WO2023281373A1

WO2023281373A1 - A liquid microbial consortium for biohydrogen generation and a process thereof

Info

Publication number: WO2023281373A1
Application number: PCT/IB2022/056166
Authority: WO
Inventors: Prashant K. Dhakephalkar; Sumit Singh DAGAR; Soham D PORE; Sai S HIVARKAR; Pranav Kshirsagar; Vikram B. Lanjekar; Shashishekhar PANDIT; Kaustubh Pathak
Original assignee: Agharkar Research Institute Of Maharashtra Association For Cultivation Of Science; Kpit Technologies Limited; Sentient Labs Pvt Ltd
Priority date: 2021-07-08
Filing date: 2022-07-04
Publication date: 2023-01-12

Abstract

The present invention relates to a liquid microbial consortium for biohydrogen generation and a process thereof. The liquid microbial consortium comprises of Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513. The invention relates to a process for two-stage microbial degradation leading to direct production of recoverable biohydrogen with the help of microbial fermentation of lignocellulosic crop residues by disclosed consortium in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I. The disclosed consortium has higher growth rate and broad optimal growth range that ensures stability, reproducibility, and scalability. The improved biohydrogen production also eliminates the requirement of harsh pretreatment of substrates, sterilization of the substrates/ containers/equipment, need for repetitive dosing of disclosed consortium, and addition of acids/ alkali to maintain pH or inert gases/ reducing agents to maintain anaerobic conditions.

Description

A LIQUID MICROBIAL CONSORTIUM FOR BIOHYDROGEN GENERATION

AND A PROCESS THEREOF

FIELD OF THE INVENTION

The present invention relates to a liquid microbial consortium for biohydrogen generation and a process thereof by microbial degradation of lignocellulosic crop residues. More particularly relates to a two-stage microbial degradation process for direct production of recoverable biohydrogen with the help of said consortium from lignocellulosic crop residues in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I.

BACKGROUND OF THE INVENTION AND DESCRIPTION OF THE PRIOR- ART

Over the past few decades, global energy demand has risen due to industrialization and population growth. As fossil fuels are the dominant energy source, heavy reliance on fossil fuels depletes them and contributes to climate change. To overcome this issue, efficient utilization of alternative energy sources, such as biomass, solar, wind, and hydro, is getting more attention. Biomass is becoming a promising alternative among all renewable sources due to its ample availability.

Hydrogen is one of the best alternatives as only water and heat are produced on combustion, making it a cleaner fuel. The calorific value of hydrogen (150 KJ/g) is also the highest among all of the widely used fuels like natural gas (52 KJ/g) and liquid petroleum gas (50 KJ/g), making it one of the highest energy-yielding fuels. At present, hydrogen is mainly produced by thermochemical processes such as pyrolysis, gasification, steam reformation of natural gas, and water electrolysis. Biohydrogen of microbial origin has been suggested as the most suitable alternative to make hydrogen production more economical and industrially viable. Biomethane produced during the process can be converted to hydrogen by steam methane reformation. Alternatively, biomethane can be used directly for internal combustion engines with minimum modification. Also, the combustion of methane occurs with a significantly lower emission of pollutants. Unfortunately, the economic and commercially viable production of these biofuels has remained a challenge. Lignocellulosic waste materials are a vast group of biomasses with biohydrogen production potential. Agricultural residues as a source of lignocellulosic waste materials such as rice straw, wheat straw, sugar cane bagasse, bamboo, Napier grass, and food waste/decomposable municipal solid waste are some waste materials that can be used for biohydrogen production. Anaerobic microbes can degrade organic wastes, including agricultural crop residues, and produce biohydrogen and biomethane as products. India generates around 500 million tons of crop residues per year, of which rice straw is the most abundant and available throughout India. Of the 140Mt rice straws, approximately 92 Mt is burnt each year which causes major environmental problems leading to health issues and contributing to global warming. The abundant availability and high cellulose (30-35%) and hemicellulose (18-20%) contents make rice straw a preferred lignocellulosic crop residue for microbial biodegradation and biohydrogen production.

Lignocellulosic materials have a complex structure made from cellulose, hemicelluloses, and lignin and consist of 50 to 80% carbohydrates in dry weight. Pretreatment methods have been used to break down the complex structure of lignocelluloses to make them a more bio degradable feedstock for biofuel production. These pretreatment methods can be divided into three major groups: physical pretreatment, chemical pretreatment, biological pretreatment or enzymatic pretreatment. Biological pretreatment is more environment-friendly than chemical pretreatment; however, it is time-consuming and cannot be considered a proper industrial method. Additionally, the requirements of biological treatment of large space for conducting the process and a controlled environment make it less attractive for large and industrial-scale production and applications. Chemical pretreatment is achieved by soaking the lignocellulosic crop residues/ rice straw in high alkali concentrations (~4% or higher NaOH w/v in water). Physical pretreatment is carried out by changing the parameters like temperature, pressure, particle size, etc. Such pretreatments have been proven to be the most effective. However, it consumes a lot of energy. The researchers have tried out milder pretreatment to overcome this issue, but they have not yet proved to be as efficient.

Thus, there exists a dire need for an efficient process for producing hydrogen directly from the abundant biomass available with the help of a microbial consortium. There is a need to produce higher amounts of recoverable biohydrogen and biomethane. There is also a need to efficiently utilize abundantly produced lignocellulosic crop residues, thereby limiting their burning and eventual pollution. Further, there is a need to utilize the microbial consortium that eliminates the requirement of expensive harsh pretreatments, which adversely affect the environment. There is also a need for a process that operates optimally compared to thermophilic processes, thus, saving a lot of energy required to heat the anaerobic bioreactors. There exists a need for a process to produce recoverable biohydrogen that prevents changes in the microbial community composition and bioreactor performance. There is a need to eliminate the requirement of repetitive dosing of microbial consortium in the anaerobic bioreactor during the continuous generation of biohydrogen gas. Maintaining the pH in a larger bioreactor is challenging and consumes a large amount of acid/alkali; consequently, there is a deviation in optimal biohydrogen and biomethane generation. There is also a need to eliminate pH maintenance during continuous hydrogen generation in an anaerobic bioreactor. Further, there is also a need for a process that utilizes the volatile fatty acids to produce biomethane, saving a lot of time and energy required in the post-process treatment of digestate before release. There is also a need for a process to produce recoverable biohydrogen, saving a lot of space and operational and capital expenditure. There is also a need for an economical and commercially viable production of biohydrogen and biomethane that can be used for various applications. All publications herein are incorporated by reference to the same extent as if each publication or patent application were specifically and individually indicated to be incorporated by reference.

