METHOD FOR FATTY ACID ALKYL ESTER SYNTHESIS AND THEIR EXTRACTION FROM OLEAGINOUS MICROBES

Abstract

The present invention relates to a method for in-situ synthesis of alkyl esters of fatty acids and their efficient extraction from oleaginous microbes. Particularly, the present invention provides a rapid method for downstream processing of oleaginous biomass for in-situ synthesis of alkyl esters of fatty acids and their efficient extraction. The method of the present invention results in increased transesterification efficiency (>95%) of FAAE production from wet biomass, while eliminating the necessity of biomass harvesting, drying and large requirement of chemical solvents.

Claims

1. A rapid downstream processing method for in situ synthesis and extraction of fatty acid alkyl esters from an oleaginous wet biomass slurry or a fermentation broth from fermenters having a oleaginous microbe, the method comprising: (i) subjecting the oleaginous wet biomass slurry or the fermentation broth to an osmotic treatment and an acidic treatment to obtain a treated biomass slurry; (ii) mixing the treated biomass slurry of step (i) with an extraction mixture to obtain a mixture, wherein the treated biomass slurry has a moisture content in a range of 80%-90%; (iii) adding a non-polar solvent to the mixture of step (ii) to obtain an extract; (iv) subjecting the extract to evaporation and treating with a polar solvent; and (v) adding a moisture adsorbent to the extract to obtain fatty acid alkyl esters (FAAE) rich in omega-3 fatty acids, wherein adding the moisture adsorbent increases the FAEE transesterification efficiency up to 95%.

2. The method as claimed in claim 1, wherein the fatty acid alkyl esters are methyl ester, ethyl ester and propyl ester.

3. The method as claimed in claim 1, wherein in step (ii) the treated biomass slurry is mixed with extraction mixture and is agitated at 100 rpm at a temperature of 90° C. for a duration of 1 hour.

4. The method as claimed in claim 1, wherein the oleaginous microbe is a heterotrophic microalga.

5. The method as claimed in claim 1, wherein the oleaginous wet biomass slurry is directly harvested from a reactor.

6. The method as claimed in claim 1, wherein the osmotic treatment in step (i) comprises treating the oleaginous wet biomass slurry with single distilled water, double distilled water, triple distilled water, or water having low TDS.

7. The method as claimed in claim 1, wherein the acidic treatment in step (i) comprises treating the oleaginous wet biomass slurry with an organic or an inorganic acid solution.

8. The method as claimed in claim 1, wherein the non-polar solvent is selected from the group consisting of hexane, heptane, chloroform, and acetone.

9. The method as claimed in claim 1, wherein the polar solvent is selected from the group consisting of acidic ethanol, methanol, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF).

10. The method as claimed in claim 1, wherein the polar solvent is acidic ethanol.

11. The method as claimed in claim 9, wherein a ratio of the oleaginous wet biomass slurry to the acidic ethanol is in a range of 1:5-1:60.

12. The method as claimed in claim 1, wherein the moisture adsorbent is selected from the group consisting of activated alumina, sodium sulphate, silica gel, and calcium chloride.

13. The method as claimed in claim 1, wherein the extraction mixture comprises ethanol and hydrochloric acid.

14. The method as claimed in claim 1, wherein the FAAEs are rich in omega-3 fatty acids, wherein the omega-3 fatty acids are palmitic acid, oleic acid, Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), or Eicosapentaenoic acid (EPA).

15. The method as claimed in claim 1, wherein the FAEEs are obtained within 4 hours from a time of harvesting of the fermentation broth.

16. The method as claimed in claim 1, wherein the method eliminates dewatering of the oleaginous wet biomass slurry.

17. The method as claimed in claim 1, wherein the osmotic treatment and the acidic treatment of the oleaginous wet biomass slurry results in a synergistic effect and reduce an ash content of the oleaginous wet biomass slurry.

18. The method as claimed in claim 14, wherein the the omega-3 fatty acids are in a range of 15%-50% of total fatty acids.

19. The method as claimed in claim 14, wherein the method reduces a time of downstream processing and eliminates oxidation or epoxidation of omega-3 fatty acids.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0043] These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings wherein:

[0044] FIG. 1 illustrates comparison of fatty acid composition processed by Folch method and direct transesterification.

