DESC:TITLE OF INVENTION

PROCESS FOR MULTISTAGE CONTROLLED BIO-DIOGESTION FOR BIOGAS PRODUCTION
FIELD OF INVENTION
The present invention relates to a multistage controlled bio digestion process for production of biogas. More particularly, the present invention relates to the process for production of the biogas with enhanced methane content and reduced carbon-dioxide and reduced hydrogen sulfide content in a gas mixture.
BACKGROUND OF THE INVENTION
Conventionally, when biomass is degraded by the natural uncontrolled process, it produces greenhouse gases like methane (CH4) and carbon-di-oxide (CO2), which affect the environment adversely. Hence, their disposal in controlled manner is essentially required. On the other hand, in the recent times a worldwide energy shortage has prompted a search for non-fossil source of energy. One obvious source of energy is production of methane through anaerobic digestion of any organic carbonaceous material which is susceptible to biodegradation like wastewater, biomass residues, municipal waste, food industry waste, animal manure etc.

Anaerobic digestion is a series of processes in which biodegradable material is broken down by microorganisms and biochemical processes in absence of oxygen. There are four biochemical metabolic processes that take place successively, but also in parallel and interacting with one another, leading to the degradation of organic fermentation substrates to the end products methane and carbon dioxide: hydrolysis, acidogenesis, acetogenesis and methanogenesis. By separately segregating these steps in the process, in presence of specific microbes the quantity and quality of biogas can be improved.

In hydrolysis, high-molecular weight insoluble organic polymers such as carbohydrates, proteins, lipids and the like are broken down into some variety of sugars and/or amino acids, and make them available for another consortium of bacteria. In this process, the gaseous products that are also formed consist mainly of carbon dioxide.

Acidogenic bacteria then convert the hydrolyzed products (e.g. mono- and disaccharides, di- and oligopeptides, amino acids, glycerol, long-chain fatty acids) to short-chain fatty acids or carboxylic acids, for example butyric, propionic and acetic acid, to short-chain alcohols such as ethanol and to the gaseous products hydrogen and carbon dioxide.

In the subsequent acetogenesis, the short-chain fatty acids and carboxylic acids formed in acidogenesis and the short-chain alcohols are taken up by acetogenic bacteria and, after ß-oxidation, are excreted again as acetic acid. By-products of acetogenesis are CO2, hydrogen sulfide (H2S) and molecular hydrogen (H2). The products of acetogenesis such as acetic acid, and also other substrates such as methanol and formate, are converted by methane-forming organisms to methane and CO2 in the anaerobic methanogenesis.

Raw biogas is composed of 50–60% CH4, 30–40% CO2 and small amount of H2S but for high calorific value of biogas, the ratio of methane to CO2 needs to be increased. Generally methane content is enriched by scrubbing of the biogas by physiochemical and ex-situ treatment methods as pressure swing adsorption (PSA), absorption and membrane separation techniques. While these techniques are effective at CO2 removal, but they not only require complicated operating systems but produce unwanted end products that need further treatment. Moreover, these techniques add cost to the process further. Hence, scrubbing after product is obtained will not be desirable but in-situ transformation of the CO2 to methane and removal of H2S will be desirable. In this direction, approach that promises biogas upgradation involves the use of bio electrochemical systems that allow microorganisms to convert CO2 into CH4 when electricity and water are provided as the only energy and electron sources. Beside CO2, removal of H2S is also important as it is harmful to human and animals. At the lower concentration H2S imparts unpleasant odor to the biogas but at a higher concentration it can even pose as a threat to the life. Hence, its removal is also necessary. Therefore, it will be desirable to have in-situ transformation H2S to some non -toxic substance.

WIPO published application WO 2010046914 A1 discloses a method for improving biogas generation by segregation of the two stages i.e. acidogenesis and methanogenesis by means of two separate reactors and semi-permeable membrane units. This document claims improved delivery of the volatile fatty acids (VFA) to methanogenesis stage but does not talk about reducing the CO2 or H2S concentration. Moreover the use of semi-permeable membrane to large scale reactor will be cost intensive and its fouling will be also an issue.

US patent application US 20140186929 A1 entitled “Compositions and methods comprising combination silage inoculants” discloses compositions comprising a combination microbial inoculant, silage produced from forage inoculated with the combination microbial inoculant, and biogas produced from the silage. Various methods are provided for increasing biogas production and decreasing dry matter loss by inoculating forage with a combination inoculant. This prior art does not teach about reducing the CO2 or H2S concentration in biogas and increasing the concentration of methane.

Research paper entitled “Direct Biological Conversion of Electrical Current into Methane by Electromethanogenesis” (Environ. Sci. Technol. 2009, 43, 3953–3958) discloses a process called electromethanogenesis where at a set applied potential, carbon dioxide was reduced to methane. This paper discusses the conversion of CO2 to methane and not about organic digestion of organic waste to methane. Moreover, it does not teach about the reduction of H2S content in the produced biogas.

US patent US 8440438 entitled “Electro methanogenic reactor and processes for methane production” discloses a biological process for producing methane gas and capturing carbon from carbon dioxide in an electro methanogenic reactor having an anode, a cathode and a plurality of methanogenic microorganisms disposed on the cathode. Electrons and carbon dioxide are provided to the plurality of methanogenic microorganisms disposed on the cathode. The methanogenic microorganisms reduce the carbon dioxide to produce methane gas, even in the absence of hydrogen and/or organic carbon sources. This invention discusses about conversion of CO2 to CH4 but does not provide a situation where organic waste material is also present in the cathode along with CO2. Moreover, it does not teach about the reduction of H2S content.

Research article entitled “Bio electrochemical removal of carbon dioxide (CO2): An innovative method for biogas upgrading (Bio resource Technology 173 (2014) 392–398)” describes a methods for biogas upgrading based on biological/in-situ concepts. Bio electrochemical removal of CO2 for biogas upgrading was proposed and demonstrated in both batch and continuous experiments in two chamber reactor. The proposed method could reduce CO2 content up to 10%. This method involves use of two chambered cells and costly membranes which is difficult to upscale. Moreover, it does not teach about the reduction of H2S content and improvement in any other stages of the anaerobic digestion process.

US patent application US 20100107872 entitled “Biogas upgrading” discloses a process and apparatus for treating a biogas stream with respect to reducing H2S content and increasing CH4 content by scrubbing. This adds a step in the biogas production system.

