Description:FIELD OF THE INVENTION
The present disclosure relates to the field of lignocellulosic biomass pre-treatment for 2G ethanol (second-generation) bio-refinery operations, specifically relating to a cost- and energy-effective, a fungal strain’s depolymerization-/whole mycelial bio-transformation-assisted, in-situ human urine’s adsorption-facilitated efficient biomass deconstruction procedure.

BACKGROUND OF THE INVENTION
2G ethanol’s bio-refinery operations, despite being mature face hurdles with respect to their cost-effectiveness and overall economic feasibility prior to establishment and commissioning, thereby creating skepticism among policy- and decision-makers of the global nations. While the Indian government has made investments to the tune of Rs. 14k crores towards the establishment of bio-refineries, the responsibility of bio-process engineers is of surmount importance related to postulating highly cost- and energy-effective unit-operations without compromising on the efficacies of any of the individual stages. A lignocellulosic biomass pre-treatment operation’s process-flow design is of paramount significance to bio-refineries, owing to its contribution to ~40% of the cost-incurred in bio-ethanol production, thereby severely impacting the marketing cost of the end product — 2G bio-ethanol. Furthermore, as is the case with India, while the price for 1G bio-ethanol has been fixed by the nation’s government, the onus of assigning the marketing price of 2G bio-ethanol is with the major oil-marketing companies of India; improving the process economy of each of the major stages of a typical 2G bio-refinery operation will assist in decreasing the price of indigenous 2G ethanol. Biomass pre-treatment, the first stage of a typical 2G bio-refinery process, involves the reduction of biomass’s recalcitrance by de-constructing and removing lignin, which is, usually, followed along by hydrolysis and removal of a C5 sugar-rich hemicellulose stream too; additionally, a pre-treatment process is treated to be highly efficient, wherein, the process facilitates the induction of sufficient porosity on the biomass’s surface, causes substantial reduction in the cellulosic crystallinity, significantly reduces the degree of polymerization of the cellulosic architecture of the biomass. Though the primary objective of a pre-treatment process is to improve the accessibility of the biomass’s interior architecture to saccharifying enzyme complexes during the stage of saccharification, a reduction in the biomass : enzyme mass-loading ratio would also positively impact the process economy involved during enzyme procurement/in-situ production/utilization. Cellulose is a water insoluble polysaccharide with a natural degree of polymerization of ~6,000–14,000, comprising β-D-glucose monomers. The intricate intra- and inter-molecular hydrogen bonding, along with cellulose’s amphiphilic nature contribute to its insolubility in majority of the common solvents. While a pre-treatment can, to an extent, effectively de-crystallise, depolymerize/reduce the degree of polymerization and solubilize the cellulosic content of biomass to shorter and soluble oligosaccharides/β-D-glucans in a non-derivatizing solvent system, it additionally reduces the enzyme loading during enzymatic saccharification as described in the earlier statement. Various chemical-based pre-treatments, such as alkali-based, acid-based, or high-pressure and high temperature-employing hydrothermal pre-treatments, though are efficient in terms of lignin degradation, frequently result in the formation of enzymatic saccharification and fermentation inhibitors. When the temperatures of pre-treatment processes exceed 160–180oC for a prolonged period of exposure, the formation of process inhibitors occurs as another major constraint in devising a pre-treatment condition. Furfural, pseudo-lignin, 5-hydroxymethyl furfural (HMF) are well-reported pre-treatment-resultant process inhibitors. At defined titer ranges, such process inhibitors are detrimental to the saccharifying enzyme complexes and are also known to hamper the enzymatic pathways of the yeast strain involved in glucose-to-ethanol fermentation. Furthermore, the dissolution or solubilization of cellulose is a major impediment, which is trying to be addressed by several researchers globally. The highly insoluble nature of cellulose (in widely used common solvents) could be attributed to the presence of several inter- and intra-molecular hydrogen bonds in its (cellulose’s) architecture. Additionally, the amphiphilic nature of cellulose, owing to its hydrophobicity, is also considered as a contributing factor to its difficulty in solubilization. A category of pre-treatment, termed the ionic liquid (IL)-based pre-treatment employs the usage of a green solvent, which possesses low volatility resulting in miniscule vapour pressures, low viscosity, along with increased ionic conductivity, and are, generally, thermally stable. ILs are usually synthesized by chemists using ions of organic/inorganic nature, and while employed as a biomass pre-treatment agent, their ionic nature (of ILs) result in the hydroxyl group of cellulose interacting with the anions of the ILs, while ILs’ cations serve electron-accepting entities. Such an interaction hinders the inter- and intra-molecular hydrogen bonding network of the cellulosic components’ elemental fibrils of the biomass, thereby resulting in the solubilization of cellulose in the ILs environment; additionally, conventional and well-reported IL pre-treatments also assist hemicellulose hydrolysis and solubilization, and considerable lignin dissolution. In summary, IL pre-treatments serve as a lignocellulosic biomass fractionation procedure. Several ionic liquid compositions have been reported by contemporary researchers, which include certain cationic groups, such as imidazolium, pyridinium, ammonium etc., comprising alkyl or allyl side groups, and bonded to anionic groups like chloride, acetate, etc. A short-coming of the IL-based pre-treatment is the detrimental impact of residual IL to the cellulolytic enzyme complex during an enzymatic saccharification process, which is reported to decrease the efficiency of an enzymatic saccharification process by 40%. Other few noted shortcomings of the IL-based method, include the substantial costing involved in industrial scaling up of the procedure, environmental toxicity of the ionic liquids present in the pre-treatment hydrolysate, lack of appropriate recycle ratio of the employed ILs, and demonstrative documentation available only at lab-scale configurations.
