DESC:FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENT RULES, 2003

COMPLETE SPECIFICATION
(See Section 10 and Rule 13)

TITLE OF INVENTION:
PROCESS FOR PRODUCING BIOGAS FROM LIGNOCELLULOSIC FEEDSTOCK

APPLICANT:
PRAJ INDUSTRIES LTD.
An Indian entity having registered address as:
PRAJ Tower, 274-275, Bhumkar Chowk- Hinjewadi Road,
Hinjewadi, Pune – 411057, India

The following specification particularly describes the invention and the manner in which it is to be performed.
CROSS-REFERENCE TO RELATED APPLICATIONS AND PRIORITY
The present specification claims priority from Provisional Patent Application 202221073755 filed on December 20, 2022, in India.

TECHNICAL FIELD
The present subject matter, in general, relates to the field of process chemistry. The present disclosure particularly relates to a process for producing biogas from lignocellulosic feedstock.
BACKGROUND
Biogas, a renewable and environment-friendly energy source, is a promising sustainable alternative to fossil fuels that can be used for the production of heat, power, and transport fuels and thus make a large contribution to reducing greenhouse gas emissions.
Anaerobic digestion (AD) for biomethanation (BM) entails a multi-step biochemical process involving microbial break down of organic matter in the absence of oxygen to generate bioenergy in the form of biogas. Biomethanation yields biogas containing about 60% methane by volume and a residual digestate of stable total solids that after appropriate treatment serves as an excellent fertilizer. Such high content of methane makes biogas a useful fuel that can replace natural gas in pipelines or be converted to electricity and heat.
AD offers several environmental and economic benefits, including waste reduction, greenhouse gas mitigation, and the generation of renewable energy. It plays a crucial role in sustainable waste management and the transition to a more circular and environmentally friendly economy.
Lignocellulosic feedstock is abundantly available in nature. It is often derived from sources like industrial waste, forestry residues, and agricultural byproducts such as crop stalks, leaves, roots, fruit peels, and seed/nut shells. It comprises of about 20-50% cellulose, 15-35% hemicellulose and 5-30% lignin, and presently poses as a significant untapped source for biogas production.
However, there are various challenges in the direct application of lignocellulosic feedstock as an energy source. The implementation and course of AD process is determined by both chemical and physical characteristics of the lignocellulosic feedstock in addition to complexity of feedstock composition, nutrient imbalance, feedstock variability, and the like. Moreover, the growth and activity of anaerobic microorganisms that directly impacts biogas production is influenced by process conditions such as particle size of the feedstock, partial pressure of oxygen, temperature stability, pH, nutrient supply, agitation as well as presence and number of inhibitors. Hence, these parameters are required to be maintained within a desirable range to ensure optimal activity of the microbes.
Nevertheless, the primary challenge hindering optimal utilization of lignocellulosic feedstock is the encasing and/or binding of hemicellulose and lignin on/to cellulose, making it difficult for cellulolytic enzymes such as cellulases to access and degrade cellulose during the fermentation step. Thus, although cellulose and hemicellulose are polysaccharides that can be hydrolysed to simple sugars, lignin hinders their exposure to microbial enzymes thus limiting their hydrolysis. Hence, the composition of lignocellulosic feedstock contributes/causes its limited biodegradability and exceptionally slow rates of sugar hydrolysis, acidolysis, and methanogenesis.
The most efficient approach to overcome the recalcitrant nature of lignocellulose material and consequently improve its digestion efficiency or degradation rate involves disrupting the chemical bonds in the lignocellulosic material making it prone to hydrolysis. This can be achieved by introducing a pretreatment step prior to the AD process. Pretreatment disrupts the lignin layer that protects the cellulose and hemicellulose and renders the feedstock more accessible for digestion.
The current major pretreatment methods include physical, mechanical, chemical, thermophysical, and thermochemical approaches. Physical pretreatment methods effectively destroy the compact structure of lignocellulose but generally require high energy and specialized equipment. Although chemical pretreatment has a good effect on improving the gas yield of anaerobic fermentation, it often leads to the formation of waste streams which cause secondary pollution. In addition, it leads to the formation of undesired impurities which may inhibit further downstream biological reactions.
Mechanical processes that fragment the lignocellulosic feedstock to shorter / incised strands increases the surface area available for digestion, thereby improving the conversion rate of the feedstock to the desired products. Thus, size reduction efficiently breaks down the complex lignocellulosic structure easing access to cellulosic sugars and increasing the contact area for the action of biocatalysts that are supplied in the form of crude or purified isolates or microbial whole cells. Additionally, it also lowers viscosity in the digesters making mixing easier and reducing the problem posed due to floating layers. Further, the preferred particle size distribution also involves finding a balance between promoting the maximum biological activity and maintaining physical and biochemical stability. Moreover, a pretreatment procedure must combine different techniques to fulfil the aforementioned requirements and demonstrate effectiveness by producing lignocellulosic feedstock of a specific size. Therefore, the present invention is directed to a mechanobiological pretreatment of lignocellulosic feedstock involving combined physical or mechanical and biological pretreatment approaches. In the present invention, the lignocellulosic feedstock is subjected to biological disintegration post-size reduction by mechanical methods rendering it more amenable to further biological treatments during the AD process for biogas production.

