A BIO-complex of anaerobic fungi cultivated on lignocellulosic material, an enzyme complex and volatile fatty acids

Abstract

A bio-complex comprising of anaerobic fungi cultivated on lignocellulosic material, an enzyme complex comprising of one or more lignocellulose-degrading enzymes, and one or more volatile fatty acids, wherein the concentration of the lignocellulosic material is 1-12%, the concentration of the enzyme complex is 5- 20 IU / ml, the concentration of the volatile fatty acids is 1000 to 10000 mg / L, and the enzyme complex is adapted to enhance degradation of the lignocellulosic material under anaerobic conditions.

Description

A BIO-COMPLEX OF ANAEROBIC FUNGI CULTIVATED ON LIGNOCELLULOSIC MATERIAL, AN ENZYME COMPLEX AND VOLATILE FATTY ACIDSFIELD OF THE INVENTION

[0001] The present invention relates to a bio-complex comprising of anaerobic fungi cultivated on a lignocellulosic material, an enzyme complex and volatile fatty acids, which can directly be used as a bio-substrate for aiding better degradation of biomass during anaerobic digestion or in a biogas system.BACKGROUND OF THE INVENTION

[0002] The increasing awareness of global warming, and its long-term effects on the environment has resulted in a shift towards clean, safe and renewable sources of energy. Biofuels, being locally produced and cost friendly, satisfy the above criteria and are therefore the commonly advocated alternative.

[0003] Biofuel is developed from biomass i.e. plant and animal waste. In so far as plant waste is concerned, lignocellulosic material i.e. dry plant matter is abundantly available, and a popular raw material used in the production of biofuel. Lignocellulosic material primarily comprises of cellulose, hemicellulose and lignin in varying ratios. They differ chemically but are interconnected in an intricate manner. Cellulose and hemicellulose, being polysaccharides, can be biologically hydrolyzed to simple sugars, and converted into high-value biofuels. However, lignin is a complex polymer of phenylpropane, making its biochemical decomposition a challenge. Due to the intricate interconnection of cellulose and hemicellulose with lignin in lignocellulosic material, not all the available cellulose and hemicellulose can be hydrolyzed. In particular, the combination of hemicellulose and lignin forms a protective barrier around the cellulose, which must be removed before the hydrolysis of cellulose. Lignin is made up of a large macromolecular and heterogenous polymer and its degradation is difficult as it does not contain any hydrolysable linkages.

[0004] The complex structure of lignocellulosic material restricts its sufficient utilization for biofuel production and therefore pre-treatment of this material is necessary as it increases the availability of lignocellulosic biomass for cellulase enzymes, their digestibility, and productyield. Since lignocellulosic material is used as an environmentally benign raw material for biofuel production, it is essential to ensure that its pre-treatment is eco-friendly, efficient and cost-effective and the resultant product is an environment friendly biofuel. Hence, there is a need for a bio-complex as a degradation aid which overcomes the disadvantages of the prior art, is cost-effective, environment friendly and boosts biogas production and can be directly used in a biogas system or plant.SUMMARY OF THE INVENTION

[0005] Accordingly, one or more embodiments of the present invention is to provide a biocomplex comprising of anaerobic fungi cultivated on a lignocellulosic material; an enzyme complex comprising of one or more lignocellulose-degrading enzymes; and one or more volatile fatty acids, wherein the concentration of the lignocellulosic material is 1-12%, the concentration of the enzyme complex is 5-20 lU / ml, the concentration of the volatile fatty acids is 1000 to 10000 mg / L; and the enzyme complex is adapted to enhance degradation of the lignocellulosic material under anaerobic conditions (“the bio-complex”).

[0006] Other features and aspects of this invention will be apparent from the following description and the accompanying drawings. The features and advantages described in this summary and in the following detailed description are not all-inclusive, and particularly, many additional features and advantages will be apparent to one of ordinary skill in the relevant art, in view of the drawings, specification, and claims hereof. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposes and may not have been selected to delineate or circumscribe the inventive subject matter, resort to the claims being necessary to determine such inventive subject matter.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The accompanying drawings, which are incorporated herein, and constitute a part of this disclosure, illustrate exemplary embodiments of the disclosed bio-complex. Components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure.

