Simultaneous saccharification and fermentation of starch to lactic

acid
R. Anuradha, A.K. Suresh, K.V. Venkatesh *
Department of Chemical Engineering, Indian Institute of Technology, Bombay, Powai, Mumbai 400076, India

Abstract

Batch experiments were conducted to establish optimum operating conditions for the simultaneous saccharification and
fermentation (SSF) of starch to lactic acid acid using Lactobacillus delbrueckii. A predictive model was developed for SSF by
combining the kinetics of saccharification and fermentation. Saccharification kinetics were determined through experiments on
starch hydrolysis in which the effects of temperature, pH and different fermentation products as inhibitors were studied.
Fermentation kinetics was studied using glucose as substrate and effect of initial lactate on growth of Lactobacillus delbrueckii was
also examined. The saccharification kinetics were fitted using a Michaelis – Menten type equation, while the growth kinetics of L.
delbrueckii was represented by a Monod expression incorporating lactic acid inhibition. The kinetic model was used to predict the
performance SSF accurately. The saccharification rate was always higher SSF than in simple saccharification (SS) at all substrate
concentrations. Lactate productivity was 1.21 g/l h for SSF conducted under optimum conditions with 250 g/l potato starch,
higher than that of lactic acid productivity by fermentation after saccharification. Potato tuber and pearl tapioca are good raw
materials for the production of lactic acid using SSF with yields up to 70%.

