METHOD OF PRODUCING BIOGAS FROM LIGNOCELLULOSIC BIOMASS

Field of the invention:

The present invention relates to the method of producing biogas from lignocellulosic biomass, such as crop residues, grasses and vegetable waste via Anaerobic Digestion process under anaerobic and thermophilic conditions. More particularly, the present invention relates to the production of biogas comprising methane with or without hydrogen from lignocellulosic substrate using thermophilic microbial consortium.

Background of the invention:

Approximately 200 billion tons of lignocellulosic biomass is annually produced worldwide. The main components of lignocellulosic biomass are hemicellulose and cellulose, primarily consisting of C5 and C6 sugars, which could be used for the production of fuels and chemicals. Biomethane and biohythane production using lignocellulosic biomass has been intensely investigated. However, long hydraulic retention time (HRT) and low gas production are observed when using lignocellulosic biomass for anaerobic digestion. This is mainly due to the natural recalcitrance of the lignocellulosic structure, which makes it difficult to use directly and effectively.

The production of biomethane and biohythane depends on the type and biochemical composition of the substrate. However, challenges arise where the biomass contains difficult- to-degrade substrates that might hinder biomethane and biohythane production. Besides, technical challenges are also related to the scaling up of the process for lignocellulosic biomass conversion technologies, such as the stability of the dual-stage fermentation process, its energy efficiency and also the ability to deal with soluble toxic/inhibitory products formed. Sugar-rich substrate is known to be ideal for biohythane production as it is easily accessible for fermentative bacteria. In contrast to such easily digestible substrate, a cellulose-based substrate (e.g. lignocellulosic waste) has a rigid three-dimensional polysaccharide sugars structure and it imparts crystallinity and poor solubility; thus, making it difficult to degrade and transform into biofuels or other value-added products by the microbial consortia. Thus, during the anaerobic transformation of lignocellulosic materials, the microorganisms require long start-up time to initiate their growth. Moreover, the presence of lignin does not favor high yields for biomethane and biohythane production because only few bacterial strains can degrade lignin. Hence, the nature of the lignocellulosic substrate and the compositional and structural features of lignocellulosic biomass such as crop residue, grasses, vegetable waste are major challenges that should be overcome in order to achieve high biomethane and /or biohythane yields.

In most cases, the carbohydrate sources for hydrogen and methane production can be recovered from agricultural wastes, food processing wastes, or grasses. Most agricultural wastes are composed of complex polymers such as cellulose (38% to 50%), hemicellulose (23% to 32%) and lignin (15% to 25%).

Additionally, the presence of polyphenols and essential oils in food waste such as oil, fruit and vegetable remain one of the major challenges because they have shown to limit the biohythane yield.

In the final step of Anaerobic Digestion (AD), strictly anaerobic methanogenic archaea transform hydrogen together with acetic acid and carbon dioxide into methane. In comparison to this conversion, that is restricted to a small group of microorganisms inside the archaea domain, the hydrogenotrophic methanogenesis is conducted by a wide variety of species. In this case, the reduction of carbon dioxide with hydrogen leads to the formation of methane. Growth of acetogens and methanogens is favored by mesophilic conditions and neutral pH-values. Since the anaerobic archaea show the slowest growth among most of the microbes in AD, they are the most fragile towards varying conditions and the presence of inhibitors like ammonia. Thus, in the widespread use of single-stage AD applications, the physical and chemical parameters of the system are adjusted in favor to keep these organisms vital.

It has been suggested that thermophilic temperature should be applied to restrain hydrogenotrophic methanogens for obtaining a higher biogas (biohythane or biomethane). However, it was reported that mesophilic temperature is more efficient for methanogenic activities due to the prevention of ammonia inhibition. This difference in optimal temperature leads to a challenge for biohythane production.

In general, from the kinetic point of view, the degradation velocity, promoted by higher temperature, allows for better gas yields. Valdez-Vazquez et al. changed the operative condition within a CSTR (Continuous-flow Stirred-Tank Reactor) system, fed with organic waste, from mesophilic to thermophilic temperatures finding a higher hydrogen production.

Different anaerobic microorganisms, such as Clostridium spp., Thermoanaerobacterium spp., Enterobacter, and Bacillus, collaborate in a chain of simultaneous biological activities for the carbohydrates-rich substrates' degradation to produce biohydrogen by dark fermentation under favorable conditions of pH, HRT, OLR, and temperature, as previously commented. Instead, biomethane production is assured by microorganisms, such as Methanosarcinabarkeri and Methanococcus, which have a less thick cellular membrane and, consequentially, request more stable temperature, pH conditions, and a less vigorous agitation.

Thermophilic conditions accelerate microorganism's kinetics, allowing for the reduction of the HRT, of the working volume of the reactor and, consequently, the capital and installations costs.

AD at extreme thermophilic temperatures (70-90°C) showed favorable kinetics and stoichiometry of Hydrogen production compared to the mesophilic systems. Fermentation under extreme thermophilic condition was involved with Thermotogas . and Caldicellulosiruptors . The hydrogen production ability of Caldicellulosiruptoi . was explored at extreme temperatures. These microbes are known to have various kinds of hydrolytic enzymes that can utilize a wide range of substrate such as cellulose, cellobiose, and xylan. Caldicellulosiruptors . has high potential to use lignocellulosic waste for Hydrogen production with high yields. The predominant metabolites formed by these organisms are acetic acid and lactic acid.

However, an extremely thermophilic process requires a higher energy input compared to mesophilic process and, therefore, significant increase in the energy recovery from enhanced biogas production is required to compensate reactor heating requirements to make the extremely thermophilic process more economical. Considering this hindrance there is a need to devise process for production of biomethane and biohythane which is not required to be run at extremely thermophilic conditions and yet demonstrates high kinetics of microbial fermentation as well as gives high yields of biogas comprising methane with or without hydrogen.

Further, as opposed to the singular microbial species, it has been felt that use of microbial consortium or mixed cultures provides more enzymes for the utilization of complex substrates than pure cultures. Industrially, the use of mixed cultures for hydrogen production from organic wastes in the first stage could be more advantageous than pure cultures. Thus, using mixed cultures is more practical than using pure cultures due to the easy operating and control under the non-sterile conditions. Hence, there is a need to develop new microbial consortium for the effective production of biogas comprising methane with or without hydrogen.

Object of the Invention:

The primary object of the present invention is to provide a novel and effective method of producing biogas from crop residues such as paddy straw via Anaerobic Digestion process under anaerobic and thermophilic conditions.

Further object of the present invention is to provide a novel and effective method of producing biogas from grasses via Anaerobic Digestion process under anaerobic and thermophilic conditions.

Further object of the present invention is to provide a novel and effective method of producing biogas from vegetable waste via Anaerobic Digestion process under anaerobic and thermophilic conditions.

Further object of the present invention is to provide the novel and effective method for production of biogas comprising methane with or without hydrogen from lignocellulosic substrate using thermophilic microbial consortium.

Another object of the present invention is to provide the novel microbial consortium for effective production of biogas comprising methane with or without hydrogen.

Summary of the Invention:

In order to obviate the drawbacks of the prior art and achieve the aforesaid objectives, the present invention provides a method for production of biogas comprising methane with or without hydrogen.

According to the first aspect, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein said method comprises treating a lignocellulosic substrate of reduced particle size with a microbial consortium in an anaerobic digester at a temperature of 50-60°C; wherein the microbial consortium is developed by anaerobic digestion of water hyacinth and soil from cow dung and manure dumping sites.