SUMMARY
An embodiment of the instant disclosure relates to a process (100) for producing biogas from lignocellulosic feedstock comprising, selecting lignocellulosic feedstock (101); processing the lignocellulosic feedstock by reducing their size to obtain processed lignocellulosic feedstock (102); pre-treating the processed lignocellulosic feedstock with at least one micro-organism to obtain pre-treated lignocellulosic feedstock (103); and anaerobically digesting the pre-treated lignocellulosic feedstock to obtain biogas (104); such that, yield of the biogas is improved.
This summary is not intended to identify all the essential features of the claimed subject matter, nor is it intended to be used in determining or limiting the scope of the claimed subject matter.
BRIEF DESCRIPTION OF DRAWINGS
A detailed description of the drawings is outlined with reference to the accompanying figures. In the figures, the left-most digit (s) of a reference number identifies the Figure in which the reference number first appears. The same numbers are used throughout the drawings to refer to features and components.
Figure 1 illustrates the process flow diagram (PFD) for producing biogas from lignocellulosic feedstock.
Figure 2 (a, b) illustrates the scanning electron microscopic (SEM) images of shredded rice straw.
Figure 3 (a, b) illustrates the scanning electron microscopic (SEM) images of milled rice straw.
Figure 4 (A) illustrates the scanning electron microscopic (SEM) images of extruded rice straw; (B) plug formation; and (C) phytolith
Figure 5 illustrates the scanning electron microscopic (SEM) image of rice straw material after pretreatment with at least one microorganism.
Figure 6 illustrates the scanning electron microscopic (SEM) image of the morphological changes after complete degradation of rice straw during anaerobic digestion.
Figure 7 illustrates effect of HRT on biogas production.
DETAILED DESCRIPTION
Reference throughout the specification to “various embodiments,” “some embodiments,” “one embodiment,” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases “in various embodiments,” “in some embodiments,” “in one embodiment,” or “in an embodiment” in places throughout the specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
Reference throughout the specification to “components”, “component”, “features”, or “feature” means a constituent or group of constituents embodying the process.
Before the present process is described, it is to be understood that this disclosure is not limited to the particular process as described, as there can be multiple possible embodiments which are not expressly illustrated in the present disclosure but may still be practicable within the scope of the present disclosure.
Also, the technical solutions offered by the present disclosure are clearly and completely described below. Examples in which specific reagents or conditions may not have been specified have been conducted under conventional conditions or in a manner recommended by the manufacturer.
An aspect of the instant disclosure relates to a process for producing biogas; particularly from lignocellulosic feedstock.
For the purpose of instant disclosure, the term “lignocellulosic feedstock” or “feedstock” pertains to conventionally known feedstock or biomass material.

An aspect of the instant disclosure relates to a process for producing biogas from lignocellulosic feedstock comprising, selecting lignocellulosic feedstock; processing the lignocellulosic feedstock by reducing their size to obtain processed lignocellulosic feedstock; pre-treating the processed lignocellulosic feedstock with at least one micro-organism to obtain pre-treated lignocellulosic feedstock; and anaerobically digesting the pre-treated lignocellulosic feedstock to obtain biogas; such that, yield of the biogas is improved.
An embodiment relates to selecting lignocellulosic feedstock.
In a related embodiment, lignocellulosic feedstock is selected from conventionally known feedstock/biomass material; and preferably selected from rice straw, wheat straw, cotton stalk, Juli flora, groundnut shell, palm oil empty fruit bunch, bagasse, and soyabean straw.
In a further related embodiment, lignocellulosic feedstock comprises total solids (TS), total volatile solids (TVS), lignin, carbohydrates, and ash in a range of about 55– 95 % w/w, 50-90% w/w, 11-32% w/w, 28-55%, and 3-13%, respectively.

Another embodiment relates to processing lignocellulosic feedstock; preferably, by reducing their size; and particularly to obtain processed lignocellulosic feedstock.
In a related embodiment, the processing of the lignocellulosic feedstock is carried out through at least one of shredding, milling, extrusion, and plug screw.
In a further related embodiment, the processing of the lignocellulosic feedstock is carried out through shredding or milling with at least one of extrusion or plug screw.
In yet another related embodiment, the lignocellulosic feedstock is processed to particle size distribution of = 20 mm; and preferably, to particle size distribution of = 5 mm.
In a further related embodiment, shredded/milled lignocellulosic feedstock having 10 - 90% w/w total solids; and preferably, around 15 - 60% w/w total solids is used as inlet.

