Bioresource Technology 188 (2015) 214–218
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil Jyoti Singh, Manoj Kumar Singh, Madan Kumar, Indu Shekhar Thakur ⇑ School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India
h i g h l i g h t s Novel lipase from Schizophyllum commune ISTL04 was immobilized onto Celite. Immobilized lipase was studied for transesterification of cyanobacterial oil. Crucial reaction conditions were optimized using single factor method. Immobilized lipase produced 94% FAMEs yield. Immobilized lipase produced >90% relative FAMEs yield after 4 repeated cycles.
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Article history: Received 12 December 2014 Received in revised form 20 January 2015 Accepted 22 January 2015 Available online 31 January 2015 Keywords: Schizophyllum commune Immobilized lipase Biodiesel FAMEs Celite
a b s t r a c t Novel lipase from model mushroom Schizophyllum commune strain ISTL04 produced by solid state fermentation of Leucaena leucocephala seeds, was immobilized onto Celite for enzymatic FAMEs production from cyanobacterial endolith Leptolyngbya ISTCY101. The isolate showed vigorous growth and produced remarkable lipase activity of 146.5 U g 1 dry solid substrate, without any external lipase inducer. Singlefactor experiments were carried out to study the effects of various reaction parameters on the FAMEs yield. The best conditions for enzymatic transesterification as revealed by the results were: 1:3 oil to methanol molar ratio, added at 3 h intervals, 12% water content, 1581.5 U g 1 immobilized lipase, temperature 45 °C, and time 24 h. Under these conditions, the maximum FAMEs yield reached 94%. The immobilized lipase was able to produce >90% of the relative FAMEs yield after four repeated transesterification cycles. This immobilized lipase exhibited potential for application in biodiesel industry. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In the past few years, microalgae have been highlighted as promising feedstock for biofuel generation as they surpass other energy crops in terms of photosynthetic efficiency, areal biomass productivity, growth rate and low nutritional requirements (Mata et al., 2010). Despite of the numerous advantageous facts about microalgae, the process of converting microalgal biomass into biodiesel is faced by multitude economical and practical challenges arising at many steps. One such capital and energy intensive step is transesterification of the extracted microalgal oil accounting for 30–40% of the total biodiesel processing cost. Transesterification is carried out in presence of a catalyst, conventionally chemical catalysts including bases, acids and biological catalysts; lipases. The chemical catalysts have several demerits such as difficult ⇑ Corresponding author. Tel.: +91 11 26704321 (O), +91 11 26191370 (R); fax: +91 11 26717586. E-mail addresses: [email protected], [email protected] (I.S. Thakur). http://dx.doi.org/10.1016/j.biortech.2015.01.086 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
catalyst and products separation, undesirable product formation, energy intensiveness and potential environmental threats (Tan et al., 2010). On the other hand, lipase catalysed biodiesel production seems promising as it allows the synthesis of specific alkyl esters, easy recovery of glycerol, and the transesterification of triglycerides with high free fatty acid content (Lee et al., 2011). However, the cost and stability of lipases hinder its potential industrial application. Immobilizing lipases on a suitable substrate is postulated to increase the enzyme stability and reduce the overall production cost. Not only that, lipase immobilization facilitates the product separation thus enabling easy recovery of biocatalyst for further use as well as easy purification of biodiesel. Anchoring the enzyme onto an appropriate support is one of the most crucial steps of the ladder to the successful application of the biocatalyst. Adsorption matrices such as Celite being cheap, chemically inert with unique porous structure have been widely used as a support for enzyme immobilization (Hanefeld et al., 2009). With the surge in finding alternative fuels, it has become indispensable to develop
J. Singh et al. / Bioresource Technology 188 (2015) 214–218
biocatalysts for transesterification with high activity, stability and usability for feasible applications in biodiesel production. Rhizopus arrhizus, Rhizopus japonicus, Rhizopus niveus, Mucor miehei, Candida rugosa, Aspergillus niger and Aspergillus terreus are predominant among the commercially important lipase producing fungi (Shu et al., 2006). Recently we have reported the purification and characterization a novel thermo-alkalotolerant lipase for the first time from Basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds (Singh et al., 2014a). Whole genome of S. commune has been recently sequenced and is being studied as a model system to explore the genetics and proteomics of these potential mushrooms/higher fungi, which have not got attention heretofore for industrial enzymes other than ligno-cellulolytic enzymes (Ohm et al., 2010). The S. commune ISTL04 (SCI) lipase exhibited a lipolytic activity of 146.5 U g 1 dry solid substrate, and showed remarkable thermo-alkalostability. Also, it was appreciably stable in the presence of non-polar and polar organic solvents (50% v/v) including methanol (Singh et al., 2014a). Moreover, L. leucocephala seeds proved to be a cheap, readily available and highly suitable substrate inducing remarkable enzyme production in absence of any added supplement and inducer i.e. the seed constituents only in presence water were sufficient to provide vigor to fungal growth and lipase production attenuating the cost factor to a great extent (Singh et al., 2014a). In this study, we are reporting the continual part of the former report describing the immobilization of SCI lipase onto Celite and its transesterification potential. Cyanobacterial oil was extracted from marble endolith Leptolyngbya sp. ISTCY101 (Singh et al., 2014b) and was then subjected to enzymatic transesterification using immobilized SCI lipase. To the best of our knowledge, this is the first report of lipase from S. commune able to produce FAMEs. This study would widen the prospects of exploiting these organisms to their full untapped potential. 2. Methods 2.1. Materials All chemicals and reagents including Celite 545, were procured from Sigma–Aldrich (St. Louis, MO, USA). All solvents (HPLC grade) were purchased from Merck (Darmstadt, Germany). 2.2. Preparation of Celite for immobilization Celite 545 was prepared for immobilization by pre-treating it with 0.1 M HCl for 1 h, then washing with distilled water to neutrality. It was then suspended in 50 mM sodium phosphate buffer (pH 8) stirred at room temperature for 1 h. The fine particles in the suspension were removed by decantation and the process was repeated thrice. Celite was then filtered and dried under vacuum. 2.3. Lipase production and immobilization The solid state fermentative production of extracellular lipase from S. commune ISTL04 was done using L. Leucocephala seeds (Singh et al., 2014a). The dried seeds were crushed in a grinder. 100 g of well milled L. leucocephala seeds was taken into 2 L Erlenmeyer flask, moistened with 400 ml of distilled water (1:4 w/v) and inoculated with 30 ml of the homogenous inoculums of S. commune ISTL04. The inoculated flasks were incubated at 30 °C for 5 days at humidity (65%). For extraction of lipase the solid fermented medium was mixed with sodium phosphate buffer (50 mM, pH 8.0) in a ratio of 1:10 w/v. The mixture was shaken at 180 rpm, for 1 h at 30 °C. The suspension was then centrifuged at 12,000g for 15 min
