Claims:1. A process for obtaining one or more halophilic bacteria for use in high salt wastewater treatment, comprising steps of:
a) incubating marine sediment in nutrient broth comprising sea water to obtain a culture,
b) incubating the culture in media comprising sea water optionally along with high salt wastewater to obtain a second culture, and
c) incubating the second culture in media comprising high salt wastewater and sea water to obtain halophilic bacteria.

2. The process as claimed in claim 1, wherein the ratio of the sea water to the nutrient broth ranges from about 1:1 to about 1:5, preferably from about 1:1 to about 1:3; and wherein the incubating of the marine sediment is carried out at a temperature ranging from about 28°C to about 32°C, preferably about 30°C for a period of about 68 h to about 74 h, preferably about 72 h.

3. The process as claimed in claim 1, wherein the high salt wastewater in step b) or step c) is Hydrothermal Liquefaction-Raffinate (HTL-R); wherein the media in step b) or step c) further comprises one or more nutrient supplement; and/or wherein the nutrient supplement is selected from a group comprising source of nitrogen, carbon, micro-nutrients, minerals, salts and phosphate or any combination thereof.

4. The process as claimed in claim 1, wherein the ratio of the wastewater to the seawater ranges from about 1:15 to about 1:20, preferably about 1:19; and wherein the incubating in step b) or c) is carried out at a temperature ranging from about 28°C to 32°C, for about 140 to 240 h.

5. The process as claimed in claim 1, wherein the halophilic bacteria is isolated by technique selected from a group comprising spread plate, steak plate and pour plate or any combination thereof, the incubating is carried out in a shaker, and wherein the culture of step a), b) or c) is obtained by separating the culture from the nutrient broth or the media by technique selected from a group comprising centrifugation, settling or a combination thereof.

6. The process as claimed in claim 5, wherein the halophilic bacteria is a pure culture or a consortium of two or more isolated halophilic bacteria.
7. Halophilic bacteria for use in high salt wastewater treatment obtained by the process as claimed in any of the preceding claims.

8. A process for treating high salt wastewater comprising steps of:
a) diluting the high salt wastewater with seawater, and
b) subjecting the diluted wastewater with the halophilic bacteria as claimed in claim 6 to obtain treated wastewater.

9. The process as claimed in claim 8, wherein the diluting is carried out with 2X to 20X seawater, preferably 20X seawater; and wherein the wastewater is further supplemented with one or more nutrient supplement prior to or post contacting with the halophilic bacteria.

10. The process as claimed in claim 8, wherein the subjecting is carried out at a temperature ranging from about 28°C to about 32°C for a period ranging from about 140 h to about 260 h at a speed ranging from about 200 to about 250 rpm; wherein the halophilic bacteria is a consortium of two or more bacteria; and wherein the halophilic bacteria is at an amount ranging from about 25% to 3.0%, preferably 2.5% (v/v %).

11. The process as claimed in claim 1 or claim 8 or the halophilic bacteria as claimed in claim 7, wherein the high salt wastewater has Chemical Oxygen Demand (COD) ranging from about 30000 to 50000 ppm, Biochemical Oxygen Demand (BOD) ranging from about 20000 to 30000 ppm, chloride ranging from about 2 to 3%, Total Solid (TS) ranging from about 60000 to 70000 ppm, Total Suspended Solids (TSS) ranging from about 900 to 1000 ppm, Total Dissolved Solids (TDS) ranging from about 60000 to 70000 ppm, Total Organic Carbon (TOC) ranging from about 14000 to 20000 ppm, Total Nitrogen (TN) ranging from about 20000 to 25000 ppm, total protein ranging from about 5000 to 8000 ppm or total carbohydrates ranging from about 30 to 100 ppm, or any combination thereof.

12. The process as claimed in claim 1 or claim 8 or the halophilic bacteria as claimed in claim 7, wherein the high salt wastewater is Hydrothermal Liquefaction-Raffinate (HTL-R).

13. Treated wastewater obtained by the process as claimed in any of claims 8 to 12.

14. A process for growth of algae comprising step of:
diluting the treated wastewater obtained from the process as claimed in any of claims 8 to 12 and recycling the same into algal pond for growth of algae.

15. The process as claimed in claim 14, wherein the diluting is carried out with at least 5X of water, preferably sea water; and wherein the treated wastewater is supplemented with nutrient supplement.

16. The process as claimed in claim 13, wherein the growth of algae is carried out at temperature ranging from about 25°C to about 30°C; for a time period ranging from about 160 h to about 260 h; and/or at a relative humidity ranging from about 65 to about 75%.
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Description:TECHNICAL FIELD
The present disclosure relates to the fields of wastewater management and particularly relates to a process for treating wastewater. Particularly, the present disclosure relates to halophilic bacteria or consortium for use in high salt wastewater treatment and process of producing the same. The present disclosure also relates to process of treating high salt wastewater by the halophilic bacteria or consortium and the treated wastewater thus obtained. The treated wastewater is suitable for re-use or environment friendly disposal. The present disclosure also provides for a process for growth of algae employing the treated wastewater.

BACKGROUND & PRIOR ART
Hydrothermal liquefaction (HTL) is a thermal depolymerization process which is used to convert wet biomass into crude-like oil under moderate temperature and high pressure. Hydrothermal Liquefaction of algae is a promising process for the production of biofuel from algae. However, the wastewater or “raffinate” generated after Hydrothermal Liquefaction of the algae is of large volume, highly complex and extremely harsh for disposal or to support algal growth on recycling. It has high Total Organic Carbon (TOC), salinity/chloride and is dark in colour. It cannot be re-used for algal growth or disposed off unless it is diluted ~1000 X, which is economically inefficient. The raffinate is extremely harmful to bacteria because of its high salinity, not to mention it’s high Chemical Oxygen Demand (COD)/TOC, which causes osmotic stress to the cells resulting in disruption of cell wall and inhibiting reaction pathways in the organic degradation process. Hence it needs to be diluted excessively before recycle/re-use or even for disposal purposes.

