Description:FIELD OF INVENTION
[001] The present invention relates to a method of biofuel production from a lignocellulosic biomass. Particularly, the present invention relates to a method of green pretreatment of rice straw coupled with alkaline wastewater followed by autoclave pretreatment, anaerobic digestion and fermentation for efficient energy and carbon conversion of rice straw into biofuels.
BACKGROUND OF THE INVENTION
[002] Rice is second major crop worldwide which provides a large amount of dry lignocellulosic biomass. Rice straw is a major agricultural waste which is casually discarded during rice grains harvesting. Rice straw is removed with the rice grains during harvest, and it ends up being piled or spread out in the field. The annual production of rice straw is between 800 and 1,000 million tons worldwide, with about 600 to 800 million tons per year produced in Asia. Rice Straw is generated in large amounts and its disposal is problematic. Due to lack of proper management for agricultural wastes, rice straw is burnt in open fields which leads to an increase in carbon footprint, which has a devastating effect on the atmosphere. Average 92 million tons of crop residue is burnt yearly in India, which contributes to major share of agricultural GHG emission. Rice straw alone accounts for up to 40% of this volume, and its generation is expected to increase up to 45% by year 2050, which raises a concern and therefore, there is a need for several sustainable alternatives to effectively utilize rice straw.
[003] Due to rapid depletion in fossil fuels with increasing demand and due to the hazardous environmental effect associated with their use, there is a need for clean sources of energy. We can produce the required energy with clean energy without releasing greenhouse gases or having a negative impact on the environment. Biofuels are one such source of clean energy and are regarded as one of the most viable options for reducing CO2 emissions, produced from biomass such as plant or algae material or animal waste.
[004] Although, the presence of cellulose and hemicellulose in rice straw makes it a promising source for biofuel production, its biodegradation is restricted due to the presence of tough lignin layers which sandwich cellulose and hemicellulose. Hence, pretreatment of the lignocellulosic biomass is essential as it breaks the lignin layers and dissolves cellulose and hemicellulose. Different techniques of pretreatment are known such as acid pretreatment, oxidative pretreatment, ozonolysis, ionic liquid pretreatment, organic solvent pretreatment and alkaline pretreatment. Alkaline pretreatment is the most preferred technique.
[005] There are several patent applications that disclose pretreatment of lignocellulosic materials for biofuel production. One such United States patent application US9243364B2 relates to a method of producing ethanol from lignocellulosic biomass via enzymatic saccharification and fermentation that utilize the pretreatment method. The cited patent application discloses a two-stage pretreatment process. In the first stage, the lignocellulosic biomass is treated in a mildly acidic or near pH-neutral solution to promote delignification and lignin sulfonation. However, the cited patent application requires toxic chemicals such as sulfites at first stage of pretreatment, which is not recommended.
[006] Another Australian patent application AU2014228977B2 relates to an alkaline treatment of lignocellulosic biomass. The cited patent application discloses a method for the treatment of lignocellulosic biomass comprising (a) incubating a biomass in a sufficient amount of an ionic liquid (IL) b) applying radio frequency (RF) to heat the biomass to a target temperature range of 80°C to 140°C;(c) washing the pretreated biomass;(d) subjecting said biomass to mild alkaline treatment comprising adding an alkaline agent and incubating for about 1-60 minutes at about 40°C-70°C. Alkaline agent used in this cited document is NaOH, aqueous ammonia, LiOH, Mg(OH)2, A1(OH)3, Ca(OH)2, H2O2, NaS, Na3CO3, or a combination thereof. However, this technique utilizes harmful chemicals for pretreatment method.
[007] Another non-patent literature titled “Two-stage pretreatment of rice straw using aqueous ammonia and dilute acid” discloses two-stage pretreatment process using aqueous ammonia and dilute sulfuric acid in a percolation mode to improve production of fermentable sugars from rice straw. Aqueous NH3 was used in the first stage which removed lignin selectively but left most of cellulose (97%) and hemicellulose (77%). Dilute acid was applied in the second stage which removed most of hemicellulose, partially disrupted the crystalline structure of cellulose, and thus enhanced enzymatic digestibility of cellulose in the solids remaining. Evidently, chemical pretreatment is more expensive as a large amount of chemicals are used for pretreating the lignocellulosic substrate.
[008] Further, traditional methods of pretreatment of biomass and their conversion into biofuels known in the art utilizes chemicals that are hazardous to humans and have adverse effect on environment. Hence an innovative and novel solution is needed that does not employ chemicals for production of biofuels from rice straw.
[009] In view of the problems associated with the above state of art, there is a need for an economic, chemical-free method of production of biofuels from rice straw employing a step of green pretreatment of rice water with alkaline wastewater, ensuring waste stabilization along with efficient energy generation.
OBJECTIVES OF THE INVENTION
[010] A primary objective of the present invention is to provide a chemical-free coupled pretreatment of rice straw with alkaline wastewater for efficient biofuel production.
[011] Another objective of the present invention is to provide a method of coupled pre-treatment of lignocellulosic waste such as rice straw, water hyacinth, and fallen leaves with alkaline wastewater obtained from food processing which is further used for efficient biofuel production.
[012] Another objective of the present invention is to provide a method of coupled green pretreatment of rice straw with petha wastewater, stabilizing highly alkaline petha wastewater casually discarded without pretreatment and thus, ensuring waste stabilization and its further conversion to biofuels.
[013] Another objective of the present invention is to provide a combined two steps pretreatment technique, enabling production of biofuels.
[014] Yet another objective of the present invention is to provide an economic and environment friendly method, ensuring efficient energy generation and waste stabilization simultaneously.
[015] Yet another objective of the present invention is to provide alkaline wastewater treated rice straw for feeding cattle.
[016] Other objects and advantages of the present invention will become apparent from the following description taken in connection with the accompanying drawings, wherein, by way of illustration and example, the aspects of the present invention are disclosed.
SUMMARY OF THE INVENTION
[017] The present invention relates to an efficient method of biofuel production from a lignocellulosic biomass. Particularly, the present invention relates to a method of green pretreatment of rice straw coupled with alkaline wastewater comprising the step of soaking 5% w/v of Rice Straw (RS) in Petha Wastewater (PWW) for 7 days followed by autoclave pretreatment; anaerobic digestion and fermentation using livestock excreta as mixed microbial culture for efficient energy and carbon conversion of rice straw into biofuels. Thus, the present invention provides an economic, chemical-free method of production of biofuels from rice straw employing a step of green pretreatment of rice water with alkaline wastewater, ensuring waste stabilization along with efficient energy generation.

