxianus, were determined in a pure culture.
Based on this information, a cascade pH control system in a batch
mixed-culture process was developed. Nisin production in a mixed
culture was compared with anaerobic and aerobic production in pure
cultures of L. lactis with and without pH control by
addition of NaOH.
| |
MATERIALS AND METHODS |
Microorganisms and media.
L. lactis subsp.
lactis ATCC 11454 (American Type Culture Collection,
Rockville, Md.) was used as a nisin-producing microorganism. K. marxianus MS1 was isolated from kefir grains in our laboratory and
identified by its morphological and biochemical properties (13). S. aureus IFO12732, which was obtained from
the collection at the Institute for Fermentation Osaka (IFO), Osaka,
Japan, was used as an indicator microorganism in the bioassay used for
measurement of nisin concentrations. The compositions of media used for
growth of microorganisms are summarized as follows. Medium a, used for seed culture and preculture of L. lactis (pH 7.0), contained
(per liter) 5 g of maltose, 5 g of polypeptone (Nihonseiyaku,
Tokyo, Japan), and 5 g of yeast extract (Difco Laboratories,
Detroit, Mich.). Medium b, used for seed culture and preculture of
K. marxianus (pH 7.0), contained (per liter) 10 g of
L-lactate, 10 g of polypeptone, and 10 g of yeast
extract. Medium c, used for the primary culture of L. lactis, contained maltose at 10 g/liter (in anaerobic cultures without pH control) or at 33 to 37 g/liter (in both anaerobic and
aerobic cultures with pH control [pH 6.0]), 10 g of polypeptone per liter, and 10 g of yeast extract per liter. Medium d, used for
primary cultures of K. marxianus (pH 6.0), contained (per liter) 40 g of L-lactate, 10 g of polypeptone,
and 10 g of yeast extract. Medium e, used for mixed culture of
L. lactis and K. marxianus (pH 6.0), contained
(per liter) 42 g of maltose, 10 g of polypeptone, and 10 g of yeast extract. Medium f, used for bioassay of nisin (pH 7.0),
contained (per liter) 10 g of glucose, 5 g of polypeptone,
5 g of yeast extract, and 5 g of NaCl. Selective medium g,
used for determination of L. lactis CFU (pH 7.0), contained (per liter) 5 g of maltose, 5 g of polypeptone, 5 g of
yeast extract, 5 mg of cycloheximide (Wako, Osaka, Japan), and 15 g of agar. Selective medium h, used for determination of K. marxianus CFU (pH 7.0), contained (per liter) 5 g of glucose,
5 g of polypeptone, 5 g of yeast extract, 5 mg of
streptomycin (Nacalai tesque, Kyoto, Japan), and 15 g of agar.
Initial maltose concentrations in the pH-controlled anaerobic,
pH-controlled aerobic, and mixed cultures were 33.2, 36.8, and 41.4 g/liter, respectively.
Analysis.
Cell concentrations of the pure cultures were
measured by determining the dry cell mass and optical density (OD). For
determination of dry cell mass, the cells were filtered with a membrane
filter (pore size, 0.45 µm; ADVANTEC, Tokyo, Japan) and dried in an
oven at 70°C. ODs at 660 nm (OD660s) were measured at 660 nm with a UV spectrophotometer (model UV-2000; Hitachi, Tokyo, Japan).
The viable-cell concentrations of L. lactis and K. marxianus in mixed cultures were determined as CFU on selective
media g and h, respectively. The relationship between dry-cell
concentration (dry weight [DW]) and CFU was approximated linearly by
the least-squares method-derived correlation coefficients of data, and
estimated values were also calculated by using the determined line
(5). Concentrations of L-lactate, acetate, and
formate in the medium were analyzed enzymatically by using F-Kit
Lactate, F-Kit Acetate, and F-Kit Formate (Boehringer, Mannheim,
Germany), respectively. Ethanol concentrations were measured with a gas
chromatography apparatus (Hitachi model G-3000). Glucose concentrations
were measured with a glucose analyzer (model 2700; YSI Inc., Yellow
Springs, Ohio). Maltose concentrations were measured after hydrolysis
to glucose as follows. A 100-µl volume of 2 N HCl was added to an
equal volume of the sample, and the solution was boiled at 100°C for
20 min. Then 200 µl of 1 N NaOH was added, and the glucose
concentration was measured with a glucose analyzer. The calibration
curves for ethanol and maltose concentrations were determined linearly
by the least-squares method. Accuracy of measurements was evaluated by
using correlation coefficients (5).
