A Cellulase and Lipase Producing Oleaginous Yeast ⇑ Sachin Vyas, Meenu Chhabra

Bioresource Technology 223 (2017) 250–258

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Bioresource Technology

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Isolation, identiﬁcation and characterization of Cystobasidium oligophagum JRC1: A cellulase and lipase producing oleaginous yeast ⇑ Sachin Vyas, Meenu Chhabra

Environmental Biotechnology Laboratory, Department of Biology, Indian Institute of Technology Jodhpur (IIT J), Jodhpur, Rajasthan 342011, India

highlights graphical abstract

Oleaginous yeast capable of cellulase and lipase production was isolated. It can grow on wide range of substrates including carboxylmethylcellulose (CMC). It can accumulate 36.46% (w/w) lipid on the medium with CMC as sole carbon source. Intracellular and extracellular lipase activity: 2.16 and 2.88 IU/mg respectively. The FAME composition suitable for use as biodiesel (glucose medium).

article info abstract

Article history: Oleaginous yeast closely related to Cystobasidium oligophagum was isolated from soil rich in cellulosic Received 15 August 2016 waste. The yeast was isolated based on its ability to accumulate intracellular lipid, grow on car- Received in revised form 12 October 2016 boxymethylcellulose (CMC) and produce lipase. It could accumulate up to 39.44% lipid in a glucose med- Accepted 13 October 2016 ium (12.45 ± 0.97 g/l biomass production). It was able to grow and accumulate lipids (36.46%) in the Available online 15 October 2016 medium containing CMC as the sole carbon source. The specific enzyme activities obtained for endoglucanase, exoglucanase, and b-glucosidase were 2.27, 1.26, and 0.98 IU/mg respectively. The specific Keywords: enzyme activities obtained for intracellular and extracellular lipase were 2.16 and 2.88 IU/mg respec- Cystobasidium oligophagum tively. It could grow and accumulate lipids in substrates including glycerol (42.04%), starch (41.54%), Rhodotorula oligophaga Oleaginous yeast xylose (36.24%), maltose (26.31%), fructose (24.29%), lactose (21.91%) and sucrose (21.72%). The lipid pro- Cellulase file of the organism was suitable for obtaining biodiesel with desirable fuel properties. Cellulolytic yeast Ó 2016 Elsevier Ltd. All rights reserved. Lipase FAME

1. Introduction sustainable biofuels for the future (Li et al., 2008). The photoau- totrophic nature of algae is an obvious advantage, however, the A continuous increase in the population and demands for the slower growth rates and lower lipid productivity is still a challenge transportation fuel has urged the scientists across the globe to ﬁnd for algal biofuels commercialization. Yeast, on the other hand, alternative fuel resources, of which the biomass derived fuels holds exhibit faster growth rate, high lipid productivity, can grow inde- the special place (Sitepu et al., 2014). Microbes such as algae and pendent of the climate condition and at low pH value which natu- yeasts have been extensively studied for their ability to provide rally suppresses the growth of many bacteria. Other useful characteristics of the yeast are the ability to grow on the wide range of waste substrates, lesser space requirements and easy ⇑ Corresponding author. genetic modiﬁcation (Li et al., 2008; Sitepu et al., 2014). Yeasts like E-mail address: [email protected] (M. Chhabra).

Yarrowia lipolytia, Lipomyces starkeyi, Rhodotorula glutinis, etc., for TLC, p-nitrophenyl-b-D-glucopyranoside (pNPG), tributyrin and which can accumulate cellular lipids more than 20 percent of their vitamins were purchased from Hi-media Laboratories, India. The dry cell weight (DCW) (or biomass production) are known as PCR primers, FAME standard (for GC–MS analysis) and other chem- oleaginous yeasts (Ratledge, 2004). Oleaginous yeasts have been icals [i.e. Methanolic BF3, p-nitrophenyl palmitate (pNPP), etc.] extensively studied for biodiesel production in the past few years. were purchased from Sigma-Aldrich. Yeasts primarily accumulate lipids in the form of triacylglycerol (TAG), comprising of saturated and unsaturated chains of fatty 2.2. Isolation and screening of oleaginous yeast strain acids. However, biodiesel production from yeasts is also equally challenging since these are heterotrophic organisms and rely on The soil samples rich in cellulosic wastes such as decaying an external supply of organic carbon sources. leaves, vegetable wastes etc., were collected from the five different 0 00 Cellulose, a linear chain polymer of D-glucose attached by regions around the local area, Jodhpur (coordinates: 26°16 18.2 N, b-(1,4)-glycosidic bonds, is the most abundant carbohydrate in 73°01059.200E). Samples were collected in the sterile tubes and the world (Van Soest, 1982). Unlike starch, the cellulose is not a stored at 4 °C until further use. One gram of the homogenized sam- source of the nutrition for most of the higher level organisms; ple was aseptically suspended in 100 ml of the sterilized saline making it a sustainable raw material for biofuel production. Vari- solution (0.85% w/v NaCl). 1 ml of the dilution obtained was trans- ous oleaginous yeasts of the genera like Rhodotorula, Cryptococcus, ferred into 250 ml Erlenmeyer flask containing 100 ml of YPD med- Yarrowia, Lipomyces, Rhodosporidium, are recently reported to accu- ium (g/l): yeast extract, 10; peptone, 20; glucose, 10; and mulate higher amount of lipids on the pre-treated lignocellulosic incubated at 30 °C and 120 rpm (pH 6.0). Ampicillin (100 lg/ml) substrates, low cost agro-industrial waste (paper mill, crude glyc- was used to avoid the bacterial growth. Around 139 yeast colonies erol), and other renewable feedstocks such as animal fat, municipal were isolated on YPD agar supplemented with ampicillin. The iso- solid waste, etc., (Leiva-Candia et al., 2014). An enzymatic pre- lates obtained were further plated on the lipid production medium treatment of cellulosic biomass to release utilizable hexose and (g/l): Glucose, 40; KH2PO4, 7; NaH2PO4, 2; (NH4)2SO4,2; pentose sugars normally precedes the oil production step by MgSO47H2O, 1.5; yeast extract, 1; agar powder, 20. Vitamins yeasts. Fewer reports discuss the lipid accumulation directly on (mg/l): D-biotin, 0.002; calcium pantothenate, 0.4; folic acid, the cellulosic substrate. Consolidated Bioprocessing (CBP) for the 0.002; inositol, 2; niacin, 0.4; para-aminobenzoic acid (PABA), accumulation of lipids from cellulosic materials without the use 0.2; pyridoxine HCl, 0.4; riboflavin, 0.2; thiamine, 0.4, at 30 °C of additional cellulase enzymes will be a very advantageous (pH 5.5). The yeast colonies growing on lipid production medium (Lynd et al., 2005). There are many fungi as well as bacteria, which were further screened for lipid accumulation using the nile red flu- are reported to be the excellent cellulase producers; limited stud- orescence microscopy protocol as described in the subsequent sec- ies are reported however for cellulolytic yeasts. Cellulose utiliza- tions. The yeast colonies which were showing significant lipid tion involves multi-enzyme complex consisting of three major droplets inside the cells were further grown in the production enzymes (i) endo-1,4 b-D-glucanase (or CMCase) (ii) cellobiohydro- medium described later, and the lipid outputs were determined. lase (or exoglucanase) and (iii) b-glucosidase. Certain genera of the yeast such as Rhodotorula, Cryptococcus, Candida, Sporobolomyces, 2.3. Screening of oleaginous yeasts for cellulase activity and Aureobasdium are reported to be the producer of different types of cellulases (Baldrian and Vendula, 2008; Strauss et al., The oleaginous yeasts obtained from the above step were cul-

