Isolation and screening of fungi for lactic

acid production
INTRODUCTION:-Lactic acid is a product that find several
application in food,cosmetics, pharmaceutical and chemical industries.
The main objective of this work was the development of a bioprocess to
produce lactic acid using dry grass,coconuthusk,sugarance waste and
wood chips as substrate. Among the fungal strains, Rhizopusspeciese
was selected for fermentation, due to its potential for the production of
bidegradable biocompatible polylactate fermentation was conducted
without need for supplementary for other minerals. Lactic acid can be
produced form renewable materials using various fungal species of the
Rhizopus genus. As it demands low nutrient requirement,easy and fast
growth and valuable fermentation by product fungal biomass. The
objective of the experiment is to select a suitable cheap,economiceasily
available substrate for production of lactic acid in crystal form by
examining its % acid production and xylose utilization on per day basis
of fermentation.
FungalRhizopus species have attracted a great interest and have been
recognized as suitable candidates for lactic acid production. Unlike
LAB lactic acid producing Rhizopus strains generate L-lactic acid as a
sole isomer of lactic acid.Rhizopus is a genus of
common saprophytic fungi on plants and specialized parasites on
animals. They are found on a wide variety of organic substrates,
including "mature fruits and vegetables",jellies, syrups, leather, bread,
peanuts, and tobacco. Some Rhizopus species are opportunistic agents of
human zygomycosis (fungal infection) and can be
fatal. Rhizopus infections may also be a complication of

diabetic ketoacidosis.[3] This widespread genus includes at least eight

species.

Rhizopus 400x magnification

Rhizopus species grow as filamentous, branching hyphae that generally
lack cross-walls (i.e., they are coenocytic). They reproduce by forming
asexual and sexual spores. In asexual reproduction, sporangiospores are
produced inside a spherical structure, the sporangium. Sporangia are
supported by a large apophysate columella atop a long stalk, the
sporangiophore. Sporangiophores arise among distinctive, root-like
rhizoids. In sexual reproduction, a dark zygospore is produced at the
point where two compatible mycelia fuse. Upon germination, a
zygospore produces colonies that are genetically different from either
parent.

R. microsporus var. oligosporus is used to make tempeh, a

fermented food derived from soybeans.

R. oryzae is used in the production of alcoholic beverages in parts

of Asia and Africa.

Rhizopusstolonifer (black bread mold) causes fruit rot on

strawberry, tomato, and sweet potato and used in commercial
production of fumaric acid and cortisone.

Various species, including R. stolonifer, may cause soft rot in sweet

potatoes and Narcissus.

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.oryzaeMTCC 8784 starch by
submerged fermentation (SmF). The biotechnological conversion of a
few vegetable and fruit wastes to lactic
acid by R.oryzaeMTCC 8784 is also demonstrated.
The discarded potato-waste materials as substrate offer advantages of
minimal pre-processing and supplementation. Producing lactic acid by
using waste materials instead of sugar and starch would reduce the lactic
acid production dramatically and can be both environmentally and
economically favourable.
The aim of this research was to develop a process integrating the
production of lactic acid with treatment of potato industry waste streams.
The research objectives were focused on selecting a suitable fungal
strain and determining the optimal conditions for direct fermentation to
lactic acid.

Review of literature

The effect of different carbon sources on lactic acid production

by Rhizopusoryzae was studied using glucose, sucrose, beet molasses, carob pod
and wheat bran as substrate. The highest lactic acid concentration was obtained
when 150 g/l glucose was present in the medium as the sole carbon source. In that
case, the lactic acid yield was approximately 60% by weight based on the amount
of glucose consumed.(By sule buluen,Murat Elibola)

-lactic acid production by pellet-form Rhizopus oryzaeR1021 in a

stirred tank fermentor(By Dong-Mei Bai, Min-ZeJia)
-lactic acid concentration of
was observed at the inoculum level
of
. Further studies on the effect of agitation speed on the fungal
morphology showed that increasing agitation speed caused the fungal pellets to
decrease in size but to increase in number per unit volume. Similar studies on the
effect of aeration rate on the fungal morphology showed that increasing aeration
rate caused the fungal pellets to increase in size but to decrease in number per unit
volume.
Direct fermentation of potato starch wastewater to lactic acid
by Rhizopusoryzae and Rhizopusarrhizus9(LiPing Huang)
The biochemical kinetic of direct fermentation for lactic acid production by fungal
species ofRhizopusarrhizus 3,6017 and Rhizopusoryzae 2,062 was studied with
respect to growth pH, temperature and substrate. The direct fermentation was
characterized by starch hydrolysis, accumulation of reducing sugar, and production
of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation,
biomass formation and lactic acid production were affected with the variations in
pH, temperature, and starch source and concentration. A growth condition with
starch concentration approximately 20 g/l at pH 6.0 and 30C was favourable for
both starch saccharification and lactic acid fermentation, resulting in lactic acid
yield of 0.870.97 g/g starch associated with 1.52.0 g/l fungal biomass produced

in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic

acid, while R. oryzae 2,062 produced more fungal biomass under similar condition.
L-lactic acid production by Rhizopusoryzae MBG-10 using starch-rich
waste loquat kernels as substrate

