CLIAMS:We claim:
1. A process for sequential bio-hydrogen production through integration of dark fermentation process with photo fermentation process, the process comprising:
cultivating isolated bacteria in a bioreactor for dark fermentation under specific nutritional conditions for producing hydrogen from an organic substrate, wherein nitrogen supply is optimized; and
cultivating another isolated bacteria, in the effluent of dark fermentation under specific conditions and nutritional supplementation in a photo bioreactor, thereby integrating dark and photo-fermentation for enhancing hydrogen production.
2. The process according to claim 1, wherein the organic substrate used for dark fermentation is sugarcane molasses.
3. The process according to claim 2, wherein concentration of sugar in sugarcane molasses is between 40% and 45%.
4. The process according to claim 1, wherein the bacteria used for dark fermentation is consortium of Clostridium bifermentans strain and Clostridium butyricum strain.
5. The process according to claim 3, wherein the concentration of bacterial consortium added is 10% of total nutrient media.
6. The process according to claim 1, wherein the temperature of the bio reactor is maintained between 36 ºC and 38 ºC.
7. The process according to claim 1, wherein the pH in the bioreactor is maintained between 7 and 9.
8. The process according to claim 1, wherein the bioreactor is operated at ambient pressure.
9. The process according to claim 1, wherein the bioreactor for dark fermentation is made anaerobic by sparging inert gas.
10. The process according to claim 1, wherein the effluent of dark fermentation comprises acetic acid and butyric acid.
11. The process according to claim 1, wherein the isolated bacteria used for fermentation in the photo bioreactor is Rhodobacter sphaeroides CNT 2A strain.
12. The process according to claim 11, wherein the concentration of the bacterial culture added is between 10% (v/v) and 20% (v/v) of the total nutrient media.
13. The process according to claim 1, wherein the pH for photo fermentation is maintained between 6.5 and 7.5.
14. The process according to claim 1, wherein the temperature maintained in the photo bioreactor is between 28 0C and 31 0C.
15. The process according to claim 1, wherein the photo bioreactor for photo fermentation is made anaerobic by sparging inert gas. ,TagSPECI:BACKGROUND
Field
[0001] In general, subject matter relates to the field of bio hydrogen production. More particularly, but not exclusively, to enhancing hydrogen production by adopting both dark fermentation and photo fermentation.
Discussion of related field
Biological hydrogen production is considered to be the most environment friendly route of producing hydrogen. Conventional techniques to produce bio hydrogen include photo-catalytic water splitting and microbial fermentations. Microbial fermentations can be further classified as dark fermentation process, photo fermentation process and integration of both dark and photo fermentation processes.
[0002] In one of the conventional techniques, hydrogen is produced by dark fermentation process. In this technique, suitable microbes are used to degrade the organic substrates. Further, the process is carried out in absence of light and a nitrogen source is supplemented along with the organic substrate under anaerobic condition. However, the efficiency of hydrogen production from organic substrate is observed to be relatively low.
[0003] In another conventional technique, hydrogen is produced by photo fermentation process. In this process, strains of purple non-sulfur bacteria are used to produce hydrogen from organic acids, in the presence of light. However, it has been observed that, if the purple non sulphur bacterium does not possess a good potential for acetate and butyrate conversion, the efficiency of hydrogen production is decreased.
[0004] In yet another conventional technique, hydrogen is produced by the integration of dark fermentation process and photo fermentation process. In this two stage process, initially dark fermentation is carried out on organic waste. The effluent of dark fermentation process is used as raw material for photo fermentation process to produce hydrogen. It has been observed that, effluent of dark fermentation process comprises nitrogen, which is not favourable for production of hydrogen through photo fermentation process.
[0005] Furthermore, the concentration of butyrate in the effluent also does not efficiently enhance the production of hydrogen unless the purple non sulphur bacterial strain has good potential for butyrate utilization to produce hydrogen.
[0006] In light of the foregoing discussion, there is a need to develop a more effective technique for producing bio hydrogen, wherein effluent of dark fermentation process can be used as raw material for photo fermentation process.
SUMMARY
[0007] An embodiment provides process for sequential bio-hydrogen production through integration of dark fermentation process with photo fermentation process. The process includes cultivating isolated bacteria in a bioreactor for dark fermentation under specific nutritional conditions for producing hydrogen from an organic substrate, wherein nitrogen supply is optimized; and cultivating another isolated bacteria in the effluent of dark fermentation under specific conditions and nutritional supplementation in a photo bioreactor, thereby integrating dark and photo-fermentation for enhancing hydrogen production.
BRIEF DESCRIPTION OF DRAWINGS
[0008] Embodiments are illustrated by way of example and not limitation in the Figures of the accompanying drawings, in which like references indicate similar elements and in which, FIG.1 is a flow chart of an exemplary method for production of bio hydrogen from an organic substrate by dark fermentation process followed by photo fermentation process, in accordance with an embodiment.

