Bioresource Technology 254 (2018) 284–289

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Microbial lipid production by Cryptococcus curvatus from vegetable waste T

hydrolysate
⁎
Sulogna Chatterjeea,b, S. Venkata Mohana,
a
Bioengineering and Environmental Sciences Lab, EEFF Department, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India
b
Academy of Scientific and Innovative Research (ACSIR), CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad 500 007, India

A R T I C L E I N F O A B S T R A C T

Keywords: This study primarily evaluated the effect of pre-treatment on release of reducing sugars (RS) from vegetable
Pre-treatment waste (VW). Different acids and alkalis viz., H2SO4, HCl, HNO3, H3PO4, NaOH and KOH were evaluated at varied
Reducing sugars concentration (0.5, 1.0, 1.5 and 2.0%) for pretreatment. The highest RS yield of 472.36 ± 1.89 g/l and
Oleaginous yeasts 439.13 ± 1.04 g/l was obtained with 1.5% H2SO4 and 2% HCl respectively. Secondly, pre-treated vegetable
Biodiesel
waste hydrolysates (PT-VWH) were evaluated for yeast fermentation using Cryptococcus curvatus MTCC 2698 for
FAME
lipid production. Maximum biomass (9.46 ± 0.1 g/l and 8.12 ± 0.1 g/l) and lipid (28.3 ± 0.5% and
26 ± 0.5%) was obtained with 1.5% H2SO4 PT-VWH and 2% HCl PT-VWH respectively. The FAME profiling
revealed the predominance of palmitic, stearic, oleic and linoleic acid. The presence of these fatty acids in
majority has beneficial effect on the biodiesel quality.

1. Introduction
It has been anticipated that fossil fuel reserves will be exhausted by
next 40–50 years due to rapid increase in the consumption (CPCB,
2013). More importantly, the usage of fossil fuels contributes to the
emissions of greenhouse gases and global warming that causes climate
change, rise in sea level, loss of biodiversity and urban pollution
(Faloye et al., 2014; CPCB, 2013). The global trend of energy con-
sumption from non-renewable fossil fuels and the environmental con-
cern are prompting research towards an alternative and more sustain-
able source of energy (Faloye et al., 2014). Biodiesel, produced through
transesterification of fatty acids (lipids) from biological sources offers
one of the attractive substitutes to fossil-derived fuels. Chemically,
biodiesel is a mixture of methyl esters with long-chain fatty acids.
Conventionally, biodiesel is produced from a variety of feedstock, in-
cluding vegetable oils, waste cooking oils and animal fat. But the lim-
ited supply of feedstock and cost restrains the expansion of biodiesel
industry. Oleaginous microorganisms belonging to the genera of mi-
croalgae, yeast, fungi and bacteria are reported to accumulate lipids
from 20% to 70% of dry biomass under specific cultivation conditions
(Ratledge, 2002). Among all other oleaginous microorganisms, yeast
are of special interest as it has advantages over others like short life
cycle, less labour intensive, lower cultivation area and unlike photo-
trophic algae heterotrophic process has benefits of high cell density
without light limitation (Chi et al., 2011). Some oleaginous yeast
strains, like Cryptococcus sp., Lipomyces sp., Rhodosporidium sp. and
Rhodotorula sp. can accumulate intracellular lipids up to 60% of their
dry cell weight (Ratledge, 2002). However, they represent a minor
proportion of the total yeast population and only 5% of yeasts have
been reported to be able to accumulate more than 25% lipids (Ageitos
et al., 2011).
Utilisation of organic waste resources as feed-stock for biodiesel
production is of much interest at present. Thus a variety of inexpensive
carbon sources viz., grape must (Buzzini and Martini, 2000), molasses
(Alvarez et al., 1992), radish brine (Malisorn and Suntornsuk, 2008),
hydrolyzates of agricultural residue (Yu et al., 2011) and sweet sor-
ghum bagasse (Liang et al., 2012) have been reported as feedstock for
cultivation of oleaginous yeast. Also, vegetable waste, fruit waste
(Razaghi et al., 2016) and organic fractions of MSW (Ghanavati et al.,
2015) were also reported as a potential feedstock for yeast cultivation.
Urban India generates 62 million tons of MSW per annum and has
approximate 40–60% biodegradable portion which is mainly composed
of food and kitchen waste, green waste (vegetables, flowers, leaves,
fruits) and paper (Planning Commission Report, 2014; CPCB, 2013).
Estimated production of fruits and vegetables in India is 150 million
tons, out of which a significant fraction (approx. 50 million tons) is
wasted per annum which contributes towards the generation of great
amount of pollution (Sridevi and Ramanujam, 2012).
The use of renewable raw materials, for the production of biobased
product provides sustainable options that could alleviate economic,