Nisin concentrations were measured by a bioassay method based on the
method of Matsuzaki et al. (14) as follows. Five milliliters of medium f was inoculated with S. aureus IFO12732 and
incubated on a reciprocal shaker (100 strokes/min) at 30°C for
12 h. Fifty microliters of the cell suspension of S. aureus and 50 µl of the sample solution were added to 5 ml of
fresh medium, and the cell suspension was incubated on the reciprocal
shaker under the same conditions. After 12 h, the cell
concentration was determined by measuring the OD660, using
a UV spectrophotometer (model U-2000; Hitachi). A calibration curve was
made for each new nisin concentration measurement, using commercially
available nisin as a standard (Sigma, St. Louis, Mo.; 1,000 IU/mg of
solid; nisin content, 2.5% by weight). The sample was diluted so that
the OD660 was in the range of 0.1 to 1.5 absorbance units
because in this range the nisin concentration was linearly related to
the OD660. A calibration curve for bioassay of the nisin
was prepared for each fermentation experiment by the least-squares
method (5). Nisin concentration was represented by
concentration by weight (in milligrams per liter), and a nisin
concentration of 1 mg/liter was equivalent to 40 IU/ml (15,
24).
Cultivation methods.
All microorganisms in the growth phase
were stored in 20% (vol/vol) glycerol at 
80°C. Before cultivation
in 5-liter jar fermentors was performed, culture size was scaled up by
two steps in order to increase the proportion of cells with high growth
activity; first, a culture was started in test tube (the so-called seed culture), and second, culture size was scaled up to 500-ml Erlenmeyer flasks (for preculture) and, finally, 5-liter jar fermentors (for primary cultures). For seed cultures of L. lactis, 10 ml of
medium a was inoculated with 50 µl of a stock solution and statically incubated at 30°C for 6 h. Preculture of L. lactis
was performed in 500-ml Erlenmeyer flasks containing 200 ml of medium a
and also 200 µl of the seed medium. The flask was statically
incubated at 30°C for 10 h, and harvested cells were inoculated
into the primary-culture medium. In the seed culture of K. marxianus, 5 ml of medium b was inoculated with 50 ml of stock
solution and incubated on a reciprocal shaker at (100 strokes/min) at
30°C for 6 h. Preculture of K. marxianus was
performed in 500-ml Erlenmeyer flasks with 100 ml of medium b and 100 µl of seed medium. The flask was incubated in the same way as the
seed culture. After 16 h, the cells were harvested by
centrifugation at 10,000 × g for 15 min and used to
inoculate the primary-culture medium.
Primary cultures were performed in 5-liter jar fermentors (EPC Control
Box, Eyla, Tokyo, Japan) equipped with temperature,
pH, dissolved
oxygen [DO]) concentration, and gas flow control
systems. The working
volume was 2 liters. The pCO
2 in the exhaust
gas was
measured with a CO
2 gas analyzer (model VBI-210; Horiba,
Kyoto, Japan). Air or nitrogen was supplied to the fermentor for
aerobic or anaerobic cultivation conditions, respectively. The
CO
2 production rate
(
rCO2, in moles per hour) was
determined as
shown in equation 1:
|
(1)
|
where
rAF, pCO
2,
pCO
2*,
R, and
T are the air flow rate
(in liters per hour), partial CO
2 pressure in the exhaust
gas from
the fermentor (in atmospheres), partial CO
2
pressure in the air
(in atmospheres), gas constant (0.08206 liter
· atm K
1 mol
1), and fermentation
temperature (in degrees Celsius), respectively.
The accumulation of
CO
2 was determined by integrating the CO
2 production rate. The cascade controller developed for this system
has
more than one output per manipulation (
23). In this study,
the cascade control strategy was applied to control the pH level
via DO
control by manipulation of the agitation speed. The control
strategy
was coded in N
88BASIC (NEC, Tokyo, Japan) on a personal
computer (NEC model PC-9801BX). The control strategy and inoculum
conditions in the mixed culture are described
later.
Calculation of kinetic parameters.