2001). So far there is only one report on the oleaginous yeast accu- tured in the medium (g/l): CMC, 10; yeast extract, 0.6; KH2PO4, mulating lipids using carboxymethylcellulose (CMC) as a carbon 7; K2HPO4, 2; MgSO4 7H2O, 0.1; (NH4)2SO4, 1, agar 20 (pH 6.0) source (Kanti and sudiana, 2015) to the best of our knowledge. (Goldbeck et al., 2012), and incubated at 30 °C. The colonies which However, a systematic and quantitative estimation of cellulolytic showed growth were replica plated. Extracellular cellulase enzyme activity along with lipid accumulation is missing in the majority activity was confirmed using 0.2% congo red dye method (Teather of these studies. and Wood, 1982). In addition to the use of the cellulosic substrate, we wanted the same yeast to produce lipase, a potential transesterification cata- 2.4. Screening of selected yeasts for lipase activity lyst. Lipase holds a special interest in next generation biodiesel synthesis. Studies are underway to design efficient lipases and uti- The yeast cultures which were found to be oleaginous, and lize them for transesterification instead of conventional catalysts expressing cellulolytic activity, were further tested for lipase activ- to produce biodiesel (Aguieiras et al., 2015). ity. The lipase activity was detected by qualitative plate assay A successful attempt to isolate oleaginous cellulolytic yeast using tributyrin as a lipid source. The selected yeast cultures were with both intracellular and extracellular lipase activity was made. allowed to grow on plates containing (g/l): tributyrin, 10; peptone, The isolation of the yeast was followed by its identification, char- 5; yeast extract, 3; agar, 20, pH 6.0 (Sztajer et al., 1988), and incu- acterization and lipid profiling. Isolation of such an organism opens bated at 30 °C. The expression of lipase enzyme was confirmed by a up the possibility of intracellular FAEE (fatty acid ethyl ester) syn- zone of clearance around the colony. thesis directly by few more experimental interventions. This study thus opens up the new prospect of utilization of cellulosic or indus- 2.5. Identification and characterization of the yeast strain trial wastes directly for the production of biodiesel with desirable fuel properties. Identification of the selected yeast strain was performed by determining partial 18S rRNA gene sequence. Total DNA was extracted using yeast genomic DNA isolation kit (Himedia labora- 2. Materials and methods tories, India) and the gene was PCR amplified using universal primers, nu-SSU-0817-50 (TTAGCATGGAATAATRRAATAGGA) and 2.1. Materials nu-SSU-1536-30 (ATTGCAATGCYCTATCCCCA) (Borneman and Hartin, 2000). The 843 bp product was purified and sequenced All the medium supplements and the general chemicals, i.e. (Eurofins, India). The identity of the strain was done by nucleotide n-hexane, methanol, chloroform and other chemicals like nile red BLAST (using NCBI database) assuming at least 99% identity as the (for fluorescence microscopy), triolein standard (Glycerol trioleate) best hit. The obtained sequence was submitted to GenBank and 252 S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258 accession number KX668182 was assigned. Taxonomic positioning described in Section 2.10.3) for measurement of lipid output as and construction of the phylogenetic tree of the isolate were done well as the lipid content. by MEGA 6.0 software (Tamura et al., 2013). The yeast was subse- quently named as Cystobasidium oligophagum JRC1. 2.9. Effect of carbon source on lipid production

