Description:TECHNICAL FIELD
[001] The present disclosure generally relates to the field of biogas and its use as a source of energy and its conversion to fuel precursors, and in particular to an integrated method or system for the selective production of fuel precursors from biogas.
BACKGROUND OF THE INVENTION
[002] A methane-enriched gas mixture, such as biogas, is a mixture of gases, primarily consisting of methane (CH4), carbon dioxide (CO2), and hydrogen sulphide (H2S), produced from anaerobic digestion of organic matter. It is a non-toxic, colorless, and flammable gas with an ignition temperature of 650-750°C. Unlike natural gas, biogas can be produced from abundant and renewable organic feedstock and is considered a carbon-neutral energy source. Therefore, biogas is not only a crucial part of the current energy portfolio, but it will also play an important role in achieving long-term energy sustainability. Methane-enriched gas is also found in landfill sites, wastewater treatment plants, oil exploration sites, etc. Raw biogas is usually purified by an energy-intensive purification process to remove various contaminants like CO2 and H2S and obtain purified methane for its application as transportation fuel, power generation, and heating purpose etc. In the case of transportation fuel, purified methane has specific engine requirements. Furthermore, methane is usually pressurized at high pressure (21-25 MPa) and with a boiling point of -164°C, making it costly to store, transport, and distribute. Further, all these processes also require high energy inputs and large capital expenditures.
[003] In order to address these issues, it is desirable to convert raw biogas into easily handled, high-value chemicals, and fuel precursors with high energy densities.
[004] Methane is the major component of biogas and is difficult to activate due to its high C-H bond strength, high ionization potential, and low acidity. Currently, the most commonly used method for converting methane to chemicals and fuel technology is the two-step or one-step thermochemical conversion process with syngas as the intermediate.
[005] There is a plethora of literature available for the thermochemical conversion of methane to chemicals.
[006] In the review paper entitled “Advances in methane conversion processes.” Catalysis today 285 (2017): 147-158, Wang et.al describe the direct conversion of methane into chemicals and fuels.
[007] Ge et. al. (Biotechnology advances, 32(8), 1460-1475, 2014) disclose studies on various promising groups of microorganisms that hold potential for converting methane to liquid fuels. However, in most of these processes, genetic modifications are required by conventional and recombinant techniques to produce enzymes. Mutants created after genetic manipulation by recombinant DNA technology require specific growth factors or media components for the fermentation of methane.
[008] Kim, et. al (Nature Catalysis 2, no. 4 (2019): 342-353) developed pMMO-mimetic catalytic protein constructs by genetically encoding the beneficial reassembly of catalytic domains of pMMO on apoferritin as a biosynthetic scaffold. This approach resulted in synthesis of stable and soluble protein constructs in Escherichia coli that successfully retained enzymatic activity for methanol production with a turnover number comparable to that of native pMMO.
[009] Adeel Mehmood et. al. (Photoelectrochemical Conversion of Methane into Value-Added Products Catalysts 2021, 11(11)) disclose a photoelectrochemical conversion of methane into value-added products.
[0010] CN113663623A discloses a method of bionic leaf that converts solar energy into a liquid fuel. It uses a photocatalyst to fix carbon dioxide as an intermediate product, and then selects a biocatalyst that can use the intermediate product to convert the intermediate product into liquid fuel through microbial electrosynthesis.
[0011] EP3536798A1 discloses a semi-conducting biogenic hybrid catalyst capable of reducing CO into fuel precursors. It involves a method for bio-assisted conversion of CO to fuel precursors using the semiconducting biogenic hybrid catalyst in batch and continuous modes.
[0012] Adeel Mehmood et. al. (Photoelectrochemical Conversion of Methane into Value-Added Products Catalysts 2021, 11(11)) disclose a photoelectrochemical conversion of methane into value-added products.
[0013] Although the available literature provides several methods for the bioconversion of methane, the methods available face several challenges in the direct conversion of methane containing raw biogas to chemicals. Prior literature does not disclose a method that can completely transform raw biogas enriched with methane and containing contaminants like CO2, H2S, NH4 in a single reactor setup to obtain desired chemical precursors. Most of the methods in the literature require an expensive biogas purification process, which is capital-intensive. The chemical conversion of purified methane from biogas is energy-intensive and requires a larger capital investment. Simultaneous conversion of CH2 and CO2 is not possible as it results in lower carbon efficiency. The yield of desired products obtained from the methods disclosed in the prior literature is very low. The methods described in the prior literature are not selective to produce fuel precursors from biogas, and thus, product separation and purification are tedious and expensive.
[0014] To address the problems in the art, the present disclosure provides an integrated method or system for the selective production of fuel precursors from biogas, enriched with methane and also comprising contaminants like CO2, H2S, and NH4 etc. More specifically, the present disclosure provides a bio-inorganic hybrid method for tunable production of acids, alcohols and polyhydroxyalkanoate (PHA) from raw biogas. The method and system of the present disclosure comprise a photo-bioreactor and a loop bioreactor, wherein the photo-bioreactor comprises photosensitized biocatalysts and the loop reactor comprises a microbe and an inorganic energy equivalent. The method and system of the present disclosure are able to convert raw biogas or any methane-containing gas to industrially relevant chemicals with high yield and selectivity.
SUMMARY OF THE INVENTION
[0015] This summary is provided to introduce a selection of concepts in a simplified format that are further described in the detailed description of the invention.
[0016] In a first aspect of the present disclosure, there is provided an integrated method for the selective production of fuel precursors from biogas, the integrated method comprising:
selecting and culturing an electroactive microorganism in a culture medium in a first stimulator generating bio-reactor, adding a semi-conducting precursor to the culture medium, and sparging a biogas stream through an immobilized carbon-dioxide solubilizing enzyme into the culture medium to form semi-conducting particles functionalized on the surface of the electroactive microorganism, adding a light harvesting fluorescent dye to the culture medium, irradiating the electroactive microorganism in the culture medium with a light source to metabolize a carbon source present in the biogas stream, and obtaining a first aqueous medium and a first residual biogas stream;
selecting and culturing a yeast strain in a culture medium in a second stimulator generating bio-reactor, routing the aqueous medium and the first residual biogas stream to the second stimulator generating bio-reactor, adding the semi-conducting precursor to the culture medium to form semi-conducting particles functionalized on the surface of the yeast strain, adding the light harvesting fluorescent dye to the culture medium, irradiating the yeast strain in the culture medium with a light source to metabolize the carbon source present in the first residual biogas stream, and obtaining a second aqueous medium and a second residual biogas stream; and
selecting and culturing a methanotrophic microorganism in a culture medium in a final product loop bio-reactor, adding an inorganic enzyme equivalent to the culture medium, routing the second aqueous medium and the second residual biogas stream to the culture medium of the final product loop bio-reactor to metabolize methane present in the second residual biogas stream, and recovering a final product stream having the fuel precursors,
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof.
[0017] In another aspect of the present disclosure, there is provided an integrated system for selective production of fuel precursors from biogas, the integrated system comprising:
a first stimulator generating bio-reactor, the first stimulator generating bio-reactor comprising: an inlet for receiving a biogas stream into the first stimulator generating bio-reactor, an immobilized carbon-dioxide solubilizing enzyme, a light source, an agitator, an electroactive microorganism in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the electroactive microorganism to metabolize a carbon source present in the biogas stream and produce a first aqueous medium and a first residual biogas stream, and an outlet for passing the first aqueous medium and the first residual biogas stream;
a second stimulator generating bio-reactor, the second stimulator generating bio-reactor comprising: an inlet for receiving the first aqueous medium and the first residual biogas stream into the second stimulator generating bio-reactor, a light source, an agitator, a yeast strain in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the yeast strain to metabolize the carbon source present in the first residual biogas stream and produce a second aqueous medium and a second residual biogas stream, and an outlet for passing the second aqueous medium and the second residual biogas stream; and
a final product loop bio-reactor, the final product loop bio-reactor comprising: an inlet for receiving the second aqueous medium and the second residual biogas stream into the final product loop bio-reactor, an agitator, a methanotrophic microorganism in a culture medium, and an inorganic enzyme equivalent functionalized on the surface of the methanotrophic microorganism to metabolize methane present in the second residual biogas stream and produce a final product stream having the fuel precursors,
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof,
wherein the second stimulator generating bio-reactor is placed downstream of the first stimulator generating bio-reactor, and the final product loop bio-reactor is placed downstream of the second stimulator generating bio-reactor, and
wherein the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, and the final product loop bio-reactor are configured to be operated in a sequential manner.
