DESC: FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
[See section 10, Rule 13]

TITLE: APPARATUS AND METHOD FOR LARGESCALE AND CONTINUOUS GROWTH OF ANAEROBIC MICRO-ORGANISMS

APPLICANTS:
(1) AGHARKAR RESEARCH INSTITUTE OF MAHARASHTRA ASSOCIATION FOR THE CULTIVATION OF SCIENCE, WHOSE ADDRESS IS, G.G. AGARKAR ROAD, PUNE-411004

(2) GPS RENEWABLES PVT. LTD., WHOSE ADDRESS IS: PRESTIGE PINNACLE, NO. 113, 20TH MAIN ROAD, 3RD FLOOR, 7TH BLOCK, ADUGODI, KORAMANGALA, BANGALORE, KARNATAKA – 560034

THE FOLLOWING SPECIFICATION PARTICULARLY DESCRIBES THE INVENTION AND THE MANNER IN WHICH IT IS TO BE PERFORMED

[0001] The present application claims priority from Indian Patent Application No. 202441019825 filed with the Indian Patent Office on 18th April 2024 titled “PROCESS FOR CONTINUOSULY PRODUCING ANAEROBIC FUNGAL BIOMASS ON A LARGE SCALE” and from Indian Patent Application No. 202441019826 filed with the Indian Patent Office on 18th April 2024 titled “A SYSTEM FOR CONTINUOUS PRODUCTION OF ANAEROBIC FUNGAL BIOMASS ON A LARGE SCALE”, the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION
[0002] The present invention relates to an apparatus and a method for largescale and continuous growth of anaerobic micro-organisms, particularly using lignocellulosic material as a substrate.

BACKGROUND OF THE INVENTION
[0003] Anaerobic fungi degrade lignin-rich biomass through the secretion of cellulases, hemicellulases and hydrolytic mechanisms which involve secreted enzymes and membrane anchored extracellular cellulosomes.

[0004] However, growing monoculture of anaerobic micro-organisms in a large fermenter presents several technical challenges. Some of the challenges associated with growing anaerobic micro-organisms in an apparatus or a system on a largescale and continuously are related to optimizing culture conditions, selecting suitable growth substrates, maintaining predominantly the appropriate microbial community, controlling contamination, and monitoring fermentation parameters.

[0005] There is, therefore, a need for an apparatus and a method for largescale and continuous growth of anaerobic micro-organisms that facilitates the techno-economically viable and continuous growth of anaerobic micro-organisms and produces lignocellulosic enzymes and overcomes the disadvantages of the prior art discussed above.

[0006] Accordingly, one or more embodiments of the present invention is to provide an apparatus and a method for largescale and continuous growth of a monoculture anaerobic micro-organisms.

SUMMARY OF THE INVENTION

[0007] In one aspect of the present invention, an apparatus for largescale and continuous growth of anaerobic micro-organisms is disclosed. The apparatus for largescale and continuous growth of anaerobic micro-organisms comprises of one or more containers and is adapted to sterilize a batch of lignocellulosic material along with a nutrient solution A and a nutrient solution B at a pre-determined temperature and for a pre-determined heating time. The apparatus is further adapted to purge the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with an oxygen free sterilized gas at a pre-determined flow rate. The apparatus is further adapted to cool the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The apparatus is further adapted to add a received nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cool the sterilized mixture to a pre-determined temperature. The apparatus is further adapted to add a received antimicrobial compound to the further cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a sterilized first nutrient medium. The apparatus is further adapted to add a received culture of the anaerobic micro-organism to the sterilized first nutrient medium. The apparatus is further adapted to cultivate the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined Hydraulic Retention Time (HRT) by utilizing the sterilized first nutrient medium. The apparatus is further adapted to pasteurize a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time. The apparatus is further adapted to purge the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate. The apparatus is further adapted to cool the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The apparatus is further adapted to add the received nutrient solution C to the cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cool the pasteurized mixture to a pre-determined temperature. The apparatus is further adapted to add the received antimicrobial compound to the further cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium. The apparatus is further adapted to facilitate growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of the anaerobic micro-organism cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT.

[0008] In another aspect of the present invention, a method for largescale and continuous growth of anaerobic micro-organisms is disclosed. The method includes the step of sterilizing a batch of lignocellulosic material along with a nutrient solution A and a nutrient solution B at a pre-determined temperature and for a pre-determined heating time. The method further includes the step of purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with an oxygen free sterilized gas at a pre-determined flow rate. The method further includes the step of cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The method further includes the step of adding a nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the sterilized mixture to a pre-determined temperature. The method further includes the step of adding an antimicrobial compound to the further cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a sterilized first nutrient medium. The method further includes the step of adding a culture of the anaerobic micro-organism to the sterilized first nutrient medium. The method further includes the step of cultivating the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined HRT by utilizing the sterilized first nutrient medium. The method further includes the step of pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time. The method further includes the step of purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate. The method further includes the step of cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The method further includes the step of adding the nutrient solution C to the cooled and purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the pasteurized mixture to a pre-determined temperature. The method further includes the step of adding the antimicrobial compound to the further cooled mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium. The method further includes the step of facilitating growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of the anaerobic fungi cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT.

[0009] 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

[0010] The accompanying drawings, which are incorporated herein, and constitute a part of this disclosure, illustrate exemplary embodiments of the disclosed apparatuses and methods in which like reference numerals refer to the same parts throughout the different drawings. Components in the drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the present disclosure. Some drawings may indicate the components using block diagrams and may not represent the internal circuitry of each component. It will be appreciated by those skilled in the art that disclosure of such drawings includes disclosure of connectors, components or parts, pumps, electronic components or circuitry commonly used for working of the various components together as an apparatus.

[0011] FIG. 1 is an exemplary block diagram of an environment for largescale and continuous growth of anaerobic micro-organisms according to one or more embodiments of the present invention.

[0012] FIG. 2 is an exemplary block diagram of an apparatus for largescale and continuous growth of anaerobic micro-organisms, according to one or more embodiments of the present invention.
[0013] FIG. 3 is a schematic representation of the apparatus, according to one or more embodiments of the present invention.

[0014] FIG. 4 is a flow diagram of the method for largescale and continuous growth of anaerobic micro-organisms, according to one or more embodiments of the present invention.

[0015] The foregoing shall be more apparent from the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0016] 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.

[0017] 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.

[0018] A person of ordinary skill in the art will readily ascertain that the illustrated steps detailed in the figures and herein below are set out to explain the exemplary embodiments shown, and it should be anticipated that ongoing technological development will change the manner in which particular functions are performed. These examples are presented herein for 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.

[0019] As per various embodiments depicted, the present invention discloses an apparatus and a method for largescale and continuous growth of anaerobic micro-organisms.

[0020] Referring now to the drawings, where similar reference characters denote corresponding features consistently throughout the figures, there are shown preferred embodiments.

[0021] FIG. 1 is an exemplary block diagram of an environment 100 comprising of an apparatus 108 for largescale and continuous growth of anaerobic micro-organisms, according to one or more embodiments of the present disclosure. In this regard, the environment 100 includes a source for a lignocellulosic material, a nutrient solution A, a nutrient solution B, a nutrient solution C and an antimicrobial compound 102 (hereinafter referred to as “source A”), a source for oxygen free sterilized gas 104 (hereinafter referred to as “source B”), a source for a culture of the anaerobic micro-organism 106 (hereinafter referred to as “source C”), an apparatus 108 for largescale and continuous growth of anaerobic micro-organisms (hereinafter referred to as “the apparatus”), and a digester tank 110, all communicably coupled to each other.

[0022] In an embodiment of the present invention, the lignocellulosic material includes at least one of, 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.

[0023] In one embodiment of the present invention, the anaerobic micro-organisms are at least one of, but not limited to, fungi, algae and bacteria.

[0024] In an embodiment of the present invention, the source A 102 includes, by way of example but not limitation, a shredder, hammer mill, or pulveriser. In another embodiment of the present invention, the source A 102 is a container having one or more partitions. For example, the container may also be referred to as at least one of, but not limited to, a reactor, a digestor, a vessel, a chamber, a compartment, a drum, a tank, and an enclosure that are utilized to store the lignocellulosic material, the nutrient solution A, the nutrient solution B, the nutrient solution C, and the antimicrobial compound. In another embodiment, the container is made up of at least one of, but not limited to, a stainless steel, concrete, an aluminium, a plastic, a fibre and synthetic materials or a combination thereof.

[0025] In an embodiment of the present invention, when the lignocellulosic material to be used in the present invention is brittle, the source A 102 supplies a mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B to the apparatus 108 as a first step. The mixing of the lignocellulosic material, the nutrient solution A and the nutrient solution B in the source A 102 may be conducted by any solid-liquid mixing process, including but not limited to, stirring, blending, agitation, shear mixing, convective mixing or diffusion mixing. In another embodiment of the present invention, when the lignocellulosic material to be used in the present invention is recalcitrant, the lignocellulosic material, the nutrient solution A and the nutrient solution B are not mixed in the source A 102 and are added separately to the apparatus 108 in the manner as detailed below. In yet another embodiment of the present invention, the source A 102 also separately supplies the nutrient solution C and the antimicrobial compound to the apparatus 108 in the manner as detailed below.

[0026] The environment 100 further includes the source B 104 which is communicably coupled to the apparatus 108. In particular, the apparatus 108 is configured to receive the oxygen free sterilized gas from the source B 104 to facilitate growth of the anaerobic micro-organisms inside the apparatus 108. In an embodiment of the present invention, the source B 104 includes, by way of example but not limitation, oxygen free sterilized gas cylinders, gas blowers, or low-pressure compressors.