EP published application EP 2449117A1 relates to a method of producing in a bioreactor a biogas rich in methane comprising steps of electrolyzing water in an aqueous medium at a voltage sufficient to electrolyze water without destroying microbial growth in a range of from 1.8V to 12V in the presence of electrochemically active anaerobic microorganisms that catalyse production of hydrogen gas, and contacting a species of hydrogen trophic methanogenic microorganisms with the hydrogen gas and carbon dioxide to produce methane.

US patent application US 20110111475A1 relates to a method for producing hydrocarbon and hydrogen fuels comprising steps of fermenting a biomass material containing fermentation medium with an inoculum comprising a mixed culture of microorganisms derived from the rumen contents of a rumen-containing animal and incubating under anaerobic conditions and for a sufficient time to produce volatile fatty acids in said medium. Secondly, subjecting said volatile fatty acids or their salts to electrolysis under conditions effective to convert said volatile fatty acids or their salts to hydrocarbons and hydrogen.

German patent application DE 102013001689A1 relates to a method for generation of bio-Methane from biogas or digester gas and by electrolysis with renewable energy or algae or cyanobacteria produced into hydrogen and oxygen, which produce biogas from a biogas plant or a digester without prior cleaning with hydrogen stoichiometric or lean of stoichiometry mixed a bio-methanation reactor is supplied. Additionally, the bio-methanation reactor with bacterial suspension from the main fermenter or the secondary fermenter or fermentation residue of the biogas plant or from the digester of a sewage treatment plant is fed, the suspension phase preferably in counter current, or are being undertaken in co-current to the gas phase and the carbon dioxide with hydrogen is converted by the Methane bacteria of the suspension to Methane and water.

US patent application US 20130299400 A1 relates to bio-electrochemical system for treating wastewater, comprising an anaerobic reaction chamber with an anode/cathode pair disposed therein, the anode/cathode pair comprising at least one anode and at least one cathode; at least one methanogenic microbe disposed in proximity to said cathode; and at least one power source configured to apply a voltage to the anode/cathode pair.
In the prior art, the various attempts have been made to generate the high content of methane gas in the biogas produced from biomass. However, no prior art relates to simultaneous attempts to reduce the carbon-di-oxide and hydrogen sulphide contents along with the biogas production with high methane content in a cost effective manner for its wider applications. Additionally, none of the prior arts propose specific microbes at each step for high production of methane content in the biogas.

In view of above, there remains a need for a process which performs the following:
• facilitates the hydrolysis of biomass/organic waste,
• converts the hydrolyzed product to VFA particularly to the acetic acids,
• recycles the CO2 formed at different stage of process to methane,
• reduces in-situ H2S production.

