Thraustochytrid based process for treating waste effluents

Abstract

The present invention relates to the continuous process of sequestration of nutrients from waste effluents released from gas fermentation plants or green-house gases emission managing plants by novel Thraustochytrids. Particularly this invention relates to the methods and systems to enhance sequestration rate and productivity of the process. This invention also relates to rapid biotransformation of nutrients present in waste effluents into high value omega-3 fatty acids like Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), Eicosapentaenoic acid (EPA) and lipids for biodiesel. This disclosure is about means of processing of waste streams and producing value added products out of it.

Claims

1. A process comprising two staged continuous fermentation for sequestration of nutrients from waste effluents of gas fermentation plants using Thraustochytrids for producing high value omega-3 fatty acids and lipids for biodiesel, said process comprising; (a) Continuous culturing Thraustochytrid strains in a first stage fermentation reactor comprising continuous supply of waste effluent stream and nitrogen source; (b) Continuous transferring the culture of Thraustochytrid strains of step (a) through biomass harvesting/separator system to a second stage fermentation reactor having only waste effluent stream; (c) Continuous culturing the biomass of step (b) producing a concentrated biomass in the second stage fermentation reactor optionally in absence of nitrogen; (d) Continuous separating of the biomass from broth through biomass harvesting/separator system; and recycling of the broth to gas fermentation plants; and (e) obtaining omega-3 fatty acids and lipids for biodiesel wherein the Thraustochytrid strains are selected from the group consisting of strain MTCC 5890 (DBTIOC-1), strain MTCC 5895 (DBTIOC-6), strain MTCC 5893 (DBTIOC -14) and strain MTCC 5896 (DBTIOC-18).

2. The process as claimed in claim 1, wherein waste effluents were containing mixture of organic acids and alcohol, sulphides and no sugar or carbohydrate.

3. The process as claimed in claim 1, wherein waste effluents contains acetate at a concentration of 5g/L-100g/L or more and alcohol at a concentration of 0.5 g/L or 5 g/L or more.

4. The process as claimed in claim 1, wherein omega-3 fatty acid fatty acids were Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), or Eicosapentaenoic acid (EPA).

5. The process as claimed in claim 1, wherein step (b), the culture from first fermentation reactor was directly transferred into the second fermentation reactor.

6. The process as claimed in claim 1, wherein step (c), the culture of second fermentation reactor was cultured in absence of nitrogen.

7. The process as claimed in claim 1, wherein in the absence of nitrogen, the biomass increases in the range of 8 gm/liter/day to 150 gm/liter/day.

8. The process as claimed in claim 1, wherein in the absence of nitrogen, the lipid content increases in the range of 20% to 90% from the first stage fermentation reactor.

9. The process as claimed in claim 1, wherein in the absence of nitrogen, the DHA content increases in the range of 15% of total fatty acid to 50% of total fatty acid from the first stage fermentation reactor.

10. The process as claimed in claim 1, wherein step (b) culture from the first stage fermentation reactor was continuously passed through biomass separating reactor to separate out biomass and broth.

11. The process as claimed in claim 5, wherein the transfer of biomass directly from first reactor to second reactor enhance the dry biomass in the range of 8 gm/liter/day to 200 gm/liter/day.

12. The process as claimed in claim 1, wherein the transfer of biomass directly from first reactor to second reactor enhance the lipid content in the range of 20% of dry mass to 80% of dry mass.

13. The process as claimed in claim 1, wherein the transfer of biomass directly from first reactor to second reactor enhance the DHA content in the range of 15% of total fatty acid to 50% of fatty acid.

14. The process as claimed in claim 1, wherein in step 1 (c) absence of nitrogen in the second reactor creates a continuous nitrogen stress condition to stimulate lipid accumulation in the cell.

15. The process as claimed in claim 1, wherein the broth coming out of second reactor was having zero dissolved oxygen and recycled back to gas fermenting plants.

16. The process as claimed in claim 2, wherein step 1 (a) the waste effluent was continuously purged with air to oxidize sulphides.

17. The process as claimed in claim 2, wherein the sulphides from the waste effluents increased the nutrient sequestration in the range of 55 gm/liter/day to 90 gm/liter/day.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1. Strategy employed for the Thraustochytrid isolation.

(2) FIG. 2. Phylogenetic relationship (NJ tree) of novel Thraustochytrid strains with existing Thraustochytrids

(3) FIG. 3(a). Acetate tolerance and biomass production on different concentration of acetate by Thraustochytrid strains

(4) FIG. 3(b). Acetate tolerance and lipid content on different concentration of acetate by Thraustochytrid strains

(5) FIG. 3(c). FAB and DHA content on different concentration of acetate by Thraustochytrid strains

(6) FIG. 4(a). Biomass production of Thraustochytrid strains on waste effluents

(7) FIG. 4(b). Lipid content of Thraustochytrid strains on waste effluents

(8) FIG. 4(c). FAB and DHA content of Thraustochytrid strains on waste effluents

(9) FIG. 5. Effect of addition of nitrogen source on biomass production on waste effluent stream

(10) FIG. 6. Effect of different inoculum sizes on biomass production on waste effluent stream

(11) FIG. 7(a). Cell count, Cell OD.sub.600 nm, dry biomass in bioreactor with combination of Rushton impeller and drilled pipe spargers for the cultivation of DBTIOC-18 (MTCC 5896) on waste effluent stream.

(12) FIG. 7(b). Cell count, Cell OD.sub.600 nm, dry biomass in bioreactor with combination of pitch blade and microspargers for the cultivation of DBTIOC-18 (MTCC 5896) on waste effluent stream.

(13) FIG. 8. Cell count, cell OD600 nm, cell size, dry biomass in 2 L bioreactor having 1.2 L working volume with combination of pitch blade and microspargers for the cultivation of DBTIOC-18 (MTCC 5896) on waste effluent stream continuously purged with air.

(14) FIG. 9. Cell count, cell OD.sub.600 nm, cell size, dry biomass and lipid content in two stage bioreactor system having 1.2 L working volume, First reactor was continuously supplied with waste stream having nitrogen source whereas second reactor was supplied waste streams without amending with nitrogen source for the cultivation of DBTIOC-18 (MTCC 5896)

(15) FIG. 10. Cell count, cell OD.sub.600 nm, cell size, dry biomass and lipid content in two stage bioreactor system with inline biomass harvesting system, First reactor was continuously supplied with waste stream having nitrogen source whereas second reactor was supplied waste streams without amending with nitrogen source for the cultivation of DBTIOC-18 (MTCC 5896). Biomass was continuously concentrated from broth coming out of first reactor and concentrated biomass was continuously pumped into second reactor.

