A METHOD FOR PRODUCING MICROBIAL OIL FROM LIGNIN OR LIGNIN HYDROLYSATE USING OLEAGINOUS MICROBES

TECHNICAL FIELD

[0001] The present invention relates to a method for producing microbial oil from lignin or lignin hydrolysate using oleaginous microbes.

BACKGROUND

[0002] Recalcitrance of lignocellulosic biomass hinders its complete utilization in the process of biorefining. Although, there is abundant availability of the lignocellulosic biomass in nature, it cannot be harnessed for efficient conversion into value added products. Resistance in chemical or biological disintegration of the complex and rigid framework composed of mainly cellulose, hemicellulose and lignin is the major predicament.

[0003] Fermentable sugars obtained on the hydrolysis of these components, except lignin, have been deployed widely for use in production of microbial oil using oleaginous organisms. Microbial oil provides as a promising non-crop based renewable resource; catering as a source of highly nutritious lipids, majorly in the form of triglycerides, which can be further used as a source of food as well as an energy resource (Ratledge, C. (2004), Biochimie, 86(11), 807- 815). Promising microbial yeast strains such as Lipomyces starkeyi have been reported to accumulate almost 52.6% lipids when cultivated on xylose where as it showed 50.8% lipid content when grown on sewage sludge. Also, Cryptococcus curvatus grown on glycerol accumulated 25% lipids (Li, Q., Du, W., and Liu, D. (2008), Applied Microbiology and Biotechnology, 80(5), 749-756). Rhodosporidium torruloides demonstrated lipid accumulation of 56% when grown on jerusalem artichoke (Ali Abghari and Shulin Chen (2014), Frontiers in Energy Research, 2 (21), 1-21).

[0004] A considerable amount of lignocellulosic materials as waste byproducts are being generated through agricultural practices, mainly from various agro based industries (J. Pe rez JE J. Mun oz-Dorado T. de la Rubia JE J. Marti nez. (2002), Int Microbiol, 5, 53-63). The lignocellulosic materials thus obtained are the most promising feedstock, as natural and renewable resource, essential to the functioning of modern industrial societies. However, much of the lignocellulosic biomass is often disposed of by burning, which can rather be converted into different high value products including bio-fuels, value added fine chemicals, and cheap energy sources for microbial fermentation and enzyme production (Asgher et al. (2013), BioResources, 8(1), 944-968), BioResources, 8(1), 944-968; H.M.N. Iqbal, G. Kyazze, T. Keshavarz. (2013), BioResources, 8 (2), 3157-3176; M. Irshad, Z. Anwar, H.I. But, A. Afroz, N. Ikram, U. Rashid (2013), BioResources, 8 (1), 145-157; M.R. Isroi, S. Syamsiah, C. Niklasson, M.N. Cahyanto, K. Lundquist, M.J. Taherzadeh. (2011), BioResources, 6 (4) , 5224- 5259).

[0005] The lignin component of this biomass, however, remains un-addressed owing to its rigid structure, forbidding the tranquility in deconstruction. Also, the inhibitory nature of compounds like furan, phenols and other aromatic moieties, generated during degradation, impede the growth and metabolism of microbes.

[0006] An effective strategy for depolymerization of lignin into low molecular weight compounds that can be assimilated by oleaginous organisms and converted to triglycerides and/or oleochemicals that have great commercial relevance, thus needs to be devised. These oleaginous organisms have emerged as ideal entrants for lignin degradation as they can tolerate, grow on and adapt to inhibitory compounds produced from the pretreatment processes of lignocellulosic biomass. The capability to produce vegetable plant equivalent triglycerides by the oleaginous microbes from all the different components (sugars and phenolics) of lignocellulosic biomass adds great value to the waste to value concept.

[0007] The existing methodologies uses carbohydrate -based carbon source, such as C5-C6 sugars or lignocellulosic biomass hydrolysates of rice bran, wheat bran, bagasse, corncob etc. However, the use of C5 to C6 carbon source in said processes increase cost of nutrient media used for production of microbial oil which ultimately increases the production cost.

[0008] Therefore, there is a need to develop better alternative carbon source for C5-C6 sugars which is a renewable, abundant in nature, cheap and which reduces microbial oil production cost and serves as a better alternative for oleochemical biorefinery.

OBJECTIVE OF THE INVENTION

[0009] An object of the present invention is to provide a method for producing microbial oil from lignin or lignin hydrolysate using adapted oleaginous microbes.

[0010] Another object of the present invention is to provide a method for producing lignin hydrolysate from lignin.

[0011] Yet another object of the present invention is to provide a method for producing microbial oil from lignin hydrolysate by using adapted oleaginous microbes.

SUMMARY [0012] An aspect of the present disclosure relates to a method for production of microbial oil comprising: (a) a growth phase comprising cultivating at least one of the species of oleaginous microbe in a growth media, wherein the growth media comprises of at least one first carbon source and at least one nitrogen source to obtain confluent cells; (b) a production phase comprising inoculating the confluent cells into a production media under suitable conditions to produce and accumulate microbial oil, wherein the production media comprises lignin hydrolysate; and (c) recovering the microbial oil from the production phase.

[0013] Another aspect of present disclosure provides a method for producing microbial oil from lignin hydrolysate using adapted microorganisms, wherein said method is carried out by inoculating at least one of species of adapted oleaginous microbes in the fermentation media containing lignin hydrolysate for five to seven days at 28°C to obtain wet cell biomass in the range of 10 g/1 to 100 g/1. The wet cell biomass of around 20g/l is obtained at the end of seven days with oil content of 3 to 4 g/1. The extraction of intracellular oil is carried out using solvents like, but not limited to ethyl alcohol, isopropyl alcohol etc. at temperature of 40-60°C for 30- 60 mins at agitation speed of 300-900 rpm whereas the extracellular oil is extracted using solvent mixture of nonpolar and polar solvents include but not limited to chloroform and methanol.

