Ann Microbiol (2015) 65:1473–1480
DOI 10.1007/s13213-014-0986-9
ORIGINAL ARTICLE
A new method studying the kinetics of L-lactic acid production
by pellets Rhizopus oryzae in semi-continuous fermentation
Xuefeng Wu & Shaotong Jiang & Min Zhang &
Shuizhong Luo & Xingjiang Li & Lijun Pan & Zhi Zheng &
Mo Liu
Received: 20 May 2014 / Accepted: 16 September 2014 / Published online: 22 November 2014
# Springer-Verlag Berlin Heidelberg and the University of Milan 2014
Abstract L-Lactic acid production by Rhizopus oryzae
in a semi-continuous fermentation system has been rec-
ognized as a promising technology. We studied L-lactic
acid production in ten batches of semi-continuous fer-
mentation by R. oryzae using a 7-L stirred fermentor
and found that the kinetics of the system was in good
agreement with the logistic and Luedeking–Piret model
equations. In particular, in order to mathematically cal-
culate the actual specific growth rate in each repeated
batch, we assumed that the new biomass was “zero” at
the beginning of each repeated batch, and the same
method was used to calculate the parameters of the
product formation kinetic. The biomass remaining from
the previous batches played an important role in the L-
lactic acid production in each repeated batch. During the
repeated fermentation, the values of δ (the efficiency of
the reused biomass maintained in the former batch for
producing L-lactic acid (%)) all exceeded 90 %, but the
values of QPm (maximum L-lactic acid production rate)
decreased gradually, possibly due to the breaking-down
and autolysis of the biomass concomitant with the
growth of new fungi, which led to the reduction in
the maximum specific growth rate (μm).
Keywords L-Lactic acid . Kinetics . Rhizopus oryzae .
Semi-continuous fermentation
X. Wu (*) : S. Jiang : M. Zhang : S. Luo : X. Li : L. Pan :
Z. Zheng : M. Liu
School of Biotechnology and Food Engineering, Institute of
Agricultural Products Processing Technology, the Key Laboratory
for Agricultural Products Processing of Anhui Province, Hefei
University of Technology, No.193 Tunxi Road,
Hefei 230009, Anhui, People’s Republic of China
e-mail: applewuxf@hotmail.com
Introduction
L-Lactic acid is a commonly occurring organic acid of great
economic value due to its wide use in food-related industries
and its potential applications in the production of biodegrad-
able polylactate polymers (Jin and Wang 2000; Efremenko
et al. 2006; Rojan et al. 2007; Zhang et al. 2007; Maneeboon
et al. 2010). In industrial-scale production systems, L-lactic
acid is usually produced by bacteria and fungi (Zhao et al.
2010; Mohamed et al. 2013). Rhizopus oryzae has been the
focus of much research for several years since it is one of the
organisms that produces mostly L-lactic acid as a sole isomer
of lactic acid. These studies mainly focused on the develop-
ment of reliable, efficient and cost-effective fermentation
methods, such as the fungi morphology control methods,
and on processes using fungi immobilization or free fungal
pellets to improve the concentration of the L-lactic acid pro-
duced (Bai et al. 2003; Tanaka et al. 2006; Liu et al. 2006;
Liao et al. 2007; Liu et al. 2008; Wang et al. 2010). A semi-
continuous fermentation system for L-lactic acid production
using Rhizopus species has recently been developed. Due to
the absence of the lag phase for the cell growth and product
accumulation in the repeated fermentation batches using this
fungi (Du et al. 1998; Yin et al. 1998; Martak et al. 2003; Yu
et al. 2007; Wang et al. 2013), productivity has been
improved.
A number of studies on the kinetics of batch or con-
tinuous fermentation have been published. A considerable
amount of data on L-lactic acid biosynthesis by R. oryzae
are available in these reports, but they provide little infor-
mation on the kinetics of L-lactic acid and/or other organic
acid production by fungi in semi-continuous or repeated
fermentation systems. It is becoming increasingly impor-
tant to understand the kinetics of the fermentation system
since the advantages and economics of semi-continuous
fermentation are relevant worldwide.
1474
Here, we describe L-lactic acid production in a 7-L stirred
fermentor by semi-continuous fermentation using R. oryzae
AS 3.819 pellets. The kinetics modeling was studied by