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at 4 °C and the supernatant was used as crude lipase extract (Singh et al., 2014a). Lipase activity and protein concentration of the extract was determined by p-nitrophenyl palmitate (p-NPP) spectrophotometric method (Singh et al., 2006) and Bradford method, respectively (Bradford, 1976). Enzyme was immobilized by adding 1 L crude lipase extract to 10 g of Celite with continuous shaking at 200 rpm for overnight at room temperature so that the lipase was fully adsorbed on Celite. The solution was decanted and the immobilized lipase was washed with 50 mM sodium phosphate buffer several times until complete removal of unbound lipase. Lipase loading efficiency (LLE) was calculated by determining the protein concentration before and after immobilization on Celite (Tran et al., 2012). The immobilized lipase was lyophilized and the lyophilized lipase powder was used to determine the enzyme activity and production of FAMEs from cyanobacterial lipid. 2.4. Cyanobacterial culture and oil extraction A novel marble endolithic cyanobacterial strain Leptolyngbya sp. ISTCY101 (Gene Bank accession number-KC538900) previously isolated from Jhiri marble mining site, Alwar, India which has been recently reported for prospective CO2 recycling and biodiesel production was used in this study (Singh et al., 2014b). The cyanobacterial inoculum was grown in liquid BG-11 medium amended with 50 mM NaHCO3 in a semicontinuous mesh culture system (Singh et al., 2014b). Cultures were maintained under continuous fluorescent light (50 lE s 1 m 2), provided by eight 61-cm 18 W fluorescent tubes placed 20 cm from the cell culture, at 30 °C. Lipids were extracted from cell pellets using a modified Bligh–Dyer extraction (Bligh and Dyer, 1959), followed by transesterification using free and immobilized lipase. 2.5. Transesterification using immobilized lipase Immobilized SCI lipase was used as catalyst for enzymatic transesterification of cyanobacterial oil. The cyanobacterial oil was added to water (2–20%) and immobilized lipase (316.3– 1897.8 U g 1 oil) and was put in screw-capped glass vials (25 mL). Methanol (1.04 g for1:3 M ratio of oil to methanol) was then added to the reaction mixture shaken at 200 rpm, incubated at different temperatures (25–55 °C). Effects of lipase loading, water content, methanol to oil molar ratio, reaction temperatures, reaction time and methods of methanol addition on FAMEs production were studied by single-factor experiment design (keeping other variables constant other than the one variable being studied). The resultant FAMEs were analysed by GC–MS. The detailed method has been described previously (Singh et al., 2014b), hence not described here. Experiments were also carried out to assess the reusability of the immobilized lipase, in which the reaction was carried out for 30 h and the solution was decanted to recover the immobilized lipase. After removal of the products, fresh cyanobacterial oil and methanol were added to the recycled biocatalyst. The reaction was carried out repeatedly ten times. For comparing the transesterification activity of immobilized lipase and free lipase, an equivalent amount of free lipase (1581.5 U g 1 oil) was used to carry out the transesterification reaction under optimized conditions. All observations were taken in triplicates. 3. Results and discussion 3.1. Lipase production and immobilization In this study, the lipase was produced by the fungus using cheaply and abundantly available mature L. leucocephala seeds, in presence of distilled water only. The seeds were able to support
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vigorous growth and induce appreciable lipase production (146.5 U g 1 of dry solid substrate) by the isolate even in the absence of any external inducer supplement (Singh et al., 2014a). The strain S. commune ISTL04 produced a thermo-alkalostable lipase with optimal activity at pH 11.0 and a temperature of 60 °C (Singh et al., 2014a). The activity of the free lipase was determined to be 14.65 U mL 1. The lipase loading efficiency was estimated as 87% and enzyme activity of 1265.2 U g 1of Celite. 3.2. Production of cyanobacterial biomass and fatty acid profile When grown in semicontinuous mesh culture system as described in Section 2.4, the strain Leptolyngbya sp. ISTCY101 grew well forming thick biofilm covering the mesh surface homogenously. The isolate attained maximum biomass productivity of 2.01 g m 2 d 1 in BG-11 (50 mM NaHCO3). The total lipid content was estimated to be 20%, consisting predominantly of C16:0, C16:1, C18:0, C18:1 fatty acids (>60%). The fatty acid composition of Leptolyngbya sp. ISTCY101 (C16:0 17%, C16:1 13%, C18:0 23%, C18:1 20%, C18:2 8.9%, C18:3 7%) is comparable to the most suitable vegetable oils used in biodiesel industry namely rapeseed oil (C16:0 4%, C18:0 2%, C18:1 61%, C18:2 22%, C18:3 10%), palm oil (C16:0 45%, C18:0 4%, C18:1 39%, C18:2 11%) and soybean oil (C16:0 6%, C18:0 5%, C18:1 29%, C18:2 58%, C18:3 1%) (Moser, 2009), thus making it a potential feedstock for biodiesel production (Singh et al., 2014b). 3.3. FAMEs production from extracted cyanobacterial oil via enzymatic transesterification; effect of various reaction parameters The effects of various reaction parameters are summarized in Table 1. The effect of different water contents from 2% to 20% (v/ w, cyanobacterial oil) on FAMEs production was examined while keeping other variables constant at: methanol added in a ratio of (1:6 oil: methanol molar ratio) at 6 h intervals and the total reaction time 30 h, temperature 35 °C and lipase loading 1265.2 U g 1 of oil. The FAMEs yield increased from 2% to 12% with maximum 73% at 12% water content, after which there was a slight decrease in the yield with 14%. However, as the water content increased from 14% to 20%, the FAMEs yield declined remarkably. This decline can be attributed to the fact that excess water results in lipase aggregation and also it increases the flexibility of lipase enzyme thereby promoting side reactions like hydrolysis. The best water content vary with different type of lipase, support used for immobilization and type of oil (Antczak et al., 2009). It is well reviewed that lipase acts at the interfaces of organic and aqueous phases. Insufficient water content can lead to enzyme inactivation.