SOURCE OF MATERIALS USED IN THE PRESENT INVENTION
[018] The rice straw is a waste generated after the rice harvest. Alkaline water was discarded water produced by factories making Ash gourd candy (commonly known as Petha) from Agra, UP. Although any food processing factory may generate waste water having alkalinity > pH 12. Rice is cultivated in farms nearby Dayalbagh, Agra area for the fulfillment of local rice supply. Rice straw (RS) is generated in huge amounts at the time of rice harvesting (October and November). RS was collected from nearby farms and used as substrate in the present study. PWW was collected from cottage petha industries situated at “Noori Darwaja, Agra” and used for the primary green pretreatment of RS.
BRIEF DESCRIPTION OF THE DRAWINGS
[019] The present invention will be better understood after reading the following detailed description of the presently preferred aspects thereof with reference to the appended drawings, in which the features, other aspects and advantages of certain exemplary embodiments of the invention will be more apparent from the accompanying drawing in which:
[020] Figure 1 illustrates flow chart describing the process of efficient biofuel production.
[021] Figure 2 illustrates surface morphology of untreated rice straw through SEM analysis.
[022] Figure 3 illustrates surface morphology of PWW treated rice straw through SEM analysis.
[023] Figure 4 illustrates surface morphology of autoclaved PWW treated rice straw through SEM analysis.