[0014] Yet another aspect of present invention provides a method for producing microbial oil from lignin hydrolysate using adapted microorganisms, wherein said method is carried out by simultaneous recovery of the produced microbial oil from the broth concomitant to its production using 1% (v/v) n-hexane in a two-stage extractive production process, to obtain a yield of 87.32% oil w.r.t DCW (dry cell weight).

[0015] These and other features, aspects and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

DETAILED DESCRIPTION

[0016] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively and any and all combinations of any or more of such steps or features.

Definitions

[0017] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0018] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0019] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. Throughout this specification, unless the context requires otherwise the word "comprise", and variations, such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0020] The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0021] The term "oleochemical/s" used herein refers to the chemicals derived from chemical and/or enzymatic reactions of fatty acids, fatty alcohols, fatty acid methyl esters (FAMEs), fatty amines, glycerol etc.

[0022] The term "microbial oil" or "oil" or "lipids" used herein refers to the microbial oil which is derived from oleaginous microbes, including yeast, fungi and bacteria, having distinct triglyceride profiles. The "microbial oil" or "oil" or "Lipids" can be used interchangeably in the specification.

[0023] The term "oleaginicity" refers to the ability of microbes to accumulate oil more than 20% of its dry cell weight (DCW).

[0024] The term "lignin feedstock" used herein defines the lignin fraction of any stream of lignocellulosic biomass derived from but not limited to forestry, agro based industries, paper and pulp industry (kraft lignin) etc.

[0025] The term "kraft lignin" used herein defines the lignin component of waste stream derived from the process of converting wood chips into wood pulp which mainly comprises of pure cellulose fibers used for making paper. [0026] The term "lignin" used herein refers to a complex polyphenolic polymeric material with three-dimensional network devoid of carbohydrate (sugar) moiety. The polyphenolic polymeric material is composed of complicated phenyl propane units, nonlinearly and randomly linked with each other and consist of monomers i.e. coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, hydroxybenzoic acid, hydroxycinnamic acid, protocatechuic acid, syringic acid, and the like.

[0027] The term "lignin hydrolysate" used herein refers to the material obtained by any treatment/pre-treatment of "lignin". The methods include but are not limited to pyrolysis, hydrothermal treatment, enzymatic treatment, thermochemical treatment, acid treatment, alkali treatment etc., which is able to degrade or hydrolyse lignin into its oligomers or monomers. The terms "lignin monomers", and "lignin oligomers" refer to the above-mentioned oligomers and monomers. The lignin hydrolysate consists of phenolics (e.g. coniferyl aldehyde, ferulic acid, 4-hydroxybenzoic acid, etc.), non-phenolics (e.g. cinnamic acid, benzoic acid, etc.), with/without ash and may be present in different proportions. The phenolics being part of hydroxyl benzene ring(s) and non-phenolics being part of non-hydroxyl benzene ring(s). The terms "lignin", "lignin hydrolysate", "lignin monomer", and "lignin oligomers" are interchangeably used throughout the disclosure and refer to the polyphenolic polymeric or monomeric material consisting of both the phenolics and non-phenolics part.

[0028] The term "adapted oleaginous microbe" or "adapted" used herein refers to microbes adapted/sensitized to utilize and grow on phenolics and/or non-phenolics as carbon and/or nitrogen source(s) which produce oils (lipids) and accumulate them. The said adapted oleaginous microbe includes but not limited to oleaginous yeast, oleaginous fungi, and oleaginous bacteria, oleaginous algae etc.

[0029] The term "confluent cells" refer to the cells grown during the growth phase or stage of the process of present disclosure. The confluent cells have a cell density in the range of 10 to lOOg /l.

[0030] The term "catalyst" used herein refers include chemical or biological catalyst capable of hydrolyzing or degrading lignin into its oligomers and/or monomers.

[0031] The term "yield" used herein refers to amount or concentration of the oil produced which is expressed as either 'percent oil produced with respect to dry cell weight of biomass' or 'weight of oil produced in grams per gram of carbon source utilized' or 'weight of oil in grams per litre of oil production broth. [0032] The term "COD (chemical oxygen demand)" refers to an indicative measure of the amount of oxygen that can be consumed by reactions in a measured solution. A COD test can be used to easily quantify the amount of organics in a solution.

[0033] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a temperature range of about 100°C to about 200°C should be interpreted to include not only the explicitly recited limits of about 100°C to about 200°C, but also to include sub-ranges, such as 125°C to 145°C, 130°C to 150°C, 175°C to 200°C and so forth, as well as individual amounts, including fractional amounts, within the specified ranges, such as 122.2°C, 150.6°C, and 191.3°C, for example.

[0034] As discussed previously, a cost-effective alternative for the production of microbial oil is required. The present disclosure provides a method for producing microbial oil from lignin using adapted oleaginous microbes. The present methodology uses lignin hydrolysate as an alternate carbon source for microbial oil production whereas the existing methodologies uses carbohydrate-based carbon source such as C5-C6 sugars e.g. glucose, xylose or lignocellulosic biomass hydrolysates of rice bran, wheat bran, bagasse, corncob etc. This new route utilizes lignin, which is a renewable, cheap and abundant natural polymer, in an environment friendly way for production of microbial oil using adapted oleaginous yeast strains. Therefore, the implementation of the present disclosure would be able to overcome the food v/s fuel conflict, as well as use the by-product of second generation biofuel processes. Lastly, the process exploits lignin as a carbon source for growth of microbes which serves as a better alternative for oleochemical biorefinery.

[0035] In an embodiment of the present disclosure, there is provided a method for production of microbial oil comprising: (a) a growth phase comprising cultivating at least one of the species of oleaginous microbe in a growth media, wherein the growth media comprises of at least one first carbon source and at least one nitrogen source to obtain confluent cells; (b) a production phase comprising inoculating the confluent cells into a production media under suitable conditions to produce and accumulate microbial oil, wherein the production media comprises lignin hydrolysate; and (c) recovering the microbial oil from the production phase.