[0045] FIG. 2 illustrates the effect of biomass to solvent ratio on extraction yields from dry biomass.

DETAILED DESCRIPTION OF THE INVENTION

[0046] 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.

Definition

[0047] For the purposes of this invention, the following terms will have the meaning as specified therein:

[0048] 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.

[0049] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0050] 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.

[0051] The term “including” is used to mean “including but not limited to” “including” and “including but not limited to” are used interchangeably.

[0052] The present invention relates to a rapid method for downstream processing for in-situ synthesis of alkyl esters of fatty acids and their efficient extraction from wet biomass slurry. Particularly, the present invention relates to a method to eliminate the necessity of dewatering of biomass slurry by biomass harvesting and drying before FAAEs synthesis and extraction from oleaginous biomass. The method disclosed in the present invention increases the transesterification efficiency (>95%) of FAAEs production from wet biomass without adding large volume of solvents such as ethanol or methanol to overcome the negative effect of water presence in the reaction. The method results in more than 95% FAAE transesterification efficiency from treated oleaginous biomass slurry via two step in-situ transesterification reaction. Obtained FAAEs from wet oleaginous biomass slurry can be directly used as biodiesel or in mix with diesel fuel. These alkyl esters of fatty acids also have wider applications in pharmaceutical as well as in nutraceutical industry due to presence of higher amount of omega-3 fatty acids like Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), Eicosapentaenoic acid (EPA). Accordingly, the present invention provides a means of downstream processing of wet oleaginous biomass slurry for fatty acid alkyl ester synthesis and their extraction while eliminating the necessity of biomass harvesting, drying and large requirement of chemical solvents.

[0053] Thus, in accordance with the present invention, there is provided a rapid downstream processing method for synthesis and extraction of fatty acid alkyl ester from an oleaginous microbe, the method comprising: [0054] (i) subjecting wet biomass slurry to osmotic and acidic treatment; [0055] (ii) mixing the treated biomass slurry of step (i) having moisture content in the range of 80%-90% with an extraction mixture; [0056] (iii) adding non-polar solvent to the mixture of step (ii) to obtain an extract; [0057] (iv) subjecting the extract to evaporation and treating with a polar solvent; and [0058] (v) adding moisture adsorbent to the extract to obtain fatty acid alkyl ester (FAAE) rich in omega-3 fatty acids with the non-polar solvent,
wherein the addition of moisture adsorbent at step (v) increases the FAEE transesterification efficiency up to 95%.

[0059] In an embodiment of the present invention, there is provided a method wherein the fatty acid alkyl ester is selected from methyl ester, ethyl ester and propyl ester. The method of the present invention can be used to obtain fatty acid methyl ester, fatty acid ethyl ester and fatty acid propyl esters etc. depending upon the addition of alkyl group donating solvent in the reaction.

[0060] In another embodiment of the present invention, there is provided a method wherein the oleaginous microbe is a heterotrophic microalga belonging to the order Thraustochytrids. In the present invention, the Thraustochytrids were cultivated in different volumes in culture flasks including but not limited to 50 mL, 100 mL 250 mL flasks or in fermenters including but not limited to 2 L fermenters, 10 L fermenters, 60 L fermenters, 100 L fermenters or 500 L fermenters.

[0061] In a preferred embodiment of the present invention, the oleaginous microbe is a microalga. Particularly, the microalgae is Schizochytrium sp. DBT-IOC1 (MTCC 5890). The Schizochytrium MTCC 5980 used in the present invention was isolated from Zuari-Mandovi mangroves, in Goa, India (S15°29′57.39″, E73°52′6.13″) near the Arabian Sea.

[0062] In yet another embodiment of the present invention, there is provided a method wherein the wet biomass slurry is directly harvested from the fermenter.

[0063] In still another embodiment of the present invention, there is provided a method wherein osmotic treatment in step (i) comprises treating the wet biomass slurry with single distilled water or double distilled water or triple distilled water or water having very low TDS.

[0064] In an embodiment of the present invention, there is provided a method wherein acidic treatment in step (i) comprises treating the wet biomass slurry with an organic or inorganic acid solution.

