Oleaginous Fungi: Biodiesel Generation PotentialIntroductionBiodiesel (fatty acid methyl- or ethyl-esters) is considered a promising alternative fuel and it can be defined as the mono-alkyl esters of long chain fatty acids. Fatty acid methyl esters (FAMEs) are the most-common constituents used for biodiesel preparation, which are derived from triacylglycerols (TAGs). It is biodegradability, nontoxic and low emission profiles are some key environmentally beneficial properties. For years biodiesel has been derived from oilseeds, animal fat and non-edible plants, which can replace only a small fraction of the existing demand for transport fuels.
Therefore, it is necessary to explore new raw materials that can deliver environmental benefits, are economically competitive and can be produced in quantities sufficient to meet the energy demands, and which do not compete with food production.Microbial lipids can represent a valuable alternative feedstock for biodiesel production, and a potential solution for a bio-based economy. Oleaginous microorganisms, such as yeasts, fungi, bacteria, and microalgae, can accumulate high levels of lipids and do not require arable land, so that they do not compete with food production when supplied with high carbon to nitrogen ratio (Hou et al, 2004).
Oleaginous yeast and fungi have also been considered as potential oil sources for biodiesel production because they accumulate large amounts of lipids. (Fujian et al, 2001). Eukaryotes like such as fungi, plants and animals, and some exceptional bacteria that can accumulate lipids above the 20% of their dry biomass are termed as oleaginous microorganisms. Accumulated lipids mainly comprise of triacylglycerols (TAG) and esters that these yeasts and filamentous fungi accumulate in as high amounts in their cell reservoirs. As high as 70% of their biomass dry weight has be reported to be found in Rhodosporidium sp., Rhodotorula sp., and Lipomyces species intracellular storages (Kavadia et al., 2001).Whereas in bacteria polyhydroxyalkanoates are accumaulated as their storage compounds. The actinobacterial genera bacterial species Mycobacterium, Streptomyces, Rhodococcus and Nocardia are known to produce relevant amounts of lipids (Alvarez & Steinbuchel, 2002). Molds and yeasts are the most efficient TAG accumulators. While the oleaginous potential of various yeast species has been studies vastly, fungal based biofuels have an emerging scope. This review chapter will focus on current knowledge advances in their metabolism, physiology, and in the result achieved in strain improvement, process engineering and raw material exploitation.Lignocellulosic Substrate degradation Cellulosic biofuels are derived from the cellulose in plants, some of which are being developed specifically as energy crops rather than for food production. These include perennial grasses and trees, such as switchgrass and Miscanthus. Crop residues, in the form of stems and leaves, represent another substantial source of cellulosic biomass. Of the waste materials generated, lignocellulose is of great significance because it is produced in vast quantities every year due to every increase of land cultivation. Lignocellulosic biomass, which includes materials such as agricultural residues (corn stover, crop straws, husks and bagasse), herbaceous crops (alfalfa, switchgrass), short rotation woody crops, forestry residues, waste paper and other wastes (municipal and industrial). Lignocellulosic feedstocks do not interfere with food security and are important for both rural and urban areas in terms of energy security reason, environmental concern, employment opportunities, agricultural development, foreign exchange saving, socioeconomic issues etc.Turning Agricultural Waste to Green Power in India: India being an agricultural nation produces 98 million tonnes of paddy with roughly 130 million tonnes of straw of which only about half is used for fodder. 350,000 tonnes of cane, eventually yielding approximately 50 million tonnes of cane trash can be utilised as an excellent source of biomass fuel. The table below depicts the sugarcane bagasse production statistic of the ASEAN countries, where India undoubtedly has the highest potential.These raw wastes collected from thousands of Indian farmers, their subsequent usage for biodiesel generation can beneficial with incremental income. This approach of consolidated manpower and environment empowerment is the solution for a sustainable development of the nation. In order to accomplish these goals intensive research and development is needed with intensive strategies. In this project proposal we aim to generate second generation bio-fuel in the form of biodiesel by utilising one of the most abundantly produced agricultural waste that is sugarcane bagasse as feedstock. G. Venkata Subhash, S. Venkata Mohan / Bioresource Technology (2011) conducted comparative studies with Corncob Waste Liquor (CWL) Sabourauds dextrose broth medium (SDBM) as substarte for lipid accumulation in filamentous fungus Aspergillus sp. In this report SDBM gave better accumulated lipid yields with respect to CWL, while CWL derived biodiesel showed relatively superior fuel properties. G. Venkata Subhash, S. Venkata Mohan / Fuel 116 (2014) used the Taguchi design of experimental methodology to determine the precise functional role of eight factors involved in the lipid production using A. awamori (MTCC11639). Where