measuring the biomass of the fungus, the concentration of L-
lactic acid and the concentration of substrate in each batch.
The logistic and Luedeking–Piret model (Luedeking and Piret
1959) equations were used to calculate the kinetics data in the
semi-continuous fermentation system.
Materials and methods
Microorganism and microbiological growth media
Rhizopus oryzae AS 3.819 was purchased from China General
Microbiological Culture Collection Center, preserved in the
Laboratory of the Key Laboratory for Agricultural Products
Processing of Anhui Province, Institute of Agricultural
Prod ucts Processing Technolog y, th e S chool of
Biotechnology and Food Engineering, Hefei University of
Technology and used in this study. This R. oryzae strain
was first inoculated into potato-dextrose agar (PDA) slants
and then incubated at 32 °C for about 3 days until all spores
were black. The fungal spores were collected with a platinum
loop and suspended in sterilized water, following which the
spore concentrations were adjusted to 5× l06 spores/mL (Wu
et al. 2011).
First batch of the semi-continuous fermentation system
A 200-mL [4 % (v/v) of the sterile nutrient broth] sample of the
fungi spore suspension (adjusted to 5×l06 spores/mL) was
inoculated into a 7-L stirred fermentor containing 5 L of sterile
nutrient broth [12 % (w/v) glucose; 0.4 % (w/v) ammonium
sulfate; 0.014 % (w/v) monopotassium phosphate; 0.016 %
(w/v) sodium dihydrogen phosphate; 0.044 % (w/v)
heptahydrate; 0.025 % (w/v) magnesium sulfate heptahydrate].
The parameters of the fermentor were as described by Wu et al.
(2011). The fermentation conditions for the first batch were an
approximately 42 hours fermentation period at 32 °C with an
aeration rate of 1.0 L/min and an agitation speed of 300 rpm.
Sterilized 0.6 % (w/v) calcium carbonate was added to the
culture medium at the beginning of the fermentation to prevent
a decrease in pH. Samples were taken out every 6 h.
Repeated batch of the semi-continuous fermentation system
At the end of each batch, the agitation and aeration were
stopped until the mycelial pellets had precipitated onto the
bottom of the fermentor, at which time 4.5 L of nutrient broth,
without pellets, was pumped out and then 4.5 L of sterile
nutrient broth [10 % (w/v) glucose, 0.2 % (w/v) ammonium
sulfate, 0.03 % (w/v) monopotassium phosphate, 0.022 %
Ann Microbiol (2015) 65:1473–1480
(w/v) heptahydrate, 0.025 % (w/v) magnesium sulfate
heptahydrate and 0.5 % (w/v) calcium carbonate] was added
to the fermentor under sterile conditions. When this transfer of
medium was completed, the fermentor was restarted under the
same conditions as those for the first batch, with the exception
that the fermentation period was now 20 h and samples were
taken out every 4 h.
Analytical methods
L-Lactic acid was extracted from the fermented medium with
0.5 M H2SO4, diluted with distilled water and filtered through a
0.22-μm membrane. The concentration of the extracted L-lactic
acid was analyzed using a high-performance liquid chromatog-
raphy system equipped with a Purospher STAR C18 column
[250×4.6 (5 μm); Merck & Co., White House Station, NJ] and
a UV detector at 210 nm. The eluent was 5 mM H2SO4 and the
flow rate was 0.8 mL/min. The residual glucose concentration
was determined by the dinitrosalicylic acid method. Dry cell
weight was determined by washing the mycelial pellets twice
with 4 M HCl to remove the residual calcium carbonate and
then drying the washed biomass at 80 °C for 24 h before weight
analysis (Wu et al. 2011).
Statistical analysis
The mean values of three replications and standard deviations
were calculated from the data obtained from the semi-
continuous fermentation system using Microsoft Office
Excel (ver. 2003; Microsoft, Redwood, WA). Matlab version
2007 (MathWorks, Natick, MA) was used for the simulation
and data analysis for kinetics models, using only the mean
values from the data obtained from the semi-continuous
fermentation.
Kinetics models
Cell growth
The logistic equation is known to be a substrate-independent
model that describes very well the inhibition of biomass on
growth in many batch or repeated batch fermentation systems
(Arzumanov et al. 2000; Liu et al. 2003; Liu and Liu 2004;
Zhao et al. 2010). Cell growth follows the logistic equation
 
