Journal of Applied Science and Engineering, Vol. 16, No. 2, pp. 205-210 (2013)
DOI: 10.6180/jase.2013.16.2.12
Optically Pure L-Lactic Acid Production Directly
from Leftover Bits and Pieces of Potato Starch
Using an Amylolytic Pellet-Form Complex
Rhizopus Oryzae ASC081
Gaofeng Shi1,2*, Guoying Wang 1,2, Xuefu Chen1,2 and Chunlei Li1
1
College of Petroleum and Chemical Engineering, Lanzhou University of Technology,
Lanzhou 730050, P. R. China
2
Analysis & Testing Center, Lanzhou University of Technology,
Lanzhou 730050, P. R. China
Abstract
Optically pure L-(+)-lactic acid fermentation directly from leftover bits and pieces of potato
starch (LBPPS) using an amylolytic pellet-form complex Rhizopus oryzae ASC081 was studied. The
complex ASC081, which have the capability for the simultaneously saccharification and production of
pure L-(+)-lactic acid, was mutagenized and screened from two parent Rhizopus oryzaes As1 and As2.
In the ASC081 fermentation, the variation in temperature, inoculum size, and pH obviously affected
the amylolytic capacity, the L-(+)-lactic acid production and the fungal biomass formation. The
addition of phytic acid and bitter salt (an alternate name: magnesium sulfate) significantly stimulated
the L-(+)-lactic acid fermentation process, and enabled complete bioconversion within 90 h. A
condition, LBPPS concentration 140 g/L, pH 5.2, temperature 32 °C, inoculum size 15%, phytic acid
addition 0.05 g/L and bitter salt addition 0.5 g/L, was favourable for the optically pure L-(+)-lactic acid
production, resulting in the optically pure L-(+)-lactic acid yield of 0.12 g / 1.0 g LBPPS.
Key Words: Amylolytic, Rhizopus Oryze ASC081, Leftover Bits and Pieces of Potato Starch,
Optically Pure L-Lactic Acid
1. Introduction
Lactic acid, or named as milk acid, is a chemical
compound playing roles in several chemical and biochemical processes. Lactic acid has a chiral carbon atom
and occurs naturally in two optical isomers. One is
known as L-(+)-lactic acid or (S)-lactic acid and the
other, its mirror image, is D-(-)-lactic acid or (R)-lactic
acid. L-(+)-lactic acid is the biologically important isomer. Since elevated levels of the D-(-)-lactic acid are
harmful to human beings, optically pure L-(+)-lactic
acid is the preferred isomer in pharmaceutical and food
industries [1-7]. The chemical structure of L-(+)-lactic
acid and D-(-)-lactic acid are shown in Figure 1.
*Corresponding author. E-mail: [email protected]
Lactic acid can be produced comercially by either
chemical synthesis or biotechnological production. However, most production methods result in a racemic mixture of the L-(+)- and D-(-)-isomers [1,8-10]. A biotechnological process can yield either a mixture of two isomers in different proportions, or form a single isomer
alone (L-(+)- or D-(-)-lactic acid), which depend on the
Figure 1. Chemical structures of L-(+)-lactic acid and D-(-)lactic acid.
206
Gaofeng Shi et al.
microorganism, substrate, metabolism control and growth
conditions used.
Leftover bits and pieces of potato starch is an abundant byproduct in the potato processing industry. The
main processes to produce potato starch are following:
1) the potatoes are washed and rasped into fine particles,
2) fruit juice and solid matter are separated into two
streams, 3) fresh water is added to the stream of solid
matter and pulp is separated from the starch by centrifugation 4) starch is refined in hydro cyclones and
vacuum filters, 5) the concentrated slurry is dried in a
warm air stream. The byproducts (waste products) mainly
include potato juice and potato pulp. The potato pulp is a
side product of washing the starch from the mash. The
byproducts contain (approximate composition) 0.5-1%
of potato, 25-35% of starch (not extracted because of
economical reasons), 50-60% protein, 2-3% minerals,
1-2% fat, 5-7% nitrogen and other. In this study, leftover bits and pieces of potato starch (LBPPS) were used
as an inexpensive fermentation medium. To achieve the
production of optically pure L-(+)-lactic acid directly
from LBPPS simultaneously without D-(-)-lactic acid
produced, a complex Rhizopus oryzae ASC081 (R.
