International Journal of ChemTech Research

CODEN( USA): IJCRGG ISSN : 0974-4290

Vol.6, No.1, pp 527-537, Jan-March 2014

Production of L-Lactic acid from starch and food waste by

amylolytic Rhizopus oryzae MTCC 8784

Ranjit Kumar and Srividya Shivakumar*

Department of Microbiology, Centre for PG Studies, Jain University, 18/3,

9th Main, 3rd Block, Jayanagar, Bangalore 560011,India.

*Corres.author: [email protected]
Ph: 080 43226500; Fax: 080 43226507.

Abstract: Lactic acid, commonly used in food, chemical and pharmaceutical industries, has recently received
much attention for the production of biodegradable plastics. In this study, Rhizopus oryzae MTCC 8784 was
used to convert starch into optically pure L-Lactic acid. Effect of time, pH, addition of calcium carbonate and
starch concentration were analyzed using shaker flasks in order optimize the L-lactic acid concentration and
volumetric productivity. Under optimized conditions [pH6.0, CaCO3 (10g/L), starch (10g/L) and 72 h of
cultivation] R.oryzae MTCC 8784 gave a lactic acid concentration of 50.48g /L with a volumetric productivity
(Qp) of 0.7 (g/L/h) and lactic acid yield(YP/s) of 4.54 g/g (2 fold increase), 100% starch saccharification, and 1.5
g/L mycelial biomass. To reduce the Lactic acid production costs, inexpensive raw materials such as food waste
(fruit and vegetable peel/waste) like sapota, banana, papaya, potato, corn cob and carboxymethyl cellulose were
explored. All substrates tested supported growth and lactic acid production. Efficient lactic acid concentration
(72g/L) with a biomass of 1.9 g/L; volumetric productivity (Qp) of 1g/L/h and lactic acid yield(YP/s) of 3.6g/g
was obtained with sapota peel fermentation. Lactic acid was detected in the fermentation broth by HPLC.
Key words: R.oryzae MTCC 8784, lactic acid, process optimization, starch, vegetable and fruit waste.

Introduction
Lactic acid is a valuable chemical and one of its extensive applications is for polymerization of L-lactic acid to
poly (L-lactic acid), which is an attractive polymer because it can be produced from renewable resources and is
biodegradable. These properties have strengthened

interest in developing more efficient production processes for optical purity of L-lactic acid1. In India, the
annual production capacity of Lactic acid is 6000t and an estimated gap of 2300 t in supply by the year 2015
have been predicted, if the present level of production is not increased2. Wastes containing starch generated
from food processing plants may be regarded as a viable option for meeting this growing demand for lactic
acid, if appropriate biotechnological interventions are used and specific sectors amongst the Indian food
processing industry are targeted3. Rapidly increasing prices of animal feed supplements is one of the challenges

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faced by livestock industries at a global scale. Incidentally, solid unwanted agricultural materials resulting from
postharvest activities of farmers and food processors are also growing at a faster rate due to improved farming
methods and high fruit and vegetable produce4. Therefore, fruit and vegetable residues remain source of solid
agricultural waste. Fruit residues may cause serious environmental problems, since it accumulates in agro-
industrial yards without having any significant and commercial value. Since disposal of these wastes is
expensive due to high costs of transportation and a limited availability of landfills they are unscrupulously
disposed causing concern as environmental problems. Furthermore, the problem of disposing by-products is
further aggravated by legal restrictions. A high level of BOD and COD in fruit wastes add to further difficulties
in disposal. However, inspite of their pollution and hazard aspects, in many cases, food processing wastes have
a good potential for conversion into useful products of higher value as by-product, or even as raw material for
other industries. A large quantity of the waste include peels, seeds and pulps depending on the type of fruits5.
Banana waste (peel), pineapple waste and papaya waste are examples of agricultural wastes found abundantly
in several tropical and sub-tropical areas such as Indian subcontinent and Southeast Asian countries6.