dX X
¼ μm 1− X ð1Þ
dt Xm
1 dX
μ¼ ð2Þ
X dt
Ann Microbiol (2015) 65:1473–1480
where X is biomass concentration (g/L), Xm is maximum
biomass concentration (g/L), t is fermentation time (h), dX/dt
is cell growth rate [g/(L·h)], μ is the specific growth rate (h−1)
and μm is the maximum specific growth rate (h−1). The inte-
grated form of Eq. (1) using X=X0 [when t=0, X0 is the initial
biomass concentration (g/L)] gives a sigmoidal variation of X
as a function of t, which may represent both an exponential
and a stationary phase [Eq. (3)].
X 0 e μm t
X ðt Þ ¼ ð3Þ
X0
1− ½1−eμm t 
Xm
Product formation
According to the research of Sun et al. (1999), L-lactic acid
production is a semi-growth-associated process, which means
that the L-lactic acid production rate can be approximately
depicted by the Luedeking–Piret equations, as follows
dP dX
¼α þ βX ð4Þ
dt dt
1 dP
Qp ¼ ð5Þ
X dt
where α is the growth-associated constant in the
Luedeking–Piret model (g/g), α ≠ 0, β is the biomass-
associated constant in the Luedeking–Piret model [g/(g·h)],
β≠0, P is L-lactic acid concentration (g/L), dP/dt is L-lactic
acid production rate [g/(L·h)] and Qp is the L-lactic acid
production rate [g/(L·h)]. This model was originally devel-
oped for the formation of lactic acid by Lactobacillus
delbrueckii. According to this model, the product formation
rate depends on both the instantaneous biomass concentration
(X) and the growth rate (dX/dt), in a linear manner (Luedeking
and Piret 1959; Liu et al. 2003; Liu and Liu 2004).
In this study, Eqs. (1)–(5) were used to simulate the exper-
imental results.
Results and discussion
Production of L-lactic acid by R. oryzae
in the semi-continuous fermentation system
A biomass of up to 7.39 g/L was accumulated during the first
42 h in the first batch fermentation, as shown in Fig. 1. A near
linear growth rate was found when both the last nine batches
1475
were considered together and as separate batches. The bio-
mass continued to grow slowly, with no log phase in repeated
batch fermentation, while there were 6 h of lag time for growth
in the first batch. The final biomass was about 14.9 g/L after
222 h and ten batches.
The lag phase of L-lactic acid production and glucose
consumption were both about 18 h in the first batch, but there
was no lag time of L-lactic acid production and glucose
consumption during the last nine batches, with about a near
linear increase (L-lactic acid production) or decrease (glucose
consumption) in each of the last nine batches. The L-lactic acid
concentration at the initiation of fermentation was about 20 g/
L; for each repeated batch, it was about 90 g/L after 20 h
fermentation. There was no obvious change in L-lactic acid
concentration in each repeated batch, with only a slow de-
crease from 94.56 g/L in the second batch to 90.85 g/L in the
tenth batch—i.e. L-lactic acid concentration quickly increased
by 72.54–76.32 g/L during 20 h of fermentation (Fig. 1).
Residual glucose concentrations were all less than 5 g/L at
the end of each repeated batch. When the initial glucose
concentration was about 80 g/L, in the repeated batches, the
glucose conversion rates [L-lactic acid concentration (g/L)
relative to the initial glucose concentration (g/L) as a percent-
age] were 90.92–95.19 %, and the L-lactic acid yields [L-lactic
acid concentration (g/L) relative to the used glucose concen-
tration (g/L), which is the initial glucose concentration (g/L)
subtracted from the final glucose concentration in each repeat-
ed batch), g/g] were 0.93–0.96 g/g. In comparison, in the first
batch these were 88.6 % and 0.89 g/g, respectively.
Kinetics of the first batch in the semi-continuous fermentation
system
Cell growth
To be able to use Matlab software for data processing, we re-
wrote Eq. (3) in the following form:
Xm
X ¼ ð6Þ
eð−μm tÞ ⋅C⋅X m þ 1
where C is a constant. The experimental data obtained from
the first batch of the semi-continuous fermentation system
were used in the computations made with Matlab software
to quickly and rapidly obtain the kinetics parameters
employing the least square method.
The value of the maximum specific growth rate (μm) ob-
tained from the parameter estimation was 0.1802 h−1; in
comparison, it was 0.093 and 0.079 h−1for free cell and
immobilized cells of R. oryzae NRRL-395, respectively, in a
previous study (Efremenko et al. 2006). In a study by Anjana
and Surendra (2008) using Enterococcus faecalis RKY1 for
1476 Ann Microbiol (2015) 65:1473–1480