Yet another embodiment relates to pre-treating the processed lignocellulosic feedstock to obtain pre-treated lignocellulosic feedstock.
In an embodiment, the pre-treatment is carried out with at least one of conventionally known enzymes and/or at least one micro-organism; and preferably, at least one microorganism.
In a related embodiment, the pretreatment of the processed lignocellulosic feedstock increases total volatile acid (TVA); preferably, by 3 times.
In another related embodiment, the pretreatment of the processed lignocellulosic feedstock is carried out at 37°C to 55°C.
In yet another related embodiment, the pretreatment of the processed lignocellulosic feedstock is carried for 12 to 96 h; and preferably, for 24 to 96 h.
In a further related embodiment, concentration of the micro-organism is 0.5-10%.
In an embodiment, the microorganism belongs to genus Bacillus, Lactobacillus, Pediococcus, and Thermobifida; preferably, the microorganism belongs to genus Thermobifida; and particularly, the microorganism is Thermobifida fusca.
In another embodiment, the processed lignocellulosic feedstock is added to a pretreatment section (PTR); and preferably, 0.5% - 10% Thermobifida fusca is added and mixed using aeration; particularly, at about 37-55°C and continued for about 12-96 h.
In yet another embodiment, high yield of total volatile acids (TVA) is produced with Thermobifida fusca pretreatment.
In a related embodiment, TVA produced with Thermobifida fusca pretreatment is about 2000-4500 ppm.
For the purpose of this disclosure and as is perceivable to a person skilled in the art, the term PTR, and/or digester pertain to fermenter, generator, bioreactor, vessel, jar, tank, or any manufactured device or system that supports a biologically active environment.
In an embodiment, the PTR, and/or digester is rectangular, cylindrical, or circular shaped; and preferably, cylindrical shaped. Furthermore, in a related embodiment, the PTR, and/or digester is horizontally, diagonally, or vertically angled.
In another embodiment, the PTR, and/or digester is a small, medium, or large capacity apparatus.
In a further embodiment, volume of the PTR, and/or digester is any volume from 1L to several thousands of L, as per requirements.

A further embodiment relates to anaerobically digesting the pre-treated lignocellulosic feedstock; preferably, to obtain biogas.
In a related embodiment, the anaerobic digestion of the pre-treated lignocellulosic feedstock is carried out using acclimatized cow dung.
For the purpose of instant disclosure, the term “acclimatized cow dung” pertains to commercially available cow dung diluted with water and incubated for microbial enrichment and activation.
In a related embodiment, cow dung is diluted to about 4-5% with water.
In another related embodiment, cow dung is incubated at about 35°C to 40°C.
In yet another embodiment, cow dung is incubated for 2.5 to 4 weeks; and preferably, for about 2 weeks.
In another related embodiment, the anaerobic digestion of the pre-treated lignocellulosic feedstock provides at least one of biomethanated solids and biomethanated liquid.
In a further related embodiment, total volatile solids (TVS) of the biomethanated solids is up to 20% w/w.
In yet another further related embodiment, total volatile solids (TVS) of the biomethanated liquid is up to 2% w/w.
In an embodiment, the acclimatized cow dung comprises 4-5% of total solids.
In another embodiment, hydraulic retention time (HRT) for producing the biogas is 10 to 28 days; and preferably, is 14 to 28 days.
In a further embodiment, conventionally implemented digester, as per requirements, is filled initially with anaerobic sludge having about 15-20% total solids and acclimatized cow dung slurry containing about 4-5% total solids. The anaerobic digester is allowed to stabilize for a period two to three weeks. Digester temperature is maintained at about 37±1? during stabilization and overall bio methanation process. pH is stabilized/maintained between 6.5 to 7.0.
In yet another further embodiment, the digestate subjected to separation; and preferably, solid liquid separation using conventionally known separation means.
For the purpose of instant disclosure and as is conventionally known to a person skilled in the art, the term “digestate” pertains to the material remaining/retentate after the anaerobic digestion of pre-treated feedstock.

An embodiment of the instant disclosure relates to selecting (101) and processing lignocellulosic feedstock (102), wherein, the lignocellulosic feedstock is subjected to shredding and/or milling to obtain processed lignocellulosic feedstock with particle size distribution of around = 30 mm, and preferably, ranging from 10 mm to 30 mm. Further, the shredded and/or milled lignocellulosic feedstock is subjected to extrusion or plug screw (formation) (with single/double screw) for further size reduction to obtain processed lignocellulosic feedstock with particle size distribution of around = 10 mm, and particularly = 5 mm.
For the purpose of instant disclosure, the term “about”/ “around” pertains to fundamentally perceivable ± 2.
Another embodiment relates to pre-treating lignocellulosic feedstock with at least one microorganism to obtain pre-treated lignocellulosic feedstock (103) wherein, the processed lignocellulosic feedstock is added to a PTR. To this, about 0.5 % to 10% Thermobifida fusca culture is added and mixed using agitator and aeration. The pretreatment reaction is carried out at about 37°C to 55°C and for about 24 to 96 h. Post treatment, part of pretreated lignocellulosic feedstock is transferred to anaerobic digester. For continuous pretreatment process, once the pretreated lignocellulosic feedstock is transferred, same volume of processed lignocellulosic feedstock is added to PTR.
Further embodiment relates to anaerobically digesting/biomethanation the pre-treated lignocellulosic feedstock to obtain biogas (104), wherein, the digester is filled initially with anaerobic sludge having about 15-20% total solids and acclimatized cow dung slurry containing about 4-5% total solids and anaerobic digestion is carried out as described previously.
In another further embodiment, after HRT, the entire digestate is subjected to solid liquid separation to obtain wet biomethanated solids having moisture, and biomethanated liquid having solids. Raw biogas is obtained.
In a related embodiment, digestate obtained is further subjected to solid/liquid separation using screw press to obtain biomethanated solids and biomethanated liquid.