Keywords: SSF; Starch; L. delbrueckii; Lactic acid; Kinetic model

1. Introduction starch has attracted attention for various reasons. In

Lactic acid has gained prominence in research and
industry as a potential source for biodegradable and
biocompatible lactic acid polymers. Polylactate poly-
mers could be an environment friendly alternative to
plastics derived form petrochemical materials. Antitu-
mor and antimicrobial effects of these polymers have
also been reported. For the production of L-( + )-lactic
acid, the direct microbial synthesis of the pure isomer is
preferred, since the chemical route in which it is pre-
pared by the hydrolysis of lactonitrile, produces a
racemic mixture, whose resolution is difficult. The car-
bon sources for lactic acid fermentation process can be
sucrose or such raw materials as molasses, cheese whey
(containing, respectively glucose and lactose) and
starch. Among the raw materials used for fermentation,
many cases extensive local cultivation of renewable
starch sources such as potato and tapioca make its use
economically attractive. Usually, however starch has to
be first hydrolyzed to glucose before it can be fer-
mented. Amylase synthesis is a rare characteristic of
lactic acid bacteria. Some strains like Lactobacillus
plantarum A6 derived from rhetted cassava, Lactobacil-
lus amylo6orus, Lactobacillus amylophillus are known to
degrade soluble starch [1]. Conventionally, gelatiniza-
tion and liquefaction of starch is carried out enzymi-
cally at high temperatures of 90–130°C for 15 min
followed by enzymic saccharification to glucose. The
glucose is subsequently converted to lactic acid by
fermentation using lactobacillis. This two step process
involving consecutive enzymic hydrolysis and microbial
fermentation makes utilization of starch as a fermenta-
tion substrate economically unattractive [2]. Microbial
conversion of starch to lactic acid can be made much
more economical by coupling the enzymic hydrolysis of
starchy substrates and microbial fermentation of the
368
derived glucose, into a single step. A direct benefit of
such a simultaneous saccharification and fermentation
(SSF) process is a decrease in the inhibition caused by
glucose accumulation leading to an increase in sacchar-
ification rates and a consequent reduction in reactor
volume and capital costs.
SSF has been utilized for the conversion of cellulose
to ethanol by yeast [3]. Extensive studies on SSF have
since been conducted focussing on the production of
ethanol and single cell protein from cellulosic sub-
strates. Phillipidis et al. [4] have studied the enzymic
hydrolysis of cellulose in an attempt to optimize SSF
performance. Ghose et al. [5] have increased ethanol
productivity by employing a vacuum cycling in an SSF
process using lignocellulosic substances. In addition to
producing power fuels like ethanol from cellulo-
biomass, the bioconversion of agricultural byproducts
to single cell protein has attracted worldwide attention.
Only a few studies have explored the possibility of
producing lactic acid from agricultural biomass. Abe
and Takagi [6] explored the SSF of cellulose powder to
lactic acid in a media containing cellulases and Lacto-
bacillus delbrueckii cells. Venkatesh [7] developed a
kinetic model for SSF of cellulose to lactic at varying
pH using Trichoderma reesei cellulases and Lactobacil-
lus bulgaricus. Hang [8] and Yu & Hang [9] have
reported that Rhizopus oryzae is capable of simulta-
neously saccharifying and fermenting corn to L-(+)-
lactic acid. However, in industry, Lactobacillus is the
preferred genus for fermentation because of greater
rates of metabolism and higher yields. Barley starch has
been used to produce lactic acid without prior hydroly-
sis using L. amylo6orus but with yields as low as 61%
[10]. L. amylo6orus and Lactobacillus casei have been
used in mixed culture with barley starch as substrate
with no additional nutrients. Linko and Javanainen [11]
have reported the production of lactic acid from barley
starch by simultaneous liquefaction and saccharification
using a-amylase and glucoamylase enzymes and fer-
mentation employing L. casei. A lactic acid concentra-
tion as high as 162 g/l was obtained and yields were
90 – 98%. Hence the SSF process is seen to be a more
comprehensive yet a simple process for utilization of
renewable resources. A review of literature reveals that
no SSF studies have been reported on starchy sub-
stances using L. delbrueckii. Modelling studies have
also not been reported on SSF of starch to lactic acid.
In the present work, the performance of SSF of
analytical grade starch to lactic acid has been evaluated
and optimum conditions of SSF operation determined.
The SSF process was then used for the production of
lactic acid using commercial native potato tubers and
pearl tapioca (Sabudana). A predictive model for SSF
has been developed from the separate kinetic expres-
sions for fermentation and saccharification.
2. Materials and methods
2.1. Materials
2.1.1. Microorganism
Homeofermentative Lactobacillus delbrueckii (NCIM
2365), a strain producing mainly L-(+)-lactic acid, was
obtained from National Collection of Industrial Mi-
croorganisms, National Chemical Laboratory (NCL),
Pune, India. Cultures were maintained at 4°C on slants
containing 3% glucose along with other essential nutri-
ents. Lactic acid bacteria were revived by two succes-
sive propagations at 45°C for 12–18 h in the modified
MRS broth.
2.1.2. Enzymes
Commercial a-amylase and glucoamylase form Novo
Nordisk, Denmark, were employed in this present
study.
2.1.3. Fermentation medium
Shake flask experiments were carried out using MRS
broth containing Yeast extract (0.5%), Urea (0.5%),
dipotassium hydrogen phosphate (0.1%), potassium di-
hydrogen phosphate (0.1%), Sodium Acetate (0.5%),
magnesium sulphate (0.03%), and varying glucose
concentrations.
2.1.4. SSF medium
The SSF medium consisted of Yeast extract (0.5%),
Urea (0.5%), dipotassium hydrogen phosphate (0.1%),
potassium phosphate (0.1%), Sodium acetate (0.5%),
magnesium sulphate (0.03%), and varying quantities
(10, 30, 100, 150 and 250 g/l of liquefied analytical
grade potato starch (containing 20% moisture).
2.2. Methods
2.2.1. Saccharification
Potato starch slurry (50 g/l) was used as substrate
and the pH adjusted to 6 using dilute HCl. a-Amylase
was added [0.1% on a dry substrate basis] and 200 ppm
of calcium added to stabilize a-amylase activity. The
starch suspension was charged into a reactor kept in a
water bath at 100°C. The reaction was carried out for 2
h. After liquefaction, saccharification was carried out
using glucoamylase after changing the pH to 4.2. The
temperature was maintained at 60°C. Experiments were
carried out to determine the effect of various inhibitors
(citric acid, propionic acid, acetic acid, lactic acid and
ethanol) on saccharification kinetics at 60°C and pH
4.2. The inhibitors were added in required concentra-
tions after the liquefaction step. The concentrations
studied were 10, 20 and 40 g/l. The pH for saccharifica-
tion was adjusted after adding the inhibitor in appro-
priate concentration. Samples were withdrawn every
hour and frozen immediately to arrest enzyme action.
 369