According to one embodiment, invention provides a method for production of biogas comprising methane with or without hydrogen wherein the microbial consortium is developed by anaerobic digestion of water hyacinth and soil from cow dung and manure dumping sites at 52±2°C for 30 days.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the lignocellulosic substrate comprises grasses, vegetable waste and crop residues.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the lignocellulosic substrate has a particle size of 0.7 to 5.6 mm.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the lignocellulosic substrate has C/N ratio of 10:1 to 30:1.

In an alternate embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the lignocellulosic substrate comprises 3 to 17% total solids.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the lignocellulosic substrate comprises 5% total solids.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the temperature in anaerobic digester is 52 to 60°C.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the temperature in anaerobic digester is 55 to 60°C.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the method comprises adding minerals to the lignocellulosic substrate in the anaerobic digester wherein the minerals comprise iron, copper and nickel at a concentration of 5 to 10 ppm for iron and copper, 20 to 40 ppm for nickel. In an alternate embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the method comprises adding minerals to the lignocellulosic substrate in the anaerobic digester wherein the minerals comprise iron, copper, nickel at a concentration of 10 ppm for iron and copper, 40 ppm for nickel.

In another embodiment, the present invention provides a method for production of biogas comprising methane with or without hydrogen wherein the method comprises adding the microbial consortium at a concentration of 10 to 30% of the lignocellulosic substrate.

In another embodiment, the present invention relates to a microbial inoculum for production of biogas comprising methane with or without hydrogen, wherein the microbial inoculum is developed by anaerobic digestion of water hyacinth and soil from cow dung and manure dumping sites at 52±2°C for 30 days.

In another embodiment, the present invention provides a method for preparation of a microbial inoculum for production of biogas comprising methane with or without hydrogen, comprising: a. mixing soil samples collected from cow dung and manure dumping sites with water hyacinth, b. adding urea to the mixture of step a to maintain C/N ratio of 20:1, c. incubating the mixture obtained in step bin a bacterial incubator at a temperature of 52±2°C for 30 days for anaerobic digestion, d. collecting the digestate produced at the end of 30 days of anaerobic digestion of step c, e. storing the digestate collected in step d at 4°C.

Further, aspects of the present invention will become clear through the subsequent description of the invention.

Description of the drawings:

The following drawings form part of the present specification and are included to further illustrate aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein. It is to be noted, however, that the appended drawings illustrate only typical embodiments of the present subject matter, and are therefore, not to be considered for limiting of its scope, for the subject matter may admit to other equally effective embodiments.

Fig. 1 illustrates the diagrammatic representation of biogas production in lab-scale plant of Anaerobic Digestion.

Fig. 2 illustrates the daily biogas production from freshwater hyacinth at 50°C.

Fig. 3 illustrates the daily biogas production from dried water hyacinth at 50°C.

Fig. 4 illustrates the daily biogas production from dried water hyacinth at 55°C.

Fig. 5 illustrates the daily biogas production from dried water hyacinth at optimum temperature (52°C).

Fig. 6 illustrates the daily biogas yield from paddy straw for experimental runs selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (PS1-13 as given in Table 3).

Fig. 7 illustrates the cumulative yield of products for biogas production from paddy straw in experimental batches selected by software ((RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (PS1-13 as given in Table 3).

Fig. 8 illustrates the daily biogas yield from paddy straw at different C/N ratio (Seed-10%; Solid loading-5.0%).

Fig. 9 illustrates the cumulative yield of products for biogas production from paddy straw at different C/N ratio (Seed-10%; Solid loading-5.0%).

Fig. 10 illustrates the Daily biogas yield from paddy straw at different particle size (Seed- 10%; Solid loading-5.0%; C/N-25: l).

Fig. 11 illustrates cumulative yield of products for biogas production from paddy straw at different particle size (Seed-10%; Solid loading-5.0%; C/N-25:l).

Fig. 12 illustrates the daily biogas yield from paddy straw in batches conducted at different temperatures (Seed-10%; Solid loading-5.0%; C/N-25: l; particle size: 5.6-0.71 mm).

Fig. 13 illustrates the cumulative yields of products for biogas production from paddy straw in batches conducted at different temperatures (Seed-10%; Solid loading-5.0%; C/N- 25: 1; particle size: 5.6-0.71 mm).

Fig. 14 illustrates the daily biogas yield from paddy straw in batches conducted with addition of minerals (Seed-10%; Solid loading-5.0%; C/N-25: l; particle size: 5.6-0.71 mm).

Fig. 15 illustrates the cumulative yields of products for biogas production from paddy straw in batches conducted with addition of minerals (Seed-10%; Solid loading-5.0%; C/N- 25: 1; particle size: 5.6-0.71 mm).

Fig. 16 illustrates the plot of daily production of biogas production from grass as a comparative study under mesophilic and thermophilic conditions. Fig. 17 illustrates the cumulative yield of products from grass for G-M and G-T (G-M: batch under mesophilic conditions; G-T: batch under thermophilic conditions).

Fig. 18 illustrates the daily biogas yield from grass for experimental runs selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (Gl-13 as given in Table 9).

Fig. 19 illustrates the cumulative yield of products for biogas production from grass in experimental batches selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (Gl-13 as given in Table 9).

Fig. 20 illustrates the daily yields of products for biogas production from grass in batches conducted at different temperatures (G-35 at 35°C; G-45 at 45°C; G-50 at 50°C; G- 52 at 52°C; G-55 at 55°C; G-60 at 60°C).

Fig. 21 illustrates the cumulative yields of products for biogas production from grass in batches conducted at different temperatures (G-35 at 35°C; G-45 at 45°C; G-50 at 50°C; G-52 at 52°C; G-55 at 55°C; G-60 at 60°C).

Fig. 22 illustrates the products yield vs. time for biogas in 7.5 L bioreactor.

Fig. 23 illustrates the plot of daily production of biogas production from vegetable waste as a comparative study under mesophilic and thermophilic conditions.

Fig. 24 illustrates the cumulative yield of products from Vegetable Waste for VW-M and VW- T (VW-M: batch under mesophilic conditions; VW-T: batch under thermophilic conditions).

Fig. 25 illustrates the daily yield of biohythane (biohydrogen + biomethane) from vegetable waste for experimental runs selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (VW1-13 as given in Table 13).

Fig. 26 illustrates the daily yield of biohydrogen for experimental runs selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (VW1-13 as given in Table 13).

Fig. 27 illustrates the daily yield of biogas from vegetable waste for experimental runs selected by software (RSM, Design Expert, STATE-Ease version 8.0, U.S.A) (VW1-13 as given in Table 13).

Fig. 28 illustrates the cumulative yield of products for biohythane production from vegetable waste in experimental batches selected by software ((RSM, Design Expert, STATE- Ease version 8.0, U.S.A) (VW1-13 as given in Table 13).

Fig. 29 illustrates the cumulative yields of products for biohythane production from vegetable waste at different temperatures (VW-35 at 35°C; VW-45 at 45°C; VW-50 at 50°C; VW-52 at 52°C; VW-55 at 55°C; VW-60 at 60°C).

Fig. 30 illustrates the products yield vs. time for biohythane from vegetable waste in 7.5 L bioreactor. These and other features of the invention will become apparent from a consideration of the detailed description.

Detailed description of invention:

The following description is provided to assist in a comprehensive understanding of exemplary embodiment of the invention. It includes various specific details to assist the understanding, but these are to be regarded as merely exemplary.

Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, description of well-known functions / constructions is omitted for clarity and consciousness.

Those skilled in the art will be aware that the invention described herein is subject to variations and modifications, other than those specifically described. It is to be understood that the invention described herein includes all such variations and modifications. The invention also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The present invention is not to be limited in scope by the specific embodiments described herein, which is intended for the purposes of exemplification only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention, as described herein.