Unlike the Lactic acid bacteria (LAB), lactic acid producing Rhizopus strains generate L-lactic acid as a sole
isomer of lactic acid7-10. The production of L-lactic acid using a surface culture of Rhizopus was reported in
191111. An efficient submerged fermentation using fungal species for the production of L-lactic acid was first
reported in 193612-13. However, an increased research interest has been given to lactic acid fermentation by
fungal species in recent decades14.

The present study attempted at identifying the process parameters in terms of time, pH, neutralizing agents
(CaCO3), starch concentration for maximizing lactic acid production by R.oryzae MTCC 8784 starch by
submerged fermentation (SmF). The biotechnological conversion of a few vegetable and fruit wastes to lactic
acid by R.oryzae MTCC 8784 is also demonstrated.

Experimental
Microbial strain

The pure culture of Rhizopus oryzae MTCC8784 (Microbial Type Culture Collection, Chandigarh) was
obtained and it was cultured on solid Sabouraud’s agar. Mycelia of freshly grown culture was used as inoculum
for Lactic acid production.

Media and fermentation conditions

The fermentations at shake flask level (50 mL in a 250 mL Erlenmeyer flask) were carried out using the media
as described by Huang et al. (2003)15, and contained carbohydrates 40g/L, yeast extract 10g/L, peptone 20g/L,
K2HPO4 0.5g/L, KH2PO4 0.5g/L, MgSO4.7H2O 0.05g/L, (NH4)2SO4 2g/l, pH 6.0, agitation at 160 rpm,
temperature at 30°C. Starch was used as carbohydrate source unless otherwise stated. Peptone, yeast extract,
ammonium sulphate were used as nitrogen substrates. Magnesium sulphate (MgSO4.7H2O), dipotassium
phosphate (K2HPO4), monopotassium phosphate (KH2PO4) were used as inorganic minerals.

Agro-Food Waste Substrates

A total of six different types of agro-food wastes viz. Sapota peel, banana peel, papaya peel, potato peel, corn
cob powder and carboxymethyl cellulose (CMC) (procured from the local markets of Bangalore), and the
material from one single batch was used in all the studies in order to minimize any possible interference due to
variation in composition of residues. The samples were dried in an oven at 60°C for two days, grounded and
screened to collect the particles of the size between 1.2 and 1.6 mm. Steam explosion treatment was given to
the substrates, amount of sugar content was determined by preparing their hydrolysate and used for lactic acid
production by R.oryzae MTCC 8784.

Steam explosion

The modified method of Pumiput et al. (2008) was used for substrate hydrolysate preparation16. 40 g of each
agro-food waste was steam-exploded in 100 L capacity autoclave at 121°C for 20min. Water was added to the
wet pre-treated material to make up the volume of 1 L and boiled at 80°C for 30 min. Later the hydrolysate was
recovered by filtration with cheese cloth.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 529

Acid Hydrolysis

Acid post hydrolysis of hemicellulose hydrolysate was carried out to cleave the xylooligosaccharides into
monomeric sugars by autoclaving at 121°C with concentrations of HCl varied from 2% v/v for 30 min16.
Chemical pre-treatment is to remove chemical barriers, so the enzymes can have access to cellulose for
microbial destruction. The hydrolysate from acid post hydrolysis was adjusted with CaO to pH 6- 6.8 and the
CaSO4 precipitates were removed by filtration with Whatmann filter paper No.115.

Direct hydrolysis

Direct infusion was carried out by drying and pulverizing the agro-wastes. The powdered substrates were added
as the sole carbon source and the media was supplemented with minimal salts.

Analytical methods

Screening for Lactic acid production

Czapeck dox agar (HIMEDIA, INDIA) plates with bromocresol green (0.05%, w/v) as the indicator or
supplemented with 1% CaCO3 were inoculated with a loopful of fungi and incubated for three to five days for
the formation of either yellow zone or clearance around the mycelial growth, respectively, indicating the
presence of Lactic acid production.

DNS method for reducing sugar estimation

Total reducing sugars in the fermentation broth was determined by Miller method using dinitrosalicylic acid
reagent17.