In an embodiment, the yield of the biogas is improved.
In a related embodiment, the yield of the biogas is improved to = 200 AM3/ton; preferably, to = 250 AM3/ton, and particularly, to = 400 AM3/ton.

In an embodiment, biomethanated solids and/or biomethanated liquids are applied as biofertilizers.

Various modifications to the embodiment will be readily apparent to those skilled in the art and the generic principles herein may be applied to other embodiments. However, one of ordinary skill in the art will readily recognize that the present disclosure is not intended to be limited to the embodiments illustrated but is to be accorded the widest scope consistent with the principles and features described herein.
The foregoing description shall be interpreted as illustrative and not in any limiting sense. A person of ordinary skill in the art would understand that certain modifications could come within the scope of this disclosure. The features and properties of the present disclosure are described in further detail below with reference to examples.
Example 1
Processing lignocellulosic feedstock by reducing their size to obtain processed lignocellulosic feedstock
A) Selecting lignocellulosic feedstock:
Lignocellulosic feedstock was selected from rice straw, wheat straw, cotton stalk, Juli flora, groundnut shell, palm oil empty fruit bunch, bagasse, and soyabean straw comprising total solids (TS), total volatile solids (TVS), lignin, carbohydrates, and ash in a range of about 55– 95 % w/w, 50-90% w/w, 11-32% w/w, 28-55%, and 3-13%, respectively for the purpose of the present disclosure.
B) Processing lignocellulosic feedstock
About 100 kg of rice straw (90% total solids) sourced from Raigad, Maharashtra, India, was subjected to at least one of shredding and milling to obtain processed lignocellulosic feedstock with particle size distribution of around = 20 mm. Alternatively, the shredded and/or milled lignocellulosic feedstock was further subjected to at least one of extrusion or plug screw (with single/double screw) for further size reduction to obtain processed lignocellulosic feedstock with particle size distribution of around = 5 mm.
In case of extrusion, shredded/milled rice straw having 40 - 60% w/w total solids was used as inlet feed, and rice straw having 45 - 55% w/w total solids was used as outlet feed. In case of plug screw, shredded/milled rice straw having 18 - 30% w/w total solids was used as inlet feed, and rice straw having 45 - 55% w/w total solids was used as outlet feed. The processed lignocellulosic feedstock obtained from plug screw (single) was without steam and chemical.
Morphological study of the processed lignocellulosic feedstock was carried out using conventionally applied field emission scanning electron microscope (see Figs. 2-6). As per Fig. 2 (a, b), in case of shredded rice straw inter and intra wall were intact including phytolith structure. As per Fig. 3 (a, b), in case of milled rice straw, inter, and intra cell wall of rice straw were completely opened up providing accessibility to cellulosic layer for further degradation of cellulosic layer during microbial pretreatment process. The silica containing phytolith structure were intact. As per Fig. 4, in case of extrusion (A) and plug formation (B) mechanical size reduction, intra and inter cell was broken down including phytolith structure (C). Occluded organic matter and silica was released after disruption of phytolith layer providing higher accessibility for cellulosic layer during the pre-treatment of processed lignocellulosic feedstock (see Fig. 5) (described below). The released organic matter from phytolith structure can also be utilized during anaerobic digestion for enhanced bio methanation process resulting in higher biogas yield (see Fig. 6).

Example 2
Selection of microorganism to obtain pre-treated lignocellulosic feedstock.
Microorganisms such as Bacillus subtilis (BS) MTCC 2414, Bacillus subtilis (BS) MTCC 2415, , Bacillus licheniformis (BL) NCIM 2051, Lactobacillus fermentum (LF) MTCC 9748, Lactobacillus bifermentans (LBF) MTCC 3818, Lactobacillus plantarum (LP) MTCC 9495, Pediococcus pentosaceous (PP) ATCC 43200 (USA), and Thermobifida fusca (TF) NBRC 14071 (Japan), were screened for their ability to grow in liquid media comprising 1% carboxy methyl cellulose (CMC) as sole carbon source. Total viable count (TVC, cfu/ml) was quantified using serial dilution method on an agar plate comprising about 1% CMC as the sole carbon source (see Table 1). De Man, Rogosa and Sharpe (MRS) medium was implemented for culturing Lactobacillus and nutrient broth (NB) solid agar medium was implemented for culturing Bacillus and T. fusca.