(16) FIG. 11. Schematic diagram of the process for the conversion of waste effluents to lipid and DHA

DETAILED DESCRIPTION

(17) While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of example in the drawings, graphs and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims. Before the present methods and the products are described, it is to be understood that this invention is not limited to particular method, product and experimental conditions described; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting.

(18) The graphs, tables, figures and protocols have been represented where appropriate by conventional representations in the drawings, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

(19) The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

(20) The terms comprises, comprising, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by comprises . . . a does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.

(21) The terms, alone or in combination or any other variations thereof, are intended to described and/or cover a non-exclusive inclusion, wherein the molecules or the oligonucleotides exist individually or together with any one or all of the other oligonucleotides.

Definitions

(22) For the purposes of this invention, the following terms will have the meaning as specified therein:

(23) As used herein, the terms Two stage fermentation process or Two stage fermentation system or Two stage continuous fermentation process or Two stage continuous fermentation system when used in the context of the present invention refers a process or method which comprises of two continuous fermentation reactions taking place in two separate reactors. The first fermentation takes place in the first stage fermentation reactor followed by second fermentation that takes place in the second stage fermentation reactor. Although both first and second fermentation reactions take place in separately in two different reactors but the overall process is continuous.

(24) As used herein, the terms Novel strains when used in the context of the present invention refers to those strains that were created out of adaption of to extreme waste effluent streams of gas fermentation plants. The said Novel Strains are capable of multifold expression of multiple enzymes related for example Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc. and capable of degrading high acetate containing waste effluents from gas fermentation plants.

(25) As used herein, the terms Omega-3 fatty acids or ?-3 fatty acids or n-3 fatty acids refers to polyunsaturated fatty acids (PUFAs) with a double bond (C?C) at the third carbon atom from the end of the carbon chain. More specifically there are three types of Omega-3 fatty acids such as eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) which have been considered, identified, studied and included as Omega-3 fatty acids in context of the present invention.

(26) The present invention describes method and systems for continuous processing of waste effluents from gas fermentation or green-house gases managing plants for rapid conversion into omega-3 fatty acids and lipids using novel strains of Thraustochytrids. The novel strains of Thraustochytrid biomass is suitable feedstock for biodiesel production as well.

(27) The present invention provides for novel wild strains of Thraustochytrid i.e. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC5893 (DBTIOC-14), MTCC 5896 (DBTIOC-18) that were isolation from mangrove ecosystem of the Indian coastline from degrading leaves or soil rich in dead organic matter from Mandovi Zuari mangroves, (Coordinates of Collection: S15?2957.39,E73?526.13, Panjim, Goa) and identified and their characterization based on 18S rDNA gene sequencing was described. These 18S rDNA sequences have been deposited with NCBI database with accession Nos. KF668624, KF668627, KF668629 and KF668632.

(28) In another aspect the present invention also provides for novel strains of thraustochytrid capable of expressing multifold expression of Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc. and capable of degrading high acetate containing waste effluents from gas fermentation plants. Waste effluents discharged from gas fermentation plants also contained alcohol (0.5 g/L or 2 g/L or 5 g/L or more).

(29) One aspect of the present invention provides for novel Thraustochytrid strains that are capable of hydrolyzing or degrading high acetate waste effluent. In one aspect the present invention provides for novel Thraustochytrid strains that can use acetate as carbon source present in the high acetate waste effluent in addition to yeast extract, peptones, corn steep liquor, nitrates, ammonium salts as nitrogen sources for the production of lipids and fatty acids.

(30) In one aspect the present invention provides novel Thraustochytrid strains can tolerate wide range of acetate concentration from 5 g/L to 30 g/L to 50 g/L or more [FIG. 3 (a)-(c)]. In general the biomass was found in the range of 1.5 g/L to 6.5 g/L and lipid content was 14% to 54.5% w/w of biomass in the strains [FIG. 3 (a)-(c)]. For example the thraustochytrid strain MTCC 5896 (DBTIOC-18) has been found to utilize acetate in the range of 15 g/L which yielded 6.5 g/L biomass, 54.5% lipid and 15% DHA content.

(31) Another aspect of the present invention provides for using effluent waste but not limited to the fermentation or green-house gases managing plants. In another aspect the present invention provides for novel Thraustochytrid strains as herein described which are capable of using both carbon and nitrogen source from various waste effluents. The nutrients in waste effluent or waste stream included organic acids and alcohol. The most common nutrients in the waste effluent/waste stream were found to be Acetic acid, formic acid and other acids along with alcohol. Thus with such a medium the biomass production was found to be ranging from 1.5 g/L to 3.3 g/L, lipid content was 21% to 50% of biomass and DHA content was 10% to 30% of total fatty acids (FIG. 4).

(32) Another aspect of the present invention provides for the method of adapting the wild or parent strains to develop capability for hydrolyzing or degrading waste effluents. For adaptation of the wild or parent strains were cultured on waste effluents for longer duration for example one month, two month or three month or in some cases 6 months. Once nutrients are totally consumed in waste effluent streams, cultures were transferred to fresh stream. Transferring of culture was repeated 10 times, or 20 times, 30 times or in some cases 100 times. This enabled the wild or parent strains to selectively utilize nutrients from these streams and gradually adapt on waste effluents.

(33) The novel strains showed higher nutrient sequestration i.e. 30 g/L, 50 g/L or 100 g/L from waste effluents and higher biomass and lipid production. These novel strains were able to tolerate very high concentration of organic acids particularly acetate/acetic acid and other growth inhibitors present in these streams. A Molecular analysis of the genes for example Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc. associated with acetate metabolism showed multi fold increase in expression level in the novels strains compared to native strains.

(34) In another aspect the novel strains were found to be efficient in respect of high robustness and productivity parameters such as biomass production, lipid content, TFA content, nutrient sequestration from stream remained higher as compare to native or control strains [(FIG. 4 (a)-(c) and (Table (1a)-(1b)]. These novel strains were later cultivated in Erlenmeyer flasks, baffle flasks or bioreactors.