DETAILED DESCRIPTION OF THE INVENTION
[024] The following is a detailed description of embodiments of the disclosure. The embodiments are in such detail as to clearly communicate the disclosure. However, the amount of detail offered is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure.
[025] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.
[026] Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
[027] The terms and words used in the following description and claims are not limited to the bibliographical meanings but are merely used to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention.
[028] It is to be understood that the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
[029] It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps, or components but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.
[030] Accordingly, the present invention relates to a method of pretreatment of lignocellulosic biomass for efficient biofuel production. Particularly, the present invention relates to a method of coupled green pretreatment of lignocellulosic waste such as rice straw, water hyacinth and fallen leaves etc., with alkaline wastewater followed by autoclave pre-treatment, anaerobic digestion and fermentation for efficient energy and carbon conversion of rice straw into biofuels.
[031] In an exemplary embodiment of the present invention, Petha wastewater pretreatment followed by autoclave pretreatment of rice straw is done for production of various biofuels (value-added products), such as methane, ethanol, volatile fatty acids (acetic acid, propionic acid etc.). Biofuel production via anaerobic digestion of rice straw is an energy extensive process and leads to waste stabilization. Hence it is doubly beneficial i.e. both energy generation and waste stabilization can be achieved in a single process, and also can solve the disposal problem of both rice straw (global problem) and petha wastewater (local problem). The digestate obtained at the end of the reactor can be utilized as good organic fertilizer. The complete idea would hence aim towards zero waste strategy.
[032] An important green aspect of the process of the present invention is the use of another agricultural waste, Ash gourd candy (commonly known as Petha) waste water. This waste water is a major environmental concern because of its highly alkaline (~ pH 12-14) nature and is at present drained off untreated. Petha waste-water in return gets stabilized by the present process, as its pH becomes near neutral after pretreating rice straw. The geographical region where the study was conducted (Agra City, UP, India) is the prime centre for Ash Gourd Candy production, containing about 500 cottage units which produce 700-800 tons of candy daily, producing about 2000 Litre waste water/day. This Petha wastewater pretreated crop residue (rice straw) is subjected to anaerobic digestion for biofuel production using livestock excreta as microbial culture.
[033] Green Coupled Pretreatment of Rice Straw with Petha Wastewater- High lignocellulosic content in rice straw (RS) and high alkalinity of Petha wastewater (PWW) makes them difficult to be utilized and consumed. Due to high alkalinity of PWW, lignocellulosic bonds of RS break down into cellulose, hemicellulose and lignin on pretreatment and thus, makes RS suitable for anaerobic pretreatment. Further, coupled pretreatment of PWW decrease its alkalinity as RS absorbs CaCO3 present in PWW. Both RS and PWW are then used for extraction of energy (biofuels) without using any chemical in the treatment process. PWW treated RS also would be suitable for feeding cattle as on pretreatment, calcium present in PWW combines with oxalate present in RS and hence absence of free oxalate in RS makes it suitable to be used as a fodder.
[034] Autoclave Pretreatment- In this step, PWW pretreated RS was autoclaved in an autoclave operated at 121oC and 15 to 20 psi pressure for secondary pretreatment at different time intervals of 0, 10, 20, 30, 40, 50 and 60 minutes to optimize the sugar release from RS. Optimum sugar was released after 40 minutes of autoclave which was almost eight times the sugar released by RS pretreated with PWW without autoclave.
[035] Anaerobic Digestion and Fermentation- The finally pretreated RS (after secondary pretreatment) can be then subjected to anaerobic digestion for biofuel production using livestock excreta as mixed microbial culture. Eco-friendly cow dung and soil derived mixed microbial cultures can be used as inoculum sources for anaerobic digestion process.
[036] In an embodiment of the present invention, a method for biofuel production from Rice Straw comprises:
• a step of coupled green pretreatment of RS with PWW;
• a step of autoclave pretreatment ;
• a step of anaerobic digestion and fermentation.
• a step of testing physical and chemical parameters such as TS, TDS, VS, TSS, Ash, COD and Glucose.
• a step performing chromatographic analysis of biofuel produced.
[037] The step of coupled green pretreatment comprises:
• Soaking 5% w/v of Rice Straw (RS) in Petha Wastewater (PWW) for 7 days.
• Performing compositional analysis of pretreated PWW before soaking RS and after soaking RS are then performed [Shown in Table 1 and Table 2].
[038] The step of autoclave pretreatment comprises:
• Optimizing sugar release from RS by secondary pretreatment of PWW pretreated RS in an autoclave operated at 121oC and 15 psi pressure at different time intervals of 0, 10, 20, 30, 40, 50 and 60 minutes.
[039] The step of anaerobic digestion comprises:
• Adding 100 ml of inoculum derived from soil or cow dung in the ratio 80: 20 in a 1000 ml batch reactor (working volume 500 ml) at room temperature 37 oC.
• All reactors were purged with nitrogen gas for 5 minutes to ensure anaerobic environment in the reactors.
[040] Chromatographic analysis of biofuel produced:
Biogas and biohydrogen production from the reactors are estimated and monitored regularly using Gas Chromatograph (5765 Nucon, India) equipped with thermal conductivity detector (TCD). Concentrations of bioethanol, biomethanol and VFAs are measured using GC equipped with flame ionization detector (FID). Nitrogen is used as the carrier gas with a flow rate of 30 ml/min.
[041] Examples of lignocellulosic biomass are selected from a group of but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, water hyacinth and fallen leaves etc., or a combination thereof.
[042] CHARACTERIZATION AND RESULTS

