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Life Science Archives (LSA)

ISSN: 2454-1354
Volume 1; Issue - 1; Year 2015; Page: 1 5

Research Article
SINGLE CELLL PROTEIN PRODUCTION FROM MARINE YEAST
Yarrowia lipolytica USING FRUIT WASTE
S. Brammavidhya*
PG Department of Microbiology, A.V.C College (Autonomous), Mayiladuthurai, Tamil Nadu, India.
Abstract
Extracts of fruit wastes such as banana, orange and apple were used in the present research as a
substrate for single cell protein production using a marine yeast Yarrowia lipolytica. This strain isolated from
the marine sediment sample and they were identified and cultivated using yeast extract potato dextrose agar
(YEPD) medium. Yarrowia lipolytica gives more protein content (42%) using fruit waste as cheaper source
for its cultivation. The nucleic acid content, especially RNA, is very high on a dry weight basis and was
reported to be 1.7 - 2.5%. The protein bands were observed using SDS-PAGE and their molecular weight
ranging from 15-79 kDa. The amino acid profiling was also performed and its revealed that the 18 amino
acids were present.
Article History
Received : 03.02.2015
Revised : 18.02.2015
Accepted: 23.02.2015

Keywords: Yarrowia lipolytica, Fruit waste and

Single cell protein.

1. Introduction

The growing shortage of protein and other

protein rich food supplies has stimulated the effort
in searching new and alternate source of protein
rich food and feed. The increased production of
wastes in the world is of great concern. Various
alternatives are exercised to diminish this increase
by elimination, purification and recycling (Saadia
et al., 2008; Saleem et al., 2008). Biomass is an
alternative natural source for chemical and feed
stock with a replacement cycle short enough to
meet the demands of the world fuel market
(Ahmed et al., 2009). Single cell proteins (SCP)
are used as protein sources in food and animal
feed. Single cell proteins are produced by
culturing different microbes on different substrates
such as whey starch, cellulose, hydrocarbon,
* Corresponding author: S. Brammavidhya
Tel.: +91-9944547060
E-mail: brammav@yahoo.com

alcohols and molasses. Fermentable sugarcanes

are used as a substrate for the growth of microbes
and production of single cell protein. Agricultural
wastes are the most abundant raw materials
consists cellulose as a major component, which is
suitable for the growth of microbes and production
of SCP biomass.
Today yeast SCP is considered as a
potential protein source for humans as well as for
animals. Food grade yeasts can provide proteins,
carbohydrates, fats, vitamins, minerals and
essential amino acid. Generally, the lysine content
in yeasts is higher than that of bacteria and algae.
Moreover, yeast contains low amounts of nucleic
acid. The acceptability of particular microbes as
food or feed depends on its nutritional value and
safety issue including nucleic acid content,
presence of toxins and residual undesirable
compounds such as heavy metals. Today, the only
species fully acceptable as food for humans is

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S.cerevisiae (bakers and brewers yeast). At this

juncture the present study aimed at production of
SCP from marine yeast using fruit wastes as
cheaper substrate.

culture was inoculated and kept in a shaker (120

rpm) for incubation and the absorbance was
measured at 600 nm at 12 hrs interval.

2. Methodology

The effect of temperature on SCP

production was studied at various temperatures
like 25C, 30C, 35C, and 40C for 3 days
incubation. The absorbance was measured at 600
nm at 12 hrs interval.

2.1. Isolation and identification of yeast

For isolation of yeast, marine sediment
sample was collected from Parangipet coastal
area. One g sediment sample was transferred to
99ml diluent blank (50% aged sea water) and
serially diluted up to 10-7. After serial dilution, the
samples were inoculated into Potato dextrose agar
plates using spread plate technique and incubated
for 3 - 5 days at room temperature.
Yeast colonies appeared with different
morphology was isolated and pure cultures were
stored on YEPD slants. The isolated yeast colonies
were identified based on colony color, shape,
texture, microscopic morphology, physiological,
biochemical test and various sugar assimilations
(Wickerham, 1951; Sanni and Lonner, 1993;
Yarrow, 1998; Barnett et al., 2000). At log phase
cells were harvested and their protein level was
estimated.
2.2. Antimicrobial activity of Yarrowia lipolytica
Yarrowia lipolytica were checked for
antimicrobial activity against pathogens such as
Staphylococcus aureus, Salmonella typhi,
Salmonella paratyphi, Klebsiella oxytoca,
Pseudomonas aeruginosa, E.coli, Proteus
mirabilis, Lactobacillus vulgaris, Vibrio cholerae
and Klebsiella pneumoniae were determined by
the Agar well diffusion method.
2.3. Optimization of Yarrowia lipolytica
2.3.1. Effect of pH
Optimization of pH for the growth of the
potential strain, medium was prepared with
different pH range 5-10. Then loopful of culture
was inoculated and kept in a shaker (120 rpm) for
incubation and the absorbance was measured at
600 nm at 12 hrs interval.
2.3.2. Effect of salt concentration
Different concentration (0.5-3 %) of salt
was added in to the medium. Then, loopful of