Generally, to treat high saline wastewater by activated sludge it is diluted with fresh water. However, this is also unsustainable considering the pressure on industries to reduce freshwater intake. Further, biodegradation studies have been carried out with salt tolerant microorganisms only at lower COD ranging from about 1400 to 5000 ppm.

Thus, there is a need for providing efficient means and methods for overcoming the constraints of the prior art and efficiently treating wastewater in conditions of high salinity, COD and/or toxic pollutants.

SUMMARY OF THE DISCLOSURE
The present disclosure relates to a process for obtaining one or more halophilic bacteria for use in high salt wastewater treatment, comprising steps of a) incubating marine sediment in nutrient broth comprising sea water to obtain a culture, b) incubating the culture in media comprising sea water optionally along with high salt wastewater to obtain a second culture, and c) incubating the second culture in media comprising high salt wastewater and sea water to obtain halophilic bacteria; halophilic bacteria for use in high salt wastewater treatment obtained by said process; a process for treating high salt wastewater comprising steps of: a) diluting the high salt wastewater with seawater, and b) subjecting the diluted wastewater with said halophilic bacteria to obtain treated wastewater; treated wastewater obtained by the said process; and a process for growth of algae comprising step of diluting the treated wastewater obtained from the aforesaid process and recycling the same into algal pond for growth of algae.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure where:
Figure 1(A) depicts schematic representation of an exemplary embodiment of the present disclosure for preparation of halophilic bacteria or consortium.
Figure 1(B) depicts % of COD removal at the three stages of acclimatization of RDMT (Reliance Dahej Marine Terminal) consortium in HTL-R.
Figure 2 depicts TOC removal from HTL-R Composite water by the HTL culture mix at different hours of incubation depicted as remaining TOC conc. (ppm) in water (A), and % of TOC removal (B).
Figure 3 depicts growth measured in different HTL-R Composite feed compositions.
Figure 4 depicts the pH profile during growth period (or incubation) in different HTL-R Composite feed compositions.
Figure 5 depicts individual components analyzed by GC/MS in biotreated (A) and untreated (B) samples of HTL-R Composite wastewater.
Figure 6 depicts TOC removal in HTL-R 420 water by HTL mixed culture at different hours of incubation depicted as remaining TOC conc. (ppm) in water and % of TOC removal.
Figure 7 depicts individual components analyzed by GC/MS in untreated (upper chromatogram) and biotreated (lower chromatogram) HTL-R 420 wastewater.
Figure 8 depicts analysis of COD removal by individual and mixed HTL cultures.
Figure 9 depicts algal growth in biotreated HTL-Composite water.

DETAILED DESCRIPTION
The present disclosure overcomes the drawbacks of the prior art and provides for an efficient and cost-effective means for treatment of highly saline wastewater of >35000 ppm chloride with halophilic bacteria.

In an embodiment, the present disclosure provides for halophilic bacteria or consortium for use in high salt wastewater treatment and process of producing the same and application thereof.

In embodiments of the present disclosure, the halophilic bacteria or consortium thereof is capable of sustaining harsh environmental conditions and degrading diverse organic compounds (having high COD ranging from about 30,000 to 50,000 ppm), toxic pollutants and salinity > 35000 ppm.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The use of the expression “at least” or “at least one” suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” or “containing” or “has” or “having” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

As used herein, the terms ‘method’ and ‘process’ have the same scope and meaning and are used interchangeably.

As used herein, the term ‘high salt wastewater’, ‘ww’ and ‘wastewater’ refers to water produced as a by-product of industrial or commercial activities and having a salt content greater than at least 30000 ppm, preferably at least 35000 ppm. In an exemplary embodiment, the high salt wastewater employed in the present disclosure is Hydrothermal Liquefaction-Raffinate.

As used herein, the terms ‘raffinate’, ‘Hydrothermal Liquefaction-Raffinate’ (HTL-R), ‘HTL-Raffinate’, are used interchangeably. The said terms refers to the liquid/wastewater resulting from Hydrothermal Liquefaction after extraction of oil. The raffinate generated after Hydrothermal Liquefaction of the algae is of large volume, highly complex and extremely harsh for disposal or to support algal growth on recycling. It has high TOC/COD, salinity/chloride and is dark in colour, and unsuitable for bacterial growth. In an exemplary embodiment, the raffinate is produced from Hydrothermal Liquefaction of Algae.

As used herein, the term “impurities” or “impurity” refers to the less soluble residue of the raffinate. In an exemplary embodiment, impurities present in the raffinate include but are not limited to high boiling hydrocarbons (> 200 MW), such as but not limiting to oxygenates including phenols, diols, acetate & esters; high molecular weight (MW) paraffins (C26 – C33); nitrogenous organic compounds; complex carbohydrates; major feed stock nutrients etc.

In an exemplary embodiment of the present disclosure, the Hydrothermal Liquefaction-Raffinate (HTL-R) employed in the present invention is selected from group comprising HTL-R Composite and HTL-R 420.

As used herein, HTL–R Composite refers to the pooled aqueous phase of HTL experiments from a number of algal species collected during the screening of best algal species. In an exemplary embodiment of the present disclosure, salinity of HTL-R composite is about 52 ppt (29200 ppm Chloride), TOC of the HTL-R composite is about 14050 ppm, and Total dissolved solids (TDS) is in the range of about 62,000-68,000 ppm.