(35) TABLE-US-00001 TABLE 1(a) Biomass production of different Thraustochytrid strains on waste effluents. Dry biomass (g/L) Dry biomass (g/L) of of novel strains Control strains on Strains on waste effluent waste effluent MTCC5890 (DBTIOC-1) 2.1-3.1 0.21-0.53 MTCC 5895 (DBTIOC-6) 2.4-3.3 0.23-0.61 MTCC 5893 (DBTIOC-14) 3.9-5.2 0.15-0.32 MTCC 5896 (DBTIOC-18) 2.5-3.4 0.25-0.61

(36) TABLE-US-00002 TABLE 1(b) Lipid content of different Thraustochytrid novel strains on waste effluents. Lipid content (%) Lipid content (%) of novel strains of Control strains Strains on waste effluent on waste effluent MTCC5890 (DBTIOC-1) 18-28 15-24 MTCC 5895 (DBTIOC-6) 28-35 19-32 MTCC 5893 (DBTIOC-14) 45-55 25-29 MTCC 5896 (DBTIOC-18) 35-46 25-30

(37) In yet another aspect of the present invention it has been found that that novel thraustochytrid strains have unique and unexpected property of thriving both in presence and absence of nitrogen source. It was found in the present invention that lacking of adequate nitrogen supply triggered high lipid accumulation in the thraustochytrid strains. This enabled to adapt various strategies for use of novel thraustochytrid strains in production of biomass, lipid and DHA, for example amendment of waste effluent with nitrogen sources, increased inoculum size and adaptation of the selected strains on these waste effluents were applied to enhance biomass and lipid production. Moreover such a unique property of novel thraustochytrids enabled the applicants to develop and arrive at unique and novel method comprising of a two staged continuous fermentation for sequestration of nutrients from waste effluents of gas fermentation plants using Thraustochytrids for producing high value omega-3 fatty acids and lipids for biodiesel.

(38) The various organic and inorganic nitrogen sources comprise of but are not limited to corn steep liquor, nitrates like sodium nitrate, potassium nitrates, ammonium nitrates, ammonium sulfate, ammonium chloride, and urea. The aforesaid nitrogen sources were added in the waste effluent medium. Thus in one aspect the present invention provides that the novel thraustochytrid strains when grown in medium comprising of waste effluent along with and nitrogen source resulted in higher growth of the novel thraustochytrid strains. Accordingly, in another aspect the of the present invention it was found that addition of sodium nitrate in the medium resulted into significant improvement in lipid content and lipid productivity in all the strains. Interestingly it was found that there was a two to three fold increase in the biomass concentration with the addition of nitrogen source [(FIG. 5) and (Table 2)]. For example in strain MTCC 5896 (DBTIOC-18) with addition of sodium nitrate in the effluent medium there was a two to three fold increase in the biomass concentration.

(39) TABLE-US-00003 TABLE 2 Effect of addition of nitrogen source on biomass production on waste effluent stream Dry biomass (g/L) Dry biomass (g/L) without with Strains nitrogen source nitrogen source MTCC5890 (DBTIOC-1) 2.1-3.1 5.1-6.4 MTCC 5895 (DBTIOC-6) 2.4-3.3 3.2-5.5 MTCC 5893 (DBTIOC-14) 2.9-3.2 3.9-5.1 MTCC 5896 (DBTIOC-18) 2.5-3.4 6.1-7.3

(40) A further aspect of the present invention provides that higher inoculum sizes for example 5%, 10%, 15%, 20% (v/v) was used to increase the removal of nutrients from waste effluents and increase the productivity of the process. Increase in inoculum sizes resulted into significant increase in biomass production with maximum reported almost 9 g/L with 20% v/v inoculum size (FIG. 6). All the nutrients (organic acids and alcohols) were totally removed from the stream.

(41) In another aspect the present invention it has been found that the need of adding sea salt or equivalent (such as sea water) was not required for the processing of waste effluents by novel strains. These strains were able to actively grow without the addition of sea salt or equivalent in the stream and producing almost equal biomass and lipid content. This was significant improvement in the process since this will not only avoid corrosion of steal fermenters but also extra cost associated with replacement of sea salt with other non-chloride sodium salts. This also underlines the fact that avoiding addition of sea salt in the media will not increase the salinity of effluent stream thus this stream water was recycled for gas fermentation.

(42) In another aspect the present invention provides for novel thraustochytrid strains that were able to tolerate increase in pH for example up to 9 or 9.5 without compromising the nutrient sequestration rate and productivity of the process thus reducing the need of pH balance during the cultivation of strains which will further reduce the cost associated with processing of waste effluents.

(43) In another aspect the present invention also provides for a reactor system comprising of reactor impellers that could prevent damage to delicate cells under lipid accumulation stage by shear force. Accordingly, on aspect of the present invention the large scale cell lysis was observed during lipid accumulation phase. Thus to avoid this different type of impellers for example Rushton, pitch blade or marine impellers were used. It was observed that use of pitch blade impeller significantly reduce the cell lysis during lipid accumulation phase thus enhancing the number of active cells present in the vessel. This in turn translated into faster nutrient sequestration from waste effluent streams and prevented loss of products such as lipid and DHA.

(44) In another aspect of the present invention the reactor was run in batch, fed batch, semi continuous and continuous mode. To make reactor continuous, effluent was continuously pumped in with one tube while culture was simultaneously removed from other overflow tube installed in the reactor Nitrogen source was supplied continuously or intermittently along with effluent stream. Sometimes effluent stream was supplied without addition of nitrogen source.

(45) In another aspect the present invention also provide another unique design in the reactor system wherein the reactor was fitted with microspargers instead of drilled pipe spargers. Combination of different types of spargers and impellers were applied to enhance masse transfer rate, biomass, lipid and DHA productivity and simultaneously decreasing agitation rate, shearing and power consumption. Combination of microspargers with pitch blade resulted into faster nutrient sequestration rate, higher biomass production rate. Combination of microspargers and pitch blade impeller improved nutrient sequestration from 2 grams/litre/day to 18 grams/litre/day to 30 grams/litre/day or 55 grams/litre/day more from waste streams, which resulted into higher biomass production from 0.5 grams/litre/day to 4.8 grams/litre/day to 8 grams/litre/day or 40 grams/litre/day more [FIGS. 7(a)-(b)].