2.6. Batch cultivation of C. oligophagum JRC1 for total cellulase The yeast was cultivated on different types of carbon sources production like starch, CMC, glycerol, xylose, maltose, fructose, sucrose, and lactose instead of glucose and incubated at optimized temperature The yeast was inoculated into 250 ml Erlenmeyer flask contain- and pH, at 120 rpm. ing 100 ml of the sterile broth medium (g/l): CMC, 10; yeast 2.10. Biochemical analysis of lipid production extract, 0.6; KH2PO4, 7; K2HPO4, 2; MgSO4 7H2O, 0.1; (NH4)2SO4, 1 (pH 6.0) and was allowed to grow at 28 °C and 120 rpm. 10 ml of the inoculum was transferred to the new sterile broth as produc- 2.10.1. Biomass production, residual sugar, and nitrogen estimation tion medium and incubated at 28 °C and 120 rpm. 10 ml of the cul- Biomass production (or total dry cell weight, or cell mass pro- ture from the production flask was harvested and centrifuged for at duction) was measured by harvesting 10 ml of the sample at the 8000 rpm for 10 min at intervals of 12 h. The supernatant obtained intervals of 24 h and centrifuging at 8000 rpm for 10 min and was tested for the presence of extracellular enzymes. The total cel- determination of weight gravimetrically after drying in an oven ° lulase assay was performed according to the methods of Ghose at 80 C for overnight or till the constant weight was achieved. (1987). The released sugar in both CMCase and FPase activity Residual sugar was estimated by the DNS method (Miller, 1959). was estimated by the 3, 5-Dinitrosalicylic acid (DNS) method Ammonium nitrogen was determined by Nessler’s method (Miller, 1959). b-Glucosidase (BG) activity was estimated by using (APHA, 1992). pNPG as substrate. One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 lmol of glucose or p- 2.10.2. Nile red fluorescence microscopy 500 ll of the culture sample at different time intervals was pel- nitrophenol from the appropriate substrates per minute under the l assay conditions. Total protein was measured by the Bradford leted, washed and suspended in PBS (pH 6.0). 2 l of nile red stock method (Bradford, 1976). Bovine serum albumin was used for solution (0.1 mg/ml in DMSO) was then added and mixed with the preparing the protein standard. cells. The dye and cells mixture was allowed to incubate at 100 rpm for 20 min in the dark. After the incubation period, it was pelleted and washed twice with PBS. The cell pellet was re- 2.7. Batch cultivation of C. oligophagum JRC1 for lipase production suspended in 40 lL PBS and slides were prepared (Fei et al., 2009). Fluorescence imaging was performed using cell imaging The yeast C. oligophagum JRC1 was inoculated into 250 ml station-Floid (Life technologies, India) at 20 magnification. Erlenmeyer flask containing 100 ml of the sterile medium broth as (g/l): glucose 10; tributyrin, 10; peptone, 20; yeast extract, 10; 2.10.3. Total lipid extraction ° (pH 6.0) and was allowed to grow at 28 C and 120 rpm. The crude The total cellular lipids were extracted using a modified proto- enzyme for extracellular lipase was obtained as described in Sec- col by Bligh and dyer (Bligh and Dyer, 1959). The total lipid content tion 2.6. To obtain crude intracellular enzyme, the residual cell pel- (%) and lipid output (g/l) were determined gravimetrically. let was washed twice with phosphate buffer saline (PBS) and ultrasonicated at 40 kHz for five minutes in an equal volume of 2.10.4. FTIR and TLC analysis of the lipid extract PBS buffer and used further. The lipase enzyme assays were per- The extracted lipids were analyzed by Fourier transform infra- formed according to Vorderwulbecke et al., (1992). Lipase activity red spectroscopy (FTIR) (Bruker-70V, USA) using triolein (Glycerol was estimated by using pNPP as substrate. 0.1 ml of crude enzyme trioleate) as a standard for TAG. The transmittance for the samples was mixed with 0.9 ml of pNPP and was incubated for 30 min at was recorded over the range of the spectrum from 400 to 50 °C. One unit (U) of enzyme activity was defined as the amount 1 l 4000 cm . TAG analysis was also done by thin layer chromatogra- of enzyme required to liberate 1 mol of p-nitrophenol from the phy (TLC) using 0.25-mm-thick silica gel G-60, F254, Merck, India. pNPP substrate per minute under the assay conditions. Total pro- The chromatograms were developed by hexane: diethyl ether: tein was measured by the Bradford method (Bradford, 1976). acetic acid (85:15:1, v/v/v). The TLC plate was dipped into a