The kinetic parameters,
such as specific rates, were evaluated by the least-squares method
(5). An example for the specific production rate of nisin is
as follows. The material balance of nisin production is represented as
shown in equation 2:
![<FR><NU>d([<UP>N</UP>]V)</NU><DE>dt</DE></FR>=&rgr;<SUB><UP>N</UP></SUB>(VX<SUB>L</SUB>)](/content/vol65/issue7/fulltext/3134/img002.gif)
|
(2)
|
where [N],
V,
XL, and
N
are the nisin concentration, culture volume, cell concentration of
L. lactis, and specific production
rate of nisin,
respectively. Equation
2 was integrated as shown
in equation 3:
![<LIM><OP>∫</OP><LL>[N]<SUB>0</SUB>V<SUB>0</SUB></LL><UL>[<UP>N</UP>]V(t)</UL></LIM>d([<UP>N</UP>]V)=<LIM><OP>∫</OP><LL>0</LL><UL>t</UL></LIM> &rgr;<SUB><UP>N</UP></SUB>(VX<SUB>L</SUB>)dt](/content/vol65/issue7/fulltext/3134/img003.gif)
|
(3)
|
where 0 indicates the initial value of a variable. If
N is constant, equation 3 can be rewritten equation 4:
![[<UP>N</UP>]V−[<UP>N</UP>]<SUB><UP>0</UP></SUB>V<SUB>0</SUB>=&rgr;<SUB><UP>N</UP></SUB> <LIM><OP>∫</OP><LL>0</LL><UL>t</UL></LIM> (VX<SUB>L</SUB>)dt](/content/vol65/issue7/fulltext/3134/img004.gif)
|
(4)
|
If the plot of [N]
V versus the integral of
VXL is a linear relationship, it indicates that
N is constant and can be estimated
from the slope of the
line by the least-squares
method.
| |
RESULTS |
Statistical evaluation of regression lines for measurement and
determination of kinetic parameters.
The relationship between DW
and CFU of L. lactis is described by the equation DW = 2.02 × 109 CFU + 0.012 (correlation
coefficient of data and estimated values [r2] = 0.99). The relationship between DW and OD660 of
L. lactis is described by the equation DW = 0.404 × OD660 + 2.9 × 103
(r2 = 0.99). Theses relationships were
obtained from the experiment using a pure culture of L. lactis. The relationship between DW and CFU of K. marxianus is described by the equation DW = 1.86 × 108 CFU (r2 = 0.99). The
relationship between DW and OD660 of K. marxianus is described by the equation DW = 0.270 × OD660 + 0.103 (r2 = 0.99).
These relationships were obtained from the experiment using a pure
culture of K. marxianus. All of the correlations with
measured and standard values of calibration curves for ethanol and
maltose concentrations were evaluated with acceptable accuracy by
chemical analysis (r2 = 0.95 to 0.99). The
correlation coefficient of the regression line for nisin concentration
was always larger than 0.995, which was also acceptable statistically.
All of the kinetic parameters of L. lactis and K. marxianus are shown in Tables 1 and
2, respectively. The correlation
coefficients for all of the parameter estimations obtained by the
least-squares method are also shown in these tables. The basis for
determining the kinetic parameters over the first 5 h and from
5 h to the completion of the experiment at 8, 12, or 11 h was
mainly due to the fact that growth under anaerobic conditions without
pH control ceased after 5 h. However, as a result, the
determinations reflected the differences between the log and stationary
phases of growth. Moreover, the consistency of the basis for these
determinations was checked by determining the correlation coefficients
of the linear line because the data included lag, log, and stationary
phases of growth if they existed. Almost all of the correlation
coefficients of the kinetic parameters were above 0.9, and it was
concluded that the estimated values of the kinetic parameters were
consistent.
Nisin production without and with pH control.
The time course
of nisin production by L. lactis under growth under
anaerobic conditions without pH control is shown in Fig. 1. The pH of the medium fell below 5.0 within 3 h. Cell growth was completely terminated after 6 h,
at which time the concentration of nisin was 7.4 mg/liter. After the
cessation of growth, the nisin concentration decreased slightly. When
maltose was used as the carbon source, not only lactate but also
ethanol and acetate were produced (data not shown), even under
anaerobic conditions. The change in carbon metabolism due to the carbon
source and aeration is discussed later. The kinetic parameters obtained
from this experiment are summarized in Table 1. The specific growth
rate of L. lactis (µL) and the
specific rate of production of nisin (N) without pH
control were 0.30 h1 and 4.0 mg of nisin/g of cells/h,
respectively.
The time course of nisin production by
L. lactis under
anaerobic conditions at pH 6.0 is shown in Fig.
2. The cell concentration
and the nisin
concentration increased during cultivation to a
greater extent than
they did without pH control; however, µ
L was
decreased after 5 h and nisin production reached a maximum value
of 58 mg/liter (Fig.
2). µ
L was 0.73 h
1 for 0 to 5 h of batch culture, which was much
higher than the
value attained without pH control (Table
1); however,
after 5
h, µ
L decreased to 0.25 h
1.
N changed in proportion to
µ
L (Table
1).
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|
FIG. 2.
Nisin production by L. lactis with pH control
under anaerobic conditions. The pH was controlled at 6.0 by the
addition of NaOH.
|
|
Aerobic culture of L. lactis.
Aerobic cultivation of LAB
is not common, but the aerobic growth of, and production of lactate by,
some species have been reported (3). For lactate to be
effectively assimilated by K. marxianus, aerobic conditions
must be used. Hence, growth of L. lactis under aerobic
conditions was investigated. The pH and dissolved oxygen (DO)
concentration were maintained at 6.0 (by addition of NaOH) and 2.0 mg/liter, respectively. The results are shown in Fig.