[043] Physical Parameters (total solids (TS), total dissolved solids (TDS), total suspended solids (TSS), volatile solids (VS), COD and Ash Content were estimated before and after pretreatment.
A. Total Solids (TS): Clean porcelain crucibles were taken and ignited at 550oC for 1 hour in a muffle furnace. They were allowed to cool and weighed just before use. A fixed volume of well-mixed samples was taken in the pre-weighed crucibles. The crucibles were kept in a hot air oven at 98°C overnight. The evaporated sample was then dried at 105-110oC in hot air oven for 1 hour. The crucibles were cooled up to ambient temperature and then weighed again.
TS in the sample was calculated as: TS (g/L) = (A-B) x 1000/ sample volume (mL)
Where, A = weight of dried residue + crucible (in grams)
B = weight of empty crucible (in grams)
B. Total Dissolved Solids (TDS): Clean porcelain crucibles were heated at 180 ± 2oC for 1 hour in a hot air oven. They were then allowed to cool and weighed for constant weight. The samples were centrifuged at 500 rpm for 10 minutes and the supernatant was taken for total dissolved solids analysis. A fixed volume of the supernatant was added to the pre-weighed crucibles and kept in the hot air oven at 98oC overnight. The evaporated samples were then dried at 180 ± 2oC in an oven for 1 hour. The crucibles were allowed to cool up to ambient temperature and were then weighed. TDS was calculated as:
TDS (g/L) = (A-B) x 1000/ sample volume (mL)
Where, A = weight of dried residue + crucible (in grams)
B = Weight of crucible (in grams
[044] Glucose Estimation(Miller, 1959): For analysis of glucose concentration, DNS (3, 5-Dinitrosalicylic acid) test was done. Reducing sugars have property to reduce DNS in alkaline solution by formation of 3-amino, 5-nitrosalicylic acid and result in development of red colour development.
a. Reagents Required for Glucose Analysis:

I. 3, 5-dinitrosalicylic acid (DNS) Solution: DNS reagent solution was prepared by dissolving 1g of DNS with 200 mg of phenol and 50 mg of sodium sulphite in 100 mL of 1% NaOH solution.
II. Sodium Potassium Tartrate Solution or (40% Rochelle salt Solution): It was prepared by dissolving 40 g sodium potassium tartrate in 100 mL of water.
III. NaOH Solution (1%): 1 g of NaOH was dissolved in 100 ml of water to prepare 1% NaOH solution for dissolution of DNS reagent.
IV. Glucose Stock Solution: 100 mg of glucose was dissolved in double distilled water and made up the volume to 100 mL in a volumetric flask.
V. Working Solution: 10 mL from the stock solution was taken and made up to 100 mL to form working solution of glucose.
b. Procedure: The samples were centrifuged at 500 rpm and 1 mL of each centrifuged sample was taken in separate test tube. All samples were diluted to 3 mL by adding distilled water which was followed by addition of 3 mL DNS reagent. The test tubes were covered with aluminium foil and kept for 5 minutes in a boiling water bath. Development of reddish brown colour indicated the completion of the reaction. The intensity of the colour depends upon the concentration of glucose present in the sample. Test tubes were cooled to room temperature and then 1mL of sodium potassium tartrate (40% Rochelle salt) was added to each test tube. Absorbance was taken at 540 nm using UV-visible spectrophotometer. Calibration curve was plotted to calculate the unknown glucose concentration in the samples. Reducing sugar yield was calculated using the following formula (Gabhane et al., 2011):