Fig. 1 L-Lactic acid production 140 18

1 batch 2-10 batches
curves of the different batches in 120 15
semi-continuous fermentation by
Rhizopus oryzae. Open circle L- 100
12
Lactic acid concentration (LAC),

/RSC (g/L)
LAC (g/L)
80

DCW (g/L)
filled diamond biomass (dry cell 9
weight, DCW), filled triangle 60
residual glucose concentration 6
40
(RSC). Standard deviations (SD)
were calculated from duplicate 20 3
measurements of each batch
0 0
0 30 60 90 120 150 180 210
Time (h)

lactic acid production, μm was reported to be 1.6 h−1, which is the QPm was 3.00 g/(g·h) in the Enterococcus faecalis RKY1
much greater than the value reported here. The maximum bio- fermentation study of Anjana and Surendra (2008).
mass and the C constant were 7.39 g/L and 9.2085, respectively, The fitted curves of the fungal cell growth and production
in our study. The final kinetic model was as follows. formation kinetics and their 95 % confidence intervals were
  shown in Figs. 2 and 3, respectively. The verification test
dX X carried out under the same conditions obtained a coefficient
¼ 0:1802 1− X ð7Þ
dt 7:39 determination (R2) of the correlation between the verification
test data and the fitted curve from the model equation of
0.9915 and 0.9913, respectively. Therefore, we concluded that
Where X was. the logistic equation and the Luedeking–Piret model equation
provided good descriptions of the kinetics of this fungal
7:39 fermentation system.
X ¼ ð8Þ
1 þ 68:0508eð−0:1802tÞ
Kinetics of the repeated batch in the semi-continuous
fermentation system

Product formation In the repeated batch fermentation, the accumulation of L-

The growth-associated constant (α) and biomass-associated
constant (β) obtained using the Luedeking–Piret model were
3.2631 g/g and 0.5980 g/(g·h), respectively. The α value was
much larger than that reported for lactic acid production from
lactic acid and cell growth were synchronous, and the curves
in all batches were close to being linear, as shown in Fig. 1.
The initial biomass and L-lactic acid concentration were not
zero; for example, in the second batch the initial biomass and
L-lactic acid concentration was 7.39 and 20.7 g/L, respectively