⁎
Corresponding author.
E-mail address: svmohan@iict.res.in (S.V. Mohan).

https://doi.org/10.1016/j.biortech.2018.01.079
Received 15 November 2017; Received in revised form 9 January 2018; Accepted 15 January 2018
Available online 31 January 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
S. Chatterjee, S.V. Mohan
ecological, and societal problems worldwide and also encourage waste
valorisation in a closed-loop system (Venkata Mohan et al., 2016a,b).
Vegetable waste (VW) are mainly composed of carbohydrate polymers
(starch, cellulose and hemicellulose), proteins and fats are present re-
latively in low concentrations. The majority of the carbohydrates can be
broken down into simple sugars through chemical hydrolysis (sac-
charification), and then converted to lipids by fermentation. Pre-treat-
ment (PT) of VW alters the physio-chemical and biological character-
istics resulting in release of simple sugars and other essential nutrients
for supporting the growth of microorganism. Sugars like glucose,
fructose, galactose and ribose are mostly mined during the hydrolytic
PT of the VW (Dahiya et al., 2018). Thus, the method of PT is to be
selected to achieve the following viz., acceleration of sugar yields,
minimization of the sugar degraded compounds formation or losses,
elimination of inhibitory by-products formation and finally economic
viability of the process. There is a need to evaluate the comprehensive
PT method to extract the maximum reducing sugars (RS) from VW. PT
can be either done with physical, chemical, physio-chemical, biological
and enzymatic methods or combination of them. Among the various PT
strategies dilute acid and alkaline PT has received numerous research
interests.
There are very few studies reported the utilisation of VW for yeast
based biodiesel production. This study aims to evaluate the effect of
various PT strategies on release of RS from VW and further valorising it
towards lipid production by oleaginous yeast. Six different acids and
alkalis were evaluated for hydrolysis of VW. All the acids and alkalis
were studied at different concentration ranged from 0.5% to 2%.
Further, the pre-treated vegetable waste hydrolysate (PT-VWH) were
used for cultivating oleaginous yeast Cryptococcus curvatus MTCC 2698
for lipid production. The results are compared and discussed on the
basis of RS yield from VW and biolipid accumulation by C. curvatus.
2. Material and methods
2.1. Feed stock-composite vegetable waste (VW)
The composite VW of approximately 2.6 kg was collected from our
institute canteen which caters 500–700 people per day. Collected VW
was mainly composed of left over potatoes (~35%), carrot (~18%),
cucumber (~13%), tomato (~12%), brinjal (~7%), lady finger (~6%),
cabbage leafs (~5%) and bottle gourd peels (~4%). The collected VW
was grinded using electrical blender and filtered through stainless steel
sieve to remove coarse materials and the resulted slurry was stored in
an air tight container at 4 °C. The VW was analyzed for pH (APHA,
1998) and reducing sugars (RS) (Miller, 1959). pH, RS and moisture
content of the resulted VW slurry was found to be 7.56, 48.67 ± 0.5 g/
l and 65% respectively.
2.1.1. Hydrolysis
1 l of slurry (VW) was taken and mixed with water in 1:2 ratio.
Then, 100 ml of slurry was transferred into 250 ml Erlenmeyer flasks
and varied concentration of acids (H2SO4, HCl, HNO3, H3PO4) and al-
kali (NaOH and KOH) [0.5, 1, 1.5, 2% (v/v)] was added. The contents
were mixed and autoclaved at 120 °C at 15 psi for 15 min and further
cooled to room temperature. The hydrolysate was filtered to remove the
residue and the liquid fraction was analyzed for content of RS by DNSA
method (Miller, 1959). Glucose was used as standards (0.2–1 mg/ml).
2.2. Yeast
Cryptococcus curvatus strain MTCC 2698 was obtained from
Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh,
