Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1

Bioresource Technology 197 (2015) 91–98

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Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 Alok Patel, Dev K. Sindhu, Neha Arora, Rajesh P. Singh, Vikas Pruthi, Parul A. Pruthi ⇑ Molecular Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee (IIT R), Roorkee, Uttarakhand 247667, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Non-edible lignocellulosic biomass

(CAE) as TAG feedstock for biodiesel production.
CAE have high total carbohydrate (58.12 g/l) and total reducing sugars (47.25 g/l).
R. kratochvilovae HIMPA1 grown on CAE accumulates 53.18% (w/w) lipid content.
High TAG content visualized as large sized lipid droplets (4.35 ± 0.54 lm).
FAME contain high SFA content C16:0 (43.06%), C18:0 (28.74%) and C18:1 (17.34%).

a r t i c l e

i n f o

Article history: Received 22 June 2015 Received in revised form 5 August 2015 Accepted 7 August 2015 Available online 22 August 2015 Keywords: Biodiesel Lignocellulosic biomass Cassia fistula Oleaginous yeast Rhodosporidium

a b s t r a c t This study explored biodiesel production from a low cost, abundant, non-edible lignocellulosic biomass from aqueous extract of Cassia fistula L. (CAE) fruit pulp. The CAE was utilized as substrate for cultivating novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. This oleaginous yeast accumulates high amount of triacylglycerides as large intracellular lipid droplets (4.35 ± 0.54 lm) using CAE as sole nutritional source. Total lipids (4.86 ± 0.54 g/l) with lipid content of 53.18% (w/w) were produced by R. kratochvilovae HIMPA1 on CAE. The FAME profile obtained revealed palmitic acid (C16:0) 43.06%, stearic acid (C18:0) 28.74%, and oleic acid (C18:1) 17.34% as major fatty acids. High saturated fatty acids content (72.58%) can be blended with high PUFA feedstocks to make it an industrially viable renewable energy product. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Progressive exhaustion of conventional fossil fuels and ever increasing demand of energy attracted the researcher and scientists to explore for alternative renewable sources of energy especially biomass based biofuels (Ioelovich, 2015). They can be ⇑ Corresponding author. Tel.: +91 1332 285530 (office), +91 1332 285110 (resi.), mobile: +91 09760214585; fax: +91 1332 273560. E-mail address: [email protected] (P.A. Pruthi). http://dx.doi.org/10.1016/j.biortech.2015.08.039 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

derived from various biomass feedstocks which are classified according to the source of the raw materials used such as triglyceride-based biomass, starch and sugar-derived biomass, and non-edible lignocellulosic biomass consisting of lignin, hemicellulose and cellulose as main structural units (Huber and Corma, 2007). The triglycerides feedstocks obtained mainly from vegetable oils which are either edible (Soybean, palm oil seeds, rapeseed) or non-edible (Jatropha curcas, Calophyllum inophyllum, Pongamia glabra, Madhuca indica) are usually considered as prominent feedstock for biodiesel production (Sawangkeaw and