(46) In another aspect of the present invention during high density culture, excessive foam was being generated, which was controlled by either continuous or intermittent addition of antifoam in the reactor. Sometimes antifoam negatively affected the growth and biomass production rate. Most of the antifoam tested, was found negatively affecting the growth and biomass production.

(47) In another aspect the black coloured waste effluent coming from gas fermentation plants was continuously purged with air to remove traces of sulphides which gives black colour to the effluent, which later fed into the reactor. Sometimes purging effluent stream with air resulted into higher nutrient sequestration rate and biomass production rate. This resulted into further increase in nutrient sequestration from 55 g/L/d to 90 g/L/d or more and biomass production from 40 g/L/d to 55 g/L/d or more. However comparing to batch or fed batch, lipid content decreased from 55% of dry biomass or 70% or more of dry biomass to 20% or 30% or more of dry biomass (FIG. 8).

(48) In another aspect of the present invention the supply of nitrogen source was stopped to increase lipid content of the cell, whereas in other case nitrogen source was continuously supplied to support growth. Continuous supply of nitrogen source resulted into reduced lipid production but higher growth. To support lipid production along with growth, two reactor systems was set up. One reactor was designated to promote growth while another for lipid accumulation in the cell. First reactor is kept at high aeration rate to maintain higher dissolve oxygen whereas second reactor is kept at low dissolve oxygen level.

(49) In another aspect of the present invention the culture coming out from first reactor was directly transferred into second reactor to induce lipid production under nitrogen stress. First reactor was continuously supplied with effluent stream containing nitrogen source whereas second reactor was supplied with effluent stream only. Culture coming out of second reactor showed improved biomass and lipid production. Biomass increased from 8 g/L/d to 30 g/L/d or 65 g/L/d or 95 g/L/d or 100 g/L/d or more, whereas lipid content increased from 20% of dry biomass to 40% or 50% or 60% or 70% or more of dry biomass. DHA production also improved from 15% of total fatty acid to 25% or 30% or 35% of total fatty acids (FIG. 9).

(50) In another aspect of the present invention a biomass separating system was installed between two reactors for continuous separation of cells and broth to transfer concentrated biomass to second reactor to induce lipid accumulation in the cell.

(51) In yet another aspect of the present invention the culture broth coming out from first reactor was continuously passed through biomass separating system, where cells are concentrated and transferred to second reactor. In second reactor residence time for cells are higher to promote lipid accumulation in the cell under low dissolve oxygen level and deplete the oxygen completely from the broth. Dry biomass increased from 8 g/L/d to 30 g/L/d or 65 g/L/d or 95 g/L/d or 150 g/L/d or more, whereas lipid content increased from 20% of dry biomass to 40% or 50% or 60% or 80% or more of dry biomass. DHA production also improved from 15% of total fatty acid to 25% or 30% or 45% or more of total fatty acids (FIG. 10).

(52) In yet another aspect of the present invention the alcohols particularly ethanol present in waste effluent stream caused increment in DHA production in the biomass. DHA content increased from 15% of total fatty acid to 25% or 30% or 45% or more of total fatty acids. Doping of waste effluent stream with ethanol resulted into further rise in DHA production in the biomass from 15% of total fatty acid to 25% or 30% or 45% or 50% or more of total fatty acids.

(53) In yet another aspect of the present invention the second reactor was installed with overflow tube for the continuous harvesting of the lipid rich biomass. Culture coming out of second reactor was continuously passed through second biomass separating system to separate out biomass and broth. This broth was totally devoid of oxygen therefore this broth was totally or partly recycled back for the gas fermentation process.

(54) In yet another aspect of the present invention the broth water, continuously separated out from the biomass, was recycled back for gas fermentation process thus drastically reducing the need of water for whole process. Sometimes broth water was purged with nitrogen to flush out oxygen from it (FIG. 11).

(55) In yet another aspect of the present invention one or two or three or more novel strains of thraustochytrids were co-cultivated to enhance nutrient sequestration rate, biomass, lipid and DHA productivity. In another aspect the present invention provides one or more novel strains alone or in combination to enhance nutrient sequestration rate, biomass, lipid and DHA productivity. In another aspect the present invention provides a single strain to enhance nutrient sequestration rate, biomass, lipid and DHA productivity. In another aspect the present invention provides two or more novel strains alone or combination to enhance nutrient sequestration rate, biomass, lipid and DHA productivity.

(56) Another aspect of the present invention provides for a process that was developed in way to synchronize it with any gas fermentation process to produce high value coproduct from green-house gases at significantly higher rate than conventional phototrophic algae driven green-house gas sequestration process. A person skilled in art understands that waste effluents from other sources, rich in organic acids/alcohols/sugars can be used for nutrients sequestration using this process.

(57) Accordingly the main embodiment of the present invention provides a process comprising two staged continuous fermentation for sequestration of nutrients from waste effluents of gas fermentation plants using Thraustochytrids for producing high value omega-3 fatty acids and lipids for biodiesel, said process comprising; (a) culturing Thraustochytrids strains in a first stage fermentation rector comprising of waste effluent stream and nitrogen source; (b) transferring the Thraustochytrids strains of step (a) through biomass harvesting system to second stage fermentation reactor having only waste effluent stream; (c) culturing the biomass culture of step (b) in the second stage fermentation reactor optionally in absence of nitrogen; (d) Separation of biomass and liquid broth and recycling of clear broth to gas fermentation plants; and (e) obtaining value added omega-3 fatty acids and lipids.

(58) Another embodiment of the present invention provides a process as herein described, wherein waste effluents were containing mixture of organic acids and alcohol and no sugar or carbohydrate.

(59) Another embodiment of the present invention provides a process as herein described, wherein the selected Thraustochytrid strains are MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18).

(60) Another embodiment of the present invention provides a process as herein described, wherein waste effluents contains acetate at a concentration of 5 g/L-100 g/L or more and alcohol at a concentration of 0.5 g/L or 5 g/L or more.

(61) Another embodiment of the present invention provides a process as herein described, wherein omega-3 fatty acid fatty acids were palmitic acid, oleic acid, Docosahexaenoic acid (DHA), Docosapentaenoic acid (DPA), or Eicosapentaenoic acid (EPA).

(62) Another embodiment of the present invention provides a process as herein described, wherein the step 1(b), the culture from first fermentation reactor was directly transferred into the second fermentation reactor.

(63) Another embodiment of the present invention provides a process as herein described, wherein the step 1(c) culture of second fermentation reactor was cultured in absence of nitrogen.