methanolic-MnCl2 solution (0.63 g MnCl24H2O in 60 ml water, 2.8. Batch cultivation of C. oligophagum JRC1 for lipid production 60 ml methanol and 4 ml concentrated sulphuric acid) followed by drying and heating at 120 °C for 20 min for quantification of The optimum pH and temperature for the yeast were deter- TAGs (Fei et al., 2009). mined in the lipid production medium (g/l): Glucose, 40; KH2PO4, 7; NaH2PO4, 2; (NH4)2SO4, 2; MgSO4 7H2O, 1.5; yeast extract, 1. 2.10.5. Transesterification of lipids and GC–MS analysis Vitamins (mg/l): D-biotin, 0.002; calcium pantothenate, 0.4; folic Transesterification of the lipid extract was performed by the acid, 0.002; inositol, 2; niacin, 0.4; PABA, 0.2; pyridoxine HCl, methods given by Morrison and Smith, (1964) using boron trifluo- 0.4; riboflavin, 0.2; thiamine, 0.4. pH was tested in the range of ride methanol solution. The FAME obtained by the process of trans- 2.0–7.0 and temperature was tested in the range of 18–32 °C. In esterification was further analyzed by GC–MS chromatography order to quantify lipid production, the yeast C. oligophagum JRC1 (GC–MS model QP2010 plus, Shimadzu, Japan) (column specifica- was inoculated into the 100 ml of lipid production medium and tions: Agilent DB-Wax 30 m 0.25 mm 0.25 lm). GC–MS analy- incubated at optimized temperature and pH. 10 ml of the inoculum sis was done by injecting 1 ll of the sample at 250 °C in split was transferred to the new sterile broth as the production medium injection mode at 50:1 split ratio using helium (99.999% pure) as and incubated. 10 ml of the culture was harvested and centrifuged a carrier gas (1 ml/min). Initially, the column temperature was for 10 min at 8000 rpm at an interval of 24 h. The supernatant was set at 60 °C which then was increased to 150 °C at the rate of used for estimation of the remaining sugar and ammonical nitro- 12 °C/min and was held for 1 min. It was further increased to + gen (NH4–N). The cell pellet obtained by every sampling was fur- 240 °Cat50°C/min and held for 5 min and the equilibrium time ther dried and subjected to the total lipid extraction (as given was 3 min. The mass transfer line and ion source were set S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258 253 at 230 °C and 240 °C respectively. The FAMEs obtained from the and extracellular lipase activity has been attempted for the first yeast lipid were identified by comparing their retention times with time in literature. known standards (F.A.M.E. Mix C8-C24, Sigma-Aldrich, India) and confirmed against the NIST-2010 database. 3.3. Identification and characterization of the yeast strain 2.10.6. Statistical analysis All experiments were performed in triplicates. The results Only one of the yeast isolates fulfilled the screening criteria. reported are the average values with the standard deviation rang- Identification of the yeast strain was done by genotypic amplifica- ing from 4% to 12%. tion and sequencing of partial 18s rRNA gene. The isolate was identified to be closely related to Cystobasidium oligophagum [formerly called Rhodotorula oligophaga with a sequence id: AB702968.1 of 3. Results and discussion 1750 bp (Satoh et al., 2013)]. The partial sequence of 18s rRNA gene (843 bp) was assigned Genbank Accession No. KX668182. A 3.1. Isolation and screening of oleaginous yeast recent report explained that Rhodotorula oligophaga and other few types of yeast were proposed to be transferred to the Cysto- Nearly 139 microbial colonies were obtained on the YPD med- basidum genus from the Rhodotorula minuta clade and it was ium with ampicillin. 23 of the 139 were found to be oleaginous renamed as Cystobasidium oligophagum (Yurkov et al., 2015). To yeast. The oleaginous yeasts showed accumulation of lipid in the identify the taxonomic position and relationships, the phylogenetic form of intracellular droplets. Among the 23 yeast colonies, 9 yeast trees based on partial 18s rRNA gene sequence of C. oligophagum colonies were showing the high amount of lipid accumulation by JRC1 and other related strains was constructed by using mega fluorescence microscopy. Lipid outputs were determined for these 6.0 software. (sum branch length = 0.08313443) (Fig. 1). Morpho- 9 colonies and it was found that 4 out of 9 yeasts accumulated logical characterization showed that cells are ellipsoidal higher lipid content (42, 39, 38 and 32% respectively). Previously, (4–5 lm 3–4 lm), occurring as a single cell or parental bud many strategies have been reported to isolate oleaginous yeasts pairs. After 3–4 days of incubation at 28 °C, the culture turns from the natural environments. In one of the studies, 79 yeasts slightly pink to dark pink in color (pigment production). No hyphae out of 479 microbial colonies isolated from Himalaya region, were or pseudohyphae were noticed even in the older cultures. It could identified to be oleaginous (Patel et al., 2014). Generally, 3–10% of assimilate glucose, sucrose, fructose, maltose, lactose, D-xylose, the randomly selected yeast population is found to be oleaginous, glycerol, and CMC. It grew optimally in the temperature range of which was also the case in our study (Sitepu et al., 2014). 25–30 °C, growing slowly at 31–33 °C and no growth was observed beyond 33 °C. The lineage of the yeast strain was; 3.2. Screening of oleaginous yeasts for cellulase and lipase activity fungi > Dikarya > Basidiomycota > Pucciniomycotina > Cystobasid- iomycetes > Cystobasidiales > Cystobasidiacea > Cystobasidium The selected oleaginous yeast colonies were further subjected oligophagum JRC1 (Basionym: Rhodotorula oligophaga). The scan- to congo red screening on a CMC medium. Out of four selected cul- ning electron microscopy was performed for the morphological tures, two yeasts were found to produce a zone of clearance, which studies. (Supporting data Fig. 1.2) indicated that the yeasts were expressing cellulolytic activity. Fur- ther, the selected yeasts were subjected to qualitative analysis for lipase (Supporting data Fig. 1.1). One of the two yeast isolates 3.4. Nile red fluorescence microscopy showed a zone of clearance on a tributyrin agar plate. At the end of the screening, successful isolation of one yeast strain was done. Fluorescence images were acquired for the yeast grown in glu- Isolation of oleaginous cellulolytic yeast with both intracellular cose medium at various time intervals. It was found that up to

9 Cystobasidium pallidum gene for 18S rRNA partial sequence. 2 Cystobasidium lysinophilum strain KF165 18S ribosomal RNA gene partial sequence. 0 Cystobasidium sp. BG02-6-15-006A-1 18S ribosomal RNA gene partial sequence. Cystobasidium lysinophilum strain LF980 18S ribosomal RNA gene partial sequence. Cystobasidium laryngis gene for 18S rRNA partial sequence. 66 Cystobasidium pinicola gene for 18S rRNA partial sequence. Cystobasidium psychroaquaticum partial 18S rRNA gene isolate KBP3881. 51 Cystobasidium ritchiei partial 18S rRNA gene isolate KBP4220. Cystobasidium lysinophilum strain KF157 18S ribosomal RNA gene partial sequence. Cystobasidium sp. 9A-1 gene for 18S rRNA partial sequence strain: 9A-1. 75 Cystobasidium benthicum gene for 18S rRNA partial sequence. 99 Occultifur externus isolate AFTOL-ID 860 18S small subunit ribosomal RNA gene partial sequence. Occultifur externus gene for SSU rRNA partial sequence strain:CBS8732. 60 Symmetrospora marina isolate H-12 18S ribosomal RNA gene partial sequence. 79 Sakaguchia cladiensis strain CBS 10878 18S ribosomal RNA gene partial sequence. Sakaguchia sp. JCM 8162 strain JCM8162 18S ribosomal RNA gene partial sequence. 55 Rhodotorula oryzicola strain AS2.3289 18S ribosomal RNA gene partial sequence. 96 Sporobolomyces symmetricus voucher P118 18S ribosomal RNA gene partial sequence. 16 Microsporomyces magnisporus strain JCM11898 18S ribosomal RNA gene partial sequence. Buckleyzyma aurantiaca strain JCM3771 18S ribosomal RNA gene partial sequence. 21 Cystobasidiomycetes sp. MT-2015c voucher WRP8 18S ribosomal RNA gene partial sequence. 30 86 Symmetrospora gracilis strain JCM2963 18S ribosomal RNA gene partial sequence. Cystobasidium oligophagum JRC1 18s ribosomal rRNA partial sequence Cystobasidium fimetarium small subunit ribosomal RNA gene partial sequence. Cystobasidium slooffiae gene for 18S rRNA partial sequence. Cystobasidium minutum strain DBSC-5 18S ribosomal RNA gene partial sequence. 62 Rhodotorula sp. TJUWZC3 18S ribosomal RNA gene partial sequence. 13 Cystobasidium sp. nymphaeae gene for 18S rRNA partial sequence strain:18-2A(IAM14903). 72 Rhodotorula sp. AspoyJ1 18S ribosomal RNA gene partial sequence. Cystobasidium slooffiae strain DBSC-9 18S ribosomal RNA gene partial sequence. Rhodotorula sp. NyZ49 18S ribosomal RNA gene partial sequence. 1 Cystobasidium minutum isolate T 18S ribosomal RNA gene partial sequence. Cystobasidium oligophaga partial 18S rRNA gene isolate TIMM10017. Cystobasidium calyptogenae gene for 18S rRNA partial sequence. Cystobasidium sp. cassiicola gene for SSU rRNA partial sequence strain:36-2A(IAM14905). 88 Cystobasidium sp. cassiicola gene for SSU rRNA partial sequence strain:36-2B(IAM14906). Cystobasidium sp. samaneae gene for SSU rRNA partial sequence strain:33-1A-1(IAM14904).