SUMMARY OF THE INVENTION

The present invention relates to bio methanation from biodegradable materials comprising biomass and organic waste. The method comprises the steps of: (a) the biomass is hydrolyzed in presence of enzymes or microbes producing enzymes, selected from esterase, protease, lactase, cellulase, hemicellulose and a combination thereof to obtain a hydrolysed residue and gaseous effluent comprising CO2, (b) a portion of hydrolysed residue is subjected to acidogenesis, to obtain volatile fatty acids enriched with acetic acid of at least 70% of the total VFAs, and gaseous effluent comprising of CO2 and H2, (c) the volatile fatty acids enriched with acetic acid is subjected to electro-assisted methanogenesis at a potential ranging from at least 0.1-1 V to obtain biogas comprising at least 95% methane, and (d) the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%. The biomethanogenesis is carried in the presence of neutral red and nano-sized iron oxide in a ratio of 2:1.
In another embodiment, pretreatment is carried out in the process prior to step (a) by heating the biomass in presence of alkali. In another embodiment, the hydrolysis is carried for at least 1-14 days at 20-70 degree C by the enzymes produced by microbes selected from the group comprising of Ruminococcus sp., Lactobacillus sp., Bacillus licheniformis, Bacillus subtilis, Pseudomonas sp., and Streptococcus sp. In another embodiment, strains for the hydrolysis are selected from the group comprising of Bacillus subtlis (MTCC 5386), Bacillus sp. (MTCC 5662), Bacillus cereus (MTCC 5665) and Lysinibacillus sp. (MTCC 5666) alone or in any combination. In another aspect, a nutrient system fed to the hydrolysis reactor comprises of nutrients that are selected from KH2PO4 (0.5 g/l -4.8g/l), K2HPO4 (0.5 g/l -5 g/l), MgSO4 (0.01 g/l -1.0 g/l), (NH4)2SO4 (0.25 g/l -0.50 g/l), KNO3 (0.15 g/l -4.75 g/l), Urea (0.15 g/l -4.75 g/l), Di-ammonium phosphate (0.15 g/l -4.75 g/l), ZnSO4 (0.2 g/l -2.1 g/l), NaCl (0.2 g/l -10 g/l) and trace element (2ml to 10 ml of solution).
In another embodiment, the acidogenesis is carried by microbes selected from the group comprising of Clostridium aceticum; Acetobacter woodii, Ruminococcus sp., Lactobacillus species, Bacillus licheniformis and Clostridium termoautotrophicum. In another embodiment, strains for acidogenesis are selected from the group comprising of Lysinibacillus sp. (MTCC 25029), Bacillus thermoleovorans (MTCC 25023), Peudomonas aerugiosa (MTCC 5388), Pseudomonas putida (MTCC 5869) and Arthrobacter sp. (MTCC 25028). In another embodiment, the acidogenesis is carried in the acidogenesis reactor by nutrients selected from KH2PO4 (4 g/l -5g/l), K2HPO4 (4.0 g/l -15 g/l), MgSO4 (0.2 g/l -2.0 g/l), Trace element (0.5ml to 10 ml of solution), yeast extract (3-10 g/l), ammonium nitrate (5 g/l -8 g/l), thiamine ( 25-300 ppm), citrate (10-20 g/l), sorbitol ester (5-25ppm) , Oleic acid (100 -1000 ppm) and pantothenic acid (20-500 ppm).
In another embodiment, the hydrolysis and the acidogenesis are carried out in a single reactor or in two different reactors. In another embodiment, the electro-assisted methanogenesis is performed in a single cell reactor with at least one cathode and one anode connected to each other by a conductive wire. In further embodiment, the cathode and the anode is selected from carbon paper, carbon cloth, carbon felt, carbon wool, carbon foam, graphite, porous graphite, graphite powder, iron, carbon nanotubes, functionalized carbon nanotubes, carbon electrodes modified with functionalized carbon nanotubes, carbon electrodes modified with graphene, metals, stainless steel, and combinations of these.
In another embodiment, the microbes for the electro-assisted methanogenesis is selected from the group comprising of, Methanosarcina sp., Desulfovibrio sp. Clostridium sp., Methanobacterium sp., Brevibacterium sp., Methanolobus sp., Methanosaeta sp., Thermotoga sp. Methanobacterium bryantii; Methanobacterium formicum; Methanobrevibacter arboriphilicus; Methanobrevibacter gottschalkii; Methanobrevibacter ruminantium; Methanobrevibacter smithii; Methanocalculus sp.; Methanococcoides burtonii; Methanococcus aeolicus; Methanococcus jannaschii; Methanococcus maripaludis; Methanocorpusculum labreanum; Methanoculleus bourgensis; Methanogenium sp., Methanomicrobium mobile; Methanoregula boonei; Methanosaeta sp.; Methanosarcina sp;; Methanosphaera sp.; Methanothermobacte sp.; Methanothermobacter thermautotrophicus and combinations of any of these and/or other methanogenic bacteria. In another embodiment, strains are selected from the group comprising of Citrobacter sp. MTCC 25018), Serratia sp. (MTCC 25017), Bacillus stearothermophilus (MTCC 25030), Shewanella sp. (MTCC 25020), Pseudomonas fragi (MTCC 25025) are used alone or in any combination for the electro-assisted biomethanogenesis.
In another embodiment, the electro-assisted methanogenesis is carried in presence of carbonic anhydrase or carbonic anhydrase producing microbes. In another embodiment, the methane content in the biogas is enhanced at temperature of at least 5°C to 65°C and salinity range from 0-10% in the single cell reactor. In another embodiment, the nutrient system fed to the electro-assisted methanogenesis reactor is selected from NiCl2.6H2O (1.5mg/L); FeSO4.H2O (0.5mg/L); MgSO4.7H2O (0.8g/L); KH2PO4 (0.5 g/L); K2HPO4 (0.5 g/L); KCl (0.05 g/L); CaCl2.7H2O (0.05 g/L); NaCl (1.5 g/L); NH4Cl (1 g/L); MnSO4.7H2O (0.6 mg/L); ZnSO4.7H2O (0.1 mg/L); CuSO4.5H2O (0.02mg/L); H3BO3 (7µg/L); CoSO4.7H2O (4µg/L); Na2MoO4.2H2O (0.5 mg/L); Na2SeO3.5H2O (3 µg/L); Na2WO4 × 2H2O (4 µg/L); Nitrilotriacetic acid (0.15 mg/L); sodium acetate (1 g/L); trypticase (2 g/L); yeast extract (2 g/L); valeric acid (5 mM); isovaleric acid (5mM); 2-methylbutyric acid (5mM); isobutyric acid (6mM); 2-methyl valeric acid (5mM); resazurin (1 mg/L) 2% (v/v): NaHCO3, 10%.
In one embodiment, a process for enhancing biogas production is provided. The method comprises steps of: (a) a biomass is hydrolysed in presence of enzymes or microbes producing esterase, protease, lactase, cellulase, hemicellulose and a combination thereof, to obtain a hydrolysed residue and gaseous effluent comprising CO2, (b) a portion of hydrolysed residue is subjected to acidogenesis to obtain volatile fatty acids enriched with acetic acid, and gaseous effluent comprising CO2 and H2, (c) the volatile fatty acids enriched with acetic acid is further subjected to electro-assisted methanogenesis at a potential ranging from 0.1-1 V to obtain biogas comprising at least 95% methane, and (d) the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%.
In another embodiment, neutral red and nano-sized iron oxide in the single cell reactor are added in at least ratio of 2:1 that increase the methane content in the biogas.

BRIEF DESCRIPTION OF ACCOMPAINED DRAWINGS
Figures 1 is schematic diagram showing various steps of method of bio-methanation of the present invention. Referral numeral 1 represents organic waste/Biomass, referral numeral 2 represents shredding/pulverization stage in the process, referral numeral 3 represents pre-treatment stage by chemical or physical or biological means, referral numeral 4 represents pH neutralization, referral numeral 5 represents hydrolysis stage where at least 102 cfu/g biomass; 20-70 degree Celsius, referral numeral 6 represents acidogenesis stage, referral numeral 7 represents power source of at least 0.1-1 V, referral numeral 8 represents effluent, referral numeral 9 represents CO2 and referral numeral 10 represents Volatile fatty acids enriched waste and 11 represents the electro-assisted biomethanogenesis reactor.
DETAILED DESCRIPTION