[0018] These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[0019] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0020] Figure 1 illustrates a schematic representation of an integrated bioprocess for the conversion of methane-enriched gas mixture to chemicals, according to an embodiment of the present disclosure.
[0021] Figure 2 illustrates a process flow diagram of overall biogas to chemical technology, according to an embodiment of the present disclosure. SGR: Stimulator generating reactor; FPLR: Final product loop reactor.
[0022] Figure 3 illustrates a schematic representation of reactors for the conversion of biogas to chemicals, according to an embodiment of the present disclosure.
[0023] Further, the skilled in the art will appreciate that elements in the drawings are illustrated for simplicity and may not have necessarily been drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps of the process, features of the invention, referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.
[0025] Definitions: For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person skilled in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.
[0026] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”. Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.
[0027] As used in this disclosure, “biogas, biogas stream, or raw biogas” refers to the gas, or mixtures of gases, produced from the breakdown of organic material or “biomass”. The terms can be used interchangeably.
[0028] As used in this disclosure, “hydraulic retention time (HRT)” refers to the average time the broth stays in a reactor. It is crucial for effective conversion.
[0029] As used in this disclosure, “alcohols” include methanol, ethanol, and/or butanol.
[0030] As used in this disclosure, “acid” includes organic acids, such as acetic acid and/or butanoic acid.
[0031] As used in this disclosure, “polyhydroxyalkanoate” includes Polyhydroxybutyrate (PHB), Polyhydroxyvalerate (PHV), and/or Polyhydroxyhexanoate (PHH).
[0032] As used in this disclosure, an “inorganic enzyme equivalent (IEE)” refers to inorganic complexes that mimic the activity of natural enzymes.
[0033] As used in this disclosure, a “biohybrid catalyst” comprises biogenic semiconductor particles/ semi-conducting particles functionalized on electroactive microbes.
[0034] As used in this disclosure, a “bio-reactor” refers to a vessel in which one or more chemical reactions may occur between one or more reactants, optionally in the presence of one or more catalysts. In the present disclosure, a first stimulator generating bio-reactor, a second stimulator generating bio-reactor, and final product loop bio-reactor are used.
[0035] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products and methods are clearly within the scope of the disclosure, as described herein.
[0036] Methane, the predominant element in natural gas and biogas, represents a promising alternative to carbon feedstocks in the biotechnological industry due to its low cost and high abundance. The bioconversion of methane to value-added products can enhance the value of gas and mitigate greenhouse gas emissions.
[0037] Problem being addressed by the present disclosure. Conversion of raw biogas to chemicals is important for sustainable bio-refinery. The current methods for biogas/methane conversion face several challenges:
• Raw biogases contain several impurities, like H2S and CO2, which may poison the catalyst or harmful for microbial growth.
• Most of the process requires an expensive biogas purification process, which is capital intensive.
• Chemical conversion of purified methane from biogas is energy-intensive and requires a larger capital-investment.
• Simultaneous conversion of CH2 and CO2 is not possible resulting in lower carbon efficiency.
• The yields of desired products described in the prior literature are very low.
• The processes described in the literature are not selective, and as a result, the product separation and purification are tedious.
[0038] The solution to the problem is provided by the present disclosure. The present disclosure relates to the direct conversion of raw biogas to industrially relevant chemicals and fuel precursors with high energy density. More specifically, the present disclosure provides a bio-inorganic hybrid method for tunable/selective production of acids, alcohols and polyhydroxyalkanoate (PHA) from raw biogas or any methane-containing gases. The present disclosure also provides an integrated bio-reactor system for biogas conversion that consists of a stimulator generating bio-reactor (SGR) and a final product loop bio-reactor (FPLR), wherein the SGR contains a photosensitized biocatalyst and the FPLR contains a microbe and inorganic enzyme equivalent (IEE). The methods and system of the present disclosure are able to convert raw biogas or any methane-containing gas to bio-based chemicals with high yield and selectivity. The current invention can convert raw biogas, or any methane-containing gas to bio-based chemicals and may reduce reliance on petroleum-derived feedstock.
[0039] The present disclosure involves two steps. In step-1, conversion of CO2 and H2S components in the raw biogas to chemicals/fuels takes place. In step-2, conversion of methane in the raw biogas to chemicals/fuels takes place. Finally, after the completion of the reaction, the liquid containing the desired product was collected. The residual gas was recalculated in reactors for its complete conversion.
[0040] The advantages of the present disclosure, but not limited to, are:
• Complete conversion of raw biogas to tunable production of acids, alcohols, and polyhydroxyalkanoate (PHA).
• There is no requirement for an expensive and energy intensive biogas purification and compression process.
• H2S in the raw biogas used by the microbes for biogenic semiconductor synthesis and assist CO2 conversion.
• The product selectivity can be tuned based on the requirements by simply controlling the light wavelength and microbe.
• The whole process is continuous and occurs in ambient conditions.
• No gaseous or liquid waste is generated in the process.
[0041] In a first aspect of the present disclosure, there is provided an integrated method for the selective production of fuel precursors from biogas, the integrated method comprising:
selecting and culturing an electroactive microorganism in a culture medium in a first stimulator generating bio-reactor, adding a semi-conducting precursor to the culture medium, and sparging a biogas stream through an immobilized carbon-dioxide solubilizing enzyme into the culture medium to form semi-conducting particles functionalized on the surface of the electroactive microorganism, adding a light harvesting fluorescent dye to the culture medium, irradiating the electroactive microorganism in the culture medium with a light source to metabolize a carbon source present in the biogas stream, and obtaining a first aqueous medium and a first residual biogas stream;
selecting and culturing a yeast strain in a culture medium in a second stimulator generating bio-reactor, routing the aqueous medium and the first residual biogas stream to the second stimulator generating bio-reactor, adding the semi-conducting precursor to the culture medium to form semi-conducting particles functionalized on the surface of the yeast strain, adding the light harvesting fluorescent dye to the culture medium, irradiating the yeast strain in the culture medium with a light source to metabolize the carbon source present in the first residual biogas stream, and obtaining a second aqueous medium and a second residual biogas stream; and
selecting and culturing a methanotrophic microorganism in a culture medium in a final product loop bio-reactor, adding an inorganic enzyme equivalent to the culture medium, routing the second aqueous medium and the second residual biogas stream to the culture medium of the final product loop bio-reactor to metabolize methane present in the second residual biogas stream, and recovering a final product stream having the fuel precursors,
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof.
[0042] In an embodiment of the present disclosure, there is provided an integrated method for the tunable production of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof from raw biogas.
[0043] In an embodiment of the present disclosure, wherein the carbon source is CO2.
[0044] In an embodiment of the present disclosure, wherein the biogas stream comprises raw biogas or methane containing biogas, and wherein the raw biogas is obtained from anaerobic digestion of plant biomass, kitchen waste, press mud or a combination thereof, and wherein the methane containing biogas is obtained from wastewater treatment, sewage treatment, septic tanks, natural gas, biomass conversion (analogous to composting), landfill gas, stranded natural gas, silage decomposition, or a combination thereof.
[0045] In an embodiment of the present disclosure, wherein the biogas stream comprises 10-80% methane, 90-20% carbon-dioxide or equivalent, and 100-3000 ppm hydrogen sulfide or equivalent, and wherein the second residual biogas stream routed to the final product loop bio-reactor comprises methane in a range of 50-100%. In an embodiment, if the methane is in pure form, equimolar CO2 equivalent carbonates to be added. They may include Na2CO3, CaCP3 and MgCO3, acetate, or a combination thereof. In an embodiment, if H2S is absent, equimolar quantities of H2S equivalent molecules like methionine, cysteine, homocysteine, taurine, thiourea, or a combination thereof.