[0065] In the present method, the wet biomass slurry is subjected to both osmotic treatment and acid treatment. The osmotic and acidic treatment of slurry results in a synergistic effect. Further, the osmotic and acidic washing or treatment results into reduction in ash content of biomass slurry.

[0066] In another embodiment of the present invention, there is provided a method wherein the non-polar solvent used in the extraction is selected from hexane, heptane, chloroform, and acetone. In a preferred embodiment, the non-polar solvent used in the extraction is hexane.

[0067] In yet another embodiment of the present invention, there is provided a method wherein the polar solvent is selected from acidic ethanol, methanol, dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). In a preferred embodiment, the polar solvent is acidic ethanol.

[0068] In still another embodiment of the present invention, there is provided a method wherein the ratio of biomass to acidic ethanol is in the range of 1:5-1:60. In a preferred embodiment of the present invention, the ratio of biomass to acidic ethanol is 1:10.

[0069] In an embodiment of the present invention, there is provided a method wherein the moisture adsorbent is selected from activated alumina, sodium sulphate, silica gel or calcium chloride. In a preferred embodiment, the moisture adsorbent is activated alumina or sodium sulphate.

[0070] In another embodiment of the present invention, there is provided a method wherein the extraction mixture comprises ethanol and hydrochloric acid.

[0071] In yet another embodiment of the present invention, there is provided a method wherein the obtained FAAEs are rich in omega-3 fatty acids selected from palmitic acid, oleic acid, Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA) or Eicosapentaenoic acid (EPA). Further, the omega-3 fatty acid content in the range of 15%-50% or more of total fatty acids. The present method also reduces the chances of oxidation or epoxidation of omega-3 fatty acids.

[0072] In still another embodiment of the present invention, there is provided a method wherein the FAAEs are obtained within 4 hours from the time of broth harvesting.

[0073] In an embodiment of the present invention, there is provided a method which further comprises washing FAAEs with 2% KHCO.sub.3 and passing through anhydrous Na.sub.2SO.sub.4 for removal of moisture.

[0074] The present invention discloses a rapid and less energy intensive downstream processing method for obtaining FAAEs directly from wet oleaginous biomass slurry without harvesting and drying of wet biomass, which helps in reducing the downstream processing time and chances of oxidation or epoxidation of omega-3 fatty acids, since omega-3 fatty acids are very prone to oxidation/epoxidation due to multiple double bonds. Further, the present method results in more than 95% of FAAE (biodiesel) from wet oleaginous biomass. The present method eliminates the need of cost and energy intensive steps of dewatering of biomass including harvesting and drying. The slurry or fermentation broth from fermenters can directly processed with this method. Moreover, since this method can be also deployed as continuous method of downstream processing of wet oleaginous biomass therefore, it can be integrated with continuous fermentation system, where fermentation broth after exiting fermenters is continuously processed to obtain FAAEs. Therefore, the method disclosed in the present invention provides a downstream processing method for in-situ synthesis of alkyl ester of fatty acids and their extraction directly from wet oleaginous biomass slurry without involving any biomass harvesting methods. Also, the method eliminates the need of biomass drying before lipid/oil extraction and transesterification without compromising fatty acid alkyl ester (FAAE) extraction yield from wet oleaginous biomass slurry.

EXAMPLES

[0075] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. 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, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to methods, and experimental conditions described, as such methods and conditions may vary.

Example-1: Cultivation of Oleaginous (Thraustochytrid) Biomass

[0076] A continuous Thraustochytrid fermentation system was set up in 100 L fermenter having working volume of 60 L. Fermenter, having 60 L acetate rich minimal growth media, was inoculated with 48 hours old 3 L culture of Schizochytrium sp. DBTIOC-1 (MTCC 5890). The microalgal strain Schizochytrium MTCC 5980 was isolated from Zuari-Mandovi mangroves, in Goa, India (S15°29′57.39″, E73°52′6.13″) near the Arabian Sea.

[0077] Fermenter was continuously aerated with air and oxygen along with agitation to maintain required dissolved oxygen level. Fermenter was continuously fed with media while broth was continuously taken out through level control. Broth was collected in 200 L tank. This broth was then centrifuged at 4000 rpm for 15 minutes to remove depleted media components and water. Pellets were washed with distilled water twice before drying. This biomass slurry was still containing more than 75% of moisture therefore, the biomass slurry was dried at 80° C. in large trays in hot air oven for 4-5 days. Dried biomass flakes were then powdered with mixer grinder for lipid extraction and fatty acid ethyl ester synthesis. Some part of fermentation broth was stored at −20° C. to study in-situ transesterification of lipids into fatty acid ethyl esters later from wet algal biomass slurry.