2.3.3. Effect of temperature

2.3.4. Effect of carbon source

The effect of carbon source on single cell
protein production was assessed by using various
carbon sources are glucose, sucrose, maltose,
starch and cellulose. Then, loopful of culture was
inoculated and kept in a shaker (120 rpm) for
incubation and the absorbance was measured at
600 nm at 12 hrs interval.
2.3.4. Effect of Nitrogen source
Different nitrogen sources such as peptone,
beef extract, ammonium nitrate, ammonium
sulphate and sodium nitrate were tested. Then
loopful of culture was inoculated and kept in a
shaker (120 rpm) for incubation and the
absorbance was measured at 600 nm at 12 hrs
interval.
2.4. Mass scale cultivation of Yarrowia lipolytica
in fruit source
The fruit waste samples such as banana,
orange and apple were used as a cheaper source
for mass scale cultivation of SCP. These fruit were
taken as composite sample used to formulate 1000
ml of medium containing 5% of cheaper source.
The log phase culture was inoculated into the
medium and incubated in optimized parameters.
After 72 hrs at which maximum biomass was
observed, the cells were harvested by using
centrifugation at 8000 rpm for 15 min, and cells
were lyophilized or dried under 65C overnight.
The weight of biomass was calculated.
2.5. Biochemical composition of biomass
2.5.1. Estimation of protein
The method of Lowry et al. (1951) was
adapted for the estimation of total protein.
Hundred mg of dried yeast cells were thoroughly

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homogenized with 1 ml of deproteinizing agent

(10% TCA) by keeping the tubes in an ice bucket
and samples were centrifuged for 20 min at 3000
rpm. The precipitate was dissolved in 2 ml of 1N
NaOH and to 1 ml of this solution; freshly
prepared 5 ml of alkaline reagent was added. This
was kept at room temperature for 10 min, after
which 0.5 ml of 1N folin ciocalteu reagent was
added and mixed rapidly. All the test tubes were
kept for 30 min, at room temperature and the
optical density of the blue colour developed was
measured against the blank at 660 nm.

tubes were kept for 20 min in boiling water bath

and then cooled. The OD of the color developed
was measured at 660 nm against blank.

2.5.2. Estimation of Carbohydrate

The purity of protein was checked by using

SDS-PAGE. The standard protein marker and
purified protein were allowed to run
simultaneously for determination of molecular
weight. The protein bands on gel were visualized
by staining it with coomassie brilliant blue.

The total carbohydrate was estimated by

phenol-sulphuric acid method of Dubois et al.
(1956). About 5 mg of dried yeast cells were taken
for carbohydrate analysis. The dried yeast cells
were taken in a test tube and 1 ml of phenol (5%)
and 5 ml concentrated sulphuric acid were added
in quick succession. The tubes were kept for 30
min at 50 C and the optical density of the color
developed was measured at 490 nm against the
blank.
2.6. Estimation of DNA
The colorimetric method of Sadasivam and
Manikam (1996) was adopted for the estimation of
DNA on basis of quantitative reaction of deoxy
sugar with diphenylamine reagent. 0.5 mg of dried
yeast cells were thoroughly mixed with 1 ml of
saline citrate and made up to 3 ml distilled water,
to which 6 ml diphenyl reagent was added. After
mixing, the tubes were kept for 10 min in boiling
water bath and then cooled. The optical density of
the color developed was measured at 600 nm
against blank.
2.7. Estimation of RNA
The colorimetric method of Sadasivam and
Manikam (1996) was adopted for the estimation of
RNA on the basis of pentose determination as well
as orcinol, phloroglucinal, aniline etc. Fifty g of
dried yeast cell were mixed thoroughly mixed
with 1 ml distilled water and kept in ice chilled 10
mM Tris-acetate, 1 mM EDTA buffer (pH 7.2),
and made up to 3 ml with distilled water. To this 6
ml of orcinol acid reagent and 0.4 ml of 6%
alcoholic orcinol were added. After mixing the

2.8. Amino acid profile of protein

Bruckner (1995) method was used for
estimating amino acid in the yeast dried cells. The
samples were hydrolyzed with 6 N HCl at 110 C
for 22 hrs. The amino acids were determined by an
automatic amino acid analyzer (Lachrom E.
Merck).
2.9. Molecular weight of protein- SDS PAGE

3. Results
3.1. Isolation and identification of yeast
Totally 10 morphologically distinct strains
were isolated from marine sample. The selected
potential strain with high protein concentration
was subjected to morphological, cultural and
various sugar assimilation tests to identify the
species. On YEPD agar plate, the colony was
found to be white, shiny and round.
3.2. Antimicrobial activity of Yarrowia lipolytica
The culture pellet obtained from Yarrowia
lipolytica exhibited varying degrees of inhibitory
activity against 10 human pathogens. More than
10 mm in diameter, zone of inhibition was
observed against all the pathogens. It was evident
that the strain very well can be used as a probiotics
(Table - 1).
3.3. Optimization of potential strain Yarrowia
lipolytica
Maximum growth was observed in the
medium at pH 9, 30C and 0.5% salinity.
Regarding the substrate concentration 1% glucose
was act as a carbon source for maximum growth
and 0.5% ammonium nitrate was used as a
nitrogen source for optimum growth.