[0027] The environment 100 further includes the source C 106 which is also communicably coupled to the apparatus 108. In particular, the apparatus 108 is configured to receive the culture of the anaerobic micro-organisms from the source C 106. In one embodiment, culturing anaerobic microorganisms involves creating an environment where oxygen is excluded or minimized, ensuring that the anaerobic micro-organisms can grow and thrive. The culture of the anaerobic micro-organisms grows on the lignocellulosic material already added to the apparatus 108, by using the nutrient solution A, the nutrient solution B and the nutrient solution C in the presence of the oxygen free sterilized gas inside the apparatus 108. In an embodiment of the present invention, the source C 106 includes, by way of example but not limitation, a central laboratory which continuously maintains the culture of the anaerobic micro-organisms at the range of 35? to 40?. In a preferable embodiment of the present invention, the culture of the anaerobic micro-organisms is brought to the site only when required in an air-tight insulated container, from which the culture of the anaerobic micro-organisms may be transferred to the apparatus 108 through any automated or semi-automated means including at least one of, but not limited to, a pump or manually.

[0028] The environment 100 further includes the apparatus 108. In particular, the apparatus 108 comprises of one or more containers, configured to facilitate growth of the anaerobic micro-organisms. In an embodiment of the present invention, the apparatus 108 comprises of a plurality of containers (as shown in FIG.2 and FIG. 3), all communicably coupled to each other, and each of the plurality of containers has one or more inlets and one or more outlets. In another embodiment of the present invention, wherein the apparatus 108 comprises of a plurality of containers, the plurality of containers is arranged either in series or in parallel. In an alternate embodiment of the present invention, the one or more containers of the apparatus 108 are housed inside a vessel (as shown in FIG.1). The one or more containers of the apparatus 108 of the present invention are communicably coupled to each other by any suitable means, including at least one of, but not limited to, pipes and tubes and are equipped with a stirrer or aeration device and adapted to mix and blend the contents added to the containers. The details of the one or more containers forming part of the apparatus 108 and their operational and functional features are discussed in detail with reference to FIG.2 and FIG. 3 below.

[0029] The environment 100 further includes a digester tank 110 which is also communicably coupled to the apparatus 108. In an embodiment of the present invention, the digester tank 110 includes, by way of example but not limitation, a biogas reactor, culture tank, pre-fermenter, growth vessel, anaerobic reactor, or an anaerobic digester. The digester tank 110 is configured to receive the contents produced inside the apparatus 108 and also the gases released inside the apparatus 108 during this process, for further treatment or use in a biogas system.

[0030] Operational and construction features of the apparatus 108 and the method adapted for largescale and continuous growth of the anaerobic micro-organisms will be explained in detail with respect to the following figures.

[0031] FIG. 2 is an exemplary block diagram of the apparatus 108, according to one or more embodiments of the present invention.

[0032] As discussed above, in an embodiment of the present invention, the apparatus 108 comprises of one or more containers configured to facilitate largescale and continuous growth of anaerobic micro-organisms. In a preferable embodiment of the present invention, as illustrated in FIG.2, the apparatus 108 comprises of four containers i.e. a first container 204, a second container 208, a third container 210, and a fourth container 214, all communicably coupled to each other via lines, and having one or more inlets and outlets. In an embodiment of the present invention, the one or more containers of the present invention may be selected from but not limited to, any anaerobic chamber or any durable, non-breakable, chemical resistant, or gas tight container made of, by way of example but not limitation, one or more of, stainless steel, high-quality medical-grade plastics, aluminium, borosilicate glass, transparent polycarbonate, or FRP lined mild steel.

[0033] As illustrated in FIG.2, in an embodiment of the present invention, the first container 204 of the apparatus 108 has two inlets i.e. a first inlet 202a and a second inlet 202b, and one outlet i.e. 206. The first inlet 202a of the first container 204 is configured to receive the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B, the nutrient solution C and the antimicrobial compound from the source A 102 (shown in FIG.1). The second inlet 202b of the first container 204 is configured to receive the oxygen free sterilized gas from the source B 104 (shown in FIG.1). In an alternate embodiment of the present invention, when the lignocellulosic material is not mixed with the nutrient solution A and the nutrient solution B, and is added separately to the apparatus 108, the first container 204 is provided with three inlets (shown in FIG. 3). The embodiment of the apparatus 108 comprising of a first container 204 having three inlets and its configuration will be explained with reference to FIG. 3.

[0034] In an embodiment of the present invention, the first container 204 is a sterilization tank. In a preferable embodiment of the present invention, the size of the first container 204 is in the range of 1-5 m3. As discussed above, the first container 204 is configured to receive the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B from the source A 102 through the first inlet 202a of the first container 204. On receiving the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B from the first inlet 202a, the first container 204 is adapted to sterilize the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time. In an embodiment of the present invention, any heating system or heating means such as, at least one of, but not limited to, heating coils or immersion heater may be used for sterilizing the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B. The sterilization of the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is carried out to inactivate all microbial life forms and obtain a nutrient medium with a zero microbial count, which is essential for the growth of the anaerobic micro-organisms. In a preferable embodiment of the present invention, the pre-determined temperature for sterilizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B inside the first container 204 is in the range of 80? to 120? and the pre-determined heating time is 0-1 day. For example, the minimum pre-determined heating time for sterilizing the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is in the range of 10 to 30 minutes. Details of the types of the lignocellulosic material used and the composition of the nutrient solution A and nutrient solution B received by the first container 204 shall be provided while explaining the method for largescale and continuous growth of the anaerobic micro-organisms according to one or more embodiments of the present invention.

[0035] After sterilization of the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B, the first container 204 is configured to receive the oxygen free sterilized gas from the source B 104 through the second inlet 202b of the first container 204, and purge, within the first container 204, the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate. In a preferable embodiment of the present invention, the pre-determined flow rate for purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is in the range 40 to 60 L/min.

[0036] In one embodiment, purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B refers to the process of removing unwanted gases such as at least one of, but not limited to, oxygen, contaminants, or other impurities from the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B after it has been sterilized, usually in preparation for further processing. In particular, purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas eliminates the presence of oxygen from the sterilized mixture, which is essential for the growth of the anaerobic micro-organisms. For example, when the lignocellulosic material used in the present invention is corn stover or wood chips or any other lignocellulosic material, the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B may be purged with nitrogen to remove any residual oxygen. Further, the absence of oxygen in the sterilized mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B ensures that the aforesaid sterilized mixture and the environment inside the first container 204 is unsuitable for growth of any aerobic microbial life and prevents contamination of the contents inside the first container 204 from the outset. Details of the oxygen free sterilized gas received by the first container 204 shall be provided while explaining the method for largescale and continuous growth of the anaerobic micro-organisms according to one or more embodiments of the present invention.

[0037] Pursuant to purging, the first container 204 is adapted to cool the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The cooling of the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B refers to the process of reducing the temperature of the mixture after it has been sterilized and purged of unwanted gases. In a preferable embodiment of the present invention, the pre-determined temperature for cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 50? to 60?. In an embodiment of the present invention, the purged sterilized mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is cooled by using any suitable means for reducing the temperature inside the first container 204 including at least one of, but not limited to, air cooling, water cooling, natural cooling, or use of one or more heat exchangers. Let us consider that the lignocellulosic material is in a large container such as inside the first container 204, then the cooling may be performed with a jacket arrangement around the first container 204 with cooling water passing inside the jacket. The cooling of the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B inside the first container 204 ensures prevention of thermal degradation of the lignocellulosic material and provides optimal conditions for anaerobic micro-organisms and maintains the stability and integrity of the nutrients in the purged sterilized mixture.

[0038] In an embodiment of the present invention, once the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is cooled to a pre-determined temperature, the first container 204 is further configured to receive the nutrient solution C from the first inlet 202a of the first container 204. In particular, the received nutrient solution C is added to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B. Thereafter, the first container 204 is further configured to cool the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to a pre-determined temperature. In an embodiment of the present invention, the nutrient solution C is supplied to the first container 204 through the source A 102 (shown in FIG. 1). In an alternate embodiment of the present invention, the nutrient solution C is manually supplied to the first container 204 through the first inlet 202a of the first container 204.

[0039] In a preferable embodiment of the present invention, the pre-determined temperature for cooling the sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C inside the first container 204 is in the range 30? to 45?. The cooling of the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C facilitates cooling of the sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C and removal of any trace oxygen from the mixture, simultaneously and ensures optimal temperature for further processing.

[0040] Upon adding the nutrient solution C and cooling the sterilized mixture to the pre-determined temperature, the first container 204 is further configured to receive an antimicrobial compound from the first inlet 202a of the first container 204. In particular, the antimicrobial compound is added to the cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a sterilized first nutrient medium. In an embodiment of the present invention, the antimicrobial compound is supplied to the first container 204 through the source A 102 (shown in FIG. 1). In an alternate embodiment of the present invention, the antimicrobial compound is manually supplied to the first container 204 through the first inlet 202a of the first container 204. For example, the antimicrobial compound is added to the cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to prevent the growth of micro-organisms (such as bacteria, fungi, and molds) that could cause spoilage, degradation, or contamination of the sterilized first nutrient medium.

[0041] In one embodiment, the antimicrobial compound kills and inhibits the growth of micro-organisms, if any, in the sterilized first nutrient medium. Details of the antimicrobial compound added to the first container 204 shall be provided while explaining the method for largescale and continuous growth of the anaerobic micro-organisms according to one or more embodiments of the present invention. The aforesaid features of the first container 204 along with the temperature parameters and flow rate for purging the oxygen free sterilized gas inside the first container 204 allows the apparatus 108 to produce a sterilized first nutrient medium at an industrial scale without contamination.

[0042] Upon forming the sterilized first nutrient medium, the sterilized first nutrient medium is released from the outlet 206 of the first container 204 into the second container 208. In another embodiment of the present invention, the first container 204 is adapted to purge the first container 204 with the oxygen free sterilized gas until the temperature inside the first container 204 is reduced to the range of 40? to 45? and while transferring the sterilized first nutrient medium to the second container 208, to avoid vacuum. The operation of the first container 204 is at least partially automated and is subject to regular monitoring to ensure its proper functionality. In a preferable embodiment of the present invention, the entire contents of the first container 204 are transferred to the second container 208.