Reducing sugar yield (%) =
(Reducing sugar in mg/mL x Total volume (mL) x 1000) / Substrate in mg
[045] Chemical Oxygen Demand (COD) Analysis (APHA, 1995): The chemical oxygen demand is the amount of oxygen essential for chemical oxidation of organic matter via strong chemical oxidant such as potassium dichromate under reflux conditions. COD is an important factor to study the strength of the waste in terms of the strength of organic compounds in the waste (both solid and liquid). The method used here for the COD determination is in accordance with the Standards Methods which is mainly used for the wastewater and water samples having high percentage of suspended solids. Since some inconsistencies in the COD values of samples were taken in replicates. Modified method given by Yadav et al. (2006) was used. The main basis for the COD test is that nearly all organic compounds can be fully oxidized to CO2 in the presence of strong oxidizing agents under acidic conditions.
Sample Preparation: Liquid samples collected from the reactors were first centrifuged at 500 rpm and freshly used for COD analysis. Solid RS samples were dried in hot air oven at 105-110oC, cooled to room temperature in desiccator and constant weight was taken for analysis.
Reagents Required:
a) Standard Potassium Dichromate Solution (0.01667 M): 1.225 g of potassium dichromate, primary standard grade, previously dried at 150oC for 2 h was added to 100 mL distilled water for preparation of 0.01667 M solution.
b) Sulfuric acid Reagent: 2.25 g of silver sulphate was added to 250 ml of concentrated sulfuric acid in volumetric flask. The reagent was kept for 1 day for complete dissolution of silver sulphate before its use.
c) Ferrous Ammonium Sulphate (FAS) Solution: 24.5 g of Ferrous Ammonium Sulphate was added to 5 mL of concentrated H2SO4 and the volume was made up to 250 mL with distilled water.
d) Ferroin Indicator: This indicator is used to indicate change in oxidation-reduction potential of the solution and indicates the condition when all dichromate has been reduced by ferrous ions. It gives a very sharp reddish brown colour change which can be seen in spite of blue colour generated by the Cr3+ ions formed on reduction of the dichromate.
[046] Procedure for Liquid Sample: 1.7 mL sample was taken in COD tube and potassium dichromate solution (0.8 mL) was added to it. Sulfuric acid reagent (2.5 mL) was carefully run down inside of vessel to form an acid layer is under the sample-digestion solution layer and a pinch of mercury sulphate was added to each sample. Each sample were then taken in tightly cap tubes or seal ampules, and inverted several times to mix completely. Later, all the tubes were placed in COD digester preheated at 120oC and reflux for 2 h under a protective shield. All the tubes were then cooled at room temperature and 10 mL distilled water was added followed by 1 to 2 drops of ferroin indicator and titrate against standardized FAS reagent. The end point was observed as a sharp change in colour from blue-green to sharp red. The readings were noted and the COD was calculated according to the formula given below.
[047] Procedure for Solid Sample: The sample was digested with acidified K2Cr2O7 using open reflux method. 0.025 g of the dried solid sample chip was taken in a round bottom flask containing 50 mL of distilled water along with a pinch of HgSO4 and 20 mL of K2Cr2O7 solution was added to it (Yadav et al., 2006). 60 mL of H2SO4 /Ag2SO4 solution was added to the above mixture from the open end of the refluxing condenser. The mixture was heated for 2:30 hrs and the sample was allowed to cool to room temperature. After cooling, 50 mL of water was added and then this solution was titrated against the standard ferrous ammonium sulphate solution using ferroin indicator. COD in mg/L was calculated in sample as follows:

where, A = Volume of FAS used for blank (mL)
B = Volume of FAS used for sample (mL)
N = Normality of FAS
8000 = Milli equivalent weight of oxygen (8) ×1000 mL/L.