Authors(2013)(By SerkanOttucu)
The objective of this work was to perform production of L-lactic acid from starchrich waste loquat kernels by newly isolated Rhizopusoryzae MBG-10 fungus.
Loquat kernel flour (LKF) was used as substrate (mainly as carbon source). The
most favorable conditions for L-lactic acid production were LKF concentration of
80g/L, CaCO3 concentration of 20g/L, ammonium sulfate concentration of 3g/L
and incubation time of 108h. Under these conditions, L-lactic acid and biomass
concentrations were 45.4 and 8.2g/L, respectively, and -amylase activity was
81.6U/mL. No significant pH changes were observed in the medium thanks to the
buffering capacity of LKF.

Xylose metabolism in the fungusRhizopusoryzae: effect of growth and

respiration on L(+)-lactic acid production(2003)

Ronald H. W. Maas
, Jan Springer
, Gerrit Eggink
, Ruud A. Weusthuis

The fungus Rhizopusoryzae converts both glucose and xylose under aerobic
conditions into chirally pure L(+)-lactic acid with by-products such as xylitol,
glycerol, ethanol, carbon dioxide and fungal biomass. In this paper, we
demonstrate that the production of lactic acid by R. oryzae CBS 112.07 only occurs
under growing conditions. Deprivation of nutrients such as nitrogen, essential for
fungal biomass formation, resulted in a cessation of lactic acid production.

Complete xylose utilisation required a significantly lower C/N ratio (61/1)

compared to glucose (201/1), caused by higher fungal biomass yields that were
obtained with xylose as substrate. Decreasing the oxygen transfer rate resulted in
decline of xylose consumption rates, whereas the conversion of glucose by R.
oryzaewas less affected. Both results were linked to the fact that R. oryzae CBS
112.07 utilises xylose via the two-step reduction/oxidation route. The
consequences of these effects for R. oryzae as a potential lactic acid producer are
discussed.

Improvement of L(+)-Lactic Acid Production of RhizopusOryzae by LowEnergy Ions and Analysis of Its Mechanism
The wild type strain Rhizopusoryzae PW352 was mutated by means of nitrogen
ion implantation (15 keV, 7.8 1014 ~ 2.08 1015 ions/cm2) to find an industrial
strain with a higher L(+)-lactic acid yield, and two mutants RE3303 and RF9052
were isolated. In order to discuss the mechanism primarily, Lactate Dehydrogenase
of Rhizopusoryzae was studied. While the two mutants produced L(+)-lactic acid
by 75% more than the wild strain did, their specific activity of Lactate
Dehydrogenase was found to be higher than that in the wild strain. The optimum
temperature of Lactate Dehydrogenase in Rhizopusoryzae RF9052 was higher.
Effect of different carbon sources on l(+) -lactic acid production
by Rhizopusoryzae(2004)(Dursun ozer)
The effect of different carbon sources on lactic acid production
by Rhizopusoryzae was studied using glucose, sucrose, beet molasses, carob pod
and wheat bran as substrate. The highest lactic acid concentration was obtained
when 150 g/l glucose was present in the medium as the sole carbon source. In that
case, the lactic acid yield was approximately 60% by weight based on the amount
of glucose consumed. Wheat bran was found to be an unsuitable substrate for this
particular fermentation. Pasteurisation of molasses increased lactic acid production
rate compared to that of untreated molasses. Likewise, 58 g/l lactic acid was
obtained by using the supernatant containing sugars extracted from carob pod. This
medium could therefore be considered as an alternative carbon source for lactic
acid production

Materials and methods

MATERIALS
1. Potato extract
2. Dextrose
3. Agar
4. Lctophenol cotton blue stain
5. Soil samples
6. Glucose
7. Ammonium sulphate
8. Magnesium sulphate
9. Zinc sulphate
10. Potassium dihydrogen orthophosphate
11.Bromocresol green
12. Triton X-100
13. Calcium carbonate
14. Concentrated sulphuric acid
15. P-hydroxy biphenyl
16. 20% Coppersulphate
17. 3% Coppersulphate
18. 3,5-dinitrosalicylic acid
19. Phenol
20. Sodium sulphate
21. Sodium potassium tartarate
22. 5% Calcium hydroxide
23. 0.1N HCl
24. 0.1N NaOH
25. Isobutylketone
26. L-octanol

METHODS

ISOLATION:The suspension of soil sample of 10cm deep were made in 0.85% saline. The sample suspension
were then centrifuged at 5000rpm for 20 minutes.
The clear supernatant thus obtained was subjected to serial dilutions on Potato Dextrose
Agar(PDA) medium.the plates were then incubated for 5-7 days at 26-27 degree centigrade.the
various distinct colonies obtained were picked up and restreaked on PDA plates to pick up the
respective single colony. The isolated pure culture were stored at 4 degree in PDA slants for
further studies.