[0036] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein said method comprises of: (a) hydrolysing lignin with at least one catalyst; (b) inoculating at least one of species of adapted oleaginous yeasts in the fermentation media under suitable conditions to produce and accumulate oil, wherein fermentation media comprises of at least one carbon source and at least one nitrogen source; and (c) recovering said oil from adapted oleaginous yeasts in the fermentation media. In another embodiment, said method comprises of: (a) inoculating at least one of species of adapted oleaginous microbes in the fermentation media containing kraft lignin under suitable conditions to produce and accumulate microbial oil, wherein fermentation media comprises of at least one nitrogen source, along with kraft lignin used as carbon source; and (b) recovering said oil from adapted oleaginous microbes in the fermentation media.

[0037] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein said method comprises of: (a) hydrolysing lignin with at least one catalyst to obtain a lignin hydrolysate; (b) inoculating at least one of species of adapted oleaginous yeasts in the growth media containing glucose, yeast extract, malt extract, and peptone under suitable conditions and (c) pelleting the oleaginous yeasts from growth media; (d) inoculating the pelleted oleaginous yeasts into lignin hydrolysate to produce and accumulate microbial oil, and (e) recovering said microbial oil from adapted oleaginous yeasts in the fermentation media. Pelleting refers to processing microbial culture into pellet form (concentrate) by centrifugation.

[0038] In another embodiment of present disclosure, there is provided a method as described herein, wherein the said microbial oil was produced continuously using lignin as a sole carbon source (without at least one second carbon source) by a viable cell biomass maintained in a particular synchronous growth phase for a prolonged duration causing an effective milking.

[0039] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one of the species of oleaginous microbe is selected from the group consisting of oleaginous yeast, oleaginous fungi, oleaginous bacteria, and combinations thereof.

[0040] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one of the species of oleaginous microbe is wild type, adapted, mutagenetic or genetically engineered. In another embodiment, the at least one of the species of oleaginous microbe is adapted.

[0041] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein lignin is obtained from biomass comprises maize bran, wheat straw, paper and pulp industry waste by conventional methods (as mentioned in

VVO2011154967 A 1 ) known thereof. [0042] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the method is a two-stage extractive production of microbial oil by oleaginous yeast utilizing lignin/ lignin hydrolysate.

[0043] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein said method may be carried out with pure/derived/substituted lignin monomers or oligomers or in combination thereof.

[0044] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one of the species of oleaginous microbe is oleaginous yeast selected from Yarrowia lipolytica NCIM 3590, Rhodotorula glutinis NCEVI 3168, Rhodosporidum toruloides NCIM 3547, Lipomyces starkeyi NCIM 3440, or Lipomyces lipofer NCIM 3252.

[0045] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one first carbon source is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, oligosaccharides, C5 sugars, C6 sugars, and combinations thereof. In another embodiment of the present disclosure, the at least one first carbon source is C6 sugar.

[0046] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the production media optionally comprises at least one second carbon source and the at least one nitrogen source.

[0047] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one second carbon source is selected from the group consisting of monosaccharides, disaccharides, polysaccharides, oligosaccharides, lignin, lignin monomers, lignin oligomers, biomass hydrolysate, C5 sugars, C6 sugars, agricultural waste comprising phenolics, bio-oil, waste oil, acid oil, and industrial waste, and combinations thereof. In another embodiment, the at least first carbon source and at least second carbon source comprises glucose, fructose, sucrose, galactose, xylose, mannose, rhamnose, N-acetylglucosamine, glycerol, glucuronic acid, and acetate. In another embodiment of the present disclosure, the at least one first carbon source is C6 sugar.

[0048] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the at least one nitrogen source is selected from the group consisting of amino acids, yeast extract, peptone, protein hydrolysate, urea, corn steep liquor (CSL), and combinations thereof. In another embodiment of the present disclosure, the at least one nitrogen source is peptone. [0049] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the confluent cells have a cell density in the range of 5 to 100 g/L.

[0050] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the lignin hydrolysate is having a weight/volume percentage in the range of 0.1 to 10% with respect to the production media and an effective COD of the production media is in the range of 0.1-30,000 ppm. In another embodiment of the present disclosure, the lignin hydrolysate is having a weight/volume percentage of 0.2%. In yet another embodiment, the lignin hydrolysate is having a weight/volume percentage of 2%. In yet another embodiment, the effective COD of the production media is in the range of 50- 25,000 ppm.

[0051] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the lignin hydrolysate comprises phenolic compounds having a total phenolic content in the range of 5% to 15% w/w, and selected from the group consisting of p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol, hydroxybenzoic acid, hydroxycinnamic acid, protocatechuic acid, syringic acid, and combinations thereof.

[0052] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein lignin hydrolysate is obtained by any treatment selected from the group consisting of catalytic treatment, hydrothermal treatment, thermochemical treatment, pyrolysis, and combinations thereof. In another embodiment, said treatment is able to degrade or hydrolyse lignin into its oligomers or monomers or constituent phenolic compounds.

[0053] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the catalytic treatment is carried out in the presence of a catalyst selected from the group consisting of acid, base, ionic liquid, at least one enzyme, and combinations thereof. In another embodiment, the catalyst is an acid which may be dilute or concentrated. In yet another embodiment, the acid is a dilute acid, selected from nitric acid, sulphuric acid, hydrochloric acid, phosphoric acid, oxalic acid, and acetic acid. In yet another embodiment, the concentration of the dilute acid is nitric acid having a concentration in the range of 0.5% to 2% (v/v).

[0054] In another embodiment, the catalyst is a base selected from sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, calcium carbonate, potassium carbonate, and amines. In another embodiment of the present disclosure, the base is sodium hydroxide. [0055] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein said method comprises reacting lignin with a catalyst at a temperature in the range of 100°C to 200°C and pressure in the range of 5 to 20 bar for the time period from 24 h to 150 hrs and said catalyst is selected from nitric acid, sulphuric acid, or hydrochloric acid. The said method can be applied directly to the lignin feedstock also.