molasses using Enterococcus faecalis RKY1 (0.26 g/g; (Fig. 1), due to the presence of adhering broth from the end of
Anjana and Surendra 2008), but it was smaller than that the previous batch. On the other hand, L-lactic acid production
reported for lactic acid production from the first batch in a
8
one- and two-reactor system by Sporolactobacillus sp. strain
CASD (10.48 g/g and 15.40 g/g, respectively; Zhao et al.
2010). These same authors reported values of β of 1.36 and 6
1.29 g/(g·h), respectively , which were higher than those of DCW
(g/L)
our study. One explanation for these differences was the 4
different strains used in these studies. In our study, the value
of α was much higher than that of β, indicating that the growth 2
of individual cells of R. oryzae led to larger amounts of L-
lactic acid. The final kinetic model was
0
0 6 12 18 24 30 36 42
dP dX Time (h)
¼ 3:2631 þ 0:5980X ð9Þ
dt dt
Fig. 2 Fitted curves of fungi growth kinetics in L-lactic acid production
using the first batch experimental data in the semi-continuous fermenta-
In order to obtain the value of QP, Eq. (8) and Eq. (9) were tion system by R. oryzae. Solid circle Biomass (DCW) obtained in the
substituted into Eq. (5). The maximum L-lactic acid produc- experiment; dotted line 95 % confidence interval (CI), solid line fitted
tion rate (QPm) obtained was 1.1860 g/(g·h). In comparison, curve. All values were measured in triplicate with an uncertainty of <5 %
Ann Microbiol (2015) 65:1473–1480
120
100
LAC 80
(g/L)
60
40
20
0 was about a 17-fold difference. In this method, the increased
0 6 12 18 24 30 36 42
Time (h)
Fig. 3 Fitted curves of L-lactic acid production kinetics in L-lactic acid
production using the first batch experiment data in the semi-continuous
fermentation system by R. oryzae. Solid circle LAC obtained from the
experiment, dotted line 95 % confidence interval, solid line fitted curve.
All values were measured in triplicate with an uncertainty of <5 %
not only relied on the new biomass grown in the second batch
but also on the biomass accumulated in the first batch. Most of
the available glucose was used for the synthesis of L-lactic
acid by the biomass accumulated during the whole fermenta-
tion process, and the growth of R. oryzae in repeated batch
fermentation was very slow. Several methods were tested to
obtain the best method to mathematically describe the kinetics
of L-lactic acid production by R. oryzae in repeated fermenta-
tion, as follows.
In the first method, the new biomass was considered to be
“zero” at the beginning of each batch. The cell growth rate
(dX/dt) had no association with the initial biomass [Xi0, initial
biomass concentration (g/L) in the ith batch, i.e. the total
biomass in the end of the previous batch], and the new
biomass in the end of each batch was the maximum biomass
[Xim, maximum biomass concentration (g/L) increased in the
ith batch]. The value of dXi/dt was obtained in terms of the
new biomass, so the relationship between dXi/dt and dX/dt
was as follows.
dXi =dt ¼ dX=dt ð10Þ
The integrated form of dXi/dt was obtained by Matlab
Microsoft using X0 =0 and (ti0 − tim)=20 h. The sum of the
Xi (t) and the initial biomass was the actual X (t), and the
initial biomass was the total biomass in the end of the previous
batch. In terms of Eq. (2), the value of μm could be obtained
by Matlab Microsoft.
In the second method, the same method as that just
described was used to calculate the parameters in the
kinetics equation for the production. The new product
was considered to be “zero” at the beginning of each
batch.
In the third method, the value of δ (%) was used to evaluate
the contribution of the reused biomass (the total biomass
1477
present at the end of the previous batch, i.e. the initial biomass
of each batch, Xi0) for producing L-lactic acid in the repeated
batch. The increased biomass in each batch was much smaller
than the reused biomass. For example, in the fourth batch, the
increased biomass was 0.856 g/L and the reused biomass (the
initial biomass of the fourth batch, X40) was 9.107 g/L; this
difference was especially evident in the tenth batch where
these values were 0.798 and 14.087 g/L, respectively, which
biomass was ignored when calculating the value of δ in each
batch due to its small contribution to the whole biomass. The
initial biomass was used only to obtain the value of δ, and
specific calculation formula derived from Eq. (4) was as
follows:
dP=dt ¼ βX i0 ð11Þ
where the Xi0 was the initial biomass of each batch and also
the experimental data and the value of β had been obtained in
production kinetic. Therefore, the values of β and Xi0 were
constants. The integral of the Eq. (11) was
P ¼ 20βX i0 ð12Þ
and the calculation formula of δ was
δ ¼ P=ðPi20 −Pi0 Þ ¼ 20βX 0 =ð−Pi20 −Pi0 Þ ð13Þ
where Pi0 is the initial L-lactic acid concentration in the ith
batch (g/L) (i.e. the remaining L-lactic acid in 0.5 L broth at
the end of the previous batch was diluted by 5 L medium), P20
is the final L-lactic acid concentration in the ith batch (g/L) and
δ is the efficiency of the reused biomass maintained in the
former batch for producing L-lactic acid (%).
Kinetics of the second batch fermentation
In the second batch of the repeated fermentation, the final
biomass (X30) subtracted from the initial biomass (X20) was
the increased biomass (X2m), with a value of 0.856 g/L, as
shown in Fig. 1. The value of X2m was substituted into Eq. (3)
and the new biomass (Xi) was described as
107
Xi ¼ ð14Þ
125 þ 107⋅C⋅e−μim t
where μim is the maximum specific growth rate of
the new biomass in the ith batch (h−1). The values of
μim and C were 0.3178 h−1 and 23.4995, respectively.
1478 Ann Microbiol (2015) 65:1473–1480