Starch-Iodine method for residual Starch estimation

The method developed by Tomas and Chamberlain (1980)18 was used which is based on color development that
results from iodine binding to starch polymers. The initial as well as residual starch was estimated by using this
method.

Titratable acidity

Every third day, unless otherwise stated, the flasks were removed and the fermentation product was centrifuged
at 8000 x g for 10mins, the centrifuged supernatant was heated at 70°C , to this 5% Ca(OH)2 was added and
filtered with Whatman filter paper1, the precipitant was collected and dissolved with 0.1N HCL and then
titrated with 1M NaOH. The acidity as total titrable acidity was determined titrating the samples with 1N NaOH
according to the method given by AOAC (2000)19. Every 1ml of of NaOH is equal to 90.08 mg of Lactic acid.

HPLC analysis

The qualitative analysis of lactic acid was analyzed using reverse phase high pressure liquid chromatography
(HPLC)20. HPLC analysis for lactic acid excreted from the metabolic activities of Rhizopus oryzae MTCC 8784
were performed on a Waters 518 model series comprised of a quaternary pump with auto-sampler injector,
micro-degassers, column compartment equipped with thermostat and a diode array detector. The column used
was a C18 (Waters 518) end capped 5 µm, 4.8x250 mm reverse phase column. The eluant used was acetonitrile
: water (7:3 v/v) and the column separation was allowed at a flow rate of 1.0 mL/min for 15 minutes. The
temperature of the column was maintained at 25°C.

1 ml of fermentation sample was taken after 72 h of fermentation, centrifuged at 14,000 rpm for 10 minutes in
Remi centrifuge in order to separate the cell mass and other insoluble materials. Supernatants were diluted 10
times to get more precise results from HPLC. Samples and standards (10µl) were injected using an autoinjector.
Lactic acid was detected at 210nm by the 410 Water UV detector.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 530

Results and Discussion

R.oryzae MTCC 8784 produced organic acid as revealed by the yellow coloration of Bromocresol indicator
plate (Plate 1 c & d) and precipitation of CaCO3 (Plate 1 a & b). The one factor at a time is the most frequently
used operation in optimization process. This technique involves changing one independent variable while
keeping the other factors constant. In the present study, we described optimized fermentation medium and
conditions [time, pH, CaCO3, starch] to obtain maximum lactic acid production with Rhizopus oryzae MTCC
8784.

Plate 1. Plate 1: Screening of R.oryzae MTCC 8784 for acid production using CaCO3(1%,w/v) plates a)
Control and b) Growth of R.oryzae showing precipitation of CaCO3; Bromocresol plates (0.05%,w/v) c)
Control plate and d) Growth of R.oryzae showing yellow coloration

Time course production of L-Lactic acid by R.oryzae MTCC 8784

The biochemical kinetics of simultaneous saccharification and fermentation (SSF) for lactic acid production by
fungal species of Rhizopus oryzae 8784 was studied with respect to time of incubation.

Rhizopus oryzae MTCC 8784 produced 50.48 g/L of L-lactic acid by 72h from starch (10g/L) (Fig.1). The
fungal biomass gradually increased from day one (1.2g/L) to day three (1.6g/L) with concomitant utilization of
starch and then remained constant. By day five, starch was completely utilized. The production of L-lactic
gradually increased from day one (19.97g/L), peaked by day three (50.48g/L) and the production gradually
decreased on day four and five.

Fungal Rhizopus species have attracted a great interest, and have been recognized as suitable candidates for
lactic acid production. Rhizopus oryzae can produce large amounts of L-lactic acid and utilize both various
sugars and starch as carbon sources8-10. Lactic acid production using Rhizopus oryzae seems to be a viable
alternative because it can grow on minimal liquid medium and on solid medium7.

Starch has been considered for use as a raw material for various fermentation because of its abundance and low
price. However, when high concentration of starch is used in medium, an increase in viscosity of the medium
due to gelatinization by heat will reduce the microbial growth.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 531

Fig. 1. Time course production of L-Lactic acid by R.oryzae MTCC 8784.