ASC081) was breeded and screened by induced mutagenesis method. Direct fermentation process using the
amylolytic pellet-form complex R.ASC081 was developed. The R.ASC081 was found to be an excellent fungus for the direct degradation of LBPPS. The culture pH,
temperature, and an addition of phytic acid and minerals
of medium were the main influential factors on the productivity of optically pure L-(+)-lactic acid.
2. Material and Methods
2.1 Bacterial Strain, Media and Instruments
R.ASC081, a highly pure L-(+)-lactic acid producing strain bred by using Rhizopus oryzae As1 and Rhizopus oryzae As2 as parents, was used in the experiments. The strain was maintained on potato dextrose
agar slants at 4 °C. For fermentation study, agar slant
containing sporulated fungus was washed by sterile
water to obtain spore suspension. Slant preservation medium consisted of (g/L): potato starch 20, refined glucose 20, KH2PO4 0.3, MgSO4 0.2, agar 15, pH of 6.0.
The pre-culture medium consisted of (g/L): potato starch
40, refined glucose 20, (NH4)2SO4 4, KH2PO4 0.4,
MgSO4 0.5, ZnSO4 0.5. The fermentation medium had the
following components (g/L): LBPPS 140, (NH4)2SO4 4,
KH2PO4 0.4, MgSO4 0.5, ZnSO4 0.5, phytic acid 0.05.
The pH of medium lies between 5.7-5.8 after sterilizing.
A sterilization time of 30 min at 121 °C is recommended.
The experimental instruments used included YLD-6000
incubator (Hongdu, Yangzhou, China), SHZ-82A rotary
shaker (Guohua, Changzhou, China), BIOF-2005 5-liter
automatic fermenter (Gaoji, Shanghai, China), JHDCB2058 cleaning table (Hongdu, Yangzhou, China),
YX280A high pressure steam sterilizer (Shanghai, China),
RE-52A vacuum rotary evaporator (Sanshen, Shanghai,
China), FD-IB vacuum freeze drying machine (Boyikang,
Beijing, China), U-2001 uv-vis spectrophotometer
(HITACHI, Tokyo, Japan), CR22G high-speed freezing
centrifuge (HITACHI, Tokyo, Japan), B204LEDR microscope (Aote, Chongqing, China) and LCQ DECA XP
PLUX high performance liquid chromatography (Thermo
Finnigan, San Jose, USA).
2.2 Inoculum Preparation
Spores were grown on a potato dextrose agar slants
at 32 °C for 60 h. They were collected with an inoculating loop and suspended in distilled sterilized water. 5 mL
of spore suspension was transferred to a new growth medium (100 mL) in a 250 mL vial every 12 h. A 25 mL of
this culture was then transferred to a 500 mL vial that
contained 400 mL sterile growth medium. The inoculum
was incubated at 32 °C for 8 h on an orbital shaker at 200
rpm before inoculation at 15 % (v/v) to the fermenter.
2.3 Fermentation Conditions
Fermentations were performed in BIOF-2005 5-liter
automatic fermenter with 3.5 liter working volume. The
experiments were carried out at 32 °C, agitation speed 600
rpm, aeration rate 30 L/h, and the pH was maintained at
5.7 by automatic addition of neutralization agent. The
samples were withdrawn at intervals for analysis.
2.4 Analytical Methods
The culture broth was first centrifuged, and then the
resulting supernatant was used for analysis. The measurements of glucose, lactic acid, malate, fumarate, acetic and citric acid were carried out by high-performance
liquid chromatography (HPLC) equipped with a PDA/
UV dectetor at 210 nm. A Thermo Hypersil Gold C18 column (150 * 2.1 mm, 3 mm) was used with methanol and
0.1% phosphoric acid as a mobile phase at a flow rate of
Optically Pure L-Lactic acid Production from LBPPS
0.2 ml/min, while the column temperature was maintained at 25 °C. Dry cell weight was determined by harvesting the culture samples, filtering and then washing
the mycelia three times with distilled water, and drying at
95 °C until constant weight was achieved.
L-(+)-lactic acid was determined using an enzymeUV method. In the presence of L-lactate dehydrogenase
(L-LDH), L-(+)-lactic acid is oxidized to pyruvate by
nicotinamide-adenine dinucleotide (NAD), L-Lactate +
NAD+ ƒ pyruvaet + NADH + H+. The equilibrium of this
reaction lies on the side of lactate. The product, pyruvate,
was trapped by hydrazine. The amount of NADH formed
in the above reaction is stoichiometric to the amount of
L-(+)-lactic acid. The increase in NADH is quantified by
measuring its light absorbance at 340 nm.