[0008] FIG. 1A shows the presence of Orpinomyces joyonii mycelia as one of the examples on straw particles forming part of the bio-complex of the present invention grown in a 300 ml reactor, on microscopic examination.

[0009] FIG. IB shows the presence of Orpinomyces joyonii mycelia as one of the examples on straw particles forming part of the bio-complex of the present invention grown in a 200 litre (L) reactor, on microscopic examination.

[0010] FIG. 1C shows the presence of Orpinomyces joyonii mycelia as one of the examples on straw particles forming part of the bio-complex of the present invention grown in an 8 kilo litre (KL) reactor, on microscopic examination.

[0011] The foregoing shall be more apparent from the following detailed description of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0012] Some embodiments of the present disclosure, illustrating all its features, will now be discussed in detail. It must also be noted that as used herein and in the appended claims, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0013] 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 including the definitions listed herein below are 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.

[0014] A person of ordinary skill in the art will readily ascertain that the images and their details herein below are set out to explain the exemplary embodiments shown. The examples are presented herein for the purposes of illustration, and not limitation. Alternatives (including equivalents, extensions, variations, deviations, substitutes etc., of those described herein) will be apparent to persons skilled in the relevant art(s) based on the teachings contained herein. Such alternatives fall within the scope and spirit of the disclosed embodiments.

[0015] As per various embodiments depicted, the present invention discloses a biocomplex comprising of anaerobic fungi cultivated on a lignocellulosic material; an enzyme complex comprising of one or more lignocellulose-degrading enzymes; and one or more volatile fatty acids, wherein the concentration of the lignocellulosic material is 1-12%, theconcentration of the enzyme complex is 5- 20 lU / ml, the concentration of the volatile fatty acids is 1000 to 10000 mg / L; and the enzyme complex is adapted to enhance degradation of the lignocellulosic material under anaerobic conditions. Current methods for degradation of lignocellulosic materials prominently cover the use of strong alkali or acids followed by use of commercially available cellulase enzyme making the pretreatment energy intensive and costly.

[0016] In an embodiment of the present invention, the lignocellulosic material forming part of the bio-complex is selected from, but not limited to, paddy straw, cellobiose, wheat straw, wheat bran, bagasse, sucrose, cotton stalk, mustard stalk, or sugarcane trash or a combination thereof. In a preferable embodiment of the present invention, paddy straw is used as the lignocellulosic material. Lignocellulosic material serves as a rich source of carbohydrates, making them a promising feedstock for biofuel production. Further, since it is abundantly and naturally available, it serves as an environment friendly substrate.

[0017] In another embodiment of the present invention, the culture of the anaerobic fungi cultivated on the lignocellulosic material belongs to the phylum Neocalimastigomycota. In a preferable embodiment of the present invention, the anaerobic fungi cultivated on the lignocellulosic material is Orpinomyces joyonii, Piromyces sp., Anaeromyces sp., or Caecomyces sp. The anaerobic fungi cultivated on the lignocellulosic material are commonly found in the digestive tracts of herbivores, such as cows, sheep and horses and commercially available. In a preferable embodiment of the present invention, a culture of Orpinomyces jyoyonii MCMB 1461 anaerobic fungi is cultivated on the lignocellulosic material and obtained from MACS Collection of Microorganisms (MCM), Pune.

[0018] To enable the anaerobic fungi to cultivate on the lignocellulosic material, the lignocellulosic material is treated with a nutrient solution A, a nutrient solution B, a nutrient solution C, and an anti-microbial compound, details whereof are provided below.