Reference may be made to WO2018088884A1, which relates to a method for obtaining a stable and robust microbial consortium or inoculum, which produces hydrogen and hydrolysates for the generation of methane as an energy source in biological production systems using complex substrates, specifically agro-industrial waste, industrial wastewater, urban solid organic waste, and lignocellulosic materials. The method of the invention ensures the production of an inoculum having a microbial structure with the following advantages: reproducible on a large scale; highly robust, predominance over the microflora native to the substrates; high hydrolytic capacity; generates natural anaerobic conditions without requiring the injection of an inert gas or the addition of a chemical reducing agent; highly stable, viable for up to 2 years when refrigerated; and reactivation time measured in hours. In contrast, the present invention discloses a liquid microbial consortium comprising of pure and isolated microbes, which will help ensure the stability and reproducibility of the process. The said consortium used in the present invention operates optimally at 30-degree C for biohydrogen production without requiring additional energy to heat up the reactor and help make the process economically viable.

The retention time of the said process is only 10 days in Stage I and 20 days in Stage II, and the broad optimal growth range of said consortium helps it achieve the stability, reproducibility, and scalability. It is noteworthy to mention here that the said process does not require any harsh pretreatment of substrates or sterilization of the substrate/ containers/equipment. Additionally, there is no need for the repetitive dosing of the microbial consortium, only one-time addition as a start-up inoculum is sufficient. The present invention does not involve any addition of acids/ alkali to maintain pH or inert gases.

Reference may be made to AU2016297929B2, which relates to isolated and biologically pure microorganisms that have application, inter alia, in agriculture. The disclosed microorganisms can be utilized in their isolated and biologically pure states, as well as being formulated into agriculturally acceptable compositions. Further, the disclosure provides agriculturally beneficial microbial consortia, containing at least two members of the disclosed microorganisms, as well as methods of utilizing said consortia in agricultural applications. This document teaches that the individual microbes, or microbial consortia, or microbial communities, developed according to the disclosed methods-including any single microorganism or combination of microorganisms disclosed in Tables 1-4 of the specification-can be combined with any plant bio stimulant.

Thus, the cited document does not overlap with the teachings of the present invention, and it only mentions microbes having applications in agriculture. Furthermore, the said microbes in the cited art are aerobic, while the ones used in the present invention are anaerobic strains.

Reference may be made to WO2014033345A1, which relates to a microbial consortium comprising a Clostridium roseum strain with Spanish Type Culture Collection access number CECT8187 and a Streptomyces sp. strain with Spanish Type Culture Collection access number CECT8185. The invention also relates to the use of said consortium for the production of hydrogen, organic acids, solvents or biofilms, and to a method for producing said consortium. This document therefore, provides a mixture of strains, one belonging to the species Clostridium roseum with access number to the Spanish Type Culture Collection (CECT) 8187 which has been isolated by an isolation method under conditions of under vacuum (which allows a better selection of highly producing strains by eliminating the limitation caused by the partial pressure of H 2 in its production) and another belonging to the species Streptomyces sp. with access number to the Spanish Type Crop Collection (CECT) 8185, isolated from the granular sludge in aerobic conditions. The consortium of the invention, as shown in the examples, has the characteristic that by joining the strain of H5 Clostridium roseum with access number to the Spanish Crop Collection Type CECT8187 (producer of ¾) with the strain EJ1 of Streptomyces sp. The consortium used in the present invention is different and does not use Clostridium roseumor Streptomyces sp. Besides, the substrate is not disclosed in the said prior art. There is a lack of clarity on whether the consortium has the potential to degrade the recalcitrant lignocellulosic biomass without pretreatment. Further to this, it is clearly evident that there is no disclosure on if the digestate that comes out after hydrogen production is suitable for biomethane production.

Reference may be made to WO2011159924A2, which relates to a natural gas and methylotrophic energy generation, bio-generated fuels and microbiology. In alternative embodiments, the invention provides nutrient amendments and microbial compositions, e.g., consortia, that are both specifically optimized to stimulate methanogenesis, or for "methylotrophic" or other conversions. In alternative embodiments, the invention provides methods to develop nutrient amendments and microbial compositions that are both specifically optimized to stimulate methanogenesis in a given reservoir. The invention also provides methods for the evaluation of potentially damaging biomass formation and scale precipitation resulting from the addition of nutrient amendments. In other embodiments, the invention provides methods for simulating biogas in sub-surface conditions using a computational model. The said invention relates only to increasing methane production in subsurface reservoirs and does not relate to hydrogen production or utilization of agricultural wastes.

Although various pieces of literature have reported the usage of microbial consortia in generating biohydrogen, most of them disclose the use of a natural microbial consortium, which may change or get affected by inherent/ native microbes of substrates, thus affecting the reproducibility of the process. Most of the cited prior arts cannot degrade the untreated lignocellulosic biomass and require harsh pretreatment methods. Further, the cited documents involve the inability of microbes to work at a broad temperature range or work optimally only at high-temperature conditions. The cited invention requires the sterilization of substrates, containers or other equipment and also requires the addition of inert gases/ reducing agents to maintain anaerobic conditions. These documents also have the disadvantages such as low yields of hydrogen and / or methane, low substrate loading rates or longer hydraulic retention times, requiring repetitive dosing of microbial consortium and lower yield of hydrogen and / or methane.

Therefore, keeping the drawbacks of hitherto reported prior-art, the inventors of the instant invention have derived a simple and inexpensive process for direct generation of hydrogen gas by microbial degradation of lignocellulosic crop residues and, more particularly, relates to a two-stage microbial degradation process for direct production of recoverable biohydrogen from lignocellulosic crop residues with the help of liquid microbial consortium in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I. The said process is an economically and commercially viable production of biohydrogen and biomethane, which can be used for various applications.

The said process is carried out in a range of reactor sizes and designs and is not restricted to reactors with some specific size and design or requirements.

OBJECTIVES OF THE INVENTION

The main objective of the present invention is to provide a process for direct generation of biohydrogen from lignocellulosic crop residues (such as agro-wastes), and more particularly relates to a process for a two-stage microbial degradation for the production of recoverable biohydrogen from lignocellulosic crop residues with the help of a liquid microbial consortium in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I.

Yet another object of the present invention is to provide a process for developing a liquid microbial consortium using lignin and xylan that serves as a carbon source for the isolation of pure cultures of biohydrogen producing bacteria.