The calculated formulation of dXi/dt and the biomasses taken into account, the fungi specific growth rate was sharply
(X) were reduced.
  In the same way, the values of α and β obtained were
dX i Xi
¼ 0:3178 1− Xi ð15Þ 0.9610 g/g and 0.4929 g/(g·h), respectively, and the value of
dt 0:856 α was much smaller than that in the first batch (3.2631 g/g).
These results suggest that the new biomass did not have a
major effect on the production. The final formulation of the
production kinetics was
107
X ¼ X i þ 7:39 ¼ þ 7:39 ð16Þ
125 þ 107⋅C⋅e−0:3178t
dP dX
¼ 0:9610 þ 0:4929X ð18Þ
dt dt
where 7.39 g/L was the initial biomass in the second batch
and, in addition, the biomass at the end of the first batch. The
integral of Eq. (16) was

dX 85502:7474e−0:3178t dX i Equation for QP was obtained by substituting Eq. (17) and

¼ 2
¼ ð17Þ
dt ð125 þ 2514:4465e −0:3178t Þ dt
The value of μm obtained by Matlab Microsoft was
0.0087 h−1 when Eq. (16) and Eq. (17) were substituted into
Eq. (2). In comparison, it was 0.009 h−1 in the study of
Efremenko et al. (2006). The value of μ2m (0.3178 h−1) was
much larger than that of both μm in the second batch
(0.0087 h−1) and in the first batch (0.1802 h−1). The difference
was mainly dependent on the participation of initial biomass.
In the second batch, 0.856 g/L of biomass was rapidly accu-
mulated within 20 h, and the specific growth rate was rela-
tively large, possibly due to the lack of lag phase and decline
phase in the repeated batch. However, when both the initial
biomass and the change in the actual fermentation time were
Eq. (18) into Eq. (5). The value of QPim {maximum L-lactic
acid production rate [g/(L·h)]} obtained was 0.5013 g/(g·h),
which is smaller than that in the first batch.
The R2 values of the correlation between the verification
test data and the fitted curve of the biomass and L-lactic acid
production from the model equation were 0.9899 and 0.9976,
respectively. This high correlation indicates that using the
methods above, the logistic and Luedeking–Piret model equa-
tions described very well the kinetics of L-lactic acid produc-
tion by Rhizopus oryzae in the repeated fermentation system.
Given the value of β, the value of δ obtained in terms of
Eq. (13) was 98.63 %; on the other hand, 98.63 % of the L-
lactic acid synthesized in the second batch was associated with
the biomass from the first batch fermentation. These results
suggested that the reused efficiency of the former biomass was
very high.

Table 1 Fitted values of parameters in kinetics models in L-lactic acid production by Rhizopus oryzae in semi-continuous fermentation

Batch Cell growth kinetic models Production kinetic models

Xim (g/L) Xm (g/L) μim (h−1) μm (h−1) C R2 α (g/g) β [g/(g·h)] QPm (g/(g·h)) δ (%) R2

1 ─ 7.39 ─ 0.1802 9.2085 0.9915 3.2631 0.5980 1.1860 — 0.9913

2 0.856 8.246 0.3178 0.0087 23.4995 0.9899 0.9610 0.4929 0.5013 98.63 0.9976
3 0.861 9.107 0.3193 0.0079 19.1439 0.9901 1.2142 0.4481 0.4577 96.83 0.9846
4 0.856 9.963 0.2916 0.0065 21.9179 0.9905 2.8049 0.4008 0.4192 97.97 0.9903
5 0.846 10.809 0.2886 0.0059 19.1055 0.9894 3.3009 0.3587 0.3781 95.90 0.9953
6 0.831 11.640 0.2841 0.0053 19.0266 0.9876 5.0569 0.3309 0.3575 96.02 0.9903
7 0.824 12.464 0.2838 0.0049 18.7130 0.9871 7.7597 0.3018 0.3395 95.03 0.9858
8 0.819 13.283 0.2823 0.0045 19.4506 0.9880 7.8544 0.2768 0.3121 93.32 0.9933
9 0.804 14.087 0.2805 0.0041 16.1022 0.9857 8.6540 0.2558 0.2915 93.00 0.9864
10 0.789 14.876 0.2778 0.0038 16.8136 0.9853 9.1181 0.2387 0.2732 92.71 0.9913