[0019] In an embodiment of the present invention, the nutrient solution A used for treating the lignocellulosic material in the present invention comprises of one or more of the minerals and nitrogen sources disclosed in the table 1 below, dissolved in water.Quantity in gChemical name (per lit)TABLE 1

[0020] In a preferable embodiment of the present invention, yeast extract is used as the nitrogen source in the nutrient solution A, in the range of 0.05-0.2%, and more preferably 0.1%.

[0021] In an embodiment of the present invention, the nutrient solution B used for treating the lignocellulosic material in the present invention comprises of one or more of the minerals disclosed in the table 2 below, dissolved in water.TABLE 2

[0022] In an embodiment of the present invention, the nutrient solution C used for treating the lignocellulosic material in the present invention comprises of one or more of the minerals disclosed in the table 3 below, dissolved in water.TABLE 3

[0024] In an embodiment of the present invention, any commercially available antimicrobial compound is used for treating the lignocellulosic material. In a preferable embodiment of the present invention, any commercial antimicrobial compound selected from, but not limited to, chloramphenicol, ampicillin, streptomycin, bactoferm, or tetracyclin are used as antimicrobial compounds at the stage of treatment of the lignocellulosic material. The antimicrobial compound is added to inhibit the growth of any unwanted micro-organisms, which will otherwise interfere with the cultivation of the anaerobic fungi on the lignocellulosic material. The antimicrobial compound is selected based on its specificity of action thus ensuring it will not impact growth of anaerobic fungi

[0025] In an embodiment of the present invention, the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C are purged with an oxygen free sterilized gas to make the lignocellulosic material suitable for anaerobic digestion by the anaerobic fungi. In a preferable embodiment of the present invention, the oxygen free sterilized gas used for purging the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C includes by way of example but not limitation N2 (Nitrogen), H2 (Hydrogen), CO2 (Carbon Dioxide), Ar (Argon), He (Helium), or CH4 (Methane) or mixture of two or more of these gases. Since the anaerobic fungi do not require oxygen for their physiological processes, the purging of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C makes the substrate suitable for growth of the anaerobic fungi, which in turn releases useful enzymes and produces a by-product which may be used as a precursor for producing other value added products such as volatile fatty acids and soluble sugars.

[0026] In an embodiment of the present invention, the concentration of the enzyme complex in the bio-complex is 5-20 lU / ml. In a preferable embodiment of the present invention, the enzyme complex in the bio-complex comprises of hydrolytic enzymes endoglucanase, exoglucanase, beta glucosidase and xylanase, wherein endoglucanase and xylanase enzymes cumulatively make up 80-95% of the enzyme complex forming part of the bio-complex. The enzyme complex in the bio-complex acts on the carbohydrate fraction of the bio-complex i.e. on the lignocellulosic material and breaks down the lignocellulosic bonds. Due to this, the lignocellulosic material is not required to be separately treated with enzymes for breakdown of the lignin, thereby saving cost and time taken during pre-treatment of the lignocellulosic material. The focus of the pre-treatment processes of the prior art is on degrading lignin using chemicals or high temperature systems, however, in the present invention, the focus is on breaking lignocellulosic bonds and releasing cellulose and hemi cellulose and their degradation products.

[0027] In an embodiment of the present invention, the concentration of the volatile fatty acids in the bio-complex is 1000 to 10000 mg / L. In a preferable embodiment of the present invention, the volatile fatty acids forming part of the bio-complex include acetic acid, propionic acid, and butyric acid.

[0028] The bio-complex of the present invention includes a high concentration of high- activity enzymes like endoglucanase, exoglucanase, beta-glucosidase and xylanase capable of efficiently breaking down crystalline cellulose, a challenging substrate for many conventional enzymatic systems. This enzymatic system significantly improves the overall efficiency of the conversion of the lignocellulosic material into valuable products. The bio-complex as per the present invention represents a blend of partially decomposed biomass, microscopically visible penetrative growth of anaerobic fungi cells and extracellularly secreted enzyme complex and fermentative products by anaerobic fungi during their growth for the efficient degradation of lignocellulosic waste. The use of the bio-complex of the present invention as a biofuel boosts biogas production by up to 20%.