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Ngamprasertsith, 2013). Currently, more than 350 oil-bearing crops recognized as prospective sources for biodiesel production (Atabani et al., 2013), but their sustainability due to food crisis worldwide is a major challenge. This led to search for non-edible, cost effective renewable source of energy which can substitute fossil fuel. In this context, oleaginous microorganisms seem to overcome this hurdle as a promising substitute for triglyceride feedstock for biodiesel production (Koutinas et al., 2014). These microbial lipids have an edge over their vegetable oil counterparts in terms higher lipid yield, devoid of any seasonal and climatic changes, less labor intensive, easy to scale up and genetically modify to achieve higher production yields (Meng et al., 2009). Microbial lipids by these oleaginous microorganisms, involving bacteria, algae, fungi, and yeasts can utilize organic carbon to synthesize oils in their intracellular compartment and enhanced productivity than oil producing crops (Liang and Jiang, 2013). Among different microorganisms, oleaginous yeasts of genera Rhodosporidium, Rhodotorula, Yarrowia, Cryptococcus, Candida, Lipomyces and Trichosporon have distinct advantage of high lipid productivity and can be cultivated on a large number of renewable feedstocks, even on low-cost materials, such as nutritional residues from agricultural and industrial wastes (Huang et al., 2009; Ageitos et al., 2011; Freitas et al., 2014; Sitepu et al., 2014; Leiva-Candia et al., 2014). The specific benefit of using oleaginous yeast for the production of TAG feedstock is that they can grow on various kind of simple (glucose, glycerol and lactose) and complex sugars (lignocellulose hydrolysates, polysaccharides, agriculture residues, rice straw hydrolysates, wheat straw hydrolysate, sugarcane molasses) as substrates and convert them into TAG at optimized conditions (Huang et al., 2009; Yu et al., 2011; Leiva-Candia et al., 2014). The conversion of complex hydrolysates into neutral lipids is the primary target for achieving techno economic utility of cheaper sources like agriculture stover, forest residues, energy plants (Yu et al., 2011; Sitepu et al., 2014) for biodiesel production. Pioneering research work in this direction revealed enhanced TAG accumulation by simultaneous utilization of fermentable: glucose (Glu), fructose (Fru), sucrose (Suc), and non-fermentable carbon sources: glycerol (Gly), arabinose (Arb) and xylulose (Xyl) individually as well as on the mixture of both carbon sources together as substrate for cultivation of Rhodosporidium kratochvilovae HIMPA1 (Gene Bank accession No. KF772881) screened from Himalayan permafrost soil (Patel et al., 2015). This yeast accumulate total lipid content of 9.26 g/l in mixed carbon sources as compared to glucose (6.2 g/l). Earlier, investigations on this yeast demonstrated the usage of nontoxic aqueous seed hydrolysate of Cannabis sativa L. (hemp), for accumulation of TAG in the lipid bodies signifying industrial Hemp seeds as the cheapest triglyceride feedstock raw material (Patel et al., 2014). In the present investigation, we have explored Cassia fistula L. fruit pulp as an easily available, unutilized, non-edible lignocellulosic biomass feedstock as a raw material for biodiesel production. The aqueous extract of C. fistula L. fruit pulp (CAE) was used as the lipid production media for the cultivation of R. kratochvilovae HIMPA1 oleagenic yeast cells. These cells accumulate high amounts of neutral lipids (TAG) in their intracellular lipid droplets which were visualized by epi-fluorescence microscopy. The accumulated lipids were harvested and transesterified into FAME as biodiesel which were characterized using TLC, FTIR and GCMS analysis. This dual step production leads to conversion of non-edible lignocellulosic biomass to triacylglycerides feedstock for biodiesel production by oleagenic yeast cells. C. fistula L. is known as Golden Shower worldwide and ‘‘Amaltas” locally for Indian laburnum. It is abundantly distributed especially in Asian countries, South Africa, West Indies and Brazil. It is a popular medium sized deciduous ornamental tree (8–24 m height) which belongs to family-Fabaceae, genus-Cassia, species-Fistula. The fruits are dark brown, pendulous, cylindrical, septate with long

pods (25–50 cm) having 1.5–3.0 cm diameter, possessing 25–100 seeds and almost 400–500 fruiting bodies per tree (Rajagopal et al., 2013). The seeds contain glycerides with major fatty acids (palmitic acids, stearic, oleic and linoleic) along with trace amounts of caprylic and myristic acids (Sayeed et al., 1999). Laburnum fruits pulp are known to be a rich in Fe, Ca, K, Mn, and also contains aspartic acid (15.3%), glutamic acid (13%) and lysine (7.8%) amino acids. However, the major portion of large number of pods remains unutilized and tons of dried material gets wasted every year. This investigation confirms the feasibility for utilization of cheaper, copious, unexploited waste lignocellulosic biomass of C. fistula L. fruit pulp as feedstock for biodiesel production. 2. Methods 2.1. Materials Analytical grade solvents viz., n-hexane, chloroform, and methanol were purchased from Merck Ltd., Mumbai, India. Sugars (glucose, fructose, xylose) and other chemicals (NaCl, KCl, anhydrous sodium sulfate, 14% methanolic BF3, Castor oil) were procured from Hi-Media Laboratories, Mumbai, India. FAME standard (AOCS low erucic rape seed oil O7756-1AMP) for GC– MS analysis and standard for TLC (Triolein) were obtained from Sigma Aldrich (St. Louis, USA). BODIPY 493/505 nm (4,4-difluro-1, 3,5,7-tetramethyl-4-bora-3a, 4a-diaza-s-indacene) was purchased from Invitrogen (Life Technology, USA) for fluorescence staining. 2.2. Yeast strain and growth condition Oleaginous yeast, R. kratochvilovae HIMPA1 isolated from Himalayan permafrost soil (Gene Bank Acc. No. KF772881) was used to investigate the effect of C. fistula L. fruit pulp aqueous extract (CAE) on the growth and lipid accumulation. The yeast was grown on YEPD broth for 48 h on 200 rpm at 30 °C. To obtain the seed culture, cells were harvested as reported previously (Patel et al., 2014). The culture was maintained on slants of Malt Extract Agar at 4 °C. 2.3. Preparation of C. fistula L. fruit pulp aqueous extract for lipid production media Fresh green unripe pods of C. fistula L. were collected from IIT Roorkee (Uttarakhand, India) campus and were used for preparing an aqueous extract of fruit pulp obtained by removing the shell from fresh pods with scalpel. The green pods were harvested before they hardened to dark brown woody biomass. A single green pod contains yellowish fruit pulp along with approximate 100 white young seeds (50 g) was chopped into fine pieces (1–2 mm) and boiled in a Erlenmeyer flask (500 ml) for 30 min with 100 ml of sterile distilled water in the ratio 1:2 w/v. The suspension was then centrifuged at 5000 rpm for 20 min at 4 °C. Supernatant obtained was filtered with a 0.22 mm membrane filter. The filtrate (100%; w/v) was then diluted with distilled water to prepare the different strengths (25, 50 and 75%) of CAE (w/v) was used as lipid producing media for the cultivation of R. kratochvilovae HIMPA1. 2.4. Batch cultivations of R. kratochvilovae HIMPA1 for biomass and lipid production Batch cultivation of R. kratochvilovae HIMPA1 for lipid production was performed in conical flasks (250 ml) containing 25, 50, 75 and 100% CAE (w/v) and inoculated with 2% of seed culture for 144 h at 30 °C with 200 rpm. Glucose synthetic medium