[0046] In an embodiment of the present disclosure, wherein the electroactive microorganism has an acetyl-CoA pathway and wherein the electroactive microorganism is selected from the group consisting of Ochrobactrum anthropi, Acidiphilium cryptum, Rhodopseudomonas palustris, Rhodoferax ferrireducens, Cupriavidus necator, Shewanella oneidensis, Shewanella putrefaciens, Pseudomonas aeruginosa, Pseudomonas alcaliphila, Pseudomonas fluorescens, Azotobacter vinelandii, Escherichia coli, Aeromonas hydrophila, Actinobacillus succinogenes, Klebsiella pneumonia, Klebsiella sp. ME17, Klebsiella terrigena, Enterobacter cloacae Citrobacter sp. SX-1, Geopsychrobacter, electrodiphilus, Geobacter sulfurreducens, Geobacter metallireducens, Geobacter lovleyi, Desulfuromonas acetoxidans, Desulfovibrio desulfuricans, Desulfovibrio paquesii, Desulfobulbus propionicus, Arcobacter butzleri, Acidithiobacillus ferrooxidans, Sporomusa ovate, Sporomusa sphaeroides, Sporomusa silvacetica, Thermincola sp. JR, Geothrix fermentans, Clostridium ljungdahlii, Clostridium aceticum, Clostridium sp. EG3, Moorella thermoacetica, Thermincola ferriacetica, Bacillus subtilis, Lactococcus lactis, Lactobacillus pentosus, Enterococcus faecium, Brevibacillus sp. PTH1, Corynebacterium glutamicum.
[0047] In an embodiment of the present disclosure, wherein the electroactive microorganism is selected from the group consisting of Enterobacter aerogenes MTCC 25016, Serratia sp. MTCC 25017, Shewanella sp. MTCC 25020, and Alcaligenes sp. MTCC 25022.
[0048] In an embodiment of the present disclosure, wherein the culture medium of the first stimulator generating bio-reactor comprises micro nutrients such as 0.40 g/L NaCl, 0.40 g/L NH4Cl, 0.33 g/L MgSO4·7H2O, 0.05 g/L CaCl2, 0.25 g/L KCl, 0.64 g/L K2HPO4, 2.50 g/L NaHCO3, trace mineral (1000.0 mg/L MnSO4·H2O, 200.0 mg/L CoCl2·6H2O, 0.2 mg/L ZnSO4 7H2O, 20.0 mg/L CuCl2·2H2O, 2000.0 mg/L nitriloacetic acid), and vitamin such as, pyridoxine·HCl 10.0 mg/L, thiamine·HCl 5.0 mg/L, riboflavin 5.0 mg/L, nicotinic acid 5.0 mg/L, biotin 2.0 mg/L, folic acid 2.0 mg/L, and vitamin B12 0.1 mg/L.
[0049] In an embodiment of the present disclosure, wherein the biogas stream is passed to the culture medium through a nano or micro bubbler at a flow rate of 30-100 ml/min.
[0050] In an embodiment of the present disclosure, wherein the semi-conducting precursor is selected from the group consisting of a metal halide, a metal nitrate, a metal perchlorate, a metal carbonate, and a metal sulfate.
[0051] In an embodiment of the present disclosure, wherein the semi-conducting precursor is obtained from one or more types of metals in ionic form. Some examples of precursor metal compounds applicable herein include the metal halides (e.g., CuCl2, CdCl2, ZnCl2, ZnBr2, GaCl3, InCl3, FeCl2, FeCl3, SnCl2, and SnCl4), metal nitrates (e.g., Cd (NO3)2, Ga (NO3)3, In (NO3)3, and Fe(NO3)3), metal perchlorates, metal carbonates (e.g., CdCO3), metal sulfates (e.g., CdSO4, FeSO4, and ZnSO4).
[0052] In an embodiment of the present disclosure, wherein the concentration of the semi-conducting precursor depends on the concentration of H2S in the biogas stream. For every 10-100 ppm of H2S, 1-10 ppm of semi-conducting precursor to be added.
[0053] In an embodiment of the present disclosure, wherein the semi-conducting particles are selected from the group consisting of CdS, ZnS, CdSe, CuS/ZnS, CuS-ZnS/CNTF, CuS/TiO2, ZnS:Ag2S, Bi2S3/ZnS, MoS2 /graphene-CdS, ZnSe, CdTe, TiC, Graphene/CdS, and ZnTe. In one embodiment, the biogenic semiconductors or semiconducting biogenic hybrid catalysts are obtained from semi-conducting particles.
[0054] In an embodiment of the present disclosure, wherein the light harvesting fluorescent dye is selected from the group consisting of a neutral red, an azo-dye, a porphyrin complex, a Schiff base complex, a multi walled CNT, Fe, Cd (II) or Cu (II) imidazole complex, fluoresce rhodamine or rhodamine dye, and a ruthenium complex.
[0055] In an embodiment of the present disclosure, wherein the light harvesting fluorescent dye is added in a concentration of 10-20 ppm to the culture medium of the first stimulator generating bio-reactor or the second stimulator generating bio-reactor.
[0056] In an embodiment of the present disclosure, wherein the immobilized carbon-dioxide solubilizing enzyme comprises a carbonic anhydrase immobilized on a solid support.
[0057] In an embodiment of the present disclosure, wherein the carbonic anhydrase is obtained from Bacillus thermoleovorans IOC-S3 (MTCC 25023) and/or Pseudomonas fragi IOC S2 (MTCC 25025), and/or Bacillus stearothermophilus IOC S1 (MTCC 25030) and/or Arthrobacter sp. IOC-SC-2 (MTCC 25028).
[0058] In an embodiment of the present disclosure, wherein the solid support is selected from the group consisting of silica, alumina, carbon, and any solid support in the first stimulator generating bio-reactor.
[0059] In an embodiment of the present disclosure, wherein concentration of the carbonic anhydrase is in a range of 50-100 mg/g of the solid support.
[0060] In an embodiment of the present disclosure, wherein the wherein the light source in the first stimulator generating bio-reactor has a wavelength of 400-800 nm, and the light source in the second stimulator generating bio-reactor has a wavelength of 380-620 nm, and wherein the light source enables continuous or intermittent light irradiation.
[0061] In an embodiment of the present disclosure, wherein the light source in the second stimulator generating bio-reactor has a wavelength selected from the group consisting of 380-450nm, 495-570 nm, 620-750nm, and 590-620.
[0062] Light, which is an essential substrate for the phototrophic performance of the microbial culture, must be supplied continuously. Both the spectral quality and the intensity of light are important for microbial performance.
[0063] In an embodiment of the present disclosure, the light source is direct sunlight, LED lights, or any light sources. In some embodiments continuous and flashing light was provided to the culture medium. The flashing light was provided in a dark light ratio of 1:6 second to 1:10. It was found that the overall yield has been significantly enhanced with intermittent light sources. So, the continuous light probably saturates the electron confinement in microbes and results in a decrease formation. On the other hand, intermittent light is suitable due to the fraction of the dark-cycle, which is essential for complete use of the electron by the microbe cells.
[0064] In an embodiment of the present disclosure, wherein the light source provides continuous or flashing light, and wherein the flashing light has a dark light ratio of 1:6 to 1:10 seconds.
[0065] In an embodiment of the present disclosure, wherein the light source provides intermittent light.
[0066] In an embodiment of the present disclosure, wherein the temperature of the culture medium of the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, or the final product loop bio-reactor is in a range of 25 to 55oC and the effect on the microbial population and hydrocarbon formation is essentially unaffected by a variation in temperature. Duration for the reaction in the first stimulator generating reactor is in a range of 1-10 min. Duration for the reaction in the second stimulator generating bio-reactor is in a range of 2-20 min (i.e., 2 times more than that of HRT of SGR-1). Duration for the reaction in the final product loop bio-reactor is in a range of 20 -30 minutes.