In one prior art, the pre-treatment of date palm wastes is studied using 50 g/L sea-water as the solvent and employed various temperatures between 180–200oC with the simultaneous utilization of catalysts such as H2SO4, Na2CO3, and NaOH; the pre-treatment was performed within a specialized high-pressure high-temperature reactor. Around 80% glucan recovery was recorded for a temperature of 210oC and the utilization of H2SO4 as the catalyst. Another prior art reported the utilization of seawater, treated with 5% sulfuric acid, and an autoclaving regimen to result in 64.63%, 69.19%, and 63.03% sugar release from brown, green, and red algae. In contrary to the techniques reported in prior arts above, in the present inventive procedure, there does not exist a phase of addition of exogenous chemical catalysts unlike the above methods. Another prior art reported the production of methane in ~10 days while using synthetic human urine-based pre-treatment of grain straw; the method typically relies on the urea component of the synthetic human urine. In another prior art, source separated human urine was used as a buffering agent to result in the production of a biogas volume of 37–101 mL/gVS. Another prior art reported the utilization of 5–25% (w/w) urine to improve the production of medium chain fatty acids by 3.3-times in an anaerobic waste-activated sludge fermentation, along with ethanol as the electron donor. It has been reported that the direct utilization of NaCl in a combination with dicarboxylic acid and CO2 gas resulted in a 20-fold increase in soluble oligomer release. Another prior art have utilized 20% (w/w) NaCl in combination with 0.1 M H2C2O4 alongwith a 40-min cycle of sonication followed by heating at 200oC and 70 bar to result in a maximum reducing sugar concentration of 3.2 g/L. The ions of salt will help in cleaving the intra-molecular hydrogen bonds of the cellulose chain. Another prior art have reported the employment of lithium chloride, zinc chloride, calcium chloride, and ferric chloride, in combination with various ratios of water to analyse their potential of bringing through cellulose dissolution. Among the tested combinations, ZnCl2.3H2O and FeCl3.6H2O resulted in swift and highly-efficient solubilization of cellulose. A similar prior art used a system of NaCl-H2O to cause ~99% of solubilization of cellulose into oligomers within the molecular weight range of ~200–400 Da. They utilized a 20% (w/w) of NaCl, and performed the treatment at 220oC to result in the above-described solubilization.
In comparison with relevant existing techniques described and known in the prior-art hitherto, it is evident that the present disclosure describes a first-of-its-kind and completely new biomass pre-treatment for a 2G bio-ethanol production bio-refinery, wherein, post a stage of fungal bio-porosity induction and a fungal strain-assisted controlled cellulose depolymerization, an in-situ adsorption of a stipulated volume of human urine is performed on lignocellulosic biomass within a customized urine-concentrator device, followed by heat treatment at a lesser inhibitor-generating temperature regimen, resulting in the solubilization of a significant volume of cellulose, complete hydrolysis and solubilization of hemicellulose, and a substantial degradation of the lignin content of the biomass as well. The present disclosure is of significant interest as majority of the earlier reports had concluded that NaCl, as an individual chemical agent, was ineffective in solubilizing cellulose. In the present invention, human urine, which is majorly constituted by NaCl, is adsorbed/concentrated in situ onto the surface of the biomass and used as the pre-treatment agent.
From the provided background of the invention and the above review of the state-of-the-art solutions, it is very much evident that bio-refinery operations are in requirement of a cost- and energy-effective yet efficient process for the deconstruction of lignocellulosic biomass, along with no/less process inhibitor generation and additionally favouring a lesser enzyme procurement/loading/utilization during the subsequent step of enzymatic saccharification.

SUMMARY OF THE INVENTION
The present disclosure relates to fungal strain-catalyzed controlled depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for second-generation bio-ethanol production. The present invention relates provides an alternative, cost- and energy-effective, and innovative process for lignocellulosic biomass pre-treatment, which, conventionally, is a highly cost-incurring unit-operation involved in a 2G bio-ethanol refinery process. The disclosed pre-treatment operation involves the technique of in-situ adsorption of human urine-derived total dissolved solids (TDS), majorly comprising a consortium of various inorganic salts, followed by a stipulated heating regime. Additionally, prior to subjecting the biomass to the urine-derived salt-based heat treatment, the biomass is subjected to a stage of endolithic fungus-assisted exogenous bio-porosity induction. Subsequently, the biomass is subjected to a whole-mycelial bio-transformation using a fungal strain to perform a controlled depolymerization/reduction in the degree of polymerization of the cellulosic component of the biomass.
The present disclosure seeks to provide aprocess for fungal-strain-catalyzed controlled-depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for 2G-bio-ethanol production. The process comprises: pre-preparing biomass and applying eggshell paste to the pre-prepared biomass thereby preparing a pre-inoculum and inoculum followed by inoculating the biomass with the pre-inoculum and inoculum to induce exogenous bio-porosity; reducing a degree of polymerization of a cellulose architecture of the biomass by performing a fungal strain-assisted controlled biomass depolymerization process using a whole-mycelial bio-transformation technique; performing an in-situ adsorption of human urine onto depolymerized biomass and evaporatively concentrating adsorbed human urine in a human urine concentrator reactor; and treating salt-adsorbed biomass by subjecting the salt-adsorbed biomass to a heating element for enhancing cellulose solubilization, resulting in a reduction in a degree of polymerization of a cellulose thereby positively reducing cellulase enzyme complex-loading during saccharification and subsequently serving as a significant breakthrough in reduction of a final production cost of 2G bio-ethanol.