For convenience, before further description of the present invention, certain terms employed in this specification, examples and appended claims are collected here. These definitions should be read considering the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way or more other embodiment and/or in combination with or instead of the features of other embodiments.

The articles "a", "an" and "the" are used to refer to one or 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 element or steps but not the exclusion of any other element or step or group of element or steps.

The term "including" is used to mean "included but not limited to". "Including" and "including but not limited to" are used interchangeably.

The term "under anaerobic conditions", used in the specification means that reactions are conducted in absence or near absence i.e. trace amounts of oxygen for example, biomass is loaded into the plant or vessel containing microorganisms-rich seed (also referred as consortium or inoculum or culture or seed) and digested anaerobically.

By using the term "AD or anaerobic digestion", it is meant that breaking down the complex molecules of a substrate or feedstock (e.g. biomass or organic waste) by the action of a range of microbial community to generate H2, CFhand CO2 along with other products in traces under anaerobic conditions. In an embodiment, AD is accomplished through four phases; hydrolysis of complex organic molecules (carbohydrates, fats or proteins) of substrate into simpler molecules (e.g. sugars, fatty acids or amino acids) carried by hydrolytic bacteria; Flproducing phase comprising breakdown of simpler molecules to organic acids (e.g. butyric acid, propionic acid), H2, ammonia and CO2 carried out by acidogens; acetogenesis, which involves further breakdown of organic acids into acetic acid, more H2, ammonia, and CO2 carried out by acetogens and finally during methanogenesis, acetoclastic methanogens utilize the acetic acid and CO2 or hydrogenotroph methanogens utilize H2 and CO2 to generate CH4 as main product along with H2, CO2 and hydrogen sulphide (H2S) in traces.

By "Biogas", it is meant a mixture of gaseous fuel with "biomethane" as major component followed by CC and other components in traces produced in a biological process particularly from biomass or organic waste (Hans and Kumar, 2019). During CH4 generation, the methane content in vessel may vary between 55-80% for example, maximal CH4 content in vegetable waste is about 70% (Dong et al., 2015). Other components may include CO2 (15-40%), H2S and air (in traces). By "biohythane", it is meant the two fuels, "biohydrogen and biomethane" produced in a simultaneous manner in one biological process particularly from biomass or organic waste. In an embodiment, biohythane is produced by two-stage AD of biomass by integration of reactors. In an alternative embodiment, biohythane can be produced in one-stage AD, wherein H2 is produced selectively by keeping moisture concentrations at low level; collecting the gas; producing CH by increasing moisture concentration at high level and collection of gas (US20110033908A1). Two-stage AD involves the inoculum pretreatment by heat-shock (e.g., dry or wet heat) or chemical agents (e.g., acids, antibiotics or methanogen inhibitors) or load-shock (providing high loadings of nutrients to inoculum at slightly acidic pH) to deactivate the H2 consuming methanogens (hydrogenotrophs). During biohythane production, the biohydrogen is produced in H2 vessel or by selective H2 production in single vessel, which contain about 10-50% H2 content and 45-85% CO2 (H2:CO2 may depend upon the reaction time).

The term herein used as 'consortium', it is meant a seed to initiate the process that consists of an anaerobic mixed microbial community of hydrolytic, acidogens, acetogens, hydrogenogens, methanogens, sulfate-reducing, and iron-reducing bacteria or other species, which grow together in a syntrophic and symbiotic relationship. In some embodiments, anaerobic consortium or seed or inoculum may be acquired as soil sample from a dump yard (maintained under anaerobic conditions in nature), and developed anaerobically using some biomass (e.g. water hyacinth, grass or organic waste) or sludge from bottom of an anaerobic digester or spent slurry or effluent from wastewater-treatment plants, industrial sites, municipal waste-treatment facilities, anaerobic lagoons or biogas plants. In some embodiments, anaerobic sludge from multiple anaerobic digesters or sites are collected and merged to develop a mixed microbial community.

The term "crop residue" or "agro-residue" refers to any plant matter left after harvesting, cutting and utilization of useful parts of crops for food or other products, which may be anaerobically digested to produce biogas. Crop residues may be burnt open or dumped into soil or served as cattle feed, which are inappropriate management of largely generated agrowastes world-wide in environment concerns. Crop residues may include, but not limited to residual plant or part of important largely generated crops for example, wheat straw, paddy straw, rice husk, maize husk and corn cob may also be covered in this context. In certain embodiments, a carbohydrate-rich substrate or pentose or hexose products or volatile fatty acids or sugar acids or organic solvent may be used for biogas production. In an alternative embodiment, crop residues are rich in biodegradable matter, which lead to easy and rapid degradation, thus potential substrate for biogas production. Moreover, utilizing waste plant matter is sustainable in terms of economy due to lower costs, and for commercial uses.

The term "grasses" refers to a plant matter or biodegradable waste or energy crops, which may be anaerobically digested to produce biogas. Grasses may include, but not limited to whole plant or silage or one or mixed species of grasses for example, Bermuda grass, Elephant grass, Napier grass and Kans grass may also be covered in this context. In certain embodiments, a carbohydrate-rich substrate or pentose or hexose products or volatile fatty acids or sugar acids or organic solvent may be used for biogas production. In an alternative embodiment, grasses are rich in biodegradable matter, which lead to easy and rapid degradation, thus potential substrate for biogas production. Moreover, utilizing waste plant matter is sustainable in terms of economy due to lower costs, and for commercial uses.

The term "VW or vegetable waste" refers to biodegradable waste or discarded vegetables, which may be anaerobically digested to produce biohythane. VW may include, but not limited to rotten vegetables or residues leftover after using edible parts of vegetables for example, pea peels, cauliflower stems, rotten brinjal, rotten tomatoes, etc. In an embodiment, "KW or kitchen waste" and "FW or fruit waste" may also be covered in this context. In certain embodiments, a carbohydrate-rich substrate or pentose or hexose products or volatile fatty acids or sugar acids or organic solvent may be used for biohythane production. In an alternative embodiment, vegetables are rich in organic acids, which lead to easy and rapid H2 production, thus potential substrate for biohythane production. Moreover, utilizing wastes (e.g., KW, FW or VW) is sustainable in terms of economy due to lower costs, and for commercial uses.

Term herein, "total solids or TS" or "solid loading" refers to the total content of dry biomass, other than moisture content present in it. VW may contain large amount of water as 70-80% or 80-90% or 90-92% or 93-99%.

In an embodiment, biogas yield may not be affected by amount of substrate loaded but by percentage of TS digested during the process (Dhanalakshmi and Ramanujam, 2012). Therefore, biogas yield may be determined based on consumption of TS or dry biomass. Rate of formation of products is highly dependent on loading rate of substrate for example, CH yield may be increased with reduction in loading rate because high substrate loading may lead to VFA accumulation resulting into halt in process (Vartak et al, 1997; Li et al., 2011). In an embodiment, solid loading, or TS play an important role during digestion process, affect the stability and biogas yield. For example, <6% or about 5% TS or solid loading or dry biomass loading favors high volatile solids (VS) degradation, and process stability (Bouallagui et al, 2003; Alkanok et al, 2014). In an alternative example, biogas production from agricultural wastes is maximal at optimum value of TS as 9% (Yavini et al, 2014).

In an alternative embodiment, in particular, biogas yield is determined on the basis of consumption of VS present in biomass or in total solids of biomass.

The term "VS or volatile solids" refers to the total biodegradable organic matter or biodegradable fraction and refractory fraction contained in the TS content in biomass. In an embodiment, high biodegradable fraction with low refractory fraction may be more appropriate for anaerobic digestion.