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(GSM) containing 40 g/l glucose with yeast nitrogen base (YNB) was used in control experiment. The cells were harvested for lipid extraction using protocol described earlier (Patel et al., 2014). The cell dry weight (g/l), lipid content (%), and total lipid yield (g/l) of R. kratochvilovae HIMPA1 were calculated. All experiments were done in triplicate. The equation used to calculate percentage lipid content in the oleaginous yeast was:

Y L ¼ W L =DCW

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proportions of 1:5 and heated at 80 °C for 20 min. Extraction of methyl esters were then done by addition of hexane: water (2:1). The samples were then centrifuged (3500g) and supernatant were analyzed by GC–MS (Agilent, Santa Clara, CA, USA) using DB-5MS capillary column (30 m  0.25 mm  0.25 lm). Splitless injection mode was used for injecting the sample (1 lL at 250 °C) using helium (1 mL/min) as a carrier gas as reported earlier (Patel et al., 2015).

where WL = total lipid weight (g/l), DCW = dry cell weight (g/l).

3. Results and discussion

2.5. Fluorescence microscopy analysis for TAG accumulation in R. kratochvilovae HIMPA1

3.1. Biomass and lipid production by R. kratochvilovae HIMPA1 using aqueous extract of C. fistula L. fruit pulp (CAE)

The TAG accumulated in lipid droplets (LDs) of R. kratochvilovae HIMPA1 cells were examined by live fluorescence microscopy as reported previously (Patel et al., 2014). Briefly, the size of cells and intracellular LDs imaging of BODIPY stained cells were analyzed using ImageJ 1.48a software. The length and width of LDs present in approx.100 oleaginous yeast cells that shows spherical geometry were counted to calculate its average size, represented as mean ± standard deviation.

Cost reduction of the raw feedstocks is the major challenge for biodiesel production from microbial lipids. The fermenting media imparts almost 60–70% of the total cost is always being in the focus for researchers (Leung et al., 2010). In this investigation CAE, which is a non-edible lignocellulosic biomass is used as cost effective medium for cultivating the novel oleaginous yeast, R. kratochvilovae HIMPA1 isolated from Himalayan range for biodiesel production. Lignocellulosic biomass (agricultural waste and forest biomass) signifies a renewable and largely untapped source of raw feedstock utilized mainly for thermochemical conversion (pyrolysis, gasification and combustion) into liquid, gas fuels and other energy-related end products (Huber and Corma, 2007). It contains 55–65% carbohydrate and hemicelluloses, cellulose and lignin which have major potential for biological conversion into C6 and C5 sugars widely recognized as a promising feedstock for liquid bio-fuel production. Since the carbohydrates in lignocellulose are usually refractory to hydrolysis, pretreatment are required to make the carbon sources accessible to microorganisms. However, existing thermochemical pretreatments leads to byproducts such as furfural, hydroxylmethyl furfural (HMF), weak acids and phenolic which are inhibitory to growth of microorganisms. Organisms that can utilize both C-5 and C-6 sugars for fermentation and can tolerate these inhibitors are highly desired in order to increase the efficiency of lipid production from lignocellulosic materials. Oleaginous yeast from Rhodosporidium spp. genera do have such potential and have shown utilization of sugars produced from the lignocellulosic hydrolysates as substrate and convert them into triglycerides that accumulates as lipid bodies for further biodiesel production (Zhan et al., 2013; Zhao et al., 2010; Yu et al., 2011). This investigation endorses the feasibility of this novel combined approach. The oleaginous yeast R. kratochvilovae HIMPA1 showed potential for utilizing aqueous boiled extracts of the fruit pulp of C. fistula L. (100% CAE) as direct lipid producing medium for their cultivation. The chemical composition of fruit pulp of C. fistula L. were analyzed for its nutritional value as growth substrate for lipid production medium is listed in (Table 1). It depicts high amount of total carbohydrates (58.12 ± 0.89 g/l) and soluble sugars such as glucose (29.45 ± 0.17 g/l), fructose (17.98 ± 0.39 g/l), xylose (3.87 ± 0.73 g/l) and 329 ± 1.87 mg/l total nitrogen in aqueous extract which is in agreement with the work done by Barthakur et al. (1995). The lignocellulosic content in the dried pod sample showed presence of lignin (26.89 ± 0.78%), holo-cellulose (46.68 ± 0.91%) and a-cellulose (41.26 ± 0.86%) which get diminuted in the boiled and filtered aqueous extract (100% CAE) and further consumed by R. kratochvilovae HIMPA1 for its growth (Table 1). The lignocellulosic biomass contents isolated from the dried pod sample showed the comparison of various compositional changes in freshly prepared boiled extract (100% CAE) from the fruit pulp and extract remained after cultivation of the yeast strains. Both C6 (Glucose and fructose) and C5 sugars were consumed rapidly and simultaneously showed maximum consumption of total carbohydrates (94%), reducing sugars (98%)