Yet another object of the present invention is to provide a liquid microbial consortium comprising of defined microbial species in a specified proportion which ensures the stability, reproducibility, and scalability of the process.

Yet another object of the present invention is to provide a process for the generation of biohydrogen that eliminates the sterilization/autoclaving of substrates, containers, equipment and transfer lines/piping, thus reducing the time, energy, capital, and operational expenditure.

Yet another object of the present invention is to provide a process that eliminates the use of thermochemical pretreatment, enzymatic pretreatment or metal catalyst.

Yet another objective of the present invention is to facilitate ¾ production in Stage I and eliminate C¾, ¾S, and SO ₂ in the headspace gas.

Yet another object of the present invention is to provide a process that eliminates repetitive dosing of the microbial cultures.

Another object of the present invention is to provide a two-stage process to directly produce significantly higher amounts of recoverable biohydrogen followed by biomethane. Yet another object of the present invention is to provide a process for soaking of the lignocellulosic crop residues such as rice straw, wheat straw, sorghum straw, Napier grass, etc., as opposed to the currently and routinely used harsh pretreatment methods that are not only expensive but also adversely affect the environment. The process of soaking, as disclosed in the foregoing disclosure, is economical, efficient, and environmentally friendly.

Another object of the present invention is to provide a process using a low NaOH concentration for soaking the lignocellulosic crop residues to prevent floating and eventual scum formation while maintaining the desired pH for the optimal performance of Stage I. The low concentration will also help to make the process more environmentally friendly as it eliminates the use of high concentrations of acid and alkali.

Still another object of the present invention is to provide a process operational at optimal pH without adding or pumping acid or alkali in the digesters, thus reducing the time, energy, capital, and operational expenditure.

Yet another object of the present invention is to provide a process for operating the bioreactor at optimal conditions, saving a lot of energy required to heat the anaerobic bioreactor, saving a lot of space required, thus reducing capital expenditure and operational expenses.

SUMMARY OF THE INVENTION

The present invention relates to a liquid microbial consortium for biohydrogen and a process thereof by microbial degradation of lignocellulosic crop residues. More particularly relates to a two-stage microbial degradation process for direct production of recoverable biohydrogen with the help of said consortium from lignocellulosic crop residues in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I. The said consortium has a higher growth rate and a broad optimal growth range that ensures the stability, reproducibility, and scalability of the process. The said consortium’s robustness also eliminates the requirement of harsh pretreatment of substrates, sterilization of the substrate/ containers/equipment, the need for repetitive dosing of the said consortium, and the addition of acids/ alkali to maintain pH or inert gases/ reducing agents to maintain anaerobic conditions. The efficiency of the said consortium also ensures biomethane production from the digestate in Stage II, eliminating the requirement of any post-process treatments and is directly used as organic fertilizers. In an embodiment of the present invention, the instant invention provides a liquid microbial consortium for biohydrogen and a process thereof, wherein the process of obtaining the liquid microbial consortium capable of producing hydrogen is characterized in that it comprises the steps:

• Extracting guts of the worker termites of genus Odontotermes;

• Homogenizing the guts immediately in a sterile anaerobic diluent medium comprising (per litre) of salt solution 1 (0.3% K2HPO4), and salt solution 2 [0.3% KH2PO4, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1 ml resazurin (0.1%, v/v), 5 ml sodium carbonate (8%, v/v), 0.5 gm L-cysteine hydrochloride at the pH 7.0 ± 0.1;

• Inoculating of gut homogenates in a nutrient medium which comprises(per litre) of salt solution 1 (0.3% K₂HP0₄),and 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS04.7H20 and 0.06% CaCl2-2H20], 1 ml resazurin (0.1%, v/v), 0.5 gm of yeast extract, 1 ml Pfennig’s trace mineral solution(0.03% H₃BO₃, 0.01% ZnS0₄.7H₂0, 0.003% MnCl₂.4H₂0, 0.002% COC1₂.6H₂0, 0.003% Na₂Mo0₄.2H₂0, 0.001% Na₂Se0₃, 0.002% NiCl₂, 0.001% CUC1₂.2H₂0, 0.015% FeCl₂.4H₂0), 2 ml haemin solution (0.05%, v/v), 0.5 gm L-cysteine hydrochloride and carbon source selected from either of 0.05%:0.8% (v/v) lignin: xylose, 1% w/v xylan, 1% w/v cellulose, 5% w/v rice straw, at pH 7.0 ± 0.1;

• Incubating the inoculum at 20-45°C, preferably 30°C, for 7-8 weeks;

• Maintaining the positive enrichments in the nutrient media with the xylan and the lignin as the carbon source by periodic subculturing into the respective fresh sterile nutrient medium.

• Sub-culturing the inoculum periodically for two years at the oxidation-reduction potential of -200 to -500 millivolts under anaerobic conditions provided by removing dissolved oxygen, cooling on ice under the stream of oxygen-free nitrogen, dispersing into pre-gassed bottles and sealing by aluminium crimps;

• Pooling the inoculum in the isolation medium with rice straw as a substrate for microbial succession;

• Incubating and sub-culturing the inoculum at 20-45°C, preferably at 30°C for 3-5 days at the oxidation-reduction potential of -200 to -500 millivolts under the anaerobic conditions, and • Isolating and cultivating individually the selectively enriched microbial species under the anaerobic conditions in the nutrient medium consisting of the rice straw for 48-72 hours at 20-45°C, preferably at 30°C.

• Screening, selecting and combining the highest hydrogen producing pure cultures to produce the liquid microbial consortium

According to the one embodiment of the present invention, the process of generation of biohydrogen and biomethane comprises of a feeder (101), a soaking container/containers (103), a stage I reactor (107), a Stage II reactor (109), and a post biomethanation treatment (121). The present invention process comprises soaking of the lignocellulosic crop residues in the soaking container /containers (103), which is then fed to the Stage I reactor (107) containing the liquid microbial consortium of the present invention. In the Stage I reactor (107), the liquid microbial consortium disclosed in the present invention enhances the enzyme activity for degradation of lignocellulosic crop residues and produces recoverable biohydrogen. The digestate from the Stage I reactor (107) is further processed in the Stage II reactor (109) to produce biomethane. The digestate of the Stage II reactor is rich in nutrients that can be dewatered and used for organic fertilizer. The liquid part separated can be recycled in a Stage II reactor. The biohydrogen and biomethane generated further undergo purification and enrichment to provide pure hydrogen and biomethane that can be used for various applications, including but not limited to fuel for vehicles, industrial applications, chemical applications, consumer applications, etc.

