Description:
FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE OF THE INVENTION – A MICROBIAL ENZYMATIC PRETREATMENT PROCESS FOR BIOGAS GENERATION FROM LIGNOCELLULOSE BIOMASS
2. Applicant (s)
NAME: CHEM PROCESS SYSTEMS PVT LTD
NATIONALITY: INDIAN
ADDRESS: UNIT 1: 15, NATRAJ INDUSTRIAL ESTATE. IYAVA VASNA,SANAND-VIRMGAM HIGHWAY, TA.SANAND, AHMEDABAD, GUJARAT -382170, INDIA.

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A MICROBIAL ENZYMATIC PRETREATMENT PROCESS FOR BIOGAS GENERATION FROM LIGNOCELLULOSE BIOMASS

FIELD OF THE INVENTION
The present invention provides a novel approach of a pretreatment process for biogas production. More particularly, the present invention is a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass.

BACKGROUND OF THE INVENTION
The growing emphasis in light of global warming and mitigating greenhouse gas emission and promoting sustainable and renewable energy solutions has positioned biogas as a vital contender in the global transition to green energy. Recognized for its environmentally-friendly attributes, biogas is increasingly replacing convnentional fossil fuels in various applications such as electricity generation, heating and vehicle fuel.

Biogas is a renewable energy source produced from raw materials such as agricultural waste, manure, municipal waste, plant material, sewage, green waste, wastewater, and food waste. Biogas production is generally produced by anaerobic digestion with anaerobic organisms or methanogens inside an anaerobic digester, biodigester or a bioreactor. The biogas production relies primarily on anaerobic digesters, which are sophisticated systems engineered mechanisms enhancing microbial activity to optimize the breakdown of organic matter in the absence of oxygen ensuring efficicent biogas production and maximizing energy yield. The performance of these systems depends significantly on the characteristics of the substrates used, making substrate selection and pretreatment critical factors in achieving high biogas output. One promising substrate category is lignocellulosic biomass.

Lignocellulose is the major structural unit of all plants. It consists of three components, viz., cellulose, hemicelluloses and lignin. Large quantities of lignocellulosic wastes are produced as a result of agricultural activities and it is also produced by many related industries. While lignocellulose is an excellent source of fermentable sugars, its tight bound components pose significant challenges for enzymatic and microbial accessibility, limiting the efficiency of biogas production. Converting lignocellulose raw materials into methane by means of anaerobic digestion is an effective way for recycling such substances, which not only can alleviate the energy crisis, but also alleviate the environmental problems caused by excessive use of fossil energy. However, so far, the lignocellulose raw material still has the problems of low utilization rate and low gas production rate because of its compact structure.

To overcome this limitation, advanced pretreatment techniques are essential to disrupt the lignin structure and expose cellulose and hemicellulose for effective microbial digestion. However available physical and chemical pretreatment methods are cost effective and may produce toxic by-products, making biological pre-treatment a more sustainable alternative. These innovations can significantly enhance the digestibility of lignocellulosic substrates, contributing to improved biogas yields and making anaerobic digestion an even more viable technology for renewable energy production.

US20180298286 patent application describes a method of treating a lignocellulosic biomass with a combined chemical and microbial process.

US11702711B2 patent application describes a method of treating biomass including lignocellulosic polymers. The biomass is treated in a mixture of water with at least one oxidizing agent and steam. The pH of the mixture is controlled and periodically measured for substantially an entire duration of the treating step.

The above patent applications describe the methods of treatment of lignocellulosic biomass, but the method mentioned in the above process is completely chemical based, whereas the present invention is a fully microbial consortium based pretreatment process.

There is a constant need of developing an easy and microbial process for such pretreatment. The inventors of the present invention have developed a novel pretreatment process for microbial digestion of lignocellulose that significantly enhance the digestibility of the lignocellulosic substrates, contributing to improved biogas yields and making anaerobic digestion an even more viable technology for renewable energy production.

OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass.

Another objective of the present invention is to provide a microbial consortium comprising bacteria for pretreating the lignocellulose biomass.

Another objective of the present invention is to provide a microbial consortium acting in synergy to efficiently degrade lignocellulosic biomass from various natural products.

Another objective of the present invention is to provide a microbial enzymatic pretreatment to significantly enhance the digestibility of lignocellulosic biomass.

Another objective of the present invention is to provide a microbial enzymatic pretreatment making the anaerobic digestion more viable and easy process for renewable energy production.

Another objective of the present invention is to provide a microbial enzymatic pretreatment to improve biogas yield.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass.