(64) Another embodiment of the present invention provides for a process as herein described, wherein in the absence of nitrogen increases the biomass in the range of 8 gm/litre/day to 150 gm/litre/day, preferably in the range of 8 gm/litre/day to 100 gm/litre/day.

(65) Another embodiment of the present invention provides for a process as herein described, wherein in the absence of nitrogen increases the lipid content in the range of 20% to 90% from the first stage fermentation reactor, preferably in the range of 20% to 70% from the first stage fermentation reactor.

(66) Another embodiment of the present invention provides for a process as herein described, wherein in the absence of nitrogen increases the DHA content in the range of 15% of total fatty acid to 50% of total fatty acid from the first stage fermentation reactor, preferably in the range 15% of total fatty acid to 35% of total fatty acid from the first stage fermentation reactor.

(67) Another embodiment of the present invention provides for a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the dry biomass in the range of 8 gm/litre/day to 200 gm/litre/day, preferably in the range of 8 gm/litre/day to 150 gm/litre/day

(68) Another embodiment of the present invention provides for a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the lipid content in the range of 20% of dry mass to 100% of dry mass, preferably in the range of 20% of dry mass to 80% of dry mass.

(69) Another embodiment of the present invention provides for a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the DHA content in the range of 15% of total fatty acid to 50% of fatty acid, preferably enhance the DHA content in the range of 15% of total fatty acid to 45% of fatty acid.

(70) Another embodiment of the present invention provides for a process as herein described wherein the removal of sulphide increased the in nutrient sequestration in the range of 55 gm/litre/day to 90 gm/litre/day, preferably in the range of 40 gm/litre/day to 55 gm/litre/day.

(71) Another embodiment of the present invention provides for a process as herein described, wherein the strains produce (a) dry biomass in the range of 8 gm/litre/day to 150 gm/litre/day; (b) lipids in the range of 20% of dry mass to 80% of dry mass; and (c) DHA content in the range of 15% of total fatty acid to 45% of fatty acid.

(72) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the biomass in the range of 8 gm/litre/day to 150 gm/litre/day.

(73) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the biomass in the range of 8 gm/litre/day to 100 gm/litre/day.

(74) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the lipid content in the range of 20% to 90% from the first stage fermentation reactor.

(75) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the lipid content in the range of 20% to 70% from the first stage fermentation reactor.

(76) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the DHA content in the range of 15% of total fatty acid to 50% of total fatty acid from the first stage fermentation reactor.

(77) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the DHA content is in the range of 15% of total fatty acid to 35% of total fatty acid from the first stage fermentation reactor.

(78) Another embodiment of the present invention provides a process as herein described wherein the culture from the first stage fermentation reactor was continuously passed through biomass separating reactor to separate out biomass and broth.

(79) Another embodiment of the present invention provides a process as herein described wherein the concentrated biomass obtained from separating system was continuously transferred to second stage fermentation reactor.

(80) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the dry biomass in the range of 8 gm/litre/day to 200 gm/litre/day.

(81) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the dry biomass in the range of 8 gm/litre/day to 150 gm/litre/day.

(82) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the lipid content in the range of 20% of dry mass to 100% of dry mass.

(83) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the lipid content in the range of 20% of dry mass to 80% of dry mass.

(84) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the DHA content in the range of 15% of total fatty acid to 50% of fatty acid.

(85) Another embodiment of the present invention provides a process as herein described wherein the transfer of biomass directly from first reactor to second reactor enhance the DHA content in the range of 15% of total fatty acid to 45% of fatty acid.

(86) Another embodiment of the present invention provides a process as herein described wherein the second reactor was used to create continuous nitrogen stress condition to stimulate lipid accumulation in the cell.

(87) Another embodiment of the present invention provides a process as herein described wherein the culture coming out of second fermentation reactor was continuously passed through biomass separating system to separate out biomass and broth water and wherein the water was having zero dissolved oxygen was continuously recycled back for gas fermentation process.

(88) Another embodiment of the present invention provides a process as herein described wherein the waste effluent was continuously purged with air to remove traces of sulphide.

(89) Another embodiment of the present invention provides a process as herein described wherein the removal of sulphide increased the in nutrient sequestration in the range of 55 gm/litre/day to 90 gm/litre/day.

(90) Another embodiment of the present invention provides a process as herein described wherein the removal of sulphide increased the biomass production in the range of 40 gm/litre/day to 55 gm/litre/day.

(91) Yet another embodiment of the present invention provides Novel strains of thraustochytrid having Accession No. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18).

(92) Yet another embodiment of the present invention provides novel strains as herein described capable of producing omega-3 fatty acids and lipids for use in biodiesel.

(93) Yet another embodiment of the present invention provides novel strains as herein described wherein the strains produce dry biomass in the range of 8 gm/litre/day to 150 gm/litre/day.

(94) Yet another embodiment of the present invention provides novel strains as herein described wherein the strains produce lipids in the range of 20% of dry mass to 80% of dry mass.

(95) Yet another embodiment of the present invention provides novel strains as herein described wherein the strains produce DHA content in the range of 15% of total fatty acid to 45% of fatty acid.

(96) Yet another embodiment of the present invention provides Use of novel strains of thraustochytrids having Accession No. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18) for producing omega-3 fatty acids and lipids for use in biodiesel.

(97) One embodiment of the present invention provides a process as herein described wherein the novel strains are able to produce lipid content in the range of 14% to 54.5% w/w of biomass in the strains.

(98) In another embodiment the present invention provides a thraustochytrid strain MTCC 5896 (DBTIOC-18) capable of utilizing or reducing acetate to yield 6.5 g/L biomass, 54.5% lipid and 15% DHA content.

(99) Another embodiment of the present invention provides a process as herein described wherein the biomass production is in the range of 1.5 g/L to 3.3 g/L, lipid content is in the range of 21% to 50% of biomass and DHA content is in the range of 10% to 30% of total fatty acids (FIG. 4).

(100) Another embodiment of the present invention provides thraustochytrid strains having Accession No. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18) wherein the strains show multi fold increase in the expression of Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc.

(101) Another embodiment of the present invention provides thraustochytrid strains having Accession No. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18) wherein the strains show multi fold increase in the expression of enzymes associated with acetate metabolism, wherein the enzyme may be selected from group of one or more Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc.