3. As shown in Table 1,
µL and N from 0 to 5 h of
aerobic batch culture were slightly lower than the values obtained
under anaerobic conditions. Furthermore, µL
after 5 h under aerobic conditions was lower than it was under
anaerobic conditions, but N under aerobic conditions was
higher than it was under anaerobic conditions. As a result, the
production yield of lactate with respect to maltose and the lactate
concentration at 13 h were lower than the values obtained under
anaerobic conditions (Table 1). Lactate, formate, acetate, and ethanol
were formed from maltose under anaerobic conditions. On the other hand,
lactate, acetate, ethanol, and CO2 were formed from maltose
under aerobic conditions.
Aerobic fermentation of K. marxianus.
The specific
lactate consumption rate of K. marxianus was determined
under aerobic conditions. The effect of the DO concentration on the
rate of lactate consumption is shown in Fig.
4. The maximum specific rate of lactate
consumption by K. marxianus (
L)
was about 0.7 g of lactate/g of cells/h (Table 2), which was higher than the maximum specific rate of lactate production by L. lactis under aerobic conditions. Thus, it was expected that the
lactate produced by L. lactis would be completely consumed
by K. marxianus. L decreased
linearly as the DO concentration decreased in the range below 2 mg/liter, as shown in Fig. 4. When the lactate concentration was
expected to be decreased, the DO level should have been increased and
the specific rate of lactate consumption by K. marxianus
would have been enhanced. On the other hand, when the lactate
concentration was expected to be increased, the DO level should have
been decreased and the specific rate of lactate consumption by K. marxianus would have been attenuated. These strategies were
realized by a cascade controller of pH with DO concentration control in
the mixed culture (see below). The maximum specific growth rate of
K. marxianus (µK) in pure culture
at a high DO level was lower than µL (Table 2).
Dynamics of pH in the mixed culture of L. lactis and
K. marxianus with a cascade controller.
The dynamics
of concentrations of lactate and acetate ([L] and [A],
respectively) are represented by equations 5 and 6, respectively:
![<FR><NU>d[<UP>L</UP>]</NU><DE>dt</DE></FR>=&rgr;<SUB><UP>L</UP></SUB>X<SUB>L</SUB>−&ngr;<SUB><UP>L</UP></SUB>X<SUB>K</SUB>](/content/vol65/issue7/fulltext/3134/img005.gif)
|
(5)
|
![<FR><NU>d[<UP>A</UP>]</NU><DE>dt</DE></FR>=&rgr;<SUB><UP>A</UP></SUB>X<SUB>L</SUB>](/content/vol65/issue7/fulltext/3134/img006.gif)
|
(6)
|
where
XL,
L, and
A are the cell concentration and the specific
production rates of lactate and acetate of
L. lactis,
respectively,
and X
K and
L are the cell concentration and specific
lactate consumption rate of
K. marxianus, respectively. For
overall
electroneutrality balance, the total cation concentration in
the
medium has to be equal to the anion concentration. The
H
+ ion concentration (10
pH), OH
ion concentration (10
pH 14), dissociated lactate
ion concentration, and dissociated acetate
ion concentration were
balanced with the ionic concentrations
of acid and base, except lactate
and acetate, as shown in equation
7:
![X<SUB><UP>acid</UP></SUB>−X<SUB><UP>base</UP></SUB> =10<SUP><UP>−pH</UP></SUP> −10<SUP><UP>pH</UP> −14</SUP> − <FR><NU><FR><NU>[<UP>L</UP>]</NU><DE><UP>MW<SUB>L</SUB></UP></DE></FR></NU><DE>1+10<SUP><UP>−pH</UP>+<UP>pK<SUB>L</SUB></UP></SUP></DE></FR> (<UP>Term1</UP>) − <FR><NU><FR><NU>[<UP>A</UP>]</NU><DE><UP>MW<SUB>A</SUB></UP></DE></FR></NU><DE>1 + 10<SUP><UP>−pH + pK</UP><SUB><UP>A</UP></SUB></SUP></DE></FR> (<UP>Term 2</UP>)](/content/vol65/issue7/fulltext/3134/img007.gif)
|
(7)
|
where X
acid and X
base are the total
dissociated ion concentrations of acid and base, except lactate and
acetate; MW
A and
MW
L are molecular weights of
acetate and lactate, respectively;
and pK
L and
pK
A are the pK values of lactate and acetate, respectively.