2.6. Determination of sugar concentration in aqueous extract of C. fistula L. fruit pulp (CAE) DNS and phenol sulfuric acid methods were used to estimate reducing sugars and total carbohydrate content present in CAE (Dubois et al., 1956) used for lipid production medium for growing oleaginous yeast. The amount of sugar consumption (%) were determined as C = St0  St/St0  100, where C is the amount of sugar consumption, St0 is the amount of initial sugar (g/l) added and St is the residual sugar. 2.7. TLC and FT-IR of extracted lipid from R. kratochvilovae HIMPA1 Analysis of TAG in the extracted total lipid was carried out by TLC using a solvent system hexane: diethyl ether: acetic acid in the ratio 85:15:1, v/v/v with triolein as standard as reported earlier (Patel et al., 2014). For qualitative analysis of SEs and TAGs, plates were dipped into methanolic MnCl2 solution (0.63 g MnCl24H2O, 60 ml water, 60 ml methanol and 4 ml concentrated sulfuric acid), dried and heated at 120 °C for 15 min (Fei et al., 2009). The TAG spots were visualized on TLC plate by charring it at 135 °C after spraying it with sulfuric acid 50% (w/w). Conversion of TAG into transesterified product (FAMEs) was detected by double development method of TLC. Briefly, the plate was firstly developed to 3.5 cm from the origin using solvent system chloroform/metha nol/water/acetic acid (65:35:4:1, v/v/v/v), and after air dried, redeveloped to 8 cm from the origin with hexane/tert-butyl methyl ether/acetic acid (90:10:0.5, v/v/v) as described by (Ichihara and Fukubayashi, 1996). FAME of castor oil was used as standard. Lipids separated were visualized by spraying 50% (w/w) sulfuric acid and then heating at 135 °C for FAME analysis. FTIR spectrometer (Thermo Nicolet NEXUS, Maryland, USA) was used to analyze the extracted lipid samples. Spectra were collected over the wave number range 400–4000 cm1 and triolein was used as standard. The baseline of the spectra was corrected using the automatic baseline correction algorithm and samples were analyzed in triplicate. 2.8. Transesterification of extracted fatty acid and GC–MS analysis Extracted yeast lipid samples were transesterified by using the modified protocol of Morrison and Smith (1964). Briefly, BF3–methanol reagent was added to the lipid samples in the

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Table 1 The nutrient composition of Cassia fistula L. fruit pulp as in dry sample, aqueous extract (100% CAE) before and after consumption by R. kratochvilovae HIMPA1. Chemical composition

Dry green pod sample of Cassia fistula L. fruit pulp

Boiled 100% CAE (g/l)

100% CAE after consumption (g/l)

Lignin Holo-cellulose a-Cellulose Hemicellulose Total reducing sugars Total carbohydrate Glucose Fructose Xylose Total nitrogen Total Kjeldahl nitrogen Total phosphorus Ortho-phosphorous Nitrate–nitrogen (NO3–N) Ammonia–nitrogen (NH3–N)

26.89 ± 0.78% 46.68 ± 0.91% 41.26 ± 0.86% 5.42 ± 0.65% – – – – – – – – – – –

2.89 ± 0.23 g/l 7.23 ± 0.34 g/l 4.67 ± 0.43 g/l 0.125 ± 0.06 g/l 47.25 ± 0.34 g/l 58.12 ± 0.89 g/l 29.45 ± 0.17 g/l 17.98 ± 0.39 g/l 3.87 ± 0.73 g/l 329 ± 1.87 mg/l 325 ± 1.6 mg/l 7.8 ± 0.43 mg/l 5.3 ± 0.23 mg/l 4.6 ± 0.41 mg/l 196 ± 0.98 mg/l