0.001

Fig. 1. The phylogenetic tree of C. oligophagum JRC1: Constructed by neighbour-joining method (MEGA 6.0 software) (sum branch length = 0.08313443). 254 S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258

96 h, no or little size of droplets were present. After 120 h, the lipid et al., 2015). Several yeasts such as Lipomyces starkeyi, Yarrowia droplets of a considerable size were observed inside the cells. lipolytica, etc. have been reported to produce extracellular as well as intracellular lipase and their utilization as a catalyst for biodie- 3.5. Batch cultivation of C. oligophagum JRC1 for total cellulase sel (FAME) production has also been studied (Ribeiro et al., 2011). production The kinetics of lipase catalysed transesterification can be enhanced by combining it with physicochemical methods such as ultrasoni- The cellulase enzymes include the endoglucanase (CMCase), cation (Malani et al., 2014). Further experimental interventions exoglucanase (cellobiohydrolase/FPase) and b-glucosidase. The will be needed to utilize the enzymatic machinery of the isolated assays for all three enzymes as well as protein concentration yeast in the present study for FAEE/Biodiesel synthesis in vivo or (100 ml medium) at 28 °C were recorded after every 12 h as shown in vitro. in Fig. 2. When the culture was cultivated on the CMC medium, the highest specific enzyme activity for endoglucanase (CMCase), 3.7. Batch cultivation of C. oligophagum JRC1 for lipid production and exoglucanase and b-glucosidase achieved were 2.27 ± 0.007, extraction 1.26 ± 0.0408 and 0.98 ± 0.0408 IU/mg respectively (enzyme activity 0.072 ± 0.013, 0.040 ± 0.006, 0.031 ± 0.011 IU/ml respectively) The optimization of the cultivation conditions in the lipid pro- after 72 h of incubation. Till date, very few studies are reported duction medium was done at various temperature and pH values. on the production of cellulases by yeasts. Rhodotorula muciliginosa After the experiments, the temperature 28 °C and pH 6.0 was found was reported to produce endoglucanase as well as b-glucosidase to be giving highest lipid production in a glucose medium (Baldrian and Vendula, 2008; Strauss et al., 2001). In a recent (Fig. 4A and B). The biomass production, lipid content and the lipid report, a marine yeast Aureobasdium pullulans produced maximum output (at 28 °C and 6.0 pH) were monitored at every 24 h inter- CMCase activity of 4.51 IU/mg in an optimized medium (Rong vals till 192 h, and their time courses were recorded as shown in et al., 2015). b-Glucosidase activity of the given yeast isolate was Fig. 4C and D. At 72 h the biomass production, lipid output, and comparable with the recently reported yeast Pseudozyma brasilien- lipid content were 5.31 ± 0.94 g/l, 1.26 ± 0.26 g/l and 23.79% sis (0.14 IU/ml) (Adalberto et al., 2015). Cryptococcus flavus and respectively. In the post stationary phase, the lipid accumulation Rhodotorula muciliginosa species have also been reported to secrete phase started and the lipid content increased steadily from 96 h endoglucanase enzyme naturally (Oikawa et al., 1998). In one of to 192 h. At 168 h, the highest biomass production, lipid output, the study, the gene encoding endoglucanase from C. flavus yeast and lipid content were 12.45 ± 0.97 g/l, 4.92 ± 0.52 g/l and 39.44% was cloned and expressed in the Saccharomyces cerevisiae for CBP respectively. The profile of residual sugars as well as nitrogen of cellulosic biomass to bioethanol (Hatano et al., 1994). The ability (ammonical nitrogen) available in the medium was obtained every to produce of cellulase, as well as oleaginous nature, create a 24 h (Fig. 4C and D). Most of the oleaginous yeasts accumulate lipid unique combination which makes the C. oligophagum JRC1 yeast in post stationary phase. Generally, the incubation period for a strong candidate for CBP of cellulosic waste to biodiesel. achieving the highest lipid content varies among different yeasts (120–192 h). Various other genera like Candida, Cryptococcus, 3.6. Batch cultivation of C. oligophagum JRC1 for lipase production Rhdosporidium, Trichosporon, Yarrowia, Rhodotorula etc., of the yeast species, have also been reported to accumulate lipids at dif- Lipases are either extracellular or intracellular. As shown in ferent levels (values of which are mentioned in Table 1 for compar- Fig. 3, the highest extracellular lipase activity achieved was ison). The values for lipid productivity in case of glucose as a 0.141 ± 0.009 IU/ml and specific enzyme activity was carbon source were comparable with C. viswanathii Y-E4, 2.88 ± 0.166 IU/mg after 84 h of incubation. The intracellular crude R. toruloids, and C. vishniacci and lower than Y. lipolytica and lipase was recovered by sonication and pNPP assay was performed, R. graminis (Ayadi et al., 2016; Deeba et al., 2016; Fontanille as shown in Fig. 3, the highest activity achieved was et al., 2012; Galafassi et al., 2012; Patel et al., 2014). The lipid pro- 0.085 ± 0.001 IU/ml and specific enzyme activity was ductivity was comparable with C. viswanathii Y-E4, R. bajevae 2.16 ± 0.04 IU/mg. The efficiency of ultrasonication is affected by (Ayadi et al., 2016; Munch et al., 2015) and lower than R. graminis the medium composition, dissolved gas, temperature etc., (Galafassi et al., 2012) while using glycerol as a carbon source. The (Chakma and Moholkar, 2013, 2016). Thus ultra sonication was lipid productivity using xylose and starch as a carbon source were carried out at conditions used in the previous studies (Patel comparable with other yeasts such as C. viswanathii and C. terricola