[0029] A table containing the results of the bio-complex and its components when produced for batch modes of 300 ml and 200 litre and continuous mode of 8 kilolitres is provided below:

[0030] The growth of Orpinomyces joyonii on the lignocellulosic material as examined under a microscope at 40X in respect of the bio-complex produced in a 300 ml reactor is shown in FIG.1A; the growth of Orpinomyces joyonii on the lignocellulosic material as examined under a microscope at 40X in respect of the bio-complex produced in a 200 L reactor is shown in FIG.1B; and the growth of Orpinomyces joyonii on the lignocellulosic material as examined under a microscope at 40X in respect of the bio-complex produced in a 8KL reactor is shown in FIG.1C.

[0031] Further, a study evaluating the biogas yield (L / kg VS) from a pulverized and shredded paddy straw at three inclusion levels (5%, 7.5%, and 10% or 10.5%) is provided below:In a 60L reactor scale:1. At 5% Paddy Straw Inclusion:A continuous reactor with 60 L working volume was operated at an HRT of 20 days using paddy straw as a substrate. The paddy straw was pulverized (size of 0.5 to 7 mm) and added to the reactor. The bio-complex of the present invention was added at 1% of volume of feed to be added each day (30 ml). The TS in the digester was fed at 5%. Similarly, another 60 L reactor was operated with similar parameters but using shredded straw (size of 1 to 10 cm). The average biogas yield obtained was 472 to 487 L / kgVS for pulverized material and 423 to 479 L / kg VS for shredded material. Different sizes of the straws were taken to determine impact of the anaerobic fungi on different sizes of straw.2. At 7.5% Paddy Straw Inclusion:A continuous reactor with 60 L working volume was operated at an HRT of 20 days using paddy straw as a substrate. The paddy straw was pulverized (size of 0.5 to 7 mm) and added to the reactor. The bio-complex of the present invention was added at 1% of volume of feed to be added each day (30 ml). The TS in the digester was fed at 7.5%. Similarly, another 60 L reactor was operated with similar parameters but using shredded straw (size of 1 to 10 cm). The average biogas yield obtained was 600 to 697 L / kgVS for pulverized material and 542 to 579 L / kg VS for shredded material.3. At 10.5% Paddy Straw Inclusion:A continuous reactor with 60 L working volume was operated at an HRT of 15 days using paddy straw as a substrate. The paddy straw was pulverized (size of 0.5 to 7 mm) and added to the reactor. The bio-complex of the present invention was added at 1% of volume of the feed to be added each day (30 ml). The TS in the digester was fed at 10.5%. Similarly, another 60 L reactor was operated with similar parameters but using shredded straw (size of 1 to 10 cm). The average biogas yield obtained was 590 to 611 L / kgVS for pulverized material and 575 to 582 L / kg VS for shredded material.In an 1800L reactor scale -1. At 5% Paddy Straw Inclusion:A continuous reactor with 1800 L working volume was operated at an HRT of 20 days using paddy straw as a substrate. The paddy straw was pulverized (size of 0.5 to 7 mm) and added to the reactor. The bio-complex of the present invention was added at 1% of volume of the feedto be added each day (30 ml). The TS of the digester was maintained at 5%. Similarly, another 1800 L reactor was operated with similar parameters but using pulverized straw and without the bio-complex. The average biogas yield obtained from the paddy straw mixed with the biocomplex was 490 L / kgVS and that for the pulverized straw without the bio-complex was 403 L / kg VS.2. At 7.5% Paddy Straw Inclusion:A continuous reactor with 1800 L working volume was operated at an HRT of 20 days using paddy straw as a substrate. The paddy straw was pulverized and added to the reactor. The biocomplex of the present invention was added at 1% of volume of the feed to be added each day (30 ml). The TS of the digester was maintained at 7.5%. Similarly, another 1800 L reactor was operated with similar parameters but using pulverized straw and without the bio-complex. The average biogas yield obtained from the paddy straw mixed with the bio-complex was 477 to 485 L / kgVS and that of the pulverized straw without the bio-complex was 418 to 437 L / kg VS.3. At 10% Paddy Straw Inclusion:A continuous reactor with 1800 L working volume was operated at an HRT of 20 days using paddy straw as a substrate. The paddy straw was pulverized and added to the reactor. The biocomplex of the present invention was added at 1% of volume of the feed to be added each day (30 ml). The TS of the digester was maintained at 10%. Similarly, another 1800 L reactor was operated with similar parameters but using pulverized straw and without the bio-complex. The average biogas yield obtained from the paddy straw mixed with the bio-complex was 426 to 561 L / kg VS and that of the pulverized straw without the bio-complex was 376 to 481 L / kg VS.A summary table of the above results is provided below:1. 60L Digester Table (Pulverized & Shredded Paddy Straw) with the bio-complex:2. 1800L Digester Table (with and without the bio-complex)