On the other hand, excessive water can decrease the resistance of mass transfer arising from glycerol formation, which in turn may decrease the lipase activity. Most of the studies have reported the optimal water content to vary between 10% and 20% of the oil weight (Tan et al., 2010). High concentrations of methanol generally have detrimental effect of lipase activity (Bajaj et al., 2010). Experiments were conducted to assess the influence of oil to methanol molar ratios, taking four different oil to methanol molar ratios into account i.e. 1:3, 1:6, 1:9 and 1:12. Other reaction parameters were kept constant at; methanol added at 6 h intervals, total reaction time 30 h, temperature 35 °C and lipase loading 1265.2 U g 1 of oil, water content 12% (v/w). Highest FAMEs yield (76%) was obtained with oil to methanol molar ratio of 1:3. Nevertheless, ratio of 1:6 also yielded more than 71% FAMEs yield. However, when the ratio was increased from 1:6 to 1:9, there was a sharp decline in the FAMEs yield implying the negative effect of excess methanol on enzyme activity. Shimada et al. also reported a similar trend with Candida antarctica lipase on addition of methanol in a ratio greater than 1:3 (Shimada et al., 2002). Tran et al. studied the transesterification potential of immobilized Burkholderia sp. C20 lipase on microalgal oil and reported an increase in biodiesel yield with the increase of the oil: methanol molar ratio from 1:3 to 1:5, followed by a reverse trend on further increase in methanol concentration (Tran et al., 2012). In our earlier report also we found that extracellular SCI lipase retained 45% of its activity in presence of methanol (50% v/v) (Singh et al., 2014a). In this study it was found that best oil: methanol molar ratio for transesterification reaction was 1:3. Experiments were also conducted to assess the influence of ways of methanol addition; I: methanol was added in one go at the onset (1.04 g of MeOH added to 10 g of oil to achieve a oil to methanol molar ratio of 1:3), II: methanol (in equal parts to reach a final oil to methanol molar ratio of 1:3 at the end of the reaction time) was added in 3 h intervals, III: methanol was added in 6 h intervals, IV: methanol was added in 9 h intervals. Except for the parameter being studied, other parameters were kept constant at; oil: methanol 1:3, total reaction time 30 h, temperature 35 °C, lipase loading 1265.2 U g 1 of oil and water content 12% (v/w). The addition of methanol at 3 h intervals gave the maximum FAMEs yield. The results suggest that addition of small quantities of methanol at short intervals helps in attaining the maximum transesterification activities. Approximately 80% FAMEs yield was acquired after 30 h of the reaction. Method III i.e. addition of methanol in 6 h intervals also resulted in a fair deal of FAMEs yield of approximately 70%. However, adding methanol in 9 h intervals showed significant decrease in FAMEs yield. The results imply that addition of methanol at 3 h and 6 h intervals minimised the toxic effects of methanol upon the lipase. Finding
Table 1 Effect of various reaction parameters on FAMEs yield (%) by immobilized SCI lipase (experiments were carried out in triplicates and values are expressed as mean of three values). Effect of water content
Effect of methanol concentration
Effect of ways of methanol addition
Effect of catalyst loading
Effect of reaction temperature
Effect of reaction time
Lipase reusability
Water content (% v/w)
FAMEs yield (%)
Methanol:oil molar ratio
FAMEs yield (%)
Way
FAMEs yield (%)
Immobilized lipase (U g 1 oil)
FAMEs yield (%)
Temperature (°C)
FAMEs yield (%)
Time (h)
FAMEs yield (%)
Cycle
Relative FAMEs yield (%)
2 4 6 8 10 12 14 16 18 20
44.08 48.28 57.40 66.71 70.92 73.85 68.47 60.53 55.80 43.20
3 6 9 12
76.41 71.45 54.70 46.77
I II III IV
53.11 80.30 70.33 53.53
1897.8 1581.5 1265.2 632.6 316.3
91.03 89.73 82.36 64.11 28.31
25 30 35 40 45 50 55
54.51 70.44 90.27 93.97 94.41 81.88 63.56
6h 12 h 18 h 24 h 30 h 36 h 42 h
54.73 72.5 88.1 94.43 94.58 94.29 94.07
I II III IV V VI VII VIII IX X
99.42 98.66 96.66 90.83 79 68 62.73 50.14 40.5 31.23
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the appropriate amount of enzyme yielding maximum product formation is often important to minimise the production costs. The effects of different lipase loading concentrations per unit weight of oil, i.e. 1897.8 U g 1, 1581.5 U g 1, 1265.2 U g 1, 632.6 U g 1 and 316.3 U g 1 were assessed keeping other reaction conditions as: methanol added in a ratio of (1:3 oil:methanol molar ratio) at 3 h intervals, reaction time 30 h, temperature 35 °C, and water content 12% (v/w). The FAMEs yield showed an increasing trend with increasing enzyme concentration. The maximum FAMEs yield (91%) was obtained with 1897.8 U g 1 lipase loading. Nevertheless, catalyst concentration of 1581.5 U g 1 also yielded comparable FAMEs yield (>89%). Higher concentrations of the biocatalyst may allow the interaction of substrate molecules and enzyme active sites to a greater extent, thereby, increasing the bioconversion. Previous reports have also confirmed the enhancing effect of enzyme amount on biodiesel yield (Bajaj et al., 2010; Tran et al., 2012). Taking into account, together the cost and bioconversion efficiency, the optimal catalyst loading concentration in our study is 1581.5 U g 1 of oil. The effect of temperature from 25 to 60 °C on the enzyme catalysed transesterification of cyanobacterial oil by immobilized lipase was studied. The other reaction conditions were set as: methanol added in a ratio of (1:3 oil: methanol molar ratio) at 3 h intervals, reaction time 30 h, lipase loading 1581.5 U g 1 of oil and water content 12% (v/w). The maximum FAMEs yield (94%) was obtained at a reaction temperature of 45 °C. With a gradual increase in the reaction temperature from 25–45 °C, the FAMEs yield also increased. Nevertheless, remarkable transesterification activity yielding FAMEs (>80%) was retained even at 50 °C. It has been reported that rise in temperature usually has a positive effect on immobilised lipase activity (Bajaj et al., 2010). However, the enzyme can also undergo thermal denaturation at very high temperatures (Bajaj et al., 2010) and high temperature may also favour hydrolysis reaction (Bajaj et al., 2010). The SCI lipase has been found to have a remarkable thermal stability, retaining more than 50% of its lipolytic activity at 70 °C, as reported in the former publication (Singh et al., 2014a). The oil to alcohol molar ratio, thermostability of the enzyme, operational stability and transesterification rate co-act together and govern the optimum temperature (Antczak et al., 2009). In this study, the optimal temperature for transesterification reaction was found to be 45 °C. The effect of reaction time on the FAMEs yield was assessed in range 6–48 h. Other reaction conditions were set as: methanol added in a ratio of (1:3 oil: methanol molar ratio) at 3 h intervals, temperature 35 °C, lipase loading 1581.5 U g 1 of oil and water content 12% (v/w). Most of the previous studies on lipase catalyzed transesterification and biodiesel production have reported that longer reaction time results in highest biodiesel yields (Tran et al., 2012). The FAMEs yield increased as the reaction time increased from 6 h to 30 h. The maximum yield (94%) was observed with a reaction time of 24 h, after which the FAMEs yield levelled off. Thus, the optimal reaction time for immobilised SCI lipase was considered as 24 h, as the reaction is known to proceed with an increase in water content, and this increasing water content can lead to hydrolysis of biodiesel (Bajaj et al., 2010). Moreover, the lesser time a reaction takes the more economical and industrially sustainable it becomes. The biodiesel productions catalyzed by immobilized lipase and free lipase were conducted under optimized conditions as derived from the aforementioned experiments. The FAMEs yield reached 94% for immobilized lipase compared to 70% for free lipase, implying that immobilized lipase could have been more efficient owing to increased stability and better substrate interaction. However, the possibilities of optimum conditions being different for free lipase cannot be ruled out. Moreover, free lipase dissolved in the reaction medium is often difficult to retain, separate and reuse, rendering immobilization anyways a better option for the sustainability of the process.