Fig. 2. Cellulase activity profile of C. oligophagum JRC1 with time: Showing profile Fig. 3. Lipase activity profile of C. oligophagum JRC1with time: Showing extracel- of endoglucanase, exoglucanase and b-glucosidase activity (IU/ml) and total protein lular and intracellular lipase activity (IU/ml) and protein concentration (lg/ml) (lg/ml) with time in medium containing CMC as a carbon source. with time in medium containing tributyrin and glucose as a carbon source. S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258 255

Fig. 4. (A) Effect of temperature on lipid production: Profile of biomass production (g/l), lipid output (g/l), and lipid content (%) of C. oligophagum JRC1 in lipid production medium at various temperatures. (B) Effect of pH on lipid accumulation: Profile of biomass production (g/l), lipid output (g/l), and lipid content (%) of C. oligophagum JRC1 in lipid production medium at various pH ranging from pH value 2–7. (C) Profile of substrate utilization; biomass production (g/l), residual glucose (g/l), ammonical nitrogen (g/l) of C. oligophagum JRC1with time in lipid production medium. (D) Profile of lipid output (g/l) and lipid content (%) of C. oligophagum JRC1 with time in lipid production medium.

respectively (Ayadi et al., 2016; Tanimura et al., 2014). Further the lipid accumulation in medium containing various different car- optimization of the culture conditions and cultivation strategies bon sources. Table 2, summarizes different lipid production param- using these carbon sources can enhance the lipid productivity from eters obtained on individual carbon sources. Calculations were the isolate. done as per the definitions given by Sitepu et al., 2014. Although the lipid content (%) in the case of starch and glycerol was observed 3.8. Effect of carbon source on lipid production to be higher than that of glucose, the lipid productivity was low which demands further optimization studies. The ability of the iso- The yeast C. oligophagum JRC1 showed a wide range of substrate late to utilize different types of carbon sources reflects upon its utilization when grown on the different types of carbon sources ability to utilize industrial wastes such as those obtained from bio- like maltose, fructose, sucrose, lactose, starch, CMC, xylose, and diesel transesterification plants, dairy industry, starch processing glycerol. In all the experiments the biomass production (g/l), lipid industries, sugar mill effluents etc. Most of the basidiomycetes output (g/l) and lipid content (%) were measured as shown in Fig. 5. yeasts are able to grow and accumulate lipids on the pentose and

By using individual carbon sources the highest biomass production hexoses (C5 as well as C6 sugars) (Sitepu et al., 2014). The different obtained with glucose (12.45 ± 0.97 g/l) followed by fructose studies so far show that the yeasts of the genus like Yarrowia, Rho- (11.50 ± 0.79 g/l), xylose (11.09 ± 0.75 g/l), lactose (9.74 ± 0.83 g/l), dotorula, Rhodosporidium are reported to be grown on the carbon sucrose (8.98 ± 0.03 g/l), glycerol (6.48 ± 0.57 g/l), CMC sources derived from the lignocellulosic biomass (Leiva-Candia (6.25 ± 0.78 g/l), starch (4.72 ± 0.55 g/l) and maltose et al., 2014). Also, glycerol a byproduct of the biodiesel industry (2.26 ± 0.45 g/l). The lipid output (g/l) was highest for glucose is widely employed as a substrate for lipid accumulation. (4.91 ± 0.52 g/l) followed by xylose (4.02 ± 0.32 g/l), fructose Cryptococcus terricola was reported to be an oleaginous yeast that (2.79 ± 0.20 g/l), glycerol (2.72 ± 0.30 g/l), CMC (2.28 ± 0.33 g/l), can grow directly on starch wastes (Tanimura et al., 2014). The lactose (2.13 ± 0.13 g/l), starch (1.96 ± 0.22 g/l), sucrose lipid profile of oleaginous yeast changes with the change in the car- (1.95 ± 0.17 g/l) and maltose (0.59 ± 0.14 g/l). Thus, the variation bon source and other conditions (Sitepu et al., 2013). The fatty acid was observed in the accumulation of lipid as the carbon source constitution is important as it affects the biodiesel properties and was varied. The lipid content (%), on the other hand was highest determines its usability as fuel. The carbon sources other than glu- for glycerol (42.04 ± 1.71%) followed by starch (41.54 ± 0.34%), glucose are assimilated via different metabolic pathways and thus cose (39.44 ± 1.19%), CMC (36.46 ± 1.49%), xylose (36.24 ± 1.09%), leads to different fatty acid composition in lipids (Fakas et al., maltose (26.31 ± 1.82%), fructose (24.29 ± 0.90%), lactose 2009). Leiva-Candia et al., 2014, have reviewed the effect of carbon (21.91 ± 0.63%) and sucrose (21.72 ± 1.77%). The fluorescence sources derived from agro-industrial wastes on the yeast oil fatty microscopy images obtained showed similar results which confirm acid profile. Yeast lipid profiles obtained from various carbon 256 S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258

Table 1 The summary of the lipid production parameters for some of the oleaginous yeast reported in the literature.