 
2.2.2. Fermentation with L. delbrueckii dG S
= Vm (1)
Batch experiments (100 ml) were carried out in 250 dt G
ml flasks, shaken at 250 rpm on a Neolab shaker at the Km 1+ +S
KG
required temperature. Samples of 2 ml of broth were
Using the stoichiometric relation for the conversion
removed for analysis every 2 h and the pH was checked
of starch to glucose (1 g starch yields 1.1 g glucose on
every 30 min. The pH was controlled by the addition of
complete saccharification), the rate of glucose forma-
a slurry of CaCO3. The samples were centrifuged and
tion via starch saccharification can be given by the
washed after which they were analyzed for biomass,

 
expression:
glucose and lactate content. The effect of pH was
studied at a temperature of 45°C, by conducting fer- G
S0 −

   
mentations at a pH of 4, 5, 5.5 and 6.5. The effect of dG 1.11
= Vm (2)
temperature was studied, at the pH of 5.6, by conduct- dt G G
ing fermentations at 40, 45, 50, 55 and 60°C. Fermenta- Km 1+ + S0 −
KG 1.11
tion kinetics was studied at substrate concentrations of
The parameters in this expression (Eq. (2)), Vm, Km
10, 30 and 100 g/l.
and Ki, are to be regarded as functions of pH and
temperature. In SSF, Eq. (2) may also need modifica-
2.2.3. SSF of starch with L. delbrueckii tion in view of possible inhibition by fermentation
The medium containing starch was autoclaved along products.
with a-amylase (0.15 ml/100 g Starch) at 121°C and 15
psi for 15 min. The starch concentration was varied 3.2. Fermentation
from 10 to 250 g/l. Yeast extract and urea were propor-
tionally increased to avoid nitrogen limitation. The For the growth kinetics of L. delbrueckii, a typical
medium was inoculated with the second generation of Monod’s expression was modified to include substrate
L. delbrueckii cultivated at 45°C for about 14 –18 h. inhibition an lactate inhibition. The rate equation for

: ;
The inoculum size was fixed at 10% and SSF was biomass production can be given as:
carried out after adding 0.15% (based on dry starch) of
glucoamylase.
dX G
rx = = mm e − KLP X (3)
dt G2
2.3. Sample analysis Ks + G+
KI
To model lactic acid formation, the expression
Samples withdrawn from either fermentation or SSF
developed originally by Leudeking and Piret [15],
medium were centrifuged at 10 000 rpm for 15 min, the
which takes into account both growth associated
supernatent frozen for further analysis and the pellet
and non-growth associated product formation, was
analyzed for biomass. Biomass was determined by mea-
used:
suring the absorbance using a standard curve of ab-
sorbance against dry cell weight. Absorbance was dP dX
rP = =a +b X (4)
measured at 600 nm in a Shimadzu spectrophotometer dt dt
(model UV160). Lactate and glucose concentrations
Similarly, the equation for glucose consumption can
were determined by the LDH [12] and o-Toluidine [13]

 
be given as:
methods, respectively.
dG dX
rg = =− a +b X (5)
dt dt
3. Model description
3.3. Simultaneous saccharification and fermentation
Independent kinetic models for saccharification and
The model equations developed in the previous
fermentation were developed, and these models were
sections for saccharification and fermentation were
integrated to model SSF. The models are discussed
combined to form the model equations to predict
below.
SSF. The rate of saccharification, as given by Eq. (3),
involves the substrate concentration expressed in
3.1. Saccharification terms of equivalent glucose obtained stoichiomet-
rically (G*). The actual glucose concentration that ac-
The kinetic model for the enzymic saccharification of cumulates in the system (G) inhibits saccharification.
starch [14] with glucoamylase allowing for competitive Therefore, the rate of saccharification in SSF is given
inhibition can be given by: by:
370