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3.4. Reusability of SCI immobilised lipase The reusability and stability of immobilized enzymes is also a prime cause of concern that can attenuate the cost of production. To assess the reusability of the immobilized lipase, each reaction was terminated after 30 h and the solution was decanted to recover the immobilized lipase. After removal of the reacted mixture, fresh cyanobacterial oil and methanol were added to the recycled biocatalyst. The reaction was carried out in the same manner repeatedly for ten times. The immobilized lipase was able to maintain more than 90% of relative FAMEs yield for 4 repeated cycles, implying that it retained 90% of its relative transesterification activity till the 4th cycle. After the 4th cycle the relative yield declined continuously to 50% at the 7th cycle (Table 1). The enzyme is immobilized on Celite by the means of physical adsorption only. A significant demerit of immobilizing by adsorption is that when aqueous media is used, the enzyme tends to leach readily from the carrier. The loss of activity can be attributed to enzyme loss during recovery steps and leakage of the enzyme from the support on continuous use (Hanefeld et al., 2009; Zhao et al., 2010). Exploring different supports for immobilization and more optimization experiments would surely improve the bioconversion efficiency of the SCI lipase. Nevertheless, the results imply that the stability of the immobilized SCI lipase is promising. 4. Conclusion The study is the first ever report of a novel lipase from model mushroom S. commune being immobilized for FAMEs production from microalgal oil. The best conditions for transesterification reaction were: methanol addition at 3 h intervals in a ratio of 1:3 (oil:methanol), 12% water, lipase loading 1581.5 U g 1 of oil and temperature 45 °C attaining a FAMEs yield of around 94% after 24 h. The SCI lipase retained 90% of its transesterification activity after 4 repeated cycles of use. The immobilized SCI lipase exhibited potential for industrial application. Acknowledgements We thank Mr. Ajai Kumar of Advanced Instrumentation Research Facility (AIRF) Jawaharlal Nehru University, New Delhi for GC-MS analysis and Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for providing Research Fellowship. References Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis – key factors affecting efficiency of the process. Renewable Energy 34, 1185–1194. Bajaj, A., Lohan, P., Jha, P.N., Mehrotra, R., 2010. Biodiesel production through lipase catalyzed transesterification: an overview. J. Mol. Catal. B Enzyme 62 (1), 9–14. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification, Canadian. J. Biochem. Physiol. 37, 911–917. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 242–254. Hanefeld, U., Gardossi, L., Magner, E., 2009. Understanding enzyme immobilisation. Chem. Soc. Rev. 38, 453–468. Lee, J.H., Kim, S.B., Kang, S.W., Song, Y.S., Park, C., Han, S.O., Kim, S.W., 2011. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process. Bioresour. Technol. 102, 2105– 2108. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renewable Sustainable Energy Rev. 14, 217– 232. Moser, B.R., 2009. Biodiesel production, properties, and feedstocks. In Vitro Cell. Dev. Biol. Plant 45, 229–266. Ohm, R.A. et al., 2010. Genome sequence of the model mushroom Schizophyllum commune. Nat. Biotechnol. 28, 957–965.
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Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B Enzym. 17, 133–142. Shu, C.-H., Xu, C.-J., Lin, G.-C., 2006. Purification and partial characterization of a lipase from Antrodia cinnamomea. Process Biochem. 41, 734–738. Singh, J., Tripathi, R., Thakur, I.S., 2014a. Characterization of endolithic cyanobacterial strain, Leptolyngbya sp. ISTCY101, for prospective recycling of CO2 and biodiesel production. Bioresour. Technol. 166, 345–352. Singh, M.K., Singh, J., Kumar, M., Thakur, I.S., 2014b. Novel lipase from basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds. J. Mol. Catal. B Enzyme 110, 92–99.
Singh, R., Gupta, N., Goswami, V.K., Gupta, R., 2006. A simple activity staining protocol for lipases and esterases. Appl. Microbiol. Biotechnol. 70, 679–682. Tan, T.W., Lu, J.K., Nie, K.L., Deng, L., Wang, F., 2010. Biodiesel production with immobilized lipase: a review. Biotechnol. Adv. 28, 628–634. Tran, D.T., Yeh, K.L., Chen, C.L., Chang, J.S., 2012. Enzymatic transesterification of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour. Technol. 108, 119–127. Zhao, Q., Hou, Y., Gong, G.H., Yu, M.A., Jiang, L., Liao, F., 2010. Characterization of alcohol dehydrogenase from permeabilized Brewer’s yeast cells immobilized on the derived attapulgite nanofibers. Appl. Biochem. Biotechnol. 160, 2287– 2299.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil Jyoti Singh, Manoj Kumar Singh, Madan Kumar, Indu Shekhar Thakur ⇑ School of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India
h i g h l i g h t s Novel lipase from Schizophyllum commune ISTL04 was immobilized onto Celite. Immobilized lipase was studied for transesterification of cyanobacterial oil. Crucial reaction conditions were optimized using single factor method. Immobilized lipase produced 94% FAMEs yield. Immobilized lipase produced >90% relative FAMEs yield after 4 repeated cycles.