By using term "seed loading", it is meant, the percentage of seed or inoculum or microbial culture or consortium added to the reaction mixture. Start and progress of a microbial process is affected by the amount of starting amount of microorganisms introduced in the vessel containing substrate (e.g. biomass), which may adapt themselves according to the new surroundings, consume the organic matter of substrate, grow in size, double in amount or multiply themselves and produce the byproducts (e.g. H2, CH4, ethanol, organic acids). A perfect balance of percentage of seed or inoculum and TS may lead to process stability with significant yields and rates. Further, performance of AD dependent on temperature conditions of reaction mixture contained in the vessel/reactor.

In some embodiments, AD could be performed well under psychrophilic, mesophilic and thermophilic conditions, which are basically working conditions of responsible microbial community. For example, optimum temperatures for psychrophiles, mesophiles, thermotolerants, and thermophiles (microbial communities) are <20, 35, 45 and 55 °C, respectively (Bouallagui et al, 2004). Although performing AD under mesophilic conditions is feasible from economical point of view (already maintained as ambient conditions), however in some embodiments, thermophilic conditions result into higher metabolic rates of microorganisms, improved kinetics, energy production, avoid the risk of contamination in reaction mixture and short hydraulic retention time (HRT).

In an embodiment, under thermophilic conditions, cellulose hydrolysis rate increases up to 5- 6 times higher than mesophilic conditions. For example, extreme thermophiles (e.g. Thermotoga maritima, Caldicellulosiruptor saccharo/yticus, or Caldicellulosiruptor bescii possess high rate of hydrolysis, and release thermostable (stable at high temperatures) cellulases and xylanases to hydrolyze the complex structure of cellulose and xylan, respectively in plant cell wall (biomass herein), and responsible for co-digestion of both pentose (e.g. xylose, arabinose) as well as hexose (e.g. glucose, galactose) sugars (de Vrije et al, 2009; Frock and Kelly, 2012). Further, C. bescii is capable of biomass degradation without pretreatment (Basen et al, 2014).

In an alternative embodiment, thermophilic inoculum also does not require the need of feedstock pretreatment, which is essential for mesophillic inoculum, thus avoid the extra time and cost of inoculum pretreatment. Therefore, thermophilic conditions could be adapted for biogas production in terms of high biogas production rate in spite of energy consumption.

"HRT or Hydraulic retention time" refers to the time required for complete degradation of the organic matter of substrate or biomass. HRT of an AD depends upon varies process factors such as temperature, composition of substrate and microbial activity in seed. In an embodiment, HRT of AD under mesophilic and thermophilic processes vary between 15-60 or more and 12-14 days, respectively.

"C/N ratio" refers to the relative amounts of carbon (C) and nitrogen (N) present in organic material in reaction mixture in AD. In an embodiment, required C/N ratio in AD digester should be 20-30. During AD, microorganisms utilize C as 25-30 times faster than N, thus they need a 20-30:1 ratio of C to N. Therefore, it is mandatory to maintain C/N ratio of substrate (by adding chemicals or co-digestion of two or more substrates) before loading into digester.

The present invention provides a method of producing biogas from crop residues, grasses via well-known AD process under anaerobic and thermophilic conditions. A method of combined production of biohydrogen and methane (in form of biogas), called as biohythane from VW in one process called as one-stage AD under anaerobic conditions is also discloses in the present invention. Fig. 1 represents the diagrammatic representation of lab-scale set-up of AD for biogas production. The method consists of loading feedstock (paddy straw, grass, VW herein) into a lab-scale AD plant or vessel or bioreactor maintained under anaerobic conditions; keeping C/N as 20-30 by adding urea; introducing seed into the vessel to facilitate digestion of the feedstock; collecting and analyzing biogas after the starting of production.

Efficiency of biogas production may be enhanced by optimizing the various controlling process factors such as seed loading, solid loading, temperature, etc. The invention provides the optimization of seeding, solid loading and temperature for enhanced product yield. The invention extends the small-scale batch study to large bioreactor-batch for better understanding of the process.

The present invention is further illustrated by following examples:

Example I

Isolation and development of the thermophilic consortium

Different soil samples were collected from cow dung and manure dumping sites, and stored at 4°C. The collected soil samples were mixed in similar proportions, and added into the slurry of dried water hyacinth. After that, the slurry was used to prepare and develop the consortium via AD of water hyacinth. To conduct the above experiment, freshwater hyacinth biomass was collected from Kanjli Wetland, Kapurthala (Punjab). One fraction of the collected biomass was air dried openly under sunlight, ground to fine particles using a grinding mill, while another fraction was kept fresh without drying. About 200 g of freshwater hyacinth (10% total solids) (F-WH-50) and 20 g (10%) of dried water hyacinth (D-WH-50) were loaded into separate 1-L biogas set-ups, and 0.76 g of urea was added to maintain C/N ratio of 20: 1. The set-ups were incubated at 50°C in a bacteriological incubator for 30 days. One set-up of dried water hyacinth (D-WH-55) was also installed and kept at 55°C under similar conditions. After the complete digestion of water hyacinth, the biogas formation reduced. The digestate was stored at 4°Cand used as a seed (inoculum) for further experiments. Lab-scale biogas set-up consisted of 3 screw capped reagent bottles assembled as shown in Fig. 1, consisting of digester, gas collecting and water collecting chamber. The first bottle i.e., digester was loaded with the prepared feedstock and seed. The second bottle (gas collecting chamber) was filled with water and third bottle (water collecting chamber) was kept empty. All bottles were closed with rubber corks, sealed with wax to maintain the anaerobic conditions, and to prevent gas leakage. The gas generated in the digester displaced the water placed in the second bottle (gas collecting chamber) and occupied the space. The displaced water was collected in the third bottle. Sampling port 1 was used for sampling the digestate for pH and VFA analysis, and sampling port 2 was used for sampling the generated biogas for analysis every day. This thermophilic consortium constitutes-hydrolytic, acidogenic, acetogenic and methanogenic bacteria to carry out the anaerobic digestion process efficiently. The list of bacteria and archea present in the consortium and their population is summarized in Table 1. From day one, the trend of biogas production with time was in an increasing order. The production of biogas was fast at the beginning and decreased at the end. Table 2 summarizes the results of biogas production from different batches. Figs. 2-4 display the daily trend of biogas production from all the batches. Total biogas yield of 288.2, 361.18 and 245.96 l/kg-TS was obtained from F- WH-50, D-WH-50 and D-WH-55, respectively in HRT of 42, 53 and 30 days, respectively. Thus, dried water hyacinth was concluded to provide better results than fresh and wet one that too at 50°C than 55°C. Further, biogas production from dried water hyacinth was extended to analyze for improved yield at optimum temperature in the range of 50-55°C. Thus, an average of the range (50-55°C) was chosen as 52°C, and a plant was set-up (D- WH-52) with dried water hyacinth under similar conditions. Fig. 5 represents the daily production of biogas along with its components. Biogas production from batch kept at 52°C shown better production than that of 50°C with a total biogas and methane yield of 408.1 and 244.86 l/kg-TS, respectively in 33 days (Table 2). The average contents of methane and carbon-dioxide was observed as 63.32 and 35.67%, respectively. The change in methane percentage was observed during the first weeks from 30% to 72% and then further its content was moreover stable as 60-65%. Hence, it was observed that the higher biogas yield could be obtained in a short digestion period or retention time using the developed thermophilic consortium. Table 1: List of bacteria/ archaea present in the developed thermophilic consortium

Table 2: Biogas production from water hyacinth using slurry with collected soil samples (Slurry- 10%; solid loading- 10%; C/N -20:1; temperature- 50-55°C). Example II

Optimization of operational parameters for enhanced production of biogas from crop residues (paddy straw)