As used herein, HTL-R 420 refers to aqueous phase from HTL of single algal species. In an exemplary embodiment of the present disclosure, salinity of HTL-R 420 is about 37 ppt (20674 ppm Chloride), TOC of HTL-R 420 is about 16875 ppm, and Total dissolved solids (TDS) is in the range of about 62,000-68,000 ppm.
In embodiments of the present disclosure, the high salt wastewater has one or more of Chemical Oxygen Demand (COD) ranging from about 30000 to 50000 ppm, Biochemical Oxygen Demand (BOD) ranging from about 20000 to 30000 ppm, chloride ranging from about 2 to 3%, Total Solid (TS) ranging from about 60000 to 70000 ppm, Total Suspended Solids (TSS) ranging from about 900 to 1000 ppm, Total Dissolved Solids (TDS) ranging from about 60000 to 70000 ppm, Total Organic Carbon (TOC) ranging from about 14000 to 20000 ppm, Total Nitrogen (TN) ranging from about 20000 to 25000 ppm, total protein ranging from about 5000 to 8000 ppm or total carbohydrates ranging from about 30 to 100 ppm, or any combination thereof.

In embodiments of the present disclosure, the high salt wastewater has chloride ranging from about 2 to 3%.

In embodiments of the present disclosure, the high salt wastewater has at least about 20000 ppm chloride, preferably at least about 35000 ppm.

In an exemplary embodiment of the present disclosure, the high salt wastewater has chloride ranging from about 20000 ppm to about 40000 ppm, preferably about 35000 ppm.

In embodiments of the present disclosure, the raffinate needs to be diluted only 20x with sea water (SW) for biodegradation. Dilution of the wastewater with 20X sea water provides high salinity environment to the halophilic bacteria/consortium of the present disclosure which results in better COD removal of HTL-R.

The ‘halophilic bacteria’ of the present disclosure refers to bacteria capable of efficiently reducing the COD/TOC of wastewater in conditions of high salinity, such as >30000 ppm preferably >35000 ppm. The halophilic bacteria of the present disclosure may be a pure culture or a consortium of two or more bacterial groups living symbiotically.

As used herein, the abbreviation JSW refers to Jamnagar Gagva sea water employed for dilution in the present disclosure.

As used herein, the term "about" means to be nearly the same as a referenced number or value. As used herein, the term "about" should be generally understood to encompass ± 10% of a specified amount or value.

The present disclosure provides for a process for developing halophilic bacteria or a consortium of halophilic bacteria that can effectively degrade a raffinate generated after hydrothermal liquefaction of algal biomass making it recyclable to algal ponds for re-use in algal growth or suitable for disposal.

In an embodiment, the present disclosure provides for developing bacteria exposed to saline conditions such as but not limiting to bacteria originating from marine mud/sediment. The bacteria thus obtained is further subjected to 3 phase acclimatization by (i) adding sediment in nutrient broth, enumerating the culture in the 2nd & 3rd acclimatization by adding 20x diluted HTL-R in sea water and finally isolating, purifying and remixing the cultures to get the halophilic consortium suitable for biodegradation.

In an embodiment, the process for obtaining one or more halophilic bacteria for use in high salt wastewater treatment, comprises steps of:
a) incubating marine sediment in nutrient broth comprising sea water to obtain a culture,
b) incubating the culture in media comprising sea water optionally along with high salt wastewater to obtain a second culture, and
c) incubating the second culture in media comprising high salt wastewater and sea water to obtain halophilic bacteria.

In an embodiment, the halophilic bacteria obtained by the aforesaid process is further isolated and/or purified by conventional techniques.

In an alternate embodiment, the process for obtaining one or more halophilic bacteria for use in high salt wastewater treatment, comprises steps of:
a) incubating marine sediment in nutrient broth comprising sea water to obtain a culture,
b) incubating the culture in media comprising sea water optionally along with high salt wastewater to obtain a second culture,
c) incubating the second culture in media comprising high salt wastewater and sea water to obtain halophilic bacterial culture, and
d) isolating and optionally purifying the culture to obtain the halophilic bacteria.

In another embodiment, the ratio of the sea water to the nutrient broth employed for incubating the marine sediment ranges from about 1:1 to about 1:5, preferably from about 1:1 to about 1:3.

In yet another embodiment, the incubation of the marine sediment is carried out at a temperature ranging from about 28°C to about 32°C, preferably about 30°C for a period of about 68 h to about 74 h, preferably about 72 h.

In still another embodiment, the high salt wastewater employed in the process for obtaining one or more halophilic bacteria is Hydrothermal Liquefaction-Raffinate (HTL-R).

In still another embodiment, the media comprising sea water optionally along with high salt wastewater or the media comprising sea water and high salt wastewater may further comprise one or more nutrient supplement. The nutrient supplement includes one or more source of nitrogen, carbon, micro-nutrients, minerals, salts and/or phosphate. In a non-limiting exemplary embodiment of the present disclosure, the said nutrient supplement is selected from a group comprising yeast extract, phosphoric acid, urea, peptone, beef extract and sodium chloride or any combination thereof.

In still another embodiment, the ratio of the wastewater to the seawater employed in the process for obtaining one or more halophilic bacteria ranges from about 1:15 to about 1:20, preferably about 1:19

In still another embodiment, the incubation of the culture or the second culture is carried out at a temperature ranging from about 28°C to 32°C, preferably about 30°C for about 140 to 240 h.

In still another embodiment, the halophilic bacteria is isolated by employing any conventional technique such as but not limiting to plating. In an exemplary and non-limiting embodiment, the halophilic bacteria is isolated by technique selected from a group comprising spread plate, steak plate and pour plate or any combination thereof.

In still another embodiment, the incubation in the process for obtaining one or more halophilic bacteria is carried out in a shaker.

In still another embodiment, the culture of any step of the process for obtaining one or more halophilic bacteria is obtained by separating the culture from the nutrient broth or the media by technique selected from a group comprising centrifugation, settling or a combination thereof.