In an embodiment, the biomass preparation claimed in step a comprises: manually removing visible impurities from the biomass and rinsing the biomass with jets of ambient temperature-bearing municipal water;packing the biomass into heavy-gauge polypropylene bags capable of autoclaving; and tightly sealing the bags and freezing the biomass at an ultra-low temperature of -80°C for 3–4 hours.

In an embodiment, the egg shell application onto the biomass comprises:rinsing egg shells using a water jet washing method;pulverizing the egg shells using a multi-blade mixer/blender to produce powder with an average particle size of 2–6 µm; mixing the pulverized egg shell powder with autoclaved distilled water to form a paste, maintaining a temperature between 80–90°C;introducing the shell paste into the frozen biomass within a laminar air-flow chamber through a top of an opened poly-propylene bag and allowing it to trickle down so as to evenly disperse the shell powder’s paste throughout various sections of the packed biomass;applying a shell powder to biomass weight ratio of 4:1;subjecting the paste-coated biomass, placed within thick-walled glass beakers, to ultrasonication in batches using a sonicator probe, operating between 20–25 kHz at an amplitude of 50–60%, for a duration of ~30 minutes;sealing the polypropylene bag containing the shell-infused biomass;
subjecting the biomass to autoclave treatment at a temperature of 100°C, at 15 psi, for 24 minutes; draining excess calcium carbonate-rich paste through a bottom of the polypropylene bag, and removing the biomass from the bag within a laminar air-flow chamber; and evenly distributing the biomass on trays within a solid-state reactor for further processing.

In an embodiment, the preparation of pre-inoculum of the bio-porosity-inducing fungus comprises: culturing Aspergillussydowii MTCC 6693, an endolithic fungus by preparing a solid medium by infusing a fungus growth-specific minimal medium with 1.5–2.4% (w/v) calcium carbonate and using seawater as solvent; introducing a drop starter culture of Aspergillussydowii MTCC 6693 from a preserved glycerol stock onto the prepared calcium carbonate agar plates; wrapping agar plates with aluminum foil to create a light-deprived environment for fungus; and incubating wrapped plates at a temperature range of ~36–45°C for approximately 25 days to promote fungal growth.

In an embodiment, the preparation of the inoculum/inoculum carpets of the exogenous bio-porosity-inducing fungus comprises: harvesting developed hyphal mats from calcium carbonate agar plates grown under a light-deprived environment at ~36–45°C for approximately 25 days; preparing a fungus growth-specific minimal medium containing 4–5% (w/v) exogenous calcium carbonate, using seawater as the solvent; introducing harvested hyphal mats as floating layers into prepared medium within a large-bottom Haffkine flask, ensuring that the mats do not sink to the bottom to maintain a floating condition; wrapping the Haffkine flask with aluminum foil to create a light-deprived environment to further support a growth of the fungus; incubating the wrapped flask in a temperature-programmable shaker incubator for approximately 15–20 days at a controlled temperature of ~37–45°C; setting a shaker to perform gentle shaking at a speed of ~10–15 RPM to maintain homogeneity of the culture medium while preventing shear stress on the hyphal mats; wearing appropriate personal protective equipment, including gloves, lab coat, and face mask, to ensure safety during handling of the culture; and conducting all handling and culturing activities within a bio-safety level 2 (BSL-2) facility to minimize contamination and ensure safety of experimentalist.

In an embodiment, the endolithic fungus-assisted exogenous bio-porosity induction comprises: transferring developed hyphal carpets, previously cultured in a Haffkine flask, into a laminar air-flow chamber to ensure an aseptic environment; laying the hyphal carpets over surface of biomass beds placed within a solid-state reactor; maintaining the reactor at a temperature of approximately 45°C during the bio-porosity induction phase for a duration of 18–24 days; periodically introducing autoclaved minimal medium-infused seawater into the reactor to provide essential nutrients and maintain moisture content throughout induction phase, wherein the biomass is subjected to a decontamination process within an autoclave prior to washing, wherein the biomass is washed with jets of water until the egg shell paste is visually assessed to be completely removed; monitoring environmental conditions within the reactor to ensure optimal growth conditions for the endolithic fungus; after completion of an incubation period, removing the hyphal carpets under aseptic conditions to prevent contamination; and storing the removed hyphal carpets in seawater within a Haffkine flask for future use in further experiments or applications.