India. The yeast culture was maintained on yeast extract peptone dex-
trose (YPD) medium (20 g/l glucose, 20 g/l peptone, 10 g/l yeast ex-
tract) at 25 °C, 180 rpm and pH 6. Media was sterilized at 121 °C for
15 min before use. Regular sub-culturing was carried out in 1–2 weeks.
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Bioresource Technology 254 (2018) 284–289
24–48 h grown cultures were used for the study.
2.3. Lipid production
For lipid production 100 ml of 1.5% H2SO4 and 2% HCl pre-treated
vegetable waste hydrolysate (PT-VWH) was taken as carbon source in
250 ml flasks. The hydrolysate was neutralized using sodium hydroxide
(NaOH). The medium for lipid production was also supplemented with
0.25% of yeast extract and 0.25% of peptone as a nitrogen source and
0.15% of MgSO4·7H2O considering the work previously done by Chang
et al. (2015). The media was sterilized at 121 °C for 15 min and allowed
to cool to room temperature. Further the autoclaved PT-VWH was in-
oculated with 10% of pre-culture of yeast and incubated on a rotary
shaker (180 rpm; 28 °C). Before fermentation, the pH value of PT-VWH
was adjusted to 6.5. pH, volatile fatty acids (VFA), RS, biomass and
lipid were analyzed regularly. All the experiments were performed in
triplicate parallels.
2.4. Biomass growth and kinetics
Yeast dry biomass was analyzed gravimetrically. 10 ml of culture
broth was taken in pre-weighed vial and centrifuged at 8000 rpm for
5 min. Supernatant was collected for further analysis and the dry cell
weight was determined by subtracting the initial weight after drying
the samples at 60 °C overnight. Volumetric biomass productivity
(PBiomass) was calculated (Eq. (1)), where, X1 and X2 were the dry
biomass weight (g/l) on day T1 (start point of cultivation) and T2
(endpoint of cultivation).
PBiomass (g/l. d) = (X2−X1)/(T2−T1) (1)
Growth rate of Cryptococcus curvatus was estimated using (Eq. (2)),
where, μ represents specific growth rate, Ny and Nx are biomass con-
centration at Tx (initial) and Ty (end) days respectively and (ln) is
natural log.
μ = ln (Ny/Nx)/(Ty−Tx) (2)
2.5. Lipid extraction
To determine total fatty acid, lipids were extracted from cells by
modified Bligh and Dyer method using chloroform and methanol (2:1)
as solvents. Entire biomass was separated at the end of the experiment
by centrifuging the culture broth (8000 rpm; 10 min). Supernatant was
discarded and pellet was dried at 60 °C for 24 h to obtain a dry biomass.
Prior to extraction of lipid yeast biomass was grounded into fine
powder using mortar and pestle. 100 mg of dried biomass were sus-
pended in chloroform/methanol (2:1 v/v) followed by cell disruption
using probe sonicator for 2 min at 40 kHz (Labtech) and centrifugation
(8000 rpm, 10 min). The resulting solvent-lipid layer was transferred to
pre-weighed glass vial and the solvent was evaporated. Total lipids
were quantified by gravimetry. Lipid content (%) and lipid productivity
(mg/l.d) were calculated using Eqs. (3) and (4).
Lipid content (%) = (Weight of lipid/Weight of sample) × 100 (3)
Lipid productivity(mg/l. d) = Biomass productivity × Lipid content
(4)
2.6. Fatty acid methyl esters (FAME)
The transesterification process was performed by refluxing lipids in
the presence of H2SO4 as acid catalyst. 100 mg dried biomass was
subjected to methanol-sulfuric acid (2%) mixture and refluxing was
performed at 65–70 °C for 4 h in a glass vial and later washed in a retort
funnel with 25 ml distilled water to maintain the neutral pH. After
washing ethyl acetate was added to the reaction mixture and the Fatty
S. Chatterjee, S.V. Mohan Bioresource Technology 254 (2018) 284–289

Acid Methyl Esters (FAME) were collected from the organic phase 525 H2SO4 HCl H3PO4 HNO3 NaOH KOH

which was separated from aqueous phase using separating funnel.