Organism Carbon source Type of culture Biomass Lipid Lipid Lipid Reference production (g/l) output (g/l) content (%) productivity g/ l/day Candida viswanathii Y-E4 Glucose Batch-7L stirred tank 13.62 3.45 25.33 0.69 Ayadi et al. (2016) Xylose bioreactor 12.19 3.24 26.57 0.64 Glycerol 9.61 2.50 32.72 0.50 Cryptococcus vishniaccii Glucose Batch Shake-flask 13.59 5.5 40.44 0.91 Deeba et al. (2016) Rhodosporidium bajevae Glycerol Batch Shake-flask 9.9 2.4 24.1 0.48 Munch et al. (2015) Rhodosporidium Glycerol Batch Shake-flask 14.1 7.1 50.3 1.42 Munch et al. diobovatum (2015) Batch reactor 13.6 6.9 50.7 0.98 Rhodosporidium Glucose Batch Shake-flask 14.46 6.2 41.92 0.88 Patel et al. (2014) kratochvilovae HIMPA1 Yarowia lipolytica Glucose + acetic acid Batch 0.5 L 30.83 12.36 40.69 4.56 Fontanille et al. MUCL28849 bioreactor (2012) Rhodotorula graminis Glucose Batch Shake-flask 15.75 NM* 45.00 2.40 Galafassi et al. Glycerol 16.25 NM* 53.00 3.12 (2012) Cryptococcus terricola JCM Starch Batch Shake-flask 4.88 3.02 61.96 0.302 Tanimura et al. 2452 Glucose NM* NM* 61.53 NM* (2014) Rhodosporidium toruloids Glycerol + surfactants Batch Shake-flask 10.7 7.1 66.00 1.25 Xu et al. (2016) Cryptococcus curvatus Crude glycerol Batch Shake-flask 29.2 7.7 26.0 NM* Leiva-Candia et al. ATCC20509 (2015) Lipomyces starkeyi DSM Crude glycerol Batch Shake-flask 16.6 2.6 16.0 NM* Leiva-Candia et al. 70296 (2015) Rhodosporidium toruloides Crude glycerol Batch Shake-flask 28.5 7.6 27 NM* Leiva-Candia et al. DSM 4444 (2015) Candida freyschussii ATCC Glycerol Batch Bioreactor 13.9 4.6 33 3.6 Raimondi et al. 18737 (2014) Trichosporonoides Glycerol Batch-5 L Bioreactor 11.3 5.01 44.30 NM* Kitcha and spathulata Cheirsilp (2013) Candida ortholopsis Carboxymethylcellulose Batch Shake-flask NM* NM* 63.75 NM* Kanti and Sudiana (CMC) (2015) Cystobasidium Glucose Batch Shake-flask 12.34 4.91 39.44 0.70 Present study oligophagum JRC1 Carbxymethylcellulose 6.25 2.28 36.46 0.32 Glycerol 6.48 2.72 42.04 0.38 Starch 4.72 1.96 41.54 0.28 Xylose 11.09 4.02 36.24 0.57

* Not mentioned.

of culture conditions and/or genetic modification is needed to obtain the desirable fatty acid profile which in turn determines the biodiesel quality. In a lipid production medium containing CMC as a carbon source, the organism exhibited a simultaneous lipid accumulation, cellulase production, and lipase production. The maximum specific activities for endoglucanase, exoglucanase, and b-glucosidase were 1.39 ± 0.0132, 0.60 ± 0.0436, and 0.44 ± 0.0447 IU/mg respectively after 72 h. The specific activity of the extracellular lipase was 1.48 IU/mg after 84 h.

3.9. FTIR and TLC analysis of the lipid extract

Transmission spectra for the extracted lipids from the yeast culture were obtained and showed similarity with triolein, used as a standard (Supporting data Fig. 1.3). The peak assignment to the respective functional group was done according to the previous reports (Patel et al., 2014). The absence of the peaks in the lipid extract from 4000 cm1 to 3010 cm1 indicated the absence of Fig. 5. Effect of carbon sources on lipid accumulation: proﬁle of biomass production (g/l), lipid output (g/l), and lipid content (%) of C. oligophagum JRC1 in lipid the free hydroxyl group (–OH) and an amine group. The three char- 1 1 production medium containing various carbon sources. acteristic absorbance peaks at 3007 cm , 2925 cm , and 2854 cm1 show the presence of stretching vibration of –CH, –

CH2, and –CH3 respectively, while three peaks observed at 1463– sources were found to be similar to palm oil or rapeseed oil (com- 1377 cm1, 1370–1098 cm1 and 722 cm1 were bending vibra- monly used substrates for ﬁrst generation biodiesel). It was also tions for –CH3, –CH2 and –CH respectively. One peak observed at found that majority of the substrates leads to higher degree satu- 1745 cm1 show the presence of carbonyl (AC@O) group in the ration in lipids which leads to higher oxidation stability and better crude lipid extract. The TLC of the lipid extract was run against cetane number (Leiva-Candia et al., 2014). Therefore, manipulation the triolein standard which showed Rf values similar to it. It was S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258 257

Table 2 Summary of the various cell growth and lipid production parameters obtained for C. oligophagum JRC1 on different carbon sources.

Type of Biomass Biomass yield (YX) Biomass conversion Lipid content (CL)(% Lipid yield/ Co-efﬁcient (EL) Lipid output Lipid

carbon production (cell g/g of carbon (cell g/100 g of carbon w/w) (g lipid/100 g (g lipid/100 g of carbon (YL) (g lipid/ productivity (g