DETAILED DESCRIPTION
I. OVERVIEW
II. EXEMPLARY DARK FERMENTATION PROCESS
III. PROCESSING OF EFFLUENT FROM DARK FERMENTATION PROCESS
IV. EXEMPLARY PHOTO FERMENTATION PROCESS
V. EXEMPLARY METHOD
VI. CONCLUSION

I. OVERVIEW
[0009] In general, subject matter relates to the field of bio hydrogen production. More particularly, but not exclusively, to an integrated approach of producing hydrogen, wherein effluent of dark fermentation process is used as raw material for photo fermentation process.
[0010] In an embodiment, production of hydrogen is carried out in a two stage process, wherein dark fermentation process is followed with photo fermentation process. In the first stage, organic substrate is utilized by the bacteria during the dark fermentation process, thereby resulting in formation of biomass, volatile fatty acids, H2 and CO2. Further, the temperature, pH and pressure of a bioreactor used for dark fermentation are maintained within desired ranges to carry out fermentation. In the second stage, the volatile fatty acids resulting from dark fermentation are used as raw material for the photo fermentative process. The photosynthetic bacteria used in the process produces hydrogen, thereby fermenting the raw material in a photo bioreactor.
[0011] The following detailed description includes references to the accompanying drawings, which form part of the detailed description. The drawings show illustrations in accordance with example embodiments. These example embodiments are described in enough detail to enable those skilled in the art to practice the present subject matter. However, it will be apparent to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures and components have not been described in detail so as not to unnecessarily obscure aspects of the embodiments. The embodiments can be combined, other embodiments can be utilized or structural and logical changes can be made without departing from the scope of the invention. The following detailed description is, therefore, not to be taken as a limiting sense.
[0012] In this document, the terms “a” or “an” are used, as is common in patent documents, to include one or more than one. In this document, the term “or” is used to refer to a nonexclusive “or,” such that “A or B” includes “A but not B,” “B but not A,” and “A and B,” unless otherwise indicated.

II. EXEMPLARY DARK FERMENTATION PROCESS
[0013] Bio hydrogen is produced from organic waste materials by dark fermentation process. In this process, organic substrate and bacterial culture are fermented in a bioreactor provided with nutritional supplements. The nutrient medium enhances the growth of bacterial culture added in the bioreactor. The hydrogen produced upon completion of dark fermentation process is passed through 40% w/w KOH solution for purification.

MEDIUM FOR DARK FERMENTATION
[0014] In an embodiment, the medium used for dark fermentation process is buffer based medium for hydrogen production (BMH medium).
[0015] In an embodiment, the BMH medium is composed of [g/L]:Sodium bicarbonate(NaHCO3) 4 - 4.5 gram; 4 ml trace element solution; and 4 ml of vitamin solution.
[0016] In an embodiment, the trace element solution is composed of (mg/L): MnO4.7H2O – 0.03-0.04 milligram; ZnSO4.7H2O – 0.1-0.25 milligram; H3BO3 – 0.03-0.04 milligram; N(CH2COOH)3 – 17-20 milligram; CaCl2.2H20 – 0.01-0.03 milligram; Na2MoO4 – 0.01-0.03 gram; CoCl2.6H2O – 0.7 -0.8 milligram; AlK(SO4)2 – 0.02 -0.04 milligram; MgCl.6H2 – 0.4-0.8 milligram; FeCl2 – 0.2-0.5 milligram; and CuCl2.6H2O – 0.01-0.3 milligram.
[0017] In an embodiment, the vitamin solution added to the aforementioned fermentation medium is composed of [mg/L]: riboflavin – 0.06 -0.07 milligram; citric acid – 0.020-0.04 milligram; folic acid – 0.01-0.02 milligram; and para-amino benzoic acid – 0.01-0.03 milligram.
[0018] In an embodiment, the media is prepared anaerobically by simultaneous heating and sparging with an inert gas.