Terms 1 and 2 in equation 7 correspond to the dissociated lactate
ion
concentration and the dissociated acetate ion concentration,
respectively. By rewriting the right-hand side of equation 7 as
a
nonlinear function of pH, L, and A as f(pH, L, A) and differentiating
equation 7 with respect to time (
t), equation 8 is obtained:
![<FR><NU>d(<UP>X<SUB>acid</SUB> − X</UP><SUB><UP>base</UP></SUB>)</NU><DE>dt</DE></FR>=<FENCE><FR><NU>∂<UP>f</UP></NU><DE>∂<UP>pH</UP></DE></FR></FENCE><FENCE><FR><NU>d<UP>pH</UP></NU><DE>dt</DE></FR></FENCE>+<FENCE><FR><NU>∂<UP>f</UP></NU><DE><UP>∂</UP>[<UP>L</UP>]</DE></FR></FENCE><FENCE><FR><NU>d[<UP>L</UP>]</NU><DE>dt</DE></FR></FENCE>](/content/vol65/issue7/fulltext/3134/img008.gif)
|
(8)
|
Because the changes in X
acid and X
base are
negligible compared with the changes in the concentrations of lactate
and acetate,
the dynamics of the pH change with time is described as
shown
in equation 9:
![<FR><NU>d<UP>pH</UP></NU><DE>dt</DE></FR>=<FR><NU>1</NU><DE><FENCE><FR><NU>∂<UP>f</UP></NU><DE>∂<UP>pH</UP></DE></FR></FENCE></DE></FR><FENCE>−<FENCE><FR><NU>∂<UP>f</UP></NU><DE>∂[<UP>L</UP>]</DE></FR></FENCE><FENCE><FR><NU>d[<UP>L</UP>]</NU><DE>dt</DE></FR></FENCE>−<FENCE><FR><NU>∂<UP>f</UP></NU><DE><UP>∂</UP>[<UP>A</UP>]</DE></FR></FENCE><FENCE><FR><NU>d[<UP>A</UP>]</NU><DE>dt</DE></FR></FENCE></FENCE>](/content/vol65/issue7/fulltext/3134/img010.gif)
|
(9)
|
By substituting equations 5 and 6 into equation 9, equation 10 is
obtained:
![<FR><NU>d<UP>pH</UP></NU><DE>dt</DE></FR>=<FR><NU>1</NU><DE><FENCE><FR><NU><UP>∂f</UP></NU><DE><UP>∂pH</UP></DE></FR></FENCE></DE></FR><FENCE><FR><NU><UP>−</UP><FENCE><FR><NU>∂<UP>f</UP></NU><DE><UP>∂</UP>[<UP>L</UP>]</DE></FR></FENCE></NU><DE><UP>MW</UP><SUB><UP>L</UP></SUB></DE></FR>(<UP>&rgr;<SUB>L</SUB></UP>X<SUB>L</SUB>−&ngr;<SUB><UP>L</UP></SUB>X<SUB>K</SUB>)<UP> − </UP><FR><NU><FENCE><FR><NU>∂f</NU><DE>∂[<UP>A</UP>]</DE></FR></FENCE></NU><DE><UP>MW<SUB>A</SUB></UP></DE></FR>&rgr;<SUB>A</SUB>X<SUB>L</SUB></FENCE>](/content/vol65/issue7/fulltext/3134/img011.gif)
|
(10)
|
The terms (
f/
pH), (
f/
[L]), and (
f/
[A]) in
equation 10 are positive, and the rate of production of acetate at the
beginning
of the batch culture is negligible. Thus, it is easily
understood
that if the rate of lactate production by
L. lactis is higher
than the rate of lactate consumption by
K. marxianus
that is,
the term
(
LXL L
XK) is positive
the pH decreases. On the other
hand, when the rate of consumption of lactate is higher than its
rate
of production
that is, the term
(
LXL L
XK) is negative
the
pH increases. Then, the
rate of lactate consumption by
K. marxianus must be
increased or decreased depending on whether the pH is
above or below
the set point of 6.0, as long as lactate exists
in the medium. The
specific rate of lactate consumption by
K. marxianus
(
L) can be controlled within a limited range (0 to
0.7 g of lactate/g of cells/h) by changing the DO level (0 to
2 mg/liter) as shown in Fig.
4. The DO concentration in the medium
can be
controlled by manipulating the agitation speed of the impeller.
Finally, it was found that the pH of the medium of the mixed-culture
system could be controlled by changing the DO control set point.
This
type of controller is categolized as a cascade controller
(
23). The cascade controller of pH coupled with DO control
developed
here is shown below (see Fig.
5). Proportional and integral
(PI)
and proportional, integral, and differential (PID) controllers
were used as precompensators of DO and pH in the cascade controller,
respectively.
The dynamic response resulting from the change in pH due to the DO
change, shown in box A of Fig.