3. Results and Discussion
3.1 Chemical Composition of LBPPS Samples
In order to determine the probability of producing
L-(+)-lactic acid from LBPPS, the variation in chemical
composition of LBPPS collected were first characterized.
The compositions of samples are presented in Table 1.
The average contents of starch, moisture, crude protein, crude fat and crude fiber were 48.27%, 45.42%,
3.05%, 0.11% and 0.25%, respectively. The C/N ratios
(proportion between carbon content and nitrogen content) were in a range from 8.78 to 23.58. The contents of
carbohydrates in LBPPS ranged from 15.9% to 80.5%.
In those samples, over 15% of carbohydrate existed as
starch. LBPPS is an inexpensive and abundantly available raw material compared to sucrose, glucose and hydrolyzed starch. In comparison with other strains, R.
ASC081 showed more efficiency in direct fermentation
of LBPPS to L-(+)-lactic acid avoiding the conventional
multi-step process. The R.ASC081 organism also showed
obvious amylolytic efficiency. This result suggested that
the LBPPS have the potentiality as fermentative substrate for producing L-lactic acid. This result further implied that the amylase expressed by R.ASC081 during
207
fermentation could become a key factor for the direct
L-(+)-lactic acid production.
3.2 Effect of pH Control on Fermentation
In order to determine the impact of pH on the fermentation of L-(+)-lactic acid by R.ASC081, the pH was controlled at 4.0, 4.5, 5.0, 5.5, 6.0 and 7.0. Without pH control, 2.7 g lactic acid was produced from 55 g starch after
120 h fermentation, and culture pH finally dropped to 3.0.
With pH control at 4.0 to 7.0, production of lactic acid varied correspondingly (See Table 2). When the pH lower
than 4.5, the productivity of L-(+)-lactic acid was restrained. When the pH higher than 6.0, the productivity of
total lactic acid was raised; however, D-(-)-lactic acid was
detected in the medium, and the yield of D-(-)-lactic acid
increased with increasing pH up to pH at 7.5. Therefore,
the pH for optically pure L-(+)-lactic acid production
ranged from 4.5 to 6.0 was recommended. The results of
L-(+)-lactic acid production and biomass increase under
different pH value are summarized in Table 2. The fermentation conditions of the experiments are as follows:
the fermentation medium had the following components
(g/L) : LBPPS 320, (NH4)2SO4 6, KH2PO4 0.7, MgSO4
0.3, ZnSO4 0.2, phytic acid 0.06. The pH of medium lies
between 5.7-5.8 after sterilizing. A sterilization time of 30
min at 121 °C is recommended. Inoculum size: 15 %
(v/v). The experiments were carried out at 32 °C, agitation
speed 600 rpm, aeration rate 30 L/h, and the pH was maintained at 4.8, 5.0, 5.2, 5.4, 5.6, 6.0 6.5 and 7.0, respectively, by automatic addition of neutralization agent.
The results also indicated that R.ASC081 had a high
enzymatic capability for saccharification of LBPPS in
the pH range of 4.8-7.0. The starch hydrolysis and lactic
acid accumulation were influenced obviously by growth
pH. When the pH was controlled at 5.2, the highest production of L-(+)-lactic acid was achieved after 120 h fermentation, and at the same time, no D-(-)-lactic acid was
found in the medium. If the pH was controlled higher than
6.0, D-(-)-lactic acid was found in the medium after 80 h
fermentation. Because in the culture controlled at pH ³
Table 1. Major chemical compositions and its contents in LBPPS samples (m/m, %)
Sample 1
Sample 2
Sample 3
Sample 4
Starch
Moisture
Crude protein
Crude fat
Crude fibre
Ash
Other components
23.6
15.8
80.2
73.5
74.4
81.3
13.0
13.0
1.1
1.7
3.1
6.3
0.06
0.02
0.23
0.14
0.1
0.1
0.3
0.5
0.54
0.92
2.01
4.06
0.2
0.16
1.16
2.50
208
Gaofeng Shi et al.