[0067] In an embodiment of the present disclosure, wherein the biogas stream has a hydraulic retention time (HRT) of 1-10 minutes in the first stimulator generating bio-reactor, after which it should be transferred to SGR-2.
[0068] In an embodiment of the present disclosure, wherein the HRT of the first residual biogas stream in the second stimulator generating bio-reactor is at least 2 times more than that of HRT of SGR-1.
[0069] In an embodiment of the present disclosure, wherein the HRT of the second residual biogas stream present in the final product loop bio-reactor is 20 to 30 minutes.
[0070] In an embodiment of the present disclosure, wherein the first aqueous medium obtained from the first stimulator generating bio-reactor is subjected to a cell separator before routing to the second stimulator generating bio-reactor, wherein the cell separator recycles the cells present in the first aqueous medium and routing back to the first stimulator generating bio-reactor.
[0071] In an embodiment of the present disclosure, wherein the yeast strain is selected from the group consisting of Candida vini, Candida entamophila, Candida blankie, Pichia farinosa, and Candida tropicalis (Castellani) Berkhout (ATCC 750).
[0072] In an embodiment of the present disclosure, wherein the yeast strain is Candida tropicalis (Castellani) Berkhout (ATCC 750).
[0073] In an embodiment of the present disclosure, wherein the culture medium of the second stimulator generating bio-reactor is similar to that of the first stimulator generating bio-reactor or different.
[0074] In an embodiment of the present disclosure, wherein the culture medium of the second stimulator generating bio-reactor comprises micro nutrients such as 0.40 g/L NaCl, 0.40 g/L NH4Cl, 0.33 g/L MgSO4·7H2O, 0.05 g/L CaCl2, 0.25 g/L KCl, 0.64 g/L K2HPO4, 2.50 g/L NaHCO3, trace mineral (1000.0 mg/L MnSO4·H2O, 200.0 mg/L CoCl2·6H2O, 0.2 mg/L ZnSO4 7H2O, 20.0 mg/L CuCl2·2H2O, 2000.0 mg/L nitriloacetic acid), and vitamin such as, pyridoxine·HCl 10.0 mg/L, thiamine·HCl 5.0 mg/L, riboflavin 5.0 mg/L, nicotinic acid 5.0 mg/L, biotin 2.0 mg/L, folic acid 2.0 mg/L, and vitamin B12 0.1 mg/L.
[0075] In an embodiment of the present disclosure, wherein the second aqueous medium obtained from the second stimulator generating bio-reactor is subjected to a cell separator before routing to the final product loop bio-reactor, and wherein the cell separator recycles the cells present in the second aqueous medium and routing back to the second stimulator generating bio-reactor.
[0076] In an embodiment of the present disclosure, wherein the methanotrophic microorganism is selected from the group consisting of Methanococcus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylococcus capsulatus Foster and Davis (ATCC-33009), Methylococcus capsulatus Foster and Davis (ATCC-19069), Methylomonas sp. (ATCC TSD-253), Methylomonas sp. (ATCC 43722), Methylomonas paludis (DSM 24973), and Methylophilus methylotrophus (DSM 6330).
[0077] In an embodiment of the present disclosure, wherein the inorganic enzyme equivalent is prepared using transition metals and ligands.
[0078] In an embodiment of the present disclosure, wherein the inorganic enzyme equivalent comprises a transition metal and a ligand, wherein the transition metal is selected from the group consisting of copper, iron, and nickel, and wherein the ligand is selected from the group consisting of salen, Schiffs base, amino-bis (benzimidazole), bis(2- hydroxy-4-octadecyl-oxybenzal)ethylenediimine, 1,2-bis(2-hydroxy-4-octadecyl-oxybenzal)phenylenediimine, 2,4-dihydroxybenzaldehyde, and combinations thereof.
[0079] In an embodiment of the present disclosure, wherein the concentration of the inorganic enzyme equivalent is present in a range of 200-500 ppm and deepens with the concentration of methane in the feed gas.
[0080] In an embodiment of the present disclosure, after the completion of the reaction, the cells from the final product loop bio-reactor are separated using a cell separator. In an embodiment, the product from the final product loop bio-reactor is collected using crystallization, solvent extraction, or membrane-based separation methods.
[0081] In an embodiment of the present disclosure, the appropriate operational pH of the medium has been estimated by varying pH using phosphate buffer. In an embodiment of the present disclosure, the pH of the culture medium of the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, or the final product loop bio-reactor can be varied from 3 to 11. The microbes can grow suitably in the pH range, and there is no effect on product formation.
[0082] In another aspect of the present disclosure, there is provided an integrated system for the selective production of fuel precursors from biogas, the integrated system comprising:
a first stimulator generating bio-reactor, the first stimulator generating bio-reactor comprising: an inlet for receiving a biogas stream into the first stimulator generating bio-reactor, an immobilized carbon-dioxide solubilizing enzyme, a light source, an agitator, an electroactive microorganism in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the electroactive microorganism to metabolize a carbon source present in the biogas stream and produce a first aqueous medium and a first residual biogas stream, and an outlet for passing the first aqueous medium and the first residual biogas stream;
a second stimulator generating bio-reactor, the second stimulator generating bio-reactor comprising: an inlet for receiving the first aqueous medium and the first residual biogas stream into the second stimulator generating bio-reactor, a light source, an agitator, a yeast strain in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the yeast strain to metabolize the carbon source present in the first residual biogas stream and produce a second aqueous medium and a second residual biogas stream, and an outlet for passing the second aqueous medium and the second residual biogas stream; and
a final product loop bio-reactor, the final product loop bio-reactor comprising: an inlet for receiving the second aqueous medium and the second residual biogas stream into the final product loop bio-reactor, an agitator, a methanotrophic microorganism in a culture medium, and an inorganic enzyme equivalent functionalized on the surface of the methanotrophic microorganism to metabolize methane present in the second residual biogas stream and produce a final product stream having the fuel precursors,
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof,
wherein the second stimulator generating bio-reactor is placed downstream of the first stimulator generating bio-reactor, and the final product loop bio-reactor is placed downstream of the second stimulator generating bio-reactor, and
wherein the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, and the final product loop bio-reactor are configured to be operated in a sequential manner.
[0083] In an embodiment of the present disclosure, wherein the first stimulator generating bio-reactor and the second stimulator generating bio-reactor is made with stainless steel (SS) or glass having arrangement for light, autoclaving, and stirring. Its outlet should be equipped with microbial filters.
[0084] In an embodiment of the present disclosure, wherein the final product loop bio-reactor is a loop reactor having provision for mixing microbes with feed gas. It is made with stainless steel or glass having arrangement for light, autoclaving, and stirring.
[0085] In an embodiment of the present disclosure, wherein the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, and the final product loop bio-reactor are configured to be operated in a sequential manner or independently depending on the product profile.
[0086] In an embodiment of the present disclosure, wherein the electroactive microorganism is selected from the group consisting of Enterobacter aerogenes MTCC 25016, Serratia sp. MTCC 25017, Shewanella sp. MTCC 25020, and Alcaligenes sp. MTCC 25022,
[0087] In an embodiment of the present disclosure, wherein the yeast strain is selected from the group consisting of Candida vini, Candida entamophila, Candida blankie, Pichia farinosa, and Candida tropicalis (Castellani) Berkhout (ATCC 750).
[0088] In an embodiment of the present disclosure, wherein the methanotrophic microorganism is selected from the group consisting of Methanococcus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylococcus capsulatus Foster and Davis (ATCC-33009), Methylococcus capsulatus Foster and Davis (ATCC-19069), Methylomonas sp. (ATCC TSD-253), Methylomonas sp. (ATCC 43722), Methylomonas paludis (DSM 24973), and Methylophilus methylotrophus (DSM 6330).
[0089] In an embodiment of the present disclosure, wherein the light source in the first stimulator generating bio-reactor has a wavelength of 400-800 nm, and the light source in the second stimulator generating bio-reactor has a wavelength of 380-620 nm, and wherein the light source is configured to enable continuous or intermittent light irradiation.