While the invention is susceptible to various modifications and/or alternative processes and/or compositions, specific embodiment thereof has been shown by way of examples and tables and will be described in detail below. It should be understood, however that it is not intended to limit the invention to the particular processes and/or compositions disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.
The examples, tables, and protocols have been represented where appropriate, showing only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.
The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that one or more processes or composition/s or systems or methods proceeded by “comprises... a” does not, without more constraints, preclude the existence of other processes, sub-processes, composition, sub-compositions, minor or major compositions or other elements or other structures or additional processes or compositions or additional elements or additional features or additional characteristics or additional attributes.
In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:
It must be noted that, as used in the specification/description and the appended claims and examples, the singular forms “a”, “an” and “the” may include plural referents unless the context clearly dictates otherwise.
Ranges may be expressed herein as from “about” one particular value, and or “to about” another particular value. When such a range is expressed, another aspect includes from the one particular value and or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about”, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
Definitions:
As used herein, the terms “Biodegradable materials” when used in context of the present invention refers to an organic matter including, biomass, organic waste, kitchen waste, sewerage, municipal waste, refinery waste water, petrochemical industry waste water or any organic material which is susceptible to biodegradation. As used herein, the terms “microbial consortium” when used in context of the present invention refers to two or more microbial groups living symbiotically. Consortiums can be endosymbiotic or ectosymbiotic.
As used herein, the terms “hydrolysis” when used in context of the present invention refers to the chemical breakdown of a compound due to reaction with water.
As used herein, the terms “acidogenesis” when used in context of the present invention refers to a biological reaction where simple monomers are converted into volatile fatty acids.
As used herein, the terms “electro-assisted methanogenesis” when used in context of the present invention refers to a formation of methane by microbes known as methanogens in presence of electric current.
As stated before, there remains a need to develop a cost effective method for a process which could facilitate the hydrolysis of biomass, convert the hydrolyzed product to volatile fatty acid particularly to the acetic acids, recycles carbon-di-oxide formed at different stage of process to methane and reduces in-situ hydrogen sulfite during the process. The present invention provides a multistage controlled bio digestion process for production of usable energy in the form of methane from waste biomass materials. The present invention particularly provides a process which facilitates the hydrolysis of biomass/organic waste, converts the hydrolyzed product to VFA particularly to the acetic acids, recycles the CO2 formed at different stages of the process to methane, converts CO2 to methane in-situ, enhances the methanogenesis process by electric current and reduces the in-situ H2S concentration. More specifically, the recycled CO2 is converted in-situ to the methane in the process which results in higher methane content and reduced CO2 content. The invention can be used for cooking where LPG can be replaced, for generating electrical power by using gas engines, for lightening purposes in gas fired lanterns and as a transport fuel in automobiles by replacing CNG.
Accordingly, in an embodiment of the present invention, an integrated process for bio methanation from biomass is provided. The method comprising the steps for bio digestion for producing methane includes the steps of:
(a) the biomass is hydrolysed in presence of enzymes or microbes producing enzymes selected from the group comprising of esterase, protease, lactase, cellulase, hemicellulose and a combination thereof, to obtain a hydrolysed residue and gaseous effluent comprising CO2,
(b) a portion of hydrolysed residue is subjected to acidogenesis to obtain volatile fatty acids enriched with at least 70% acetic acid of the total VFAs, and the gaseous effluent comprising CO2 and H2,
(c) the volatile fatty acids enriched with acetic acid is subjected to electro-assisted methanogenesis at a potential ranging from 0.1-1 V to obtain biogas comprising at least 95% methane, and the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%. The biomethanogenesis is carried in the presence of neutral red and nano-sized iron oxide in a ratio of 2:1.
In another embodiment, the pretreatment of biomass is prior to the hydrolysis by heating the biomass in presence of alkali. According to present invention the pre-treatment of the biomass is done by chemical, physical or biological means. It also includes activities like shredding, pulverization etc. The gaseous emission, such as carbon-di-oxide if produced during this stage is transferred to electro-assisted biomethanogenesis reactor.

In another embodiment of the present invention, irrespective of the chemical difference between various organic waste product, a simple heat treatment and in particular heat treatment in combination of alkali treatments, such as with NaOH, KOH, etc., improves the hydrolysis and the acidogenesis step in the process by making sites for bacterial action in subsequent stages so that microbes work effectively.

In another embodiment, of the present invention, the hydrolysis step is to convert pre-treated waste to low molecular weight components selected from the group but not limited to monomeric forms of sugars, amino acids and fatty acids by action of selective microbial consortia in an aqueous media comprising an optimized nutrient system. In another embodiment, the hydrolysis of pretreated wastes is carried for at least 1-14 days at temperature ranging from 20- 70 degree C by mixing under micro-aerophilic conditions and/or anaerobic conditions.
In another embodiment, at least 70% of the hydrolysed residue is transferred to the acidogenesis stage and remaining is used as inoculum for next batch of the organic waste. The gaseous effluent, such as carbon-di-oxide produced during this stage is transferred to electro-assisted biomethanogenesis reactor. In another embodiment, the microbial consortium for the hydrolysis is selected from the group comprising of but not limited to, combination of the microbes that are adapted to produce esterase, proteases, lactase, cellulase and hemicellulase enzymes. In further embodiment the microbial consortium includes, but is not limited to, Ruminococcus sp., Lactobacillus sp., Bacillus licheniformis, Bacillus subtilis, Pseudomonas sp., Streptococcus sp. or consortium thereof. Additionally, the microbial consortium of hydrolysis is effective at least if 102 cfu/g of the biomass are present in the hydrolysis reactor. In another embodiment, the free or immobilized enzymes obtained from above mentioned microbial species or any other sources are used in the hydrolysis step. In another embodiment, strains for hydrolysis are selected from the group comprising of but not limited to, Bacillus subtlis (MTCC 5386), Bacillus sp. (MTCC 5662),Bacillus cereus (MTCC 5665) and Lysinibacillus sp. (MTCC 5666) used alone or in any combination.
In another embodiment of the present invention, a nutrient system fed to the hydrolysis reactor is selected from the group comprising of but not limited to (g/L) KH2PO4 (0.5-4.8), K2HPO4 (0.5-5), MgSO4 (0.01-1.0), (NH4)2SO4 (0.25-0.50), KNO3 (0.15-4.75), Urea (0.15-4.75), Di-ammonium phosphate (0.15-4.75), ZnSO4 (0.2-2.1), NaCl (0.2-10) Trace element (2ml to 10 ml of solution). The trace element solution (gram per liter) comprises Nitrilotriacetic acid (0.1-1.0), FeSO4.7H2O (0.01-0.15), MnCl2.4H2O (0.001- 0.005), CoCl2.6H2O (0.005-0.02), CaCl2.2H20 (0.01-0.5), ZnCl2 (0.01-0.15), CuCl2.H2O (0.01-0.03), H3BO3 (0.002-0.02), Na2MoO4 (0.001-0.02), Na2SeO3 (0.005-0.02), NiSO4 (0.01-0.03), SnCl2 (0.01-0.03).
In another embodiment of the present invention, in acidogenesis step of the process, the hydrolyzed biomass is subjected to the microbes convert the hydrolyzed biomass to the Volatile organic acids (VFA). In another embodiment, the concentration of the acetic acid is at least 70% of the total VFAs. This acidogenesis reactor is carried out in micro-aerophilc conditions and/or anaerobic conditions with total solid loading of more than 50% that result in higher gas production. The gaseous effluent, such as carbon-di-oxide and hydrogen that are produced during this stage is further transferred to electro-assisted methanogenesis reactor. In another embodiment, the microbes for acidogenesis are selected from group comprising of but is not limited to, Clostridium aceticum; Acetobacter woodii, Ruminococcus sp., Lactobacillus species, Bacillus licheniformis, Clostridium termoautotrophicum. In yet another embodiment, strains for acidogenesis are selected from the group comprising of Lysinibacillus sp. (MTCC 25029), Bacillus thermoleovorans (MTCC 25023), Peudomonas aerugiosa (MTCC 5388), Pseudomonas putida MTCC 5869, Arthrobacter sp. (MTCC 25028) used alone or in combinations thereof.
In another embodiment of the present invention, a nutrient system fed to the acidogenesis reactor selected from the group comprising but not limited to K2HPO4 (4-5g/l), KH2PO4 (4-15 g/l) , MgCl2 (0.2-2 g/l), 0.5-10 ml trace elements, yeast extract (3-10 g/l), ammonium nitrate (5-8 g/l), thiamine ( 25-300 ppm), citrate (10-20 g/l), sorbitol ester (5-25ppm) , Oleic acid (100 -1000 ppm), pantothenic acid (20-500 ppm). The trace element solution (gram per liter) comprises nitrilotriacetic acid (1.0), FeSO4.7H2O (0.01), CoCl2.6H2O (0.09), CaCl2.2H20 (0.9), ZnCl2 (0.55), CuCl2.H2O (0.03), H3BO3 (0.02), Na2MoO4 (0.02).
In another aspect of the present invention, the contents of the hydrolysis reactor and acidogenesis reactor are adapted to circulate at 20-60 degree C at a rate that entire volume is completely re-circulated about 1-5 times in a day.