Example-2 Comparison of Folch Method with Direct Transesterification or In-Situ Ethyl Ester Synthesis Method

[0078] Total lipid content of biomass was estimated by the modified Folch method using chloroform and ethanol (2:1) solvent mixture in the ratio of 1:60 (dry biomass:solvent mixture). Dried powder of biomass (20 g) was added in 3 L round bottom flasks having 1.2 L solvent mixture and agitated at 100 rpm at 70° C. for 1 hour. Whole mixture was centrifuged at 4500 rpm for 15 minutes for solid liquid separation. Supernatant was transferred in separating funnel followed by addition of hexane for phase separation. Upper layer containing lipid was carefully collected followed by solvent evaporation in Rotary evaporator. All the steps were repeated thrice for complete extraction of lipid from biomass. Lipid was measured gravimetrically followed by FAEE synthesis and analysis as per protocol given by Gupta et. al. in 2016. Total FAEE content was calculated gravimetrically as well as using GC-FID data and these values were taken as a reference to calculate FAEE extraction yield of different samples (FIG. 1).

[0079] In Direct transesterification method, 20 g of dry biomass powder was mixed with 1.2 L of extraction mixture having 1.18 L ethanol and 20 ml of HCl (biomass to solvent ratio 1:60) and agitated at 100 rpm at 90° C. for 1 hour. Whole mixture was transferred in separating funnel followed by addition of hexane for phase separation. Upper layer containing FAEE was carefully harvested followed by hexane evaporation in Rotary evaporator. FAEE was calculated gravimetrically as well as with GC-FID to quantify FAEE extraction yield and FAEE transesterification efficiency. FAEE content (5.25 g) was found to be comparable with that of Folch method (5.4 g) suggesting complete extraction of FAEE from biomass in direct transesterification with FAEE transesterification efficiency of more than 97% (Table 1). This reflects that almost all of lipid, present in biomass, is converted into ethyl esters of fatty acid without any significant change in fatty acid composition (FIG. 1). Biomass to ethanol ratio was further optimized to reduce the amount of ethanol needed for the reaction. 20 g of biomass was added in 1.18 L or 0.78 L or 0.38 L or 0.18 L or 0.08 L of ethanol while amount (20 ml) of acid catalysts i.e., HCl was same in all the reactions. FAEE extraction yield was almost equal till biomass to solvent ratio of 1:10, but there here was significant reduction in FAEE extraction yield when biomass to solvent ratio was reduced to 1:5 suggesting almost complete FAEE extraction when biomass to solvent ratio of 1:10 was applied for the reaction (FIG. 2).

TABLE-US-00001 TABLE 1 Comparison of Folch method and Direct transesterification method Folch method Direct Parameters (Reference) transesterification FAEE Content (g) from 20 g 5.4 5.3 dry biomass FAEE extraction yield (%) 100 98.14 FAEE Extraction 100 97.5 efficiency (%) C16:0 (%) 30.68 30.08 C22:6n3 (%) 15.86 17.64

Example-3: Production of FAEE from Present Method (Direct Transesterification or In-Situ Ethyl Ester Synthesis of FAEE) from Dry and Wet Biomass

[0080] 100 g to 110 g of wet biomass paste or slurry, having 80%-90% moisture content, was taken for in-situ synthesis of FAEE and their FAEE extraction yields were compared with dry biomass as per direct transesterification method given in Example 2. FAEE extraction yield from wet biomass was found to be almost 30% less than dry biomass suggesting incomplete extraction (Table 2). This implies the fact that cell wall is acting as barrier in solvent accessibility of lipid hence needs to be disrupted for better extraction. To overcome the issue of incomplete extraction, wet biomass pastes or slurry or fermentation broth was directly washed with 0.3N HCl (for acidic treatment) or low TDS water (for osmotic shock) or both. There was not much improvement in FAEE extraction yield when slurry was washed with acid or water alone however when both were combined there was significant improvement in FAEE extraction yield (Table 3). Subsequently this step was added before proceeding for FAEE extraction from wet biomass slurry. Although FAEE extraction yield was equal to that from dry biomass, but FAEE transesterification efficiency was still very low from 50% to 60% of total lipid suggesting incomplete transesterification reaction and making it unsuitable for biodiesel application (Table 4).