In some embodiments, the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, temperatures, timings, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.”

In a preferred embodiment of the present invention, the biomass used is a lignocellulosic crop residue, for example, rice straw, wheat straw, sugarcane bagasse, sorghum and Napier grass. The recoverable biohydrogen and biomethane are produced by treating the lignocellulosic crop residues with the specialized microbial consortium of the present invention.

In a preferred embodiment of the present invention, there is a process for making a defined, and specialized liquid microbial consortium wherein the process includes enrichment and acclimatisation of organisms in the consortium to enhance enzyme activity and biohydrogen production potential. The biohydrogen thus produced is used as fuel for fuel cell electric vehicles. The biomethane produced is used to generate hydrogen, which can be further used to fuel cell electric vehicles.

The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

The terms biohydrogen and hydrogen, and biomethane and methane, although used interchangeably throughout the patent, basically refer to hydrogen and methane generated from lignocellulosic crop residues using biological means. The biomass used is of any type known in the art, including, but not limited to, agriculture residue, wastes, etc. In a preferred embodiment of the present invention, the biomass used is the lignocellulosic crop residue, for example, rice straw, wheat straw, sorghum, sugarcane bagasse and Napier grass. Although the preferred embodiment of the present invention is described with a rice straw example, it is to be noted that the process of direct hydrogen generation of the present invention encompasses direct biohydrogen generation from all types of lignocellulosic crop residues, like, but not limited to, rice straw, cereal straw, biogases, crop residue, etc.

Therefore, the present invention provides a process of direct generation of hydrogen gas by microbial degradation of lignocellulosic crop residues and, more particularly, relates to a two- stage microbial degradation process for direct production of recoverable biohydrogen with the help of the liquid microbial consortium in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I.

An important aspect of the present invention is to provide a two-stage process for the generation of biohydrogen and biomethane characterized such that the Stage I of the process comprises the steps of: • Soaking the lignocellulosic crop residue, like rice straw, wheat straw, sugarcane bagasse, sorghum and Napier grass in 0.5% NaOH in a soaking container (103) at room temperature for 72h under open soaking conditions;

• Hydrolyzing and deconstructing the individual lignocellulosic polymers like cellulose and hemicellulose to their respective monomers like glucose and xylose; and

• Carrying out subsequent fermentation by the disclosed liquid microbial consortium to produce biohydrogen, along with carbon dioxide and volatile fatty acids.

Yet another important aspect of the present invention is to provide a two-stage process for the generation of biohydrogen and biomethane such that Stage II of the process comprises the steps of:

• Using the disclosed inoculum, leftover digestate and co-metabolites, which are produced in Stage I as a substrate;

• Mixing the said digestate and co-metabolites of Stage I with cattle dung based start-up inoculums;

• Operating the bioreactor optimally at an optimal temperature of 37°C and a pH of 6-7;

• Obtaining biomethane and CO2;

• Separating the output of Stage II in a solid-liquid separator; and

• Using the solid digestate as biofertilizer and part of the liquid for recycling.

An important aspect of the present invention is that the microbial consortium is a defined one, and comprises five anaerobic bacteria cultures, viz. Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513 that were isolated following selective enrichment of the termite gut microflora.

Yet another important aspect of the present invention is to provide a process for direct production of recoverable biohydrogen in a semi-continuous mode.

Still another aspect of the present invention is to provide a microbial consortium for biohydrogen generation, and a process wherein the oxidation-reduction potential of the liquid culture medium remains in a range between -200 to -500 millivolts during culturing under anaerobic conditions. Yet another important aspect of the present invention is to process for direct production of recoverable biohydrogen wherein the digestate/co-metabolites produced alongside biohydrogen are not discarded but used as a substrate for the Stage II biomethanation process. Yet another important aspect of the present invention is to provide a process for direct production of recoverable biohydrogen and biomethane, which utilizes the volatile fatty acids, thus, saving time and energy required in the post-process treatments of the effluent before release.

Still another important aspect of the present invention is a process for making a liquid microbial consortium for effective degradation of lignocellulosic crops to produce recoverable biohydrogen directly.

Yet another important aspect of the present invention is to provide a process for enrichment of the microbial consortium for an enhanced enzymatic activity for the effective degradation of lignocellulosic crops. Yet another important aspect of the present invention is to provide a process for an economical and commercially viable production of biohydrogen and biomethane, which can be used for various applications.

Another important aspect of the present invention is to utilize the solid and liquid effluent generated through the process as organic manure and fertilizers, creating an additional stream of revenue.

An important aspect of the present invention is to utilize the abundantly available lignocellulosic crop residues for biohydrogen and biomethane production, thus, limiting the lignocellulosic crop residues burning the eventual pollution while also contributing to the farmer’s income. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1 illustrates the workflow for enrichment, isolation and combination of microbial species to get a liquid microbial consortium useful for the generation of biohydrogen.

Figure 2 illustrates the block diagram of the two-stage microbial degradation process with the help of a disclosed liquid microbial consortium to produce recoverable biohydrogen from lignocellulosic crop residues in Stage I, followed by the generation of biomethane in Stage II according to an embodiment of the present invention.

Figure 3 illustrates the biohydrogen production during semi-continuous mode with a 2 L working volume during 50 days of operation in Stage I.

Figure 4 illustrates the Volatile Fatty Acid (VFA) production during semi-continuous mode with 2 L working volume during 50 days of operation in Stage I.

Figure 5 illustrates the Biohydrogen production during semi-continuous mode with 14 L working volume during 50 days of operation in Stage I.

Figure 6 illustrates the Biomethane production during semi-continuous mode with 10 L working volume during 49 days of operation in Stage II.

Figure 7 illustrates a comparison of hydrogen production by different consortia and the liquid microbial consortium of the present invention utilizing a lignocellulosic residue as a rice straw.

Figure 8 illustrates a comparison of the hydrogen production by various lignocellulosic crop residues like rice straw, wheat straw, sugarcane bagasse, maize straw, a Napier grass using the liquid mial cocrobinsortium disclosed in the present invention

DETAILS OF BIOLOGICAL RESOURCES USED IN THE INVENTION

The microbial consortium comprises five anaerobic bacteria cultures viz. Clostridium chromiireducens strain [CTS0513], Clostridium chromiireducens strain [STS0514], Clostridium chromiireducens strain [XTS0511], Clostridium diolis strain [STS0519], and Clostridium diolis strain [XTS0513] that were selectively enriched and isolated from the termite gut of genus Odontotermes. The NBA-ABS filing number is INBA3202203704.