[0043] Further, as per the illustrated embodiment, the apparatus 108 includes the second container 208 coupled to the first container 204 on one side. In an embodiment of the present invention, the second container 208 has two inlets i.e. a first inlet 208a and a second inlet 208b and one outlet 208c. The first inlet 208a of the second container 208 is configured to receive the sterilized first nutrient medium from the outlet 206 of the first container 204. Further, the second inlet 208b of the second container 208 is configured to receive a culture of the anaerobic micro-organism from the source C 106 (shown in FIG.1). In an embodiment of the present invention, on receiving the sterilized first nutrient medium from the first container 204 and the culture of the anaerobic micro-organism from the source C 106, the second container 208 is adapted to cultivate the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined Hydraulic Retention Time (HRT) by utilizing the sterilized first nutrient medium. Details of the culture of the anaerobic micro-organism received in the second container 208 shall be provided while explaining the method for largescale and continuous growth of the anaerobic micro-organisms according to one or more embodiments of the present invention.

[0044] In an embodiment of the present invention, the second container 208 is a culture tank. In another embodiment of the present invention, the size of the second container 208 is in the range of 5-50 m3. In a preferable embodiment of the present invention, the pre-determined temperature for cultivating the culture of the anaerobic micro-organism inside the second container 208 is in the range 25? to 45? and the pre-determined HRT of the second container 208 is 2-10 days. In an embodiment of the present invention, the everyday volumes in the range of 50% to 10% of the contents in the second container 208 is replaced with the sterilized first nutrient medium from the first container 204 and an equivalent volume of the culture of the anaerobic micro-organism is cultivated in the second container 208.

[0045] The resultant culture of the anaerobic micro-organism cultivated inside the second container 208 is released through the outlet 208c of the second container 208 to a fourth container 214, for further processing. During the process of cultivation of the culture of the anaerobic micro-organism inside the second container 208, gases such as H2 and CO2 are also released inside the second container 208. In an embodiment of the present invention, the second container 208 is provided with two outlets (shown in FIG.3), wherein the gases released inside the second container 208 are also transferred to the fourth container 214 via a second outlet of the second container 208 (shown in FIG.3). The embodiment of the apparatus 108 comprising of a second container 208 having two outlets and its configuration will be explained with reference to FIG. 3.

[0046] The operation of the second container 208 is at least partially automated and is subject to regular monitoring to ensure its proper functionality. The aforesaid features of the second container 208 along with the temperature parameters and the HRT for cultivating the anaerobic micro-organism inside the second container 209 allows sufficient time to the anaerobic micro-organism to degrade the lignocellulosic material by utilizing the nutrient solution A, the nutrient solution B and the nutrient solution C forming part of the sterilized first nutrient medium and partially pre-treat the lignocellulosic material. The size of the second container 208 further allows the cultivation of the anaerobic micro-organism inside the second container 208 at an industrial scale. In one embodiment, for example, the culture of the anaerobic micro-organism cultivated inside the second container 208 is used for its intended purpose (e.g., degradation of the lignocellulosic material, for biogas production, fermentation, or bioremediation). The culture may be transferred to other containers or processed to extract metabolites including but not limited to enzymes, hydrogen, carbon dioxide, or volatile fatty acids.

[0047] As per the illustrated embodiment, the apparatus 108 further includes the third container 210, having two inlets i.e. a first inlet 210a and a second inlet 210b and one outlet i.e. 212. The first inlet 210a of the third container 210 is configured to receive the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B, the nutrient solution C and the antimicrobial compound from the source A 102 (shown in FIG.1). The second inlet 210b of the third container 204 is configured to receive the oxygen free sterilized gas from the source B 104 (shown in FIG.1). In an alternate embodiment of the present invention, when the lignocellulosic material is not mixed with the nutrient solution A and nutrient solution B and is added separately to the apparatus 108 and water is additionally added to the third container 210, the third container 210 is provided with four inlets (shown in FIG. 3). The embodiment of the apparatus 108 comprising of a third container 210 having four inlets and its configuration will be explained with reference to FIG. 3.

[0048] In an embodiment of the present invention, the third container 210 is a nutrient tank. In a preferable embodiment of the present invention, the size of the third container 210 is in the range of 5-50 m3. As discussed above, the third container 210 is configured to receive the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B from the source A 102 through the first inlet 210a of the third container 210. On receiving the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B from the first inlet 210a, the third container 210 is adapted to pasteurize the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time. In a preferable embodiment of the present invention, the pre-determined temperature for pasteurizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B inside the third container 210 is in the range 50? to 80? and the pre-determined heating time is 0-1 day. For example, pasteurizing a mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B involves the process of heating the aforesaid mixture to a pre-determined temperature for a certain amount of time to reduce the presence of micro-organisms (bacteria, fungi, and other pathogens) in the mixture while minimizing damage to the lignocellulosic material. In an embodiment of the present invention, any heating system or heating means may be used for pasteurizing the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B.

[0049] In yet another preferable embodiment of the present invention, the minimum pre-determined heating time for pasteurizing the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is in the range of 10 to 30 minutes. In an embodiment of the present invention, the pasteurization of the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is carried out to obtain a nutrient medium with a low microbial count. The lignocellulosic material and the composition of the nutrient solution A and the nutrient solution B received by the third container 210 are the same as those received by the first container 204. In an embodiment of the present invention, the third container 210 is also adapted to sterilize the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B. However, to reduce the overall energy consumption and cost of the apparatus 108, the third container 210 is adapted to pasteurize the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B.

[0050] After pasteurization of the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B, the third container 210 is configured to receive the oxygen free sterilized gas from the source B 104 through the second inlet 210b of the third container 210, and purge the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate. In a preferable embodiment of the present invention, the pre-determined flow rate for purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is in the range 200 to 400 L/min. Purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas eliminates the presence of oxygen from the pasteurized mixture, which is essential for the growth of the anaerobic micro-organisms. Further, the absence of oxygen in the pasteurized mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B ensures that the aforesaid pasteurized mixture and the environment inside the third container 210 is unsuitable for growth of any aerobic microbial life and prevents contamination of the contents inside the third container 210 from the outset. The oxygen free sterilized gas received by the third container 210 is the same as that received by the first container 204.

[0051] Pursuant to purging, the third container 210 is adapted to cool the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. The cooling of the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature refers to the process of reducing the temperature of the mixture after it has been pasteurized and purged of unwanted gases. In an embodiment of the present invention, the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B is cooled by using any suitable means for reducing the temperature inside the third container 210 including at least one of, but not limited to, air cooling, water cooling, natural cooling, or use of one or more heat exchangers. In a preferable embodiment of the present invention, the pre-determined temperature for cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 40? to 45?. The cooling of the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B inside the third container 210 ensures prevention of thermal degradation of the lignocellulosic material and provides optimal conditions for anaerobic micro-organisms, preserves microbial health, and maintains the stability and integrity of nutrients in the purged pasteurized mixture.

[0052] In an embodiment of the present invention, once the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is cooled to the pre-determined temperature inside the third container 210, the third container 210 is configured to receive the nutrient solution C from the first inlet 210a of the third container 210. In particular, the received nutrient solution C is added to the cooled and purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B. Thereafter, the third container 210 is further configured to cool the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to a pre-determined temperature. In an embodiment of the present invention, the nutrient solution C is supplied to the third container 210 through the source A 102. In an alternate embodiment of the present invention, the nutrient solution C is manually supplied to the third container 210 through the first inlet 210a of the third container 210. In a preferable embodiment of the present invention, the pre-determined temperature for cooling the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C is in the range 30? to 45?. The cooling of the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C ensures prevention of thermal degradation of the lignocellulosic material and is necessary to bring the pasteurized mixture to an appropriate temperature suitable for the growth of culture of the anaerobic micro-organisms.

[0053] The third container 210 is further configured to receive the antimicrobial compound from the first inlet 210a of the third container 210 and add it to the cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium. In an embodiment of the present invention, the antimicrobial compound is supplied to the third container 210 through the source A 102. In an alternate embodiment of the present invention, the antimicrobial compound is manually supplied to the third container 210 through the first inlet 210a of the third container 210. The antimicrobial compound will kill and inhibit the growth of micro-organisms, if any, in the pasteurized second nutrient medium. The antimicrobial compound added to the third container 210 is the same as that added to the first container 204.

[0054] The pasteurized second nutrient medium is released from the outlet 212 of the third container 210 into the fourth container 214. In an embodiment of the present invention, the third container 210 is adapted to purge the third container 210 with the oxygen free sterilized gas until the temperature inside the third container 210 is reduced to 40? to 45? and while transferring the pasteurized second nutrient medium to the fourth container 214, to avoid vacuum. The operation of the third container 210 is at least partially automated and is subject to regular monitoring to ensure its proper functionality. The aforesaid features of the third container 210 along with the temperature parameters and flow rate for purging the oxygen free sterilized gas inside the third container 210 allows the apparatus 108 to produce a pasteurized second nutrient medium at an industrial scale without contamination. Further, since the third container 210 is adapted to pasteurize the second nutrient medium, the overall cost and energy consumption by the apparatus 108 is reduced.

[0055] As per the illustrated embodiment, the apparatus 108 further includes the fourth container 214, coupled to the second container 208 on its one side and to the third container 210 on its other side. The fourth container 214 has two inlets i.e. a first inlet 214a and a second inlet 214b and has two outlets i.e. a first outlet 216 and a second outlet 218. The fourth container 214 is configured to receive the cultivated culture of the anaerobic micro-organism from the second container 208 through the first inlet 214a of the fourth container 214 and is configured to receive the pasteurized second nutrient medium from the third container 210 through the second inlet 214b of the fourth container 214, and facilitate growth of the anaerobic micro-organism resulting from the anaerobic digestion of the lignocellulosic material by the anaerobic micro-organism by utilizing the pasteurized second nutrient medium at a pre-determined temperature and for a pre-determined HRT.

[0056] In a preferable embodiment of the present invention, the pre-determined temperature for facilitating growth of the anaerobic micro-organism inside the fourth container 214 is in the range 25? to 45? and the pre-determined HRT is 2-10 days. Since the culture of the anaerobic micro-organism is cultivated in the second container 208, when this culture of the cultivated micro-organism is mixed with the pasteurized second nutrient medium in the fourth container 214, the anaerobic micro-organisms utilize the additional nutrient medium added to the fourth container 214 and degrade the lignocellulosic material forming part of the pasteurized second nutrient medium at an exponential rate, thereby substantially reducing the overall time for growing the anaerobic micro-organisms at an industrial scale. Further, using the lignocellulosic material in pasteurized second nutrient medium enables in faster adaptation of culture of the anaerobic micro-organism to this type of material.