Table 1: TVC medium containing 1% CMC
Medium/ Microbe Lactobacillus fermentum MTCC 9748 Lactobacillus bifermentans MTCC 3818 Lactobacillus plantarum MTCC 9495 Pediococcus pentosaceous ATCC 43200 Bacillus subtilis (BS) MTCC 2414 Bacillus subtilis (BS) MTCC 2415 Bacillus licheniformis NCIM 2051 Thermobifida fusca NBRC 14071
MRS without sugar + 1% CMC 40 0 0 0 NT NT NT NT
Minimal medium +1%CMC 0 0 0 0 NT NT NT NT
NB+1% CMC NT NT NT NT 2x106 3.2x105 1.2x104 3.4x105
Minimal medium with +1%CMC NT NT NT NT 1.8x104 2.2x104 4.5x102 4.2x104
*NT- Not tested since test microbes were not growing on minimal MRS and NB media respectively.
Microorganisms growing on minimal media with CMC as a carbon source were further implemented to screen strain development on minimal media supplemented with about 1% rice straw (RS) and 1% cotton stalk (CS) media. The strains developed through feedstock adaption were then banked in cell bank as pretreatment microorganisms for further evaluation in bio methanation/biogas production (see Table 2).
Table 2: Growth of microorganisms used for pretreatment of rice straw and cotton stalk media
TVC (cfu/ml) Bacillus subtilis (BS) MTCC 2414 Bacillus subtilis BGSC 1A1 Bacillus licheniformis NCIM 2051 Thermobifida fusca NBRC 14071
Minimal medium with 1% RS 1.1x102 2.4x102 1.7x101 7.2x108
Minimal medium with 1% CS 4.1x102 3.7x101 1x101 5.4x107
As per Table 2, Thermobifida fusca demonstrated better proliferative and degradative activity. So, Thermobifida fusca was implemented for pre-treating the processed lignocellulosic feedstock to obtain pre-treated lignocellulosic feedstock in the present disclosure.
Example 3
Preparation of Inoculum for carrying out pre-treatment of lignocellulosic feedstock.
Thermobifida fusca glycerol stock was added to about 100 ml nutrient broth and incubated at about 45? for about 48 h to obtain pre-grown culture which was used as the inoculum for carrying out optimization studies.
A) Optimization of Thermobifida fusca % inoculum
About 8% w/w processed rice straw achieved through Example 1 was pretreated with varying Thermobifida fusca inoculum percentage of 0.5 to 10% at about 45? for 48 h (see Table 5). Acclimatized cow dung as inoculum, Thermobifida fusca pretreated rice straw and water was added to make 3L reaction volume. The anaerobic biomethanation process was carried out at 37±1? and 70 rpm agitation. Methane and carbon dioxide were analysed by biogas analyser. Biogas volume was measured by water displacement method (see Table 3).
Table 3: Effect of Thermobifida fusca % inoculum on biogas generation
Thermobifida fusca inoculum (%) for rice straw pretreatment Biogas Parameters
Biogas Volume (L) Methane (%v/v) CO2 (%v/v)
0.5 22 46 30.1
1 25 52.1 34.7
2 30 56.2 36.1
5 32 55.6 36.8
10 32 55.1 35.2
As per Table 3, optimum biogas yield of about 30 L and methane concentration of about 56.2% v/v were obtained using about 2% inoculum. So, about 2% inoculum was implemented for further experimentation considering the requirements of biogas volume and methane concentration.
B) Optimization of Thermobifida fusca pretreatment temperature
About 8% w/w processed rice straw achieved through Example 1 was pretreated with about 2% Thermobifida fusca at varying temperatures ranging from 37°C to 55°C (see Table 4). Acclimatized cow dung as inoculum, Thermobifida fusca pretreated rice straw, and water was added to make 3L reaction volume. The anaerobic biomethanation process was carried out at 37±1? and 70 rpm agitation. Methane and carbon dioxide were analyzed by biogas analyzer. Biogas volume was measured by water displacement method (see Table 4).
Table 4: Effect of Thermobifida fusca pretreatment temperature on biogas generation
Temperature (?) Biogas Parameters
Biogas Volume (L) Methane (%v/v) CO2 (%v/v)
37 25 53.1 34.7
45 30 56.2 36.1
55 26 54.1 37.1
As per Table 4, maximum biogas generation of about 30L and methane concentration of about 56.2% v/v were achieved at 45?. Hence, 45? was selected for further experimentation.
C) Optimization of Thermobifida fusca pretreatment time
About 8% w/w processed rice straw achieved through Example 1 was pretreated with about 2% Thermobifida fusca at varying time intervals from 12 h to 96 h (see Table 5), and total volatile acids (TVA) were analyzed.
Table 5: Effect of Thermobifida fusca pretreatment time on TVA
Time (hr) TVA (ppm)
12 218
18 945
24 2097
30 3003
48 4500
60 749
72 600
96 550
As seen in Table 5, maximum total volatile acid formation was observed at about 48 h. Hence, Thermobifida fusca pretreatment time was optimized to 48 hours.

Example 4
Pre-treating processed lignocellulosic feedstock.
Processed lignocellulosic feedstock obtained in Example 1 was added to pretreatment reactor (PTR). To this, about 2% Thermobifida fusca culture screened through Example 2, and optimized through Example 3 was added using agitator and aeration. The pretreatment reaction was carried out at about 45? for about 48 h, as optimized through Example 3.