source (PX)(cell g/l) source) source) yeast) source) l) lipid/ l/day) Glucose 12.45 ± 0.97 0.31 ± 0.02 31.3 ± 2.43 39.44 ± 1.19 12.29 ± 1.31 4.91 ± 0.52 0.70 ± 0.07 CMC 06.25 ± 0.78 0.312 ± 0.02 31.2 ± 3.93 36.46 ± 1.49 11.41 ± 1.68 2.28 ± 0.33 0.32 ± 0.04 Starch 04.72 ± 0.55 0.118 ± 0.02 11.8 ± 1.39 41.54 ± 0.34 4.90 ± 0.57 1.96 ± 0.22 0.28 ± 0.03 Xylose 11.09 ± 0.75 0.27 ± 0.01 27.7 ± 1.89 36.24 ± 1.09 10.05 ± 0.80 4.02 ± 0.32 0.57 ± 0.04 Glycerol 06.48 ± 0.57 0.16 ± 0.01 16.2 ± 1.44 42.04 ± 1.71 06.81 ± 0.77 2.72 ± 0.30 0.38 ± 0.04 Fructose 11.50 ± 0.79 0.28 ± 0.01 28.7 ± 1.97 24.29 ± 0.90 06.98 ± 0.51 2.79 ± 0.20 0.39 ± 0.02 Sucrose 08.98 ± 0.03 0.22 ± 0.01 22.4 ± 0.76 21.72 ± 1.77 04.87 ± 0.44 1.95 ± 0.17 0.27 ± 0.02 Maltose 02.26 ± 0.45 0.05 ± 0.01 05.6 ± 1.14 26.31 ± 1.82 01.49 ± 0.36 0.59 ± 0.14 0.08 ± 0.02 Lactose 09.74 ± 0.83 0.24 ± 0.02 24.3 ± 2.08 21.91 ± 0.63 5.33 ± 0.34 2.13 ± 0.13 0.30 ± 0.01

Note: All carbon sources were added at 40 g/l, except CMC at 20 g/l. All the experiments were carried out at pH 6.0, and 28 °C (cultivation time 168 h). noticeable that the lipid extracts in the present study showed only strategies, to test lipid accumulation in real industrial wastes, to one band attributed to TAG (Supporting data Fig. 1.4) indicating test the simultaneous utilization of C6 and C5 sugars and to check that the lipid bodies of the isolate are essentially in the form of resistance to phenolic and nonphenolic inhibitors. In vivo synthesis TAG. The composition on TAG, Diacylglycerol (DAG) and Monoacyl- of FAEE can be attempted by over-expressing ethanol pathway and glycerol (MAG) are species speciﬁc (Sitepu et al., 2014). other genetic alterations as previously reported for Escherichia coli (Steen et al., 2010). 3.10. Determination of FAME composition by GC–MS analysis

The GC–MS analysis of the Fatty Acid Methyl Ester (FAME) pre- Acknowledgements pared from the lipid extracts of C. oligophagum JRC1 cultivated in Author MC thanks IIT Jodhpur for providing various research the glucose medium was performed. The palmitic acid (C16:0) content was 26.02 ± 0.60% higher than jatropha (14.66%) and soyabean facilities. Author SV thanks the Ministry of Human Resource Devel- opment (MHRD) India, for his junior research fellowship (JRF). oil (11%) (Deeba et al., 2016). While the palmitoleic acid (C16:1), Authors thank Mr. Jaymin Jadav (Research Associate at Department was found to be 0.43 ± 0.02%. The stearic acid (C18:0) content was found to be 4.15 ± 0.11% which was also matching palm oil of Biotechnology, Junagadh Agricultural University, Junagadh), for GC–MS analysis. (4–6.3%) and jatropha oil (6.86%), while the oleic acid (C18:1) content was found to be 42.79 ± 0.51%, which was higher than jatropha (39.08%), and other vegetable oils such as palm oil (37– Appendix A 53%) and soyabean oil (23.4%) indicating a better lipid proﬁle than other commonly used feedstock. Further, the linoleic acid (C18:2) content was found to be 24.22 ± 1.30% which was lower as compared to jatropha (32.48%) and other similar type of oils (Deeba g cell dry mass et al., 2016). A small portion of linolenic acid (C18:3) was observed Biomass production ðP Þ¼ ðg=lÞðA1Þ X litre of culture at 0.83 ± 0.05%. The myristic acid (C14:0), pentadecanoic acid (C15:0) and C17:0, were present in an amount of 0.72 ± 0.007%, 0.19 ± 0.014%, and 0.24 ± 0.02% respectively. The total saturated g lipid mass Lipid output ðY Þ¼ ðg=lÞðA2Þ fatty acid (SFA) (31.33 ± 0.70%) was high as compared to other L litre of culture types of oils (jatropha (21.52%) > soyabean oil (15%) > sunﬂower oil (4.5%) > rapeseed oil (6.6%) but lower than the palm oil (44.41%). Certain important properties of biodiesel such as cetane YL Lipid content ðCLÞ¼ 100 ð%ÞðA3Þ number, oxidative stability, and kinematic viscosity etc., depend PX on chain length and degree of saturation (Sitepu et al., 2014). The MUFA (Mono-Unsaturated Fatty Acid) and PUFA (Poly- ð Þ¼ Px ð = ÞðÞ unsaturated fatty acid) were present in an amount of Biomass yield YX w w A4 CCS 43.22 ± 0.48% and 25.05 ± 1.24% respectively. According to the reports, the lower degree of saturation is better for melting point v ¼ ð = ÞðÞ as well as kinematic viscosity (Sitepu et al., 2014). The overall Biomass con ersion Y X 100 Cellg 100 g of c source A5 FAME data shows that the extracted lipid from the yeast C. oligophagum JRC1 can be used for a production of biodiesel with desirable YL Lipid coefficient=Yield ðELÞ¼ fuel properties. The results show that it can be a better alternative Ccs to the vegetable and jatropha oils, thereby making it a suitable can- 100 ðg lipid=100 g of c sourceÞ didate for biodiesel production. ðA6Þ

4. Conclusions Y g lipid Lipid productivity ¼ L Oleaginous yeast capable of cellulase and lipase production was Days required to achieve maximum lipid litre=day successfully isolated. Simultaneous expression of these traits indi- ðA7Þ cated that in a single step both the feedstock and catalyst can be obtained from renewable resources like cellulose. Further studies where Ccs = Carbon source concentration in the culture medium (g/ are needed to enhance the lipids through optimal cultivation l) (Sitepu et al., 2014). 258 S. Vyas, M. Chhabra / Bioresource Technology 223 (2017) 250–258

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