BACTERIAL CULTURE
[0019] In an embodiment, the bacterial consortium used to carry out dark fermentation process is a mixture of Clostridium bifermentans strain and Clostridium butyricum strain isolated from river bed sediment. In an embodiment, the isolated bacterial strain comprises Clostridium bifermentas strain deposited in NCBI nucleotide sequence database under the accession number FJ656099 and Clostridium butyricum strain deposited in NCBI nucleotide sequence database under the accession number FJ656098.
PROCESS
[0020] In an embodiment, dark fermentation is carried out in anaerobic and sterile condition, in the absence of light. In this process, a bioreactor receives BMH media, feedstock, thereby forming a nutrient media required to carry out the process. Further, isolated bacterial culture is cultivated into the nutrient media, wherein the nutrient media enhances the growth of the bacteria within the bioreactor.
[0021] In an embodiment, the feedstock includes an organic substrate. The organic substrate, for example, can be sugarcane molasses, which serves as a source of carbon in the process. It shall be noted that the concentration of sugar in sugarcane molasses is between 40% and 45%.
[0022] In an embodiment, 2 to 4 % of molasses with respect to the BMH media is added into the bioreactor.
[0023] In an embodiment, the pH of the nutrient medium is maintained between 7 and 9, but more preferably at 8. The pH can be maintained at the desired range, for example, by adding 1N NaOH or 1N HCl solution prior to autoclaving.
[0024] Further, bacterial consortium comprising mixture of Clostridium butyricum and Clostridium bifermentans is used for dark fermentation process.
[0025] In an embodiment, about 10% of the bacterial culture with respect to the total volume of nutrient media is inoculated into the bioreactor. It shall be noted that nitrogen required to carry out the fermentation process is obtained from the organic substrate.
[0026] In an embodiment, nitrogen is sparged into the bioreactor to create an anaerobic condition.
[0027] In an embodiment, the bioreactor is operated at ambient pressure.
[0028] In an embodiment, the temperature of the bio reactor is maintained between 36 ºC and 38 ºC, more preferably at 37 ºC.
[0029] Further, in an embodiment, an agitating device is configured to mix the contents inside the bioreactor for 24 hrs at an approximate speed between 80 rpm and 100 rpm
[0030] In an embodiment, bioreactors are operated in batch mode.
[0031] The dark fermentation process yields hydrogen, carbon dioxide and organic acids as the end effluents.
[0032] In an embodiment, acid products comprise acetic acid and butyric acid at a concentration between 3000 – 4500 milligram per litre, which can be used as raw material for photo fermentation process.
[0033] In an embodiment, the gas generated during fermentation of molasses is allowed to pass through 40% w/w KOH solution for selective absorption of carbon dioxide.
[0034] In an embodiment, optimization of the above mentioned parameters result in the production of 80-115 mmole of hydrogen /7-9g of COD reduction at a scale of 100 -1000 litre.

III. PROCESSING OF EFFLUENT FROM DARK FERMENTATION PROCESS
[0035] The effluents obtained at the end of the dark fermentation process are organic acids namely, acetic acid and butyric acid.
[0036] In an embodiment, the effluents obtained at the end of the dark fermentation process is processed and subsequently used in photo fermentation process.
[0037] In an embodiment, separation techniques may be employed to obtain cell free effluents. The separation techniques can be, for example, size exclusion and centrifugation, among others.
[0038] In an embodiment, the cell-free effluent can also be obtained by subjecting the said effluents to a settling tank. Further, the cell free effluents are temperature treated for facilitating light penetration.
[0039] In an embodiment, the pH of the effluent medium is maintained between 6.5 and 7.5. The pH of the effluent medium can be adjusted, for example, by adding 1M NaOH.
[0040] Further, a buffering agent can be added to the effluent medium to maintain constant pH. The buffering agent can be, for example, phosphate buffer among others.
[0041] In an embodiment, iron source is added to the effluent medium. The iron can be, for example, Fe-citrate among others.
[0042] In an embodiment, molybdenum source is added to the effluent medium (Na2MoO4.2H2O). The molybdenum source can be, for example, sodium molybdate, which acts as an enzyme activator.
[0043] Further, in an embodiment, trace solution and vitamins of MHP medium is added.