Xim, maximum biomass concentration (g/L) increased in the ith batch, Xm is maximum biomass concentration (g/L), μim is the maximum specific growth rate of
the new biomass in the ith batch (h-1 ), μm is maximum specific growth rate (h-1 ), C is a constant, R2 is the coefficient determination, α is the growth-associated
constant in Luedeking-Piret model (g/g), β is the biomass-associated constant in Luedeking-Piret model (g/(g·h)), QPm is the maximum L-lactic acid production
rate in the ith batch (g/(L·h)), δ is the efficiency of the reused biomass maintained in the former batchfor producing L-lactic acid (%).
Ann Microbiol (2015) 65:1473–1480
Kinetics of the repeated fermentation
Fitted values of the parameters in the kinetics models of L-
lactic acid production by R. oryzae using semi-continuous
fermentation were obtained using the methods described
and were pesented in Table 1. The maximum of biomass
(Xm) in each batch increased continuously, from 7.39 g/L in
the first batch to 14.876 g/L in the tenth batch. Concomitantly,
the value of increased biomass (Xim) in each batch decreased
slowly, from 0.856 g/L in the second batch to 0.789 g/L in the
tenth batch, with the exception of the third batch, where the
biomass was 0.861 g/L and greater than that in the other
batches (Table 1).
The value of μm in each repeated batch was much smaller
than that in the first batch, and it fell gradually from
0.0087 h−1 in the second batch to 0.0038 h−1 in the tenth
batch. There was a concomitant decrease in the value of μim,
from 0.3178 h−1 in the second batch to 0.2778 h−1 in the tenth
batch. The drop in μm during the semi-continuous fermenta-
tion could possibly be explained by mechanical damage from
the stirring fermentor and the aging of the fungi, i.e. the old
pellets were breaking down and autolysing while the new
fungi were growing (Bai et al. 2003; Rojan et al. 2007; Wu
et al. 2011).
The kinetics parameters of product formation changed with
good regularity in the semi-continuous fermentation. The
value of α was much larger than that of β in each batch, and
the value of α gradually increased from 0.9610 (g/g) in the
second batch to 9.1181 (g/g) in the tenth batch, while the value
of β decreased from 0.4929 to 0.2387 g/(g·h), as shown in
Table 1. These trends suggested that a large amount of bio-
mass accumulated in the first batch but that this biomass
subsequently declined continuously due to aging in the re-
peated fermentations and that the biomass which accumulated
in previous batches also broke down in following batches. The
decreased value of β also indicates that the contribution of
biomass to the product decreased with increasing number of
fermentation batches.
The values of QPm decreased gradually, from 1.1860 g/(g·
h) in the first batch, which was about two fold higher than that
in the second batch, to 0.2732 g/(g·h )in the tenth batch, and
the results indicated that the capacity of producing L-lactic
acid by biomass reduced gradually in the repeated fermenta-
tion batches. This results is in agreement with those of previ-
ous studies (Yin et al. 1998; Bai et al. 2003; Zhang et al.
2007).
R2 values of the simulation were all >0.98, indicating that
the logistic and Luedeking–Piret model equations provided
good descriptions of the kinetics for L-lactic acid production
by Rhizopus oryzae in the repeated fermentation system.
The value of δ decreased gradually, from 98.63 % in the
second batch to 92.71 % in the tenth batch; however, all values
were >90 %. The reused biomass played an important role for
1479
L-lactic acid production in the repeated fermentation. In the
last nine batches, part of the fungal biomass decayed, but with
the accumulation of more new biomass the production capac-
ity of biomass remained high.
Conclusions
Based on our results, R. oryzae AS 3.819 is a good fungi for
use in industrial-scale systems to produce high-quality L-lactic
acid, and the semi-continuous fermentation system is an effi-
cient method to improve the productivity of L-lactic acid in
terms of volume. The logistic and Luedeking–Piret model
equations have been shown to well describe the kinetics of
the L-lactic acid production by R. oryzae. We assumed that the
new biomass was “zero” at the beginning of each batch, and
the parameters of the kinetics, all calculated by Matlab soft-
ware, showed that L-lactic acid production relied more on the
biomass maintained from the former batches than on the new
growth of R. oryzae in the repeated fermentation.
Acknowledgment Support from the Hi-tech Research and Develop-
ment Program of China (863 Program) (2007AA10Z361), National Nat-
ural Science Foundation of China (31171741, 31101352, 31470002) and
the Key Laboratory for Agricultural Products Processing of Anhui Prov-
ince, the Institute of Agricultural Products Processing Technology, the
School of Biotechnology and Food Engineering, Hefei University of
Technology are gratefully acknowledged.
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