[0056] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein said method comprises reacting corncob with a catalyst at a temperature in the range of 100°C to 200°C and pressure in the range of 5 to 20 bar for the time period from 24 h to 150 hrs and said catalyst is selected from nitric acid, sulphuric acid, or hydrochloric acid.

[0057] In another embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the catalyst is an enzyme catalyst used for hydrolysis of lignin that may include but not limited to laccases. The said catalyst may be used as such and/or in combination with one or more chemical catalysts as described herein.

[0058] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the production phase is carried out in the presence of medium-chain alkanes as liquid extractant. In another embodiment, the medium-chain alkane is hexane. In yet another embodiment, n-hexane is having a concentration in the range of 0.1% to 20% (v/v).

[0059] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein wherein the production phase is carried out in the presence of medium-chain alkanes selected from the group consisting of hexane, heptane, octane, nonane, decane, dodecane, and combinations thereof.

[0060] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil yield is in the range of 25% to 87% (on dry cell weight basis).

[0061] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein microbial oil recovery is carried out by a method selected from a group consisting of extraction, precipitation, decantation, adsorptive separation, and combinations thereof. In another embodiment, the adsorptive chromatographic separation may be carried out with/without capturing agent/s.

[0062] In another embodiment of present disclosure, there is provided a method as described herein, wherein recovery/isolation of microbial oil may be done by extraction method. The extraction method may be carried out using solvent/s selected from group such as but not limited to polar organic solvent such as but not limited to alcohols e.g. branch chain or long chain alcohols, esters e.g. ethyl acetate, ketone, acids and nonpolar organic solvent such as but not limited to chloroform, dichloromethane, n-hexane, n-heptane etc.

[0063] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the said recovery of the produced microbial oil is carried out in the production broth simultaneously with the oil production.

[0064] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the said recovery of the produced microbial oil is carried out using an in-situ liquid extractant in a two-stage extractive production process.

[0065] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the method is carried out in batch mode, fed-batch mode, or continuous mode.

[0066] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil extraction is carried out using solvent selected from polar and non-polar organic solvents.

[0067] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the polar organic solvent is selected from the group consisting of branched chain alcohol, long chain alcohol, ester of branched chain alcohol, ester of long chain alcohol, and combinations thereof, and the nonpolar organic solvent is selected from the group consisting of n-hexane, chloroform, dichloromethane, n-heptane, decane, undecane, dodecane, and combinations thereof.

[0068] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein polar organic solvent is selected from the group consisting of branched chain alcohol, long chain alcohol, methanol, isopropyl alcohol, ester of branched chain alcohol, ester of long chain alcohol, ethyl acetate, ketone, and combinations thereof, and nonpolar organic solvent is selected from the group consisting of n-hexane, chloroform, dichloromethane, n-heptane, decane, undecane, dodecane, and combinations thereof. In another embodiment of the present disclosure, the polar organic solvent is methanol, isopropyl alcohol. In yet another embodiment, the non-polar organic solvent is chloroform, dichloromethane .

[0069] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil is intracellular, extracellular, or a combination thereof. [0070] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil was produced from lignin, lignin hydrolysate, lignin derived phenolic or aromatic compounds as a substrate causing an effective reduction in the COD of the substrate feed.

[0071] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil is converted into a wide variety of useful products including fuels, hydrocarbons, esters, polymers, oleochemicals, and value- added products.

[0072] In an embodiment of the present disclosure, there is provided a method for production of microbial oil as described herein, wherein the microbial oil is converted into a wide variety of useful products including fuels, hydrocarbons, esters, polymers, or oleochemicals.

EXAMPLES

[0073] The following examples are given by way of illustration of the present invention and should not be construed to limit the scope of present disclosure. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the claimed subject matter.

[0074] The working examples of the present disclosure would now be illustrated. These examples are neither restrictive nor intended to be limiting to the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and composition, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

[0075] While, there are reports about tolerance or bioremediation of the phenolic or aromatic compounds by the oleaginous microorganisms, the tolerance beyond a certain concentration is not reported. The present disclosure describes a method for sequential acclimatization or sensitization of the oleaginous microbes for the increased tolerance to the lignin or phenolic derived from lignin, i.e., lignin hydrolysate or lignin monomers and oligomers.

[0076] In addition to the increase in tolerance, the cell viability and the oleaginicity (oil production > 20% D.C.W) was maintained through process engineering for prolonged duration (more than 300hrs) in stressed conditions when lignin (or lignin hydrolysate) was the sole source of carbon.

[0077] The oleaginicity was at par with that obtained with glucose or fermentable sugars (greater than 80% DCW) through funneling pathways.

[0078] Moreover, a step wise feeding of the lignin/hydrolysate to the glucose-based system suggest that the oleaginous organism can utilize a mixed carbon source thus avoiding discrepancies due to recalcitrance of the substrate stream. Likewise, as there is an observed cellular/ lipid homeostasis in the stressed conditions while using sole carbon source as the substrate. The membrane fluidity as indicated by the unsaturation in the free fatty acid content allowed for expulsion and dynamics of the lipids out of the cells, necessitating its timely recovery.

[0079] There was no disintegration in the cell observed during stressed conditions suggesting their acclimatization or sensitization to the stressed conditions and increased oil production.

[0080] The efflux of the produced oil and influx of the lignin/monomer was marked to be equilibrated after a certain saturation limit achieved in the broth.

[0081] This was addressed using an applicable extractive production system which allowed for a docile and concomitant recovery of the oil with its production. This further enhanced the oil production.

[0082] Thus, the overall process efficiency can be increased by the suggested methodology, wherein the near complete conversion of all the components of LBM hydrolysate is achieved, with an effective reduction in COD.