Effect of growth pH

In order to determine the impact of growth pH (uncontrolled) on the starch saccharification and fermentation of
lactic acid by the R. oryzae MTCC8784, in absence of CaCO3, the initial growth pH was set at 4.0, 5.0, 6.0, 7.0
and 8.0 by adding 4N NaOH solution.

The time course study proved that the fungus species demonstrated the highest lactic acid production by 72 h,
the variations in starch, reducing sugar concentrations and lactic acid yield in the culture were measured on
day 3 (Fig. 2).

The experimental results revealed that R. oryzae MTCC 8784 had a metabolic capability to saccharify starch,
produce lactic acid and fungal biomass using starch (20g/L) with a pH range between 4.0 and 7.0 with
maximum growth, starch utilization and lactic acid yield at pH 6.0 (with the residual pH dropped to pH 4.5),
after which all these parameters were affected at pH 7.0 and pH 8.0. Similar observations have been reported by
Domínguez and Vázquez (1999)20 for R.oryzae wherein pH 3.5-6.0 supported lactic acid production.

Effect of supplementation of CaCO3

Lactic acid is known to be a strong inhibitor for both cell growth and lactic acid production 21.Calcium
carbonate is a commonly used reagent to neutralize lactic acid during fermentation. Its low solubility in water
makes it possible to neutralize lactic acid and maintain the pH at certain level automatically22.

Addition of CaCO3 in the concentration of 0.5, 1.0, 1.5 and 2.0% (w/v) did not have any profound effect on
biomass, starch utilization and lactic acid production as compared to the control set at an initial pH 6.0 without
any CaCO3 addition in the fermentation broth (Fig.3). This indicates that R oryzae MTCC8784, addition of
CaCO3 1% (w/v) was found to be sufficient for maintaining a growth pH for one batch, to achieve an optimum
fungal cell growth and lactic acid production.

Soccol et al., (1994)7 demonstrated that the addition of CaCO3 increases the lactic acid production from
concentrations lower than 3g litre-1 to 65g litre-1. Other chemicals have also been recommended. Friedman &
Gaden, (1970)23 proposed the use of sodium hydroxide meanwhile Stieber & Gerhardt (1995)24 worked with
ammonium hydroxide. In a study by Huang et al., (2005)21 on Simultaneous saccharification and fermentation
of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizusi, CaCO3 addition of 1%
(w/v) was found to be sufficient for maintaining a growth pH to achieve an optimum fungal cell growth and
lactic acid production using potato starch in both Rhizopus cultures. This is in contrast to our results.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 532

Fig.2. Effect of initial media pH on LA production by R.oryzae MTCC 8784.

Fig.3. Effect of CaCO3 supplementation on LA production by R.oryzae MTCC 8784.

Effect of starch concentration

The effect of starch concentration varying from 10 to 50 g/l in submerged fermentation was investigated, and
the results are shown in Fig.4. The concentration of lactic acid produced by R. oryzae MTCC 8784 increased
from 50 to 66.6 g/l with increasing initial starch concentration from 10 to 40 g/l with a concomitant increase in
biomass from 1.3g/L to 1.6g/L (Fig.4). A further increase in initial starch concentration (50g/L) resulted in a
gradual decrease in lactic acid production (64.8g/L) and biomass (1.5g/L). Starch was completely utilized in all
the concentrations indicating good amylolytic potential of the strain.

Decrease in yield with further increase in starch concentration beyond 40g/L may be due to substrate inhibition
during later stages of fermentation at high concentration of substrates.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 533

Fig.4. Effect of starch concentration on LA production by R.oryzae MTCC 8784.

Kinetic characteristics of lactic acid production

The investigation of kinetic characteristics in the SmF process was conducted in shake flask fermentation using
starch (20 g/l). Cultivation conditions were set up at an initial pH of 6.0 with supplementation of 1% (w/v)
CaCO3 at 30°C. Samples were taken at the end of 72 h fermentation. Table 1 summarizes the kinetics of LA
production by R.oryzae MTCC 8784 under un-optimized and optimized conditions.