In an embodiment, the fungal strain-/whole mycelial bio-transformation-assisted controlled cellulose depolymerization/reduction in the cellulose’s degree of polymerizationcomprises:preserving the fungal culture on solid agar plates infused with 1% (w/v) micro-crystalline cellulose in a minimal medium, prepared using approximately 50% (v/v) seawater, to mimic fungus's natural habitat;preparing a primary inoculum by introducing a spore load of 9 x 10¹² spores into 100 mL of fungal growth-specific minimal medium containing 1% (w/v) micro-crystalline cellulose;incubating the inoculum at a temperature of 25–28°C for 6–10 days with shaking at 120 RPM in a temperature-programmable shaker incubator;adding approximately 46% (w/v) of the prepared primary inoculum to the bio-porosity-induced biomass and gently blending the mixture with gloved hands within a BSL-1 category laminar air-flow chamber to ensure even distribution of the fungus throughout the biomass;transferring the inoculum-blended biomass into the main vessel of a urine concentrator reactor, equipped with an aeration line for oxygen transfer, and securing the top and bottom flanges with screw-locks while keeping the steam valve open and plugged with cotton to facilitate ambient air flow;filling the reactor jacket with distilled water and placing the reactor on a hot plate to maintain a temperature range of 25–28°C for a standardized period of 5–7 days;monitoring the biomass to prevent over-incubation, which may lead to complete exhaustion of its cellulosic and hemicellulosic content due to excessive fungal growth;utilizing the thigmotropic effector response of the fungal hyphal network to promote penetration into the interior cellulose/hemicellulose-rich regions of the biomass, enhanced by previously induced bio-porosity;achieving approximately 44.7% degradation of the biomass's lignin content, with an observed degradation of approximately 82.3% hemicellulose and approximately 6% (w/w) cellulose consumption by the fungus during the controlled depolymerization phase;extracting the pre-treatment hydrolysate post-fungal growth while wearing appropriate safety gear and working within a laminar air-flow hood, ensuring that random cleavage of glycosidic bonds does not result in soluble oligomer release into the pre-treatment hydrolysate;subjecting the biomass to in-situ adsorption of human urine-derived salt followed by heat treatment to facilitate the efficient breakage of hydrogen bonds due to previously delinked glycosidic bonds and reduction of biomass crystallinity; and
removing the cotton plug and aeration lines from the reactor, screw-locking the flange plates, and proceeding to the subsequent stage of in-situ evaporative concentration of urine onto the biomass.

In an embodiment, the in-situ evaporative concentration of human urine onto biomass comprises:fabricating a stainless steel urine concentrator reactor comprising a main vessel with an external jacket for controlled heating and a steam release valve;filling the external jacket of the reactor with distilled water until overflow is observed from designated valve, followed by closing the valves to maintain water levels;loading the main reaction vessel with bio-porosity-induced biomass to a volume of approximately 1/4th of the reactor’s total volume;securing the top and bottom flange plates of the reactor with screws to prevent leaks;
subjecting the loaded reactor to pre-heating at a temperature range of approximately 50–60°C for 2 hours on a commercially available hot plate to enhance the interaction and activation of the cellulase enzyme complex with the biomass, thereby promoting microbial depolymerization;heating the reactor to a temperature range of approximately 100–112°C, while keeping the steam release valve, urine-inlet line, and steam riser line closed until the target temperature is attained in the main reaction vessel;monitoring steam pressure within the jacket throughout the operation, opening the steam release valve as needed to relieve excess pressure, and maintaining uniform water levels within the jacket by periodically adding distilled water;collecting human urine from healthy volunteers in a container stored at 4°C, wherein the total dissolved solids (TDS) content is determined to be 5.4% (w/v) during a demonstrative trial;filtering the collected urine using a motorized 0.22 µm filter cartridge assembly to remove particulate matter while wearing appropriate personal protective equipment (PPE);subjecting the filtered urine to autoclaving to ensure sterilization before use in the evaporation process;utilizing a peristaltic pump to intermittently feed the autoclaved urine into the main concentrator vessel at a pre-calculated rate based on the observed evaporation rate, approximately every 10–15 minutes for an initial 200 mL quantity;feeding the urine until the TDS content reaches approximately 27% (w/v) in the biomass, wherein the TDS is predominantly composed of salts such as sodium chloride, potassium chloride, and magnesium sulfate as confirmed by diagnostic laboratory analysis;keeping the steam riser valve open after the first batch of urine is added, allowing steam to rise through silicone tubing wrapped with stainless steel bellows to maintain cold temperatures via surrounding frozen gel packs;collecting condensed steam in a distillate harvest vessel equipped with an ice-cooled jacket, allowing the distillate to be utilized for biomass rinsing and microbial culture purposes, thereby creating a circular bio-economy;performing the in-situ adsorption of approximately 27% (w/w) TDS content from human urine onto the biomass over a period of approximately 2.5–4 hours while maintaining reactor temperatures between 100–120°C; andaseptically harvesting the treated biomass post-process using appropriate PPE and transferring it to a heating element-containing oven or heater for subsequent processing.