a r t i c l e
i n f o
Article history: Received 12 December 2014 Received in revised form 20 January 2015 Accepted 22 January 2015 Available online 31 January 2015 Keywords: Schizophyllum commune Immobilized lipase Biodiesel FAMEs Celite
a b s t r a c t Novel lipase from model mushroom Schizophyllum commune strain ISTL04 produced by solid state fermentation of Leucaena leucocephala seeds, was immobilized onto Celite for enzymatic FAMEs production from cyanobacterial endolith Leptolyngbya ISTCY101. The isolate showed vigorous growth and produced remarkable lipase activity of 146.5 U g 1 dry solid substrate, without any external lipase inducer. Singlefactor experiments were carried out to study the effects of various reaction parameters on the FAMEs yield. The best conditions for enzymatic transesterification as revealed by the results were: 1:3 oil to methanol molar ratio, added at 3 h intervals, 12% water content, 1581.5 U g 1 immobilized lipase, temperature 45 °C, and time 24 h. Under these conditions, the maximum FAMEs yield reached 94%. The immobilized lipase was able to produce >90% of the relative FAMEs yield after four repeated transesterification cycles. This immobilized lipase exhibited potential for application in biodiesel industry. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In the past few years, microalgae have been highlighted as promising feedstock for biofuel generation as they surpass other energy crops in terms of photosynthetic efficiency, areal biomass productivity, growth rate and low nutritional requirements (Mata et al., 2010). Despite of the numerous advantageous facts about microalgae, the process of converting microalgal biomass into biodiesel is faced by multitude economical and practical challenges arising at many steps. One such capital and energy intensive step is transesterification of the extracted microalgal oil accounting for 30–40% of the total biodiesel processing cost. Transesterification is carried out in presence of a catalyst, conventionally chemical catalysts including bases, acids and biological catalysts; lipases. The chemical catalysts have several demerits such as difficult ⇑ Corresponding author. Tel.: +91 11 26704321 (O), +91 11 26191370 (R); fax: +91 11 26717586. E-mail addresses: [email protected], [email protected] (I.S. Thakur). http://dx.doi.org/10.1016/j.biortech.2015.01.086 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
catalyst and products separation, undesirable product formation, energy intensiveness and potential environmental threats (Tan et al., 2010). On the other hand, lipase catalysed biodiesel production seems promising as it allows the synthesis of specific alkyl esters, easy recovery of glycerol, and the transesterification of triglycerides with high free fatty acid content (Lee et al., 2011). However, the cost and stability of lipases hinder its potential industrial application. Immobilizing lipases on a suitable substrate is postulated to increase the enzyme stability and reduce the overall production cost. Not only that, lipase immobilization facilitates the product separation thus enabling easy recovery of biocatalyst for further use as well as easy purification of biodiesel. Anchoring the enzyme onto an appropriate support is one of the most crucial steps of the ladder to the successful application of the biocatalyst. Adsorption matrices such as Celite being cheap, chemically inert with unique porous structure have been widely used as a support for enzyme immobilization (Hanefeld et al., 2009). With the surge in finding alternative fuels, it has become indispensable to develop
J. Singh et al. / Bioresource Technology 188 (2015) 214–218
biocatalysts for transesterification with high activity, stability and usability for feasible applications in biodiesel production. Rhizopus arrhizus, Rhizopus japonicus, Rhizopus niveus, Mucor miehei, Candida rugosa, Aspergillus niger and Aspergillus terreus are predominant among the commercially important lipase producing fungi (Shu et al., 2006). Recently we have reported the purification and characterization a novel thermo-alkalotolerant lipase for the first time from Basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds (Singh et al., 2014a). Whole genome of S. commune has been recently sequenced and is being studied as a model system to explore the genetics and proteomics of these potential mushrooms/higher fungi, which have not got attention heretofore for industrial enzymes other than ligno-cellulolytic enzymes (Ohm et al., 2010). The S. commune ISTL04 (SCI) lipase exhibited a lipolytic activity of 146.5 U g 1 dry solid substrate, and showed remarkable thermo-alkalostability. Also, it was appreciably stable in the presence of non-polar and polar organic solvents (50% v/v) including methanol (Singh et al., 2014a). Moreover, L. leucocephala seeds proved to be a cheap, readily available and highly suitable substrate inducing remarkable enzyme production in absence of any added supplement and inducer i.e. the seed constituents only in presence water were sufficient to provide vigor to fungal growth and lipase production attenuating the cost factor to a great extent (Singh et al., 2014a). In this study, we are reporting the continual part of the former report describing the immobilization of SCI lipase onto Celite and its transesterification potential. Cyanobacterial oil was extracted from marble endolith Leptolyngbya sp. ISTCY101 (Singh et al., 2014b) and was then subjected to enzymatic transesterification using immobilized SCI lipase. To the best of our knowledge, this is the first report of lipase from S. commune able to produce FAMEs. This study would widen the prospects of exploiting these organisms to their full untapped potential. 2. Methods 2.1. Materials All chemicals and reagents including Celite 545, were procured from Sigma–Aldrich (St. Louis, MO, USA). All solvents (HPLC grade) were purchased from Merck (Darmstadt, Germany). 2.2. Preparation of Celite for immobilization Celite 545 was prepared for immobilization by pre-treating it with 0.1 M HCl for 1 h, then washing with distilled water to neutrality. It was then suspended in 50 mM sodium phosphate buffer (pH 8) stirred at room temperature for 1 h. The fine particles in the suspension were removed by decantation and the process was repeated thrice. Celite was then filtered and dried under vacuum. 2.3. Lipase production and immobilization The solid state fermentative production of extracellular lipase from S. commune ISTL04 was done using L. Leucocephala seeds (Singh et al., 2014a). The dried seeds were crushed in a grinder. 100 g of well milled L. leucocephala seeds was taken into 2 L Erlenmeyer flask, moistened with 400 ml of distilled water (1:4 w/v) and inoculated with 30 ml of the homogenous inoculums of S. commune ISTL04. The inoculated flasks were incubated at 30 °C for 5 days at humidity (65%). For extraction of lipase the solid fermented medium was mixed with sodium phosphate buffer (50 mM, pH 8.0) in a ratio of 1:10 w/v. The mixture was shaken at 180 rpm, for 1 h at 30 °C. The suspension was then centrifuged at 12,000g for 15 min