IV. EXEMPLARY PHOTO FERMENTATION PROCESS
[0044] Production of hydrogen can also be carried out by photo fermentation process. In this process, feedstock and bacterial culture is fermented in a photo bioreactor provided with the MHP medium. Further, a source of light is required for fermentation process to be carried out. The hydrogen produced upon completion of photo fermentation process is passed through 40% w/w KOH.

MEDIUM FOR PHOTO FERMENTATION
[0045] In an embodiment, the media used for hydrogen production process is MHP medium. MHP media used for photo fermentation process is a modified form of Sistrom’s medium. The MHP media is composed of (g/L): K2HPO4-6-7 gram; KH2PO4 - 3.0 -3.50 gram; CaCl2.2H2O - 0.03 -0.05 gram; MgSO4.7H2O - 0.150.25 gram; Fe-citrate - 0.01-0.042 gram; NaCl - 0.3-0.5gram; Na-glutamate - 0.5-0.7 gram; trace solution - 1ml/ L.
[0046] In an embodiment, the trace solution is comprised of (mg/l); EDTA - 0.004-0.006 milligram; ZnSO4.7H2O - 0.07-0.15 milligram;H3BO3 - 0.2-0.4 milligram; CoCl2.6H2O – 0.1-0.3 milligram; Na2MoO4.2H20 – 0.02-0.04 milligram; MnCl2.4H2O.4H2O – 0.02-0.04 milligram; NiCl2.6H2O – 0.01-0.03 milligram; CuCl2.2H2O – 0.01-0.02 milligram.
[0047] In an embodiment, the media is prepared anaerobically by simultaneous heating and sparging with an inert gas.
BACTERIAL CULTURE
[0048] In an embodiment, bacteria used in the photo fermentation process is isolated and selected to enable robust hydrogen production during the photo fermentation process.
[0049] Bacterial strain used to carry out photo fermentative hydrogen production is isolated from fresh water sediment samples. Purification of the bacterial strains used for photo fermentative hydrogen production is carried by streaking method on modified Biebel and Pfennig’s agar slants medium. The purity of the isolated colonies is confirmed by gram staining and subsequent microscopic examination. The Individual colonies so obtained is inoculated in liquid medium and further maintained by routine sub culturing.
[0050] Identification of the purified bacterial strain for photo fermentative process is based on 16S r RNA gene sequencing. DNA extracted from the bacterial cells is purified and as template for PCR technique. In this technique, amplification of 16S rRNA gene is performed using 27F and 1492R primers. Further, the amplification conditions for PCR include denaturation at 940 C, annealing at 500C and elongation at 720C. The PCR amplicons so obtained are sequenced using cycle sequencing system. The nucleotide sequence on both the strands is determined by comparing it with the reference sequences available in the NCBI database using BLAST search.
[0051] Phylogenetic tree for CNT-2A strain is constructed and it is observed that CNT-2A fell in the cluster with the known Rhodobacter sphaeroides species. Rhodobacter sphaeroides CNT-2A obtained is deposited in NCBI nucleotide sequence database under the accession number FR731160.
PROCESS
[0052] In an embodiment, photo fermentation is carried out in anaerobic, sterile condition, in presence of light. In this process, a photo bioreactor receives MHP media, raw material, and photosynthetic bacterial culture required to carry out the process.
[0053] In an embodiment, nutrient media to carry out hydrogen production is formed by supplementing MHP media, raw material, and sodium glutamate into the photo bioreactor. The effluents obtained from dark fermentation process, which comprises organic acids, namely, acetic acid and butyric acid is used as the raw material to carry out photo fermentation process.
[0054] In an embodiment, the pH of the nutrient medium is maintained between 6.5 and 7.5. The pH can be maintained at the desired range, for example, by adding 1N NaOH as buffering agent.
[0055] In an embodiment, the photo bioreactor is flushed with argon gas thoroughly to obtain an anaerobic atmosphere.
[0056] In an embodiment, the photosynthetic bacterial strain used to carry out photo fermentation is R. sphaeroides CNT-2A. It shall be noted, that the bacterial strain used for the current process has potential to utilize acetate as well as butyrate as substrate. Further, the bacterial culture used for the photo fermentation process is cultivated in the nitrogen free effluents of dark fermentation process in presence of nutritional supplements maintaining specific conditions.
[0057] In an embodiment, 10% to 20% (v/v) of the above mentioned photosynthetic bacterial culture with respect to the total nutrient media is inoculated into the photo bioreactor.
[0058] In an embodiment, the temperature of the photo bioreactor is maintained between 280C and 310C, more preferably at 300C.