The effect of seed (5-35%) and solid loading or dry biomass loading (3.0-17%) on biogas production from crop residues (paddy straw) by AD under thermophilic conditions was evaluated using RSM of design expert software version 8.0 (STAT-EASE Inc., Minneapolis, U.S.A) keeping temperature constant as 52°C. Figs. 6 and 7 display the daily and cumulative biogas yield in the experiential batches (PS1-13). Although figures display the daily production of all batches however, biogas with volume > 0.15 L were considered for the calculations hence HRT was also calculated by taking days of production under consideration only. All batches resulted into biogas yield of 95-350 l/kg-TS or 113.63-418.66 l/kg-VS (Fig. 7). The highest cumulative biogas yield was found as 3501/kg-TS in batch with 20% seed and 2.93% solid loading (PS8) in HRT of 13days. However, we chosen batch PS 3 as optimum due to lower value of seed loading i.e. 10% as compared to the 20% and 30% loading in case of batch PS8 and PS4, respectively but with comparable total biogas yield i.e. 309.79 l/kg-TS or 370.56 l/kg-VS in HRT of 12 days. Cumulative CH4 content in biogas was found as 170.4 l/kg- TS or 203.82 l/kg-VS corresponding to 58.23% of theoretical yield (350 l/kg-VS). Average CH and CO2 contents in biogas were found as 55.00 and 35.99%, respectively. T.S and V.S conversion were found as 69.8% and 71.59%, respectively. The optimized conditions for biogas production selected by software were found to be 10 and 5.0% seed and solid loading. In model validity experiment, cumulative biogas yield was found as 320 l/kg-TS or 382.77 l/kg-VS in 12 days. Table 3 summarizes the product profile of 13 experimental (PS1-13) runs along with optimized batch. Average CH4 (maximum 75.15%) and CO2 contents were found as 55.00 and 35.00%, respectively. T.S and V.S conversion were found as 70.00% and 72.00%, respectively.

Table 3: Cumulative biogas yields along with methane and carbon dioxide content in experimental batches selected by software (RSM, Design Expert, STAT-EASE version 8.0, U.S.A). Example III

Optimization of C/N ratio for enhanced production of biogas from crop residues (paddy straw)

Optimized solid (5.0%) and seed (10%) loading were further used to conduct experiments for C/N ratio optimization (10:1-30: 1) for enhanced biogas production at 52°C. Table 4 summarizes the results of this study. Fig. 8 represents the daily yield of biogas and its components obtained from all batches. It was observed that biogas production increased with rise in C/N ratio upto 25: 1 with cumulative biogas yield from 235.75 to 330 l/kg-TS or 281.99 to 394.73 l/kg-VS then it started to decrease up to 319 l/kg-TS at C/N - 30:1 in HRT of 13 days. Fig. 9 shows the plot of cumulative biogas yields for all the batches. The highest biogas yield of 330 l/kg-TS or 394.73 l/kg-VS was observed in the batch with C/N: 25: 1 with an HRT of 13 days. Cumulative CH4 yield was found to bel82.64 l/kg-TS or 218.47 l/kg-VS. Average CH4 (maximum 69%) and CO2 contents were 55.34 and 33.68%, respectively. T.S and V.S conversions were 67% and 70%, respectively.

Table 4: Results obtained from optimization of C/N ratio for crop residues

Example IV

Optimization of particle size for enhanced production of biogas from crop residues (paddy straw)

Optimized solid loading (5.0%), seed loading (10%) and C/N ratio (25: 1) were further used to conduct experiments for optimization of particle size (<0.15->5.6 mm) of crop residues (paddy straw herein) for enhanced biogas production. Figs. 10 and 11 represent the daily and cumulative yield of biogas and its components obtained from all batches. Table 5 summarizes the results obtained from all the batches. It was observed that biogas production increased with rise in particle sizes from >5.6 to 0.7 mm with cumulative biogas yield of 283.5-403 l/kg-TS or 339.11-482.06 l/kg-VS but after which it was observed to be decline up to 203.5 l/kg-TS in <0.15 mm (Table 5; Fig. 11). Cumulative CH4 yield was found to be 238.509 l/kg-TS or 285.29 l/kg-VS. Average CH4 (maximum 69%) and CO2 contents were 59.18 and 34.57%, respectively. T.S and V.S conversions were 68% and 70.54%, respectively. Though, the maximum biogas yield was obtained for particle size 0.7 mm however, for economical process, we have taken a range of particle sizes from 5.6 mm to 0.71 mm as the optimized range.

Table 5: Cumulative biogas, methane and carbon dioxide obtained in C/N batches

Example V

Optimization of temperature for enhanced production biogas from crop residues (paddy straw)

Optimized solid loading (5.0%), seed loading (10%), C/N ratio (25:1) and particle size (5.6-0.71 mm) were further used to conduct experiments for temperature optimization (35-60°C) for enhanced biogas production keeping batch at 52°C as control. Fig. 12 represents the daily yield of biogas and its components obtained from all batches (PS-35 ^o C-PS-60°C). It was observed that biogas production increased with rise in temperature from 35 to 52°C but it was declined after 55°C which was reduced to 353.42 l/kg-TS (Table 6). There was drastic decline in biogas production at 60°C (PS-60°C) with a yield of 136.49 l/kg-TS with only 4.56% average CH4 content. Out of the trend, hydrogen was also observed at 60°C with average H2 content of 4.82%. The reason for this trend might be that methanogens (acetoclastic herein) get deactivated at temperature as higher as 60°C, while hydrogen formers started to activate at this temperature. Fig. 13 shows the plot of cumulative biogas yields for all the batches. The highest biogas yields were observed in the batch PS-55 at 55°C as 353.42 l/kg-TS or 422.751/kg-VS but its methane yield 184.5 l/kg-TS (average CH4, 52.35%) only compared to batch, PS-52at 52°C with 189 l/kg- TS (average CH4, 57.27%) with an HRT of 12 days. Therefore, batch PS-52 at 52°C temperature considered highest biogas yield as 3301/kg-TS or 394.731/kg-VS among other batches due to its highest methane yield. Average CH4 (maximum 69%) and CO2 contents were 57. J and 31.85%, respectively. TS and VS conversions were 66.8% and 70%, respectively. Considering present investigation as batch process, and as crop residues as sole substrate without co-digestion, observed results were higher than previous studies based on grass in terms of product yield, average contents (%) and TS/VS conversions. Zhang et al. (2020) investigated AD of rice straw with nitrogen additions of swine manure and/or urea at different ratios (0:0, 1:0, 4:1, 7:3, 6:4, 5:6, 4:7, 0:1) through batch experiments, and reported the cumulative biogas yield of 190 l/kg- VS for optimal ratio of 7:3. In another study, the effect of (0.8, 0.5 and 0.3, VS basis) on biogas yield and biodegradation of rice straw (chopped up to 5-15 mm size) was conducted in a batch study using inoculum obtained from microbial sample of cattle rumen, at room temperature (25- 27°C). The Maximum biogas yield of 410 l/kg-VS was obtained with optimal inoculum/substrate ratio of 0.8 of I/S ratio with CH4 content over 70% in HRT of 60 days (Candia-garcia et al., 2018). Kainthola et al. (2019) studied CH4 potential of co-digested rice straw (grinded to < 1 cm size) and food waste for different carbon to nitrogen ratios followed by interactive effect of initial pH and food/microorganism ratio using central composite design- response surface methodology with 3% TS loading using digested cow dung as inoculum (16% w/v). Significant interaction and validation experiment were obtained for the optimum C/N ratio of 30:1, pH 7.32 and food/microorganism ratio of 1.87 with a CH4 yield of 323.78 l/kg-VS in HRT of 50 days. In another study, the effect of trace elements (low, medium, and high concentration) addition to the AD of rice straw (pulverized up to 1-mm size) was investigated with 6% TS loading using inoculum (from a biogas plant based on cow manure) for 20 days.