In another embodiment, the hydrolysis and the acidogenesis steps of the process are adapted to be carried out in a single reactor or in two different reactors.

In another embodiment, the microbes with hydrolytic and acidogenesis acitivity are obtained from organic waste damping sites, effluent plant, cattle dung slurry, wood decaying sites etc. The general characteristics, growth and cultivation protocol of acetogenic bacteria is provided in Drake, Harold L. Acetogenesis (Ed) Diversity, Ecology, and Isolation of Acetogenic Bacteria Schink, Bernhard. Pages 197-235) (doi 10.1007-2F978-1-4615-1777-1_7)

In an embodiment of the present invention, acetic acid enriched Volatile fatty acids (VFA) from acidogenesis are subjected to the electro-assisted methanogenesis. pH is adjusted in a single chambered cell reactor that is provided with at least one cathode and one anode connected to each other by at least one conductive wire.
In another embodiment, electrolysis in the electro-assisted methanogenesis is poised at a potential ranging from 0.1 -1V. The leachate of the biomass from acidogenesis stage works as electrolyte and is inoculated with microbes. The 0.1-1V potential applied to the cell facilitates the conversion of VFAs to methane in faster way and the microbes present in the cells reduce the CO2 at cathode to methane and oxidized the H2S at anode to the SO4. This results in increased concentration of CH4 and reduced concentration of the CO2 and H2S. In another embodiment, the biogas produced in this reactor is collected in suitable container and it comprises of at least 95% methane and less than 2% CO2 and H2S less than 2 ppm.
In another embodiment, the strains for the electro-assisted biomethanogenesis are selected from the group comprising of but not limited to, Citrobacter sp. MTCC 25018), Serratia sp. (MTCC 25017), Bacillus stearothermophilus (MTCC 25030), Shewanella sp. (MTCC 25020), Pseudomonas fragi (MTCC 25025) and methanogenic bacteria etc.
In another embodiment, the microbes for the electro-assisted biomethanogenesis is selected from the group comprising of , but not limited to, Methanosarcina sp., Desulfovibrio sp. Clostridium sp., Methanobacterium sp., Brevibacterium sp., Methanolobus sp., Methanosaeta sp., Thermotoga sp. Methanobacterium bryantii; Methanobacterium formicum; Methanobrevibacter arboriphilicus; Methanobrevibacter gottschalkii; Methanobrevibacter ruminantium; Methanobrevibacter smithii; Methanocalculus sp.; Methanococcoides burtonii; Methanococcus aeolicus; Methanococcus jannaschii; Methanococcus maripaludis; Methanocorpusculum labreanum; Methanoculleus bourgensis; Methanogenium sp., Methanomicrobium mobile; Methanoregula boonei; Methanosaeta sp.; Methanosarcina sp;; Methanosphaera sp.; Methanothermobacte sp.; Methanothermobacter thermautotrophicus and combinations of any of these and/or other methanogenic bacteria.
In further embodiment, the methanogenic bacteria is obtained from methane producing coal bed, a manure digestor, a municipal waste, an activated sludge from wastewater treatment, sewer, sludge ponds, any other running old biogas plant, from cattle dung or an isolated culture of individual methanogenic bacteria from such sources. Methanogenic bacteria and conditions for their growth and maintenance are known, as exemplified in M. Dworkin et al., The Prokaryotes, Springer; 3rd edition, 2007 and in J. G. ZEIKUS The Biology of Methanogenic Bacteria, Bacteriological Reviews, June 1977, Vol. 41, No. 2 , pp. 514-541.
In another embodiment, the electrodes in the electro-assisted biomethanogenesis reactor provide electrons for the steering of the fermentation process leading metabolic shift to produce methane and for carrying out redox reaction that result in in-situ conversion of the CO2 and H2S. In another embodiment of the present invention, a source of external electric current wherein such source can be renewable electric source; for example, electric current is generated from solar power, wind turbines, hydroelectric or biomass-fired electrical generators, or other renewable energy sources of electricity.
In another embodiment of the present invention, an exemplary conductive electrode materials includes, but are not limited to, carbon paper, carbon cloth, carbon felt, carbon wool, carbon foam, graphite, porous graphite, graphite powder, iron, carbon nanotubes, functionalized carbon nanotubes, metals, stainless steel, and combinations of these. The physical orientation of the electrodes is any convenient mutual orientation relative to each other. In another embodiment of the present invention, the cathode and anode are of equal dimensions and oriented with surfaces parallel to each other. In yet another embodiment, the electrodes particularly the cathode, are gas diffusion electrodes. Gas diffusion electrodes (GDE) provide a conjunction at interface of solid, liquid and gas, where an electrical conducting catalyst helps in carrying out an electrochemical reaction between the liquid and the gaseous phase. GDE helps in biotransformation of organic materials as well as CO2 to methane.
In another embodiment of the present invention, the electric sources comprises of renewable and non-renewable sources of electricity. The renewable source of the energy are selected from group comprising of but are not limited to solar energy, wind turbines, hydroelectric or biomass-fired electrical generators, or other renewable energy sources of electricity. In another embodiment, the source of electrical current is selected from any convenient source of electrical current, including a grid-connected power supply, a battery, a fuel-powered generator, an electrochemical cell, microbial fuel cells, and a bio electrochemical cell with anodic chamber having bio-oxidation of wastewater. In yet another embodiment, the reactor may contain carbonic anhydrase, its mimic or carbonic anhydrase producing microbes in free and/or immobilized form improving the solubility of the CO2 in the reactor.