Table 2. Effects of growth pH on L-(+)-lactic acid production and fungal biomass formation
pH value
L-Lactic acid production (g)
Fermentation time 20 h
40 h
60 h
80 h
100 h
120 h
D-Lactic acid production
Fermentation time 20 h
40 h
60 h
80 h
100 h
120 h
Fungal biomass formation (g)
Fermentation time 20 h
40 h
60 h
80 h
100 h
120 h
4.8
5.0
5.2
5.4
5.6
6.0
6.5
7.0
2.0
4.2
12.0
25.0
28.0
29.0
2.5
5.0
13.0
26.5
30.0
30.8
3.1
6.0
14.0
29.0
33.8
34.0
2.9
8.0
15.2
28.1
33.0
33.0
3.0
7.5
16.0
27.0
31.0
31.5
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
-------
-------
-------
-------
-------
-+
+
+
+
+
-+
+
+
+
+
-+
+
+
+
+
0.2
0.3
1.5
2.4
3.0
3.5
0.3
0.5
2.0
2.8
3.5
3.8
0.3
0.6
2.2
3.0
3.8
4.1
0.35
0.8
2.6
3.8
4.0
4.3
0.3
1.0
2.8
3.6
4.3
4.5
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
Note: --: not found; +: found; /: no data.
6.0, the heterofermentation occurred, resulting in the accumulations of D-(-)-lactic acid and acetic acid. However,
if pH was controlled lower than 4.5, higher Ca2+ was injected, and the higher concentration of Ca2+ was inhibitory to the organism, leading to lower production of
L-(+)-lactic acid, consequently, a growth pH higher than
6.0 or lower than 4.5 was unfavourable for the production
of L-(+)-lactic acid with high optical purity.
3.3 Effect of Inoculum Size on L-(+)-lactic Acid
Fermentation
The influences of inoculated spore concentration on
the L-(+)-lactic acid production and biomass increase
were investigated. The results are shown in Figure 2. In
the fermentation on 5 L fermenter, an inoculum size 15%
(spore concentration of 1×107 spores per micro liter) yield
the highest L-(+)-lactic acid production (32.3 g) after 100
h fermentation. When inoculum size was lower than 10%
(spore concentration < 1 ´ 104 spores per micro liter),
L-(+)-lactic acid yield decreased, due to inadequacy of enzymatic efficiency. When inoculum size was more than
20% (spore concentration more than 1 ´ 108 spores per
micro liter), irregularly shaped masses or pieces of spores
were formed in production medium, while the L-(+)-lactic
acid production was lower than that of inoculum size 10%
after 60 h fermentation (see Figure 2A). The productivity
of L-(+)-lactic acid was weakened perhaps due to the nutrient and dissolved oxygen limitation in the irregularly
shaped masses or pieces. However, the biomass concentration increased correspondingly with the inoculum size
(see Figure 2B). Therefore, with inoculum level of 5×107
spores per micro liter was the optimum for L-(+)-lactic
acid production and biomass formation.
3.4 Effect of Phytic Acid on L-(+)-lactic Acid
Fermentation
The influence of adding phytic acid on L-(+)-lactic
acid production and mycelial morphology by R.ASC081
was investigated. The results are shown in Table 3. In the
fermentation culture, an addition of phytic acid (0.05 g/L),
made for forming a great deal of mycelial pellets in fermentation medium during 40-80 h, yields the highest
L-(+)-lactic acid production (31.0 g) after 90-h culture. In
the L-(+)-lactic acid fermentation by R.ASC081, the
growth of mycelium, formation of mycelial pellet and accumulation of irregularly shaped masses or pieces were
observed during the growth of R.ASC081. The formation
of mycelial pellet was crucial for optically pure L-(+)-lac-
Optically Pure L-Lactic acid Production from LBPPS
209
Figure 2. Effect of inoculum size on L-(+)-lactic acid fermentation and fungal biomass formation.