Example 1

Method for Hydrolysis of lignin

[0083] Yarrowia lipolytica NCIM 3590 was cultivated on kraft lignin hydrolysed with different methods namely, chemical and enzymatic. The chemical method involved treatment of kraft lignin with 2% HNO3 (pH = 2) in a High-Pressure Reactor (Amar Equipment, HAST- C276) at 130°C and 8 bar pressure for 30mins. The enzymatic method used laccase enzyme for hydrolysis for 24 hrs at 60°C temperature. The Total Phenolic Content of Kraft lignin was found to increase by 6.8% and 13.8% by chemical and enzymatic hydrolysis respectively.

Example 2

Adaptation of oleaginous yeasts to lignin hydrolysate [0084] Stock cultures of yeast strains maintained at -80°C in MGYP (Malt extract, Glucose, Yeast extract and Peptone) medium with 40% (v/v) glycerol were revived in 50mL MGYP medium and was incubated at 28°C for 48hrs. This culture was used for pre-inoculum development in the MGYP growth medium with composition per litre: 3g malt extract, 20g glucose, 3g yeast extract, 5g peptone. The seed culture was transferred to the experimentation flasks with a MLYP medium having composition per litre: 3g malt extract, 20g lignin hydrolysate, 3g yeast extract, 5g peptone making a 10% (v/v) inoculum concentration and the culture was incubated at 28°C, 200rpm for seven days. All the solutions were prepared in sufficient amount of water.

[0085] The lignin hydrolysate was prepared by hydrolysis of Kraft Lignin (20g/L) by 2% HNO3 (pH = 2). This was further processed in the High-Pressure Reactor (Amar Equipment, HAST-C276) at 130°C and 8 bars pressure for 30 mins.

[0086] In order to adapt or sensitize the strains to the growth inhibitory phenolic compounds present in the lignin hydrolysate, the yeast cultures were transferred to a fresh media after every seven days. This process of adaptive evolution was carried out for seven generations leading to the adapted grown strains. The broth was centrifuged at 7000 rpm in refrigerated ultracentrifuge at 20°C to obtain a cell pellet. The cell density was thus estimated gravimetrically considering the moisture content. Intracellular lipid extraction from the wet cell biomass was carried out using isopropyl alcohol (IP A) (polar solvent) in a thermomixer at 45°C at 500 rpm. The process was repeated several times to ensure complete extraction of the intracellular oil.

[0087] Five strains of oleaginous microbes were tested for the production of microbial oil, viz., Yarrowia lipolytica, Lipomyces starkeyi, Rhodosporidum toruloides, Lipomyces lipofer and Rhodotorula glutinis. Out of the five microbes taken, Yarrowia lipolytica demonstrated the maximum intracellular oil content of 41.25% after seven generations whereas Lipomyces starkeyi showed the minimum intracellular oil content of 26.14% while Rhodosporidum toruloides, Lipomyces lipofer and Rhodotorula glutinis accumulated 33.10% 38.37% and 28.02% intracellular oil with respect to their Dry Cell Weight (DCW). The intracellular oil accumulation for all the yeast strains after seven generation can be seen in Table 1.

Table 1. Intracellular Oil content of oleaginous yeast strains before and after adaptation to lignin hydrolysate Intracellular Oil

Intracellular Oil content content w.r.t DCW

Strain w.r.t DCW after

before adaptation

adaptation (%)

(%)

Yarrowia lipolytica (NCIM

21.23 41.25

3590)

Rhodosporidium toruloides

20.04 33.10

(NCIM 3547)

Rhodotorula glutinis (NCIM

20.15 28.02

3168)

Lipomyces lipofer

20.68 38.37

(NCIM 3440)

Lipomyces starkeyi

20.46 26.14

(NCIM 3252)

[0088] The percentage values summarized in the table specifically indicate the intracellular oil content. The increase in the intracellular oil accumulation in all the oleaginous yeast strain imply the assimilation of lignin hydrolysate as a carbon source, thereby suggesting their successful adaptation to lignin hydrolysate.

Example 3

Growth associated oil production in oleaginous yeasts with variation in lignin hydrolysate concentration

[0089] All the oleaginous yeast strains were cultivated with media consisting of 3g/L malt extract, 3g/L yeast extract and 5g/L peptone along with concentration of lignin hydrolysate varying between 0.2% and 2% keeping rest of the experimental conditions constant. The cell density and intracellular oil content were gravimetrically estimated after 48hrs as shown in Table 2.

Table 2. Microbial oil production by oleaginous yeast strains with variation in lignin concentration Intracellular Intracellular

Increase

Oil content Increase in Oil content in DCW

w.r.t. DCW DCW (g/L) w.r.t. DCW

(g L)

(%) (%)

Yarrowia

2.0 22.07 3.2 23.56 lipolytica

Rhodosporidium

1.8 20.12 3.0 21.23 toruloides

Rhodotorula

1.0 20.33 2.8 22.41 glutinis

Lipomyces lipofer 1.4 21.52 2.6 21.73

Lipomyces starkeyi 1.2 20.68 2.6 22.62

[0090] As seen in Table 2, there was a considerable increase in the intracellular oil content and thereby the oleaginicity of the organism in 48hrs with an increased lignin hydrolysate concentration. This indicates that the availability of the excess carbon source in the growth medium promotes oil production by inducing necessary stress conditions to channel the carbon flux towards oil production.

Example 4

Assimilation of carbon source in the growth medium

[0091] Amongst the available oleaginous microbial (yeast) strains Yarrowia lipolytica demonstrated maximum oil production after the adaptation, while the other strains demonstrated an identical behaviour as that of their natural variants. Therefore, further studies were carried out using Yarrowia lipolytica.

[0092] The fermenter study was carried out in a fully instrumented and automatically controlled BIOSTAT® B plus fermenter (Sartorius Stedim Biotech S.A, Germany) using a working volume of 1 L. Temperature and stirring rate were monitored and controlled at 28 °C +/- 1°C, 200 rpm, respectively. The bioreactor was sparged with atmospheric air maintaining aeration of 1 volume per volume per minute (vvm) (1 standard litre per minute (slpm)). Partial oxygen pressure (p0 ₂ ) was constantly monitored online.