0.11 ± 0.09 g/l 0.61 ± 0.10 g/l 0.046 ± 0.003 g/l 0.002 ± 0.001 g/l 1.23 ± 0.76 g/l 3.96 ± 0.25 g/l 0.64 ± 0.24 g/l 1.22 ± 0.31 g/l 0.12 ± 0.41 g/l 0.18 ± 0.01 m g/l 25 ± 2.3 mg/l 0.38 ± 1.6 mg/l 0.17 ± 0.09 mg/l 1.2 ± 0.8 mg/l 17 ± 1.2 mg/l

respectively. The data confirms that the cellulosic content was solubilized by mild boiling treatment without inhibiting or delay in growth of the organism and sugars were consumed simultaneously as reported in Fig. 1B. The low nitrogen content obtained through Nitrate Nitrogen estimation (NO3–N, 4 ± 0.41 mg//) and Ammonia–Nitrogen estimation (NH3–N, 196 ± 0.98 mg/l) may be conducive of natural Nitrogen stress condition which may cause enhancement in lipid accumulation. Although, the earlier reports suggests that the seeds C. fistula L are rich in protein and fatty acid content (Sayeed et al., 1999). Comparative study of dry cell weight (g/l), total lipid yield (g/l), and lipid contents (%) of other newly found oleaginous yeasts growing on various low cost lignocellulosic materials are listed in Table 2. The data showed that when CAE was used as a sole carbon source, it yielded higher lipid content (53.18%) as compared to other low cost substrate reported recently such as: carob pulp syrup (17.27%), waste sweet potato vine hydrolysate (35.6%), wheat straw hydrolysate (33.5%), corn fiber hydrolysate (45.7%), industrial fats (44%), monosodium glutamate waste water (20%), as reported earlier (Xue et al., 2008, 2010; Yu et al., 2011; Economou et al., 2010; Xing et al., 2012; Huang et al., 2013; Zhan et al., 2013; Freitas et al., 2014). The cell dry weight (g/l), total lipid yield (g/l) and lipid content (%) observed during the batch cultivation of the oleaginous yeast R. kratochvilovae HIMPA1 on 100% CAE and glucose synthetic medium (GSM) are depicted in Fig. 1A. The maximum cell dry weight obtained was 8.9 ± 0.49 g/l when the yeast was grown on 100% CAE. The total lipid yield, when R. kratochvilovae HIMPA1 utilized 100% CAE as carbon source was recorded to be 4.75 g/. The maximum cell dry weight (g/l), total lipid yield (g/l), and lipid content (%) of R. kratochvilovae HIMPA1 grown on GSM were 6.78 g/l, 3.12 g/l, and 46.02% respectively after 144 h (Fig. 1A). The composition of prepared 100% CAE was determined by DNS and PSA methods and it showed presence high sugar content as it has high amount of soluble carbohydrates (58.12 ± 0.89 g/l) and total reducing sugars (47.25 ± 0.34 g/l) which is utilized by the oleaginous yeast for TAG accumulation. The sugar consumption data showed that total carbohydrates (80.67%) and reducing sugars (70.83%) were utilized simultaneously by R. kratochvilovae HIMPA1 and consumed rapidly in the early stationary phase (96 h) of growth. During late stationary phase (144 h), the consumption of total carbohydrates and reducing sugars were recorded to be 97.88% and 91.61% respectively (Fig. 1B). The results correlate with previous investigations on R. kratochvilovae HIMPA1 which suggests synergistic utilization of sugars (Patel et al., 2015). The data obtained revealed maximum total lipid yield (4.73 ± 0.34 g/l), lipid content (53.18%) and cell dry weight (8.9 ± 0.12 g/l) by R. kratochvilovae HIMPA1 when grown on 100% CAE for 144 h at 30 °C (Fig. 1B). Similar increase in lipid accumulation was also observed during late log growth phase of other