In another embodiment of the present invention, the methanogenic microbes produce maximum methane content in biogas at a temperature of at least 5°C to 65°C and salinity range from about 0-10%. In another embodiment, the microbial consortium disclosed in the present invention produces stable biogas production without seasonal variation impact. In another embodiment of the present invention, addition of some electron donor like metal (s) may be added to methanogenic reactor. In another embodiment, the effluent from the methanogenic reactor comprises of microbes and some unutilized feedstock that is recycled to the hydrolysis reactor to recover the residual BOD content and recycles the water resulting to reduced water requirements.
In another embodiment, the electro-assisted methanogenesis reactor is supplemented with the nutrient mixture selected from the group comprising but not limited to NiCl2.6H2O, 1.5 mg/L; FeSO4.H2O, 0.5 mg/L; MgSO4.7H2O, 0.8 g/L; KH2PO4, 0.5 g/L; K2HPO4, 0.5 g/L; KCl, 0.05 g/L; CaCl2.7H2O, 0.05 g/L; NaCl, 1.5 g/L; NH4Cl, 1 g/L; MnSO4.7H2O, 0.6 mg/L; ZnSO4.7H2O, 0.1 mg/L; CuSO4.5H2O, 0.02 mg/L; H3BO3, 7 µg/L; CoSO4.7H2O, 4 µg/L; Na2MoO4.2H2O, 0.5 mg/L; Na2SeO3.5H2O, 3 µg/L; Na2WO4 × 2H2O, 4 µg/L; Nitrilotriacetic acid, 0.15 mg/L; sodium acetate, 1 g/L; trypticase, 2 g/L; yeast extract, 2 g/L; valeric acid, 5mM; isovaleric acid, 5mM; 2-methylbutyric acid, 5mM; isobutyric acid, 6mM; 2-methyl valeric acid, 5mM; resazurin, 1 mg/L. 2% (v/v): NaHCO3, 10%; Na2S, 2%; methanol, 4M; sodium format, 8M.
In one embodiment, a process for enhancing biogas production comprises steps of: (a) a biomass is hydrolyzed in presence of enzymes or microbes producing enzymes selected from esterase, protease, lactase, cellulase, hemicellulose and a combination thereof, to obtain a hydrolysed residue and gaseous effluent comprising CO2, (b) a portion of hydrolysed residue is subjected to acidogenesis to obtain volatile fatty acids enriched with acetic acid, and gaseous effluent comprising CO2 and H2, (c) the volatile fatty acids enriched with acetic acid is subjected to electro-assisted methanogenesis at a potential ranging from at least 0.1-1 V to obtain the biogas comprising at least 95% methane, and (d) the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%.
In another embodiment, addition of neutral red and nano-sized iron oxide in the electro-assisted biomethanogenesis reactor in ratio of 2:1 at the final concentration ranging from 2-10 ppm further improves the methane gas content and yield in the biogas.
In another embodiment, the process disclosed in the present invention is effective in temperature 5-65 degree C, pH 4-10 and salinity 0-10% on wide range of the feedstock like biomass, kitchen waste, sewerage, municipal waste, refinery wastewater, petrochemical industry wastewater etc.
In another embodiment, the process disclosed in the present invention can be used in batch wise, semi -continuous or continuous process of biomethanation under septic conditions.
According to the state of the art, the anaerobic degradation of organic substance takes place in an aqueous medium with contents of dry substance of normally less than 30%. In the present invention the solid loading is more than 50%.
In another embodiment, the nutrients are added to obtain optimum carbon: nitrogen: phosphorus: sulfur ratio is 300:10:4:2 for hydrolysis and acidogenesis, and 500:10:5:3 for methanogenesis. The optimum pH-value for hydrolysis and acidogenesis is in the range of pH 4.2 to 6.3; the optimum pH-value for methanogenesis is in the range of pH 6.7 to 8.5.
In another embodiment, trace elements containing manganese and/or copper and/or tungsten and/or zinc and/or nickel are added at concentration of 5-100 ppm at various stages of the process at different concentration.
In another embodiment of the present invention the electron efficiency of the methanogenesis stage is more than 97%. More specifically, 97% of the energy supplied to the methanogenesis reactor is converted to Methane.
In another embodiment of the present invention the Methane production ability of the present process is at least 99% of theoretical yield based on the COD/BOD content of the feed.
Having described the basic aspects of the present invention, the following non-limiting examples illustrate specific embodiment thereof.

EXAMPLES
The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration to the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention various changes to the described embodiments may be made in the functions and arrangement of the elements described without departing from the scope of the invention.
Example 1
The organic waste (kitchen waste) (5 Kg) was pulverized and subjected to hydrolysis using the microbial blend consisting of Bacillus sp. and Lactobacillus sp. at pH 7 and temperature 65°C with stirring at 10 rpm for 5 hrs. The hydrolysed material was transferred to acidogenesis reactor using microbial blend consisting of Clostridium sp. and Acetobacter for 12 hrs under microaerophilic conditions it produces the VFA (2 g/L) with around 1.5 g/L acetic acid. The acetic acid was continually fed to the methenogenesis reactor with hydraulic retention time (HRT) of 16 hrs.
The electro-assisted methanogenesis reactor (10L) was having the graphite stick cathode and anode (both 10 × 30 cm) were suspended in the reactor which are connected to each other by a conductive wire. Electrodes were separated to avoid short circuit. Voltage was applied to circuit through the electric source and cell poised at -700 mV. The gaseous effluent from all reactors was continuously sparged at 20ml/min rate to reactor. This assembly was kept under stirring at temperature 55°C. It was inculcated by microbial consortium consisting of Shewanella sp. IOC-EA-106 ( MTCC 25020), Pseudomonas fragi IOC-S2 (MTCC 25025), and microbial innoculum obtained from any operational biogas plant, comprising of bacteria such as Methanosarcina sp, Desulfovibrio sp., Clostridium sp., Methanobacterium sp., Brevibacterium sp. etc. The electro -methanogenesis reactor was dosed with neutral red and nano-sized iron oxide in a ratio of 2:1 at final concentration of 10 ppm .
A control reactor without electric current was also run parallel. The result showed methane content of 97% and CO2 content of 2% in the biogas produced with no detectable H2S. The control reactor contained 54% CH4, 100 ppm H2S and 45% CO2.