Pinch of anhydrous sodium sulphate was added to the organic layer to
450
remove traces of water and finally the organic phase was subjected to
evaporation using rotavapour (G3 Heidolph) for solvent recovery

Reducing sugar (g/l)

375
leaving FAME content in the tube. Finally 1 ml of chloroform was added
to dissolve FAME content which was further analyzed for FAME com-
position by Gas chromatography, GC with FID (Agilent-7988) through 300
capillary column [Valcobond (VB) 30 mm] using nitrogen as carrier gas
(1 ml/min). The temperature of the oven was initially maintained at 225
140 °C (for 5 min), later increased to 240 °C at a ramp of 4 °C/min for
10 min. The injector and detector temperatures were maintained at 150
280 °C and 300 °C respectively with a split ratio of 1:10. FAME com-
position was compared with the standard FAME mix (C8-C22; LB66766,
75
SUPELCO).

0
3. Result and discussion 0.5 1.0 1.5 2.0
Concentration (%)
3.1. Reducing sugar yields from VW
Fig. 1. Reducing sugar concentration after acid and alkaline pre-treatment of municipal
vegetable waste.
Six combinations of acid and alkalis (H2SO4, HCl, HNO3, H3PO4,
NaOH and KOH) at varied concentration (0.5%, 1%, 1.5% and 2% v/v)
were evaluated for pretreatment of VW (Table 1). The effectiveness of 2016; Tasić et al., 2009; Del Campo et al., 2006). Alkaline PT yielded
the hydrolysis approach was determined indirectly based on release of relatively less RS in comparision to acid PT. Concentration above 1%
RS. All the acids yielded significantly good amount of RS in comparison showed inhibitory effect on the yield. Concentration of 0.5% NaOH and
to alkali. Control samples (untreated VW) without any acid or alkali KOH yielded 129.81 ± 0.22 g/l and 162.96 ± 0.39 g/l of RS respec-
showed RS yield of 210.8 ± 0.5 g/l. The results of RS yield with dif- tively. Alkaline PT disrupts the lignin structure and breaks the linkage
ferent acid and alkali are depicted in Fig. 1. Optimised concentration of between lignin and the other carbohydrate fractions in lignocellulosic
acid used for pre-treatment of VW was observed to be in the range of biomass, thus making the carbohydrates in the hetero-matrix more
1.5–2%. Acid hydrolysis with 1.5% H2SO4 yielded highest amount of RS accessible. In alkaline PT a significant amount of hemicellulose is hy-
(472.36 ± 1.89 g/l) followed by 2% HCl (439.1 ± 1.0 g/l). PT with drolysed into monomer (Zheng et al., 2009).
1.5% H3PO4 yielded comparatively less RS (296.9 ± 0.8 g/l) and 1.5% Results suggested that acid hydrolysis catalyzes the breakdown of
HNO3 treated VW yielded 409.0 ± 0.9 g/l which is comparable with complex sugars into monosaccharides more efficiently in comparison to
H2SO4 and HCl. Presence of high amount of potato waste in the initial alkali. PT with dilute sulphuric acid (H2SO4) and hydrochloric acid
VW could be the probable reason behind the significant amount of RS. (HCl) is reported in several studies for the hydrolysis of biomass (Yu
Similar results were observed from starch based VW (Kumar et al., et al., 2011; Nguyen et al., 2000). Zheng et al. (2009) reported PT of
biomass using dilute acids viz., dilute sulphuric acid (H2SO4), dilute
Table 1 nitric acid (HNO3), dilute hydrochloric acid (HCl), dilute phosphoric
Concentration of RS in pre-treated vegetable waste hydrolysates. acid (H3PO4), and peracetic acid (C2H4O3) where the mode of action of
dilute acid was reported to solubilize hemicelluloses. Several studies
Acid/Alkali Concentration (%) Temperature (°C) Time Reducing Sugars
(min) (g/l) reported that dilute acid PT has been applied to a wide range of feed-
stocks, including softwood, hardwood, herbaceous crops, agricultural
Control No Acid/Alkali 120 15 210.78 ± 0.5 residues, wastepaper, and municipal solid waste (Gosavi et al., 2017;
H2SO4 0.5 120 15 398.78 ± 0.47 Zheng et al., 2009). PT using hydrochloric acid (HCl) and sulphuric acid
1 409.18 ± 0.93 (H2SO4) at low concentrations and high temperature are commonly
1.5 472.36 ± 1.89 applied to avoid the formation of sugar degraded compounds (fur-
2 452.16 ± 1.83
furals/hydroxyl methyl furfurals) (Dahiya et al., 2018; Zheng et al.,
HCl 0.5 120 15 262.7 ± 2.4 2009). Acid PT requires the use of an alkali to neutralize the hydro-
1 393.7 ± 1
lysate prior to fermentation.
1.5 410.9 ± 0.4
2 439.1 ± 1.0