[048] Table 1: Physical and Chemical characteristics of RS before (1) and after soaking (2) in PWW:
Parameters Values in PWW before soaking RS (1) Values in PWW after soaking RS (2)
TS (g/L) *5.44 ± 0.74 *6.14 ± 0.14
TDS (g/L) *5.22 ± 0.69 *4.86 ± 0.06
TSS (g/L) *0.22 ± 0.04 *1.31 ± 0.84
VS (g/L) *1.64 ± 0.1 *1.84 ± 0.08
Ash (g/L) *1.76 ± 0.23 *3.54 ± 0.5
pH *12.3 ± 0.5 *6.5 ± 0.5
Glucose (mg/L) *10.1 ± 1.0 *710 ± 0.12
COD (mg/L) *5882 ± 23.2 *353 ± 5.26
DO (mg/L) *3.8 ± 0.1 ND
BOD5 (mg/L) *580 ± 3.1 ND
Alkalinity (mg/L) *2400 ± 1.3 0
Chloride (mg/L) 1174.66 ± 7.09 ND
Hardness (mg/L) 2760 ± 12.87 ND
Sodium (mg/L) 19.8 ± 0.23 ND
Potassium (mg/L) 26.3 ± 0.65 ND
Calcium (mg/L) 3.2 ± 0.17 ND
Phosphorus (µg/mL) 5.47 ± 0.27 ND
Sulphur ?(mg/gDW) 0.037 ± 0.005 ND

[049] Inference: From the given table 1, it is evident that alkalinity of the pretreated biomass gets significantly reduced after coupled green pretreatment of RS with PWW. As evident from the table above, the chemical oxygen demand (COD) significantly decreases and biological oxygen demand becomes non-detectable. pH of PWW treated RS is reduced to < 7. Further, presence of different elements such as sodium, potassium, calcium and phosphorus become non-detectable after the pretreatment and glucose level increases.
[050] Table 2: Composition of sample components (in percentage) of PWW before soaking RS (1), after soaking RS (2) and Autoclaved PWW treated RS (3)

Samples?
Components (%) 1 2 3
Cellulose *42.64±2.1 *52.26±1.94 61.32±1.49
Hemicellulose *28.89±1.8 *21.66±2.56 16.14±2.08
Lignin *18.26±1.3 *9.46±1.36 6.21±0.57
Ash content *10.14±0.65 *15.81±0.83 15.16±0.36
Extractives *1.95±0.30 *1.61±0.21 1.02±0.04
Reducing sugars *3.26±0.34 *16.83±1.23 49.33±3.28
Volatile Solids *89.86±4.5 *84.18±4.25 84.84±3.12
Moisture content 4.29±0.14 3.99±0.13 4.97±0.21
Protein *4.28±0.20 *3.02±0.14 ND
Phosphorus (µg/mL) 5.82±0.02 9.10±0.08 4.56±0.09
Iron (µg/gDW) 727.62±1.32 501.4±3.23 ND
Zinc (µg/gDW) 14.96±1.02 18.07±0.82 ND
Sulphur (µg/gDW) 290.91±1.01 327.27±0.52 117.5±0.27
Sodium (ppm) 19.80±0.75 20.80±0.11 16.70±0.39

[051] Inference: From the given table 2, it is evident that lignocellulosic bonds of RS break down into cellulose, hemicellulose and lignin on pretreatment due to high alkalinity of PWW and thus, makes RS suitable for anaerobic pretreatment.
[052] Percent Crystallinity (CI%): PWW pretreatment significantly reduced the crystallinity of RS to 41.42% which was further reduced to 56.37% after autoclaving it. Reduction in crystallinity indicates increase in amorphous nature of RS which is favourable for its anaerobic digestion for biofuel production.
[053] Table 3: Percentage Crystallinity Index (CI%) of the untreated RS (1), PWW treated RS (2) and autoclaved PWW treated RS (3)