significant role of, pH individually was seen on lipid synthesis, followed by other factors such as incubation temperature, glucose concentration, incubation time, NaCl concentration, nitrate concentration, phosphorous concentration and protein source. Validation and optimization experiments showed enhanced lipid production by 31% and with higher number of saturated fatty acids in this FAME obtained had better biodiesel properties.Below various reports showing the successful utilization of lignocellulosic agro waste for biodiesel has been enlisted. Strain Lignocellulosic Carbon source Lipid (g /L) Lipid content (%) (%) Lipid productivity(g/L/ h) mediaReferenceMicrosphaeropsis sp. Wheat straw + bran 80 (mg gds) 10.2 0.33 (mg/g/h) Peng and Chen (2008)A. oryzae A-4 Wheat straw + bran 62.9 (mg gds) N.A. 0.44 (mg/g/ h) Lin et al. (2010)Mortierella isabellina ATHUM2935 Sweet sorghum 0.11 (g/ gds) 25 0.055 (g/g/h) Economou et al. (2010)Colletotrichum sp. DM06 Rice straw + wheat bran 68.2 (mg gds) N.A. 0.28 (mg/g/ h) Dey et al. (2011)Alternaria sp. DM09 Rice straw + wheat bran 60.3 (mg gds) N.A. 0.25 (mg g_1 h_1) Dey et al. (2011)Mortierella isabellina ATCC42613 Alkali treated corn stover hydrolysate2.48 29.47 0.027 Z. Ruan et al. (2012)Fungal Lipid assimilationFor an oleaginous microbe to be considered as a suitable strain for biodiesel, the single cell oil (SCO content, > 20%) and the type of fatty acids (long chain saturated and/or monounsaturated fatty acids) are important criteria. The oleaginous microorganisms accumulate lipids via two classified biochemical processes termed as ex novo lipid accumulation and the growth on sugar-based (hydrophilic) substrates follows de novo lipid accumulation pathway. Nitrogen-limiting conditions support the excretion of citrate from the mitochondrial matrix into the cytoplasm and its subsequent break down into acetyl-CoA and oxaloacetic acid by the enzyme ATP citrate lyase. In ex novo pathway, yeasts like Yarrowia, Candida and Torulopsis rely on the active transportation of hydrophobic substrates (HS) and assimilation occurs in an unchanged or modified form at different rates for substrate fatty acids (Papanikolaou andAggelis 2003). The presence of ATP-citrate lyase is an important marker to determine the oleagenicity of a given microorganism with the absence of the enzyme leading to an accumulation of citric acid rather than lipid (Papanikolaou and Aggelis 2011a).The SCO produced via ex novo process contains lower TAG compared with de novo synthesized lipid (Papanikolaou and Aggelis, 2011). Lipid content and composition rely on the organism’s particular metabolic machinery and the rate of lipid accumulation in these microorganisms in general increases during nitrogen-starvation while cells continue to assimilate excess carbon and cell division is hindered. The excess of carbon is channelled into triacylglycerides (TAGs) [1, 2″4]. Oleaginous organisms have very few metabolic variations from the non-oleaginous counter parts. This specialised capability to accumulate excess lipds is related to mainly the fatty acids biosynthesis, Biosynthesis of triacyl-glycerol, andc the biogenesis of lipid bodies, as reviewed by Maddalena Rossi et. al, Biodiesel ” Feedstocks and Processing Technologies.The ability to accumulate high amounts of lipid relies mainly on the regulation the biosynthetic pathway enzymes and the availibilty of the precursors like acetyl-CoA, malonyl-CoA, and glycerol-3- phosphate and the cofactor NADPH.Getting Lipids for Biodiesel:Chavanne of the University of Brussels (Belgium), in 1937 explained the alcoholysis reaction (often referred to as transesterification of vegetable oils) using methanol and ethanol for the separation of the fatty acids from the glycerol by the replacement glycerol by short linear alcohols. This work titled as “Procedure for the transformation of vegetable oils for their uses as fuels” (fr. ‘Proc©d© de Transformation d’Huiles V©g©tales en Vue de Leur Utilisation comme Carburants’) was granted a Belgian Patent 422,877 on August 31, 1937 as it is the first known report on biodiesel production. The previously used vegetable oils were not transestrified, thus are not categorised as biodiesel. Biodiesel (fatty acid methyl- or ethyl-esters) is considered a promising alternative fuel and it can be defined as the mono-alkyl esters of long chain fatty acids. Fatty acid methyl esters (FAMEs) are the most-common constituents used for biodiesel preparation, which are derived from triacylglycerols (TAGs). It is biodegradability, nontoxic and low emission profiles are some key environmentally beneficial properties (Krawczyk, 1996).FAME i.e Fatty acid aceyl methyl esterase or biodiesel derived from microbial lipids can be extracted via two principles:Direct Transestrification Indirect Transestrification. The traditional Indirect Transestrification process consists of fungal cell wall disruption by grinding of dried mycelia were in a mortar lipid extraction by various enzymatic, chemical or physical methods to obtain the lipids stored within the cell (Bligh and Dyer, 1959). This extracted lipid is then chemically transestrified by methanolysis to form fatty acid methyl esters (Indarti et al.,2005). In this the total lipid (SCO) extraction is performed after cell disruption of the dried fungal biomass. Lipid extraction method laid daown by Schneiter and Daum (2006) was future investigated by Khot et