One more aspect of the present invention is to provide a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass, wherein said microbial consortium comprises bacterial species named Cytobacillus kochii (CPSNB2401), Klebsheilla pneumonia (CPSNB2402), Bacillus altitudinus (CPSNB2403) and Brucella pseudintermedia (CPSNB2404).

One more aspect of the present invention is to provide a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass, wherein the DNA sequence of the bacterial species have SEQ ID No:1 for Cytobacillus kochii, SEQ ID No:2 for Klebsheilla pneumonia, SEQ ID No:3 for Bacillus altitudinus and SEQ ID No:4 for Brucella pseudintermedia.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: illustrates screening of enzyme producing microbes.
Figure 2: illustrates colony characteristics of the microbes.
Figure 3: illustrates gram staining of the microbes
Figure 4: illustrates zone of clearance in Cellulose containing Plates.
Figure 5: illustrates acetic acid production in the hydrolysed sample where control has no peak and experimental samples show peaks for acetic acid in the hydrolysed media.
Figure 6: illustrates the process flow of biogas production using the microbial consortium.
Figure 7: illustrate wheat germination studies fo the microbial consortium.

DESCRIPTION OF THE INVENTION
The main embodiment of the present invention is to provide a microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass.

The detailed description set forth below is intended as a description of exemplary embodiments and is not intended to represent the only forms in which the exemplary embodiments may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and/or operating the exemplary embodiments. However, it is to be understood that the same or equivalent functions and sequences which may be accomplished by different exemplary methods are also intended to be encompassed within the spirit and scope of the invention.

As defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Although any process and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

As stated in the present invention herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to means approximately, in the region of, roughly, or around.

As stated herein, that it follows in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components but may also include additional features or components.

Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term "comprising" means "including, but not limited to.
The term used herein, “agar and broth” refers to types of growth medium used to grow microorganisms.

The term used herein, “microorganism” refers to a a very small living thing that you can only see with a special piece of equipment (a microscope).

The term used herein, “microbial consortium” refers to a group of diverse microorganisms that have the ability to act together in a community.

Another main embodiment of the present invention is to provide a microbial enzymatic process for biogas generation from lignocellulose biomass, wherein said process comprises following steps:
a. Growing the isolated microbes of microbial consortium at pH 6.5 to 7.2 for 08 to 24 hours in growth media;
b. Shredding the lignocellulosic biomass into 1-3 mm of particle size;by mechanical means.
c. Adding shredded biomass of step (b) in grown microbial culture of step (a);or vice versa.
d. Incubating or agitating the mixture of step (c) at 150 to 200 rpm for 48 hours at 32 to 36 deg C; for hydrolysis of sugars present in biomass into total volatile acids (TVA).
e. Feeding the hydrolyzed pre-treated biomass of step (d) into the anaerobic bio-digesters for anaerobic digestion of the pretreated biomass to generate biogas;
f. Commissioning of the anaerobic biodigesters of step (e) by acclimatized inoculum from existing anaerobic digester or cowdung.
g. Carrying out the anaerobic digestion of step (e) at hydraulic retention time (HRT) 18 to 30 days.

As per another embodiment, the said lignocellulose biomass is selected from agrowastes and energy crops such as cotton stalks, cotton residues, juli flora, paddy straw, cellulosic pulp, Bana grass, cattail grass, Napier grass, most preferably Napier grass and cotton stalks more preferably napier grass.

As per another embodiment, the said pretreatment of the lignocellulosic biomass is a bio-based microbial pretreatment.

As per another embodiment, the said microbial consortium comprises bacterial species named Cytobacillus kochii (CPSNB2401), Klebsheilla pneumonia (CPSNB2402), Bacillus altitudinus (CPSNB2403) and Brucella pseudintermedia (CPSNB2404).

As per another embodiment of the present invention, the DNA sequence of Cytobacillus kochii is as per SEQ ID No.1.

As per another embodiment of the present invention, the DNA sequence of Klebsheilla pneumonia is as per SEQ ID No.2.

As per another embodiment of the present invention, the DNA sequence of Bacillus altitudinus is as per SEQ ID No.3.

As per another embodiment of the present invention, the DNA sequence of Brucella pseudintermedia is as per SEQ ID No.4.