[0057] In another preferable embodiment of the present invention, the fourth container 214 is configured to first receive the pasteurized second nutrient medium from the third container 210 through the second inlet 214b of the fourth container 214 and thereafter receive the cultivated culture of the anaerobic micro-organisms from the second container 208 through the first inlet 214a of the fourth container 214. In yet another embodiment of the present invention, the fourth container 214 is configured to receive the pasteurized second nutrient medium from the third container 210 through the second inlet 214b of the fourth container 214 and the cultivated culture of the anaerobic micro-organisms from the second container 208 through the first inlet 214a of the fourth container 214, simultaneously. The simultaneous addition of the contents of the third container 210 and the second container 208 into the fourth container 214 is for ease of operation. The growth of the anaerobic micro-organisms in the fourth container 214 is monitored periodically by microscopic examination of a sample of the contents inside the fourth container 214 retrieved from inside the fourth container 214 through one of the one or more outlets of the fourth container 214, followed by conducting enzyme assay for cellulase enzyme in the retrieved sample. In an embodiment of the present invention, when the cellulase activity in the sample was found to be below 5 IU/ml, the fourth container 214 is configured to receive the sterilized first nutrient medium from the first container 204 through a third inlet of the fourth container 214 (shown in FIG.3) and receive gases released inside the second container 208 through a fourth inlet of the fourth container 214 (shown in FIG.3). The embodiment of the apparatus 108 comprising of a fourth container 214 having four inlets and its configuration will be explained with reference to FIG. 3.

[0058] In an embodiment of the present invention, the fourth container 214 is an inoculum tank. The inoculum tank is a vessel used in various industrial processes to grow and maintain an inoculum such as a population of micro-organisms (such as bacteria, yeast, or fungi) that are added to support at least one of, but not limited to, fermentation, biogas production, or other biological transformations. In another embodiment of the present invention, the size of the fourth container 214 is in the range of 30-150 m3.

[0059] In an embodiment of the present invention, the first outlet 216 of the fourth container 214 is configured to release the contents inside the fourth container 214 to the digester tank 110 (shown in FIG.1), and the second outlet 218 of the fourth container 214 is configured to release the gases produced inside the fourth container 214 also to the digester tank 110 (shown in FIG.1). In an embodiment of the present invention, the fourth container 214 has means for separation of gases released inside the fourth container 214 such as, by way of example but not limitation, molecular sieves or copper heating or any absorbent or adsorbent or employs any membrane separation method for separating the gases released inside the fourth container 214 and transfer it to the digester tank 110. The aforesaid features of the fourth container 214 along with the temperature parameters inside the fourth container 214 allows the apparatus 108 to facilitate growth of the anaerobic micro-organisms and produce a bio-complex which may be directly used in a biogas system.

[0060] The operation of the fourth container 214 is at least partially automated and is subject to regular monitoring to ensure its proper functionality. In a preferable embodiment of the present invention, the everyday volume in the range of 50% to 10% of the contents in the fourth container 214 is replaced with the combined volume of the cultivated culture of the anaerobic micro-organisms from the second container 208 and the pasteurized second nutrient medium from the third container 210.

[0061] In an embodiment of the present invention, the apparatus 108 is managed and operated from a central facility equipped with a programming system and may be fully automated, semi-automated or operated manually.

[0062] FIG. 3 illustrates a schematic diagram according to a preferred embodiment of the present invention. As mentioned earlier in FIG. 2, the apparatus 108 comprises of the first container 204 having one or more inlets and one or more outlets. In a preferable embodiment of the present invention, as illustrated in FIG. 3, the apparatus 108 includes a first container 204 having three inlets i.e. the first inlet 202a for receiving the lignocellulosic material, the second inlet 202b for receiving the oxygen free sterilized gas, and a third inlet 202c for receiving the nutrient solution A, the nutrient solution B, the nutrient solution C and the antimicrobial compound. In an embodiment of the present invention, the nutrient solution A, nutrient solution B, nutrient solution C and the antimicrobial compound are manually added to the first container 204 through the third inlet 202c of the first container 204. The lignocellulosic material comprises of 1-5% of the total volume inside the first container 204.

[0063] In another embodiment of the present invention, the first container 204 of the apparatus 108 has two outlets i.e. the first outlet 206 for releasing the sterilized first nutrient medium from the first container 204 into the second container 208, and a second outlet 206a for releasing the sterilized first nutrient medium from the first container 204 into the fourth container 214. As discussed above, the growth of the anaerobic micro-organisms in the fourth container 214 is monitored periodically by measuring the enzyme activity for cellulase enzyme in the contents inside the fourth container 214. When the enzyme activity of the contents inside the fourth container 214 is found to be below 5 IU/ml, the first container 204 is configured to release the sterilized first nutrient medium from the second outlet 206a of the first container 204 to the fourth container 214.

[0064] In an embodiment of the present invention, the release of the sterilized first nutrient medium from the first container 204 to the fourth container 214 is a semi-automated process and the volume of the sterilized first nutrient medium to be added to the fourth container 214 is calculated and monitored by at least one of, but not limited to, a level sensor or indicator. The other operations, features and functions of the first container 204 are already explained in FIG. 2. For the sake of brevity, a similar description related to the working and operation of the apparatus 108 as illustrated in FIG. 2 has been omitted to avoid repetition. The limited description provided for the apparatus 108 in FIG. 3 should be read with the description as provided for the apparatus 108 in the FIG. 2 above and should not be construed as limiting the scope of the present disclosure.

[0065] In an embodiment of the present invention, and as mentioned earlier in FIG. 2, the apparatus 108 further includes the second container 208 having one or more inlets and one or more outlets. In a preferable embodiment of the present invention, as illustrated in FIG. 3, the apparatus 108 includes the second container 208 having two inlets i.e. the first inlet 208a and the second inlet 208b and having two outlets i.e. the first outlet 208c and a second outlet 208d. The first inlet 208a of the second container 208 is configured to receive the sterilized first nutrient medium from the first container 204 and the second inlet 208b of the second container 208 is configured to receive a batch of the culture of the anaerobic micro-organism from the source C 106 (shown in FIG.1). As stated above, the second container 208 is configured to cultivate a batch of the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined HRT by utilizing the sterilized first nutrient medium received from the first container 204.

[0066] In an embodiment of the present invention, the first outlet 208c of the second container 208 is configured to release the cultivated culture of the anaerobic micro-organism into the fourth container 214. The cultivation of the culture of the anaerobic micro-organism involves anaerobic digestion of the lignocellulosic material in the sterilized first nutrient medium by the anaerobic micro-organism inside the second container 208. During this process, certain gases are released inside the second container 208. In an embodiment of the present invention, the gases released inside the second container 208 comprise of H2 and CO2. These gases are suitable for growing anaerobic micro-organisms. Hence, in a preferable embodiment, the second container 208 is provided with the second outlet 208d which is configured to transfer the gases released inside the second container 208 into the fourth container 214.

[0067] In an embodiment of the present invention, the second container 208 has means for separation of gases released inside the second container 208 such as, by way of example but not limitation, molecular sieves or copper heating or any absorbent or adsorbent or it employs any membrane separation method for separating the gases released inside the second container 208 and transfer it to the fourth container 214. When the gases from the second container 208 are released into the fourth container 214, they facilitate the anaerobic digestion of the lignocellulosic material forming part of the pasteurized second nutrient medium. The release of the gases from the second container 208 to the fourth container 214 is periodically monitored via sampling and analysing gas content by an external analyser. The other operations, functions and features of the second container 208, are already explained in FIG. 2. For the sake of brevity, a similar description related to the working and operation of the apparatus 108 as illustrated in FIG. 2 has been omitted to avoid repetition. The limited description provided for the apparatus 108 in FIG. 3 should be read with the description as provided for the apparatus 108 in FIG. 2 above and should not be construed as limiting the scope of the present disclosure.

[0068] In an embodiment of the present invention, and as mentioned earlier in FIG. 2, the apparatus 108 further includes the third container 210 having one or more inlets and one or more outlets. In a preferable embodiment of the present invention, as illustrated in FIG. 3, the apparatus 108 includes the third container 210 having four inlets i.e. the first inlet 210a configured to receive the lignocellulosic material, the second inlet 210b configured to receive the oxygen free sterilized gas, a third inlet 210c configured to receive the nutrient solution A, the nutrient solution B, the nutrient solution C and the antimicrobial compound and a fourth inlet 210d configured to receive water from an external source of water (not shown). In an embodiment of the present invention, the nutrient solution A, nutrient solution B, nutrient solution C and antimicrobial compound are manually added to the third container 210. In another embodiment of the present invention, water is added to the third container 210 to dilute the volume of the pasteurized second nutrient medium, based on requirement. In an embodiment of the present invention, the supply of water to the third container 210 is a semi-automated process and the volume of the water to be added to the third container 210 is calculated and monitored by at least one of but not limited to a water flow meter or totaliser.

[0069] In an embodiment of the present invention, the lignocellulosic material comprises of 1-5% of the total volume inside the third container 210. The other operations, features and functions of the third container 210, are already explained in FIG. 2. For the sake of brevity, a similar description related to the working and operation of the apparatus 108 as illustrated in FIG. 2 has been omitted to avoid repetition. The limited description provided for the apparatus 108 in FIG. 3 should be read with the description as provided for the apparatus 108 in FIG. 2 above and should not be construed as limiting the scope of the present disclosure.