[0093] Yarrowia lipolytica was first grown with MGYP medium as described in Example 1, for 120h in a 1L fermenter. Later, a Stepwise Continuous Fed Batch (SCFB) mode of operation was applied wherein 0.5L of the broth was harvested and 0.5L of media was supplemented with lignin hydrolysate (20g/L) so as to have a mixed carbon source in the medium and then Yarrowia lipolytica was grown for 120h along with other media components having the same concentration as earlier. Finally, the entire volume of media (1L) was replaced with lignin hydrolysate (20g/L) i.e now having MLYP medium as stated in Example 1 and Yarrowia lipolytica was grown for another 120h keeping the other media components constant.

Table 3. Production of microbial oil from glucose in combination with lignin

hydrolysate using Yarrowia lipolytica

[0094] As can be observed from Table 3, the oleaginous yeast strain of Yarrowia liplytica NCEVI 3590 was found to accumulate intracellular oil in the growth media containing glucose, lignin or a mixed carbon source containing glucose and lignin hydrolysate. The highest microbial oil was derived from media containing exclusively lignin (MLYP). Thus, it can be inferred that lignin and lignin derived phenolic compounds can be similarly assimilated as a carbon source by Yarrowia lipolytica for growth and oil accumulation at fermenter scale. Further, the co-utilization of glucose and lignin in a mixed carbon source containing growth medium suggest the possibility of using lignocellulosic biomass hydrolysates directly into the growth medium, without any pre-treatment for separation of lignin-based components.

Example 5 Mode of operation for growth associated production of microbial oil

[0095] All the studies performed above were carried out for all the above-mentioned oleaginous yeast strains. However, it was evident that among all the strains Yarrowia lipolytica NCEVI 3590 showed better adaptability for utilization of lignin and also gave comparatively high intracellular oil content. Therefore, hereafter Yarrowia lipolytica NCEVI 3590 was considered for further studies.

Batch Cultivation

[0096] Yarrowia lipolytica NCIM 3590 was cultivated for cell growth in 2L fully instrumented and automatically controlled BIOSTAT® B plus fermenter (Sartorius Stedim Biotech S.A, Germany) using a working volume of 1 L. Temperature and stirring rate were monitored and controlled at 28°C +/- 1°C, 200 rpm, respectively. The bioreactor was sparged with atmospheric air maintaining aeration of 1 volume per volume per minute (vvm) (1 standard litre per minute (slpm)). Partial oxygen pressure (p02) was constantly monitored online. The growth medium consisted of MLYP medium and the growth conditions and oil extraction methodologies were kept same as that described in Example 1.

Continuous cultivation

[0097] The cell biomass production of the oleaginous yeast strain Yarrowia lipolytica NCEVI 3590 was carried out in 2L fermenter containing 20g/L lignin hydrolysate along with 3g/L Malt extract, 3g/L Yeast extract and 5g/L Peptone. Culturing was carried out in a batch mode, at 28°C, agitation of 200 rpm and aeration of 1 volume per volume per minute (vvm) for 48h, till a cell density of 8g/L was achieved. Later, a continuous system was set up at 0.18 h ^"1 dilution rate and biomass yield was observed to be 20g/L. The oil content for the continuous system was estimated after 48h.

[0098] The comparative study of batch and continuous mode of operation with respect to oil content and cell density has been summed up in Table 4.

Table 4. Modes of operation for production of microbial oil

[0099] As seen in Table 4, Yarrowia lipolytica demonstrated an increased cell density and oleaginicity in continuous mode of operation. The increase in the cell density can be ascribed to the continuous media replenishment achieved in a chemostat mode, whereas the effective increase in an intracellular oil accumulation can be essentially attributed to the sequential adaptation of the yeast for assimilating lignin as a carbon source. The continuous replenishment of the broth with fresh lignin helped in inducing the necessary stress conditions due to increased concentration of carbon source in the growth medium, causing an increase in the intracellular oil accumulation.

Example 6

Utilization of various lignin hydrolysates Yarrowia lipolytica NCIM 3590

[00100] Yarrowia lipolytica was cultivated on feedstocks with different lignin hydrolysates. These included Kraft lignin hydrolysate, maize bran hydrolysate and wheat straw hydrolysate. Yarrowia was grown on these feeds for 48h at 28°C and 200rpm. For these feeds, total phenolic assay was also performed to estimate the lignin consumption throughout the study which has been shown in Table 5.

[00101] It was inferred that lignin hydrolysate obtained from different sources resulted in the production of different percentages of the intracellular oil. It was found that kraft lignin hydrolysate obtained by the hydrolysis of kraft lignin produced the highest percentage of the intracellular oil. Therefore, kraft lignin hydrolysate is working best among the other sources. Table 5. Utilization of various lignin hydrolysates by Yarrowia lipolytica NCIM 3590

Example 7 Utilization of lignin monomers by Yarrowia lipolytica NCIM 3590

[00102] Along with various lignin sources, lignin monomers namely syringic acid and benzoic acid were used for the growth of Yarrowia lipolytica NCIM 3590 with a concentration of 20g/L keeping all the experimental conditions constant as example 5. Similar to earlier studies, the cell density, total phenolic content and oil content were estimated as shown in Table 6. It was inferred from Table 6 that the monomers of the lignin hydrolysate like syringic acid and benzoic acid are assimilated by the organism and the intracellular oil is produced by using these molecules as sole carbon source.