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3.4. Mass scale cultivation of Yarrowia lipolytica

in fruit source

a 25% protein enrichment of the basic substrate

(Skogman, 1976).

Mass cultivation of the strain using

cheaper sources like fruit source was done in
shake flask to get enough biomass for further
study and also to assess the minimum production
of biomass compared to the artificial YEPD
medium.

Table - 1: Antimicrobial activity of Yarrowia

lipolytica

Staphylococcus aureus

23

3.5. Biochemical qualities analysis

Salmonella typhi

17

Salmonella paratyphi

12

Klebsiella oxytoca

16

Pseudomonas aeruginosa

20

Escherichia coli

18

3.6. SDS-PAGE

Proteus mirabilis

23

Protein profile obtained from the results of

SDS-PAGE revealed that Yarrowia lipolytica
contain proteins of different molecular weight
.Totally, 13 bands were present in this species.
The molecular weight of different protein band
was compared with standard protein markes and
the results showed that the molecular weight of the
protein range from 15-79 kDa.

Lactobacillus vulgaris

20

Vibrio cholera

25

Klebsiella pneumoniae

25

The biochemical estimation of the

Yarrowia lipolytica strain was done to assess its
potential to use as SCP. The biochemical
constituents like protein, lipid, carbohydrate and
nucleic acid content of the samples were assessed
and the results were presented in Table - 2.

Pathogens

Table-2: Biochemical composition of microbial

biomass

Composition

Mass
cultivation in
optimized
YEPD
medium (%)

Mass
cultivation
using cheaper
sources (%)

Crude protein

37

42

Total
carbohydrate

26.4

29.1

Total
acid

7.1

3.2

3.7. Amino acid profiling

In the amino acid profiling, the following
amino acids such as Aspartic acid (46 mg/g),
Glutamic acid (74 mg/ g), Alanine (40.5mg/g),
Valine (41 mg/g), Lysine (50 mg/g), Phenyl
alanine (40 mg/g), Glycine (33 mg/g), Serine,
Therionine and Tyrosin (30.5 mg/g), Isoleucine
(30 mg/g), Leucine (35 mg/g), Prolin (34 mg/g),
cysteine (20 mg/g) were present in the Yarrowia
lipolytica strain.

Zone of Inhibition
(mm in diameter)

nucleic

4. Discussion

RNA

5.0

1.7

In the yeast strain Yarrowia lipolytica

isolated from the marine sediment sample, have
high protein contents and has an antimicrobial
activity. Potato starch is generally used for feeds
but might also be fermented for protein
enrichment (Rusendi and Sheppard, 1995). After
fermentation for about 24 hrs, it was estimated
that a 50% biomass yield would be attainable,
based on starch dry matter, which corresponded to

DNA

2.6

1.2

Considering both biomass yield and

protein content, best results were obtained with
Candida utilis ATCC 9256 which was easy to
grow and gave 15.9 g of yeast protein per 100 g of
glucose. In the present study, Yarrowia lipolytica
analyzed showed high protein and certain essential
amino acids. The crude protein content obtained,
using cheaper sources is around 42% of the total
dry weight which seems to be high compared to

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fish and shell fishes. The biomass production

using optimized YEPD medium the crude protein
content was 37%. It shows as the best result for
SCP production using cheaper sources like fruit
wastes.
5. Conclusion
The production of single cell protein from
Yarrowia lipolytica gives more protein content
(42%) using fruit waste as cheaper source for its
cultivation. The results of chemical analysis of the
dry yeast cell show that the yeast protein was high
nutritional value. It has been given good result for
single cell protein production. So this biomass is
used for cattle feed and poultry feed.
6. Reference
1) Ahmed, S., Bashir, A., Saleem, M. and Jamil,
A., 2009. Production and purification of
cellulose - degrading enzymes from a
filamentous fungus Trichoderma harzianum.
Pak. J. Bot., 4:1411-1419.
2) Ahmed,S., Riaz, S and Jamil, A., 2009
Molecular cloning of fungal xylanases: an
overview. Appl. Microbiol. Biotechnol., 84:1935.
3) Bruckner, H., 1995. Estimation of Aminoacid.
J. Chrom., 697:295-307.
4) Dubios, M., Gilles, K.A., Hamilton, J.K.,
Rebers, P.A and Smith, F., 1956. Colorimetric
method for determination of sugars and related
substances. Anal.chem., 28:305-356.
5) Lowry, O.H., Farr, N.J., Rosenbrough, A.L.,
Randall .R.J., 1951. Protein measurement with
the folin phenol reagent. J. Biol. Chem., 193:
265-275.
6) Rusendi, D and Sheppard, J.D., 1995.
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Isolation and cloning of crel gene from a
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9) Sadasivam, S and Manickam (Eds) A., 1996.

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