Example 5
Comparative study of Total volatile acids (TVA) with and without Thermobifida fusca pre-treatment
About 8% w/w of processed rice straw achieved through Example 1 was pre-treated with and without Thermobifida fusca at about 45±2? for about 48 h and analyzed for TVA (see Table 6).
Table 6: Total TVA with and without Thermobifida fusca pretreatment
Pretreatment Total volatile acids (ppm)
Without T. fusca 300-1500
With T. fusca 3000-4500

As per Table 6, TVA produced without Thermobifida fusca pretreatment was about 300-1500 ppm and with Thermobifida fusca pretreatment was about 3000-4500 ppm; thereby deducing that the pretreatment of the processed lignocellulosic feedstock with Thermobifida fusca increases total volatile acid (TVA) by 3 times.

Example 6
Comparative study of enzyme and Thermobifida fusca pretreated lignocellulosic feedstock on biogas generation
About 8% w/w of processed rice straw achieved through Example 1 was pretreated with cellulase enzyme sourced from Novozymes, India at a dose of about 30 mg/g of cellulose at about 37? for about 24 h. In another set, the processed rice straw achieved through Example 1 was pretreated with about 2% Thermobifida fusca culture at about 45? for about 48 h. Acclimatized cow dung as inoculum, Thermobifida fusca pretreated rice straw and water was added to make 3L reaction volume. The anaerobic biomethanation process was carried out at 37±1? and 70 rpm agitation. The pretreated rice straw was analysed for total volatile acids (TVA) and biogas generation (see Table 7).
[Table 7]
Sr. No. Pretreatment TVA (ppm) Biogas yield (M3/DMT) Biogas volume
1. Enzymatic 1500-2000 230-250 22
2. BMSolve 3000-3500 350-400 30
As per Table 7, maximum volatile acids and biogas generation was achieved through Thermobifida fusca pre-treatment.
Example 7
Anaerobically digesting pre-treated lignocellulosic feedstock to obtain biogas
The digester was filled initially with anaerobic sludge having about 15-20% total solids and acclimatized cow dung slurry containing about 4-5% total solids. The anaerobic digester was allowed to stabilize for a period two to three weeks. Digester temperature was maintained at about 37±1? during stabilization and overall bio methanation process. Digester pH was stabilized/maintained between 6.5 to 7.0. Digester was stabilized within 10-12 days of inoculum addition.

Example 8
Optimization of cow dung inoculum ratio for biogas generation
Acclimatized cow dung was obtained by diluting cow dung to 4-5% with water and incubated at about 37? for two weeks for microbial enrichment and activation.
Acclimatized cow dung ranging from 0.1 to 50% and comprising about 4-5% w/w total solids was used as inoculum with Thermobifida fusca pretreated milled rice straw achieved through Example 4, and water to make the final reaction volume of 3L (see Table 8). ¬The initial pH was adjusted to about 7.5±0.2. Nitrogen was flushed into flasks to maintain the anaerobic environment and biogas was collected in the attached balloon. Incubation was carried out at about 37±0.2? and about 70 rpm. Methane and carbon dioxide were analysed using a biogas analyser. Biogas volume was measured by water displacement method.
Table 8: Optimization of cow dung inoculum ratio bio methanation (biogas production)
Sr. No. Parameters Cow dung
(50% inoculum) Cow dung
(10% inoculum) Cow dung
(1% inoculum) Cow dung
(0.1% inoculum)
1 Cow dung (gm) 1500 300 30 3
5 Biogas Volume (L) 27 8 1 0.5
6 CH4 (%v/v) 55 45 3.1 1.0
7 CO2 (%v/v) 38 30 10.2 6.2
As per Table 8, maximum biogas volume and methane concentration was achieved at about 50% inoculum. So, about 50% cow dung inoculum was selected for further experimentation.

Example 9
HRT optimization
About 8% w/w pretreated rice straw was added to the anaerobic digester containing acclimatized cow dung. The pH of anaerobic digester was maintained to about 7±0.2. Digester incubation was carried at about 37±0.5?, and at around 70 rpm agitation. Methane and carbon dioxide were analyzed by biogas analyzer and biogas volume was measured by water displacement method (see Table 9).
Table 9: Effect of HRT on biogas production
HRT (Days) Biogas Yield (M3/DMT)
28 425
25 420
20 396
14 350
10 263
As per Table 9, and Fig. 7, biogas yield of about 350 - 420M3/DMT was achieved through about 14-25 days HRT with an average methane concentration of about 55% v/v and carbon dioxide concentration of about 40% v/v.

