Converting paper mill sludge into neutral lipids by oleaginous yeast Cryptococcus vishniaccii for biodiesel production
Graphical abstract
Introduction
Worldwide rapid progression in fuel demand and current fossil fuel crisis act as a driving force behind the search for alternative renewable energy sources such as agro-industrial waste based biofuels (Leiva-Candia et al., 2014). As relevance, current demand for biodiesel production has executed triacylglycerol (TAG) into an ever-blooming and valuable consumption resource (Meher et al., 2006, Chang et al., 2015). Hence, production of biodiesel from single cell oil seems to overwhelm this difficulty as a promising replacement for other feedstock (Sitepu et al., 2014). These microbial lipids have inherent benefit compare to vegetable oils in terms of higher lipid yield, easy maintenance of growth conditions, devoid of any seasonal and climatic changes, less labor intensive and genetically modify to accomplish higher production yields (Meng et al., 2009, Sitepu et al., 2014). Various oleaginous yeasts of genera Cryptococcus, Rhodosporidium, Rhodotorula, Candida, Yarrowia, Lipomyces and Trichosporon have incomparably high lipid productivity utilizing number of renewable feedstock’s for cultivation, such as nutritional residues from low-cost industrial and agricultural wastes (Ageitos et al., 2011, Freitas et al., 2014, Sitepu et al., 2014, Leiva-Candia et al., 2014). The yeasts accumulate oil mainly in triacylglycerols form that are principally palmitic (16:0), palmitoleic acids (C16:1), stearic (18:0), oleic (18:1) or linoleic (18:2) acids (Meng et al., 2009, Fei et al., 2009) with only a small amount of Steryl esters. Recently, intensive research on improving lipid production and high cell density cultivation in Cryptococcus and Rhodosporidium species has been carried out (Li et al., 2007, Wu et al., 2011, Liang et al., 2012). The specific benefit for the TAG feedstock production by using oleaginous yeast is that they can be cultivated on numerous kinds of complex and non-complex sugars as substrates and at optimized conditions convert them into TAG (Patel et al., 2014, Sitepu et al., 2014, Leiva-Candia et al., 2014). Therefore, oleaginous yeast would assign a competitive avenue for the biodiesel production utilizing different sugars from the substrate, when the cost of these substrates can be minimized to economically adequate level.
Paper mill sludge (PMS), rich in organic matters and nutrients, was analyzed as an economic replacement for organic substances. According to Working Group report of 12th FYP (Fifth Year Plan) India’s paper manufacturing industry produces 10.92 million tons of paper annually and about 1 tonne of paper generates 40–50 kg of sludge (dry). The biodiesel production from PMS can lessen reliance on fossil fuels and simultaneously resolving the environmental issues related with PMS disposal. However, a major part of PMS is lignocellulosic biomass, predominantly consisting of cellulose, followed by lignin and hemicelluloses contribution (Jackson and Line, 1997) which could be hydrolyzed to monosaccharide’s including xylose, glucose, and arabinose that are suitable carbon sources for production of microbial lipid (Zhang et al., 2011). In addition, other non-lignocellulosic compounds, e.g., lipids and proteins, can be hydrolyzed and used by microorganisms (Ghanavati et al., 2015). The PMS has an advantage as a carbon source for microbial utilization compared to other lignocellulosic materials as during the pulping step of the paper manufacturing process most of the lignin gets eliminated (Prasetyo et al., 2011). Therefore, PMS can be used as carbon source directly to hydrolyse PMS organic component. To separate insoluble materials such as other biomass and clay, simple separation processes are required. PMS are a widely available resource to be utilized as feedstock by microbe and contain significant amounts of nutrients; which can be used for recycling of organic by-products, thereby curtailing environmental pollution such as controlling greenhouse gas emissions, limiting land filling, and direct land applications. The sludge containing carbon moieties could be transformed into lipids by the oleaginous micro-organisms. These converted lipids can then aid as a raw material for the biodiesel production (Angerbauer et al., 2008). For efficient conversion of lignocelluloses into bio products by fermentation, pre-treatment plays a significant role. Among various pre-treatment methods, ultrasonication has been verified to be an inexpensive and fast method for production of sugars from various residues (Angerbauer et al., 2008). In our investigation, we have explored the utilization of sonicated PMSE prepared for intracellular TAG (neutral lipids) accumulation in the lipid bodies of oleaginous yeast, Cryptococcus vishniaccii to produce biodiesel. For oleaginous yeast cultivation in paper mill sludge extract, the modest pre-treatment method of ultrasonication was prosecuted for effective extraction of sugar, protein and lipid components (Saha et al., 2011, Patel et al., 2014). The cells of this yeast accumulate high amounts of neutral lipids (TAG) in their intracellular lipid droplets which were visualized by using epi-fluorescence microscope. The accumulated lipids were extracted and transesterified into FAME as biodiesel which were characterized by TLC, FT-IR and GC–MS analysis. This leads to conversion of industrial waste biomass to triacylglycerides feedstock for biodiesel production by oleagenic yeast cells. Furthermore, investigation confirms the feasibility for utilization of cheaper, substantial, unexploited industrial waste biomass of paper mill sludge as a promising resource for biodiesel production. This is the first elaborated report on biodiesel production from the C. vishniaccii oil using paper mill sludge as a substrate.
Section snippets
Materials
Analytical grade solvents viz., n-hexane, methanol, and chloroform were taken from Merck Ltd., Mumbai, India. Nile red 493/505 nm (9-diethylamino-5H-benzo[α-phenoxa]phenoxazine-5-one) and other chemicals (NaCl, KCl, anhydrous sodium sulfate, 14% methanolic BF3, castor oil) were obtained from Hi-Media Laboratories, Mumbai, India. For GC–MS analysis, FAME standard (AOCS low erucic rape seed oil O7756-1AMP) was obtained from Sigma Aldrich (St. Louis, USA) and Triolein used as standard for TLC was
Biomass and lipid production by C. vishniaccii using paper mill sludge extract (PMSE)
The organic component of the sludge is vitally high in carbohydrates and low in lignin. It contains 45–55% carbohydrate and hemicelluloses, cellulose and lignin which have potential for conversion into C6 and C5 sugars broadly recognized as a favorable feedstock for production of biofuel (Prasetyo et al., 2011, Liang et al., 2012, Leiva-Candia et al., 2014). Its small size and high surface area make it susceptible for biological conversion. On a dry matter basis, the used PMS material consisted
Conclusions
Paper mill sludge extract containing all essential nutrients components is a prudential triacylglycerides feedstock for growing oleaginous yeast C. vishniaccii to enhanced maximum accumulation of TAG in LDs compartments of yeast cells. Sonicating pre-treatment in PMSE boosted total carbohydrates and reducing sugars and yield of lipid production was 15% higher than GSM. Compared to jatropha and other vegetable oil, FAME profile of yeast oil shows more oxidative stability which is related to
Acknowledgements
Authors are thankful to Ministry of Human Resource Development (MHRD), Govt. of India for their financial support.
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