 S0 −
G* Table 2

   
dG* 1.11 Effect of inhibitors on maximum saccharification rate and inhibition
r1 = =Vm (6) parameters
dt G G*
Km 1 + + S0 −
KG 1.11 Inhibitor n K Vm/V 0m at 40 g/l

The rate of glucose accumulation is given by: Acetic acid 0.5497 0.032 0.338
Citric acid 0.4101 0.044 0.294
dG Ethanol 0.3027 0.3039 0.132
r2 = = r1 − rG (7)
dt Propionic acid 0.892 0.033 0.323
Lactic acid 0.3998 0.0203 0.40
The rates of lactic acid production (rp ) and biomass
production (rx ) is given by Eqs. (5) and (6). Eqs.
(5) – (8) form the model equations to predict the perfor- decrease in temperature and the decrease was up to
mance of the SSF process. During SSF it is assumed 72% at a temperature of 30°C as compared with the
that L. delbrueckii grows on glucose alone, although it maximum obtained at a temperature of 60°C. Similarly,
is known that the organism can grow on liquified starch Vm decreased on either side of the optimum pH of 4.
(for example on maltose). But the assumption is This demonstrates that the rate of saccharification was
justified since the growth rate on glucose will be far substantial at the temperature and pH at which fermen-
greater than other oligosaccharides. tation is normally conducted.

4.2. Inhibition of saccharification by fermentation

4. Results and discussion
4.1. Simple saccharification (SS)
Saccharification was conducted at various pH and
temperatures to characterize the saccharification kinet-
ics. The percentage decrease in the maximum saccharifi-
cation rate (Vm) at various temperatures and pH are
listed in Table 1. The fit of the experimental data show
that the Michaelis–Menten constant (Km) and the glu-
cose inhibition constant (KG ) were not altered due to
products
Different fermentation products like ethanol, lactic
acid, citric acid, proponic acid and acetic acid were
tested for their inhibitory action on starch saccharifica-
tion. All the products affected the value of Vm in the
rate expression (Eq. (2)). The inhibition effect by all the
products exhibited a saturation type of behaviour with
increase in concentration. The following empirical ex-
pression was used to fit the effect of fermentation
products on saccharification.
n

either temperature or pH. The values of Km, KG and Vm
are also listed in Table 1. Extensive saccharification
experiments were conducted with different initial starch
concentrations at various pH and temperatures.
Lineweaver–Burk plots were used to establish the val-
ues of Km, KG and Vm. The saccharification rate can be
calculated based on the parameters established experi-
mentally. The effect of pH and temperature are ac-
counted for by varying the maximum rate (Vm). It was
seen that Km and KG did not vary with pH and temper-
ature. It is clear from Table 1 that Vm decreases with
Table 1
Effect of pH and temperature on maximum saccharification ratea
Vm = V 0me − (kP) (8)
The values of n, k and Vm/V0 for 40 g/l of fermenta-
tion products are listed in Table 2. It can be seen that
ethanol exhibits the maximum inhibitory effect and
lactic acid, the minimum effect. Therefore, in terms of
saccharification rate, lactic acid is a prefered product of
SSF. Saccharification was inhibited more by glucose
than by any of the fermentation products. Therefore, it
is advantageous to produce any of the above products
from a SSF process. Since lactic acid demonstrated the
least inhibition, it was chosen as the product of interest.
4.3. Fermentation