Example 10
Process for producing biogas from lignocellulosic feedstock (see Fig. 1)
Selecting lignocellulosic feedstock and processing of lignocellulosic feedstock by reducing their size to obtain processed lignocellulosic feedstock (101) and (102):
About 100 kg of rice straw (90% total solids) sourced from Raigad, Maharashtra, India, was subjected to shredding as well as milling to obtain processed lignocellulosic feedstock with particle size distribution of = 15 mm. Further, the shredded and/or milled lignocellulosic feedstock was subjected to extrusion as well as plug screw (formation) (with single/double screw) for further size reduction to obtain processed lignocellulosic feedstock with particle size distribution = 5 mm. The effect of processing (shredder and milling), as well as the effect of further processing (extrusion or plug screw (formation)) on final product (biogas) yield, particle size, and PTR TVA is illustrated through Tables 10, and 11.
Table 10: Particle size, PTR TVA and biogas yield after shredding and milling with particle size.
Description Units Shredder Mill
Particle Size mm 30-50 =15
PTR TVA ppm 2200-2700 2500-3000
Biogas Yield M3/Ton 280-320 330-360

Table 11: Effect of extrusion and plug screw mechanical treatment using shredded and milled material and its impact on PTR TVA and biogas yield of rice straw.
Description Units Shredded/milled material with
Extrusion Plug Screw
Particle size mm 1-5 1-5
PTR TVA ppm 3000-4500 3000-4500
Biogas Yield M3/Ton 350-400 350-400
As seen in Tables 10, and 11, maximum size reduction and biogas yield was achieved for milled and shredded with extrusion or plug screw processed rice straw.

Pre-treating the processed lignocellulosic feedstock with at least one microorganism to obtain pre-treated lignocellulosic feedstock (103): Pretreatment was carried out in a -pretreatment reactor with approximately 3.2 M3 capacity. Considering about 3.2 M3 reaction volume and about 7-7.5% -total solids, the processed lignocellulosic feedstock obtained above was added to a PTR. To this, about 2% Thermobifida fusca culture was added and mixed using agitator and aeration. The pretreatment reaction was carried out at about 45°C and for about 48 h. Post pretreatment, part of pretreated lignocellulosic feedstock was transferred to anaerobic digester. For continuous pretreatment process, once the pretreated lignocellulosic feedstock was transferred, same volume of processed lignocellulosic feedstock was added to PTR. It was found that the pretreated rice straw comprised total solids in the range of 7.0-7.5%w/w and total volatile acids in the range of about 3000-4500ppm.
Anaerobically digesting/biomethanation the pre-treated lignocellulosic feedstock to obtain biogas (104): The digester was filled initially with anaerobic sludge having about 15-20% total solids and acclimatized cow dung slurry containing about 4-5% total solids and anaerobic digestion was carried out as described in Examples 7, 8, and 9. TVA, biogas volume, methane and carbon dioxide concentration was analysed on daily basis. HRT of 20-22 day was maintained for all types of rice straw material during bio methanation process.
During the entire bio methanation, raw biogas composed of about 55-57% v/v methane, about 35-37% v/v carbon dioxide was obtained.
After about 25 days of HRT, the entire digestate having a volume of 1120 L and containing around 4% total solids was subjected to solid liquid separation to obtain about 95 kg wet biomethanated solids having about 70 w/w moisture, and about 1025 L biomethanated liquid having about 1.6% solids. Raw biogas of about 40M3 (about 46 Kg weight of biogas) was obtained.
The digestate obtained was further subjected to solid/liquid separation using screw press to obtain biomethanated solids and biomethanated liquid that were analysed (see Table 12). Wet biomethanated solids were further composted to obtain dry biomethanated solids that were further analysed (see Table 13) as per FCO norms and were certified by NOCA for its applicability as a solid fertilizer. Similarly, biomethanated liquid was further analysed (see Table 14) as per FCO norms and was certified by NOCA for its applicability as a liquid fertilizer. The biomethanated liquid (75%) obtained was also recycled for volume makeup during rice straw feeding to the digester and remaining 25% liquid was implemented to make up volume during rice straw pre-treatment.
Table 12: Comparative analysis of biomethanated solids and liquid
Sr. No. Parameter Biomethanated liquid Biomethanated solids
Physical Parameters
1 TS (%w/w) 2.5±0.5 30±2
2 Ash (%w/w) 1±0.5 5±1
3 TDS (%w/w) 0.7±0.5 2±0.5
4 TSS (%w/w) 1.8±0.4 18±2
5 TVS (%w/w) 1.5±0.3 17±2
6 Total Nitrogen (%w/w) 0 8±3
7 pH (%w/w) 7.7±0.3 7.7±0.2

Table 13: Analytical results for biomethanated solids
Parameter Test Values Permissible limit on dry wt basis Remarks
Physical properties
Colour Blackish Black to dark green, parent material slightly visible. Normal
Odour Odorless Smells like rich humus from the forest floor; no ammonia or anaerobic odor. Normal
Chemical Properties
Organic Carbon ( %) 36 14.00 (min) Normal
Moisture (%) 89.14 25% (max) -
Particle Size more than 4mm Fine texture (all below 1/8" mesh or 4mm) powder or granular form Normal
Bulk Density 0.98 1g/cm3 (max) -
pH 8.26 6.5 - 7.5 Slightly Alkaline
C:N 61.02:1 20:1 (max) Normal
Conductivity (mmhos/cm) 0.83 4.0 (max) Normal
Major Nutrients
Total Nitrogen (N) (%) 0.59 0.8 (min) Excellent
Total Phosphorus (P2O5) (%) 0.57 0.4 (min) Excellent
Total Potassium (K2O) (%) 0.48 0.4 (min) Normal
Sum total of (N+P+K) (%) 1.64 1.5% (min) Excellent