[0090] In an embodiment of the present disclosure, wherein the semi-conducting precursor is selected from the group consisting of a metal halide, a metal nitrate, a metal perchlorate, a metal carbonate, and a metal sulfate, wherein the light harvesting fluorescent dye is selected from the group consisting of a neutral red, an azo-dye, a porphyrin complex, a Schiff base complex, a multi walled CNT, Fe, Cd (II) or Cu (II) imidazole complex, fluoresce rhodamine or rhodamine dye, and a ruthenium complex, and wherein the inorganic enzyme equivalent comprises a transition metal and a ligand.
[0091] In an embodiment of the present disclosure, wherein the immobilized carbon-dioxide solubilizing enzyme comprises a carbonic anhydrase immobilized on a solid support.
[0092] The present disclosure relates to an integrated method for the conversion of methane-enriched gas mixtures into industrially relevant chemicals. The present disclosure provides a bio-inorganic hybrid method for tunable production of acids, alcohols, and polyhydroxyalkanoate (PHA) from raw biogas or any methane-containing gases. In particular, the present disclosure provides an integrated method for the selective production of fuel precursors from biogas. The present disclosure also provides an integrated system for selective production of fuel precursors from biogas comprising a SGR and a FPLR, wherein the SGR comprises a photosensitized biocatalyst and the FPLR comprises a microbe and IEE. The method involves the following steps.
[0093] Step-1: Bioconversion of CO2 and H2S components in the raw biogas in SRG. Step-1 occurs in SGR. The SRG contains a biohybrid catalyst. The biohybrid catalyst comprises a biogenic semiconductor functionalized on the surface electroactive microbes (EAB). The H2S is used to synthesize biogenic semiconductors on the surface of the microbe in the presence of an inorganic precursor. The biohybrid used energy from light to produce a redox equivalent for the conversion of CO2 to product. SRG consists of 2 consecutive vessels exposed to light of different wavelengths for efficient conversion of metabolites responsible for CO2 conversion.
[0094] The various activities in step-1 are as follows:
a. Selection of electroactive microbes having an acetyl-CoA pathway and growing in culture media in SRG-1.
b. Addition of a semiconducting precursor and light-harvesting fluorescent dye (10-20 ppm) to the microbe developed in step1a.
c. Addition of an immobilized CO2 solubilizing enzyme to SRG-1.
d. Development of a semiconducting biogenic hybrid catalyst on the microbe surface by passing raw biogas with H2S to the process described in step1b.
e. Illuminating light of the desired wavelength.
f. Providing the desired hydraulic retention time (HRT) to raw biogas in SRG-1.
g. Transferring the liquid from SRG-1 to SRG-2.
h. Separating and recycling the cells in step-1g.
i. The SRG-2 contains a yeast, biogenic semiconductor, and light-harvesting fluorescent dye (10-20 ppm).
j. SRG-2 was illuminated with light of the desired wavelength at a specified temperature and duration.
k. Separation of cells and transfer of the broth and residual gas to FPLR.
[0095] Step-2: Bioconversion of methane in FPLR. The residual methane gas after CO2 and H2S removal enters FPLR. Step-2 occurs in FPLR. The FPLR is a loop reactor and contains a methanotroph. An IEE is functionalized on the microbe surface to improve its oxidizing potential.
[0096] The various activities in step-2 are as follows:
l. Selection of microbes from methanotrophs and growing in culture media.
m. Preparing IEE.
n. Functionalizing IEE on the surface of the microbe described in step-2l surface by a suitable method, as described above.
o. Passing the residual gas from SRG-2 and maintaining desired temperature and HRT.
p. Separation of cells and obtaining broth with at least one product from the culture broth.
q. Recycling of the cells and residual gases.
r. Analyzing the product.
[0097] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.
EXAMPLES:
[0098] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary. Person skilled in the art will be aware of the fact that the present examples will further subject to variations and modifications specifically described herein based on the technical requirement of the experiment and shall not be limiting what specifically mentioned.
Example 1: Selective production of alcohols form biogas fermentation
1.1 Selective production of methanol
[0099] In the first stimulator generating bio-reactor (SGR-1), an electroactive Enterobacter aerogenes microbe was cultivated in an optimized medium (M1) (2L). Prior to the experiment, the reactor was sterilized by autoclaving at 121oC for 15 min. The media composition (M1) was as follows: (0.40 g/L NaCl, 0.40 g/L NH4Cl, 0.33 g/L MgSO4·7H2O, 0.05 g/L CaCl2, 0.25 g/L KCl, 0.64 g/L K2HPO4, 2.50 g/L NaHCO3, trace mineral (1000.0 mg/L MnSO4·H2O, 200.0 mg/L CoCl2·6H2O, 0.2 mg/L ZnSO4·7H2O, 20.0 mg/L CuCl2·2H2O, 2000.0 mg/L nitriloacetic acid), and vitamin (pyridoxine·HCl 10.0 mg/L, thiamine·HCl 5.0 mg/L, riboflavin 5.0 mg/L, nicotinic acid 5.0 mg/L, biotin 2.0 mg/L, folic acid 2.0 mg/L, vitamin B12 0.1 mg/L, 0.5% glucose). Cultures were grown at 30oC. Cell growth was monitored by measuring OD at 660 nm with a UV-visible spectrophotometer.
[00100] Cadmium sulfide (CdS) semiconducting particles have been synthesized on the surface of the microbes by passing raw biogas containing 80% CH4, 300 ppm H2S and balance CO2 with a flow rate of 30 ml/min followed by adding 10 ppm of fluoresce rhodamin. After 3 days of inculcation, the active microbial population was found to be CFU=2.3*108.
[00101] White light of wavelength (? > 400-800 nm) was employed to provide photon flux. Provision has been made to provide light at different conditions to the biogenic hybrid catalyst to utilize CO2 in their metabolic activity. In one set of experiments, constant solar light was given for 24 h and in another set, intermittent light source was given with light: dark ratio of 1:6s.
[00102] The hydraulic retention time (HRT) of raw biogas was maintained for 5 min in SGR-1 and after completion of the fermentation, the broth and biogas were transferred to SGR-2. The cells were separated by a membrane cell separator and circulated back to SGR-1.
[00103] The second stimulator generating bio-reactor (SGR-2) contains a fully grown yeast strain Candida tropicalis (Castellani) Berkhout (ATCC 750) having CFU=2.8*1011. CdNO3 was supplied to the broth solution at a rate of 2 ml/min for the formation of CdS on yeast strain followed by 10 ppm of fluoresce rhodamin. LED light (495-570 nm) provision was used to supply energy to the SGR-2.
[00104] The HRT of biogas in SGR-2 was maintained as 10 min followed by transfer of broth to the final product loop bio-reactor (FPLR). Using a membrane cell separator all the cells were separated and transferred back to SGR-2.
[00105] The loop reactor contains grown microbes of Methylomonas paludis (DSM 24973) having CFU=1.8*1011 and 300 ppm of inorganic enzyme equivalent (IEE). The temperature of the loop reactor was maintained at 40-45oC. IEE was synthesized by the following method:
[00106] A mixture of amino-bis (benzimidazole) (1.5 mmol), CH3NH2·HCl (1.5 mmol), dimethylaminoethanol (0.1 mL) and methanol (10 mL) in a 50 mL conic flask was stirred magnetically at 60°C for half an hour. CuCl2·6H2O (0.5 mmol) dissolved in methanol (5 mL) was added to the yellow solution of the formed schiff base, and the stirring continued for another 40 mins at the same temperature. After that, 0.03 g (0.5 mmol) of Zn powder was added to the flask and the mixture was stirred for an hour to achieve the total dissolution of the metal powder. Filtration and evaporation of the resultant brown solution at room temperature afforded dark-red crystals of IEE.
[00107] The broth and biogas from SGR-2 was transferred to FPLR. The HRT of biogas in the loop reactor was maintained for 20 min.
[00108] After completion of the process, the final broth was separated, and the cells were recalculated. The product was analyzed by gas chromatography (GC).