[0070] In an embodiment of the present invention, and as mentioned earlier in FIG. 2, the apparatus 108 further includes the fourth container 214 coupled to the second container 208 on its one side and to the third container 210 on its other side, and having two inlets i.e. the first inlet 214a and the second inlet 214b and two outlets i.e. the first outlet 216 and the second outlet 218. In a preferable embodiment of the present invention, as illustrated in FIG. 3, the apparatus 108 includes the fourth container 214 having four inlets i.e. the first inlet 214a configured to receive the cultivated culture of the anaerobic micro-organism from the second container 208, the second inlet 214b configured to receive the pasteurized second nutrient medium from the third container 210, a third inlet 214c configured to receive the sterilized first nutrient medium from the first container 204, and a fourth inlet 214d configured to receive the gases released inside the second container 208. In an embodiment of the present invention, the supply of the contents from the first container 204, the second container 208, and the third container 210 to the fourth container 214 is a semi-automated process and is periodically monitored. The other operations, features and functions of the fourth container 214, are already explained in FIG. 2. For the sake of brevity, a similar description related to the working and operation of the apparatus 108 as illustrated in FIG. 2 has been omitted to avoid repetition. The limited description provided for the apparatus 108 in FIG. 3 should be read with the description as provided for the apparatus 108 in FIG. 2 above and should not be construed as limiting the scope of the present disclosure.

[0071] The apparatus 108 of the present invention replicates the natural rotting process of the lignocellulosic material in a closed container using anaerobic micro-organisms under controlled conditions to create access to holocellulose in the lignocellulosic material that is being shielded by lignin, on an industrial scale.

[0072] The present invention further discloses a method for largescale and continuous growth of anaerobic micro-organisms using an apparatus (hereinafter referred to as “the method”). FIG. 4 is a flow diagram of the method 400 for largescale and continuous growth of the anaerobic micro-organisms using an apparatus according to one or more embodiments of the present invention. For the purpose of description, the method 400 is described with the embodiments as illustrated in FIG. 2 and FIG. 3 and should nowhere be construed as limiting the scope of the present disclosure.

[0073] At step 402, the method 400 includes the step of sterilizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B to a pre-determined temperature and for a pre-determined heating time. In an embodiment of the present invention, the apparatus 108 (shown in FIG.2) having the first container 204 receives the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B through the first inlet 202a of the first container 204. In an alternate embodiment of the present invention, when the lignocellulosic material used in the present invention is recalcitrant, the lignocellulosic material is not mixed with the nutrient solution A and the nutrient solution B, and the lignocellulosic material is supplied to the first container 204 through the first inlet 202a of the first container 204 (as shown in FIG.3), and the nutrient solution A and the nutrient solution B are supplied to the first container 204 through the third inlet 202c of the first container 204 (as shown in FIG.3). In an embodiment of the present invention, the pre-determined temperature for sterilizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 80? to 120? and the pre-determined heating time is 0-1 day. In a preferable embodiment of the present invention, the minimum pre-determined heating time for sterilizing the lignocellulosic material, the nutrient solution A and the nutrient solution B at step 402 is in the range of 10 to 30 minutes.

[0074] In an embodiment of the present invention, the lignocellulosic material used in the method 400 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.

[0075] In an embodiment of the present invention, the nutrient solution A used in the present invention comprises of one or more of the minerals and nitrogen sources disclosed in the table 1 below, dissolved in water.

Chemical name Quantity in g
(per lit)
Potassium hydrogen phosphate (K2HPO4) 0.3-1
Potassium dihydrogen phosphate (KH2PO4) 0.3-1
Sodium phosphate dibasic (Na2HPO4) 0.2-1
Sodium phosphate monobasic (NaH2PO4) 0.2-1
Ammonium dihydrogen phosphate (NH4H2PO4) 0.1-2
Diammonium hydrogen phosphate (NH4)2HPO4 0.1- 2
Yeast Extract 0.5-10

Tryptone
0.5-20

TABLE 1

[0076] 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%.

[0077] In an embodiment of the present invention, the nutrient solution B used in the present invention comprises of one or more of the minerals disclosed in the table 2 below, dissolved in water.

Chemical name Quantity in g
(per lit)
Sodium chloride
(NaCl) 0.5-1.5
Magnesium sulphate
(MgSO4) 0.08-1.3
Sodium sulphate (Na2SO4) 0.08-1.3
Calcium sulphate (CaSO4) 0.06-1.3
Calcium chloride (CaCl2) 0.06-1.3
Hemin 0.0005-0.001
Nitrilotracetic acid (NTA) 0.001-0.01
Manganese sulphate monohydrate (MnSO4. H2O) 0.0001-0.01
Ferrous sulfate heptahydrate (FeSO4 .7H2O) 0.00001-0.00015
Cobalt chloride hexahydrate (CoCl2. 6 H2O) 0.00001-0.0005
Zinc sulphate heptahydrate (ZnSO4. 7 H2O) 0.00001-0.0005
Copper sulphate pentahydrate (CuSO4. 5 H2O) 0.0000001-0.0001
Potassium alum dodecahydrate (AlK(SO4)2 .12H2O) 0.0000001-0.0001
Boric acid (H3BO3) 0.0000001-0.0001
Sodium molybdate dihydrate (Na2MoO4 .2H2O) 0.0000001-0.0001
Nickel sulfate hexahydrate (NiSO4 .6H2O) 0.0000001-0.0001
Sodium selenite (Na2SeO3) 0.0000001-0.0001
Sodium tungstate dihydrate (Na2WO4 .2H2O) 0.0000001-0.0001

TABLE 2

[0078] In another embodiment of the present invention, the minerals dissolved in the water to form the nutrient solution A and the nutrient solution B, which are added to the first container 204 are in the concentration range of 1% to 4% of the total volume of the nutrient solution A and the nutrient solution B used in step 402 of the method 400.
[0079] At step 404, the method 400 includes the step of purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with an oxygen free sterilized gas at a pre-determined flow rate. In an embodiment of the present invention, the apparatus 108 having the first container 204 receives the oxygen free sterilized gas through the second inlet 202b of the first container 204 (shown in FIG.2 and FIG.3). In a preferable embodiment of the present invention, the pre-determined flow rate for purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is in the range 40 to 60 L/min. In another preferable embodiment of the present invention, the oxygen free sterilized gas used in the present invention is selected from, but not limited, to N2 (Nitrogen), H2 (Hydrogen), CO2 (Carbon Dioxide), Ar (Argon), He (Helium), or CH4 (Methane) or a mixture of two or more of these gases.

[0080] At step 406, the method 400 includes the step of cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. In a preferable embodiment of the present invention, the pre-determined temperature for cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 50? to 60?.

[0081] At step 408, the method 400 includes the step of adding the nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the sterilized mixture to a pre-determined temperature. In an embodiment of the present invention, the apparatus 108 having the first container 204 receives the nutrient solution C through the first inlet 202a of the first container 204 (shown in FIG.2). In an alternate embodiment of the present invention, when the lignocellulosic material used in the present invention is recalcitrant, the nutrient solution C is supplied to the first container 204 through the third inlet 202c of the first container 204 (as shown in FIG.3). In a preferable embodiment of the present invention, the pre-determined temperature for cooling the sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C is in the range 30? to 45?.

[0082] In an embodiment of the present invention, the nutrient solution C used in the present invention comprises of one or more of the minerals disclosed in the table 3 below, dissolved in water.

Chemical name Quantity in g
(per lit)
L-Cysteine hydrochloride monohydrate (C3H7NO2S·HCl·H2O) 0.5-1.5
Sodium bicarbonate (NaHCO3) 4-15

TABLE 3

[0083] At step 410, the method 400 includes the step of adding an antimicrobial compound to the further cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a sterilized first nutrient medium. In an embodiment of the present invention, the method 400 uses any commercial antimicrobial compound selected from, but not limited to, chloramphenicol, ampicillin, streptomycin, bactoferm, or tetracyclin.

[0084] In a preferable embodiment of the present invention, the concentration of the lignocellulosic material in the sterilized first nutrient medium is in the range of 1% to 5% of the total volume of the sterilized first nutrient medium. Sterilization of the first nutrient medium results in a microbe free nutrient media. Further, purging of the sterilized first nutrient medium with oxygen free sterilized gas ensures that the sterilized first nutrient medium is unfit for any aerobic microbial growth. The addition of the antimicrobial compound serves as an added measure to produce a microbe free sterilized first nutrient medium.

[0085] In an embodiment of the present invention, the step 402 of sterilization of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to the pre-determined temperature and for the pre-determined heating time, the step 404 of purging of the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at the pre-determined flow rate, the step 406 of cooling of the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to the pre-determined temperature, the step 408 of addition of the nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the sterilized mixture to the pre-determined temperature; and the step 410 of addition of the antimicrobial compound to the further cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form the sterilized first nutrient medium constitutes a first stage of the method 400.

[0086] At step 412, the method 400 includes the step of adding a culture of the anaerobic micro-organism to the sterilized first nutrient medium. In an embodiment of the present invention, the apparatus 108 having the second container 208 receives the culture of the anaerobic micro-organism from the source C 106 (shown in FIG.1) through the second inlet 208b of the second container 208 (shown in FIG.2). The step of supply of the culture of the anaerobic micro-organism to the second container 208 is either fully automated, semi-automated or manual.

[0087] In an embodiment of the present invention, the culture of the anaerobic micro-organism added to the second container 208 belongs to the Phylum Neocallimastigacea which has genera Orpinonmyces, Piromyces, Anaeromyces, or Caecomyces commonly found in the digestive tracts of herbivores, such as cows, sheep and horses. In a preferable embodiment of the present invention, the anaerobic fungi added to the second container 208 are selected from Orpinonmyces spp. and are commercially available. In yet another preferable embodiment of the present invention, the culture of Orpinomyces jyoyonii MCMB 1461 anaerobic fungi is used in the present invention and available at MCM, Pune.