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at 4 °C and the supernatant was used as crude lipase extract (Singh et al., 2014a). Lipase activity and protein concentration of the extract was determined by p-nitrophenyl palmitate (p-NPP) spectrophotometric method (Singh et al., 2006) and Bradford method, respectively (Bradford, 1976). Enzyme was immobilized by adding 1 L crude lipase extract to 10 g of Celite with continuous shaking at 200 rpm for overnight at room temperature so that the lipase was fully adsorbed on Celite. The solution was decanted and the immobilized lipase was washed with 50 mM sodium phosphate buffer several times until complete removal of unbound lipase. Lipase loading efficiency (LLE) was calculated by determining the protein concentration before and after immobilization on Celite (Tran et al., 2012). The immobilized lipase was lyophilized and the lyophilized lipase powder was used to determine the enzyme activity and production of FAMEs from cyanobacterial lipid. 2.4. Cyanobacterial culture and oil extraction A novel marble endolithic cyanobacterial strain Leptolyngbya sp. ISTCY101 (Gene Bank accession number-KC538900) previously isolated from Jhiri marble mining site, Alwar, India which has been recently reported for prospective CO2 recycling and biodiesel production was used in this study (Singh et al., 2014b). The cyanobacterial inoculum was grown in liquid BG-11 medium amended with 50 mM NaHCO3 in a semicontinuous mesh culture system (Singh et al., 2014b). Cultures were maintained under continuous fluorescent light (50 lE s 1 m 2), provided by eight 61-cm 18 W fluorescent tubes placed 20 cm from the cell culture, at 30 °C. Lipids were extracted from cell pellets using a modified Bligh–Dyer extraction (Bligh and Dyer, 1959), followed by transesterification using free and immobilized lipase. 2.5. Transesterification using immobilized lipase Immobilized SCI lipase was used as catalyst for enzymatic transesterification of cyanobacterial oil. The cyanobacterial oil was added to water (2–20%) and immobilized lipase (316.3– 1897.8 U g 1 oil) and was put in screw-capped glass vials (25 mL). Methanol (1.04 g for1:3 M ratio of oil to methanol) was then added to the reaction mixture shaken at 200 rpm, incubated at different temperatures (25–55 °C). Effects of lipase loading, water content, methanol to oil molar ratio, reaction temperatures, reaction time and methods of methanol addition on FAMEs production were studied by single-factor experiment design (keeping other variables constant other than the one variable being studied). The resultant FAMEs were analysed by GC–MS. The detailed method has been described previously (Singh et al., 2014b), hence not described here. Experiments were also carried out to assess the reusability of the immobilized lipase, in which the reaction was carried out for 30 h and the solution was decanted to recover the immobilized lipase. After removal of the products, fresh cyanobacterial oil and methanol were added to the recycled biocatalyst. The reaction was carried out repeatedly ten times. For comparing the transesterification activity of immobilized lipase and free lipase, an equivalent amount of free lipase (1581.5 U g 1 oil) was used to carry out the transesterification reaction under optimized conditions. All observations were taken in triplicates. 3. Results and discussion 3.1. Lipase production and immobilization In this study, the lipase was produced by the fungus using cheaply and abundantly available mature L. leucocephala seeds, in presence of distilled water only. The seeds were able to support
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vigorous growth and induce appreciable lipase production (146.5 U g 1 of dry solid substrate) by the isolate even in the absence of any external inducer supplement (Singh et al., 2014a). The strain S. commune ISTL04 produced a thermo-alkalostable lipase with optimal activity at pH 11.0 and a temperature of 60 °C (Singh et al., 2014a). The activity of the free lipase was determined to be 14.65 U mL 1. The lipase loading efficiency was estimated as 87% and enzyme activity of 1265.2 U g 1of Celite. 3.2. Production of cyanobacterial biomass and fatty acid profile When grown in semicontinuous mesh culture system as described in Section 2.4, the strain Leptolyngbya sp. ISTCY101 grew well forming thick biofilm covering the mesh surface homogenously. The isolate attained maximum biomass productivity of 2.01 g m 2 d 1 in BG-11 (50 mM NaHCO3). The total lipid content was estimated to be 20%, consisting predominantly of C16:0, C16:1, C18:0, C18:1 fatty acids (>60%). The fatty acid composition of Leptolyngbya sp. ISTCY101 (C16:0 17%, C16:1 13%, C18:0 23%, C18:1 20%, C18:2 8.9%, C18:3 7%) is comparable to the most suitable vegetable oils used in biodiesel industry namely rapeseed oil (C16:0 4%, C18:0 2%, C18:1 61%, C18:2 22%, C18:3 10%), palm oil (C16:0 45%, C18:0 4%, C18:1 39%, C18:2 11%) and soybean oil (C16:0 6%, C18:0 5%, C18:1 29%, C18:2 58%, C18:3 1%) (Moser, 2009), thus making it a potential feedstock for biodiesel production (Singh et al., 2014b). 3.3. FAMEs production from extracted cyanobacterial oil via enzymatic transesterification; effect of various reaction parameters The effects of various reaction parameters are summarized in Table 1. The effect of different water contents from 2% to 20% (v/ w, cyanobacterial oil) on FAMEs production was examined while keeping other variables constant at: methanol added in a ratio of (1:6 oil: methanol molar ratio) at 6 h intervals and the total reaction time 30 h, temperature 35 °C and lipase loading 1265.2 U g 1 of oil. The FAMEs yield increased from 2% to 12% with maximum 73% at 12% water content, after which there was a slight decrease in the yield with 14%. However, as the water content increased from 14% to 20%, the FAMEs yield declined remarkably. This decline can be attributed to the fact that excess water results in lipase aggregation and also it increases the flexibility of lipase enzyme thereby promoting side reactions like hydrolysis. The best water content vary with different type of lipase, support used for immobilization and type of oil (Antczak et al., 2009). It is well reviewed that lipase acts at the interfaces of organic and aqueous phases. Insufficient water content can lead to enzyme inactivation.