Table 6. Utilization of lignin monomers by Yarrowia lipolytica NCIM 3590

Example 8

Estimation of oil from various sources (Analysis of lignin and microbial oil)

[00103] Analysis of lignin utilization has been a vital part of the study. The qualitative analysis was performed using High performance liquid chromatography (HPLC) (The Agilent 1260 Infinity Quaternary LC, C I S Column). It was observed that the lignin monomers namely, benzoic acid, syringic acid, proto-catechuic acid present in lignin were assimilated by Yarrowia and accumulated majorly in the form of triglycerides. The fatty acid composition predominantly showed the presence of, inyristic acid (C14), penta-decanoic acid (C15), palmitic acid (C I 6), palmit-oleic acid (C16: l ), margaric acid (CI 7), heptadecenoic acid (C17: l), heptadeca-dienoic acid (C17:2), stearic acid (C18:0), oleic acid (C 18: l) and linoleic acid (CI 8:2), as shown in Table 7 below.

Table 7. Fatty acid composition of microbial oil

96 - - 41.36 3.47 - - 25.3 2.25 25.8 1.87

120 - - 40.5 12.17 - - 27.54 1.67 16.28 1.87

24 - - 42 - 3.89 1.13 38.72 2.2 12.06 -

Mixed

48 - - 53.87 - 2.79 - 37.31 1.19 4.83 - source

72 - - 53.13 - 2.79 - 30.76 1.16 2.5 9.7 (Glucose

96 - - 44.59 - 3.27 - 38.57 3.06 10.52

and Lignin)

120 - - 34.18 4.4 - - 16.33 2.01 16.22 26.86

24 12.34 - 42.67 6.15 - - - 0.07 7.56 31.182

48 9.44 - 42.79 5.04 - - - 0.26 9.47 33

72 Lignin 8.42 - 42.7 7.44 - - - 0.22 11.25 29.97

96 6.23 - 38.73 9.46 1.42 - 10.54 0.99 17.52 15.11

120 5.74 - 30.32 9.97 0.07 - 9.03 1.57 29.15 14.15

Maize bran

120 5.28 48.79 14.22 2.54 8.85 20.32 Hydrolysate

Wheat

120 straw 9.16 8.93 24.33 9.53 0.78 3.77 - 1.9 31.5 10.1 hydrolysate

[00104] As can be observed from Table 7 above, the fatty acid profile of the microbial oil produced using lignin from different sources was comparable to that obtained by fermentable sugars.

[00105] Over the period of time, it was observed that there was an increase in the elongation and unsaturation in the produced lipids. This indicates that the lipid homeostasis is being maintained in the stressed conditions by expulsion of the produced lipid, thereby suggesting the mobility and dynamics of the lipid. The elongation and unsaturation in the lipids was consistent with glucose-based system, glucose system sequentially fed with lignin and solely lignin-based system. This observation depicts an acclimatization or sensitization of the oleaginous yeast strain towards the stress conditions.

Example 9

Estimation of extracellular oil

[00106] The oleaginous yeast strains were found to expel a considerable amount of oil into the broth. The broth containing kraft lignin hydrolysate contained significant extracellular oil i.e., oil present in the aqueous medium. This phenomenon can be regarded as a natural tendency of the organism to maintain an equilibrium under stress conditions imposed by the culturing conditions. The increase in the carbon source concentration in the broth compels the organism to accumulate oil while the other growth processes are altered due to limited nitrogen.

However, to maintain an equilibrium inside the cell or lipid homeostasis, the organisms expels the surplus lipid. Thus, in order to promote the assimilation of lignin and enhance the oil production in the oleaginous yeast, the excretion of extracellular oil and its timely recovery becomes important. For the process of extraction of this extracellular oil, a solvent mixture of chloroform and methanol in the ratio of 1 : 1 with solvent to medium ratio of 3 : 1 was used. After rigorous shaking, the mixture was allowed to separate for a while and then the two layers thus formed were carefully separated. Each layer was collected, and the solvent was distilled in Rotary vacuum evaporator at high temperature and vacuum. The oil thus obtained was gravimetrically estimated for its content. The extracellular oil was found to be constant at 3.15g/L and hence the total microbial oil yield was found to be 0.18 g of oil/g of lignin.

[00107] Thus, the extracellular oil production by oleaginous yeast needs to be considered while estimating total oil production capacity. The total oil produced thus can be regarded as a cumulative of Intracellular and extracellular oil.

Example 10

Two-stage oil production process

Growth Phase

[00108] Yarrowia lipolytica NCIM 3590 was cultured in a chemostat mode at a dilution rate of 0.12 h ^"1 for cell growth in 2L fully instrumented and automatically controlled BIOSTAT® B plus fermenter (Sartorius Stedim Biotech S.A, Germany) using a working volume of 1 L. Temperature and stirring rate were monitored and controlled at 28°C +/- 1°C, 200 rpm, respectively. The bioreactor was sparged with atmospheric air maintaining aeration of 1 volume per volume per minute (vvm) (1 standard litre per minute (slpm)). The growth medium consisted of Yeast extract (10 g/L), Peptone (20g/L) and Glucose (100 g/L). High cell biomass productivity of 20g/L/h was obtained while operating at this critical dilution rate. Production phase or Oil accumulation phase (Estimation of both intracellular and extracellular oil)

[00109] An ammonia treated wheat straw lignin rich stream was treated with 70% cone.

HNO3 to carry out acid precipitation of the polymeric lignin. The precipitated lignin was separated using filtration and centrifugation to obtain a solid mass of polymeric lignin weighing 1.2g/L, while the monomeric lignin rich supernatant was further neutralized and filtered through 0.2μ filer to obtain a clarified feed stream having COD 8053.33 ppm.

[00110] The obtained monomeric lignin rich stream along with the high cell biomass

(obtained from the growth phase) was fed to another fermenter to have an effective cell load of 8gDCW/L. The oil production was carried keeping in a batch mode at 28 °C and 200 rpm with an aeration of 1 vvm. Increase in oil content was observed with decrease in COD of the medium, thus suggesting effective assimilation of monomeric lignin (Table 8). Moreover, an increase in intracellular lipid content with a steady increase in the increase in extracellular oil content was observed over the period of time. Maintenance of the cell density was observed throughout the operation suggesting the viability of the cells and bioconversion of available carbon into oil. Further an insignificant increase in the extracellular oil titre imply the oil saturation in the aqueous medium, necessitating its recovery.