(102) Another embodiment of the present invention provides thraustochytrid strains having Accession No. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14) and MTCC 5896 (DBTIOC-18) wherein the strains show multi fold increase in the expression of enzymes selected from group of one or more Acetyl Co-A synthase, malic enzyme, ATP Citrate lyase, Isocitrate dehydrogenase etc, wherein the multi fold expression of the said enzymes may be alone or in combination with each other.

(103) Another embodiment of the present invention provides novel strains as herein described highly efficient in respect of high robustness and productivity parameters such as biomass production, lipid content, TFA content and nutrient sequestration

(104) Yet another embodiment of the present invention provides novel strains as herein described are capable of producing biomass in the range of 2 g/L to 6 g/L, preferably 2.1-5.2 g/L [Table 1(a)].

(105) Another embodiment of the present invention provides novel strains as herein described capable of producing biomass in the range of 2.1 to 3.1 g/l, 2.4 to 3.3 g/l, 3.9 to 5.2 g/l or/and 2.5 to 3.4 g/l [Table 1(a)].

(106) Yet another embodiment of the present invention provides novel strains as herein described are capable of producing lipid content in the range of 18% to 60%, preferably 18%-55% [Table 1(b)].

(107) Another embodiment of the present invention provides novel strains as herein described are capable of producing lipid content in the range of 18% to 28%, 28% to 35%, 45% to 55% 1 or/and 35% to 46% [Table 1(b)].

(108) Yet another embodiment of the present invention provides a process as herein described wherein the biomass production is in the range of 2 g/L to 6 g/L, preferably 2.1-5.2 g/L.

(109) Another embodiment of the present invention provides a process as herein described wherein the biomass is in the range of 2.1 to 3.1 g/l, 2.4 to 3.3 g/l, 3.9 to 5.2 g/l or/and 2.5 to 3.4 g/l [Table 1(a)].

(110) Yet another embodiment of the present invention provides a process as herein described wherein the lipid content is in the range of 18% to 60%, preferably 18%-55% [Table 1(b)].

(111) Another embodiment of the present invention provides a process as herein described wherein the lipid content is in the range of 18% to 28%, 28% to 35%, 45% to 55% 1 or/and 35% to 46% [Table 1(b)].

(112) Yet another embodiment of the present invention provides novel strains as herein described which enhance the biomass by two to three folds with addition of nitrogen source.

(113) Yet another embodiment of the present invention provides novel strains as herein described wherein addition of nitrogen source enhances the biomass in the average range of 3-8 g/l [Table 2].

(114) Yet another embodiment of the present invention provides novel strains as herein described wherein addition of nitrogen source enhances the biomass in the range of 3.3-5.5 g/l, 3.9-5.1 g/l, 5.1-6.4 g/l and/or 6.1-7.3 g/l [Table 2].

(115) Yet another embodiment of the present invention provides a nitrogen source as herein described selected from any of the known conventional naturally occurring or artificial known nitrogen sources.

(116) Yet another embodiment of the present invention provides a process as herein described wherein there is two to three fold increases in the biomass with addition of nitrogen source.

(117) Yet another embodiment of the present invention provides a process as herein described wherein addition of nitrogen source enhances the biomass in the average range of 3-8 g/l [Table 2].

(118) Yet another embodiment of the present invention provides a process as herein described wherein addition of nitrogen source enhances the biomass in the range of 3.3-5.5 g/l, 3.9-5.1 g/l, 5.1-6.4 g/l and/or 6.1-7.3 g/l [Table 2].

(119) In another embodiment the present invention provides a process as herein described wherein biomass production is increased is upto 9 g/L (FIG. 6).

(120) In another embodiment the present invention provides novel strains as herein described wherein the strains are capable of producing biomass upto 9 g/L (FIG. 6).

(121) Another embodiment of the present invention provides a process as herein described wherein the combination of microspargers and pitch blade impeller enhanced nutrient sequestration from 2 gm/litre/day to 55 gm/litre/day, which resulted in increase in biomass in the range of 0.5 gm/litre/day to 40 gms/litre/day [FIGS. 7(a)-(b)].

(122) Another embodiment of the present invention provides a process as herein described wherein the combination of microspargers and pitch blade impeller enhanced the in the range of 2 gm/litre/day to 18 gm/litre/day to 30 gm/litre/day or 55 gm/litre/day, which resulted in increase of biomass in the range of 0.5 gm/litre/day to 4.8 grams/litre/day to 8 grams/litre/day or 40 grams/litre/day more [FIGS. 7(a)-(b)].

(123) In another embodiment the present invention provides a process as herein described wherein the removal of sulphides increased the in nutrient sequestration in the range of 55 gm/litre/day to 90 gm/litre/day.

(124) In another embodiment the present invention provides a process as herein described wherein the removal of sulphides increased the biomass production in the range of 40 gm/litre/day to 55 gm/litre/day. (FIG. 8).

(125) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase of biomass in the range of 8 gm/litre/day to 150 gm/litre/day (FIG. 9).

(126) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase of biomass in the range of 8 gm/litre/day to 30 gm/litre/day or 65 gm/litre/day or 95 gm/litre/day or 100 gm/litre/day (FIG. 9).

(127) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase of lipid content in the range of 20% of dry biomass to 90% of dry biomass (FIG. 9).

(128) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase of lipid content in the range of 20% of dry biomass to 40% or 50% or 60% or 70% or more of dry biomass (FIG. 9).

(129) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase in DHA in the range of 15% of total fatty acid to 50% of total fatty acids (FIG. 9).

(130) In another embodiment the present invention provides a process as herein described wherein supply of nitrogen to only first reactor and absence of nitrogen in the second reactor resulted in increase in DHA in the range of 15% of total fatty acid to 25% or 30% or 35% of total fatty acids (FIG. 9).

(131) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the dry biomass in the range of 8 gm/litre/day to 200 gm/litre/day (FIG. 10).

(132) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the dry biomass in the range of 8 gm/litre/day to 30 gm/litre/day or 65 gm/litre/day or 95 gm/litre/day or 150 gm/litre/day (FIG. 10).

(133) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the lipid content in the range of 20% of dry biomass to 100% of dry biomass (FIG. 10).

(134) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the lipid content in range of 20% of dry biomass to 40% or 50% or 60% or 80% or more of dry biomass (FIG. 10).