On the other hand, excessive water can decrease the resistance of mass transfer arising from glycerol formation, which in turn may decrease the lipase activity. Most of the studies have reported the optimal water content to vary between 10% and 20% of the oil weight (Tan et al., 2010). High concentrations of methanol generally have detrimental effect of lipase activity (Bajaj et al., 2010). Experiments were conducted to assess the influence of oil to methanol molar ratios, taking four different oil to methanol molar ratios into account i.e. 1:3, 1:6, 1:9 and 1:12. Other reaction parameters were kept constant at; methanol added at 6 h intervals, total reaction time 30 h, temperature 35 °C and lipase loading 1265.2 U g 1 of oil, water content 12% (v/w). Highest FAMEs yield (76%) was obtained with oil to methanol molar ratio of 1:3. Nevertheless, ratio of 1:6 also yielded more than 71% FAMEs yield. However, when the ratio was increased from 1:6 to 1:9, there was a sharp decline in the FAMEs yield implying the negative effect of excess methanol on enzyme activity. Shimada et al. also reported a similar trend with Candida antarctica lipase on addition of methanol in a ratio greater than 1:3 (Shimada et al., 2002). Tran et al. studied the transesterification potential of immobilized Burkholderia sp. C20 lipase on microalgal oil and reported an increase in biodiesel yield with the increase of the oil: methanol molar ratio from 1:3 to 1:5, followed by a reverse trend on further increase in methanol concentration (Tran et al., 2012). In our earlier report also we found that extracellular SCI lipase retained 45% of its activity in presence of methanol (50% v/v) (Singh et al., 2014a). In this study it was found that best oil: methanol molar ratio for transesterification reaction was 1:3. Experiments were also conducted to assess the influence of ways of methanol addition; I: methanol was added in one go at the onset (1.04 g of MeOH added to 10 g of oil to achieve a oil to methanol molar ratio of 1:3), II: methanol (in equal parts to reach a final oil to methanol molar ratio of 1:3 at the end of the reaction time) was added in 3 h intervals, III: methanol was added in 6 h intervals, IV: methanol was added in 9 h intervals. Except for the parameter being studied, other parameters were kept constant at; oil: methanol 1:3, total reaction time 30 h, temperature 35 °C, lipase loading 1265.2 U g 1 of oil and water content 12% (v/w). The addition of methanol at 3 h intervals gave the maximum FAMEs yield. The results suggest that addition of small quantities of methanol at short intervals helps in attaining the maximum transesterification activities. Approximately 80% FAMEs yield was acquired after 30 h of the reaction. Method III i.e. addition of methanol in 6 h intervals also resulted in a fair deal of FAMEs yield of approximately 70%. However, adding methanol in 9 h intervals showed significant decrease in FAMEs yield. The results imply that addition of methanol at 3 h and 6 h intervals minimised the toxic effects of methanol upon the lipase. Finding
Table 1 Effect of various reaction parameters on FAMEs yield (%) by immobilized SCI lipase (experiments were carried out in triplicates and values are expressed as mean of three values). Effect of water content
Effect of methanol concentration
Effect of ways of methanol addition
Effect of catalyst loading
Effect of reaction temperature
Effect of reaction time
Lipase reusability
Water content (% v/w)
FAMEs yield (%)
Methanol:oil molar ratio
FAMEs yield (%)
Way
FAMEs yield (%)
Immobilized lipase (U g 1 oil)
FAMEs yield (%)
Temperature (°C)
FAMEs yield (%)
Time (h)
FAMEs yield (%)
Cycle
Relative FAMEs yield (%)
2 4 6 8 10 12 14 16 18 20
44.08 48.28 57.40 66.71 70.92 73.85 68.47 60.53 55.80 43.20
3 6 9 12
76.41 71.45 54.70 46.77
I II III IV
53.11 80.30 70.33 53.53
1897.8 1581.5 1265.2 632.6 316.3
91.03 89.73 82.36 64.11 28.31
25 30 35 40 45 50 55
54.51 70.44 90.27 93.97 94.41 81.88 63.56
6h 12 h 18 h 24 h 30 h 36 h 42 h
54.73 72.5 88.1 94.43 94.58 94.29 94.07
I II III IV V VI VII VIII IX X
99.42 98.66 96.66 90.83 79 68 62.73 50.14 40.5 31.23
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the appropriate amount of enzyme yielding maximum product formation is often important to minimise the production costs. The effects of different lipase loading concentrations per unit weight of oil, i.e. 1897.8 U g 1, 1581.5 U g 1, 1265.2 U g 1, 632.6 U g 1 and 316.3 U g 1 were assessed keeping other reaction conditions as: methanol added in a ratio of (1:3 oil:methanol molar ratio) at 3 h intervals, reaction time 30 h, temperature 35 °C, and water content 12% (v/w). The FAMEs yield showed an increasing trend with increasing enzyme concentration. The maximum FAMEs yield (91%) was obtained with 1897.8 U g 1 lipase loading. Nevertheless, catalyst concentration of 1581.5 U g 1 also yielded comparable FAMEs yield (>89%). Higher concentrations of the biocatalyst may allow the interaction of substrate molecules and enzyme active sites to a greater extent, thereby, increasing the bioconversion. Previous reports have also confirmed the enhancing effect of enzyme amount on biodiesel yield (Bajaj et al., 2010; Tran et al., 2012). Taking into account, together the cost and bioconversion efficiency, the optimal catalyst loading concentration in our study is 1581.5 U g 1 of oil. The effect of temperature from 25 to 60 °C on the enzyme catalysed transesterification of cyanobacterial oil by immobilized lipase was studied. The other reaction conditions were set as: methanol added in a ratio of (1:3 oil: methanol molar ratio) at 3 h intervals, reaction time 30 h, lipase loading 1581.5 U g 1 of oil and water content 12% (v/w). The maximum FAMEs yield (94%) was obtained at a reaction temperature of 45 °C. With a gradual increase in the reaction temperature from 25–45 °C, the FAMEs yield also increased. Nevertheless, remarkable transesterification activity yielding FAMEs (>80%) was retained even at 50 °C. It has been reported that rise in temperature usually has a positive effect on immobilised lipase activity (Bajaj et al., 2010). However, the enzyme can also undergo thermal denaturation at very high temperatures (Bajaj et al., 2010) and high temperature may also favour hydrolysis reaction (Bajaj et al., 2010). The SCI lipase has been found to have a remarkable thermal stability, retaining more than 50% of its lipolytic activity at 70 °C, as reported in the former publication (Singh et al., 2014a). The oil to alcohol molar ratio, thermostability of the enzyme, operational stability and transesterification rate co-act together and govern the optimum temperature (Antczak et al., 2009). In this study, the optimal temperature for transesterification reaction was found to be 45 °C. The effect of reaction time on the FAMEs yield was assessed in range 6–48 h. Other reaction conditions were set as: methanol added in a ratio of (1:3 oil: methanol molar ratio) at 3 h intervals, temperature 35 °C, lipase loading 1581.5 U g 1 of oil and water content 12% (v/w). Most of the previous studies on lipase catalyzed transesterification and biodiesel production have reported that longer reaction time results in highest biodiesel yields (Tran et al., 2012). The FAMEs yield increased as the reaction time increased from 6 h to 30 h. The maximum yield (94%) was observed with a reaction time of 24 h, after which the FAMEs yield levelled off. Thus, the optimal reaction time for immobilised SCI lipase was considered as 24 h, as the reaction is known to proceed with an increase in water content, and this increasing water content can lead to hydrolysis of biodiesel (Bajaj et al., 2010). Moreover, the lesser time a reaction takes the more economical and industrially sustainable it becomes. The biodiesel productions catalyzed by immobilized lipase and free lipase were conducted under optimized conditions as derived from the aforementioned experiments. The FAMEs yield reached 94% for immobilized lipase compared to 70% for free lipase, implying that immobilized lipase could have been more efficient owing to increased stability and better substrate interaction. However, the possibilities of optimum conditions being different for free lipase cannot be ruled out. Moreover, free lipase dissolved in the reaction medium is often difficult to retain, separate and reuse, rendering immobilization anyways a better option for the sustainability of the process.