In still another embodiment, the halophilic bacteria of the present disclosure is a pure culture or a consortium of two or more isolated halophilic bacteria.

In an embodiment, the process of developing the halophilic bacteria consortium of the present disclosure comprises:
(i) sample collection of bacteria originating from marine sediment,
(ii) enumerating by a process of 3 phase acclimatization., wherein the a) first phase of acclimatization comprises inoculating the marine mud/sediment into a medium comprising sea water & Nutrient broth, incubating by shaking (orbital), centrifuging the sample, and obtaining the pellets, b) the second phase of acclimatization is carried out with adding a mixture of HTL-R wastewater and sea water and optionally along with yeast extract to the pellet, incubating by shaking (orbital), centrifuging the sample, and obtaining the pellets, c) the third acclimatization is carried out similar to the second acclimatization, and
(iii) isolating and purifying to obtain halophilic bacterial culture.

In an embodiment, the mixed cultures obtained from the sludge by the 3 phase acclimatization process are halophilic and capable of tolerating salinity as high as 35000 ppm.

In an embodiment, after the 3rd acclimatization, the slurry which is a mix of bacterial cultures from the biosludge is isolated and incubated to obtain purified cultures which are optionally transferred to nutrient media for preparing individual stock culture of uniform optical density (OD) for biodegradation studies and for morphological characterization.
In an embodiment, the process of the present disclosure comprises:
(i) sampling of bacteria from a marine ecosystem,
(ii) developing the halophilic bacteria by the process of acclimatization wherein
a) acclimatization step 1: the sediment (about 1 g) is placed/inoculated in a medium comprising seawater (about 50-100 ml) and nutrient broth (about 100-150 ml) comprising Yeast extract, Peptone and Glucose. This is incubated by shaking (orbital) for about 48 to 96 h, preferably about 72 h. The sample is centrifuged, the supernatant measured for COD & discarded. The pellets obtained after centrifugation is used for the second acclimatization step. In an embodiment, COD reduction is observed in the sample at this stage.
b) acclimatization step 2: HTL-R composite wastewater (cww) diluted 20x with sea water and 0.12% yeast extract is added to the pellets obtained in the acclimatization step 1. Mixed liquor suspended solids (MLSS) concentration (from centrifuged pellet) was kept at 7000 ppm. This is incubated on the shaker for about 144 - 216 h and centrifuged and the supernatant was optionally analyzed for COD.
c) acclimatization step 3: is carried out with the centrifuged pellet obtained in the acclimatization step 2. COD reduction here was 66%
(iii) isolating and purifying the acclimatized bacterial cultures obtained as per standard methods (such as but not limiting to spread plate & streak plate techniques) and preserved on a suitable media such as but not limiting to TYG (Tryptone, Yeast Glucose) agar slants, and
(iv) the pure cultures thus obtained are again mixed at uniform individual OD to give the HTL culture mix.

In embodiments of the present disclosure, the bacteria isolated from marine environment is already exposed to hydrocarbon pollution and salinity which are made further effective by a process of acclimatization to HTLWW having high salinity (35000 ppm) & TOC (800-2900 ppm) with slow increase in concentration. The first step here has grown all those bacterial culture which are capable of degrading hydrocarbon and sustain the salinity gradients .The second step has selectively allowed only those from the first who are capable of degrading the diverse nature of organics under high salinity, automatically eliminating the non-functional. So this is enrichment of HTL_R capable and high salinity resistant bacterial consortia. The third step is the enumeration of the enriched bacterial consortium capable of growing in high salinity >35000 ppm, more efficiently degrading the HTL-R as their numbers increased.

The pure or mix halophilic bacterial cultures of the present disclosure are capable of degrading the diverse organic compounds (high COD range 30,000 to 50,000 ppm), toxic pollutants and salinity > 35000 ppm.

In an embodiment, the scheme for an exemplary embodiment of the present disclosure is provide in Figure 1(A).

In an exemplary embodiment, for biodegradation studies about 3-5 pure cultures are mixed at equal OD (at about 620 nm) values and used throughout the experimentation. They are also referred to as HTL mixed culture.

In an embodiment, 16SrDNAs analysis carried out for identification of the bacterial consortia developed.

In embodiments of the present disclosure, the individual cultures obtained as per the present disclosure are compatible, do not have detrimental/negative effect when combined and exhibit a synergistic effect in the bacterial consortium/HTL mixed culture.

In embodiments of the present disclosure, the HTL mixed culture has higher COD removal compared to individual cultures and have diverse metabolic potential.

In an exemplary embodiment, in a bacterial consortium the multiple cultures are mixed equally and the most potential one will dominate and govern the biodegradation while each one of the culture imparts some beneficial role in achieving the performance according to their metabolic potential. The mixed culture of the present disclosure provides better results in COD removal due to their diverse metabolic potential.

The present disclosure also pertains to the halophilic bacteria for use in high salt wastewater treatment obtained by the process of the present disclosure.

The present disclosure also pertains to a process for treating high salt wastewater comprising steps of:
a) diluting the high salt wastewater with seawater, and
b) subjecting the diluted wastewater with the halophilic bacteria of the present disclosure to obtain treated wastewater.

In an embodiment, the high salt wastewater is diluted with about 2X to 20X seawater, preferably about 20X seawater.

In another embodiment, the high salt wastewater to be treated may further be supplemented with one or more nutrient supplement, prior to or post contacting with the halophilic bacteria. The nutrient supplement includes one or more source of nitrogen, carbon, micro-nutrients, minerals, salts and/or phosphate. In a non-limiting exemplary embodiment of the present disclosure, the said nutrient supplement is selected from a group comprising yeast extract, phosphoric acid, urea, peptone, beef extract and sodium chloride or any combination thereof.