(135) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the DHA production in the range of 15% of total fatty acid to 50% of total fatty acids (FIG. 10).

(136) In another embodiment of the present provides a process as herein described wherein the cell culture of the first reactor was concentration and then transferred to the second reactor thereby increasing the DHA production in the range of 15% of total fatty acid to 25% or 30% or 45% or more of total fatty acids (FIG. 10).

(137) Another embodiment of the present invention provides for use of novel strains of thraustochytrids as herein described for the process of production of biofuels.

(138) Another embodiment of the present invention provides for use of novel strains of thraustochytrids as herein described for the process of production of (a) biomass in the range of 8 gm/litre/day to 150 gm/litre/day, preferably in the range of 8 gm/litre/day to 100 gm/litre/day; (b) increases the lipid content in the range of 20% to 90% from the first stage fermentation reactor, preferably in the range of 20% to 70% from the first stage fermentation reactor; and (c) DHA content in the range of 15% of total fatty acid to 50% of total fatty acid from the first stage fermentation reactor, preferably in the range 15% of total fatty acid to 35% of total fatty acid from the first stage fermentation reactor.

(139) Another embodiment of the present invention provides for use of novel strains of thraustochytrids as herein described for the process of production of (a) dry biomass in the range of 8 gm/litre/day to 200 gm/litre/day, preferably in the range of 8 gm/litre/day to 150 gm/litre/day; (b) lipid content in the range of 20% of dry mass to 100% of dry mass, preferably in the range of 20% of dry mass to 80% of dry mass; and (c) DHA content in the range of 15% of total fatty acid to 50% of fatty acid, preferably enhance the DHA content in the range of 15% of total fatty acid to 45% of fatty acid.

(140) Another embodiment of the present invention provides a process as herein described wherein in the absence of nitrogen increases the (a) biomass in the range of 8 gm/litre/day to 150 gm/litre/day, preferably in the range of 8 gm/litre/day to 100 gm/litre/day; (b) increases the lipid content in the range of 20% to 90% from the first stage fermentation reactor, preferably in the range of 20% to 70% from the first stage fermentation reactor; and (c) DHA content in the range of 15% of total fatty acid to 50% of total fatty acid from the first stage fermentation reactor, preferably in the range 15% of total fatty acid to 35% of total fatty acid from the first stage fermentation reactor.

(141) The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration to the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention various changes to the described embodiments may be made in the functions and arrangement of the elements described without departing from the scope of the invention.

EXAMPLES

Example 1

Isolation of Thraustochytrids from Indian Marine Biodiversity

(142) Large number of samples (Soil, water, degraded leaves etc) was collected from Indian marine sites in March 2013 over 30-35 km stretch in mangrove areas of Ribandar across Mandovi-Zuari mangroves (Collection Coordinates: S15?2957.39,E73?526.13, Panjim Goa). Physical parameters such as geographic position, pH, temperature and humidity of the collection site were recorded with GPS, pH meter, thermometer and hygroscope respectively. 100 ?l of selective antibiotic mixture was added into each falcon and samples were stored in dry ice packs and brought to lab along with natural sea water within 24 hours for processing. Direct plating or baiting method were the two protocols followed for the isolation. Soil samples were diluted 1000 times or leaf samples were washed, with sterile sea water before spreading on agar plates. Water samples were directly spread on agar plates. These agar plates were supplemented with selective antibiotic mixture. All the samples were baited with sterile pine pollens or other selected material (25-30 mg) and incubated at 25? C. for 8-10 days until colonization appears on the periphery of pollens. Colonization was checked daily after 5 days under light microscope. Once pollens are colonized, 100 ?l of the water was taken from surface of falcon carefully and spread on antibiotic containing agar plates. Media for these plates were containing glucose (10 g/L), Yeast extract (1 g/L), peptone (1 g/L), agar (10 g/L) filtered natural sea water (100% v/v) and antibiotic mixture. These plates were incubated at 25? C. for 7-10 days. Colonies appeared on the plates were observed under microscope (40?) for morphological study. Thraustochytrid like colonies were picked up and further streaked on agar plates having above described media composition with 70% Natural sea water. Thraustochytrid like strains were purified after 3-4 streaking on agar plates having selected antibiotic mixture. Presence of Omega-3 fatty acids particularly DHA is one of the key parameters for quick identification of Thraustochytrids (FIG. 1). Therefore lipids from the biomass of all the strains were extracted and converted in to fatty acid methyl esters by adopting known methods. These FAMEs were analyzed by GC-FID especially for presence of DHA.

Example 2

Molecular Characterization of Isolated Thraustochytrid Strains

(143) For genetic identification of the strains, 1 ml of 5 day old culture was harvested and genomic DNA was extracted according to the guidelines described in DNeasy blood and tissue kit (Qiagen, USA). Genomic DNA was used for PCR amplification of 18S rRNA gene using primers T18S1F 5-CAACCTGGTTGATCCTGCCAGTA-3 and T18S5R 5-TCACTACGGAAACCTTGTTACGAC-3 (Honda et al. 1999). 25 ?L PCR reaction was setup having 12.5 ?L PCR master mix (Applied Biosystem, USA), 0.5 ?L each primer (T18S1F, T18S5R), 1 ?L genomic DNA, 10.5 ?L milliQ water. PCR program included 3 min at 94? C. for initial denaturation, 45 sec at 94? C. for final denaturation, 30 sec at 64? C. for annealing, 2 min at 72? C. for extension, 10 min at 72? C. (final extension) for 30 cycles. PCR product was purified from 1% agarose gel using QiAquick gel extraction kit (Qiagen, USA) and mixture for PCR product and primers was sent to Macrogen (South Korea) for sequencing. The resulting 18S rRNA gene sequence was compared with known Thraustochytrids 18S rRNA gene sequences in NCBI gene bank database using basic local alignment search tool (BLAST). strains sequence along with other known sequences of Thraustochytrids were used to construct phylogenetic tree (NJ tree) using MEGA 6 software (FIG. 2). The resulting sequences were deposited in NCBI database (with accession no. KF668624, KF668627, KF668629, KF668632). Strains i.e. MTCC 5890 (DBTIOC-1), MTCC 5895 (DBTIOC-6), MTCC 5893 (DBTIOC-14), MTCC 5896 (DBTIOC-18) were deposited at Microbial Type Culture Collection and Gene Bank (MTCC) Institute of Microbial Technology (IMTECH), Chandigarh, India

Example 3

Screening of Thraustochytrid Strains for their Ability to Acetate Utilization and Acetate Tolerance

(144) Different concentration of acetate such as 5 g/L, 30 g/L, 50 g/L or 100 g/L were added in the nutrient rich medium as sole carbon source or mixed with other carbon sources. pH of acetic acid was raised to 7 with sodium hydroxide followed by addition of 10 g/L yeast extract, 1 g/L peptone. 18 g/L artificial sea salt was added in the medium to mimic 50% strength of sea water. Residual acetate was measure by HPLC and it was found that maximum acetate utilization does not go beyond 15 g/L in shake flask culture. However strain tolerance towards higher concentration of acetate was tested up to 50 g/L or 100 g/L. Strain DBTIOC-18 (MTCC 5896) performed better on acetate as carbon as compare to other strains irrespective of amount of acetate in the medium (FIG. 3).