Under optimized conditions [pH6.0, CaCO3 (10g/L), starch (10g/L) and 72 h of cultivation] R.oryzae MTCC
8784 gave a lactic acid concentration of 50.48g /L with a volumetric productivity (Qp) of 0.7 (g/L/h) and lactic
acid yield (YP/s) of 4.54 g/g (2 fold increase), 100% starch saccharification and 1.5 g/L mycelial biomass.

Lactic acid detection by HPLC

The retention time for lactic acid was around 2.61 minutes which overlapped with the lactic acid standard (2.65
mins) (Fig.5). Other peaks indicate presence of other organic acids like citric acid and fumaric acid, as R.oryzae
is a heterofermentative organism.

R. oryzae produces mainly lactic acid from glucose with yields of 60–80% and also ethanol, carbon dioxide and
minor amounts of malic acid, fumaric acid and citric acid25,26. Product formation depends on cultivation
conditions; it has been shown that, under oxygen-limiting conditions, product formation shifts from lactic acid
to ethanol26,27.

Table 1. Comparison of fermentation kinetics of LA production by R.oryzae MTCC 8784 under un-
optimized and optimized conditions of cultivation by SmF.
Condition Starch (g/L) Lactic acid Volumetric Lactic acid
concentration (g/L) productivity of yield (YP/S) (g/g)
Lactic acid (g/L/h)
Unoptimized 20(~22.2g 50.48 0.7 (72h) 2.27
glucose)
Optimized 10(~11.1g 50.48 0.7(72h) 4.54
glucose)
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 534

Fig.5. HPLC chromatogram of L-Lactic acid produced by R.oryzae MTCC 8784.

Lactic acid production from different agro-food wastes

All complex substrates tested supported appreciable growth and lactic acid production by R.oryzae MTCC 8784
(Fig.6). Highest lactic acid concentration (72g/L) with a biomass of 1.9 g/L; volumetric productivity (Qp) of
1g/L/h and lactic acid yield (YP/S) of 3.6g/g was obtained with sapota peel fermentation followed by corn cob
powder [70.2g/L; QP (0.98g/L/h); YP/S (3.51g/g) and biomass (1.7g)].

Cheap raw materials, such as starchy and cellulosic materials, whey, and molasses, have been used for lactic
acid production28. Among these, starchy and cellulosic materials are currently receiving a great deal of
attention, because they are cheap, abundant, and renewable29. The starchy materials used for lactic acid
production include sweet sorghum, wheat, corn, cassava, potato, rice, rye, and barley30. These materials have to
be hydrolyzed into fermentable sugars before fermentation, because they consist mainly of α (1,4)- and α (1,6)-
linked glucose. This hydrolysis can be carried out simultaneously during fermentation with amylase-producing
L. amylophilus and L. amylovorus which are often used for the direct fermentation of starchy materials into
lactic acid31. Cellulosic materials have been used for lactic acid production in similar ways as starchy materials.
These materials consist mainly of β (1,4)-glucan, and often contain xylan, arabinan, galactan, and lignin.
Venkatesh (1997) and Yáñez et al. (2005) have previously attempted to produce lactic acid from pure cellulose
through simultaneous saccharification and fermentation (SSF)32,33. The utilization of corncob, waste paper and
wood, has been reported as well34. Sreenath et al. (2001) investigated the production of lactic acid from
agricultural residues such as alfalfa fiber, wheat bran, corn stover, and wheat straw35. They suggested that,
during SSF of alfalfa fiber, lactic acid production was enhanced by adding pectinase and cellulase together.
Garde et al. (2002) used hemicellulose hydrolyzate from wheat straw for lactic acid production by co-culture of
L. brevis and L. Pentosus36.