[0032] One of the primary advantages of the present invention, is that the bio-complex of the present invention includes an in situ produced mixture of high-activity enzymes like endoglucanase, exoglucanase, beta-glucosidase and xylanase capable of efficiently breakingdown crystalline cellulose, a challenging substrate for many conventional enzymatic systems. This enzymatic system significantly improves the overall efficiency of lignocellulosic material conversion into valuable products. The commercially available pure cellulolytic and hemi cellulolytic enzymes are cost prohibitive.

[0033] Another advantage of the present invention is that the release of volatile fatty acid (VFA) by the anaerobic fungi in the composition serves as the precursor of other value-added products including methane.

[0034] The bio-complex of the present invention can be directly used for biofuel production by adding it to the digester of the biogas plant, thereby resulting in the biogas plant being operated in a single stage and saving cost and time. It can also be used for pre-treatment of lignocellulosic material for use in biomanufacturing of various products such as sustainable aviation fuel.

[0035] All the above advantages of the present invention contribute to the costeffectiveness of the biomethane production process, which is part of biogas production, thereby making the technology more accessible and economically viable for a wider range of agricultural applications.

[0036] The present invention offers multiple advantages over the prior art and the above listed are a few examples to emphasize on some of the advantageous features. The listed advantages are to be read in a non-limiting manner.

[0037] The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since, modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of appended claims.

Claims

CLAIMSWe Claim:

1. A bio-complex comprising of: anaerobic fungi cultivated on a lignocellulosic material; an enzyme complex comprising of one or more lignocellulose-degrading enzymes; and one or more volatile fatty acids; wherein the concentration of the lignocellulosic material is 1-12%; wherein the concentration of the enzyme complex is 5- 20 lU / ml; wherein the concentration of the volatile fatty acids is 1000 to 6000 mg / L; and wherein the enzyme complex is adapted to enhance degradation of the lignocellulosic material under anaerobic conditions.

2. The bio-complex as claimed in claim 1, wherein the lignocellulosic material is paddy straw, cellobiose, wheat straw, wheat bran, bagasse, sucrose, cotton stalk, mustard stalk, or sugarcane trash or a combination thereof.

3. The bio-complex as claimed in claim 1, wherein the anaerobic fungi are selected from the phylum Neocallimastigomycota, including genera Orpinomyces, Piromyces, Anaeromyces, or Caecomyces.

4. The bio-complex as claimed in claim 1, wherein the enzyme complex comprises of endoglucanase, exoglucanase, beta glucosidase and xylanase.

5. The bio-complex as claimed in claim 4, wherein the endoglucanase and xylanase enzymes cumulatively constitute 80-95% of the enzyme complex.

6. The bio-complex as claimed in claim 1, wherein the volatile fatty acids in the biocomplex comprise of acetic acid, propionic acid, and butyric acid.

7. The bio-complex as claimed in claim 1, wherein the bio-complex is formulated for use in enhancing biomass degradation in anaerobic digesters, ruminant feed applications, or composting systems as a biofuel.

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