DETAILED DESCRIPTION OF THE INVENTION

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention. Various terms as used herein are shown below. To the extent a term used in a claim is not defined below, it should be given the broadest definition persons in the pertinent art have given that term as reflected in printed publications and issued patents at the time of filing.

Therefore, the present invention relates to a liquid microbial consortium for biohydrogen and biomethane generation and a process thereof, wherein the process of obtaining the liquid microbial consortium capable of producing hydrogen is characterized in that it comprises the steps:

• Selectively enriching the lignocellulose degrading natural microbial consortia from the termite gut by periodic sub-culturing over two years, using a defined nutrient medium consisting of purified individual lignocellulosic polymer, i.e., lignin and xylan;

• Pooling the developed natural consortia obtained from lignin and xylan enrichments in a defined nutrient medium with rice straw as the sole substrate to allow the natural succession to enrich the lignocellulose degrading and hydrogen-producing natural microbial consortium selectively;

• Isolating the lignocellulose degrading and hydrogen-producing microbes in a pure form from the natural microbial consortium and screening the most efficient biohydrogen producers; and

Identifying and combining the selected pure microbes to develop a liquid microbial consortium for generating biohydrogen and volatile fatty acids from the agricultural crop residue in a defined nutrient medium, wherein the crop residue is selected from the group comprising rice straw, wheat straw, sorghum, sugarcane bagasse, and Napier grass.

The microbial strains used for the said consortium comprise of five anaerobic bacteria cultures, viz Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513 that were selectively enriched and isolated from the termite gut of genus Odontotermes.

The present invention relates to a liquid microbial consortium for direct hydrogen generation by microbial degradation/ anaerobic digestion of lignocellulosic crop residues and, more particularly, relates to a two-stage microbial degradation process for the production of recoverable biohydrogen from lignocellulosic crop residues in Stage I, followed by generation of biomethane in Stage II from the digestate of Stage I.

The process of the present invention comprises of a feeder (101), a soaking container (103), a Stage I reactor (107), a Stage II reactor (109), and a post biomethanation process/treatment (121). The present invention process comprises a soaking of the biomass in the soaking container (103), which is then fed to the Stage I reactor (107 containing the liquid microbial consortium of the present invention. In the Stage I reactor (107), said liquid microbial consortium enhances the enzyme activity for degradation of lignocellulosic crop residues and produces recoverable biohydrogen. The digestate/co-metabolites from the Stage I reactor (107) are further processed by the specialized microbial consortium in the Stage II reactor (109) to produce biomethane. The biohydrogen and biomethane further undergo purification and enrichment to provide pure biohydrogen and biomethane, which may be used for various applications.

Figure 1 illustrates the workflow of enrichment, isolation and combination of microbial species to get a liquid microbial consortium useful for the generation of biohydrogen. The process of obtaining the liquid microbial consortium involves the extraction of gut contents from the worker termites of the genus Odontotermes, which is widely reported to consist of diverse microbial groups like lignocellulolytic fungi ( Termitomyces , Trichosporon ), yeasts Candida , Pichia), methanogenic archaea ( Methanobrevibacter ), acetogenic bacteria ( Acetonema , Clostridium) and other lignocellulolytic and fermentative bacteria ( Actinobacteria , Enterococcus). Homogenizing the guts immediately in a sterile anaerobic diluent medium (pH 7.0 ± 0.1) comprising (per litre) of 150ml each of salt solution 1(0.3% K2HPO4), and salt solution 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1ml resazurin (0.1%, v/v), 5ml sodium carbonate (8%, v/v), 0.5gm L-cysteine hydrochloride; inoculating gut homogenates in a nutrient medium, followed by incubating the inoculum at 20-45°C, preferably 30°Cfor 7-8 weeks. The nutrient medium comprises (per litre) of 150 ml each of salt solution 1 (0.3% K₂HP0₄), and salt solution 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1ml resazurin (0.1 %, v/v), 0.5gm of Yeast extract, 1ml Pfennig's trace mineral solution (0.03% H3BO3, 0.01% ZnS0₄.7H₂0, 0.003% MnCl₂.4H₂0, 0.002% COC1₂.6H₂0, 0.003% Na₂Mo0₄.2H₂0, 0.001% Na₂Se0₃, 0.002% NiCl₂, 0.001% CUC1₂.2H₂0, 0.015% FeCl₂.4H₂0), 2ml haemin solution (0.05%, v/v), 0.5gm L-cysteine hydrochloride and carbon source selected from either of 0.05%:0.8%(v/v) lignin: xylose, 1% w/v xylan, 1% w/v cellulose, 5% w/v rice straw, at pH 7.0 ± 0.1. The inoculation in the nutrient medium increases the lignocellulolytic population of anaerobes and hydrogen-producing anaerobes and reduces the hydrogen-consuming microbes. The enrichments from the nutrient media with the lignin and xylan as the carbon source are selected further for maintaining the positive growths by periodic subculturing into the respective fresh sterile nutrient medium. Further, to maintain the positive growth, the sub-culturing of the inoculum is done periodically for two years at the oxidation-reduction potential of -200 to -500 millivolts under anaerobic conditions. The anaerobic conditions are maintained by initially boiling the medium to remove dissolved oxygen, followed by cooling on ice under the stream of oxygen- free nitrogen. Cysteine-HCl, if added to the medium, acts as a reducing agent, after which the medium is dispensed into pre-gassed bottles, closed with butyl rubber stoppers and sealed with aluminium crimps before autoclaving. The inoculum is then pooled in the nutrient medium with rice straw as a substrate for microbial succession, incubated and sub-cultured at 20-45°C, preferably at 30°Cfor 3-5days at the oxidation-reduction potential of -200 to -500 millivolts under the anaerobic conditions, and isolated and cultivated individually the selectively enriched microbial species under the anaerobic conditions in the nutrient medium consisting of the rice straw for 48-72 hours at 20-45°C, preferably at 30°C. The isolated pure microbial cultures are then screened for hydrogen gas production using rice straw as a substrate. The pure microbial isolates responsible for the highest hydrogen production like Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513 are selected and combined to get the desired microbial consortium for the use of biohydrogen production.