Lactic acid has also been produced from waste sisal stems37, sugarcane bagasse38 and kitchen waste39 by using
Lactobacillus isolates. Fungal production of lactic acid from pineapple waste resulted in 19.3 and 14.7g/L lactic
acid with Rhizopus arrhizus and R. oryzae40.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 535

Unlike most bacteria, lactic acid-producing fungi, for example, the genus Rhizopus, are capable of efficiently
secreting lactic acid41 and some Rhizopus strains may secrete L-lactic acid as the only product42. In addition,
fungal cultures are tolerant of low pH environments. Consequently, pH maintenance during fungal fermentation
is not as stringent as with bacterial cultures21. Furthermore, fungal cultures, such as Rhizopus spp, are
amylolytic and can produce lactic acid from starchy substrates such as potato starch without prior
saccharification43. Fungi also have advantages such as low nucleic acid contents and high levels of protein. It
has been demonstrated that the cost of separating biomass from the spent cultivated broth may be a significant
fraction of the total capital and operating costs. In the case of fungal cultivation, the mycelial and pellet forms
are easy and inexpensive to harvest. Within the Rhizopus genus, R oryzae has received the greatest interest, and
strain R oryzae NRRL 395 has been recognized as one of the most suitable lactic acid producers30. R oryzae
NRRL 395 and R oryzae IFO 4707 strains were shown to convert ground corn and potato pulp, respectively,
directly to lactic acid. R oryzae ATCC 52311 could achieve a lactic acid concentration of 83 g dm−3 with a
yield of 0.88 g lactic acid g−1 glucose consumed.

Fig.6. Lactic acid production from agro-food wastes by R.oryzae MTCC 8784.

Conclusions
Optimization of process parameters, such as time of incubation, pH, starch concentration and CaCO3 addition
resulted in improving performance of Rhizopus species in lactic acid production. Lactic acid is known to be a
strong inhibitor of cell growth, enzymatic hydrolysis and microbial activity in lactic acid fermentation. To
prevent this self-inhibition, the addition of a neutralizing agent is, therefore, necessary. In the present work a
significant yield in terms of lactic acid and biomass production was obtained without the addition of 10 g dm−3
CaCO3 to the fermentation media for one batch. Hence, this process may alleviate the extra costs for lactic acid
purification and biomass recovery.

This study records highest production of lactic acid from sapota peel (72g/L) and corn cob (70g/L) substrates as
compared to previous studies which reports around 10.1g/L for corn and 4.2g/L for potato44. Also the study
shows novel use of food wastes like vegetable and fruit peels as use for substrate in lactic acid production. The
inference drawn from this study aims for the use of food wastes for commercial and economic production of
application based products by fermentative bioconversions.

Acknowledgements:
The authors greatly acknowledge the management of Jain University for providing financial and infrastructural
support for this work.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 536