In an embodiment, the halophilic bacteria of the present disclosure is subjected to the diluted wastewater at a temperature ranging from about 28°C to about 32°C.

In an embodiment, the halophilic bacteria of the present disclosure is subjected to the diluted wastewater for a period ranging from about 140 h to about 260 h.

In an embodiment, the halophilic bacteria of the present disclosure is subjected to the diluted wastewater at a speed ranging from about 200 to about 250 rpm.

In yet another embodiment, the halophilic bacteria of the present disclosure is subjected to the diluted wastewater at a temperature ranging from about 28°C to about 32°C for a period ranging from about 140 h to about 260 h at a speed ranging from about 200 to about 250 rpm.

In still another embodiment, the halophilic bacteria employed in the process for treating high salt wastewater is at an amount ranging from about 25% to 3.0%, preferably 2.5% (v/v %).

In still another embodiment, the halophilic bacteria employed in the present disclosure is a consortium of two or more bacteria.

In an embodiment, the biodegradation studies are carried out with 2 types of HTL-R (HTL-R Composite and HTL-R 420).

In an embodiment, different dilutions of HTL-R with distilled water (DW) and Gagva SW, is studied at a pH of 8.5 ± 0.5 and the TOC and Growth (O.D. at 620nm) is measured after 48, 72, 164 and 188 h. (i) HTL-R diluted 20 x with DW (ii) HTL-R diluted 13x DW + 7x SW (iii) HTL-R diluted 7x DW + 13x SW and (iv) HTL-R diluted with 20x SW. TOC removal was maximum at 164 h in case iv. TOC removal was 76%, growth increased three-fold.

In an embodiment, the conditions employed in the present disclosure are as follows: HTL-R dilution with SW (20X), Yeast extract = about 0.05%, For a Feed COD of 200 ppm the ratio of Phosphoric acid (P) : Urea (N) is about 5 : 1, HTL mixed culture (5 Nos.) = 2.5%, Duration of exposure = 164 – 236 h, Orbital shaking at 200 to 250 rpm; HTL mixed culture (5nos) – 2.5% to 3.0%.

In an embodiment, efficacy of the HTL mixed culture to degrade HTL Raffinate is tested by biodegradation experiments using 2 batches of HTL-R (HTL-R Composite and HTL-R 420) under above mentioned conditions. In an embodiment, TOC removal in HTL-R Composite wastewater after 20x dilution with SW using the HTL mixed culture was 60% at 120 h. With HTL-R 420 wastewater the TOC removal was 80% which increased to 87% in 216 h. In the case of HTL-R 420 Total N was also measured and its removal was 45 - 47% at 120 h. Total proteins were reduced ~30% in both batches of HTL-R. Total carbohydrates increased after treatment by 3.5 to 5 fold in HTL-R Composite and 420 respectively. Individual components analysis showed significant removal in both HTL-R batches. Of the 26 components seen in the HTL-R Composite before treatment, almost all were removed, only traces of 5 components were seen after treatment. In HTL-R 420 16 components were seen initially before treatment but after biodegradation traces of 6 components were seen most of them being biodegradation by-products.

The present disclosure also pertains to the treated wastewater obtained by the process of the present disclosure.

In an embodiment, the TOC of the treated wastewater is at least less than 700 ppm preferably less than 500 ppm.

In embodiments of the present disclosure, the treated wastewater has TOC in the range of about 300-400 ppm, total protein in the range of about 300 to 400 ppm, total carbohydrate in the range of about 10 to 20ppm and/or total nitrogen in the range of about 600- 800 ppm.

In embodiments of the present disclosure, the 20X diluted HTL wastewater post treatment with bacteria has TOC in the range of about 300-400 ppm, total protein in the range of about 300 to 400 ppm, total carbohydrate in the range of about 10 to 20 ppm and/or total nitrogen in the range of about 600- 800 ppm.

In all embodiments of the present disclosure, the high salt wastewater has at least one of the characteristics selected from a group comprising Chemical Oxygen Demand (COD) ranging from about 30000 to 50000 ppm, Biochemical Oxygen Demand (BOD) ranging from about 20000 to 30000 ppm, chloride ranging from about 2 to 3%, Total Solid (TS) ranging from about 60000 to 70000 ppm, Total Suspended Solids (TSS) ranging from about 900 to 1000 ppm, Total Dissolved Solids (TDS) ranging from about 60000 to 70000 ppm, Total Organic Carbon (TOC) ranging from about 14000 to 20000 ppm, Total Nitrogen (TN) ranging from about 20000 to 25000 ppm, total protein ranging from about 5000 to 8000 ppm and total carbohydrates ranging from about 30 to 100 ppm, or any combination thereof.

In embodiments of the present disclosure, the high salt wastewater is preferably Hydrothermal Liquefaction-Raffinate (HTL-R).

In embodiments of the present disclosure, the processes of the present disclosure may comprise analysing the halophilic bacteria for degradation of impurities in the high salt wastewater and optionally monitoring component selected from a group comprising pH, optical density, total organic carbon (TOC), Chemical Oxygen Demand (COD), total protein, carbohydrate, lipid, total nitrogen, ammonia, nitrate and individual components in the wastewater or any combination thereof.

In embodiments of the present disclosure, the total protein content of a sample is analysed by Biuret method (Raymont et al 1964), total carbohydrate estimation is analysed by Phenol Sulphuric Acid method after preconcentration (Dubois et al., 1956), total nitrogen (TN) is analysed by Chemiluminescence, ammonia and nitrate are analysed by Ion Selective Electrode (Ion analyser).

The present disclosure also relates to a process for growth of algae comprising step of diluting the treated wastewater obtained from the process of the present disclosure and recycling/employing the same in algal pond for growth of algae.

In an embodiment, dilution of the treated wastewater is carried out with at least 5X of water, preferably sea water.