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3.4. Reusability of SCI immobilised lipase The reusability and stability of immobilized enzymes is also a prime cause of concern that can attenuate the cost of production. To assess the reusability of the immobilized lipase, each reaction was terminated after 30 h and the solution was decanted to recover the immobilized lipase. After removal of the reacted mixture, fresh cyanobacterial oil and methanol were added to the recycled biocatalyst. The reaction was carried out in the same manner repeatedly for ten times. The immobilized lipase was able to maintain more than 90% of relative FAMEs yield for 4 repeated cycles, implying that it retained 90% of its relative transesterification activity till the 4th cycle. After the 4th cycle the relative yield declined continuously to 50% at the 7th cycle (Table 1). The enzyme is immobilized on Celite by the means of physical adsorption only. A significant demerit of immobilizing by adsorption is that when aqueous media is used, the enzyme tends to leach readily from the carrier. The loss of activity can be attributed to enzyme loss during recovery steps and leakage of the enzyme from the support on continuous use (Hanefeld et al., 2009; Zhao et al., 2010). Exploring different supports for immobilization and more optimization experiments would surely improve the bioconversion efficiency of the SCI lipase. Nevertheless, the results imply that the stability of the immobilized SCI lipase is promising. 4. Conclusion The study is the first ever report of a novel lipase from model mushroom S. commune being immobilized for FAMEs production from microalgal oil. The best conditions for transesterification reaction were: methanol addition at 3 h intervals in a ratio of 1:3 (oil:methanol), 12% water, lipase loading 1581.5 U g 1 of oil and temperature 45 °C attaining a FAMEs yield of around 94% after 24 h. The SCI lipase retained 90% of its transesterification activity after 4 repeated cycles of use. The immobilized SCI lipase exhibited potential for industrial application. Acknowledgements We thank Mr. Ajai Kumar of Advanced Instrumentation Research Facility (AIRF) Jawaharlal Nehru University, New Delhi for GC-MS analysis and Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for providing Research Fellowship. References Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis – key factors affecting efficiency of the process. Renewable Energy 34, 1185–1194. Bajaj, A., Lohan, P., Jha, P.N., Mehrotra, R., 2010. Biodiesel production through lipase catalyzed transesterification: an overview. J. Mol. Catal. B Enzyme 62 (1), 9–14. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification, Canadian. J. Biochem. Physiol. 37, 911–917. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 242–254. Hanefeld, U., Gardossi, L., Magner, E., 2009. Understanding enzyme immobilisation. Chem. Soc. Rev. 38, 453–468. Lee, J.H., Kim, S.B., Kang, S.W., Song, Y.S., Park, C., Han, S.O., Kim, S.W., 2011. Biodiesel production by a mixture of Candida rugosa and Rhizopus oryzae lipases using a supercritical carbon dioxide process. Bioresour. Technol. 102, 2105– 2108. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: a review. Renewable Sustainable Energy Rev. 14, 217– 232. Moser, B.R., 2009. Biodiesel production, properties, and feedstocks. In Vitro Cell. Dev. Biol. Plant 45, 229–266. Ohm, R.A. et al., 2010. Genome sequence of the model mushroom Schizophyllum commune. Nat. Biotechnol. 28, 957–965.
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Shimada, Y., Watanabe, Y., Sugihara, A., Tominaga, Y., 2002. Enzymatic alcoholysis for biodiesel fuel production and application of the reaction to oil processing. J. Mol. Catal. B Enzym. 17, 133–142. Shu, C.-H., Xu, C.-J., Lin, G.-C., 2006. Purification and partial characterization of a lipase from Antrodia cinnamomea. Process Biochem. 41, 734–738. Singh, J., Tripathi, R., Thakur, I.S., 2014a. Characterization of endolithic cyanobacterial strain, Leptolyngbya sp. ISTCY101, for prospective recycling of CO2 and biodiesel production. Bioresour. Technol. 166, 345–352. Singh, M.K., Singh, J., Kumar, M., Thakur, I.S., 2014b. Novel lipase from basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds. J. Mol. Catal. B Enzyme 110, 92–99.
Singh, R., Gupta, N., Goswami, V.K., Gupta, R., 2006. A simple activity staining protocol for lipases and esterases. Appl. Microbiol. Biotechnol. 70, 679–682. Tan, T.W., Lu, J.K., Nie, K.L., Deng, L., Wang, F., 2010. Biodiesel production with immobilized lipase: a review. Biotechnol. Adv. 28, 628–634. Tran, D.T., Yeh, K.L., Chen, C.L., Chang, J.S., 2012. Enzymatic transesterification of microalgal oil from Chlorella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour. Technol. 108, 119–127. Zhao, Q., Hou, Y., Gong, G.H., Yu, M.A., Jiang, L., Liao, F., 2010. Characterization of alcohol dehydrogenase from permeabilized Brewer’s yeast cells immobilized on the derived attapulgite nanofibers. Appl. Biochem. Biotechnol. 160, 2287– 2299.