References

1. Wang L.M., Zhao B., Liu B., Yang C.Y., Yu B., Li Q.G., Ma C.Q., Xu P. and Ma Y.H., Efficient
production of L-lactic acid from cassava powder by Lactobacillus rhamnosus, Bioresour Technol.,
101, 2010, 7895–7901
2. TIFAC. Vision 2020 reports. (2001) Technology Linked Business Opportunity Publications, Code No.
TMS157, Vol. 4.
3. World Bank Group. (2002) Markets and Agro enterprises. Accessed: 21.5.05.
4. Jamal P., Saheed O.K. and Alam Z., Bio-valorization potential of banana peels (Musa sapientum): An
overview, Asian J Biotechnol., 4, 2012, 1-14.
5. Correia R., Magalhaes M. and Macedo G., Protein enrichment of pineapple waste with Saccharomyces
cerevisiae by solid state bioprocessing, J Sci. Ind Res., 66, 2007, 259-262.
6. Roslina R. Optimization and modeling of Lactic acid production from pineapple waste. Technical
Report, VOT 74263, Faculty of Chemical and Natural Engineering, Universiti Teknologi Malaysia.
7. Soccol C.R., Marin B., Raimbault M. and Lebeault J.M., Potential of solid state fermentation for
production of l(+)-lactic acid by Rhizopus oryzae, Appl. Microbiol. Biotechnol., 41, 1994, 286–290.
8. Soccol C.R., Stonoga V.L. and Raimbault M., Production of l-lactic acid by Rhizopus species, World
J. Microbiol. Biotechnol., 10, 1995, 433–435.
9. Yu R.C. and Hang Y.D., Kinetics of direct fermentation of agricultural commodities to L(+) lactic
acid by Rhizopus oryzae, Biotechnol. Lett., 11, 1989, 597–600.
10. Yin P.M., Nishina N., Kosakai Y., Yahiro K., Park Y. and Okabe M., Enhanced production of l(+)-
lactic acid from corn starch in a culture of Rhizopus oryzae using an air-lift bioreactor, J. Ferment.
Bioeng., 84, 1997, 249–253.
11. Bulut S., Elibol M. and Ozer D. Effect of different carbon sources on l(+)-lactic acid production by
Rhizopus oryzae, Biochem. Eng. J., 21, 2004, 33–37.
12. Ehrlich F., Formation of fumaric acid by means of molds, Ber. Dtsch. Chem. Ges., 44, 1911, 3737–
3742.
13. Lockwood L.B., Ward G.E. and May O.E., The physiology of Rhizopus oryzae, J. Agric. Res., 53,
1936, 849–857.
14. Mass R.H.W., Bakker R.R., Eggink G. and Weusthuis R.A., Lactic acid production from xylose by the
fungus Rhizopus oryzae, Appl. Microbiol. Biotechnol., 72, 2006, 861–868.
15. Huang J.P., Jin B., Lant P. and Zhou J., Biotechnological production of lactic acid integrated with
potato waste water treatment by R. arrhizus, J Chem Technol. Biotechnol., 78, 2003, 899- 906.
16. Pumiput P., Chuntranuluck S., Kitpreechavanich V., Punsuron V. and Vaithanomsat P., Production
process of hydrolysate from steam explosion of oil Palm trunk for xylitol fermentation, Kasetsart J.
(Nat.Sci)., 2008, 42:73-78.
17. Miller and Gail L., Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal.
Chem., 31(3), 1959, 426-428.
18. Tomas L.C. and Chamberlain G.J., Colorimetric Chemical Analytical Methods, Tintometer Ltd.,
Salisbury, UK, 1980, 3–5.
19. A.O.A.C (2000). Official methods of analysis. Association of Official Analytical Chemists
International. Maryland, USA.
20. Domínguez J.M. and Vázquez M., Effect of the operational conditions on the l-lactic acid production
by rhizopus oryzae, Cienc. Tecnol. Aliment, 2 (3), 1999, 113-118
21. Huang L.P., Jin B., Lant P. and Zhou J., Simultaneous saccharification and fermentation of potato
starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus, Biochem. Eng. J., 23 (2005)
265–276.
22. Mart´ak J., Schlosser S., Sabolov´a E., Kriˇstof´ıkov´a L. and Rosenberg M., Fermentation for lactic
acid with Rhizopus arrhizus in a stirred tank reactor with a periodical bleed and feed operation, Process
Biochem., 38, 2003, 1573–1583.
Srividya Shivakumar et al /Int.J. ChemTech Res.2014,6(1),pp 527-537. 537