5, was as
follows: when
the DO changes,
L changes according to the
relationship between
DO and
L shown in Fig.
4. Note that
Fig.
4 shows a static relationship.
However, when the DO level changes
dynamically, there is a dynamic
time delay from DO change to
L, because cell lactate assimilation
activity occurs
after changes in activities of many enzymes due
to the change in DO
concentration. The change in pH is based on
the dynamics described by
equation 10. The dynamic response of
the agitation speed of the
impeller to the DO level, shown in
box B of Fig.
5, is as follows: when
the agitation speed changes,
the mass transfer rate of oxygen from air
bubble to liquid changes,
with a very small time delay, and the
dynamics of DO are described
by the balance between oxygen supply from
air bubble to liquid
and the rate of consumption of oxygen by both
microorganisms.
The automatic control strategies of pH and DO are
described later.
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|
FIG. 5.
Scheme of a cascade pH controller incorporating DO
control. Two outputs of pH and DO were measured by pH and DO sensors,
respectively. Manipulation was only via agitation speed (AGT), which
changed the DO, and the DO changed the pH via lactate consumption by
K. marxianus. The pH was controlled by the DO set point,
RDO.
|
|
Inoculum size of mixed culture.
The ratio of L. lactis and K. marxianus inoculum sizes was determined
so that the lactate concentration was constant at the initial time of
the operation, i.e., the start of the fermentation, as shown below. If
lactate accumulates to a high level in the medium at the beginning of
mixed culture, the growth rate of L. lactis is dramatically
decreased, causing a fatal condition. For a reliable mixed-culture
operation, the L was overestimated as 2.0 g of
lactate/g of cells/h, which was three times higher than the actual data
(Table 1). On the other hand, L was initially set to
0.5 g of lactate/g of cells/h. Then, the ratio of
XL to XK was set to 0.25, which could be derived by setting the right-hand side of equation 5 to
zero, which is equivalent to the lactate concentration being constant.
For the same reasons, the right-hand side of equation 10 becomes zero,
which means that the pH does not change. After all, before the
primary-culture experiment started, the concentrations of both
microorganisms in the preculture was determined by measuring the
OD660 and the inoculum size was set as
XL/XK = 0.25 in the subsequent experiments.
Nisin production with pH cascade control in a mixed culture at pH
6.
The time course of a mixed culture of L. lactis and
K. marxianus is shown in Fig.
6. The pH set point was 6.0. The DO set point was not obtained automatically but was manually changed in this
case for the first trial. The lactate concentration was kept at almost
zero throughout the experiment. After 2 h, the DO level was
increased and the L was enhanced because the pH decreased slightly from the set point of 6.0. The cascade control of pH
succeeded. Both L. lactis and K. marxianus were
growing exponentially until 11 h. The nisin production rate
reached a maximum of 98 mg/liter.
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|
FIG. 6.
Nisin production in a mixed culture of L. lactis and K. marxianus. The pH was controlled at 6.0 by the cascade controller.
|
|
Even though acetate accumulated (4 g/liter), the decrease in pH was
less than that observed with lactate accumulation (Fig.
1), because
when maltose was used as a carbon source under anaerobic
conditions,
not only lactate but also acetate and formate were
produced. When
lactate was produced at 2 g/liter, acetate and
formate were produced at
1.5 and 2.5 g/liter, respectively. The
decrease in pH evident in Fig.
1
was due to production of lactate,
acetate, and formate. On the other
hand, formate was not produced
under aerobic conditions; instead,
CO
2 was produced. Figure
6 shows that lactate accumulated
at a rate of 0.45 g/liter in the
early stages of fermentation (at
2 h) and that it was consumed
by
K. marxianus for
6 h. The pH decreased during the 0- to 2-h
period due to the
accumulation of lactate. Consumption of lactate
after 2 h
counteracted the accumulation of
acetate.
Biomass production by the mixed culture was also compared with that of
a pH-controlled anaerobic pure culture of
L. lactis.
In an
anaerobic pure culture of
L. lactis, the dry-weight cell
concentration was 2.6 g/liter after 13 h. The dry-weight cell
concentration of
L. lactis in the mixed culture could not be
measured
directly but was estimated by using the relationship between
DW
and CFU, which was obtained from the experiment using a pure culture
of
L. lactis. The estimated dry-weight cell concentration of
L. lactis in the mixed culture was 3.4 g/liter, which was
higher
than that in the pure culture. The fact that the rate of nisin
production in the mixed culture was also higher than that in the
pure
culture indicates that nisin production was growth
associated.