As per another embodiment of the present invention, the process of preparing the microbial consortium comprises following steps:
a) Maintaing the microbial strains on nutrient agar slant and storing them until further use;
b) Inoculating the microbial isolates of step (a) into sterile nutrient broth and incubating at 32 to 36 deg C for 48 hrs under aerobic condition;
c) Measuring the optical density (OD) of the cultures to standardize cell concentrations, ensuring consistent mixing ratios.
d) Following OD normalization, the strains were combined in equal proportions to achieve a final concentration of each organism 108 cells/mL in the prepared microbial consortium.

As per another embodiment of the present invention, the said pretreatment involves screening and isolating microbes which have high enzyme activity for the pretreatment and hydrolysis of the lignocellulosic biomass enzymes like cellulase, endo-exocellulases, amylase, xylanase, laccase, esterase, lipase.

Another embodiment of the present invention is to use the isolated microbes in synergy to efficiently hydrolyse the lignocellulosic biomass into sugars and volatile acids that are feeded in the anaerobic biodigester for efficient biogas production.

According to another embodiment of the present invention, the said microbial enzymatic pretreatment is effective under mesophilic condition of temperature range 32°C to 36°C for 24 to 48 hours.

According to another embodiment of the present invention, the said microbial enzymatic pretreatment is effective under thermophilic condition of temperature range 48°C to 52°C for 24 to 48 hours.

According to another embodiment of the present invention, the mesophilic condition of temperature range at 32°C to 36°C for 24 to 48 hours is the most preferred condition for the microbial enzymatic pretreatment process for biogas generation.

According to another embodiment of the present invention, the yield of biogas improves by 20 to 50%, more preferably 40 to 50 % after the microbial enzymatic pretreatment process.

The invention is further illustrated by the following examples, which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLE 1: SAMPLE COLLECTION
Various environmental samples, such as soil, biogas digestate, silage biomass were collected from diverse geographical locations such Biogas plant, paper and pulp industries, agricultural lands. The locations were chosen based on their potential known to harbour microorganisms with the ability to degrade complex lignocellulosic biomass. Here, the sample was collected from CHEM PROCESS SYSTEMS 25 PVT LTD located at 15, natraj Industrial Estate. Iyava Vasna, Sanand-Virmgam Highway, Ta.Sanand, Ahmedabad, Gujarat 382170, India.

EXAMPLE 2: SCREENING OF MICROBES ON SELECTIVE MEDIA
The samples collected were dispenced in sterile distilled water and plated on nutrient agar containg selective media containing,0.5% peptone as nitrogen source and 0.1% di potassium phosphate as nutientsource, the carbon source was one of the these 1% carboxy methyl cellulose( for cellulase producing microbes), 1% xylose ( for xylanase producers) 1% soda lignin (for laccase) producers and 1% starch ( for amylase).

The microbes were then streaked and plates were incubated at 33 to 36 deg C for 48 to 72 hrs, the growth and hydrolysis of the substrate released the dye giving clear zone of hydrolysis for the effective enzyme producers and such microbes were selected for further studies. The four microbes thus selected were named as CPSNB2401, CPSNB2402, CPSNB2403, CPSNB2404 and stored for further identification and characterization.

The individual microbes were also tested for their enzyme activity individually and in consortium.Activity of each microbe was analysed by inoculating 0.05 to 0.1% ( OD600 =1.0) of grown culture into production media containing 5 % w/w Bone dried biomas in 100 ml broth containing 0.5% peptone and 0.3% sodium chloride and 0.1% K2HPO4. The flasks with production media were incubated at 32 to 36 deg C and agitation 150-200 rpmfor 48 hrs. After incubation the production mass was centrifuged at 10,000 rpm , and the cell free supernatant was used as crude enzyme to study the enzyme profile of the microbes mixed culture demonstrated strongest activity.

Microbes CPSNB2401 CPSNB2402 CPSNB2403 CPSNB2404 Synergy
Enzyme activity U/ml U/ml U/ml U/ml U/ml
Fpase 6.30-6.84 7.55-8.82 5.90-6.13 4.21-5.56 7.4-9.20
Cellulase 0.29-1.31 0.31-1.64 0.29-1.20 0.29-0.98 0.39-1.88
Amylase 1.70-2.20 1.90-2.00 1.60-1.65 1.30-1.45 2.04-2.56
Xylanase 0.27-1.35 0.29-1.38 0.25-1.55 0.24-0.31 0.48-1.85
Laccase 0.55-1.02 1.81-2.15 0.53-1.47 0.55-1.06 1.07-2.51
lipase 0.23-0.90 ND 0.15-0.18 ND 0.22-1.07
Table 1: Enzyme activity profile of microbes