In an embodiment, the urine-derived salt-adsorbed biomass treatment comprises: utilizing a biomass pre-treated through a process that achieves in-situ adsorption of total dissolved solids (TDS) from human urine, resulting in a biomass that contains approximately 27% (w/v) of human urine's TDS;measuring and adding a pre-calculated weight of ice flakes to the TDS-adsorbed biomass, wherein the weight of the ice corresponds to the quantity of water that has been evaporated during the previous concentration cycles;subjecting the biomass-ice mixture to a heating regimen in a conventional electric oven and implementing a first heating cycle at a temperature range of 120–150°C for 14 minutes, followed immediately by a holding phase at 180°C for 1 minute;repeating heating cycle for a total duration of 1.5 hours, ensuring that a total time for heating does not exceed this limit to prevent excessive crystallization of cellulose;adding two volumes of water relative to the biomass quantity, and manually squeezing the biomass with gloved hands to extract a hydrolysate containing soluble sugars and other byproducts;conducting subsequent washing steps, ensuring that the wash water is devoid of salts and within native TDS limits;performing proximate analysis on remaining biomass, confirming approximately 46.8% lignin degradation, complete removal of hemicellulose, and a 40% reduction in cellulose content;measuring and reporting a crystallinity index of remnant biomass using a Segal's method, confirming a decrease to approximately 31.34% crystallinity;subjecting the remnant biomass to enzymatic saccharification using 12–15 FPU/g of biomass loading of in-house cellulases, achieving a saccharification efficiency of 98±1.2%; andfermenting the resultant saccharified hydrolysate using a 10% (w/v) inoculum of Saccharomyces cerevisiae under micro-aerophilic conditions, at temperatures of 28–30°C, in a micro-aerophilic environment, with ~15% (w/v) β-D-glucose loading, to yield a bio-ethanol output of 0.37 g/g of glucose, achieving a fermentation yield of approximately 74%.

The present disclosure also seeks to providea human urine concentrator reactor. The reactor comprises: an inner vessel, cylindrical in shape, designed to hold human urine; an outer jacket surrounding the inner vessel, creating a user-defined gap between peripheries of the inner vessel and outer jacket, wherein the gap between the jacket and the inner vessel is welded at a top to prevent air exposure; a bottom flange with a cylindrical periphery, featuring four screw bores for securing with stainless-steel screws, allowing a top flange plate to be affixed, wherein the top flange plate containing four bores for introducing screws, and rests silicone gasket adhered to the bottom flange; a stainless-steel inlet pipe for human urine, located at a top of the inner vessel; a steam outlet line mounted with a ball valve at the top of the inner vessel, facilitating removal of urine-derived steam; an outlet line positioned at a height of user-defined distance from a bottom of the jacket, equipped with a circular stainless-steel pipe and customized screw threads, allowing distilled water to enter the jacket;an overflow line at a user-defined height from a top of the jacket, similar in diameter to the inlet pipe, and equipped with a ball valve for monitoring distilled water levels; a steam release valve located on an anterior of the vessel above the gap between the inner vessel and the jacket, permitting excess steam to escape during pressure build-up; a pressure-monitoring gauge on the top plate of the jacket, positioned above a user-defined gap to measure internal pressure; a silicone tubing connected to a steam outlet valve of the inner vessel, extending to a urine distillate collection vessel; a plurality of flexible stainless-steel bellows wrapped around the silicone tubing to enhance heat transfer from frozen gel packs surrounding the tube; one or more frozen gel packs fastened around the silicone tube, ensuring an optimal temperature for a condensation of water vapor from the human urine; a distillate collection vessel, surrounded by an outer jacket, featuring a user-defined gap between the vessels; a non-fully sealed gap at a top of a condensate collection vessel, allowing loading of ice flakes while maintaining structural integrity through selective welding; wherein a harvest vessel includes a bottom and top flange capable of being tightened using screws, with a 3 mm silicone-based gasket positioned between the flanges; a peristaltic pump for transfer of appropriately autoclaved, stored, and filtered human urine into the inner vessel, utilizing silicone tubing for transport; and a distillate collection line present at the top plate of the inner vessel, directly connecting the steam outlet line from the main urine concentrator device for effective collection of distilled water.
An objective of the present disclosure is to provide a process for fungal-strain-catalyzed controlled-depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for 2G-bio-ethanol production.
Another objective of the present disclosure is to provide a human urine concentrator reactor.
Another objective of the present disclosure is to to induce exogenous bio-porosity over the biomass’s surface using fungal mycelia, which are driven to proliferate to the interior of the biomass’s architecture, driven by thigmotropic effector response to a coating of pre-prepared and pulverized egg shell paste over the biomass’s surface, wherein during this stage, hemicellulose and lignin are degraded to a substantial extent; care has to be taken to limit the duration of the fungus’s cultivation to remain within a stipulated time duration so as to refrain the fungus from completely consuming the cellulosic component towards increasing its mycelial biomass proportion.
Another objective of the present disclosure is to perform a controlled depolymerization/reduction of the degree of polymerization of the cellulosic content of the biomass using a fungal strain, which catalyses the mentioned process using a whole-mycelial bio-transformation.
Another objective of the present disclosure is to efficiently deconstruct/depolymerize the lignin content of the biomass, which is substantially facilitated by the bio-porosity-inducing fungus and the cellulose-depolymerizing fungus through a process of whole-mycelial bio-transformation; such depolymerizations significantly result in reducing the enzyme-loading during the subsequent stage of enzymatic saccharification.
Another objective of the present disclosure is to perform an in-situ adsorption of appropriately processed and stored human urine in a self-designed and fabricated customized human urine-concentrator device, wherein the urine would be added on a pre-calculated weight-basis so as to increase the adsorbed TDS content of the salt in the biomass to 27% (w/w).
Yet, another object of the present disclosure is to employ the harvested distillate from the human urine (from the sugar concentrator reactor) for biomass rinsing purposes.
To further clarify advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.