1.2 Selective production of ethanol
[00109] For the selective production of ethanol, the media composition remains the same as described above. However, SGR-1 was replaced with Serratia sp. (MTCC 25017) and FPLR with Methylomonas paludis (DSM 24973).
[00110] The semiconducting particle MoS2 /graphene-CdS was used in SGR-1. MoS2 /graphene-CdS was synthesized as follows. Graphene oxide (GO) was synthesized from natural graphite powder by a modified hummers’ method. In a typical synthesis 0.5g of CdCl2, 00.1g MoCl2 and 0.05g GO were dispersed in 30 mL of deionized water. After ultrasonication for 1 h, the mixture was transferred into the microbial culture for production of MoS2 /graphene-CdS. The wavelength of light at SGR-1 was 400-800 nm and wavelength of light at SGR-2 was fixed at 380–450 nm. All the other conditions remained constant.
[00111] Table 1: Describe microbes and conditions for selective conversion of raw biogas to chemicals (alcohol).
Microbe Media compos
-ition Photosensitizer semiconductor Light wavelength Inorganic enzyme equivalent (IEE) Total Product yield/L of media and selectivity Total Product yield in Kg/Kg of biogas
SGR-1 Enterobacter aerogenes (MTCC 25016) M1 CdS 400-800 nm Not required 87.3g/L
Selectivity of
Methanol (98 %)+ Acetic acid (2%)
1.05
SGR
-2 Candida tropicalis (Castellani) Berkhout (ATCC 750) M1 CdS 495-570 nm Not required
FPLR Methylomonas paludis (DSM 24973) M1 Not required Not required Cu-amino-bis (benzimidazole)
SGR-1 Serratia sp. (MTCC 25017) M1 MoS2 /graphene-CdS 400-800 nm Not required 98.7g/L
Selectivity of
Ethanol (93 %) + Acetic acid (7%)
1.19
SGR
-2 Candida tropicalis (Castellani) Berkhout (ATCC 750) M1 MoS2 /graphene-CdS 380–450 nm Not required
FPLR Methylomonas paludis (DSM 24973) M1 Not required Not required Cu-amino-bis (benzimidazole)

Example 2: Production of polyhydroxyalkanoate (PHA) from biogas fermentation
[00112] In SGR-1, an electroactive Alicaligens sp. MTCC 25022 microbe was cultivated in an optimized medium (M2) (2L). Prior to the experiment, the reactor was sterilized by autoclaving at 121oC for 15 min. The media composition consists of (M2): 2.6 g/L HEPES, 1 g/L yeast extract, 0.5 g/L gluconic acid, 0.5 g/L mannitol, 0.22 g/L KH2PO4, 0.25 g/L Na2SO4, 0.3 g/L NH4Cl, 0.0112 g/L FeCl3·6H2O, 0.017 g/L CuCl2·2H2O, 0.18 g/L MgSO4·7H2O, NaMoO4·7H2O, 0.0021 g/L NiCl2·6H2O, 0.01 g/L CaCl2·2H2O. Cultures were grown at 30oC. Cell growth was monitored by measuring OD at 660 nm with a UV-visible spectrophotometer.
[00113] BiCl3/ZnCl2 was used to synthesize Bi2S3/ZnS semiconducting particle on the surface of the microbes by passing raw biogas containing 80% CH4, 300 ppm H2S and balance CO2, and with a flow rate of 30 ml/min followed by adding 10 ppm of fluoresce rhodamin. After 3 days of inculcation, the active microbial population was found to be CFU=5.8*108.
[00114] Light of wavelength (? > 590-620 nm) was employed to provide photon flux. Provision has been made to provide light at different conditions to the biogenic hybrid catalyst to utilize CO2 in their metabolic activity. In one set of experiments, constant solar light was given for 24 h and in another set, intermittent light source was given with light: dark ratio of 1:6s.
[00115] The HRT of raw biogas was maintained for 5 min and after completion of the fermentation, the broth and biogas were transferred to SGR-2. The cells were separated by a membrane cell separator and circulated back to SGR-1.
[00116] The SGR-2 contains a fully grown yeast strain Candida tropicalis (Castellani) Berkhout (ATCC 750) having CFU=5*1011. BiCl3/ZnCl2 was supplied to the broth solution at a rate of 2 ml/min for the formation of Bi2S3/ZnS on yeast strain followed by 10 ppm of fluoresce rhodamin. LED light (380–450 nm) provision was used to supply energy to the SGR-2.
[00117] The HRT of biogas in SGR-2 was maintained as 10 min followed by transfer of broth to the FPLR. Using a membrane cell separator all the cells were separated and transferred back to SRG-2.
[00118] The loop reactor contains grown microbes of Methylococcus capsulatus Foster and Davis (ATCC-33009) having CFU=8*1010. 300 ppm of inorganic enzyme equivalent (IEE) was synthesized by following method:
[00119] A mixture of bis(2-hydroxy-4-octadecyl-oxybenzal) ethylenediimine (1.5 mmol), and methanol (10 mL) in a 50 mL conic flask was stirred magnetically at 60°C for half an hour. FeCl2·6H2O (0.5 mmol) dissolved in methanol (5 mL) was added to the red solution of the formed Schiff base, and the stirring continued for another 40 mins at the same temperature. The mixture was stirred for an hour to achieve the total dissolution. Filtration and evaporation of the resultant red solution at room temperature afforded dark-red/black crystals of IEE.
[00120] The broth and biogas from SRG-2 was transferred to FPLR. The HRT of biogas in the loop reactor was maintained for 20 min. After completion of the process, the final broth was separated, and the cells were recalculated. The product was analyzed by GC.
[00121] Table 2: Describe microbes and condition for selective conversion of raw biogas to PHA.
Microbe* Media composition** Photosensitizer semiconductor Light wavelength Inorganic enzyme equivalent (IEE) Total Product yield/L of media and selectivity Total Product yield in Kg/Kg of biogas
SGR-1 Alicaligens sp. MTCC 25022 M2 Bi2S3/ZnS 590-620 Not required 58.4g/L
Selectivity:
PHA (87 %), 9% Acetic acid, 4% methanol 0.70
SGR
-2 Candida tropicalis (Castellani) Berkhout (ATCC 750) M2 Bi2S3/ZnS 380–450 Not required
FPLR Methylococcus capsulatus Foster and Davis (ATCC-33009) M2 Not required Not required Fe- bis(2-hydroxy-4-octadecyl-oxybenzal)ethylenediimine

Example 3: Production of organic acids form biogas fermentation
[00122] In SGR-1, an electroactive Shewanella sp. MTCC 25020 microbe was cultivated in an optimized medium (M2) (2L). Prior to the experiment, the reactor was sterilized by autoclaving at 121oC for 15 min. The media composition of M2 includes 2.6 g/L HEPES, 1 g/L yeast extract, 0.5 g/L gluconic acid, 0.5 g/L mannitol, 0.22 g/L KH2PO4, 0.25 g/L Na2SO4, 0.3 g/L NH4Cl, 0.0112 g/L FeCl3·6H2O, 0.017 g/L CuCl2·2H2O, 0.18 g/L MgSO4·7H2O, NaMoO4·7H2O, 0.0021 g/L NiCl2·6H2O, 0.01 g/L CaCl2·2H2O. Cultures were grown at 30oC. Cell growth was monitored by measuring OD at 660 nm with a UV-visible spectrophotometer.
[00123] CdS semiconducting particles have been synthesized on the surface of the microbes by passing raw biogas containing 80% CH4, 300 ppm H2S and balance CO2 with a flow rate of 30 ml/min and CdNO3, followed by adding 10 ppm of fluoresce rhodamin. After 3 days of inculcation, the active microbial population was found to be CFU=9*1010.
[00124] Light of wavelength (? = 450-495 nm) was employed to provide photon flux. Provision has been made to provide light at different conditions to the biogenic hybrid catalyst to utilize CO2 in their metabolic activity. In one set of experiments, constant solar light was given for 24 h and in another set, intermittent light source was given with light: dark ratio of 1:6s.