EXAMPLE 3: MORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF THE MICROBES OF CONSORTIUM
Characterization of the selected microbes was carried out on the basis of techniques described,colony characteristics, Gram’s staining, Urease test, sting test, catalase test were perfomed. Slants of pure cultures isolate ware sent to Microbial Type Culture Collection and Gene bank Chandigarh for identification by sequencing using 16s RNA sequencing and use of identify Analysis .EzTaxon server 2.1 Software and Genebank database. The said isolate or microorganism were identified as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 with sequence ID and having accession numbers MTCC 25880, MTCC 25881, MTCC 25882 and MTCC 25883 respectively.
Colony characteistics of microbes
Microbes CPSNB2401 CPSNB2402 CPSNB2403 CPSNB2404 Synergy
Parameters
Colony characteristics Small circular smooth edges, slightly raised creamy white in color Circular usually dome shaped and mucoid slightly greyish color White convex regular margin small translucent smooth shiny and moist colonies
Shape Rod shaped Rod shaped Rod shaped small rods
Gram characterisitc Gram positive Gram negative Gram positive Gram negative
Motility Motile Non motile Motile Non motile
Endospore Present Absent Present Absent
Capsule Absent Present Absent Absent
Urease Negative Positive Positive Positive Positive
Catalase Positive Positive Positive Positive Positive
KOH Sting Test Negative Positive Negative Positive
Growth condition Aerobic Aerobic Aerobic Aerobic Aerobic
pH range tolerance 4.0 to 7.5 4.0 to 7.5 4.0 to 7.5 4.0 to 7.5 4.0 to 7.5
Temperature tolerance 25 to 80 deg C 25 to 80 deg C 25 to 80 deg C 25 to 80 deg C 25 to 80 deg C
Groth on 1% CMCase plate and zone diameter .+nt Zone dia 150 mm .+nt Zone dia 300 mm .+nt Zone dia 180 mm .+nt Zone dia 270 mm .+nt Zone dia 355 mm
Growth on 1% Xylose plate Present Present Present Present Present
Growth on 1% soda lignin plate Present Present Present Present Present
Cell count at 1.0 OD600 on 5%w/w biomass after 48hrs 6.5 X 10ˆ8 cfu/ml 5.12 X 10ˆ8 cfu/ml 5.015 X 10ˆ8 cfu/ml 4.99 X 10ˆ8 cfu/ml 8.80 X 10ˆ8 cfu/ml
Table 2: Phynotypic Characteristics of the microbes

EXAMPLE 4: OPTIMIZATION OF INCUBATION PARAMETERS FOR NOVEL MICROORGANISM
The isolated microorganism were optimized for the growth conditions at various pH range varying from 4.0 to 8.0 and temperature range 32 to 80 deg C, growth in salinity was checked by incubating the microbes on nutrient agar plates containg increasing concentration of Sodium chloride 2 to 5% w/w. also the microbes were subjected to various aeration rpm from 100 to 200 rmp, the OD600 were measured and the optimum condition for the growth of microbes was established.

EXAMPLE 5: SYNERGISTIC APPROACH OF THE MICROBIAL CONSORTIUM
The isolated microbes were grown in nutrient broth under optimum conditions pH 6.0 to 7.0 for 48 hrs, the grown cultures OD600 1.0 . To 100 ml of grown culture in volumetric flask 25 gms of lignocellulosic biomass was added and incubated at 200 rpm for 48 hrs. After 48 hrs the mass was centrifuges solids removed and supernatant was checked for sugar and acid concentrations, the microbes synergy producing highest values of sugars and volatile acids released were taken up for further studies.
Parameters Total Volatile acids(TVA)
Microbes mg/L*
Control without innoculum 300-1660
CPSNB2401 1545-3800
CPSNB2402 1465-4510
CPSNB2403 1600-3950
CPSNB2404 1728-4230
Synergy 2000-5720
Table 3: Acid profile of filterate of experiments conducted with the microbes individually and in synergy

EXAMPLE 6: PROCESS FOR BIOGAS PRODUCTION USING MICROBIAL CONSORTIUM
Figure 6 demonstrates the process flow as per related invention 1 shows the microbial consortium grown under optimum condition and added to 4, wher proper shredded biomass is added from 2 and fresh water from 3 or recycles liquid fertilizer as per 12, from 4 the pretreated biomass lows to tank 5 where the temperature is increased to 42 to 48 deg C for further hydrolysis of the pretreated biomass by microbial enzymatic treatment.After treatment the biomass in taken to tank 6 from where it is feeded to anaerobic biodigester 7 and biogas produced in monitored at transferred to purification systems. The overflow from the digester 7 is collected in tank 9 from where it is transferred to solid liquid separator for separation of solid and liquid fertilizer.