TABLE-US-00002 TABLE 2 Comparison of extraction yields from dry biomass and wet algal biomass slurry Parameters Dry biomass Wet algal biomass slurry FAEE Content (g) from 5.21 3.69 20 g dry biomass FAEE content (%) 26.05 18.45 FAEE extraction yield 96.5 68.33 (%) in comparison to

TABLE-US-00003 TABLE 3 Effect of different washing methods on extraction yields from wet algal biomass slurry Wet algal biomass slurry With no Acid Osmotic Osmotic + Parameters treatment treatment treatment acidic FAEE Content (g) from 3.69 4.15 4.1 5.16 20 g dry biomass FAEE content (%) 18.45 20.75 20.5 25.82 FAEE extraction yield 68.33 76.85 75.92 95.55 (%) in comparison to Folch method

Example-4: Increasing the Transesterification Efficiency for In-Situ Ethyl Ester Synthesis of Fatty Acids from Wet Thraustochytrid Biomass Slurry

[0081] Low transesterification efficiency from wet biomass slurry or broth is reported to be due to the presence of water in the reaction. Therefore, the method for direct transesterification was further modified to negate the effect of water on FAEE transesterification efficiency. Particularly, moisture adsorbents such as activated alumina or sodium sulphate were added in the reaction while a second transesterification step was added in the protocol. Sequence of moisture adsorbents addition in multistep transesterification reaction was reported to have effect on FAEE transesterification efficiency. Hexane fraction was collected from direct transesterification method as given in Example 3 followed by solvent evaporation under rotary evaporator. FAEE extract was subjected to a second transesterification reaction with acidic ethanol and FAEE was subsequently extracted with hexane. FAEE transesterification efficiency increased from 52.5% to 72.9% (reaction condition 1), however this is still short of required efficiency of over 95% for biodiesel application (Table 4). In another experiment different amount of alumina or sodium sulphate i.e., 5 g or 10 g or 20 g was added in reaction mixture (180 mL ethanol plus 20 mL HCl) as given in Example 3 followed by hexane extraction. Subsequently, hexane was evaporated and FAEE was subjected to a second transesterification reaction. Addition of moisture adsorbent resulted into increase in FAEE transesterification efficiency from 52.5% to 87.8% (reaction condition 2). When moisture adsorbent was added in second transesterification reaction instead of first, FAEE transesterification efficiency significantly increased from 55% to 96.5% (reaction condition 3).

TABLE-US-00004 TABLE 4 Effect of different transesterification reaction conditions on FAEE transesterification efficiency Wet algal biomass slurry Reaction Reaction Reaction Parameters Control condition- condition-2 condition- 3 FAEE Content (g) 5.16 5.25 5.20 5.16 from 20 g dry biomass FAEE content (%) 25.82 26.25 26.00 25.82 FAEE extraction yield 95.55 97.22 96.29 95.55 (%) in comparison to Folch method FAEE Extraction 52.15 72.9 87.8 96.5 efficiency (%)

Example-5: Analysing FAEE by GC-FID

[0082] FAEEs were washed with 2% KHCO.sub.3 and passed through anhydrous Na.sub.2SO.sub.4 for removal of any moisture if present. FAEEs were concentrated under nitrogen or rotary evaporator and analyzed with GC-FID system (Perkin Elmer Clarus 680, US), equipped with fast-GC capillary column (Omegawax 100 column of dimension 15 m×0.1 mm, 0.1 μm thickness). Sample of FAEEs (1 μl) was injected while maintaining injector temperature at 250° C. Oven temperature was ramped up at the rate of 40° C./sec from 140° C. to final 280° C. and hold for 2 minutes. Detector was set at 260° C. Hydrogen was used as carrier gas with velocity rate 50 cm sec.sup.−1. Fatty acid peaks were identified and quantified with Total chrome chromatography software (Perkin Elmer, US).