[00125] The HRT of raw biogas was maintained for 5 min and after completion of the fermentation, the broth and biogas were transferred to SGR-2. The cells were separated by a membrane cell separator and circulated back to SGR-1.
[00126] The SGR-2 contains a fully grown yeast strain Candida tropicalis (Castellani) Berkhout (ATCC 750) having CFU=2.5*1010. CdNO3 was supplied to the broth solution at a rate of 2 ml/min for the formation of CdS on yeast strain followed by 10 ppm of fluoresce rhodamin. LED light (620-750 nm) provision was used to supply energy to the SGR-2.
[00127] The HRT of biogas in SGR-2 was maintained as 10 min followed by transfer of broth to the FPLR. Using a membrane cell separator all the cells were separated and transferred back to SGR-2.
[00128] The FPLR contains grown microbes of Methylococcus capsulatus Foster and Davis (ATCC-33009) having CFU=6.8*1011. 300 ppm of inorganic enzyme equivalent (IEE) was synthesized by following method:
[00129] A mixture of bis(2-hydroxy-4-octadecyl-oxybenzal) ethylenediimine (1.5 mmol), and methanol (10 mL) in a 50 mL conic flask was stirred magnetically at 60°C for half an hour. FeCl2·6H2O (0.5 mmol) dissolved in methanol (5 mL) was added to the red solution of the formed Schiff base, and the stirring continued for another 40 mins at the same temperature. The mixture was stirred for an hour to achieve the total dissolution. Filtration and evaporation of the resultant red solution at room temperature afforded dark-red/black crystals of IEE.
[00130] The broth and biogas from SGR-2 was transferred to FPLR. The HRT of biogas in the loop reactor was maintained for 20 min. After completion of the process, the final broth was separated and the cells were recalculated. The product was analyzed by GC.
[00131] Table 3: Describe microbes and condition for selective conversion of raw biogas to organic acids.
Microbe Media compos-
ition Photosens-
itizer semicond-
uctor Light wavelength Inorganic enzyme
equivalent (IEE) Total Product yield/L of media and selectivity Total
Product
yield in Kg/Kg of biogas
SGR-1 Shewanella sp. MTCC 25020 M2 CdS
450-495 Not required 112.7 g/L
Selectivity of
Acetic acid (96 %) 1.36
SGR
-2 Candida tropicalis (Castellani) Berkhout (ATCC 750) M2 CdS
620-750 Not required
FPLR Methylococcus capsulatus Foster and Davis (ATCC-33009) M2 Not required Not required Fe- bis(2-hydroxy-4-octadecyl-oxybenzal)
ethylenediimine
Example 4: Effect of light illumination on product formation
[00132] Light wavelength plays an important role for tailoring the product yield and selectivity. Light helps in producing reducing equivalents, which trigger the redox reactions at cellular level of microbes. Light of certain wavelength is highly desirable for production of desired product from biogas fermentation in SGR-1 and 2. In this example, all conditions of Example 1.1 were fixed and the light illumination condition in SGR-1 and SGR-2 was varied. Table 4 represents the total methanol yield and selectivity at various light illumination conditions.
[00133] Table 4: Describe total methanol yield and selectivity at various light illumination conditions.
Condition Light/dark Total Product yield/L of media and selectivity Total Product yield in Kg/Kg of biogas
All conditions of Example 1.1 remain constant except light illumination condition.
SGR-1=No light
SGR-2= No light 10.2g/L
Selectivity of
Methanol (23 %) 0.12
SGR-1=Continuous light (WL=400-800 nm)
SGR-2= Continuous light (WL= 495-570 nm) 52.5g/L
Selectivity of
Methanol (59 %) 0.63
SGR-1= Intermittent light (WL= 570-590 nm)
SGR-2= Intermittent light (WL= 590-620nm) 68.3g/L
Selectivity of
Methanol (61 %) 0.82
SGR-1=Intermittent light (WL=400-800 nm)
SGR-2 = Intermittent light (WL= 495-570 nm) 87.3g/L
Selectivity of
Methanol (98 %) 1.05

Example 5: Role of photosensitizer and IEE on total methanol yield and selectivity
[00134] Photosensitizer and inorganic enzyme equivalents (IEE) play crucial roles for improving product selectivity and yields. Photosensitizer harvest light and provides required redox equivalents to microbes and yeast strains in SGR-1 and 2, respectively, for formation of desired stimulating intermediates. Similarly, IEE fascinates the oxidation reaction of microbes in FPLR.
[00135] Table 5: Describe role of photosensitizer and IEE on total methanol yield and selectivity.
Condition photosensitizer and IEE Total Product yield/L of media and selectivity Total Product yield in Kg/Kg of biogas
All condition of Example 1.1 remain constant except addition of photosensitizer and IEE
SGR-1=No photosensitizer
SGR-2= CdS
FPLR = Cu-amino-bis (benzimidazole) 6.7g/L
Selectivity of
Methanol (5.6 %) 0.08
SGR-1=CdS
SGR-2= No photosensitizer
FPLR = Cu-amino-bis (benzimidazole) 15.3g/L
Selectivity of
Methanol (15 %) 0.18
SGR-1=CdS
SGR-2= CdS
FPLR = No IEE 14.7g/L
Selectivity of
Methanol (8 %) 0.17
SGR-1=CdS
SGR-2= CdS
FPLR = Cu-amino-bis (benzimidazole) 87.3g/L
Selectivity of
Methanol (98 %) 1.05
Example 6: Effect of sequential biogas fermentation process on product formation
[00136] The product formation from biogas fermentation using integrated bio-reactor is highly dependent on sequential fermentation, wherein the feed gas to enter SRG-1 followed by SRG-1 followed by FPLR. Any deviation from this sequence will result in low product yield and selectivity. This is because; the compound produced in SRG-1 stimulates the product in SRG-1 which further stimulates final product formation in FPLR.
[00137] Table 6: Describes effect of sequential operation of bio-reactor on biogas fermentation to produce PHA.
Condition Sequential treatment of Biogas in various bioreactor Total Product yield/L of media and selectivity Total Product yield in Kg/Kg of biogas
All condition of Example 2 remain constant except light illumination condition
Passing of feed gas to SGR-1 followed by FPLR (SGR-2 skipped) 12.3g/L
Selectivity: PHA (1.5 %) 0.14
Passing of feed gas to SGR-2 followed by SGR-1 (FPLR skipped) 19.3g/L
Selectivity: PHA (7.3 %) 0.23
Passing of feed gas to SGR-2 followed by SRG-1 followed by FPLR 28.9g/L
Selectivity: PHA (21 %) 0.34
Passing of feed gas to FPLR followed by SRG-1 followed by SRG-2 10.3g/L
Selectivity: PHA (5.3 %) 0.12
Passing of feed gas to SRG-1 followed SRG-2 followed by FPLR 58.4g/L
Selectivity: PHA (87 %) 0.70

[00138] It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained, and since certain changes may be made in the constructions set forth without departing from the spirit and scope of the invention, it is intended that all matter contained in the above description shall be interpreted as illustrative and not in a limiting sense. The invention has been described with reference to preferred and alternate embodiments. Modifications and alterations will become apparent to those skilled in the art upon reading and understanding the detailed discussion of the invention provided herein. This invention is intended to include all such modifications and alterations insofar as they come within the scope of the present invention. These and other modifications of the preferred embodiments as well as other embodiments of the invention will be obvious from the disclosure herein, whereby the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.