Example 4

Screening of Thraustochytrid Strains for Nutrient Sequestration from Waste Effluents

(145) Waste effluents used in this invention were discharged from gas fermentation of green-houses gases or from deacetylation of lignocellulosic biomass or pyrolysis of lignocellulosic biomass or refining of crude oil etc. Chemical composition of these effluents revealed the dominant presence organic acids (particularly acetic acid, propionic acid, butyric acid) along with other acids and alcohols such as ethanol (0.5 g/L or 2 g/L 5 g/L or more) in the stream. This stream was as such autoclaved at 121? C. for 20 minutes without any additional nutrient source including nitrogen sources. 50 ml culture in 250 ml flask was harvested after 5 day and biomass was dried overnight. Lipids were extracted and FAMEs ware analyzed with the given method. In control strains biomass was ranging from 0.17 g/L to 0.61 g/L with slow nutrient sequestration (FIG. 4). Different strategies such as adaptation of strains on waste streams, addition of nitrogen sources and higher inoculum size were applied to increase the nutrient sequestration rate from waste streams.

(146) For adaptation the strains were cultured on waste effluents for longer duration for example one month, two month or three month or in some cases 6 months. Once nutrients are totally consumed in waste effluent streams, cultures were transferred to fresh stream. Transferring of culture was repeated 10 times, or 20 times, 30 times or in some cases 100 times. This enabled the strains to selectively utilize nutrients from these streams and gradually adapt on waste effluents resulting into higher biomass production (FIG. 4). Moreover the effect of the nitrogen source was also studied on the novel strains wherein addition of nitrogen source in waste effluents stream significantly increased nutrient sequestration and biomass production. The analysis of the biomass and the lipids showed that the novel strains were efficient in the production of the high biomass and lipids (FIG. 5). Addition of different inoculum sizes i.e. 5%, 10%, 15% and 20% also helped to increase nutrient sequestration and biomass production using waste streams as nutrient media without supplementing any nitrogen source (FIG. 6).

Examples 5

Different Continuous Cultivation Methods for the Cultivation of Selected Strain on Waste Effluent in Bioreactor

(147) Inoculum of selected strains of thraustochytrid was prepared in YPD media having 30 g/L glucose, 10 g/L yeast extract, 1 g/L peptone and 18 g/L sea salt and incubated at 25? C., 150 rpm. 10% of 48 h old inoculum was added in 2 L or 7 L or 14 L reactors, half filled with waste effluents having nitrogen source. Culture was aerated with normal drilled pipe spargers or microspargers or other type of microspargers whereas culture was agitated with Rushton or pitch blade or marine impeller or other impellers at 100 rpm or 200 rpm or 300 rpm or more. Dissolve oxygen was maintained at 10% or 20% or 30% or 50% or more with combination of air and oxygen supply. Sample was taken regularly at the interval of 8 to 12 h to determine nutrient sequestration, biomass production, lipid content and DHA production. Acetate consumption measurement was used as marker for the measurement of nutrient sequestration from waste effluents. Reactor pH was maintained at 7 with acid and base. In fed batch cultivation, waste effluents augmented with organic acids and alcohol were directly fed to culture.

(148) In continuous mode of cultivation two tubes were installed in the reactor, one tube for continuous addition of waste effluent with or without nitrogen source whereas overflow tube was installed for continuous harvesting the culture (FIG. 7).

(149) In two stage reactor system, broth coming out of first reactor is directly pumped into second reactor. First reactor was continuously supplied with nitrogen and high aeration whereas second reactor was not supplied with any nitrogen source and with very low dissolve oxygen. This system helped to increase lipid content particularly DHA by many fold.

(150) In two stage reactor system with biomass separating system, a biomass harvesting system is installed between first and second reactor and another is installed after second reactor to separate biomass from liquid broth. Broth coming out from first reactor goes to biomass harvesting system where biomass is concentrated and pumped into second reactor. Obsessed cells coming out from second reactor is continuously transferred to another biomass separating system where biomass is separated out from liquid broth. This clear liquid broth is continuously purged with nitrogen and supplied back to gas fermentation reactors (FIG. 11).

Example 6

Lipid Extraction and Fatty Acid Analysis

(151) For lipid extraction definite amount of dried biomass was taken and lipid was extracted with modified Bligh and Dyer method (FIGS. 3 and 4). For FAME analysis, 500 ?l toluene was added in dried lipid extract followed by 100 ?l internal standard (methyl tricosanoate C19:0) and 100 ?l butylated hydroxytoluene (BHT). Acidic methanol (400 ?l) was added into tube and kept for 10-12 hours at 50? C. 1 ml of 5% NaCl was added followed by the addition of 1 ml hexane and upper layer was transferred into fresh tube. FAMEs were washed with 1 ml of 2% Potassium bicarbonate and moisture was removed by passing it through anhydrous sodium sulphates. FAMEs were concentrated under nitrogen and analyzed with GC-FID system (Perkin Elmer clarus680, US), equipped with fast-GC capillary column (Omegawax100, 15 m?0.1 mm, 0.1 ?m thickness). 1 ?l of FAMEs were injected at injector temperature 250? C. Oven temperature was started increasing from initial 140? C. to final 280? C. with ramping rate of 40? C./sec and hold for 2 min. Detector was set at 260? C. Hydrogen was used as carrier gas with velocity rate 50 cm sec-1. Fatty acid peaks were identified and quantified with Totalchrome chromatography software (Perkin Elmer, US).