IDENTIFICATION:Take 1 drop of lectophenolcoton blue on slide then culture is put on drop of cotton blue stain
then cover with cover slip. Cover slip covers the specimen without trapping air bubbles
underneath.
View the slide with comp[ound microscope starting with a low objective

Observation:Under the microscope,Rhizopus appears as short strands with oval shaped heads,looking like
aballoon on a string.

SCREENING FOR LACTIC ACID PRODUCTION:The various isolated cultures were subjected to primary screening for lactic acid production by
determining the acid unitage value. The mineral acid indicator medium plates inoculated with
single spore were incubated at 35 degree centigrade for 48-72 hours. After 72 hours the acid
unitage value of the colonies were determined by dividing the diameter of the yellow zone by the
diameter of the zone of the colonies.

EXTRACTION OF LACTIC ACID

Solvent extraction of lactic acid appear a promising technique.Procedure for extraction was as
described as 50ml of the clear fermentation broth and organic phase were gently mixed in a
shaker bath at 30 degree centigrade for 24 hours.The phase containing lactic acid was separated
and concentration of lactic acid was determined colorimetrically.

MAT FORMATION IN PDB:-

PH

MAT

Glucose PH 6.0

Xylose

PH 5.5

TEMPERATURE
+ (25) more

Xylose

So, PH -5.5

+ (35)more

Temperature-25

Substrate Xylose

FERMENTATION STUDIES:The final step in screening for potent lactic acid producing strain was carried out by fermentation
studies. A loopful of the inoculum containing spores was inoculated into 25 ml of fermentation
medium in 250 ml flask. The medium contained glucose,120; ammonium sulphate,3.02;
magnesium sulphate,o.25; zinc sulphate,0.04; potassium dihydrogen orthophosphate,0.15.
Flask were incubated at 35 degree centigrade in an orbital shaker at 150 rpm for 96 hours. The
level of the lactic acid produced glucose consumed and dry cell weight of mycelial mass
produced was estimated at regular periodic intervals. In the same medium xylose also mtake as a
carbon source in another flask.
SUBSTRATE

Initial PH

PHPH

Glucose

6.0

4.0

2.0

Glucose

5.5

4.0

2.0

Xylose

5.5

6.0

4.0

Xylose

6.0

4.0

2.0

Xylose(temperature 25) 6.0

GLUCOSE ESTIMATION

2.0

PROCEDURE
To a 3ml test solution similar volume of DNSA reagent was added. The mixture was heated for 5
minutes in a boiling water bath then cooled. The absorbance was measured at 575nm in a
colorimeter.
Xylose 25

absorbance reading - 0.12

Glucose PH 6

absorbance reading -0.10

ANALYSIS:-

Paper chromatography:1. Position of chromatography paper was marked out and essential data written onto it.
2. Spot of standard and extracted sample applied on mark.
3. After spotting the chromatography was developed by ascending chromatography
technique.
4. The solvent was allow to run for 2-3 hours.
5. the paper was then removed and allow to dry.
6. Distance travelled by the spot were observed and rf value was calculated.
Solvent system (n-butanol:acetic acid:water)
( 4

: 5

TITRABLE 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 70C , 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 ofNaOH is equal to 90.08 mg
of Lactic acid.

CONCLUSION : Rhizopus species has the potential of lactic acid production.

The selected culture showed maximum optimimum growth and lactic acid production at
pH6.0 ,temperature 25 degree and substrate as xylose and under shaking condition.

FUTURE PROSPECTS: To detect the isomers of lactic acid and studies on synthetic polymers of lactic acid.