H3PO4 0.5 120 15 283.0 ± 0.9 3.2. Growth of Cryptococcus curvatus

1 287.2 ± 0.9
1.5 296.9 ± 0.8
2 285.9 ± 0.6 Result suggests that 1.5% H2SO4 and 2% HCl yielded highest RS.
Therefore, 1.5% H2SO4 PT-VWH and 2% HCl PT-VWH was considered
HNO3 0.5 120 15 316.2 ± 0.1
1 377.5 ± 0.6 for growing Cryptococcus curvatus. The batch cultivation of Cryptococcus
1.5 409.0 ± 0.9 curvatus resulted in 9.46 ± 0.1 g/l and 8.12 ± 0.1 g/l of final cell
2 403.7 ± 0.6 mass with 1.5% H2SO4 PT-VWH and 2% HCl PT-VWH and specific
NaOH 0.5 120 15 129.8 ± 0.2 growth rate (µ) of 0.07 h−1 and 0.068 h−1 respectively. These values
1 41.2 ± 0.1 are in accordance with reported data (Chi et al., 2011; Chang et al.,
1.5 35.1 ± 0.1 2015). Chang et al. (2015) reported the maximum yeast cell biomass in
2 33.3 ± 1.0
the range of 13.1–11.1 g/l and specific growth rate (µ) of 2.06 d−1 and
KOH 0.5 120 15 162.9 ± 0.4 1.74 d−1 respectively with 40 g/l and 60 g/l of glucose which is similar
1 66.9 ± 0.3
to the result obtained in this study.
1.5 41.4 ± 0.6
2 37.3 ± 0.1 Initially biomass productivity for 1.5% H2SO4 PT-VWH and 2% HCl
PT-VWH was 1.82 g/l.d and 1.58 g/l.d at 24 h respectively. At 48 h

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S. Chatterjee, S.V. Mohan Bioresource Technology 254 (2018) 284–289

35 500
10 a 1.5% H2SO4 PT-VWH 2% HCl PT-VWH
30
400

Reducing sugars (g/l)

8

Lipid content (%)

25
Biomass (g/l)

6
300
20
4
15
200
2
1.5% H2 SO4 PT-VWH 10

0
2% HCl PT-VWH 100
0 2 4 6 8 10 5
Time (Days)
0 0
10 8 6 4 2 0
2.0 b 1.5% H2SO4 PT-VWH
2% HCl PT-VWH
Time (Days)
Growth (OD at 595 nm)

Fig. 3. Reducing sugars removal and lipid production during Cryptococcus curvatus cul-
1.5 tivation.

1.0 further biomass production. The reduction in the biomass productivity

reflects the onset of lipid accumulation as nitrogen source gets ex-
hausted and the available carbon will be channelled towards synthesis
0.5 of intracellular lipids. The biomass yield and growth curve of
Cryptococcus strains on PT-VWH at different operational time was de-
picted in Fig. 2a & b which showed good growth. Growth was initiated
0.0 in PT-VWH with an initial pH of 6.5. With due course of time the pH
decreased pH 6.5 to pH 3.1 with 1.5% H2SO4 and pH 6.53 to pH 3.07
0 10 20 30 40 50 60 70 80 with 2% HCl PT-VWH. VFA production was also analyzed at every 24th
h of the operation. It was observed that pH decreased with the incre-
Time (Hours)
ment in VFA values in the culture media. At the end of the experiment
7.0 4000 total VFA was observed to be 3865.23 ± 0.5 mg/l and
c 1.5% H2SO4 PT-VWH 2% HCl PT-VWH
3688.19 ± 0.1 mg/l with 1.5% H2SO4 and 2% HCl PT-VWH. The
6.5 3800 change in pH and VFA accumulation in the course of fermentation is
6.0
represented in Fig. 2c.
3600
Total VFA (mg/l)