[0088] At step 414, the method 400 includes the step of cultivating the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined HRT by utilizing the sterilized first nutrient medium. In an embodiment of the present invention, the apparatus 108 having the second container 208 receives the sterilized first nutrient medium from the first container 204 through the first inlet 208a of the second container 208 (shown in FIG. 2) and the culture of the anaerobic micro-organism from the source C 106 through the second inlet 208b of the second container 208 (shown in FIG.2). On receiving the sterilized first nutrient medium from the first container 204 and the culture of the anaerobic micro-organism from the source C 106, the second container 208 is adapted to cultivate the culture of the anaerobic micro-organism at the pre-determined temperature and for the pre-determined HRT by utilizing the sterilized first nutrient medium. In an embodiment of the present invention, the pre-determined temperature for cultivating the culture of the anaerobic micro-organism in the second container 208 is in the range 25? to 45? and the pre-determined HRT is 2-10 days.
[0089] The resultant culture of the anaerobic micro-organism cultivated at step 414 of the method 400 is used for facilitating exponential growth of anaerobic micro-organisms at an industrial scale in the subsequent steps of the method 400. In an embodiment of the present invention, the culture of the anaerobic micro-organism cultivated at step 414 inside the second container 208 is released through the outlet 208c of the second container 208 to a fourth container 214, for facilitating growth of the anaerobic micro-organism. During the step 414 of the method 400 of cultivation of the culture of anaerobic micro-organism, gases such as H2 and CO2 are also released. In an embodiment of the present invention, the gases released during the step 414 of cultivation of the culture of anaerobic micro-organism are supplied to the fourth container 214 for facilitating growth of the anaerobic micro-organism.

[0090] In an embodiment of the present invention, the step 412 of addition of the culture of the anaerobic micro-organism to the sterilized first nutrient medium and the step 414 of cultivating the culture of the anaerobic micro-organism at the pre-determined temperature and for the pre-determined HRT by utilizing the sterilized first nutrient medium constitutes a second stage of the method 400.

[0091] At step 416, the method 400 includes the step of pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B to a pre-determined temperature and for a pre-determined heating time. In an embodiment of the present invention, the apparatus 108 having the third container 210 receives the mixture of the lignocellulosic material, the nutrient solution A and the nutrient solution B through the first inlet 210a of the third container 210 (shown in FIG.2). In an alternate embodiment of the present invention, when the lignocellulosic material used in the present invention is recalcitrant, the lignocellulosic material is not mixed with the nutrient solution A and the nutrient solution B, and the lignocellulosic material is supplied to the third container 210 separately through the first inlet 210a of the third container 210, and the nutrient solution A and the nutrient solution B are added to the third container 210 through the third inlet 210c of the third container 210 (as shown in FIG.3). In an embodiment of the present invention, the pre-determined temperature for pasteurizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 50? to 80? and the pre-determined heating time is 0-1 day. For example, the minimum pre-determined heating time for pasteurizing the lignocellulosic material, the nutrient solution A and the nutrient solution B in the third container 210 is in the range of 10 to 30 minutes. The step 416 uses the same lignocellulosic material used at step 402 of the method 400. Further, the nutrient solution A and the nutrient solution B used at step 416 are the same as those used in step 402 of the method 400 and detailed in Table 1 and Table 2 above, respectively. In an alternate embodiment of the present invention, at step 416 of the method 400, the lignocellulosic material, nutrient solution A and nutrient solution B is subjected to sterilization at a pre-determined temperature and for a pre-determined heating time, instead of pasteurization. However, the step of pasteurization is preferred as it reduces overall cost of the method 400.

[0092] In an embodiment of the present invention, the minerals dissolved in the water to form the nutrient solution A and the nutrient solution B which are added at step 416 are in the concentration range of 1% to 4% of the total volume of the nutrient solution A and the nutrient solution B added to the third container 210 and utilized in step 416.

[0093] At step 418, the method 400 includes the step of purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate. In a preferable embodiment of the present invention, the pre-determined flow rate for purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is in the range 200 to 400 L/min. In another embodiment of the present invention, the oxygen free sterilized gas used in the present invention at step 418 is the same as that used at step 404 of the method 400.

[0094] At step 420, the method 400 includes the step of cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature. In a preferable embodiment of the present invention, the pre-determined temperature for cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is in the range 40? to 45?.

[0095] At step 422, the method 400 includes the step of adding the nutrient solution C to the cooled and purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the pasteurized mixture to a pre-determined temperature. In an embodiment of the present invention, the apparatus 108 having the third container 210 receives the nutrient solution C through the first inlet 210a of the third container 210 (shown in FIG.2). In an alternate embodiment of the present invention, when the lignocellulosic material used in the present invention is recalcitrant, the nutrient solution C is supplied to the third container 210 through the third inlet 210c of the third container 210 (as shown in FIG.3). In a preferable embodiment of the present invention, the pre-determined temperature for cooling the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C is in the range 30? to 45?. The nutrient solution C used at step 422 is the same as that used in step 408 of the method 400 and detailed in Table 3 above.

[0096] At step 424, the method 400 includes the step of adding the antimicrobial compound to the further cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium. In an embodiment of the present invention, the step 424 uses the same commercial antimicrobial compound as that used at step 410 of the method 400.

[0097] In a preferable embodiment of the present invention, the concentration of the lignocellulosic material in the pasteurized second nutrient medium is in the range of 1% to 5% of the total volume of the pasteurized second nutrient medium.

[0098] In an embodiment of the present invention, the step 416 of pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at the pre-determined temperature and for the pre-determined heating time, the step 418 of purging of the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at the pre-determined flow rate, the step 420 of cooling of the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to the pre-determined temperature, the step 422 of addition of the nutrient solution C to the cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the pasteurized mixture to the pre-determined temperature, and the step 424 of addition of the antimicrobial compound to the further cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form the pasteurized second nutrient medium constitutes a third stage of the method.

[0099] In an embodiment of the present invention, water is added at the third stage of the method 400, to dilute the concentration and increase the volume of the pasteurized second nutrient medium as per requirement. In an embodiment of the present invention, the apparatus 108 having the third container 210 receives water through the fourth inlet 210d of the third container 210 (shown in FIG.3).

[0100] At step 426, the method 400 includes the step of facilitating growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of the anaerobic micro-organism cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT. In a preferable embodiment of the present invention, the pre-determined temperature for facilitating the growth of the anaerobic micro-organism is in the range 25? to 45? and the pre-determined HRT is 2-10 days.

[0101] In an embodiment of the present invention, the apparatus 108 having the fourth container 214 receives the cultivated culture of the anaerobic micro-organism from the second container 208 through the first inlet 214a of the fourth container 214 and the pasteurized second nutrient medium from the third container 210 through the second inlet 214b of the fourth container 214, and facilitates growth of the anaerobic micro-organism resulting from the anaerobic digestion of the lignocellulosic material by the anaerobic micro-organism by utilizing the pasteurized second nutrient medium at a pre-determined temperature and for a pre-determined HRT. The step 426 of facilitation of growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of anaerobic micro-organism cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT constitutes a fourth stage of the method 400.

[0102] In a preferable embodiment of the present invention, the growth of the anaerobic micro-organism at step 426 of the method 400 is monitored periodically by microscopic examination of the sample of the contents inside the fourth container 214 by conducting enzyme assay for cellulase enzyme in the retrieved sample. In an embodiment of the present invention, when the cellulase activity in the sample was found to be below 5 IU/ml, the sterilized first nutrient medium from the first stage of the method 400 is added at the fourth stage of the method 400. In another embodiment of the present invention, the gases released in the second stage of the method 400 are also added at the fourth stage of the method 400. The anaerobic micro-organism degrade the lignocellulosic material by physical penetration by producing extracellular enzymes, and the gases received from the second stage of the method 400 facilitate this process.
[0103] The aforesaid parameters and steps of the method 400 enable the growth of anaerobic micro-organisms on a largescale and on a continuous mode.

[0104] In an embodiment of the present invention, the cultivation of the culture of anaerobic micro-organism at the second stage of the method 400 and the facilitation of the growth of the anaerobic micro-organism at the fourth stage of the method 400 are carried out at mesophilic temperatures under a controlled feeding rate of 3-25%.

EXAMPLE:
[0105] Example 1: In a sterilization tank measuring 2.3 m3 (with 2 m3 working volume) 20 kg of wheat bran was added as the lignocellulosic material substrate. For the purpose of preparing the nutrient solution A and the nutrient solution B, 1.7 kg of the minerals used in nutrient solution A (composition of which is provided in Table A1 below) and 2.7 kg of the minerals used in nutrient solution B (composition of which is provided in Table B1 below) were added to 1500 litres of water and pumped into the sterilization tank. The temperature inside the sterilization tank was maintained at 80? to 85? for minimum 30 minutes. After 30 minutes, CO2 was purged at 50 L/min and the temperature inside the sterilization tank was reduced to 50?. Thereafter, 7 kg of the minerals used in nutrient solution C (composition of which is provided in Table C1 below) was added to 250 litres of water. Thereafter, the temperature inside the sterilization tank was further reduced to 45?. This allowed the internal gas (air) to be replaced with CO2. Pursuant thereto, 80g of bactoferm (antimicrobial compound) was added to 218 litres of water, and this was pumped to the sterilization tank to from a sterilized first nutrient medium. The sterilized first nutrient medium was thereafter transferred to the culture tank. The purging of CO2 was maintained inside the sterilization tank while transferring the sterilized first nutrient medium to the culture tank to ensure no vacuum is created inside the sterilization tank. This entire process was repeated three times to ensure 6 m3 sterilized first nutrient medium is added to the culture tank measuring 8 m3. Since the entire volume of the culture tank is not filled with the necessary amount of sterilized first nutrient medium after three repeated cycles, in the next step additional 1.2 m3 of sterilized first medium was prepared keeping the proportions of ingredients same as in Tables A1, B1 and C1 to fill the culture tank. Thereafter, 800 litres of the culture of Orpinomyces joyonii obtained from a central laboratory was added to the culture tank and the temperature inside the culture tank was maintained at 40?, wherein the culture was allowed to grow for 5 days inside culture tank.