Table 14: Analytical results for biomethanated liquid
Parameter Test Values Requirement/Permissible limit on dry wt basis Remark
Physical properties
Colour Blackish Black to dark green, parent material not visible. Normal
Odour Odorless Smells like rich humus from the forest floor, no ammonia or anaerobic odor. Normal
Chemical Properties
Organic Carbon ( %) 1.07 14.00 (min) Normal
Moisture 98.5 25% (max) -
Particle Size Less than 0.1mm Fine Texture (all below 1/8" mesh or 4mm) powder or granular form Normal
Bulk Density 1.00 1 g/cm3 (max) -
pH 7.30 6.5 - 7.5 Slightly Alkaline
C:N 21.4:1 20:1 (max) Normal
Conductivity (mmhos/cm) 7.17 4.0 (max) Normal
Major Nutrients
Total Nitrogen (N) (%) 0.05 0.8 (min) Excellent
Total Phosphorus (P2O5) (%) 0.07 0.4 (min) Excellent
Total Potassium (K2O) (%) 0.18 0.4 (min) Normal
Sum total of (N+P+K) (%) 0.30 1.5% (min) Excellent

The post-biomethanated solids and liquid of rice straw were found to be rich in micro and macro minerals. Due to the high C/N ratio of biomethanated solids, it can be used as soil conditioner for agriculture and horticulture industries after dilution with biomethanated spent wash of rice straw thus making it a complete biofertilizer.
The instant process can be implemented at small, medium, as well as large scale.
The embodiments, examples and alternatives of the preceding paragraphs or the description and drawings, including any of their various aspects or respective individual features, may be taken independently or in any combination. Features described in connection with one embodiment are applicable to all embodiments unless such features are incompatible.
The preferred embodiments of the present invention are described in detail above. It should be understood that ordinary technologies in the field can make many modifications and changes according to the concept of the present invention without creative work. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should fall within the protection scope determined by the claims.
,CLAIMS:We claim:
1. A process (100) for producing biogas from lignocellulosic feedstock comprising:
selecting lignocellulosic feedstock (101);
processing the lignocellulosic feedstock by reducing their size to obtain processed lignocellulosic feedstock (102);
pre-treating the processed lignocellulosic feedstock with at least one micro-organism to obtain pre-treated lignocellulosic feedstock (103); and
anaerobically digesting the pre-treated lignocellulosic feedstock to obtain biogas (104);
such that,
yield of the biogas is improved.

2. The process (100) as claimed in claim 1, wherein the lignocellulosic feedstock is selected from rice straw, wheat straw, cotton stalk, Juli flora, groundnut shell, palm oil empty fruit bunch, bagasse, and soyabean straw.

3. The process (100) as claimed in claim 1, wherein the processing of the lignocellulosic feedstock is carried out through at least one of shredding, milling, extrusion, and plug screw.

4. The process (100) as claimed in claim 3, wherein the processing of the lignocellulosic feedstock is carried out through shredding or milling with at least one of extrusion or plug screw.

5. The process (100) as claimed in claim 3, wherein the lignocellulosic feedstock is processed to particle size distribution of = 20 mm.

6. The process (100) as claimed in claim 1, wherein the pretreatment of the processed lignocellulosic feedstock increases total volatile acid (TVA) by 3 times.

7. The process (100) as claimed in claim 1, wherein the pretreatment of the processed lignocellulosic feedstock is carried out at 37°C to 55°C.

8. The process (100) as claimed in claim 1, wherein the pretreatment of the processed lignocellulosic feedstock is carried for 24 to 96 h.

9. The process (100) as claimed in claim 1, wherein concentration of the micro-organism is 0.5-10%.

10. The process (100) as claimed in claim 1, wherein the microorganism belongs to genus Thermobifida.

11. The process (100) as claimed in claim 1, wherein the microorganism is Thermobifida fusca.

12. The process (100) as claimed in claim 1, wherein the anaerobic digestion of the pre-treated lignocellulosic feedstock is carried out using acclimatized cow dung.

13. The process (100) as claimed in claim 1, wherein the anaerobic digestion of the pre-treated lignocellulosic feedstock provides at least one of biomethanated solids and biomethanated liquid.
14. The process (100) as claimed in claim 13, wherein total volatile solids (TVS) of the biomethanated solids is up to 20% w/w.

15. The process (100) as claimed in claim 13, wherein total volatile solids (TVS) of the biomethanated liquid is up to 2% w/w.

16. The process (100) as claimed in claim 12, wherein the acclimatized cow dung comprises 4-5% of total solids.

17. The process (100) as claimed in claim 1, wherein hydraulic retention time for producing the biogas is 14 to 28 days.

18. The process (100) as claimed in claim 1, wherein the yield of the biogas is improved to = 400 AM3/ton.