Example 2
The horticultural biomass residue was pulverized and subjected to hydrolysis using the microbial blend consisting of Bacillus subtlis (MTCC 5386), Bacillus sp. (MTCC 5662), Bacillus cereus (MTCC 5665) and Lysinibacillus sp. (MTCC 5666) at pH 10 and temperature 65°C with stirring at 50 rpm for 5 hrs. The hydrolysed material was transferred to acidogenesis reactor using microbial blend consisting of Lysinibacillus sp. (MTCC 25029), Bacillus thermoleovorans (MTCC 25023), Peudomonas aerugiosa (MTCC 5388), Pseudomonas putida (MTCC 5869) and Arthrobacter sp. (MTCC 25028) for 12 hrs under microaerophilic conditions it produces the VFA ( 5 g/L) with around 4 g/L acetic acid. The acetic acid was continually fed to the methenogenesis reactor with hydraulic retention time (HRT) of 16 hrs.
The electro-assisted methanogenesis reactor was designed with a total/working volume of 12/10 L. Bioreactor was inserted with graphite rod wrapped with ACC as electrode for oxidation and charcoal doped graphite rod wrapped with SS mesh as electrode for reduction. Electrodes were separated to avoid short circuit. Voltage was applied to circuit through the electric source and cell poised at -700 mV. The gaseous effluent from all reactors was continuously sparged at 20ml/min rate to reactor. This assembly was kept under stirring at temperature 30 °C. It was inculcated by microbial consortium consisting of Shewanella sp. IOC-EA-106 ( MTCC 25020), Pseudomonas fragi IOC-S2 (MTCC 25025), and microbial innoculum obtained from any operational biogas plant, comprising of bacteria such as Methanosarcina sp, Desulfovibrio sp., Clostridium sp., Methanobacterium sp., etc.
The electro-methanogenesis reactor was dosed with neutral red and nano-sized iron oxide in a ratio of 2:1 at final concentration of 20 ppm. The current generation from the system was monitored which started increasing with time and in 4hrs of operation, reached 5±0.5 A/m2, and sustained afterwards at more or less similar value till 24hrs of operation. The biogas produced during these 24 hrs was collected and measured using water displacement. The biogas sample at 8hrs and 18 hrs was analyzed for methane content of biogas.
Following sets were run parallel to conduct the experiment:
Control-1:
No electric current and no Neutral red and nano-sized iron oxide mixture was provided in this condition
Control-II:
No Neutral red and nano-sized iron oxide mixture was provided, however current was given in this condition
Experimental:
Both Neutral red and nano-sized iron oxide mixture were present in this condition.
Results showed a higher biogas production in the modified process than the control along with great enhancement in methane content (as tabulated in Table 1).
Table 1: Batch mode operation using horticultural biomass residue

Control -I
(no electric current and Neutral red and nano-sized iron oxide mixture) Control-II
( in absence of Neutral red and nano-sized iron oxide mixture, but current was given ) Experimental
In presence of Neutral red and nano-sized iron oxide mixture and electric current
Total biogas produced (L) 7.6 19.69 31.09
Methane content (%) 67 84.5 95.98
H2S content (ppm) 79 Not detectable Not detectable

The electro-methanogenesis reactors was operated in continuous mode with a HRT of 12 hrs at a flow rate of 70 ml/h. Current generation in this case increased to 7±0.8 A/m2 in 3h and sustained afterwards. The biogas produced was continuously measured using water displacement and the gas samples were also analyzed for methane and H2Scontent. Results showed a higher biogas production in the modified process than control along with large increment in methane content and TOC removal (approximately 97 %) (as tabulated in Table 2).

Table 2: Continuous mode operation of bioreactor using horticultural biomass residue

Control -I
(no electric current and Neutral red and nano-sized iron oxide mixture) Control-II
( in absence of Neutral red and nano-sized iron oxide mixture, however current was given ) Experimental
In presence of Neutral red and nano-sized iron oxide mixture and electric current
Total biogas produced (L) 8.1 16.37 38.07
Methane content (%) 69 82.67 97.57
H2S content (ppm) 84 Not detectable Not detectable
,CLAIMS:We Claim:
1. A process for producing biogas comprising:
(a) hydrolyzing a biomass in presence of enzymes or microbes producing at least one enzyme selected from esterase, protease, lactase, cellulase, hemicellulose and a combination thereof to obtain a hydrolysed residue and gaseous effluent comprising CO2,
(b) subjecting a portion of hydrolysed residue to acidogenesis, to obtain volatile fatty acids (VFAs) enriched with acetic acid of at least 70% of total VFAs, and the gaseous effluent comprising CO2 and H2,
(c) subjecting the volatile fatty acids enriched with acetic acid to electro-assisted methanogenesis at a potential ranging from at least 0.1-1 V to obtain biogas comprising at least 95% methane,
wherein the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%,
wherein the biomethanogenesis is carried in the presence of neutral red and nano-sized iron oxide in a ratio of 2:1.

2. The process of claim 1, wherein the process further comprises of pre-treating the biomass prior to step (a), by heating the biomass in the presence of alkali.

3. The process of claim 1, wherein hydrolysis in step (a) is carried for at least 1-14 days at a temperate of 20-70º C.

4. The process of claim 1, wherein the enzymes used in hydrolysis of step (a) are produced by microbes selected from a group comprising of Ruminococcus sp., Lactobacillus sp., Bacillus licheniformis, Bacillus subtilis, Pseudomonas sp., Streptococcus sp or a consortium thereof.

5. The process of claim 4, wherein the microbes are selected from a group comprising Bacillus subtlis (MTCC 5386), Bacillus sp. (MTCC 5662), Bacillus cereus (MTCC 5665), Lysinibacillus sp. (MTCC 5666) and any combination thereof.