5.5
3400 3.3. Lipid synthesis
pH

5.0
4.5
4.0
3.5
3.0
0 2 4 6
Time (Days)
Fig. 2. (a) Biomass production by Cryptococcus curvatus using acid treated vegetable
waste (1.5% H2SO4 and 2% HCl PT-VWH). (b) Growth curve of Cryptococcus curvatus
grown on 1.5% H2SO4 and 2% HCl PT-VWH. (c) Effect of fermentation on pH and total
VFA accumulation.
minor decrement in the biomass productivity was observed i.e., 1.26 g/
l.d and 1.17 g/l.d for 1.5% H2SO4 PT-VWH and 2% HCl PT-VWH re-
spectively. Maximum biomass productivity was observed in between 24
and 96 h for both the PT-VWH but highest yield of 2.19 g/l.d was ob-
tained at 72 h with 2% HCl PT-VWH. Biomass production started
ceasing after 96 h in both the acidic PT-VWH residues. Biomass pro-
ductivity was comparatively less with 1.5% H2SO4 PT-VWH (0.5 g/l.d)
and 2% HCl PT-VWH (0.43 g/l.d) at 120 h. Since RS was still available
at this point it can be assumed that nitrogen was the limiting factor for
3200 Lipid accumulation by C. curvatus showed considerable amount of
total cellular lipid storage with both PT-VWH (1.5% H2SO4 and 2% HCl
3000 PT-VWH). Lipid accumulation and sugar consumption were depicted in
Fig. 3. Lipid production reached the maximum of 28.3 ± 0.5% with
2800 1.5% H2SO4 PT-VWH and 26 ± 0.5% with 2% HCl PT-VWH. After
192 h there is stable production of lipid observed with 1.5% H2SO4 PT-
2600 VWH, whereas with 2% HCl PT-VWH stability was observed after
8 10 216 h. This variation in the pattern of lipid production is due to the
varying concentration of RS present in the acid pre-treated hydrolysate.
Chang et al. (2015) reported that batch cultures of Cryptococcus sp.
SM5S05 grown in the corncob hydrolysate medium along with 60 g/l
glucose resulted in considerable high lipid content of 60.2%. Therefore,
carbon concentration has significant role in oil accumulation by olea-
ginous microorganisms. Some strains of Cryptococcus are already re-
cognized as good producers of lipids, C. curvatus has been widely stu-
died in fermentations using different substrates, such as hydrolysates of
sorghum bagasse and wheat straw, and raw glycerol (Thiru et al., 2011,
Yu et al., 2011, Liang et al., 2012). C. albidus was also evaluated for
lipid accumulation using VFA as carbon source (Fei et al., 2011).
Regarding lipid production it needs to be considered that not all
extracted lipids are necessarily derived from the process of lipid
synthesis. Since lipids are also found in cell membrane, an increasing
lipid yield can be to some extend associated with the rise of biomass
production (Mast et al., 2014). The initial increase in lipid production is

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S. Chatterjee, S.V. Mohan Bioresource Technology 254 (2018) 284–289

100 3.4. FAME composition

Others
Linolenic Acid
90 The fatty acid composition of lipid produced by C. curvatus was
Linoleic Acid
determined by gas chromatography (GC) after transesterification. The
FAME composition (%)