In another embodiment, the treated wastewater employed for growth of algae is supplemented with one or more nutrient supplement. The nutrient supplement includes one or more source of nitrogen, carbon, micro-nutrient, minerals, salts and/or phosphate. In a non-limiting exemplary embodiment of the present disclosure, the said nutrient supplement is selected from a group comprising phosphoric acid, urea, and micro-nutrient(s) or any combination thereof.

In embodiments of the present disclosure, the micro-nutrient is selected from a group comprising MnSO4.2H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuCl2.2H2O and CoSO4.7H2O or any combination thereof.

In yet another embodiment, the growth of algae is carried out at temperature ranging from 25°C to 30°C preferably about 27°C; for a time period ranging from about 160 h to about 260 h, at a relative humidity ranging from about 65 to about 75%.

In still another embodiment, the growth of algae is carried out at about 1% to about 3%, preferably about 2% CO2 and/or about 150 to about 160 µMol/m2.s of illumination.

In an embodiment, the process of the present disclosure comprises:
(i) obtaining a marine sediment sample having a bacteria which can survive in the saline environment,
(ii) developing the halophilic consortium capable of degrading the HTL-Raffinate by the afore-described process of 3 step acclimatization,
(iii) testing the culture for degradation of the HTL-R batches and monitoring the TOC, growth and removal of individual components in the wastewater and
(iv) recycling the biotreated water into the algal pond for growth of algae.

In an embodiment, the present disclosure provides for process and bacteria consortium for reducing the COD/TOC of wastewater in conditions of high salinity. HTL wastewater is highly saline (about 35000 ppm chloride) with a TOC of 800 – 2900 ppm depending on the type of algae and detrimental to bacteria per se. Hence the bacterial consortium of the present disclosure developed through the process of the present disclosure is capable of tolerating the harsh conditions of HTL-R and suitable for biodegrading the HTL-R after 20 X dilution, reducing the TOC by 60-80%.

In embodiments of the present disclosure, the present disclosure provides for a cost-effective biodegradation process for the removal of organics collectively and individually, optionally measured quantitatively by techniques including but not limiting to TOC & qualitatively by techniques including but not limiting to GC/MS, from the raffinate/wastewater resulting from the hydrothermal liquefaction (HTL) of algae grown for biofuel.

In an embodiment, HTL-R cannot be discharged as such as it is highly toxic with TOC /COD levels much higher than statutory requirements. This means it has to be diluted at least 1000 x before disposal. The cost of energy to pump sea water for such dilution is economically unattractive. The raffinate needs to be diluted only 20x with sea water (SW) for biodegradation. The biotreated effluent may also be discharged safely if required directly without any further dilution.

In an embodiment, the treated water when recycled after further dilution (of about 5X) is suitable for support the growth of algae optionally along with appropriate nutrient supplements.

In an embodiment, the biotreated water of the present disclosure has to be diluted further 5x if it is to be recycled into the algal ponds for algal growth. As this water already has low amounts of NO3- it only needs reduced supplements of nutrients to support the algal growth. By recycling into algal ponds with 5x further dilution, costs on application of full dosage of nutrients can be saved & energy costs on dilution water could also be saved.

In embodiments of the present disclosure, TOC analysis of the raffinate/wastewater samples is carried out instead of COD due to Chloride interferences.

In embodiments of the present disclosure, TYG media includes 5.0 g Tryptone, 2.5g Yeast Extract, 1.0 g Glucose and 29.0g Agar in 1L distilled water.

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

Any possible combination of two or more of the embodiments described herein is comprised within the scope of the present disclosure.

The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

Further, while the instant disclosure is susceptible to various modifications and alternative forms, specific aspects thereof has been shown by way of examples and drawings and are described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention.

Examples:
Example 1: Developing a consortium of halophilic bacteria
Marine sediment was collected at the Reliance Dahej Marine Terminal (RDMT) and was subjected to a process of 3 phase acclimatization as depicted in Figure1(A). First phase of the acclimatization was carried out by inoculating 1 g of the marine sediment/mud into a medium comprising:
Batch (a) 50 ml sea water & 100 ml Nutrient broth (Yeast extract, Peptone & Glucose); and
Batch (b) 100 ml sea water & 150 ml Nutrient broth.

This was followed by incubation at 28 to 32°C by shaking (orbital) for 72 h. After 72 h, the sample was centrifuged, and the supernatant was measured for COD. At this stage the COD removal was 33%. The pellets obtained after centrifugation was used for the second acclimatization step. The second phase of acclimatization was carried out by adding a mixture of HTL-R composite wastewater and sea water (25 ml HTL-CWW + 475 ml sea water) and 0.6 g yeast extract to the pellet. MLSS concentration (from mixed pellet) was kept at 7000 ppm. It was incubated on the shaker at 28 to 32°C for 144 - 216 h. After 144 h & 216 h, the samples were centrifuged, and the supernatant was analyzed for COD. 53% reduction in COD was observed at this stage. The centrifuged pellet was used for the third acclimatization in a similar way. After the 3rd acclimatization the COD removal was 66%. The mixed cultures obtained from the sludge by the 3 phase acclimatization process were thus halophilic, capable of tolerating salinity as high as 35000 ppm. The % of COD removal at the three stages of acclimatization is depicted in Figure 1(B).

Isolation and Purification of halophilic bacterial culture: After the 3rd acclimatization, the slurry which is a mix of bacterial cultures from the biosludge, was isolated by American Public Health Association (APHA) Standard Spread plate & streak plate techniques on TYG agar (TYG media includes 5.0 g Tryptone, 2.5g Yeast Extract, 1.0 g Glucose and 29.0g Agar in 1L distilled water). The plates were incubated at 30°C for 48-72 h. 5 Nos. of purified cultures were obtained which were transferred to TYG agar slants and used for preparing individual stock culture of uniform optical density for biodegradation studies and for morphological characterization. 16SrDNAs analysis was carried out for identification of the bacterial consortia developed and the results are tabulated in Table 1.