Temperature pH Decrease in Vm (%) Experiments dealing with growth of L. delbrueckii at

different temperatures (30–55°C and pH 4–6.5) were
30 4 72
carried out to establish fermentation rates. As reported
40 4 71
45 4 58 elsewhere [7,15], the optimum pH and temperature for
55 4 35 growth were 5.5 and 45°C, respectively. The inhibitory
60 4 0 effect of lactate on cell growth was studied at lactate
60 5 4.4 concentrations ranging from 10 to 250 g/l L. delbrueckii
60 6 13.2
was able to survive in lactate concentrations as high as
60 7 58
60 3 60 250 g/l, although its growth rate decreased. A probable
reason for this capability to withstand such high con-
a
KG = 33 g/l, Km =95.6 g/l, Vm = 68 g/l h. centrations of lactate, was that L. delbrueckii can with-
 371

Table 3
Parameters used in the simulation of fermentation of L. delbrueckii

Condition a b a b KS KI mm KL

pH (temperature: 45°C, substrate:10 g/l glucose)

4 6.9 0.019 4.2 0.017 6.65 104.5 0.05 0.0037
5 6.85 0.012 4.9 0.01 6.65 104.5 0.3616 0.0037
5.5 7.10 0.025 5.0 0.001 6.65 104.5 0.3723 0.0037
6.5 9.0 0.03 6.2 0.01 6.65 104.5 0.1175 0.0037
Temperature (°C) (pH 5.6, substrate: 10 g/l glucose)
45 6.9 0.023 5.7 0.013 6.65 104.5 0.3723 0.0037
50 6.8 0.012 5.1 0.009 6.65 104.5 0.3524 0.0037
Subtrate (g/l) (temperature: 45°C, pH: 5.6)
10 7.1 0.025 5.0 0.009 6.65 104.5 0.3723 0.0037
30 6.9 0.023 5.7 0.0126 6.65 104.5 0.25 0.0037
100 28.3 0.030 23.8 0.013 6.65 104.5 0.11 0.0037

stand lower intracellular pH without depending on the
energy consuming proton pumps [16]. The lactate effect
could be well represented by the following expression
(refer to Eq. (4))
m = mm e − KLP
The data obtained from fermentation experiments at
various temperatures, pH and lactate concentrations
were fitted to the model equations (Eqs. (4) – (6)) using
an optimization algorithm (Box method), which mini-
mizes the least square error between the simulated and
the experimental values. The model parameters ob-
tained from such a fit are listed in Table 3. The least
square regression values for the parameter estimates
were in the range of 0.9 – 0.98, indicating a good fit of
the experimental data. It can be noted that the values of
a and a (growth related constants) are greater than b
and b (maintenance related constants). This is consis-
tent with values reported in literature [7,13]. These
model parameters were used to simulate SSF.
tion rate controls the SSF process. The final lactate
yield was 82% of the net theoretical glucose concentra-
tion from 10 g/l of starch (11.1 g/l).
The model prediction for 30 g/l of starch, could not
(9) match the experimental data accurately. However,
when the value of a was changed from 5.1 to 5.7 the
prediction was good. Fig. 2 shows the comparison
between model prediction and experimental data for
SSF of 30 g/l of starch. The glucose concentration was
always below 5 g/l and was maintained close to zero for
more that half the duration of fermentation. This again
indicated that the saccharification rates were slower
than the fermentation rates. The cells would cease to
grow once the glucose concentration was close to zero.