[0106] Separately, in a nutrient tank measuring 11 m3, 110 kg of wheat bran was added as the lignocellulosic material substrate. For the purpose of preparing the nutrient solution A and the nutrient solution B to be used inside the nutrient tank, 13.42 kg of the minerals used in nutrient solution A (composition of which is disclosed in Table A2 below) and 21.78 kg of the minerals used in nutrient solution B (composition of which is disclosed in Table B2 below) were added to 4000 litres of water and pumped into the nutrient tank. The temperature inside the nutrient tank was maintained at 75? to 80? for 30 mins. After 30 minutes, CO2 was purged at 300 L/min and the temperature inside the nutrient tank was decreased to 50?. Thereafter, 55 kg of the minerals used in the nutrient solution C (composition of which is disclosed in Table C2 below) was added to the nutrient tank. Additional 7 litres of water were also added to dilute the volume of the contents inside the nutrient tank. Thereafter, the temperature was further reduced to 45?. This allowed the internal gas (air) to be replaced with CO2. Thereafter, 440g of bactoferm (antimicrobial compound) was added to the nutrient tank to form a pasteurized second nutrient medium. The pasteurized second nutrient medium was then pumped into an inoculum tank. The purging of the CO2 was maintained inside the nutrient tank while transferring the pasteurized second nutrient medium to the inoculum tank to ensure no vacuum is created inside nutrient tank. This entire process was repeated five times to ensure 55 m3 pasteurized second nutrient medium is added to inoculum tank. Since the entire volume of the inoculum tank is not filled with the necessary amount of pasteurized second nutrient medium even after five repeated cycles, in the next step, additional 5 m3 of the pasteurized second nutrient medium was prepared keeping the proportions of the ingredients same as in Table A2, B2 and C2 to match the volume of the inoculum tank.

[0107] As stated above, the culture of Orpinomyces joyonii was allowed to cultivate inside the culture tank for 5 days. The resultant volume of the culture of Orpinomyces joyonii cultivated with the sterilized first nutrient medium was 11m3/day.

[0108] Thereafter, 20 % of the volume of the culture of the Orpinomyces joyonii cultivated with the sterilized first nutrient medium and 9900 litres of the pasteurized second nutrient medium were added to an inoculum tank measuring 60m3. The temperature inside the inoculum tank was maintained at 39? and the HRT was 5 days to allow the growth of Orpinomyces joyonii. After 5 days, the growth of the Orpinomyces joyonii culture in the inoculum tank was observed by way of microscopic examination of the contents inside the inoculum tank under 40X magnification. To confirm the growth of the Orpinomyces joyonii culture enzyme assay of the bio-complex produced inside the inoculum tank for cellulase enzyme was performed. The cellulase activity of up to 5 IU/ml was obtained. The cellulase activity was observed to vary each day but an average of 5 IU/ml over a period of at least 30 days was observed.

[0109] 11m3 of the contents from the inoculum tank was transferred each day to the digester tank to ensure hydrolysis of rice straw in the hydrolyser forming part of the digester tank. The volume of 11 m3 was replenished by preparing 9.9 m3 pasteurized second nutrient medium in nutrient tank and adding it to inoculum tank and by adding 1.1 m3 of the culture of Orpinomyces joyonii cultivated in the culture tank. The 1.1 m3 volume in culture tank was replaced by 1.1 m3 of the sterilized first nutrient medium. To monitor the effect of the culture of Orpinomyces joyonii in the hydrolyser inside the digester tank, cellulase enzyme activity was monitored and observed to increase to 20 IU on an average for a period of 40 days.
TABLE A1: Composition of nutrient solution A to be added to the sterilization tank
Name In Kg
K2HPO4 0.63
KH2PO4 0.63
DAP 0.448

TABLE B1: Composition of nutrient solution B to be added to the sterilization tank
Name In Kg
NaCl 1.26
MgSO4. 7H2O 0.126
Ammonium sulphate 1.26
Calcium chloride 0.126

TABLE C1: Composition of nutrient solution C to be added to the sterilization tank
Name In Kg
L-Cysteine HCl 1.4
Sodium Bi Carbonate 5.6

TABLE A2: Composition of the nutrient solution A to be added to the nutrient tank
Name In Kg
K2HPO4 4.95
KH2PO4 4.95
DAP 3.52

TABLE B2: Composition of the nutrient solution B to be added to the nutrient tank
Name In Kg
NaCl 9.9
MgSO4. 7H2O 0.99
Ammonium sulphate 9.9
Calcium chloride 0.99

TABLE C2: Composition of the nutrient solution C to be added to the nutrient tank
Name In Kg
L-Cysteine HCl 11
Sodium Bi Carbonate 44

[0110] The method of the present invention results in the production of useful gases such as hydrogen and carbon dioxide. Another advantage of the present invention is that it reduces the requirement of extensive mechanical pre-treatment, as the anaerobic micro-organism can grow on the lignocellulose material under anaerobic conditions on a large scale and without interruption. Since the method of the present invention undertakes treatment of the lignocellulosic material, the resultant product produced by the method of the present invention can directly be 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. This simplifies the process steps for biogas production, reducing turnaround time and making it cost effective. Since the method of the present invention can be carried out on a continuous mode, the process of biogas production can be undertaken without interruption.

[0111] Yet another advantage of the present invention is that the enzymes released in the fourth container of the apparatus during the anaerobic digestion of the lignocellulosic material may be used to treat the lignocellulosic material, which increases the efficiency of degradation of the lignocellulosic material.

[0112] Further, thermal and thermochemical systems operate at high temperature and pressure conditions and therefore need costlier systems for handling chemicals and high temperature liquid. However, in the present invention, the media used is of a low-cost and the requirements for electrical energy, thermal (heat) energy or any further chemicals or a combination thereof is reduced. Moreover, since the method is carried out at ambient temperature and pressure conditions, the method is cost-effective, thereby saving on initial capital expenditure for the unit and the operating expenses. The elimination of the need for thermochemical pre-treatment of agricultural waste not only simplifies the overall process, but also significantly reduces energy consumption, operational complexity, and associated costs. By bypassing the thermochemical pre-treatment step, the apparatus and method of the present invention enhance the overall energy efficiency in the biomethane production process as it focuses on maximizing the utilization of microbial processes, optimizing energy output while minimizing input requirements. This aligns with sustainable practices by reducing the environmental impact associated with energy-intensive processes. Thus, the present invention contributes to an eco-friendly approach to biomethane production, thereby promoting sustainability in agricultural waste management.

[0113] Another advantage of the apparatus and method of the present invention is that they are engineered to efficiently utilize a wide range of substrates present in the agricultural waste. This ensures complete digestion of the agricultural waste, leading to higher methane yields and minimizing the production of undesirable by-products. The simplicity of the apparatus and method of the present invention also facilitates streamlined operation and maintenance. With fewer intricate components and processes, the apparatus is more robust, reducing downtime and operational challenges. This characteristic is essential for scalability and adaptability across various agricultural settings.

[0114] Yet another advantage of the present invention is that the apparatus and method seamlessly integrate the production of biomethane.

[0115] The apparatus and method of the present invention are conceived with scalability and adaptability in mind, allowing for efficient integration into various farm sizes, feedstock compositions, and environmental conditions. This flexibility enhances the apparatus and method’s applicability across diverse agricultural landscapes.

[0116] All the above advantages of the present invention contribute to the cost-effectiveness of the method, thereby making the technology more accessible and economically viable for a wider range of agricultural applications. To summarize, the apparatus and method of the present invention not only address the limitations of existing systems and methods but also bring forth a range of advantages, promoting sustainability, energy efficiency, and cost-effectiveness in the production of biomethane from agricultural waste.

[0117] 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.

[0118] 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 a person skilled in the art, the invention should be construed to include everything within the scope of appended claims.

REFERENCE NUMERALS

[1] Environment – 100
[2] Source A – 102
[3] Source B – 104
[4] Source C – 106
[5] Apparatus – 108
[6] Digester tank – 110
[7] First inlet of the first container – 202a
[8] Second inlet of the first container – 202b
[9] Third inlet of the first container – 202c
[10] First container – 204
[11] First outlet of the first container – 206
[12] Second outlet of the first container – 206a
[13] Second tank – 208
[14] First inlet of the second container – 208a
[15] Second inlet of the second container – 208b
[16] First outlet of the second container – 208c
[17] Second outlet of the second container – 208d
[18] Third container – 210
[19] First inlet of the third container – 210a
[20] Second inlet of the third container – 210b
[21] Third inlet of the third container – 210c
[22] Fourth inlet of the third container – 210d
[23] Outlet of the third container - 212
[24] Fourth container – 214
[25] First inlet of the fourth container – 214a
[26] Second inlet of the fourth container - 214b
[27] Third inlet of the fourth container – 214c
[28] Fourth inlet of the fourth container – 214d
[29] First outlet of the fourth container – 216
[30] Second outlet of the fourth container - 218
,CLAIMS:CLAIMS
We Claim:

1. A method for largescale and continuous growth of anaerobic micro-organisms using an apparatus, comprising the steps of:
sterilizing a batch of lignocellulosic material along with a nutrient solution A and a nutrient solution B at a pre-determined temperature and for a pre-determined heating time;
purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with an oxygen free sterilized gas at a pre-determined flow rate;
cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature;
adding a nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the sterilized mixture to a pre-determined temperature;
adding an antimicrobial compound to the further cooled sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a sterilized first nutrient medium;
adding a culture of the anaerobic micro-organism to the sterilized first nutrient medium;
cultivating the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined Hydraulic Retention Time (HRT) by utilizing the sterilized first nutrient medium;
pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time;
purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate;
cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature;
adding the nutrient solution C to the cooled and purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the pasteurized mixture to a pre-determined temperature;
adding the antimicrobial compound to the further cooled mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium; and
facilitating growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of the anaerobic micro-organism cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT.

2. The method as claimed in claim 1,
wherein the pre-determined temperature for sterilizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 80? to 120? and the pre-determined heating time is 0-1 day;
wherein the pre-determined flow rate for purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is 40 to 60 L/min;
wherein the pre-determined temperature for cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 50? to 60?;
wherein the pre-determined temperature for cooling the sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C is 30? to 45?;
wherein the pre-determined temperature for cultivating the culture of the anaerobic micro-organism is 25? to 45? and the pre-determined Hydraulic Retention Time (HRT) is 2-10 days;
wherein the pre-determined temperature for pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B is 50? to 80? and the pre-determined heating time is 0 to 1 day;
wherein the pre-determined flow rate for purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is 200 to 400 L/min;
wherein the pre-determined temperature for cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 40? to 45?;
wherein the pre-determined temperature for cooling the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C is 30? to 45?; and
wherein the pre-determined temperature for facilitating the growth of the anaerobic micro-organism is 25? to 45? and the pre-determined HRT is 2-10 days.