EXAMPLE 7: BIOGAS EXPERIMENTS
Studies were carried out in lab scale bottle biodigester of capacity one litre, pilot plant biodigester capacity 2 M3. Control and experimental batches were run where the control batch had biomass without 48 hrs pretretment while the experimental digester had the biomass pretreated for 48 hrs in the microbial consortium. Biogas generation from both the digesters was monitored for 40 days under controlled temperature 34 to 38 deg C, with sample drawn for analysis of pH, TVA, alkalinity.Total solids, total volatile solids. Gas samples were checked for Methane, CO2, H2S content. The biogas values were recorded on daily basis and the difference in both treated and untreated batches were concuded.
The Digestate obtained at the end of 40 days was analysed for solids remaining, and the nutritive values of the Digestate.

The microbial enzymatic pretreatment process of the present invention for biogas generation from lignocellulose biomass from various natural products is a treatment used to enhance the digestibility of lignocellulosic biomass before anaerobic digestion. The pretreament of the lignocellulosic biomass with the microbial consortium increases the biogas yield by 40 to 50%.
Sr.No Substrate Control Experimental Control Experimental Increase %
M3/MT(TS) M3/MT(TS) M3/MT(VS) M3/MT(VS)
1 Napier Grass 385.00 600.00 452.94 705.88 35.83
2 Super Napier 239.11 558.82 281.31 657.99 57.25
3 Napier Grass 296.29 518.00 348.58 609.09 42.80
4 Cotton Pulp 435.00 957.00 490.50 1079.00 54.55
5 Paddy Straw 222.00 602.29 261.40 707.10 63.14
Table 4: Biogas generation from lignocellulosic biomass

EXAMPLE 8: TOXICITY STUDIES OF THE MICROBIAL CONSORTIUM
The toxicity study of the prepared microbial consortium were studied on the wheat germination and it was seen that there was no toxicity and the germination of wheat was similar to that in pure water. The results for the same germination are seen in the Figure 7, where the germination was seen.
 
, C , Claims:CLAIMS
We claim,
1. A microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass, wherein said process comprises following steps:
a. Growing the isolated microbes of microbial consortium at pH 6.5 to 7.2 for 08 to 24 hours in growth media cultures;
b. Shredding the biomass into 1-3 mm of particle size;
c. Adding shredded biomass of step (b) in grown microbial culture of step (a);
d. Incubating or agitating the mixture of step (c) at 150 to 200 rpm for 48 hours at 32 to 34 deg C; for hydrolysis of sugars present in biomass into total volatile acids (TVA).
e. Feeding the hydrolyzed pre-treated biomass of step (d) into the anaerobic bio-digesters for anaerobic digestion of the pretreated biomass to generate biogas;
f. Commissioning of the anaerobic biodigesters of step (e) by acclimatized inoculum from existing anaerobic digester or cowdung.
g. Carrying out the anaerobic digestion of step (e) at hydraulic retention time (HRT) 18 to 30 days.
2. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein said microbial consortium comprises bacterial species named Cytobacillus kochii, Klebsheilla pneumonia, Bacillus altitudinus and Brucella pseudintermedia.
3. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein said lignocellulose biomass is selected from agrowastes and energy crops such as cotton stalks, cotton residues, juli flora, paddy straw, cellulosic pulp, Bana grass, cattail grass, Napier grass, most preferably Napier grass.
4. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein the DNA sequence of Cytobacillus kochii is as per SEQ ID No.1.
5. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein the DNA sequence of Klebsheilla pneumonia is as per SEQ ID No.2.
6. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein the DNA sequence of Bacillus altitudinus is as per SEQ ID No.3.
7. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein the DNA sequence of Brucella pseudintermedia is as per SEQ ID No.4.
8. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein said microbial enzymatic pretreatment is effective under mesophilic condition of temperature range 32°C to 36°C for 24 to 48 hours.
9. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein said microbial enzymatic pretreatment is effective under thermophilic condition of temperature range 48°C to 52°C for 24 to 48 hours.
10. The microbial enzymatic pretreatment process for biogas generation from lignocellulose biomass as claimed in claim 1, wherein biogas yield improves by 20 to 50%, more preferably 40 to 50 %.

Dated this 29th Jan 2024