Table 6: Cumulative product yield in biohythane batches at different temperatures.

*TS: Total solids

**HRT: Hydraulic retention time Example VI

Enhancement of biogas production from crop residues (paddy straw) by minerals addition

The study of biogas production from crop residues (paddy straw) under optimized conditions (solid loading: 5.0%; seed loading: 10%; C/N ratio: 25:1; particle size: 5.6-0.71 mm and temperature: 52 °C) was further extended to determine the effect of minerals addition on biogas/ methane yield HRT or both. The five minerals; Fe, Cu, Ni, Co and Zn were selected for the study with variable concentrations in a range of 10-50 ppm in IL lab-scale AD digester. Out of 5 selected minerals, batches with only 3 minerals that are Fe, Cu and Ni gave positive results. It was observed that iron showed 15% increased biogas as compared to the control i.e. 50 l/kg-TS of biogas produced with 10 ppm supplementation of Fe. As the concentration of Fe ⁺² increases, the biogas production started decreasing, so it was found that Fe ⁺² has stimulatory effect on biogas production but at low concentrations. Similar results found in case of Cu ⁺² ions. An increase of 14.6% in biogas was observed with 10 ppm supplementation of Cu ⁺² ions. It also showed positive effect on biogas production but at low concentrations only. Ni ⁺² showed different pattern as compared to Fe ⁺² and Cu ⁺² . At low concentration of Ni, less biogas production was observed, but at high concentration (40ppm) it showed 24% increased biogas production (485 l/kg-TS) as compared to control. Co ⁺² and Zn ⁺² showed negative effect on biogas production. At 10 ppm, Co and Zn showed almost similar result as that of control. On increasing their concentrations, biogas production started decreasing. It means Co and Zn had negative effect on the consortium and thus biogas production. Further different concentrations of Fe, Cu and Ni were optimized using RSM for enhancing biogas production with design expert software version 8.0 with 0-5 ppm concentration of Fe and Cu, and 0-40 ppm of Ni. Fig. 14 displays the daily production of biogas in different batches (PS- 1-14). Table 7 represents the coded values of concentrations of minerals selected for the study and observed values of the biogas yield as well as CH CO2 contents obtained in the study. Fig. 15 shows the cumulative yields of all the batches (PS- 1-14) selected in RSM. It was observed that the maximum biogas yield of 522.25 l/kg-TS was obtained in the batch PS-4 with Fe-10; Cu-10; Ni-0 in HRT of 15 days along with CH4 yield of 321.99 l/kg-TS with average contents of CH4 and CC as 61.65 and 30.31%, respectively with TS of 58.85%. Based on the observations, software selected the optimized combination of only 2 minerals; Fe-10 ppm and Cu-10 ppm. Table 7: Optimization of minerals (Fe, Cu, Ni) by RSM (solid loading: 5.0%); seed loading: 10%; C/N ratio: 25:1; particle size: 5.6-0.71 mm and temperature: 52°C)

*TS: Total solids **HRT: Hydraulic retention time

Example VII

Comparative study of biogas production from grass under mesophillic and thermophilic conditions

Study was carried out for the biogas production from grass under mesophilic and thermophilic conditions. Waste grass including a variety of species from institute having total solid content as 35-47% (w/v) was prepared by chopping into small pieces, and utilized for biogas production without drying (53-65% moisture) to avoid extra energy and time consumption in lab-scale three- bottle AD setups (Fig. 1). Solid content was reduced to 10% (w/v) by addition of water. Feedstock along with seed and other constituents (e.g. water, acid-base for pH, urea for maintaining C/N) is loaded into the first digester bottle maintained under anaerobic conditions at appropriate temperature; gas produced from digestion of feedstock in digester bottle enters into the second gas collecting bottle, and displaces same amount of water present in it into the third bottle. From the third bottle, displaced water is collected for measuring the volume of the biogas produced, and analyzing different gas components (CH4, CO2, H2, N2, etc.)

For mesophilic AD process, spent slurry from pilot-scale cow dung-based biogas plant operated at SSS-NIBE, Kapurthala (under ambient conditions) was used as seed, and set-up was kept at 35- 37°C. On the other hand, for thermophilic AD process, thermotolerant consortium isolated and developed at SSS-NIBE, Kapurthala was used as seed, and set-up was kept at 52°C. Fig. 16 shows the daily production of biogas from grass along with its components. Cumulative yields of products for both the conditions are represented in Fig. 17. Cumulative biogas yield was observed as 110.05 l/kg-TS with 57.27% as average CH4 content (maximum 60.98%) in HRT of 30 days under mesophilic conditions (G-M), whereas 379 l/kg-TS of biogas yield was found with an average CH4 content of 54.26% (maximum 65.78%) at an HRT of 25 days under thermophilic conditions (G- T). TS% conversion was found as 47.1% and 55.75% for mesophilic and thermophilic batches. Hence, thermophilic consortium was concluded to have potential for biogas production from grass in terms of high biogas yield, high biomass conversion and reduced HRT. Table 8 summarizes the results of this comparative study.

Table 8: Cumulative product yield in biohythane batches under mesophilic and thermophilic conditions. *TS: Total solids

**HRT: Hydraulic retention time

Example VIII

Optimization of operational parameters for enhanced production of biogas from grass The effect of seed (10-30%) and solid loading or dry biomass loading (3.0-12%) on biogas production from grass by AD under thermophilic conditions was evaluated using RSM of design expert software version 8.0 (STAT-EASE Inc., Minneapolis, U.S.A) keeping temperature constant as 52 °C.Figs.l8 and 19 display daily and cumulative biogas yield in the experiential batches (Gl- 13). Although figures display the daily production of all batches however, biogas with volume > 0.2 L with CH ₄ content > 50%, were considered for the calculations hence HRT was also calculated by taking days of production under consideration only. All batches resulted into biogas yields as 235.4-575 l/kg-TS or 262.72-641.74 l/kg-VS, respectively (Fig. 18). The highest cumulative biogas yield was found as 575 l/kg-TS or 641.74 l/kg-VS in batch with 30% seed and 3.0% solid loading (G7) in 25 days, wherein there was no biogas production observed for first 8 days, thus the production was observed to be stared on 8 ^th day concluding for total 17 days for biogas production (Fig. 19). Cumulative CH4 content in biogas was found as 362.86 l/kg-TS or 402.53 l/kg-VS corresponding to 81.09% of theoretical yield (496.34 l/kg-VS). Average CH4 (maximum 75.8%) and CO2 contents in biogas were found as 60.27 and 36.89%, respectively. T.S and V.S conversion were found as 70.75% and 70.76%, respectively. The optimized conditions for biogas production selected by software were found to be 30 and 3.0% seed and solid loading. In model validity experiment, cumulative biogas yield was found as 597.5 l/kg-TS or 666.85 l/kg-VS, respectively in 18 days. Table 9 summarizes the product profile of 13 experimental (Gl-13) runs along with optimized batch. Average CH4 (maximum 66.47%) and CO2 contents were found as 60.05 and 36.48%, respectively. T.S and V.S conversion were found as 71.25% and 71.26%, respectively.

Table 9: Cumulative biogas yields along with methane and carbon dioxide content in experimental batches selected by software (RSM, Design Expert, STAT-EASE version 8.0, U.S.A).