3. The method as claimed in claim 1,
wherein the step of sterilization of the lignocellulosic material, the nutrient solution A, and the nutrient solution B at the pre-determined temperature and for the pre-determined heating time, the step of purging of the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at the pre-determined flow rate, the step of cooling of the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to the pre-determined temperature, the step of addition of the nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the sterilized mixture to the pre-determined temperature, and the step of addition of the antimicrobial compound to the further cooled mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form the sterilized first nutrient medium constitutes a first stage of the method;
wherein the step of addition of the culture of the anaerobic micro-organism to the sterilized first nutrient medium and the step of cultivating the culture of the anaerobic micro-organism at the pre-determined temperature and for the pre-determined Hydraulic Retention Time (HRT) by utilizing the sterilized first nutrient medium constitutes a second stage of the method;
wherein the step of pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at the pre-determined temperature and for the pre-determined heating time, the step of purging of the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at the pre-determined flow rate, the step of cooling of the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to the pre-determined temperature, the step of addition of the nutrient solution C to the cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cooling the pasteurized mixture to the pre-determined temperature, and the step of addition of the antimicrobial compound to the further cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form the pasteurized second nutrient medium constitutes a third stage of the method; and
wherein the step of facilitation of growth of the anaerobic micro-organism by mixing the pasteurized second nutrient medium with the culture of the anaerobic micro-organism cultivated with the sterilized first nutrient medium at the pre-determined temperature and for the pre-determined HRT constitutes a fourth stage of the method.

4. The method as claimed in claim 3, wherein when the activity of the cellulase enzyme at the fourth stage is below 5 IU/ml, the sterilized first nutrient medium from the first stage of the method and gases released during the second stage of the method are supplied at the fourth stage of the method.

5. The method 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.

6. The method as claimed in claim 1, wherein the oxygen free sterilized gas is N2 (Nitrogen), H2 (Hydrogen), CO2 (Carbon Dioxide), Ar (Argon), He (Helium), or CH4 (Methane) or mixture of two or more of these gases.

7. The method as claimed in claim 1, wherein the culture of the anaerobic micro-organism belongs to the phylum Neocalimastigomycota.

8. The method as claimed in claim 7, wherein the culture of the anaerobic micro-organism is Orpinonmyces joyonii, Piromyces sp., Anaeromyces sp., or Caecomyces sp.

9. The method as claimed in claim 1,
wherein the nutrient solution A comprises of one or more of the minerals potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), Sodium phosphate dibasic (Na2HPO4), sodium phosphate monobasic (NaH2PO4), monoammonium phosphate (NH4H2PO4), and one or more of yeast extract, diammonium phosphate (NH4)2HPO4 or tryptone as a nitrogen source;
wherein the nutrient solution B comprises of one or more of the minerals sodium chloride (NaCl) or Potassium Chloride (KCl), magnesium sulphate (MgSO4) or sodium sulphate or calcium sulphate calcium chloride (CaCl2), hemin, nitrilotriacetic acid (NTA), manganese sulphate monohydrate (MnSO4. H2O), ferrous sulfate heptahydrate (FeSO4 .7H2O), cobalt chloride hexahydrate (CoCl2. 6 H2O), zinc sulphate heptahydrate (ZnSO4. 7 H2O), copper sulphate pentahydrate (CuSO4. 5 H2O), potassium alum dodecahydrate (AlK(SO4)2 .12H2O), Boric acid (H3BO3), sodium molybdate dihydrate (Na2MoO4 .2H2O), nickel sulfate hexahydrate (NiSO4 .6H2O), sodium selenite (Na2SeO3), and sodium tungstate dihydrate (Na2WO4 .2H2O); and
wherein the nutrient solution C comprises of one or more of the minerals L-Cysteine hydrochloride monohydrate (C3H7NO2S·HCl·H2O) and sodium bicarbonate (NaHCO3).

10. An apparatus 108 for largescale and continuous growth of anaerobic micro-organisms comprising of one or more containers, wherein the one or more containers are adapted to:
sterilize a batch of lignocellulosic material along with a nutrient solution A and a nutrient solution B at a pre-determined temperature and for a pre-determined heating time;
purge the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with an oxygen free sterilized gas at a pre-determined flow rate;
cool the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature;
add a received nutrient solution C to the cooled and purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cool the sterilized mixture to a pre-determined temperature;
add a received antimicrobial compound to the further cooled sterilized mixture of lignocellulosic material, nutrient solution A, nutrient solution B and nutrient solution C to form a sterilized first nutrient medium;
add a received culture of the anaerobic micro-organism to the sterilized first nutrient medium;
cultivate the culture of the anaerobic micro-organism at a pre-determined temperature and for a pre-determined Hydraulic Retention Time (HRT) by utilizing the sterilized first nutrient medium;
pasteurize a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B at a pre-determined temperature and for a pre-determined heating time;
purge the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas at a pre-determined flow rate;
cool the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B to a pre-determined temperature;
add the received nutrient solution C to the cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B and further cool the pasteurized mixture to a pre-determined temperature;
add the received antimicrobial compound to the further cooled pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C to form a pasteurized second nutrient medium; and
facilitate growth of the anaerobic micro-organisms by mixing the pasteurized second nutrient medium with the culture of the anaerobic micro-organism cultivated with the sterilized first nutrient medium at a pre-determined temperature and for a pre-determined HRT.

11. The apparatus 108 as claimed in claim 10, wherein the one or more containers includes at least one of:
a plurality of containers which are housed in at least one vessel; or
a plurality of containers which are independent of the at least one vessel, and each of the plurality of containers are coupled to each other,
wherein, each of the plurality of containers include one or more inlets and one or more outlets.

12. The apparatus 108 as claimed in claim 11, wherein the one or more containers include at least a first container 204, a second container 208, a third container 210 and a fourth container 214.
13. The apparatus 108 as claimed in claim 12,
wherein the first container 204 and the third container 210 are adapted to receive the lignocellulosic material, the nutrient solution A, the nutrient solution B, the nutrient solution C and the antimicrobial compound, via a first inlet of the one or more inlets in the first container and in the third container 210; and
wherein the first container 204 and the third container 210 are adapted to receive the oxygen free sterilized gas, via a second inlet of the one or more inlets in the first container 204 and the third container 210;

14. The apparatus 108 as claimed in claim 12, wherein,
wherein the first container 204 and the third container 210 are adapted to receive the lignocellulosic material, via a first inlet of the one or more inlets in the first container and the third container 210;
wherein the first container 204 and the third container 210 are adapted to receive the oxygen free sterilized gas, via a second inlet of the one or more inlets in the first container 204 and third container 210;
wherein the first container 204 and the third container 210 are adapted to receive the nutrient solution A, the nutrient solution B, the nutrient solution C, and the antimicrobial compound, via a third inlet of the one or more inlets in the first container 204 and the third container 210; and
wherein the third container 210 is adapted to receive water, from a fourth inlet of the one or more inlets in the third container 210.

15. The apparatus 108 as claimed in claim 12,
wherein the second container 208 is adapted to receive the sterilized first nutrient medium from the first container 204 via a first inlet of the one or more inlets in the second container 208; and
wherein the second container 208 is adapted to receive the batch of the culture of anaerobic micro-organism from via a second inlet of the one of more inlets in the second container 208.

16. The apparatus 108 as claimed in claim 12,
wherein the fourth container 214 is adapted to receive the cultivated culture of anaerobic micro-organism via a first inlet of the one or more inlets in the fourth container 214, from the second container 208; and
wherein a second inlet of the one or more inlets in the fourth container 214 is adapted to receive the pasteurized second nutrient medium from the third container 210.

17. The apparatus 108 as claimed in claim 12,
wherein one of the one or more outlets in the first container 204 is adapted to release the sterilized first nutrient medium into the fourth container 214;
wherein the one of the one or more outlets in the second container 208 is adapted to release gases produced inside the second container 208 into the fourth container 214;
wherein the fourth container 214 is adapted to release a resultant anaerobic micro-organism cultivated on the lignocellulosic material produced inside the fourth container 214 into a digester tank 110 via a first outlet 216 of the one or more outlets in the fourth container 214; and
wherein a second outlet 218 of the one or more outlets in the fourth container 214 is adapted to supply the gases released inside the fourth container 214 to the digester tank 110.

18. The apparatus 108 as claimed in claim 12,
wherein the size of the first container 204 is 1-5 m3;
wherein the size of the second container 208 is 5-50 m3;
wherein the size of the third container 210 is 5-50 m3; and
wherein the size of the fourth container 214 is 30-150 m3.

19. The apparatus 108 as claimed in claim 10,
wherein the pre-determined temperature for sterilizing the mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 80? to 120? and the pre-determined heating time is 0-1 day;
wherein the pre-determined flow rate for purging the sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is 40 - 60 L/min;
wherein the pre-determined temperature for cooling the purged sterilized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 50? to 60?;
wherein the pre-determined temperature for cooling the sterilized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B, and the nutrient solution C is 30? to 45?;
wherein the pre-determined temperature for cultivating the culture of the anaerobic micro-organism is 25? to 45? and the pre-determined Hydraulic Retention Time (HRT) is 2-10 days;
wherein the pre-determined temperature for pasteurizing a batch of the lignocellulosic material along with the nutrient solution A and the nutrient solution B is 50? to 80? and the pre-determined heating time is 0 to 1 day;
wherein the pre-determined flow rate for purging the pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B with the oxygen free sterilized gas is 200 to 400 L/min;
wherein the pre-determined temperature for cooling the purged pasteurized mixture of the lignocellulosic material, the nutrient solution A, and the nutrient solution B is 40? to 45?;
wherein the pre-determined temperature for cooling the pasteurized mixture of the lignocellulosic material, the nutrient solution A, the nutrient solution B and the nutrient solution C is 30? to 45?; and
wherein the pre-determined temperature for facilitating growth of the anaerobic micro-organism is 25? to 45? and the pre-determined HRT is 2-10 days.