oleaginous yeast (Ageitos et al., 2011). The genera Rhodosporidium spp have other important oleaginous yeast species R. toruloides that utilized various kind of lignocellulosic biomass hydrolysates and waste materials such as Jerusalem artichoke hydrolysates, cassava starch, and wheat straw hydrolysates to produce 56.5, 24.6 and 64.9% lipid content with 70, 9.9 and 20.1 g/l dry cell biomass, respectively, on them (Zhao et al., 2010; Yu et al., 2011; Gen et al., 2014). The accumulation of high neutral lipid content (TAG) in these organisms is already to their maximum capacity varying between 20% and 70% which can be further improved by media engineering, system engineering and genetic engineering tools. This leads to a widen horizon for biodiesel production using oleaginous yeast than their bioethanol producing counterparts. 3.2. Estimation of TAG accumulation by fluorescence microscopy To visualize, identify and quantify the lipid, fluorescent lipophilic bright green dye, BODIPY 493/505, which has exceptional properties to specifically stain TAG while relatively insensitive to changes in pH and polarity, high molar extinction coefficient, high fluorescent quantum yields and resistance to photobleaching, was used to locate neutral lipids (TAG) in intracellular lipid droplets of the R. kratochvilovae HIMPA1 via live cell imaging protocol (Elsey et al., 2007; Patel et al., 2014) Further, BODIPY 493/505 can easily stain lipid droplets in yeast cells without any pretreatment. Moreover, the uptake of this dye is not affected by cell wall compositions and structure. In the cellular compartment of oleaginous microorganisms, lipid droplets (LDs) stores energy stock required during starvation period. LDs contain hydrophobic core of neutral lipids and is covered by a phospholipid monolayer. The results of cell size and lipid droplets were statistically verified and measured using Image J software. The bright field and fluorescence images of lipid droplet formation in R. kratochvilovae HIMPA1 when grown on 100% CAE for 144 h (Supplementary Figure 1) showed direct correlation of lipid body size with the TAG accumulating ability by R. kratochvilovae HIMPA1 on consumption sugars sources present in 100% CAE. Lipid droplets formation during cultivation of oleaginous yeast at different time intervals were recorded (Fig. 2). The yeast cells grown on 100% CAE showed maximum lipid droplets size (4.35 ± 0.54 lm) and cell size (5.28 ± 0.87 lm) during early stationary phase (120 h). Stationary phase cells of the yeast showed increase in cell size from 2.47 ± 0.23 to 5.16 ± 0.67 lm as well as lipid droplet size from 0.31 ± 0.12 to 2.69 ± 0.21 lm respectively which is similar to the earlier report on the Nile red stained fluorescence microscopy images of Saccharomyces cerevisiae where endoplasmic reticulum stress stimulate LD formation (Fei et al., 2009). The graphical data of LD size with respect to different phases of the cell revealed that the maximum accumulated lipids

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Fig. 1. (A) Graph showing dry cell weight (g/l), total lipid yield (g/l) and lipid content (%) of R. kratochvilovae HIMPA1 grown on aqueous extract of Cassia fistula L. fruit pulp (100% CAE) and on glucose synthetic medium (GSM) for 144 h. (B) Consumption of reducing sugars (g/l), and total carbohydrates (g/l), dry cell weight (g/l), total lipid yield (g/ l) and lipid content (%), glucose, fructose and xylose (g/l), when R. kratochvilovae HIMPA1 was cultivated on 100% CAE for 144 h.

Table 2 Comparative study of lipid production by different oleaginous microorganisms grown on variety of low-cost non-edible lignocellulosic biomass feedstocks. Oleaginous microorganisms

Utilized carbon source

Yarrowia lipolytica Rhodotorula glutinis Trichosporon fermentans Rhodotorula glutinis Cryptococcus curvatus Mortierella isabellina Mortierella isabellina T. coremiiforme Trichosporon fermentans Rhodosporidium toruloides NCYC 921 R. toruloides 21167 Rhodosporidium kratochvilovae HIMPA1

Industrial fats Monosodium glutamate wastewater Rice straw hydrolysate Starch wastewater Wheat straw acid hydrolysate Rice hulls hydrolysate Corn fiber hydrolysate Corncob acid hydrolysate Waste sweetpotato vines hydrolysate Carop pulp syrup (CPS) Cassava starch Aqueous extract of Cassia fistula L. fruit pulp (CAE)

should be harvested at a stage of early to late stationary phase (120–144 h) beyond which there is reduction in LD size. This demarcates the best stage of lipid harvesting and would be important for large scale production. 3.3. TLC and FTIR of extracted lipid from CAE grown R. kratochvilovae HIMPA1 cells Extracted lipids from oleaginous yeast were analyzed for their TAG contents using TLC. All content of extracted lipids such as free

Dry cell weight (g/l)

Lipid yield (g/l)

Lipid content (%)

8.7 25.0 28.6 40 17.2 5.59 18.20 20.4 26.96 5.28 20.1 8.9

3.8 5.0 11.5 14 5.8 3.6 8.3 7.7 9.6 0.91 13.04 4.73

44.0 20.0 40.1 35 33.5 64.3 45.7 37.8 35.6 17.27 64.9 53.18

References Papanikolaou et al. (2001) Xue et al. (2008) Huang et al. (2009) Xue et al. (2010) Yu et al. (2011) Economou et al. (2010) Xing et al. (2012) Huang et al. (2013) Zhan et al. (2013) Freitas et al. (2014) Gen et al. (2014) This study

fatty acids, MAGs, DAGs and TAGs were resolved easily in a single step using this technique. The separation is attained using the solvent system (hexane: diethyl ether: acetic acid; 85:15:1, v/v/v) where hexane with acetic acid migrates free fatty acids while diethyl ether used to control the separation of saturated and polyunsaturated TAGs. Complex lipids such as glycosphingolipids and phospholipids do not show any migration and remain at the origin. The TLC chromatogram developed after lipid extraction from CAE grown R. kratochvilovae HIMPA1 showed the presence of TAG, DAG and MAG bands (Fig. 3A). FAMEs obtained after