Optimization of temperature for enhanced production biogas from grass

Optimized solid loading was further used to conduct experiments for temperature optimization (35-60°C) for enhanced biogas production keeping batch at 52°C as control. However, 20% seed loading was used for this study instead of the optimized 30% loading for economical feasibility of the process. Table 10 summarizes the results of this study. Fig. 20 represents the daily yield of biogas and its components obtained from all batches (G-35-G-60). It was observed that biogas production increased with rise in temperature from 35 to 52°C but it was declined after 55°C which was reduced to 328 l/kg-TS. There was drastic decline in biogas production at 60°C (G-60) with a yield of 102.5 l/kg-TS and 54.82% average CH4 content. Out of the trend, hydrogen was also observed at 60°C with a yield of 39 l/kg-TS with average H2 (maximum 20%) content of 20% for first 6 days. The reason for this trend might be that methanogens (acetoclastic herein) get deactivated at temperature as higher as 60°C, while hydrogen formers started to activate at this temperature. Fig. 21 shows the plot of cumulative biogas yields for all the batches. The highest biogas yields were observed in the batch G-52 only (at 52°C) as 458.4 l/kg-TS or 511.72 l/kg-VS with an HRT of 14 days. Average CH4 (maximum 72.49%) and CO2 contents were 65.30 and 31.85%, respectively. T.S and V.S conversions were 72.00% and 71.98%, respectively. Considering present investigation as batch process, and grass as a sole substrate without codigestion, observed results were higher than previous studies based on grass in terms of product yield, average contents (%) and TS/VS conversions. In a study, the effect of thermo-chemical pretreatment of grass on AD was studied by pre-treating dried grass silage with different NaOH loading rates (1-7.5% by VS) and temperatures (20-150°C) followed by AD using seed loading of

33.3 g-VS/l with substrate to inoculum ratio as 1:1 at 35°C. The highest CH4 yield of452.5 l/kg-VS in HRT of 40 days was obtained for grass silage pretreated with 7.5% NaOH at 100°C improved by 38.9% in comparison with untreated silage (Xie et al., 2011). Wang et al. (2014) investigated biogas production from shredded grass in co-digestion with sewage sludge (VS ratio 0.5; TS ratio 10: 1) followed by hyperthermophilic pretreatment at 80°C in CSTRs under thermophilic conditions using seed sludge as inoculum resulting into a CH4 yield of 190 l/kg-VS. In another study of biogas production from residue grass obtained from uncultivated land, riverbank and highway verge using inoculum from mesophilic poultry manure-treating biogas plant at 39°C, average biochemical biogas potential and maximum CH4 contents of 436 l/kg-TS, 348 l/kg-TS and 413 l/kg-TS, 77.5,

75.4 and 75.3%, respectively were obtained in HRT of 40 days (Bedoi et al., 2019). Haryanto et al. (2018) studied co-digestion of Elephant grass and cow dung (1: 1) for 70 days using inoculum from mesophilic pig dung based-anaerobic digester at 36°C and reported biogas yield of 111.72 l/kg-VS with 31.37% CH4 content in HRT of 15 days.

Table 10: Cumulative product yield in biogas batches at different temperatures.

**HRT: Hydraulic retention time

Example X

Biogas production from grass in 7.5 L bioreactor under optimized conditions

Study of biogas production from grass under optimized conditions was extended from three-bottle set-up to 7.5 L bioreactor, however seed was taken as 20% for economical purpose (as optimized was 30%) as in previous study with 100 g of dry biomass loading (3.0%) at 52°C in working volume of 4.33 L. However, this batch study did not show significant results as observed in three- bottle set-up studies. Fig. 22 shows the daily yield of products for batch conducted in 7.5 L bioreactor. Table 11 summarizes the results of study. Cumulative biogas yield was observed as 405 l/kg-TS or 452.01 l/kg-VS in HRT 14 days. Average CH4 (maximum 65.4%) and CO2 contents were observed as 56.56 and 42.01%, respectively. T.S and V.S conversions were 55.22 and 55.23%, respectively. It could be due to lack of proper design pH of the bioreactor design. Hence, this type of study needed a specific design for bioreactor with proper balance of pH control and agitation during initial days of process.

Table 11: Cumulative product yield in biogas batch conducted in 7.5L bioreactor

*TS: Total solids

**HRT: Hydraulic retention time

Example XI

Comparative study of biogas/biohythane production from vegetable waste (VW) under mesophilic and thermophilic conditions

Study was carried out for the biogas/biohythane production from vegetable waste (VW) under mesophilic and thermophilic conditions. Rotten vegetables such as pea peels, cucumber, tomato, brinjal, cauliflower discarding (e.g. stem), etc. having total solid content as 3-15% was prepared by chopping into small pieces, and utilized for biogas/biohythane production in wet conditions (85- 97% moisture) without drying to avoid extra energy and time consumption in lab-scale three- bottle AD setups, developed at SSS-NIBE, Kapurthala as mesophilic and thermophilic batches (Fig. 1). Process of plant set-up and AD were similar as given in previous examples.

For mesophilic AD process, spent slurry from pilot-scale cow dung-based biogas plant operated at SSS-NIBE, Kapurthala (under ambient conditions) was used as seed, and set-up was kept at 35- 37°C. On the other hand, for thermophilic AD process, thermotolerant consortium isolated and developed at SSS-NIBE, Kapurthala was used as seed, and set-up was kept at 52°C. Mespohilic AD batch had not shown any H2 production in whole duration, whereas thermophilic AD batch shown the production of H2 during initial 2-3 days followed by CH4 as the main component of biogas as shown in Fig. 23. Cumulative yields of products for the both the conditions are represented in Fig. 24. Cumulative biogas yield was observed as 45 l/kg-TS with 51.07% as average CH4 content (maximum 65.6%) in HRT of 20 days under mesophilic conditions, whereas H2 and biogas yields were found as 8.3 and 159.25 l/kg-TS, respectively with 29.44 (maximum 35.58%) and 53.48% (maximum 68.4%) of average H2 and CH4 contents, respectively at 3 and 12 days of HRT, respectively under thermophilic conditions. TS% conversion was found as 55% and 70% for mesophilic and thermophilic batches. Hence, thermophilic consortium was concluded to have potential for biogas production from VW in term high biogas yield, simultaneous production of H2, high biomass conversion and reduced HRT. Table 12 summarizes the results of this comparative study.

Table 12: Cumulative product yield in biohythane batches under mesophilic and thermophilic conditions.

*TS: Total solids

**HRT: Hydraulic retention time

Example XII

Optimization of operational parameters for enhanced production of biogas and H2 from VW