6. The process of claim 4, wherein the hydrolysis of step (a) is carried in a hydrolysis reactor fed with a nutrient system, comprising nutrients that are selected from KH2PO4 (0.5 g/l -4.8g/l), K2HPO4 (0.5 g/l -5 g/l), MgSO4 (0.01 g/l -1.0 g/l), (NH4)2SO4 (0.25 g/l -0.50 g/l), KNO3 (0.15 g/l -4.75 g/l), Urea (0.15 g/l -4.75 g/l), Di-ammonium phosphate (0.15 g/l -4.75 g/l), ZnSO4 (0.2 g/l -2.1 g/l), NaCl (0.2 g/l -10 g/l) and trace element (2ml to 10 ml of solution).

7. The process of claim 1, wherein the acidogenesis is carried by microbes selected from a group comprising of Clostridium aceticum; Acetobacter woodii, Ruminococcus sp., Lactobacillus species, Bacillus licheniformisClostridium termoautotrophicum or a consortium thereof.

8. The process of claim 1, wherein the acidogenesis is carried out with microbes selected from Lysinibacillus sp. (MTCC 25029), Bacillus thermoleovorans (MTCC 25023), Peudomonas aerugiosa (MTCC 5388), Pseudomonas putida (MTCC 5869), Arthrobacter sp. (MTCC 25028) and any combinations thereof.

9. The process of claim 1, wherein the acidogenesis is carried in an acidogenesis reactor, fed with a nutrient medium comprising nutrients that are selected from KH2PO4 (4 g/l -5g/l), K2HPO4 (4.0 g/l -15 g/l), MgSO4 (0.2 g/l -2.0 g/l), Trace element (0.5ml to 10 ml of solution), yeast extract (3-10 g/l), ammonium nitrate (5 g/l -8 g/l), thiamine ( 25-300 ppm), citrate (10-20 g/l), sorbitol ester (5-25ppm) , Oleic acid (100 -1000 ppm) and pantothenic acid (20-500 ppm).

10. The process of claim 1, wherein the hydrolysis and the acidogenesis are adapted to be carried out in a single reactor or in two different reactors.

11. The process of claim 1, wherein the electro-assisted methanogenesis is performed in a single cell reactor with at least one cathode and one anode that are connected to each other by a conductive wire.

12. The process of claim 11, wherein the cathode and the anode are selected from a group of materials comprising but not limited to carbon paper, carbon cloth, carbon felt, carbon wool, carbon foam, graphite, porous graphite, graphite powder, iron, carbon nanotubes, functionalized carbon nanotubes, metals, stainless steel, and combinations of these.

13. The process of claim 1, wherein the electro-assisted methanogenesis is carried by microbes selected from a group comprising Methanosarcina sp., Desulfovibrio sp. Clostridium sp., Methanobacterium sp., Brevibacterium sp., Methanolobus sp., Methanosaeta sp., Thermotoga sp. Methanobacterium bryantii; Methanobacterium formicum; Methanobrevibacter arboriphilicus; Methanobrevibacter gottschalkii; Methanobrevibacter ruminantium; Methanobrevibacter smithii; Methanocalculus sp.; Methanococcoides burtonii; Methanococcus aeolicus; Methanococcus jannaschii; Methanococcus maripaludis; Methanocorpusculum labreanum; Methanoculleus bourgensis; Methanogenium sp., Methanomicrobium mobile; Methanoregula boonei; Methanosaeta sp.; Methanosarcina sp;; Methanosphaera sp.; Methanothermobacte sp.; Methanothermobacter thermautotrophicus and a consortium thereof.

14. The process of claim 1, wherein the electro-assisted biomethanogenesis is carried out by microbes selected from a group comprising Citrobacter sp. (MTCC 25018), Serratia sp. (MTCC 25017), Bacillus stearothermophilus (MTCC 25030), Shewanella sp. (MTCC 25020), Pseudomonas fragi (MTCC 25025) and any combination thereof.

15. The process of claim 1, wherein the electro-assisted bio methanogenesis is carried in the presence of carbonic anhydrase or carbonic anhydrase producing microbes.

16. The process of claim 1, wherein the process is carried out at a temperature of 5°C to 65°C and salinity range of 0-10%.

17. The process of claim 1, wherein the electro-assisted methanogenesis step is fed with a nutrient medium, wherein nutrient is selected from the group comprising but not limited to nutrients selected from NiCl2.6H2O, 1.5 mg/L; FeSO4.H2O, 0.5 mg/L; MgSO4.7H2O, 0.8 g/L; KH2PO4, 0.5 g/L; K2HPO4, 0.5 g/L; KCl, 0.05 g/L; CaCl2.7H2O, 0.05 g/L; NaCl, 1.5 g/L; NH4Cl, 1 g/L; MnSO4.7H2O, 0.6 mg/L; ZnSO4.7H2O, 0.1 mg/L; CuSO4.5H2O, 0.02 mg/L; H3BO3, 7 µg/L; CoSO4.7H2O, 4 µg/L; Na2MoO4.2H2O, 0.5 mg/L; Na2SeO3.5H2O, 3 µg/L; Na2WO4 × 2H2O, 4 µg/L; Nitrilotriacetic acid, 0.15mg/L; sodium acetate, 1g/L; trypticase, 2g/L; yeast extract, 2g/L; valeric acid, 5mM; isovaleric acid, 5mM; 2-methylbutyric acid, 5mM; isobutyric acid, 6mM; 2-methyl valeric acid, 5mM; resazurin, 1 mg/L. 2% (v/v): NaHCO3, 10%.

18. A process for enhancing biogas production comprising:
(a) hydrolyzing a biomass in presence of enzymes or microbes producing esterase, protease, lactase, cellulase, hemicellulose and a combination thereof, to obtain hydrolysed residue and gaseous effluent comprising CO2,
(b) subjecting a portion of hydrolysed residue to acidogenesis to obtain volatile fatty acids enriched with acetic acid, and the gaseous effluent comprising CO2 and H2,
(c) subjecting the volatile fatty acids enriched with acetic acid to electro-assisted methanogenesis at a potential ranging from at least 0.1-1 V to obtain biogas comprising at least 95% methane,
wherein the gaseous effluent comprising CO2 from step (a) and (b) are transferred to step (c), and has a total solid loadings of more than 50%, and
wherein the biomethanogenesis is carried in the presence of neutral red and nano-sized iron oxide in a ratio of 2:1.