Table 1:
HTL pure cultures Identified as (By 16SrDNA)
HTL-1 Bacillus Species
HTL-2 Bacillus thuringiensis
HTL-3 Lysinibacillus strain
HTL-4 Bacillus cereus strain
HTL-5 Pseudomonas Xiamensis strain

For biodegradation studies, these pure cultures (5 nos.) were mixed at equal optical density values (OD at 620 nm) and used throughout the experimentation. The said mixed culture is also referred to as HTL mixed culture.

Example 2: Biodegradation of HTL-R wastewater
Different dilutions of HTL-R composite with DW and Gagva SW, was studied at a pH of 8.5 ± 0.5 and the TOC and Growth (O.D. at 620nm) was measured after 48, 72, 164 and 188 h.
(i) HTL-R diluted 20 x with DW
(ii) HTL-R diluted 13x DW + 7x SW
(iii) HTL-R diluted 7x DW + 13x SW and
(iv) HTL-R diluted with 20x SW.
TOC removal was found to be maximum at 164 h in case iv, wherein TOC removal was 76%, and growth increased threefold. Accordingly, the biodegradation studies were carried out using HTL-R dilution with 20X sea water.

Example 3: Biodegradation of HTL-R using the HTL mixed culture
The efficacy of the culture mix to degrade HTL Raffinate was tested by biodegradation experiments using 2 batches of HTL-R (HTL-R Composite and HTL-R 420) under the following conditions:
HTL-R dilution with SW (20X)
Yeast extract = 0.05%
For a Feed COD of 200 ppm the ratio of Phosphoric acid (P):Urea (N) is 5 : 1
HTL mixed culture (5 Nos.) = 2.5%
Duration of exposure = 164 – 216 h
Orbital shaking at 200 rpm

Example 3(a): Biodegradation of HTL-R Composite samples using HTL Culture mix
HTL- Raffinate Composite samples were obtained from A2O Lab at Vadodara Manufacturing Division (VMD). It was a composite of wastewater collected over several months and several batches of Algal experiments. This raffinate was diluted 20x (F4 in Figures 2 and 3) with seawater (10 ml HTL-R + 190 ml SW). The initial TOC of the HTL-R composite waste water was 14050 ppm and the TOC of the 20x diluted HTL-R composite waste water was 730 ppm. The HTL culture mix of example 1 comprising the 5 pure cultures was used for the biodegradation studies, in shake flask experiments.

10 ml HTL-R wastewater was poured into a 500 ml conical flask and 190 ml of sea water was added to it and mixed well. 0.1 g Yeast extract, phosphoric acid and urea at a ratio of 5:1 for a COD of 200 ppm were then added to the feed flask. This is followed by addition of 1 ml of the HTL culture mix to the feed flask. 4 sets of experiments were conducted at the aforesaid conditions & incubation was carried out on a shaker for 236 h. Samples were withdrawn at 0, 96, 120, 144, 164 and 236 h for measuring TOC, while for pH and growth (OD at 620nm) measurements they were withdrawn at 0, 24, 48, 72, 96, 120 and 164 h. TOC measurement was preferred over COD because of chloride interference in COD analysis giving rise to erroneous values. Total protein, carbohydrate & lipid were measured in initial feed and the treated sample 216 h. The sample at 216 h was also analyzed for individual components by GC/MS and compared with the 0 h sample.

Figure 2 depicts removal of TOC from HTL-R Composite water by the HTL culture mix at different hours of incubation using various dilutions depicted as remaining TOC conc. (ppm) in water (A), % of TOC removal (B). The TOC removal was found to be 56% after 120 h and increased to 60% after 236 h in HTL-R diluted with 20X sea water (Figure 2(B)).

Figures 3 and 4 depicts growth and pH measured in the following HTL-R Composite feed compositions:
F1: HTL-R Composite with 0x seawater dilution
F2: HTL-R Composite with 1.4x seawater dilution
F3: HTL-R Composite with 2.5x seawater dilution
F4: HTL-R Composite with 20x seawater dilution

For F4, growth improved by 3-fold (0.7 OD at 0 h to 2 OD after 72 h). After this it stabilized between 1.6 to 1.75 OD at 620nm over the remaining period (164 h) of the study (Figure 3).

The pH profile of F4 indicated marked decrease of pH during initial 50 h of period, following gradual rise upon further incubation period. The initial decrease may be due to diverse metabolic activities as the growth also observed almost 3-fold improvement during 0 to 72h.

Figure 5 depict individual components seen by GC/MS in biotreated (A) and untreated (B) samples. Of the 26 components seen in the HTL-R Composite before treatment, almost all were removed, only traces of 5 components were seen after treatment. Thus, individual components analysis showed significant removal in the HTL-R.

Example 3(b): Biodegradation of HTL-R 420 wastewater using HTL Culture mix
HTL-Raffinate 420 sample was obtained from A2O Lab at VMD. It was collected after experiments using a potent single algal strain. This raffinate was diluted 20x with seawater (10 ml HTL-R 420 + 190 ml SW). The HTL culture mix of example 1 was used for the biodegradation studies, in shake flask experiments. The feed flasks also included 0.1 g Yeast extract, phosphoric acid and urea added at a ratio of 5:1 for a COD of 200 ppm and 1 ml of the HTL culture mix as in example 2(a). The flasks were kept on shaker for 236 h incubation. Samples were withdrawn at 0, 96, 120, 144, 168, 192 and 216 h for measuring TOC. Removal of individual components in the raffinate/wastewater was monitored through GC-MS analysis of both feed and treated samples.