Figure 2 illustrates the block diagram of the two-stage microbial degradation process to produce recoverable biohydrogen from lignocellulosic crop residues in Stage I, followed by the generation of biomethane in Stage II, according to an embodiment of the present invention. The feeder (101) comprises feed storage to store the lignocellulosic crop residues, a shredder and a pulverizer to shred the lignocellulosic crop residues to specific dimensions, a feeding mechanism that feeds the shredded and pulverized biomass to the soaking container/containers (103). The feeding mechanism could be any known mechanism in the art, for example, a conveyor system. The lignocellulosic crop residues undergo a soaking in the soaking container (103). The lignocellulosic crop residues used are of any type known in the art, including, but not limited to agriculture residue, woods, wastes, etc. In a preferred embodiment of the present invention, the lignocellulosic crop residue used is rice straw. The soaking step comprises the soaking of shredded and pulverized rice straw in 0.5% of NaOH. The use of 0.5% NaOH prevents floating of the rice straw and eventual scum formation, which facilitates continuous biohydrogen production in Stage I reactor (107) containing the liquid microbial consortium of the present invention. The present invention process includes soaking said lignocellulosic crop residue in 0.5% NaOH instead of the routinely used harsh pretreatment methods that are expensive and adversely affect the environment. With the soaking method of the present invention of using milder NaOH to make the rice straw alkaline and less prone to floating and scum formation, which helps microbial degradation of the rice straw biomass take place under optimal pH conditions in the Stage I reactor(107).

The soaked rice straw is then fed to Stage I reactor 107 through the feed pump (105). In Stage I, reactor 107, a liquid microbial consortium of the present invention, acts on the lignocellulosic crop residues to effectively degrade lignocellulosic crop residue to produce recoverable biohydrogen. The microbial consortium comprises a mix of a few bacterial cultures enriched and isolated from the termite gut. In a preferred embodiment, the liquid microbial consortium comprises five anaerobic bacteria cultures, viz. Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513.The specific desired property of this consortium is the ability to metabolize lignocellulosic crop residues leading to the formation of recoverable biohydrogen.

The liquid microbial consortium of the present invention produces around 50-70 ml biohydrogen/ g TS of rice straw in Stage I, which is among the highest reported so far and is stored in the gas storage (111). As the Stage I biohydrogen reactor contains CO2 and other gases, it undergoes a purification and enrichment process (113) to extract pure and usable hydrogen. The pure and usable biohydrogen generated in Stage I may be used for various applications, including but not limited to fuel cells for vehicles, industrial applications, chemical applications, consumer applications, etc.

With the method of the present invention, the bioreactor/Stage I reactor operates optimally at 30°C in comparison to thermophilic processes, which saves a lot of energy required to heat the anaerobic bioreactor, and thus, reduces the operational expenditure of the process. Additionally, the prevailing low pH conditions (pH 5-6) prevent the growth of microbial pathogens/ contaminants associated with the substrate, thereby preventing changes in the microbial community composition and bioreactor performance. The exemplary process parameters of the Stage I reactor are mentioned in Table 1 below: Table 1 The details of various process parameters optimized for biohydrogen production from rice straw

The process of the present invention consistently produces around 50-70 ml biohydrogen/g TS of rice straw at 30°C (pH 5-6) in 10 days of hydraulic retention time at a 2 litter and 14 litter scales in Stage I.

The digestate and the co-metabolites produced alongside the biohydrogen are not discarded but used as a substrate and fed through the feed pump (105) to the Stage II reactor (109) for the biomethanation process. In Stage II reactor (109), the specialized microbial consortium further acts on digestate from Stage I to produce biomethane. The specialized microbial consortium produces around 200 ml biomethane/g TS of rice straw in Stage, which is among the highest reported so far and is stored in the gas storage (111). The Stage II reactor gives a biomethane yield of around 200 ml/g TS of rice straw at an optimal temperature of 37°C and a pH of 6-7 in 20 days hydraulic retention time at a 141iter scale. As the produced biomethane also contains CO2, it undergoes a purification and enrichment process (119) to extract pure and usable methane. With the present invention method, the Stage II digester/ reactor operates optimally at a hydraulic retention time of 20 days, which requires less space and low capital expenditure and operational expenses. Also, with the process of the present invention, the volatile fatty acids are utilized completely in Stage II, which saves a lot of time and energy required in the post-process treatment of digestate before release. The digestate from the Stage II reactor (109) undergoes solid-liquid separation, which separates the solids (silica and manure) and the liquids. The liquids are used as fertilizer or recycled in the Stage II reactor.

Figure 7 illustrates the comparison of hydrogen production by different consortia with the liquid microbial consortium of the present invention utilizing a lignocellulosic residue as rice straw. The figure depicts that high production of hydrogen, i.e., 69ml/g TS with the rice straw as a substrate, is observed by the fermentation with the disclosed liquid microbial consortium comprising of the Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513 over the other microbial consortiums (45-57ml/g TS) enriched on the xylan, lignin and rice straw as a carbon source.

Figure 8 illustrates a comparison of the hydrogen production by various lignocellulosic crop residues like rice straw, wheat straw, sugarcane bagasse, maize straw, and Napier grass using the disclosed liquid microbial consortium in the present invention. The figure depicts that hydrogen production by the microbial fermentation using disclosed microbial consortium is in the range of 44-66ml/g TS by using lignocellulosic residues like rice straw, wheat straw, sugarcane bagasse, maize straw, and Napier grass as the substrate.

EXAMPLES

The following examples are given by way of illustration only and therefore should not be construed to limit the scope of the present invention in any manner.

EXAMPLE 1

The guts of worker termites (n=15) of genus Odontotermes were extracted using forceps from the posterior region of the gut and immediately homogenized in a sterile anaerobic diluent medium. The sterile anaerobic diluent medium is made by addition of 150 ml each of salt solution 1 (0.3% K₂HP0₄), and 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1 ml resazurin (0.1%, v/v), 5 ml sodium carbonate (8%, v/v), 0.5 gm L-cysteine hydrochloride, maintenance of pH at 7.0 ± 0.1 and making the final volume to 1000 ml with distilled water. The gut homogenate was inoculated separately into anaerobic bottles containing the nutrient medium. The nutrient medium is made up of 150 ml each of salt solution 1(0.3% K₂HP0₄), and salt solution 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1ml of resazurin (0.1 %, v/v), 0.5gm of Yeast extract, 1ml of Pfennig's trace mineral solution (0.03% H3BO3, 0.01% ZnS0₄.7H₂0, 0.003% MnCl₂.4H₂0, 0.002% COC1₂.6H₂0, 0.003% Na₂Mo0₄.2H₂0, 0.001% Na₂Se0₃, 0.002% NiCl₂, 0.001% CUC1₂.2H₂0, 0.015% FeCl₂.4H₂0), 2ml of haemin solution (0.05%, v/v), 0.5gm of L-cysteine hydrochloride, carbon source from either of 0.05% v/v lignin along with along with xylose 0.8% v/v or 1% w/v xylan or l%w/v cellulose or 5% w/v rice straw, maintenance of pH at 7.0 ± 0.1 and making the final volume to 1000 ml with distilled water.