Samples Position at 2 Theta (degree) Percent Crystallinity Index (CI%)
Iam I002
1 18.42 22.14 54.55
2 18.42 22.08 31.90
3 18.06 22.09 23.08

[054] Scanning Electron Microscope (SEM) analysis: Scanning electron microscope is used for characterization of the surface of rice straw before and after pretreatment. Samples were first coated with the help of gold spin coater to make them conductive for SEM analysis for better visualization [as shown in Figures 2,3 and 4].
[055] Inference: comparing surface morphology of untreated RS, PWW treated RS and autoclaved PWW treated RS shows significant reduction in crystallinity and increase in amorphous nature of RS which is favourable for anaerobic digestion for biofuel production.

[056] ADVANTAGES OF THE PRESENT INVENTION:
1. The process of production of biofuels or value-added products using the present invention is highly environmentally friendly because the use of petha wastewater for primary treatment of plant biomass (rice straw) does not involve any type of chemical.
2. Cow dung and soil derived mixed microbial cultures can be used as inoculum sources for anaerobic digestion process which are also environmentally benign and biodegradable.
3. The output of the process obtained in the form of value-added products which have their own calorific values more than the pure substrate (rice straw). The biofuels obtained (biomethane, bioethanol and bio-methanol) are low carbon containing fuels which do not contribute to global warming.
4. The process takes care of the problem assisted with the disposal of voluminous rice straw (global problem) and highly alkaline petha wastewater (local problem).
5. The process of the present invention is economically very cheap. Tons of rice straw generated from rice straw cultivation practices which can be easily collected from fields without any cost. Petha waste water is also a waste product of Petha industry and can also be collected at the time of its discard. This ease of availability of substrate, make this process cost effective and economic. Secondly chemicals are not used in the pretreatment process which also results in reduced cost of the process.
6. The present invention provides a hybrid approach because production of four value-added products or biofuels (biomethane, bioethanol, bio-methanol and volatile fatty acids) have been monitored for a single designed reactor using mixed microbial source.
7. Rice straw has large amount of oxalate which combines with calcium present in bones of the animal consuming this straw as fodder, leading to their decalcification. Calcium presents in petha waste-water (as petha is treated with lime water) combines with oxalate present rice straw. Since this oxalate would no more be free, it would not react with calcium present in bones of the animal. Also, the petha waste water treated rice straw has lesser silica content; it can be easily used as fodder.
, Claims:WE CLAIM:
1. A method for production of biofuel by green pretreatment of lignocellulosic biomass, comprising the steps of:
i. pretreating lignocellulosic biomass with alkaline wastewater by soaking 5% w/v of lignocellulosic biomass in alkaline wastewater for a period of 7-10 days;
ii. pretreating the mixture obtained from step (1) in autoclave for 10-60 minutes at a pressure of 15psi to 20 psi and temperature of 121 oC;
iii. subjecting the pre-treated biomass to anaerobic digestion and fermentation using livestock microbial culture for biofuel production in a batch reactor;
iv. and obtaining biofuels selected from a group of biomethane, bioethanol, bio-methanol or volatile fatty acids.
2. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein the lignocellulosic biomass is selected from a group of corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy water hyacinth and fallen leaves etc., or a combination thereof.

3. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein the alkaline wastewater has a pH in the range of 12-14.

4. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein the alkaline wastewater is petha waste water.

5. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein microbial culture for the anaerobic digestion and fermentation is an inoculum derived from soil or cow-dung or a combination thereof.

6. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 4 or 5, wherein microbial culture is mixed with the pre-treated biomass in the ratio of 20:80.

7. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein alkalinity of the pre-treated biomass is reduced to zero after coupled green pretreatment.

8. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein pH of the pre-treated biomass is reduced to less than 7.

9. The method for production of biofuels by green pretreatment of lignocellulosic biomass as claimed in claim 1, wherein the pretreatment of biomass reduces elements/compounds selected from a group of sodium, potassium, iron, sulphur, calcium, phosphorus, chloride, oxalate and lignin.