4.4. Simultaneous saccharification and fermentation

The optimum temperature for saccharification was in

the range of 55–60°C, but no substantial growth was
observed at these temperatures. The optimum pH of
saccharification was in the range of 4 – 4.5, but very low
growth rates were observed at these pH values. There-
fore, SSF needs to be operated at a pH range of 5–6
and a temperature range of 40 – 50°C. The parameters
obtained form saccharification and fermentation experi-
ments were used to simulate SSF by solving the model
equations (Eqs. (4)– (8)) and later compared with the
experimental data. Fig. 1 shows the comparison be-
tween model prediction and experimental data for SSF
of 10 g/l of starch. The model equations were able to
predict the performance of SSF accurately. The glucose
was kept below 2.2 g/l and falls continuously until the Fig. 1. Comparison of model prediction and experimental data for
end of fermentation. This implies that the saccharifica- SSF of 10 g/l starch to lactic acid at 45°C and pH 5.5.
372

Fig. 2. Comparison of model prediction and experimental data for
SSF of 30 g/l starch to lactic acid at 45°C and pH 5.5.
This implies that a substantial amount of lactate is
produced due to cell maintenance rather that growth.
The final yield of lactate was 87% (as compared with
82% for 10 g/l of starch) of the net theoretical glucose
concentration from 30 g/l of starch (33.3 g/l).
The model parameters obtained for saccharification
and fermentation could not predict SSF for higher
initial starch concentrations (100 – 250 g/l). Table 4
shows the parameters used to predict SSF for higher
initial starch concentrations. Experiments indicated that
there was an initial phase, where the saccharification
rates were greater than the fermentation rates, wherein
glucose accumulated. Therefore, a lag period in the
fermentation was introduced in the simulation. This
might be due to the effects of high concentration of
starch in the broth. During this period, the cells do not
grow, but used glucose for maintenance alone. The lag
phases were 5, 10 and 100 h for 100, 150 and 250 g/l of
initial starch concentrations, respectively. L. delbrueckii
started to grow and produce lactic acid immediately
after inoculation for SSF at lower starch concentrations
Fig. 3. Comparison of model prediction and experimental data for
the variation of glucose concentration with time for SSF at high
initial starch concentrations (100 – 250 g/l potato starch) at 45°C and
pH 5.5.
of 10 and 30 g/l. Hence, there was no need to introduce
a lag period for growth. Another parameter that
seemed to change at higher starch concentrations was
the value of Km, the Michaelis–Menten substrate satu-
ration constant (refer to Eq. (3)). This indicates that the
enzyme seems to become more active in the presence of
cells and complex media. The value of Km was seen to
decrease with increase in the substrate concentration as
shown in Table 3.
Fig. 3 shows the comparison between model predic-
tion and experimental data for glucose concentration at
high initial starch concentrations. Initially glucose accu-
mulates as the fermentation step is rate limiting. Once
the cells adjust to the medium and start to grow, the
saccharification step becomes rate limiting and glucose
concentration decreases. The glucose concentration de-
creases to zero and the saccharification rate equals the
fermentation rate. At this stage, the cells are in the
stationary phase and produce lactic acid mainly
through maintenance process. The maximum concen-

Table 4
Parameters used in the simulation of SSF of starch to lactic acid using L. delbrueckii

Substrate conc. (g/l) a b a b Km lagtime

10 7.1 0.025 5.0 0.009 95.6 0

30 6.9 0.023 5.10 0.013 95.6 0
100 28.3 0.03 14.0 0.09 60.5 5
150 28.3 0.03 15.0 0.072 25.0 10
250 28.3 0.03 14.0 0.15 18.0 10
 373