All the enrichments were incubated anaerobically at 30°C for 7-8 weeks, following which xylan- and lignin-based enrichments showed growth. The positive enrichments were maintained on the respective medium and sub-cultured periodically for up to two years. In comparison, Bacteroidetes was the most dominant phylum in the xylan consortium, accounting for more than half of the total population, followed by Firmicutes and g- Proteobacteria. The genus level analysis revealed that among bacteria, Trabulsiella, Clostridium, Lachnoclostridium, Citrobacter, and Propionispora were the most dominant genera in the lignin consortium, whereas Dysgonomonas, Citrobacter, Trabulsiella, Desulfotomaculum, Clostridium, Oscillibacter and Propionispora dominated in the xylan consortium. Both these enrichments were pooled in a nutrient medium with rice straw as the sole substrate to let the microbial succession takes place, were incubated at 30°C for 3-5days, and subcultured routinely. The subsequent DNA isolation and metagenomic analysis of the rice straw consortium revealed a significant change in the bacterial population, dominated by different members of the genus Enterococcus, Clostridium, Bacteroides, Lachnoclostridium, and Dysgonomonas (Table 2). The rice straw consortium was used to isolate pure cultures of biohydrogen-producing bacteria following the standard anaerobic cultivation techniques. The nutrient medium for isolation comprises (per litres) of thel50 ml each of salt solution 1 (0.3% K₂HP0₄), and salt solution 2 [0.3% KH₂P0₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0], 1ml resazurin (0.1 %, v/v), 0.5gm of yeast extract, lml Pfennig's trace mineral solution (0.03% H3BO3, 0.01% ZnS0₄.7H₂0, 0.003% MnCl₂.4H₂0, 0.002% COC1₂.6H₂0, 0.003% Na₂Mo0₄.2H₂0, 0.001% Na₂Se0₃, 0.002% NiCl₂, 0.001% CUC1₂.2H₂0, 0.015% FeCl₂.4H₂0), 2ml haemin solution (0.05%, v/v), 0.5gm L-cysteine hydrochloride, agar powder (added individually at final concentration of 2% (w/v) and carbon source (Cellulose or Xylan or Rice Straw; added individually at final concentration of 1% (w/v), pH maintained at 7.0 ± 0.1 and making the final volume to 1000 ml with distilled water. The individual cultures were then checked for hydrogen production in a defined nutrient medium containing rice straw at 20-45°C, preferably at 30°Cfor 3-5 days at the oxidation-reduction potential of -200 to -500 millivolts under the anaerobic conditions. Overall, 66 pure bacterial cultures were obtained and screened for biohydrogen production from rice straw (Table 3). Five bacterial cultures were selected based on the highest biohydrogen production (>50%) and identified as Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513using

16S rRNA gene sequencing. These five cultures were mixed to develop the liquid microbial consortium for biohydrogen production.

Table 2 Composition of natural microbial consortia developed on lignin, xylan and rice straw as identified using a metagenomic approach

Table 3 shows the different strains of the microbial species, including Enterococcus, Clostridium, Bacteroides, Lachnoclostridium, and Dysgonomonas species with their percentage biohydrogen production

EXAMPLE 2

The liquid consortium comprising of the species Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513and the rice straw as the lignocellulosic crop residue was then used to produce recoverable biohydrogen in Stage I, followed by the generation of biomethane in Stage II, as illustrated in Figure 1. The rice straw as the lignocellulosic substrate is shredded to 20-50mm particle size, followed by pulverization to reach the particle size of l-5mm in a feeder (101). The shredded and pulverized rice straw biomass is fed to the soaking container (103) by a conveyor system for soaking at room temperature for 72h under open soaking conditions in 0.5% of NaOH. The soaked rice straw biomass is fed into a Stage I bioreactor (107) through a feed pump (105). Degradation and fermentation of the rice straw biomass take place by the liquid microbial consortium of the present invention at 20-45°C with a pH of 5-7.5 and agitation speed of 0-200rpm to produce biohydrogen, digestate and co-metabolites. The generated biohydrogen is collected in gas storage and purified to obtain pure usable biohydrogen following the separation of the CO2 and other gases in headspace gas. The Stage I reactor was fed daily with the soaked rice straw at 10 days of hydraulic retention time, and the biohydrogen production (Figures 3 and 5) was monitored. The resulting digestate from Stage I containing the said consortium, leftover substrate, and volatile fatty acids (Figure 4) was fed into the Stage II reactor containing the cattle dung-based start-up inoculum to produce biomethane (Figure 6), and CO2 and maintained at a 20 day hydraulic retention time.

ADVANTAGES OF THE INVENTION

• Simple, two-stage process for anaerobic digestion of lignocellulosic crop residues for direct production of the recoverable biohydrogen and biomethane • No addition or pumping of acid or alkali is required during biohydrogen production in Stage I and biomethane production in Stage-II reactor to maintain the pH in the reactors.

• Eliminates the sterilization/autoclaving of substrates, containers, equipment, and transfer lines/piping, thus reducing the time, energy, capital, and operational expenditure.

• Eliminates the use of thermochemical pretreatment, enzymatic pretreatment or metal catalysts.

• Facilitates ¾ production in the First Stage and eliminates CH₄, ¾S, and SO2 in headspace gas.

• Eliminates repetitive dosing of the microbial cultures.

• The bioreactor can be operated at a wide temperature range of 20°-45°C, and optimally at 30°C, thus, saving a lot of energy required to heat the anaerobic bioreactors, saving a lot of space required, and reducing the capital expenditure and the operational expenses.

• Digestate/co-metabolites produced alongside biohydrogen are not discarded but used as a substrate for the Stage II biomethanation process.

• Complete utilization of volatile fatty acids, thus, saving time and energy required in the post-process treatments of the digestate before release.

• Economically and commercially viable production of biohydrogen and biomethane, which can be used for various applications.