The effect of seed (10-30%) and solid loading or dry biomass loading (3-8%) on biohythane production from VW by one-stage AD under thermophilic conditions was evaluated using RSM of design expert software version 8.0 (STAT-EASE Inc., Minneapolis, U.S.A) keeping temperature constant as 52°C. Figures 25-27 and 28 display the daily and cumulative yield of biohythane (biohydrogen + biogas) in experiential batches (VW1-13). Although figures display the daily production of all batches however, biohydrogen containing H2 content > 20%, and biogas with volume > 0.2 L with CH content > 50%, were considered for calculations. All batches resulted into biohydrogen and biogas yields as 142.75-246.12 and 209.12-562.12 l/kg-TS -217 or 150.26- 259.05 and 220.16-591.74 l/kg-VS, respectively (Fig. 28). The highest cumulative yield of biohydrogen as 246.12 l/kg-TS or 259.05 l/kg-VS was observed in VW1 with 10% seed and 3% solid loading with cumulative H2 yield as 81.11 l/kg-TS or 85.38 l/kg-VS. Average H2 (maximum 41.3%) and CO2 contents in biohydrogen were observed as 33.18 and 38.66%, respectively. Although VW1 (10% seed; 3% TS) showed the maximum cumulative yield of biohydrogen, however it was associated with longest lag phase (phase of microbial adaptation to substrate) of 15 days when no gas production was observed, and on 16 ^th day, production of biohydrogen was started, which elongated the HRT of 22 days for product formation (Fig 25&26). The longest lag phase could be attributed to the fact that small seed (inoculum) size as 10% is not sufficient to start up the process for least solid content in feedstock as 3% because inoculum activity and the amount of readily degradable components in the substrate affect the start-up time of AD therefore, low sugar content in small biomass loading (3%, dry basis) in VW could lead to prolonged HRT (Li et al, 2019). Hence, small seed size along with lower solid contents could not be considered as significant conditions even with highest product yield due to prolonged lag phase. Similarly, small seed size of 10% with solid contents of 5.5% (VW5) also passed through the prolonged lag phase of 6 days (Fig. 25&26). Further, 3% solid loading with 30% seed in VW2 is associated with 3 days of lag phase. Therefore, it could be concluded that both seed size and solids in substrate should be balanced to impact the start-up of process. Generally, it has been observed in the present study that least quantity in both the factors lead to prolonged lag phase, while higher value of both factors lead to the short lag phase as shown in Fig. 25&26. The highest cumulative biogas yield was found as 562.12 l/kg-TS or 591.74 l/kg-VS in batch with 30% seed and 5.5% solid loading (VW6) in 31 days, wherein first 7 days were spent for biohydrogen production (206.15 l/kg-TS or 217 l/kg-VS) leaving for total 24 days for biogas production (Fig. 28). Cumulative CH4 content in biogas was found as 382.40 l/kg-TS or 402.53 l/kg-VS corresponding to 81.09% of theoretical yield (496.34 l/kg-VS). Average CH4 (maximum 75.8%) and CO2 contents in biogas were found as 67.15 and 18.49%, respectively. T.S and V.S conversion were found as 84.00% and 84.90%, respectively. The optimized conditions for biohythane production selected by software were found to be 30 and 8.0% seed and solid loading. In model validity experiment, cumulative H2 and biogas yields were found as 223.5 and 511.25 l/kg-TS or 235.26 and 538.16 l/kg-VS, respectively in 23 days (6 days for H2 and 17 days for biogas). Table 13 summarizes the product profile of 13 experimental (VW1-13) runs along with optimized batch. Average CH4 (maximum 76.56%) and CO2 contents were found as 70.6 and 21.38%, respectively, whereas average H2 (maximum 45.6%) and CO2 content were observed as 34.48 and 57.74%, respectively. T.S and V.S conversions were found as 83 % and 84.52%, respectively.

Table 13: Cumulative biohydrogen and biogas yields along with hydrogen, methane and carbon dioxide content in experimental batches selected by software (RSM, Design Expert, STATE -Ease version 8.0, U.S.A).

Example XIII

Optimization of temperature for enhanced production biohythane from VW

Optimized conditions of seed and solid loading were further used to conduct experiments for temperature optimization (30-60°C) for enhanced biohythane production keeping batch at 52°C as control. Table 14 summarizes the results of this study. It was observed that biohythane production increased with rise in temperature from 35 to 55°C but biogas production was declined at temperature 60°C which was only 17.5 l/Kg-TS on 4 ^th day with 22.2 % and 42.32% CH and CO2 content, respectively. But biohydrogen was observed in significant quantities as 107.5 L/Kg-TS (although lower than optimized) with average H2 (maximum 36.98%) and CO2 content of 32.20% and 55.86%, respectively in 4 days. The reason for this trend might be that methanogens (acetoclastic herein) get deactivated at temperature as higher as 60°C, while hydrogen formers dominated at this temperature. Fig. 29 shows the plot of cumulative biohythane yield for all the batches. The highest biohydrogen and biogas yields were observed in batch VW55 (at 55°C) as 315 and 615.5 l/kg-TS or 331.58 and 647.89 l/kg-VS, respectively with HRT of 4 and 20 days, respectively compared to the 222.5 and 516.95 l/kg-TS or 234.21 and 544.16 l/kg-VS of biohydrogen and biogas yields, respectively from control (VW52). Average H2 (maximum 48.5%) and CO2 contents were observed as 38.08 and 49.78%, respectively, whereas average CH4 (maximum 76.52%) and CO2 contents were 69.06 and 24.40%, respectively. T.S and V.S conversions were 88.48% and 89.56%, respectively. Considering present investigation as batch process, and VW as sole substrate without co-digestion, observed results were higher than previous studies based on VW or FW in terms of product yield, average contents (%) and TS/VS conversions. In a study of AD of VW co-digested with swine manure (35:65) using seed from a lab-scale glucose and fruits-based l-breactor under thermophilic conditions (55±1°C), H2 yield and average H2 content were found as 1261/kg-VS and 42%, respectively in HRT of 2 days (Tenca et al., 2011). Yeshanew et al. (2016) investigated biohythane production from FW by keeping 55±2°C in Fbreactor and 37±2°C for CH4 production using digested sludge (from buffalo manure and dairy wastewater-treating biogas plant), and reported maximum Fb and CH4 yield, as 115.2±5.6 l/kg- COD added, respectively in HRT of 3.5 and 1.5 days, respectively. In a study of continuous AD from co-digestion of FW and VW (2.2:2.8) (feeding rate: 2.8 kgvs/m ³ /d) at 35±1 °C using seed from mesophilic abattoir waste treatment-biogas plant, biogas yield was reported as 870 l/kg-VS with average CH4 Of 57.58% in HRT of about 100 days with 52 days of lag phase (Masebinu et al, 2018). In another study of co-digestion of VW with dog and cattle manure in fed-batch AD (1 kg/week of substrate loading) under ambient conditions (31-38°C), maximum biogas yield of 668.9 l/kg-TS with 44.6% of CH4 content was reported in HRT of 28 days with 5 days of lag phase (Phetyim et al, 2015). In another AD study of FW (using anaerobic sludge from an anaerobic digester as inoculum) at 55°C, cumulative biogas yield and CH4 content were reported as 322 l/kg- TS and 53.1%, respectively with 52.07% and 63.46% of T.S and V.S conversion, respectively (Yang et al., 2015).

Table 14: Cumulative product yield in biohythane batches at different temperatures.

*TS: Total solids

**HRT: Hydraulic retention time

Example XIV

Biohythane production from VW in 7.5 L bioreactor under optimized conditions

Study of biohythane production from VW under optimized conditions was extended from three- bottle set-up to 7.5 L bioreactor, however seed was taken as 20% for economical purpose (as optimized was 30%) with 200 g of dry biomass loading (8%) at 55°C in working volume of 3 L. pH was controlled during initial days of H2 production using 5 M NaOH solution up to 5.8-8.0 without pH control for methane production later on (after 5-7 days). However, this batch study did not show significant results as observed in three-bottle set-up studies. Fig. 30 shows the daily yield of products for batch conducted in 7.5 L bioreactor. Table 15 summarizes the results of study. Cumulative biohydrogen and biogas yields were observed as 105.75 and 170.5 l/kg-TS or 111.31 and 179.47 l/kg-VS, respectively in HRT of 8 and 12 days, respectively. Average H2 (maximum 45.5%) and CO2 contents were found as 36.69 and 35.11%, respectively. Average CH (maximum 68.45%) and CO2 contents were observed as 60.32 and 24.22%, respectively. T.S and V.S conversions were 65.45 and 67.27%, respectively. It could be due to lack of proper pH control and agitation in the bioreactor design. This type of study needed a specific design for bioreactor with proper balance of pH control and agitation during initial days of process.

Table 15: Cumulative product yield in biohythane batch conducted in 7.5 L bioreactor. *TS: Total solids

**HRT: Hydraulic retention time

Although the invention has been described with reference to specific embodiments, present description should not be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the invention, will become apparent to persons skilled in the art upon reference to the description of the invention. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present invention as defined.