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carbonyl group. All of the mentioned peaks confirm that the produced oil can be converted to biodiesel potentially. 3.4. GC–MS analysis of fatty acid methyl esters (FAMEs)

Fig. 2. Graph showing LD size (lM) and cell size (lM) of BODIPY493/505 stained R. kratochvilovae HIMPA1 grown on 100% CAE at different time intervals showing TAG accumulation.

transesterification of total lipids were detected by TLC using dual solvent system, first on chloroform/methanol/water/acetic acid (65:35:4:1, v/v/v/v) followed by second solvent system hexane/ tert-butyl methyl ether/acetic acid (90:10:0.5, v/v/v) which verified the conversion of TAG with 92% conversion efficiency in 100% CAE (lane 3) and 94.5% in CAE 50% (lane 2), respectively (Fig. 3B). TAGs in extracted lipids were also analyzed using Fourier transform infrared (FTIR) spectroscopy which involves the measurement of infrared absorption in relation to a range of molecular vibrational modes. The FTIR spectra of lipid extracted from 100% CAE grown R. kratochvilovae HIMPA1 showed transmittance spectral similarities with triolein used as standard (Supplementary data Figure 2). No bands were observed in the spectral region 4000–3450 cm1 indicating thereby the absence of hydroxyl and amine groups. Both triolein and extracted lipid showed infrared absorption peaks in the second spectral region, 3100–2850 cm1. The bands obtained in this region were close to the wave numbers 2923 and 2856 cm1 respectively and assigned to the symmetrical and asymmetrical C–H stretching vibration of the CH2 and CH3 aliphatic groups, which are found in large quantities in vegetable oils. The peak obtained at 1743 cm1, demonstrate the presence of

The FAME profiles of R. kratochvilovae HIMPA1 were investigated on 100% CAE, GSM and compared with other vegetable oils using GC–MS (Table 3). The fatty acids methyl esters profile contain mainly myristic acid (C14:0) 0.78%, palmitic acid (C16:0) 43.06%, stearic acid (C18:0) 28.74%, oleic acid (C18:1) 17.34%, along with 0.48% linoleic acid (C18:2) when 100% CAE was used as carbon source by R. kratochvilovae HIMPA1. The total saturated fatty acid (SFA) contents in it were recorded as 72.58% which was higher than mono-unsaturated (19.41%) and poly-unsaturated fatty acid (0.48%) contents, respectively. The contents of total fatty acids along with saturated and unsaturated fatty acids (UFA) were decreased with the diluted amount of CAE provided (100% > 75% > 50% > 25%) which is utilized by R. kratochvilovae HIMPA1 (data not shown). The content of fatty acids were changed when GSM was used as lipid production medium as it contains 1.35 ± 0.13% myristic acid (C14:0), 17.56 ± 0.43% palmitic acid (C16:0), 20.71 ± 0.47% stearic acid, 21.95 ± 0.65% oleic acid (C18:1), along with 3.93 ± 0.42% linoleic acid (C18:2). The unsaturated fatty acid contents were high in GSM, while the amount of total fatty acids was lower than that of 100% CAE. The fatty acids profiles of R. kratochvilovae HIMPA1 on 100% CAE showed high proportion of total saturated fatty acids (72.58%) than the other vegetable oil counterparts (100% CAE, 72.58% > Jatropha oil, 21.52% > soybean oil, 15% > Sunflower oil, 4.5%). Biodiesel as fuel is much influenced by the individual fatty methyl esters present in it. Reports have further stated that the cetane number, heat of combustion, melting point, and viscosity of fatty compounds shoot up with increase in chain length and decreases with unsaturation (Purohit et al., 2012). Presence of UFA in the biodiesel is more prone to autoxidation. SFA have melting point more than the UFA contents and crystallize at higher temperature than the UFA. Moreover, biodiesel fuels derived from fats or oils with significant amounts of saturated fatty compounds displayed higher cetane number (CNs), cloud points (CPs) and pour point (PPs). High amount of SFA contents such as palmitic acid (C16:0) and stearic acid (C18:0) documented in this study can be blended with other feedstocks possessing high PUFA contents to improve biodiesel qualities. Although feedstocks with