al. Microbial Cell Factories (2012) to test two different cell lysis techniques. One method using a bead-beater’ and the second was the cryo-pulverization of biomass using liquid nitrogen, for the extraction of intracellular lipids. These experiments proved lipid extraction method using liquid nitrogen to be more efficient and indicated that the extraction method selection is crucial for obtaining good amount of oil recovery from these oleaginous fungi. The key to overcome the cost and single cell oil recovery challenges faced during biodiesel generation could be over come by in situ direct transesterification. The conventional fungal biodiesel production process demands tedious steps of drying/dewatering and cell disruption to obtain SCOs from the harvested oleaginous biomass. Further subjection of these SCO’s to solvents for extended hours, turns out to be an expensive and time consuming process, which was overcome by applying in situ direct transestrification methodology, wherein the steps of cell disruption, lipid extraction and eventually transesterification all occur in a single step can prove to be beneficial (Liu and Zhao, 2007). Compared to conventional three-step transesterification, the in situ method (direct methanolysis) is faster and consumes lesser amount of solvent while achieving improved conversion to FAME (Vicente et al., 2010). Of all the previous studies reported on direct transestrification (Kakkad et. al. 2015) gave the first report of optimization of direct transesterification to generate biodiesel derived from Aspergillus terreus grown on dairy waste whey. In this the variables affecting esterification, viz., catalyst methanol and chloroform concentrations, temperature, time, and biomass were investigated under acid catalysed conditions, and decreased biomass loading gave greater efficiency of transestrification. Such findings are crucial for the development of biofuels of fungal origin with minimum wastage of solvents; time and greatly improved efficiency make it a promising development. This phenomenon was again tested by G. Venkata Subhash, S. Venkata Mohan / Bioresource Technology (2011) by subjecting the lipids extracted from Aspergillus sp. to direct (DTE) and indirect (IDTE) transesterification methods. These researchers found broadspectrum of fatty acids profile by DTE in comparision to IDTE. Hence thoughtful application of methodologies is an important to device good fuel characteristics while keeping in mind the process and time feasibility. The scope for elimination the need for costly dehydration has been reviewed by T. Dong et al. / Applied Energy 177 (2016). This review highlighted biofuel productions with special attention to cell disruption and lipid mass transfer to support extraction from wet biomass. Varied enzymatic, chemical along with unique disruption methods have been summarised in this review and the authors have concluded with the need for intensive research on industrial biofuel production and suggested use of nontoxic non-polar solvents with minimal miscibility in water and least latent heat of vaporization characteristics for more efficient lipid recovery from microbial biomass. Strain improvementEconomical and efficient designing of upstream and downstream processes of biodiesel production using filamentous fungi can be further improved by advancement of the cell’s abilities to assimilated larger quantities of lipids. This is termed as strain improvement for biodiesel manufacturing. Filamentous fungi are amenable to genetic manipulation by metabolic engineering and hence their lipid content and fatty acid profile could be modified to suit biodiesel production.Strain improvement since its inception has be done by random/ chemical mutagenesis or directed mutagenesis. .”Directed mutations”, that would grant man with “unlimited power over Nature was rightly vision by De Vries.Random mutations are induced indiscriminately in a (mostly haploid) genome by the application of chemical or a physical mutagenic agent. This technique has been applied widely advent of recombinant DNA technology. Haploid conidia is exploited as the sculpt material for mutagenic treatment, which is common in a wide range of industrially relevant filamentous fungi. Strain improvement of oleaginous fungi for improved lipid assimiliation properties is yet to be investigated to a greater extent. This review attempts at highlighting the primary studies carried out in this context. Base pair substitution, insertion, deletion etc. or any other type of mutation events causing abnormalities in the DNA or cellular metabolism define its efficiency (K.M.H. Nevalainen et. al 2001). MNNG also called as NTG (N’-methyl-N’-nitro-N’-nitrosoguanidine) is the most widely used chemical for random mutagenesis. MNNG induced point mutations and UV irradiation mediated pyrimide dimers leading to point mutations and deletions are reviewed by R.T. Rowlands (1984, 1992).References:Alvarez, H. M. & Steinbuchel, A. (2002) Triacylglycerols in prokaryotic microorganisms. Applied Microbiology and Biotechnology. Vol. 60, No. 4, pp. 367-376, ISSN 0175-7598.Cheng, K.K., Cai, B.Y., Zhang, J.A., Ling, H.Z., Zhou, Y.H., Ge, J.P., Xu, J.M., 2008. Sugarcane bagasse hemicellulose hydrolysate for ethanol production by acidrecovery process. Biochemical Engineering Journal 38, 105″109.Dey, P., Banerjee, J., Maiti, M.K., 2011. 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