tration of glucose that accumulates in the broth are 32,

60 and 80 g/l for 100, 150 and 250 g/l of initial starch
concentrations, respectively. Since the value of KI, the
glucose inhibition constant of saccharification is 33 g/l,
there is substantial glucose inhibition on saccharifica-
tion in SSF at higher initial starch concentrations.
Therefore the advantage of SSF in terms of rate is
partly lost at higher initial starch concentrations. The
comparison between model prediction and experimental
data of lactate concentration at high initial starch con-
centrations are shown in Fig. 4. It can be seen from the
figure that there are two distinct rates of lactate forma-
tion. Initially, the lactate production rate was high,
when the cells were in the exponential growth phase
(when there was high glucose concentration in the
medium; see Fig. 3), and later the rate of production
was lower, when the cells were in the stationary phase
(when there was low glucose concentration in the
medium). Yields as high as 71% (79 g/l), 78% (129 g/l)
and 68% (189 g/l) of lactate were obtained in SSF of
100, 150 and 250 g/l of initial starch concentration, Fig. 5. Comparison of glucose formation rate by saccharification in
respectively, at the end of 100 h. Since the cells would simple saccharification (SS), simultaneous saccharification and fer-
mentation (SSF) and SSF without lactate inhibition term on sacchar-
continue to maintain after 100 h, the lactate concentra-
ification (SSF-1) with 100 g/l starch.
tion would increase further, if the process were contin-
ued to operate for longer time. This means that a 4.5. Comparison of saccharification rates in SS and
higher yield of lactate could be obtained based on net SSF
theoretical glucose concentration from starch, but at a
lower productivity. In order to compare the saccharification rates in SS
and SSF, dG/dt for SS (Eq. (2)) and dG*/dt for SSF
(Eq. (7)) were plotted. Fig. 5 shows such a plot for 100
g/l initial starch. In the case of SS, glucose accumulated
in the reactor and there was a continuous drop in rate
due to glucose inhibition whereas in the case of SSF,
initially the rate decreased during the lag phase of cell
growth, due to which glucose accumulated in the reac-
tor (refer to Fig. 3). Once the cells started to grow
exponentially, the glucose concentrations fell and the
saccharification rate increased, after which there was a
steady drop in the rate due to a decrease in hydrolyz-
able starch concentration. Fig. 5 also shows the saccha-
rification rate in SSF, when the lactate inhibition term
n
e( − KP) was excluded. It is clearly evident that the rates
are higher in SSF with removal of lactate. This indi-
cates that if lactate is simultaneously removed from the
SSF broth, both saccharification rates as well as fer-
mentation rates would increase, which would result in a
much faster SSF process. The productivity of lactic acid
from SSF, as compared with a two step process of
saccharification followed by subsequent fermentation is
listed in Table 5. At all the substrate concentrations
used, the productivity of lactic acid from SSF was
Fig. 4. Comparison of model prediction and experimental data for
the variation of lactate concentration with time for SSF at high initial
higher in magnitude. The increase in productivity was
starch concentrations (100–250 g/l potato starch) at 45°C and pH in the range of 17–35% for 10–250 g/l of starch,
5.5. respectively. Although Fig. 5 demonstrated a substan-
374

Fig. 7. Simultaneous saccharification and fermentation (SSF) of pearl

tapioca at 45°C and pH 5.5.

Fig. 6. Simultaneous saccharification and fermentation (SSF) of

potato tubers at 45°C and pH 5.5.
4.6. SSF of potato tuber and pearl tapioca

SSF was also conducted successfully on commercial

tial increase in the saccharification rate, the productivi-
ties did not match the same magnitude. This is due to
the fact that both fermentation and saccharification
rates were inhibited due to lactic acid. Also, due to
non-availability of excess glucose in the medium, the
lactic acid formation rates were low due to cell mainte-
nance. Therefore, the rate advantage obtained due to
faster saccharification rates was lost due to slower lactic
acid production rate. Since saccharification rates were
higher in SSF, this resulted in complete hydrolysis of
starch as compared with SS at higher initial starch
concentrations. Starch was completely converted to cell
mass and lactic acid in SSF, as glucose did not accumu-
late in the broth. Thus there is better utilization of
carbon present in the starch.
Table 5
Comparison of lactic acid and productivity in SSF and in saccharifi-
cation followed by fermentation process (S+F)
potato tuber and pearl tapioca. Fig. 6 shows the
variation of glucose and lactate concentrations with
time for SSF at 45°C and pH 5.5 of 10 g/l of
potato tubers. The maximum glucose concentration
that accumulated was 3.7 g/l and drops to zero after
16.5 h. A maximum of 7.2 g/l lactate was formed
resulting in a yield of 70%. The productivity was 0.3 g/l,
which is comparable with SSF on analytical grade
potato starch. Fig. 7 shows the glucose and lactate time
profiles for SSF of pearl tapioca at pH 5.5 and temper-
ature 45°C. The profiles match well with SSF carried
out on analytical grade starch with the same initial
starch concentration of 100 g/l (refer to Figs. 3 and 4)
The lactate concentrations at the end of 100 h of SSF
was 69.3 g/l corresponding to a yield of 69%. The
productivity was 0.7 g/l. These experiments proved
that agricultural that agricultural biomass is indeed a
good bioresource for the production of lactic acid using
SSF.