[00139] Finally, to the extent necessary to understand or complete the disclosure of the present invention, all publications, patents, and patent applications mentioned herein are expressly incorporated by reference therein to the same extent as though each were individually so incorporated. , Claims: An integrated method for the selective production of fuel precursors from biogas, the integrated method comprising:
selecting and culturing an electroactive microorganism in a culture medium in a first stimulator generating bio-reactor, adding a semi-conducting precursor to the culture medium, and sparging a biogas stream through an immobilized carbon-dioxide solubilizing enzyme into the culture medium to form semi-conducting particles functionalized on the surface of the electroactive microorganism, adding a light harvesting fluorescent dye to the culture medium, irradiating the electroactive microorganism in the culture medium with a light source to metabolize a carbon source present in the biogas stream, and obtaining a first aqueous medium and a first residual biogas stream;
selecting and culturing a yeast strain in a culture medium in a second stimulator generating bio-reactor, routing the aqueous medium and the first residual biogas stream to the second stimulator generating bio-reactor, adding the semi-conducting precursor to the culture medium to form semi-conducting particles functionalized on the surface of the yeast strain, adding the light harvesting fluorescent dye to the culture medium, irradiating the yeast strain in the culture medium with a light source to metabolize the carbon source present in the first residual biogas stream, and obtaining a second aqueous medium and a second residual biogas stream; and
selecting and culturing a methanotrophic microorganism in a culture medium in a final product loop bio-reactor, adding an inorganic enzyme equivalent to the culture medium, routing the second aqueous medium and the second residual biogas stream to the culture medium of the final product loop bio-reactor to metabolize methane present in the second residual biogas stream, and recovering a final product stream having the fuel precursors;
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof.
2. The integrated method as claimed in claim 1, wherein the electroactive microorganism is selected from the group consisting of Enterobacter aerogenes MTCC 25016, Serratia sp. MTCC 25017, Shewanella sp. MTCC 25020, and Alcaligenes sp. MTCC 25022.
3. The integrated method as claimed in claim 1, wherein the semi-conducting precursor is selected from the group consisting of a metal halide, a metal nitrate, a metal perchlorate, a metal carbonate, and a metal sulfate.
4. The integrated method as claimed in claim 1, wherein the light harvesting fluorescent dye is selected from the group consisting of a neutral red, an azo-dye, a porphyrin complex, a Schiff base complex, a multi walled CNT, Fe, Cd (II) or Cu (II) imidazole complex, fluoresce rhodamine or rhodamine dye, and a ruthenium complex.
5. The integrated method as claimed in claim 1, wherein the yeast strain is selected from the group consisting of Candida vini, Candida entamophila, Candida blankie, Pichia farinosa, and Candida tropicalis (Castellani) Berkhout (ATCC 750).
6. The integrated method as claimed in claim 1, wherein the light source in the first stimulator generating bio-reactor has a wavelength in a range of 400-800 nm, and the light source in the second stimulator generating bio-reactor has a wavelength in a range of 380-620 nm, and wherein the light source enables continuous or intermittent light irradiation.
7. The integrated method as claimed in claim 1, wherein the methanotrophic microorganism is selected from the group consisting of Methanococcus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylococcus capsulatus Foster and Davis (ATCC-33009), Methylococcus capsulatus Foster and Davis (ATCC-19069), Methylomonas sp. (ATCC TSD-253), Methylomonas sp. (ATCC 43722), Methylomonas paludis (DSM 24973), and Methylophilus methylotrophus (DSM 6330).
8. The integrated method as claimed in claim 1, wherein the inorganic enzyme equivalent comprises a transition metal and a ligand, wherein the transition metal is selected from the group consisting of copper, iron, and nickel, and wherein the ligand is selected from the group consisting of salen, Schiffs base, amino-bis (benzimidazole), bis(2- hydroxy-4-octadecyl-oxybenzal)ethylenediimine, 1,2-bis(2-hydroxy-4-octadecyl-oxybenzal)phenylenediimine, 2,4-dihydroxybenzaldehyde, and combinations thereof.
9. The integrated method as claimed in claim 1, wherein the immobilized carbon-dioxide solubilizing enzyme comprises a carbonic anhydrase immobilized on a solid support.
10. The integrated method as claimed in claim 1, wherein the biogas stream comprises 10-80% methane, 90-20% carbon-dioxide or equivalent, and 100-3000 ppm hydrogen sulfide or equivalent, and wherein the second residual biogas stream routed to the final product loop bio-reactor comprises methane in a range of 50-100%.
11. An integrated system for the selective production of fuel precursors from biogas, the integrated system comprising:
a first stimulator generating bio-reactor, the first stimulator generating bio-reactor comprising: an inlet for receiving a biogas stream into the first stimulator generating bio-reactor, an immobilized carbon-dioxide solubilizing enzyme, a light source, an agitator, an electroactive microorganism in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the electroactive microorganism to metabolize a carbon source present in the biogas stream and produce a first aqueous medium and a first residual biogas stream, and an outlet for passing the first aqueous medium and the first residual biogas stream;
a second stimulator generating bio-reactor, the second stimulator generating bio-reactor comprising: an inlet for receiving the first aqueous medium and the first residual biogas stream into the second stimulator generating bio-reactor, a light source, an agitator, a yeast strain in a culture medium, a semi-conducting precursor and a light harvesting fluorescent dye to form semi-conducting particles that are functionalized on the surface of the yeast strain to metabolize the carbon source present in the first residual biogas stream and produce a second aqueous medium and a second residual biogas stream, and an outlet for passing the second aqueous medium and the second residual biogas stream; and
a final product loop bio-reactor, the final product loop bio-reactor comprising: an inlet for receiving the second aqueous medium and the second residual biogas stream into the final product loop bio-reactor, an agitator, a methanotrophic microorganism in a culture medium, and an inorganic enzyme equivalent functionalized on the surface of the methanotrophic microorganism to metabolize methane present in the second residual biogas stream and produce a final product stream having the fuel precursors,
wherein the fuel precursors comprise at least one of acids, alcohols, polyhydroxyalkanoate, or mixtures thereof,
wherein the second stimulator generating bio-reactor is placed downstream of the first stimulator generating bio-reactor, and the final product loop bio-reactor is placed downstream of the second stimulator generating bio-reactor, and
wherein the first stimulator generating bio-reactor, the second stimulator generating bio-reactor, and the final product loop bio-reactor are configured to be operated in a sequential manner.
12. The integrated system as claimed in claim 11, wherein the electroactive microorganism is selected from the group consisting of Enterobacter aerogenes MTCC 25016, Serratia sp. MTCC 25017, Shewanella sp. MTCC 25020, and Alcaligenes sp. MTCC 25022, wherein the yeast strain is selected from the group consisting of Candida vini, Candida entamophila, Candida blankie, Pichia farinosa, and Candida tropicalis (Castellani) Berkhout (ATCC 750), and wherein the methanotrophic microorganism is selected from the group consisting of Methanococcus, Methylobacterium aminovorans, Methylobacterium thiocyanatum, Methylobacterium zatmanii, Acidithiobacillus ferrivorans, Methylobacterium aquaticum, Methylobacterium suomiense, Methylobacterium adhaesivum, Methylobacterium podarium, Methylococcus capsulatus Foster and Davis (ATCC-33009), Methylococcus capsulatus Foster and Davis (ATCC-19069), Methylomonas sp. (ATCC TSD-253), Methylomonas sp. (ATCC 43722), Methylomonas paludis (DSM 24973), and Methylophilus methylotrophus (DSM 6330).
13. The integrated system as claimed in claim 11, wherein the light source in the first stimulator generating bio-reactor has a wavelength in a range of 400-800 nm, and the light source in the second stimulator generating bio-reactor has a wavelength in a range of 380-620 nm, and wherein the light source is configured to enable continuous or intermittent light irradiation.
14. The integrated system as claimed in claim 11, wherein the semi-conducting precursor is selected from the group consisting of a metal halide, a metal nitrate, a metal perchlorate, a metal carbonate, and a metal sulfate, wherein the light harvesting fluorescent dye is selected from the group consisting of a neutral red, an azo-dye, a porphyrin complex, a Schiff base complex, a multi walled CNT, Fe, Cd (II) or Cu (II) imidazole complex, fluoresce rhodamine or rhodamine dye, and a ruthenium complex, and wherein the inorganic enzyme equivalent comprises a transition metal and a ligand.
15. The integrated system as claimed in claim 11, wherein the immobilized carbon-dioxide solubilizing enzyme comprises a carbonic anhydrase immobilized on a solid support.