BRIEF DESCRIPTION OF FIGURES
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Figure 1 illustrates a flow chart of a process for fungal-strain-catalyzed controlled-depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for 2G-bio-ethanol production, in accordance with an embodiment of the present disclosure;
Figure 2 illustrates a block diagram of a human urine concentrator reactor, in accordance with an embodiment of the present disclosure;
Figure 3 illustrates a flowchart representing the various stages of the four-stage biomass pre-treatment process along with their critical process variables and set-points during the course of the operation in accordance with an embodiment of the present disclosure;
Figure 4 illustrates the engineering drawings of the sugar concentrator device in accordance with an embodiment of the present disclosure; and
Figure 5 illustrates schematic representation of the process of the in-situ adsorption of the human urine-derived salts onto the biomass within a self-designed and fabricated customized human urine concentrator device in accordance with an embodiment of the present disclosure.
Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. For example, the flow charts illustrate the method in terms of the most prominent steps involved to help to improve understanding of aspects of the present disclosure. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

DETAILED DESCRIPTION:
For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.
Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.
Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.
Figure 1 illustrates a flow chart of a process (100) for fungal-strain-catalyzed controlled-depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for 2G-bio-ethanol production, in accordance with an embodiment of the present disclosure.
Referring to Figure 1, the process (100) comprises a plurality of steps as described under:
At step (102), the process (100) includes pre-preparing biomass and applying eggshell paste to the pre-prepared biomass thereby preparing a pre-inoculum and inoculum followed by inoculating the biomass with the pre-inoculum and inoculum to induce exogenous bio-porosity.
At step (104), the process (100) includes reducing a degree of polymerization of a cellulose architecture of the biomass by performing a fungal strain-assisted controlled biomass depolymerization process using a whole-mycelial bio-transformation technique.
At step (106), the process (100) includes performing an in-situ adsorption of human urine onto depolymerized biomass and evaporatively concentrating adsorbed human urine in a human urine concentrator reactor.
At step (108), the process (100) includes treating salt-adsorbed biomass by subjecting the salt-adsorbed biomass to a heating element for enhancing cellulose solubilization, resulting in a reduction in a degree of polymerization of a cellulose thereby positively reducing cellulase enzyme complex-loading during saccharification and subsequently serving as a significant breakthrough in reduction of a final production cost of 2G bio-ethanol.
In an embodiment, the biomass preparation claimed in step a comprises: manually removing visible impurities from the biomass and rinsing the biomass with jets of ambient temperature-bearing municipal water; packing the biomass into heavy-gauge polypropylene bags capable of autoclaving; and tightly sealing the bags and freezing the biomass at an ultra-low temperature of -80°C for 3–4 hours.
In an embodiment, the egg shell application onto the biomass comprises: rinsing egg shells using a water jet washing method; pulverizing the egg shells using a multi-blade mixer/blender to produce powder with an average particle size of 2–6 µm; mixing the pulverized egg shell powder with autoclaved distilled water to form a paste, maintaining a temperature between 80–90°C; introducing the shell paste into the frozen biomass within a laminar air-flow chamber through a top of an opened poly-propylene bag and allowing it to trickle down so as to evenly disperse the shell powder’s paste throughout various sections of the packed biomass; applying a shell powder to biomass weight ratio of 4:1; subjecting the paste-coated biomass, placed within thick-walled glass beakers, to ultrasonication in batches using a sonicator probe, operating between 20–25 kHz at an amplitude of 50–60%, for a duration of ~30 minutes; sealing the polypropylene bag containing the shell-infused biomass; subjecting the biomass to autoclave treatment at a temperature of 100°C, at 15 psi, for 24 minutes; draining excess calcium carbonate-rich paste through a bottom of the polypropylene bag, and removing the biomass from the bag within a laminar air-flow chamber; and evenly distributing the biomass on trays within a solid-state reactor for further processing.
In an embodiment, the preparation of pre-inoculum of the bio-porosity-inducing fungus comprises: culturing Aspergillussydowii MTCC 6693, an endolithic fungus by preparing a solid medium by infusing a fungus growth-specific minimal medium with 1.5–2.4% (w/v) calcium carbonate and using seawater as solvent; introducing a drop starter culture of Aspergillussydowii MTCC 6693 from a preserved glycerol stock onto the prepared calcium carbonate agar plates; wrapping agar plates with aluminum foil to create a light-deprived environment for fungus; and incubating wrapped plates at a temperature range of ~36–45°C for approximately 25 days to promote fungal growth.
In an embodiment, the preparation of the inoculum/inoculum carpets of the exogenous bio-porosity-inducing fungus comprises: harvesting developed hyphal mats from calcium carbonate agar plates grown under a light-deprived environment at ~36–45°C for approximately 25 days; preparing a fungus growth-specific minimal medium containing 4–5% (w/v) exogenous calcium carbonate, using seawater as the solvent; introducing harvested hyphal mats as floating layers into prepared medium within a large-bottom Haffkine flask, ensuring that the mats do not sink to the bottom to maintain a floating condition; wrapping the Haffkine flask with aluminum foil to create a light-deprived environment to further support a growth of the fungus; incubating the wrapped flask in a temperature-programmable shaker incubator for approximately 15–20 days at a controlled temperature of ~37–45°C; setting a shaker to perform gentle shaking at a speed of ~10–15 RPM to maintain homogeneity of the culture medium while preventing shear stress on the hyphal mats; wearing appropriate personal protective equipment, including gloves, lab coat, and face mask, to ensure safety during handling of the culture; and conducting all handling and culturing activities within a bio-safety level 2 (BSL-2) facility to minimize contamination and ensure safety of experimentalist.