Figure 6 depicts TOC removal in HTL-R 420 water by HTL mixed culture. The TOC reduction was found to be much faster, i.e. 78% in 96 h and by 216 h 87% reduction was obtained (Figure 5). This was probably due to a less complicated feed compared to HTL-R Composite.

Figure 7 depicts individual components analysed in HTL-R 420 water prior to biotreatment (upper chromatogram) and when biotreated with the HTL culture mix (lower chromatogram). The number of individual components seen in the feed/untreated sample were 16 in number (Figure 7 upper). After biotreatment, almost all were removed, however some traces of 6 new components were seen (Figure 7 lower) which were mostly biodegradation by-products. Thus, individual components analysis showed significant removal in the HTL-R.

Example 3(c): Analysis of biotreated HTL-R composite and HTL-R 420 wastewater using HTL Culture mix
The HTL-R Composite sample and the HTL-Raffinate 420 sample of examples 3(a) and 3(b) were diluted 20x with seawater (5 ml wastewater + 95 ml SW). The samples were contacted with 2.5 ml of HTL culture mix of example 1, 0.05 g Yeast extract, the nutrients phosphoric acid and urea at a ratio of 5:1 are added for a COD of 200 ppm. The flasks were kept on shaker for 236 h incubation. Total carbon (TOCA), protein & carbohydrate were measured in initial feed and the treated sample at 216 h. Additional parameters like Total Nitrogen (TN), Ammonia & Nitrate were also measured at 0 and 168 h incubation for the HTLR Composite and 216h for the HTL-R 420 sample. The results obtained are depicted in Table 2, wherein:
Fd = HTL feed to the bioprocess;
Trtd = biodegraded water;
Rem = Removal;
n.d. = not done; and
JSW=Jamnagar sea water.

Table 2:

For the HTL-R composite sample, total carbon reduced by about 56%, total proteins reduced by about 30-33% while total carbohydrates increased 3.5 fold at 216 h.

For the HTL-R 420 wastewater, total carbon reduced by about 86%, total proteins reduced by ~30% and total carbohydrates increased after treatment by 5 fold at 216 h. Further, total N was also measured and its removal was 45 - 47% at 216 h.

Thus, the 20X diluted HTL wastewater post treatment with bacteria has TOC in the range of about 300-400 ppm, total protein in the range of about 300 to 400 ppm, total carbohydrate in the range of about 10 to 20ppm and Total nitrogen in the range of about 600- 800 ppm.

Example 4: Biodegradation using individual HTL Cultures vs. HTL Culture mix
Some of the pure HTL cultures of example 1 (viz. HTL 1, 2 and 3) were tested individually for biodegradation of the HTL raffinate composite diluted 20x with SW as per the protocol of example 2 and compared via biodegradation using the HTL mixed culture of example 1. COD and Growth (O.D. at 610 nm) was monitored at 0, 24, 48, 72 and 96 h. Figure 8 depicts COD removal by individual and mixed HTL cultures. The results showed that with HTL1 culture 35% removal of COD was achieved in 72 h after which it reduced to 33% by 96 h. HTL 2 culture removed 34% COD by 96 h. COD removal of 27% was seen in HTL3 culture in 72 h after which it stabilized.

With mixed HTL cultures, the COD removal was 37% in 72 h and remained so even at 96 h (Figure 8). Thus, HTL mixed culture achieved higher COD removal compared to individual cultures.

Example 5: Recycle of biodegraded HTL-R Composite wastewater for algal growth
The biotreated wastewater of example 2(a) was further diluted with sea water and recycled into algal tanks and tested for algal growth. Experiments on recycling of the biotreated HTL-R Composite water of example 2(a) for algal growth was carried out after (i) diluting the same 1.25x with SW (Total dilution = 25x) and no nutrient supplement (Black line in Figure 9) (ii) dilution of 5x with SW (Total dilution = 100x) without nutrient supplement (blue line) (iii) dilution of 5x with SW (Total dilution = 100x) with nutrient supplement (green line) and (iv) control with a full complement of the nutrients, i.e. the UPA medium (red dotted line).

Nutrient supplementation consisted of addition of Phosphate and Micro-nutrient mix equivalent in concentration to that added in Control Experiment with UPA medium, however No N was added only 19.94 ppm of TPO4 (TP=6.5 ppm and Micro-nutrients at 2mL/L of 500X.

The control experiment consisted of addition of the following nutrients in Gagva sea water (Standard UPA Medium):
• Urea = 200 ppm = 3.3 mM (TN = 93.3 ppm)
• Phosphoric Acid = 210uL/L (from 1M stock) = 19.94 ppm (TP=6.5 ppm)
• Micro-nutrient mix = 2 mL/L of 500X stock where the micronutrients used were MnSO4.2H2O, ZnSO4.7H2O, Na2MoO4.2H2O, CuCl2.2H2O, CoSO4.7H2O.

All experiments were carried out at 100 mL scale at 27°C, 100rpm, 2% CO2 ,70% relative humidity (RH) and illumination of 155 µMol/m2.s in Khuner shaker (Controlled Environment).

The algal growth with limited nutrient supplements was good as controls having only the UPA medium with full nutrient supplement. Figure 9 depicts the algal growth in the biotreated HTL-Composite water samples and the results obtained indicate that (iii) performs as good, if not better (5 O.D. at 750nm), than controls (4.5 O.D. at 750nm) after 234 h. However, the growth values obtained for (ii) is also good enough (1.5 O.D. at 750 nm at 162 h), for normal algal growth (Figure 9). The overall results clearly indicate that 20x dilution of the HTL-R with SW can be biotreated and the treated water is further diluted 5x with SW and recycled for algal growth. Nutrient supplements can be added to the biotreated wastewater for flourishing growth (5 O.D. at 750nm) or biotreated wastewater without nutrient supplements can be used for normal growth (1.5 O.D. at 750 nm).