Figure
7 shows the automatic cascade
control results for the coupling of pH with DO control in the mixed
culture. PI and PID
control strategies for the automatic control of DO
and pH controllers
are described in the
Appendix. The control
parameters of the PI
(DO) controller
sampling time (
t),
proportional gain (
Kp), and
integral time
(
Ti)
are set to 0.5 min, 40 rpm · liter/mg, and
2.0 min, respectively. The control parameters of the PID
(pH)
controller
pH set point (RpH), sampling time (
t),
proportional
gain (
Kp), integral time
(
Ti), and derivative time
(
Td)
are set
to 6.0, 0.5 min, 0.5 mg/liter,
12.5 min, and 6.0 min, respectively.
The control parameters of PI and
PID controllers were tuned so
that pH fluctuation was less than ±0.5
units.
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|
FIG. 7.
Control of pH and DO by the cascade controller without
addition of NaOH in the mixed-culture system.
|
|
As shown in Fig.
7, because the set point of DO at time zero for the DO
controller [RDO(0)] was 1.0 mg/liter, the actual value
of DO
decreased rapidly at the beginning of the experiment and
returned to
the set point within 2 h. When the pH decreased below
the set
point at around 3 h, the RDO increased according to equation
A4
(see
Appendix). As a result, the
L recovered
and the pH increased
again to around 4 h due to the decreasing
lactate concentration.
By changing the RDO, the pH was reliably and
accurately controlled
at the set point of 6.0. To improve the
performance of the controller,
a predictive controller with an
estimator of the lactate consumption
activity may have to be developed.
Also, to analyze the stability
of the controller in detail, the dynamic
responses of the activity
of microorganisms to changes in environmental
conditions must
be investigated
precisely.
| |
DISCUSSION |
In this study, a novel method of pH control, utilizing the
interaction between two microorganisms was developed. Nisin production in a mixed culture (98 mg/liter) was higher than that in a pure culture
(58 mg/liter). In an anaerobic pure culture of L. lactis without pH control, the amount of nisin produced was quite small. When
the pH was controlled by addition of NaOH, nisin production in
anaerobic cultivation was increased slightly compared with that
attained under aerobic conditions. However, production ceased at 58 mg/liter because of growth inhibition, not due to the decrease in pH
but rather due to the increase in the lactate concentration. In a mixed
culture with cascade pH control, the produced nisin concentration was
over 98 mg/liter (3,920 IU/ml), which was 1.7 times higher than that in
the anaerobic culture with pH control via addition of NaOH.
It was reported that the maximum production of nisin Z (3,150 IU/ml)
was obtained in batch culture with pH control via addition of NaOH
(15). It is difficult to compare these two methods directly because the species of microorganisms used and kinds of bacteriocins produced were different; however, because the undissociated lactate and
low pH strongly inhibited the growth of LAB (9), the
complete removal of lactate might be more effective for maintaining a
high rate of cell growth and production of the growth-associated
product than the system using the pH control via addition of alkali.
Nisin production by a microfiltration technique also has been reported
(24). The maximum production rate with this method was 78 IU/ml/h. The cultivation method developed in our study was a batch
method, so the production rate was evaluated as the amount produced
divided by the volume and the production time and was found to be 360 IU/ml/h. Another reason why the microfiltration method gave a low
production rate may be adsorption of nisin onto the membrane filter.
The proposed mixed-culture system was mechanically simple because the
system utilized the interaction between two microorganisms. This method
requires careful selection of the carbon source. In this study, maltose
was selected. To develop the cascade controller, aerobic conditions
were necessary. With this limitation, it might be difficult to extend
this methodology to other mixed-culture systems. Recently, we isolated
from cheese different strains of a yeast which cannot assimilate
lactose but can assimilate lactate. In this case, lactose is used as a
carbon source. Thus, it should also be stressed that by careful
selection of the system, the principle presented here can be made
available for use with another carbon source and other combinations of microorganisms.
It is expected that this mixed-culture system will become an example of
microbial interactions and will provide technical information for
further studies. For example, the difference between pH control by
addition of alkali and that by mixed culture in terms of nisin
production will be analyzed more precisely in the future. For this, the
effect of the controlled levels of lactate concentration and DO on the
lactate and nisin production rates of L. lactis as well as
the lactate consumption rate of K. marxianus in the mixed
culture should be measured directly or estimated precisely.