In an embodiment, the endolithic fungus-assisted exogenous bio-porosity induction comprises: transferring developed hyphal carpets, previously cultured in a Haffkine flask, into a laminar air-flow chamber to ensure an aseptic environment; laying the hyphal carpets over surface of biomass beds placed within a solid-state reactor; maintaining the reactor at a temperature of approximately 45°C during the bio-porosity induction phase for a duration of 18–24 days; periodically introducing autoclaved minimal medium-infused seawater into the reactor to provide essential nutrients and maintain moisture content throughout induction phase, wherein the biomass is subjected to a decontamination process within an autoclave prior to washing, wherein the biomass is washed with jets of water until the egg shell paste is visually assessed to be completely removed; monitoring environmental conditions within the reactor to ensure optimal growth conditions for the endolithic fungus; after completion of an incubation period, removing the hyphal carpets under aseptic conditions to prevent contamination; and storing the removed hyphal carpets in seawater within a Haffkine flask for future use in further experiments or applications.
In an embodiment, the fungal strain-/whole mycelial bio-transformation-assisted controlled cellulose depolymerization/reduction in the cellulose’s degree of polymerization claimed in step b comprises: preserving the fungal culture on solid agar plates infused with 1% (w/v) micro-crystalline cellulose in a minimal medium, prepared using approximately 50% (v/v) seawater, to mimic fungus's natural habitat; preparing a primary inoculum by introducing a spore load of 9 x 10¹² spores into 100 mL of fungal growth-specific minimal medium containing 1% (w/v) micro-crystalline cellulose; incubating the inoculum at a temperature of 25–28°C for 6–10 days with shaking at 120 RPM in a temperature-programmable shaker incubator; adding approximately 46% (w/v) of the prepared primary inoculum to the bio-porosity-induced biomass and gently blending the mixture with gloved hands within a BSL-1 category laminar air-flow chamber to ensure even distribution of the fungus throughout the biomass; transferring the inoculum-blended biomass into the main vessel of a urine concentrator reactor, equipped with an aeration line for oxygen transfer, and securing the top and bottom flanges with screw-locks while keeping the steam valve open and plugged with cotton to facilitate ambient air flow; filling the reactor jacket with distilled water and placing the reactor on a hot plate to maintain a temperature range of 25–28°C for a standardized period of 5–7 days; monitoring the biomass to prevent over-incubation, which may lead to complete exhaustion of its cellulosic and hemicellulosic content due to excessive fungal growth; utilizing the thigmotropic effector response of the fungal hyphal network to promote penetration into the interior cellulose/hemicellulose-rich regions of the biomass, enhanced by previously induced bio-porosity; achieving approximately 44.7% degradation of the biomass's lignin content, with an observed degradation of approximately 82.3% hemicellulose and approximately 6% (w/w) cellulose consumption by the fungus during the controlled depolymerization phase; extracting the pre-treatment hydrolysate post-fungal growth while wearing appropriate safety gear and working within a laminar air-flow hood, ensuring that random cleavage of glycosidic bonds does not result in soluble oligomer release into the pre-treatment hydrolysate; subjecting the biomass to in-situ adsorption of human urine-derived salt followed by heat treatment to facilitate the efficient breakage of hydrogen bonds due to previously delinked glycosidic bonds and reduction of biomass crystallinity; and removing the cotton plug and aeration lines from the reactor, screw-locking the flange plates, and proceeding to the subsequent stage of in-situ evaporative concentration of urine onto the biomass.
In an embodiment, the in-situ evaporative concentration of human urine onto biomass comprises: fabricating a stainless steel urine concentrator reactor comprising a main vessel with an external jacket for controlled heating and a steam release valve; filling the external jacket of the reactor with distilled water until overflow is observed from designated valve, followed by closing the valves to maintain water levels; loading the main reaction vessel with bio-porosity-induced biomass to a volume of approximately 1/4th of the reactor’s total volume; securing the top and bottom flange plates of the reactor with screws to prevent leaks; subjecting the loaded reactor to pre-heating at a temperature range of approximately 50–60°C for 2 hours on a commercially available hot plate to enhance the interaction and activation of the cellulase enzyme complex with the biomass, thereby promoting microbial depolymerization; heating the reactor to a temperature range of approximately 100–112°C, while keeping the steam release valve, urine-inlet line, and steam riser line closed until the target temperature is attained in the main reaction vessel; monitoring steam pressure within the jacket throughout the operation, opening the steam release valve as needed to relieve excess pressure, and maintaining uniform water levels within the jacket by periodically adding distilled water; collecting human urine from healthy volunteers in a container stored at 4°C, wherein the total dissolved solids (TDS) content is determined to be 5.4% (w/v) during a demonstrative trial; filtering the collected urine using a motorized 0.22 µm filter cartridge assembly to remove particulate matter while wearing appropriate personal protective equipment (PPE); subjecting the filtered urine to autoclaving to ensure sterilization before use in the evaporation process; utilizing a peristaltic pump to intermittently feed the autoclaved urine into the main concentrator vessel at a pre-calculated rate based on the observed evaporation rate, approximately every 10–15 minutes for an initial 200 mL quantity; feeding the urine until the TDS content reaches approximately 27% (w/v) in the biomass, wherein the TDS is predominantly composed of salts such as sodium chloride, potassium chloride, and magnesium sulfate as confirmed by diagnostic laboratory analysis; keeping the steam riser valve open after the first batch of urine is added, allowing steam to rise through silicone tubing wrapped with stainless steel bellows to maintain cold temperatures via surrounding frozen gel packs; collecting condensed steam in a distillate harvest vessel equipped with an ice-cooled jacket, allowing the distillate to be utilized for biomass rinsing and microbial culture purposes, thereby creating a circular bio-economy; performing the in-situ adsorption of approximately 27% (w/w) TDS content from human urine onto the biomass over a period of approximately 2.5–4 hours while maintaining reactor temperatures between 100–120°C; and aseptically harvesting the treated biomass post-process using appropriate PPE and transferring it to a heating element-containing oven or heater for subsequent processing.