ACKNOWLEDGEMENT
It give me great pleasure to express my deep sense of gratitude and immense thankfulness to
Almighty God for giving me confidence and enthusiasm to carry my work. I express my thanks
to Dr. A.G Khan, Director Dr.Rafique Zakaria centre for Higher Learning and Advanced
research Rauza Bagh, Aurangabad for his encouragement and guidance. I also express my
thanks to principal Dr. Maqdoom Farooqui, Maulana Azad College of Arts, Science and
Commerce for providing all laboratory facilities.
I express my thanks to esteemed guide Miss. Zarina Shaikh H.O.D Of
Microbiology(P.G.) Dr.Rafique Zakaria Centre for Higher Learning and Advanced Research,
Rauzabagh, Aurangabad for her constant interest, encouragement and valuable guidance that
built up my confidence and determination express my profound gratitude to Mr. Wajed Khan ,Dr.
(Mrs.) Reshma Jaweria for their help and guidance during the complete course.
I would like to thank Dr.Sadat Quazi H.O.D Of Botany and faculty members for
guiding and helping us in project . I would also express thanks to Dr. Rafiuddin Nasir,
Department of botany for guiding and providing the laboratory facility for microscopic
observations.
I would like to thanks Dr. Ashfaque Khan, Assistant professor, Department of Botany for
his valuable help and guidance during the complete project work .
I am thankful to non-teaching staff who co-operated in my laboratory work . I express my
thanks to my friends who helped me to complete my project work. Finally the most important to
mention, thanks to my beloved parents for their blessings and support in my endeavors towards
the promising future.
Thank you.

REFERENCES
1. Barnett. A.J.G. The colorimetric determination
of Lactic acid in silage. Biochem J. 1951; Sep
49(4):527529.
2. Bulut. S., Elibol. M., Ozer. D., Effect of
different carbon sources on l(+) lactic acid
production by Rhizopus oryzae. Biochem. Eng.
J. 2004; 21:3337.
3. Datta R., Tsai S.P., Bonsignor. P., Moon. S.,
Frank. J. Technological and economic potential
of poly(lactic acid) and lactic acid derivatives.
FEMS Microbiol. Rev. 1995; 16:221231. L-127
Life Science
4. Ehrlich. F., Formation of fumaric acid by
means of molds. Ber. Dtsch. Chem. Ges. 1911
44:37373742.
5. Jin. B., Huang. L.P., Lant. P., Rhizopus
arrhizusa producer for simultaneous
saccharification and fermentation of starch
waste materials to l(+)lactic acid. Biotechnol.
Lett. 2003; 25:19831987.
6. Lockwood. L.B., Ward. G.E., May. O.E., The
physiology of Rhizopus oryzae. J. Agric. Res.
1936; 53:849857.
7. Maas, R.H.W., Bakker, R.R., Eggink, G. and
Weusthuis, R. A. Lactic acid production from
xylose by the fungus Rhizopus oryzae. Applied
Microbiology and Biotechnology 2006;

Microbiology

Vol.37(5):861 868.
8. 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; 42:7378.
9. Soccol. C.R., Marin. B., Raimbault. M.,
Lebeault. J.M., Potential of solid state
fermentation for production of l(+)lactic acid
by Rhizopus oryzae. Appl. Microbiol.
Biotechnol. 1994a; 41:286290.
10. Soccol. C.R., Stonoga V.L., Raimbault. M.,
Production of llactic acid by Rhizopus species.
World J. Microbiol. Biotechnol. 1994; 10:433
435.
11. Sodeck, G., Modi, J., Kominek, J. and
Salzbrunn, G. Production of citric acid
according to the submerged fermentation.
Process Biochem. 1981; 16(6),911
12. Sreenath, H.K, Chapman, T.W. & Jeffries,
T.W. Ethanol production from Dxylose in
batch fermentation with Candida shehatae:
process variables. Applied Microbiology and
Biotechnology 1986; 24:294299.
13. Yin. P.M., Nishina. N., Kosakai. Y., Yahiro.
K., Park. Y., Okabe. M., Enhanced production
of l(+)lactic acid from corn starch in a culture

of Rhizopus oryzae using an airlift bioreactor.

J. Ferment. Bioeng. 1997; 84:249253.
14. Yu. R.C., Hang. Y.D., Kinetics of direct
fermentation of agricultural commodities to
l(+) lactic acid by Rhizopus oryzae. Biotechnol.

RESULT and DISCUSSION:-

1.Isolation

2. Identification

Microscopic Observation:-

Under the microscope,Rhizopus appears as short strands with oval shaped heads,looking like
aballoon on a string.

3. screening

Observation table
SERIAL
NUMBER

SPECIES

1
2
3
4

Fusarium species
Rhizopus species
Aspergillus Flavus
Trichoderma species

GLUCOSE ESTIMATION
PROCEDURE

Diameter of yello
zone/Diameter of zone
of colonies
2.5/5
2.3/3.5
2/3.5
2.5/4.5

Acid unitage value

0.5
0.65
0.57
0.55

To a 3ml test solution similar volume of DNSA reagent was added. The mixture was heated for 5
minutes in a boiling water bath then cooled. The absorbance was measured at 575nm in a
colorimeter.
1
2

Temperature 25
PH
6.0

Xylose
Glucose

0.12
0.10

TITRABLE ACIDITY:1
2

Temperature 25
PH 6.0

Xylose
Glucose

1.1
0.6s

O.D.
0.25
0.2
0.15
0.1
0.05
0

II

III

IV

VI

VII

VIII

IX

UKI

UKII