CO2 formation by L. lactis was observed under
aerobic conditions. A C4 compound such as acetoin or
butanediol might be produced under aerobic conditions, because
CO2 formation indicated that pyruvate dehydrogenase was
active under these conditions (17). In glucose and lactose
media, homofermentation by L. lactis was observed. It was
reported that when maltose was assimilated, heterofermentation of
L. lactis that is different from glucose metabolism occurs (19). The change of molar flux in the metabolic pathway
should be analyzed in the future.
| |
APPENDIX |
PI (DO) control strategy. For the DO controller shown
in Fig. 5, the PI controller used is represented by the equation
![<UP>AGT</UP>(t)=K<SUB>p</SUB><FENCE>[<UP>RDO</UP>(t)−<UP>DO</UP>(t)]+<FR><NU>&Dgr;t</NU><DE>T<SUB>i</SUB></DE></FR><LIM><OP>∑</OP><LL>i=0</LL><UL>t</UL></LIM> [<UP>RDO</UP>(i)−<UP>DO</UP>(i)]</FENCE>](/content/vol65/issue7/fulltext/3134/img012.gif)
|
(A1)
|
where AGT(
t) and DO(
t) are the agitation
speed of the impeller and DO at time
t, RDO(
t) is
the set point of DO control,
t is the sampling time,
Kp is the proportional gain, and
Ti is the
integral time in the controller,
respectively. In the conventional
PI controller, RDO(
t) was
usually treated as a constant value,
but it was given as the output of
the pH controller in the cascade
controller. The PI controller was
actually used as a velocity
form by rewriting equation A1 as
![+[<UP>RDO</UP>(t)−<UP>RDO</UP>(t−1)]+<FR><NU>&Dgr;t</NU><DE>T<SUB>i</SUB></DE></FR>[<UP>RDO</UP>(t)−<UP>DO</UP>(t)]}](/content/vol65/issue7/fulltext/3134/img014.gif)
|
(A2)
|
where the initial value of AGT, AGT(0), was set to 100 rpm. In
the velocity form of the controller, AGT(
t) was realized by
correcting AGT(
t 1). Although equations A1 and A2
are completely
equivalent, the overshoot would be reduced when
manipulated variables
of AGT have upper and lower
limits.
PID (pH) control strategy. For the pH controller, a
PID controller was used as shown in equation A3:
![<UP>RDO</UP>(t)=K<SUB>p</SUB><FENCE>[<UP>RpH − pH</UP>(t)]+<FR><NU>&Dgr;t</NU><DE>T<SUB>i</SUB></DE></FR><LIM><OP>∑</OP><LL>i=0</LL><UL>t</UL></LIM> [<UP>RpH − pH</UP>(i)]+<FR><NU>T<SUB>d</SUB></NU><DE>6&Dgr;t</DE></FR> [<UP>pH</UP>(t−6)−<UP>pH</UP>(t)]</FENCE>](/content/vol65/issue7/fulltext/3134/img015.gif)
|
(A3)
|
where pH(
t), RpH, and
Td are
the pH at time
t, the pH set point, and the derivative time
in the controller, respectively.
In this controller, the derivative of
the pH was estimated by
subtracting the present pH from the pH data at
time (
t 6). The
PID controller was used as a
velocity form in the same way as
the PI (DO) controller as shown in
equation A4:
![<UP>RDO</UP>(t)=<UP>RDO</UP>(t−1)+K<SUB>p</SUB><FENCE>[<UP>pH</UP>(t−1)−<UP>pH</UP>(t)]+<FR><NU>&Dgr;t</NU><DE>T<SUB>i</SUB></DE></FR> [<UP>RpH − pH</UP>(t)]+<FR><NU>T<SUB>d</SUB></NU><DE>6&Dgr;t</DE></FR> [<UP>−pH</UP>(t)+<UP>pH</UP>(t−1)+<UP>pH</UP>(t−6)−<UP>pH</UP>(t−7)]</FENCE>](/content/vol65/issue7/fulltext/3134/img016.gif)
|
(A4)
|
RDO at time zero, RDO(0), was set 1.0 mg/liter. Because there was
a lengthy delay between the DO change and the change in
the specific
lactate consumption rate of
K. marxianus responses,
the
derivative correction term in the controller was very important
for
detecting the pH change, while there was a short delay from
the
agitation change to the DO
response.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. Phone and fax: 81-6-6879-7444. E-mail: shioya{at}bio.eng.osaka-u.ac.jp.
| |
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Applied and Environmental Microbiology, July 1999, p. 3134-3141, Vol. 65, No. 7
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[Abstract]
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Top
Abstract
Introduction
![+<FENCE><FR><NU>∂<UP>f</UP></NU><DE><UP>∂</UP>[<UP>A</UP>]</DE></FR></FENCE><FENCE><FR><NU>d[<UP>A</UP>]</NU><DE>dt</DE></FR></FENCE>](/content/vol65/issue7/fulltext/3134/img009.gif)
![<UP>AGT</UP>(t)=<UP>AGT</UP>(t−1)+K<SUB>p</SUB>{[<UP>DO</UP>(t−1)−<UP>DO</UP>(t)]](/content/vol65/issue7/fulltext/3134/img013.gif)