• The process is carried out in a range of reactor sizes and designs and is not restricted to reactors with some specific size and designs or requirements.

Claims

We Claim:

1. A liquid microbial consortium for biohydrogen generation, comprising of:

Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513.

2. The liquid microbial consortium for biohydrogen generation as claimed in claim 1, wherein the process for selective enrichment of microbial species comprises of: a. extracting guts of the worker termites of genus Odontotermes; b. homogenizing the guts immediately in a sterile anaerobic diluent medium comprising (per litre) of salt solution 1 containing 0.3% K₂HPO₄, and salt solution 2 containing 0.3% KH₂PO₄, 0.6% (NPL^SC^, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0, 1 ml resazurin (0.1%, v/v), 5 ml sodium carbonate (8%, v/v), 0.5 gm L-cysteine hydrochloride at the pH 7.0 ± 0.1; c. inoculating of gut homogenates in a nutrient medium which comprises (per litre) of salt solution 1 containing 0.3% K₂HPO₄, and salt solution 2 containing 0.3% KH₂PO₄, 0.6% (NH₄)₂S0₄, 0.6% NaCl, 0.06% MgS0₄.7H₂0 and 0.06% CaCl₂.2H₂0-, 1 ml resazurin (0.1%, v/v), 0.5 gm of yeast extract, 1 ml Pfennig’s trace mineral solution (0.03% H₃BO₃, 0.01% ZnS0₄-7H₂0, 0.003% MnCl₂.4H₂0, 0.002% COC1₂.6H₂0, 0.003% Na₂Mo0₄.2H₂0, 0.001% Na₂Se0₃, 0.002% NiCl₂, 0.001% CUC1₂.2H₂0, 0.015% FeCl₂.4H₂0), 2 ml haemin solution (0.05%, v/v), 0.5 gm L-cysteine hydrochloride and carbon source selected from either of 0.05%:0.8% (v/v) lignin: xylose, 1% w/v xylan, 1% w/v cellulose, 5% w/v rice straw, at pH 7.0 ± 0.1; d. incubating the inoculum at 20-45°C, preferably 30°C, for 7-8 weeks; e. maintaining the positive enrichments in the nutrient media with the xylan and the lignin as the carbon source by periodic subculturing into the respective fresh sterile nutrient medium. f. sub-culturing the inoculum periodically for two years at the oxidation- reduction potential of -200 to -500 millivolts under anaerobic conditions provided by removing dissolved oxygen, cooling on ice under the stream of oxygen-free nitrogen, dispersing into pre-gas sed bottles and sealing by aluminium crimps; g. pooling the inoculum in the isolation medium with rice straw as a substrate for microbial succession; h. incubating and sub-culturing the inoculum at 20-45°C, preferably at 30°C for 3-5 days at the oxidation-reduction potential of -200 to -500 millivolts under the anaerobic conditions, and i. isolating and cultivating individually the selectively enriched microbial species under the anaerobic conditions in the nutrient medium consisting of the rice straw for 48-72 hours at 20-45°C, preferably at 30°C. j. screening, selecting and combining the highest biohydrogen producing pure cultures to produce the liquid microbial consortium.

3. A process for the biohydrogen and the biomethane generation from Lignocellulosic crop residues by using the liquid microbial consortium as claimed in claim 1 comprises: a. Stage I leading to recoverable biohydrogen production, and b. Stage II leading to biomethane generation.

4. The process for the biohydrogen and the biomethane generation by using the liquid microbial consortium as claimed in claim 3, wherein the Stage I of biohydrogen generation comprises the steps of: a. shredding of the lignocellulosic crop residue to 20-50 mm, followed by pulverizing it to reach the particle size at 1-5 mm in a feeder (101); b. feeding of the shredded and pulverized lignocellulosic biomass by a conveyor system into a soaking container (103) at room temperature and soaking for 72h under open conditions; c. soaking of the lignocellulosic biomass in 0.5% of NaOH in the soaking container (103); d. feeding of the soaked biomass into a Stage I bioreactor (107) through a feed pump (105); e. degrading and fermenting the lignocellulosic biomass by the liquid microbial consortium at 20-45°C with pH of 5-7.5 and agitation speed of 0-200 rpm to produce biohydrogen, digestate and co-metabolites; f. collecting the generated biohydrogen in a gas storage (111), and g. purifying and extracting the pure and useable biohydrogen from the CO2 and other gases in headspace gas.

5. The process for biohydrogen and biomethane production by using the liquid microbial consortium as claimed in claim 3, wherein the Stage II of biomethane generation comprises the steps of: a. feeding of the digestate containing the liquid microbial consortium and the leftover lignocellulosic biomass from Stage I as well as volatile fatty acids into a Stage II bioreactor (109) by the feed pump (105); b. mixing of the content with cattle dung-based start-up inoculum in an anaerobic digester at 20-45° C, preferably at 37 °C with a pH of 6-7, agitation speed of 30-50 rpm and hydraulic retention time of 20 days at a 14 litter scale; c. purifying and extracting the pure and useable biomethane from the CO2 and other gases, and d. separating and recycling the digestate from the Stage II bioreactor.

6. The process for biohydrogen and biomethane production by using the liquid microbial consortium as claimed in claim 3, wherein the biohydrogen generation in Stage I and biomethane generation in Stage II is a semi-continuous step.

7. The process for biohydrogen and biomethane production by using the liquid microbial consortium as claimed in claim 3, wherein the lignocellulosic crop residues are selected from rice straw, wheat straw, sorghum, sugarcane bagasse and Napier grass.

8. The process for biohydrogen and biomethane production by using the liquid microbial consortium as claimed in claim 3, wherein Stage I produces 50-70 ml biohydrogen/g TS and Stage II produces 200 ml biomethane/g TS by using the rice straw as the lignocellulosic crop residue.

9. The process for biohydrogen and biomethane production by using the liquid microbial consortium as claimed in claim 3, wherein the biohydrogen production in Stage I is 60-70% by using the liquid microbial consortium comprising of the species Clostridium chromiireducens strain CTS0513, Clostridium chromiireducens strain STS0514, Clostridium chromiireducens strain XTS0511, Clostridium diolis strain STS0519, and Clostridium diolis strain XTS0513 and the rice straw as the lignocellulosic crop residue.

10. Use of biohydrogen and biomethane produced by the process as claimed in claim 3 for application including but not limited to at least fuel for vehicle, industrial application, chemical application, consumer application or the like.