Fig. 3. (A) Analysis of TAGs by thin layer chromatography (TLC) in the extracted lipid from R. kratochvilovae HIMPA1 while grown on 100% CAE (Lane 2) and 50% CAE (Lane 3). Triolein used as control (lane 1). (B) Conversion of TAG into FAMEs after transesterification of obtained lipid detected by dual solvent system. Lane 1: Standard (FAME of castor oil), Lane 2: FAME of 100% CAE, Lane 3: FAME of 50% CAE. SE, Steryl Ester; FAME, fatty acid methyl esters; TAG, triacylglycerols; FFA, free fatty acids; PC, phosphatidylcholine; PE, phosphatidylethanolamine; St, sterol; CL, cardiolipin.

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Table 3 The total percentage of fatty acid methyl esters (FAMEs) produced by R. kratochvilovae HIMPA1 when grown on aqueous extract of Cassia fistula L. fruit pulp (100% CAE), GSM and its comparison with other vegetable oils (Jatropha, Soybean and Sun flower oil). Types of fatty acid methyl ester

Total amount of fatty acid methyl esters (%) 100% CAE

GSM

Jatropha oil

Soybean oil

Sunflower oil

SFA

C 14:0 (myristic acid) C 16:0 (palmitic acid) C 18:0 (stearic acid)

0.78 ± 0.21 43.06 ± 0.12 28.74 ± 0.32

1.35 ± 0.13 17.56 ± 0.43 20.71 ± 0.47

– 14.66 6.86

– 11.00 4.00

– – 4.5

MUFA

C 18:1 (oleic acid)

17.34 ± 0.27

21.95 ± 0.65

39.08

23.40

21.10

PUFA

C 18:2 (linoleic acid)

0.48 ± 0.011

3.93 ± 0.42

32.48

53.20

66.20

SFA, saturated fatty acids; MUFA, mono-unsaturated fatty acids; PUFA, poly-unsaturated.

high SFA content such as palm oil produce biodiesel fuels with poor cold flow properties; whereas feedstocks with high UFA content such as rapeseed produce fuels having better performance. For better industrial assessment in improvement of biodiesel stability it is advisable to blend two feedstocks having different levels of inherent oxidative stability, such as blending of highly stable palm FAMEs with less stable Jatropha FAMEs (Puhan et al., 2010). In future, the blend ratio of SFA: PUFA can be used to design formulation and guidelines for optimum biodiesel fuel properties. 4. Conclusions Aqueous extract of C. fistula L. fruit pulp (CAE) was investigated as non-edible, cheaper lignocellulosic biomass as substrate for the heterotrophic production of neutral lipids using novel oleaginous yeast, R. kratochvilovae HIMPA1. CAE grown R. kratochvilovae HIMPA1 exhibited high amount dry cell weight (8.9 ± 0.12 g/l) and total lipid yield (4.73 ± 0.34 g/l) as compared to glucose synthetic medium. A high concentration of palmitic acid (C16:0) 43.06%, stearic acid (C18:0) 28.74%, and oleic acid (C18:1) 17.34% in the FAMEs content makes it an industrially viable renewable energy product which can be explored for commercial biodiesel production. Acknowledgements Authors are thankful for financial support by the Department of Biotechnology, Govt. of India, Bio Care Programme, DBT Sanction No.: 102/IFD/SAN/3539/2011-2012 (Grant No.: DBT-608-BIO) and JRF fellowship to Alok Kumar Patel from University Grants Commission, India (Grant No.: 6405-35-044). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08. 039. References Ageitos, J.M., Vallejo, J.A., Veiga-Crespo, P., Villa, T.G., 2011. Oily yeasts as oleaginous cell factories. Appl. Microbiol. Biotechnol. 90, 1219–1227. http://dx.doi.org/ 10.1007/s00253-011-3200-z. Atabani, A.E., Silitonga, A.S., Ong, H.C., Mahlia, T.M.I., Masjuki, H.H., Badruddin, I.A., Fayaz, H., 2013. Non-edible vegetable oils: a critical evaluation of oil extraction, fatty acid compositions, biodiesel production, characteristics, engine performance and emissions production. Renew. Sustain. Energy Rev. 18, 211– 245. http://dx.doi.org/10.1016/j.rser.2012.10.013. Barthakur, N.N., Arnold, N.P., Alli, I., 1995. The Indian laburnum (Cassia fistula L.) fruit: an analysis of its chemical constituents. Plant Foods Hum. Nutr. 47, 55–62. http://dx.doi.org/10.1007/BF01088167. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28 (3), 350–356. Economou, C.N., Makri, A., Aggelis, G., Pavlou, S., Vayenas, D.V., 2010. Semi-solid state fermentation of sweet sorghum for the biotechnological production of

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