Table 3. Influence of adding phytic acid on L-(+)-lactic acid production and fungal biomass formation
Fermentation time (h)
10
20
30
40
50
60
80
90
L-Lactic acid
production (g)
Adding phytic acid
Without phytic acid
0.60
0.68
1.80
1.78
3.20
2.98
4.10
4.0
8.60
6.60
18.80
10.66
30.0
16.50
31.0
22.0
Fungal biomass
formation (g)
Adding phytic acid
Without phytic acid
0.08
0.09
0.12
0.11
0.66
0.68
1.65
1.56
3.21
2.88
4.05
3.15
7.55
6.55
8.28
7.02
tic acid production. Phytic acid can accelerate the formation of mycelial pellet of R.ASC081, and then stimulate
the L-(+)-lactic acid production. The results are listed in
Table 3. The fermentation conditions of the experiments
are as follows: the fermentation medium had the following
components (g/L): LBPPS 310, (NH4)2SO4 5, KH2PO4 0.6,
MgSO4 0.6, ZnSO4 0.3. The pH of medium lies between
5.7-5.8 after sterilizing. A sterilization time of 30 min at
121 °C is recommended. Inoculum size: 15 % (v/v). The
experiments were carried out at 32 °C, agitation speed 600
rpm, aeration rate 30 L/h, and the pH was maintained at
5.2 by automatic addition of neutralization agent.
When adding phytic acid (0.05 g/L) at the beginning
of fermentation, L-(+)-lactic acid production was increased by 1.4 times higher than without adding in 90 h
fermentation. Nevertheless, that can be taken effect after
40 h. For the growth of R.ASC081, addition of phytic acid
also promoted the increase of biomass, but the promotion
(1.1 times higher than that without adding phytic acid)
was less than that for L-(+)-lactic acid production. The
promoting effect of phytic acid for the growth of
R.ASC081 was also delayed. After 30 h, the promotion
took effect obviously, and the fungal biomass formation
reached 8.28 g after 90 h. If no phytic acid was added, lots
of mycelia formation led to a long passing phase, and in a
short duration, the mycelia formed irregularly shaped
masses or pieces of spores. Therefore, addition of phytic
acid was favorable not only for lactic acid production, but
for fungal biomass formation, due to the enhanced amylolytic capacity and decreased limitation of nutrients and
dissolved oxygen in the inner parts of spores. The formation of mycelial pellets during 40-50 h by adding 0.05gram phytic acid per micro-liter substrate was recommended condition for L-(+)-lactic acid production.
3.5 Effect of Mg2+
By comparing the fermentation with or without Mg2+
in the medium, it was found that Mg2+ would not directly
increase the L-(+)-lactic acid production. However, it is
a valuable factor to shorten the fermentation period of
R.ASC081. The L-(+)-lactic acid production rate after 60
h was decreased to 0.10 g/L×h in medium without Mg2+
addition. But in Mg2+ supplemented medium, the initial
production rate can reach to 0.18 g/L×h. By addition of
Mg2+ in a range of 0.1-5.0 g/L, the L-(+)-lactic acid fermentation period was shortened. It suggested the ability
of Mg2+ to act as an inducer of Mg-amylolysis activity
agent. The above analysis of LBPPS also indicated that
Mg2+ was a deficient element. But Mg2+ was an essential
fermentation factor for R.ASC081. Therefore, when Mg2+
210
Gaofeng Shi et al.
was added to fermentation medium at 0.5 g/L, L-(+)-lactic
acid fermentation was completed within 90 h cultivation.
Therefore, the addition of 0.5 gram per liter was the recommended in view of whole fermentation time.
3.6 Effect of Temperature
The impact of the cultivation temperature on the
L-(+)-lactic acid fermentation was investigated. When
the temperature was below 28 °C or above 35 °C, the
L-(+)-lactic acid production and biomass growth were
obviously varied by the temperature. The fermentation
performed by R.ASC081 at 28 °C and 35 °C appeared to
be relatively less sensitive than at 30-33 °C. The peak
L-(+)-lactic acid production was found at 32 °C. Consequently, 32 °C was regarded as optimal condition in view
of L-(+)-lactic acid production.
4. Conclusions
Optically pure L-(+)-lactic acid has been successfully produced directly from LBPPS using R.ASC081
fermentation. The formation of mycelial pellets and the
effective expression of amylase become the key factors
for direct production of optically pure L-(+)-lactic acid.
For simultaneous saccharification and selective bioconversion of LBPPS to optically pure L-(+)-lactic acid, the
optimum pH was controlled at 5.2. The magnesium
would not directly increase the L-(+)-lactic acid production, however, it is a valuable factor to shorten the fermentation period of R.ASC081. Phytic acid, as a new
growth factor for Rhizopus oryzae, can also stimulated
L-(+)-lactic acid production and biomass growth.
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Manuscript Received: Mar. 11, 2012
Accepted: Sep. 12, 2012