Substrate conc. Productivity of S+F Productivity of SSF

(g/l) (g/l h) (g/l h) 5. Conclusions
10 0.32 0.38
30 0.51 0.64 The saccharification of starch was studied at various
100 0.83 0.87 temperatures and pH in order to characterize saccharifi-
150 1.11 1.29 cation rate. Various fermentation products like citric
250 1.22 1.87
Potato tuber – 0.3
acid, propionic acid, acetic acid, ethanol and lactic acid
Pearl – 0.7 were tested for their inhibitory action on the saccharifi-
cation of starch. Lactic acid exhibited minimum in-

hibitory effect and thus is the prefered product of SSF.
It would certainly be advantageous to produce the
other products by a SSF process, since all of these
exhibited lower inhibition than glucose on saccharifica-
tion. The growth of L. delbrueckii was studied at differ-
ent pH, temperature and substrate concentration in
order to characterize fermentation rate. Although high
initial lactate concentrations decreased the growth rate,
L. delbrueckii was able to survive high lactate concen-
trations. The optimum conditions for SSF were
established as 45°C and pH 5.5. SSF was conducted at
various substrate concentrations and yields as high
as 86% were obtained. A higher yield of lactate
could be obtained by increasing the operating time, but
at a lower productivity. A kinetic model was de-
veloped by integrating both saccharification and fer-
mentation kinetics, that predicted SSF accurately. The
feasibility of production of lactic acid by SSF
from agricultural biomass (potato tuber and pearl tapi-
oca) in high yields was established. The productivity
and yields of lactic acid in SSF were always greater
than in the two step process of saccharification fol-
lowed by fermentation. Further, the productivity could
be increased by incorporating simultaneous removal of
lactate from the broth or increasing the growth associ-
ated production of lactate. Thus, the SSF process
shows promise as a better alternative to the existent
conventional process to obtain a high yield of lactic
acid.
6. Nomenclature
a parameter for growth associated glucose
consumption [g. G/g. X]
b parameter for non-growth associated glu-
cose consumption [g. G/g. X h]
G glucose concentration [g/l]
G* glucose formed stoichiometrically by sac-
charification [g/l]
k inhibition constant for saccharification by
fermentation products [g/l]
KG glucose inhibition constant for saccharifica-
tion [g/l]
KI glucose inhibition constant for fermenta-
tion [g/l]
KL lactate inhibition constant for fermentation
[g/l]
KM Michaelis–Menten constant [g/l]
KS glucose saturation constant [g/l]
n exponential constant for inhibition on sac-
charification by fermentation product
P lactic acid concentration [g/l]
r rate of reaction [g/l h]
375
S Starch concentration [g/l]
S0 initial substrate concentration [g/l]
Vm maximum rate of saccharification [g/l h]
V 0m initial saccharification rate [g/l]
X biomass concentration [g/l]
Greek:
a Parameter for growth associated lactate
formation [g. P/g. X]
b Parameter for non-growth associated lac-
tate formation [g. P/g. X]
mm Maximum specific growth rate [per h]
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