23. Friedman M.R. and Gaden E.R., Growth and acid production by Lactobacillus (L.) delbruekii in a
dialysis culture system, Biotechnol. Bioeng., 12, 1970, 961-974.
24. Stieber R.W. and Gerhardt P., Dialysis process for ammonium lactate fermentation: stimulated and
experimental dialysis-feed, immobilized-cell system, Biotechnol. Bioeng., 23, 1981, 535-59.
25. Longacre A., Reimers J.M., Gannon J.E. and Wright B.E., Flux analysis of glucose metabolism in
Rhizopus oryzae for the purpose of increased lactate yields, Fungal Genet Biol., 21, 1997, 30–39
26. Skory C.D., Lactic acid production by Rhizopus oryzae transformants with modified lactate
dehydrogenase activity, Appl Microbiol Biotechnol., 64, 2004, 237–242
27. Taherzadeh M.J., Fox M., Hjorth H. and Edebo L., Production of mycelium biomass and ethanol from
paper pulp sulfite liquor by Rhizopus oryzae, Bioresour Technol., 88, 2003, 67–177
28. Hofvendahl K. and Hahn-Hagerdal B., Factor affecting the fermentative lactic acid production from
renewable resources, Enzyme Microb Technol., 26, 2000, 87-107.
29. Oh H., Wee Y.J., Yun J.S., Han S.H., Jung S., Ryu H.W., Lactic acid production from agricultural
resources as cheap raw materials, Bioresour Technol., 96, 2005, 1492–1498.
30. Yun J.S., Wee Y.J., Kim J.N. and Ryu H.W., Fermentative production of DL-lactic acid from amylase-
treated rice and wheat bran hydrolyzate by a novel lactic acid bacterium, Lactobacillus sp., Biotechnol.
Lett., 26, 2004, 1613–1616.
31. Vishnu C., Seenayya G. and Reddy G., Direct fermentation of various pure and crude starchy
substrates to L(lactic)acid using Lactobacillus amylophilus GV6, World J. Microbiol. Biotechnol., 18,
2002, 429–433.
32. Venkatesh K.V., Simultaneous saccharification and fermentation of cellulose to lactic acid, Bioresour
Technol., 62, 1997, 91–98.
33. Yáñez R., Alonso J.L. and Parajó J.C., D-lactic acid production from waste cardboard, J. Chem.
Technol. Biotechnol., 80, 2005, 76–84.
34. Miura S., Dwiarti L., Arimura T., Hoshino M., Tiejun L. and Okabe M., Enhanced production of L-
lactic acid by ammonia-tolerant mutant strain Rhizopus sp. MK-96-1196, J Biosci Bioeng., 97, 2004,
19–23.
35. Sreenath H.K., Moldes A.B., Koegel R.K., Straub R.J., Lactic acid production from agricultural
residues, Biotechnol Lett., 23, 2001, 179–184
36. Garde A., Jonsson G., Schmidt A.S., Ahring B.K., Lactic acid production from wheat straw
hemicellulose hydrolyzate by Lactobacillus pentosus and Lactobacillus brevis, Bioresour Technol., 81,
2002, 217–223.
37. Muruke M.H.S., Hosea K.M., Palangyo A. and Heijthuijsen J.H.F.G., Production of lactic acid from
waste sisal stems using a Lactobacillus isolate, Discovery Innov., 18(1), 2006, 1-5.
38. Adsul M.G., Varma A.J. and Gokhale D.V., Lactic acid production from waste sugarcane bagasse
derived cellulose, Green Chem., 2007, doi: 10.1039/b605839f. 9: 58-62.
39. Ohkouchi Y. and Inoue Y., Impact of chemical components of organic wastes on l(+)-lactic acid
production, Bioresour Technol., 98(3), 2007, 546-553.
doi:10.1016/j.biortech.2006.02.005.
40. Jin B., Yin P., Ma Y. and Zhao L., Production of lactic acid and fungal biomass by Rhizopus fungi
from food processing waste streams, J. of Industrial Microbiology and Biotech., 32, 2005, 678-686.
41. Kalayanamitra S., Fermentation of lactic acid from cassava starch by immobilized Rhizopus oryzae
KPS 106 in airlift bioreactor. M.S. Thesis, Kasetsart University. 130 p., 2004.
42. Sun Y., Li and Bai S. Modeling of continuous L(+)-Lactic acid production with immobilized Rhizopus
oryzae in air lift bioreactor, Biochem Eng. J., 3, 1999, 87-90.
43. Anuradha R., Suresh A.K. and Venkatesh K.V., Simultaneous saccharification and fermentation of
starch to lactic acid, Proc Biochem., 35, 1999, 367–375.

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