80
70
60
50
40
30
20
10
0 (15.3%, 15.2), C18:1 (45%, 47.7%), C18:2 (7.3%, 6.42%) produced by
1.5% H2SO4 2% HCl
PT-VWH
Fig. 4. FAME composition of lipids from Cryptococcus curvatus.
associated with onset of biomass production. The second phase of lipid
production starts after the exhaustion of nitrogen from the cultivation
medium. This is the characteristic for lipid synthesis, where in nitrogen
limitation cell growth ceases and the available carbon is trafficked to-
wards formation and accumulation of storage lipids mainly in the form
of triacylglycerides (TAGs) (Mast et al., 2014). The lipid biosynthesis
starts when acetyl-CoA is transported from the mitochondria to the
cytosol executed by the ATP citrate lyase. Acetyl- CoA carboxylase
(ACC) then catalyzes the first step towards lipid biosynthesis, con-
verting cytosolic acetyl-CoA into malonyl-CoA, which is the primary
precursor for fatty acid elongation (Dahiya et al., 2018; Venkata Mohan
et al., 2016a,b; Tai and Stephanopoulos, 2013). Malonyl-CoA is then
converted to acyl-CoA chains that are transported to the endoplasmic
reticulum (ER) or lipid body membranes for the final assembly of
triacylglycerol (TAG) (Tai and Stephanopoulos, 2013). 1.5% H2SO4 PT-
VWH showed increment in the lipid production in 96 h from 14.5% to
20.1% and with 2% HCl PT-VWH the increment has been observed in
120 h from 13.3% to 18.54%. The sudden increment in the lipid pro-
duction is due to the exhaustion of nitrogen source in the cultivation
media which leads to the channeling of available carbon into storage
lipids. Nitrogen limitation also activates diacylglycerol acyl transferase,
which converts acyl-CoA to TAG (Tai and Stephanopoulos, 2013). At
the end of the experiment lipid productivity was observed to be
28.3 ± 0.5% and 26 ± 0.5% with 1.5% H2SO4 PT-VWH and 2% HCl
PT-VWH respectively.
Oleic Acid
Stearic Acid GC analysis revealed that the cellular composition of fatty acids were
Palmitic Acid predominantly palmitic acid (C16:0), stearic (C18:0), oleic (C18:1), li-
noleic (C18:2) and linolenic acids (C18:3) (Fig. 4). C. curvatus grown on
1.5% H2SO4 PT-VWH showed 28.3 ± 0.5% of lipid content and lipid
profiling depicted the majority of C16:0 (26.5%), C18:0 (9.15%), C18:1
(53.21%), C18:2 (6.5%) and C18:3 (1.7%). While C. curvatus grown on
2% HCl PT-VWH showed 26 ± 0.5% and lipid profiling showed C16:0
(25.29%), C18:0 (10.65%), C18:1 (57.67%), C18:2 (5.29%) and C18:3
(1.4%). Trace amount of other fatty acid produced were undecanoic
acid, lauric acid, myristic acid, cis-10-Pentadecanoic acid, palmitoleic
acid, Heptadecanoic and cis-10-Heptadecenoic. Yu et al. (2011) re-
ported similar fatty acid profiling of lipid C16:0 (27%, 25%), C18:0
C. curvatus grown on detoxified and non-detoxified wheat straw hy-
drolysate. C. aerius UIMC65 grown on non-detoxified pre-hydrolysate of
MSW, detoxified pre-hydrolysate of MSW and hydrolysate of MSW also
showed similar FAME composition (Ghanavati et al., 2015). FAME
composition was C16:0 (1.33–3.14%, 1.09–3.10% and 1.24–2.77%),
C18:0 (11.26–15.57%, 10.11–14.66% and 7.17–10.89%), C18:1
(55–60%, 57–63.1% and 59–65.1%) and C18:2 (1.11–3.6%,
2.63–4.45% and 5.57–8.5%) (Ghanavati et al., 2015). Similar fatty acid
profiles of lipids synthesized by Cryptococcus sp. were also reported by
(Chang et al., 2015; Castanha et al., 2014). Predominance of palmitic
(C16:0) and oleic (C18:1) acid is characteristic feature for C. curvatus
(Meng et al., 2009). Presence of C16:0, C18:0, C18:1, C18:2 and C18:3
are most suitable for biodiesel production (Meng et al., 2009).
Lipid produced by C. curvatus in this study showed majority of C:16
and C:18 fatty acids which accounts for the vast majority with over
70–80% of total fatty acids. The high share of oleic acid has beneficial
effects on biodiesel fuel quality (Sitepu et al., 2013). The presence of
linoleic acid (C18:2) represents the fact that linoleic acid is found in the
lipids of cell membranes and indicates the presence of biomass asso-
ciated lipids (Schneider et al., 2013). Fatty acids obtained from the
yeast showed the presence of both saturated fatty acids (SFA) and un-
saturated fatty acids (USFA) which is having good applicability towards
biobased products (Table 2). The fatty acids produced have economic
viability and numerous industrial applications viz., biofuel, food and
beverage manufacturing, healthcare, medicines, detergents and cos-
metics. C16:0, C18:0 and C18:1 which have fuel properties found to be
in noticeable fraction. Fatty acids which has pharmaceutical applica-
tions are Undecanoic acids (C11:0), Palmitoleic acid (C16:1), Hepta-
decanoic acid (C17:0). Cis-10-pentadecanoic acid (C15:1) and Linolenic
acid (C18:3) which are polyunsaturated fatty acid used to treat variety
of conditions viz., arthritis, skin problems, high blood pressure,