Table 8. Oil production using lignin hydrolysate as a sole nutrient source.

Example 11

Simultaneous production and recovery of the microbial oil

[00111] Yarrowia lipolytica cultivated in lignin hydrolysate for 192h (as described earlier) was further incubated in an Erlenmeyer flask for 72 h at 28°C with an addition of 1% (v/v) n-Hexane. The addition of the alkane caused for an increase in the oil production and about 60% reduction in the COD content within 72h (Table 9). Further an increase in the oil titre corresponding to an increased extracellular lipid content (as seen in Table 9) suggest sequestration of the intracellular oil in the extractant phase. Moreover, the cell density in the broth was found constant indicating the viability of the cells, biocompatibility of the extractant and an effective milking of the microbial oil from the oleaginous yeast.

Table 9. Simultaneous production and in-situ recovery of the microbial oil (h) (ppm) Total

Cell Total Oil Oil

Intracellular Extracellular

Density titre content

Lipid Lipid

(g L) (g L) (%

DCW)

0 1053.33 15.05 0.92 0.37 1.29 44.54

72 417.2 14.2 0.03 2.45 2.48 87.32

[00112] Analysis of the broth samples for the substrate consumption using showed a significant assimilative depolymerization (Table 10). Further an LC-MS analysis of the extracted oil showed the presence of Triglycerides along with Fatty acids (Table 11).

Table 10. LC-MS analysis of the broth samples

[00113] Table 10 illustrates the assimilation of a lignin constituent by the organism, which can be seen as a disappearance in the ion peak at m/z 546.2.

Table 11. LC-MS analysis of the oil

Table 11 illustrates the lipid profile of the microbial oil produced by the oleaginous yeast utilizing lignin.

Example 12 Two-stage extractive Production process

Batch process:

[00114] Yarrowia lipolytica NCIM 3590 was cultivated for 48h in a nutrient enriched glucose based medium to achieve a cell density of 25g/ L in a BIOSTAT® B plus fermenter (Sartorius Stedim Biotech S.A, Germany) using a working volume of 2 L. Temperature and stirring rate were monitored and controlled at 28°C +/- 1°C, 200 rpm, respectively, while maintaining aeration of 1 volume per volume per minute (vvm) (1 standard litre per minute (slpm).

[00115] Acid hydrolysis of Alkali Kraft lignin solution (20g/L) was carried out with cone. HNO3 to obtain precipitation of polymeric lignin (less than lg). The monomeric lignin rich stream obtained as a supernatant (2L) was fed to the fermenter for oil production with 50 g cells of Yarrowia lipolytica in a batch mode. After 192 h of incubation, 1% v/v of Hexane was added to the broth. The fermenter was monitored for the COD content, cell density and the Oil yield throughout the run.

Table 12. Extractive two-stage oil production in batch mode

(192+48h

with 1%

hexane)

288

(192+96h

703.86 22.65 0.83 2.00 2.83 62.47 with 1%

hexane)

312

(192+120h

663.86 22.5 0.68 3.00 3.68 81.78 with

hexane)

*N.E : Not Estimated

[00116] As seen in Table 12, Yarrowia lipolytica showed an intracellular accumulation of the oil with a considerable consumption in the COD. However, the use of hexane after 192h in the fermenter augmented the fluxes for oil production with a drastic decrease in COD and an attributed increase in the oil yield w.r.t DCW. This can be observed as an induced expulsion of the produced oil in Yarrowia as implied by the increased extracellular lipid and decrease intracellular content. This further suggests that the use of n-hexane aids in increasing the miscibility of the oil in aqueous medium and thereby causing the mobility of the intracellular lipid out of the cells.

Chemostat process

[00117] A 2L broth containing Yarrowia cells with a density of 20g/L was fed with a lignin hydrolysate stream at a dilution rate of 0.0075 h ^"1 (0.25 ml/min) in a chemostat mode to cause for an effective replacement of the broth for oil production. The COD of the stream was 31,386 ppm. This mode of operation provided for a sequential adaptation of the yeast to the lignin stream while increasing its oleaginicity (Table 13).

[00118] The rate of COD consumption and thereby the oil production was further enhanced by spiking 20ml of n-hexane to the system.

Table 13. Two-stage extractive production in a chemostat

(g L) content

(%)

0 0 0 20 NE NE NE NE 0 0.0

18 13.5 53.3 18 0.6 0.1 0.7 19.44 4183.81 13.3

40 30 6666 18 0.9 0.1 1 27.78 2749.8 8.8

67 50.25 6844.00 18.57 1.00 0.10 1.10 29.62 8927.46 28.4

96 72.00 12633.33 21.87 1.20 0.10 1.30 29.73 9964.58 31.7

120 90.00 13466.67 24.93 1.40 0.10 1.50 30.09 14780.73 47.1

134 100.50 14300.00 26.78 1.60 0.10 1.70 31.74 17086 54.4

182

(134+48 h

After 118.50 11053.30 21.875 1.00 0.86 1.86 42.45 26139.11 70.6 l%Hexane

addition)

206

(134+72 h

After 154.50 17544.00 22.2 1.00 1.26 2.26 50.90 30947.37 64.0 l%Hexane

addition)

230

(134+96 h

After 172.50 24860.00 21.9 0.96 1.69 2.65 60.50 29280.85 54.2 l%Hexane

addition)

*N.E: Not Estimated

[00119] Table 13 suggests the applicability of the two-stage extractive production process for microbial oil using lignin in any mode of operation.

Advantages of the present disclosure

[00120] The present disclosure describes the microbial oil production using lignin as a carbon source for assimilation and bioconversion by the oleaginous yeast into lipid. The method as described herein allows convenient recovery of microbial oil from phenolic-rich lignin, which is known to be a difficult feedstock for bioconversion due to its negligible sugar content. Since lignin is majorly present in most agricultural waste, the method can be beneficial in converting waste to useful products/fuel.