[0059] Further, in an embodiment, an agitating device is configured to mix the contents inside the photo bioreactor for 96 to 120 hrs at a speed of 30-50 rpm. It shall be noted, that homogenous distribution of nutrient is required for proper growth of the bacterial culture used for photo fermentation, which can be accomplished through agitation of the contents by the agitating device.
[0060] In an embodiment, photo bioreactor is operated in a batch mode to carry out photo fermentation process producing hydrogen, carbon dioxide and other products.
[0061] In an embodiment, the photo bioreactor is illuminated with an internal light source for efficient light transfer to the microbial culture during photo fermentation process.
[0062] In an embodiment, the photo bioreactor has internal submerged light facility with multiple photosynthetic lights.
[0063] In an embodiment, the photo bioreactor is illuminated with a light intensity of between 4500 -7000 lux, more preferably at 6500 lux, which solved the light penetration problems.
[0064] The gas so obtained after the completion of the photo fermentation is made to pass through KOH solution or any other scrubbing agent to absorb the carbon dioxide formed.
V. EXEMPLARY METHOD
[0065] FIG.1 is a flow chart of an exemplary method for production of bio hydrogen from an organic substrate by dark fermentation process followed by photo fermentation process, in accordance with an embodiment. At step 102, 2-4 % of -molasses (organic substrate), BMH media is added into the bioreactor to obtain a nutrient medium. The pH of the nutrient medium is maintained by addition of 1N NaOH or 1N HCl solution. At step 104, 10% (v/v) of bacterial consortium- mixture of Clostridium bifermentans strain and Clostridium butyricum strain is added into the bioreactor.
[0066] An anaerobic and sterile environment is maintained inside the bio reactor by sparging nitrogen gas. Dark fermentation process is carried out in the bioreactor at step 106. The temperature of the bioreactor is maintained between 36 oC and 38 oC for fermenting molasses into volatile acids along with the production of hydrogen by hydrogen producing bacteria. Effluents, CO2 and H2 are obtained on the completion of the dark fermentation process. The gas generated is allowed to pass through 40% w/w KOH solution for selective absorption of carbon dioxide.
[0067] It shall be noted that, the effluent obtained from the aforementioned process includes significantly less or no nitrogen present in it. Organic acids, such as, acetic acid and butyric acid, obtained as effluents are fed as the raw material into a photo bioreactor comprising MHP media, to carry out photo fermentation process (at step 108). In an embodiment, sodium glutamate is also added in the photo bioreactor.
[0068] At step 110, photo fermentative bacterial strain Rhodobacter sphaeroides CNT-2A is inoculated into the photo bioreactor. Further, the pH of the medium is maintained between 6.5 and 7.5 by addition of buffering agent. An anaerobic atmosphere is maintained within the photo bioreactor by sparging the reactor with inert gas.
[0069] At step 112, photo fermentation is carried out in the presence of light source provided in internally in the photo bioreactor. The fermentation process is carried out for 96-120 hrs at an agitation rate of 20-40 rpm maintaining the temperature of the photo bioreactor between 28 oC and to 31oC.
[0070] At step 114, on the completion of photo fermentation process, hydrogen is obtained along with other products. The hydrogen so obtained is made to pass through KOH solution to remove CO2.

VI. CONCLUSION
[0071] The processes described above is described as sequence of steps, this was done solely for the sake of illustration. Accordingly, it is contemplated that some steps may be added, some steps may be omitted, the order of the steps may be re-arranged, or some steps may be performed simultaneously.
[0072] Although embodiments have been described with reference to specific example embodiments, it will be evident that various modifications and changes may be made to these embodiments without departing from the broader spirit and scope of the system and method described herein. Accordingly, the specification and drawings are to be regarded in an illustrative rather than a restrictive sense.
[0073] Many alterations and modifications of the present invention will no doubt become apparent to a person of ordinary skill in the art after having read the foregoing description. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. It is to be understood that the description above contains many specifications; these should not be construed as limiting the scope of the invention but as merely providing illustrations of some of the personally preferred embodiments of this invention. Thus the scope of the invention should be determined by the appended claims and their legal equivalents rather than by the examples given.