Table 2
Properties of Fatty acid derived from Yeast cultivated on PT-VWH.

Fatty acids Fatty acid No. Fatty acid composition (%) Properties

1.5% H2SO4 PT-VWH 2% HCl PT-VWH

Undecanoic acid C11:0 ∼1 ∼1 An antifungal agent, to treat ringworm and athlete’s foot
Lauric acid C12:0 1.5 2.3 Soaps and cosmetic manufacturing
Myristic acid C14:0 2.2 1.3 Flavouring agents, soap and cosmetics manufacturing
cis-10-Pentadecanoic acid C15:1 ∼1 ∼1 Manufacturing of esters for artificial fruit flavours and perfumes, antitumor activity
Palmitic acid C16:0 26.5 25.3 Biofuel, soap and cosmetic manufacturing
Palmitoleic acid C16:1 ∼1 ∼1 Anti-thrombotic effects
Heptadecanoic acid C17:0 ∼1 ∼1 Possess antioxidant activity and anti-allergic
cis-10-Heptadecenoic acid C17:1 ∼1 ∼1 Food and Beverage and Enzyme Inhibitors
Stearic acid C18:0 9.2 10.6 Biofuel, Manufacturing of soap, cosmetics and Lubricating agents
Oleic acid C18:1 53.2 57.7 Biofuel, emulsifying agent, pharmaceutical uses
Linoleic acid C18:2 6.5 5.3 Beauty products, antioxidants, Manufacturing of oil paints
Linolenic acid C18:3 1.7 1.4 Manufacturing of soap, emulsifiers and inflammatory agents

288
S. Chatterjee, S.V. Mohan
inflammation and tumours (Hemalatha and Venkata Mohan, 2016).
These fatty acids has wide industrial application viz., flavouring agents
in food and beverage, emulsifiers, skin care products, treatment of
cardiovascular diseases and also used as an anti-inflammatory agent
(Hemalatha and Venkata Mohan, 2016). In the present study the mi-
crobial oil showed higher fractions of oleic acid, stearic acid and pal-
mitic acid which has biofuel characteristics and can be transesterified
into biodiesel. Hence, yeast cell factory could be used efficiently as a
promising alternative for the production of cheap microbial oil from
vegetable waste.
4. Conclusions
Results suggested that VW based MSW can function as good feed-
stock after proper pre-treatment for yeast based oil production. The
highest yield of RS (472.36 ± 1.895 g/l and 439.13 ± 1.045 g/l) was
obtained with 1.5% H2SO4 and 2% HCl acid PT. Further Cryptococcus
curvatus grown on 1.5% H2SO4 and 2% HCl PT-VWH showed highest
biomass (9.46 ± 0.1 g/l and 8.12 ± 0.1 g/l) and lipid yield
(28.3 ± 0.5% and 26 ± 0.5%) respectively. The lipid profiling re-
vealed the predominance of palmitic, stearic, oleic and linoleic acids
which has biofuel properties.
Acknowledgements
The authors wish to thank the Director CSIR-IICT for the support
and encouragement in carrying out this work. SC acknowledges
Department of Science and Technology (DST), Government of India for
providing Inspire Fellowship for Doctoral Studies.
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