In an embodiment, the urine-derived salt-adsorbed biomass treatment comprises: utilizing a biomass pre-treated through a process that achieves in-situ adsorption of total dissolved solids (TDS) from human urine, resulting in a biomass that contains approximately 27% (w/v) of human urine's TDS;measuring and adding a pre-calculated weight of ice flakes to the TDS-adsorbed biomass, wherein the weight of the ice corresponds to the quantity of water that has been evaporated during the previous concentration cycles;subjecting the biomass-ice mixture to a heating regimen in a conventional electric oven and implementing a first heating cycle at a temperature range of 120–150°C for 14 minutes, followed immediately by a holding phase at 180°C for 1 minute;repeating heating cycle for a total duration of 1.5 hours, ensuring that a total time for heating does not exceed this limit to prevent excessive crystallization of cellulose;adding two volumes of water relative to the biomass quantity, and manually squeezing the biomass with gloved hands to extract a hydrolysate containing soluble sugars and other byproducts;conducting subsequent washing steps, ensuring that the wash water is devoid of salts and within native TDS limits;performing proximate analysis on remaining biomass, confirming approximately 46.8% lignin degradation, complete removal of hemicellulose, and a 40% reduction in cellulose content;measuring and reporting a crystallinity index of remnant biomass using a Segal's method, confirming a decrease to approximately 31.34% crystallinity;subjecting the remnant biomass to enzymatic saccharification using 12–15 FPU/g of biomass loading of in-house cellulases, achieving a saccharification efficiency of 98±1.2%; andfermenting the resultant saccharified hydrolysate using a 10% (w/v) inoculum of Saccharomyces cerevisiae under micro-aerophilic conditions, at temperatures of 28–30°C, in a micro-aerophilic environment, with ~15% (w/v) β-D-glucose loading, to yield a bio-ethanol output of 0.37 g/g of glucose, achieving a fermentation yield of approximately 74%.
Figure 2 illustrates a block diagram of a human urine concentrator reactor (200), in accordance with an embodiment of the present disclosure.
Referring to Figure 2, the reactor (200) includes an inner vessel (202), cylindrical in shape, designed to hold human urine; an outer jacket (204) surrounding the inner vessel (202), creating a user-defined gap between peripheries of the inner vessel (202) and outer jacket (204), wherein the gap between the jacket (204) and the inner vessel (202) is welded at a top to prevent air exposure; a bottom flange (206) with a cylindrical periphery, featuring four screw bores for securing with stainless-steel screws, allowing a top flange plate (208) to be affixed, wherein the top flange plate (208) containing four bores for introducing screws, and rests silicone gasket adhered to the bottom flange (206); a stainless-steel inlet pipe (210) for human urine, located at a top of the inner vessel (202); a steam outlet line (212) mounted with a ball valve (214) at the top of the inner vessel (202), facilitating removal of urine-derived steam; an outlet line (216) positioned at a height of user-defined distance from a bottom of the jacket (204), equipped with a circular stainless-steel pipe and customized screw threads, allowing distilled water to enter the jacket; an overflow line (218) at a user-defined height from a top of the jacket (204), similar in diameter to the inlet pipe, and equipped with a ball valve for monitoring distilled water levels; a steam release valve (220) located on an anterior of the vessel above the gap between the inner vessel (202) and the jacket (204), permitting excess steam to escape during pressure build-up; a pressure-monitoring gauge (222) on the top plate of the jacket (204), positioned above a user-defined gap to measure internal pressure; a silicone tubing (224) connected to a steam outlet valve (226) of the inner vessel (202), extending to a urine distillate collection vessel; a plurality of flexible stainless-steel bellows (228) wrapped around the silicone tubing (224) to enhance heat transfer from frozen gel packs surrounding the tube; one or more frozen gel packs (230) fastened around the silicone tube (224), ensuring an optimal temperature for a condensation of water vapor from the human urine; a distillate collection vessel (232), surrounded by an outer jacket (204), featuring a user-defined gap between the vessels; a non-fully sealed gap at a top of a condensate collection vessel (234), allowing loading of ice flakes while maintaining structural integrity through selective welding; wherein a harvest vessel (236) includes a bottom and top flange capable of being tightened using screws, with a 3 mm silicone-based gasket positioned between the flanges; a peristaltic pump (238) for transfer of appropriately autoclaved, stored, and filtered human urine into the inner vessel, utilizing silicone tubing for transport; and a distillate collection line (240) present at the top plate of the inner vessel (202), directly connecting the steam outlet line from the main urine concentrator device (200) for effective collection of distilled water.
The present invention relates to a alternative, cost- and energy-effective, and innovative process for lignocellulosic biomass pre-treatment, which, conventionally, is a highly cost-incurring unit-operation involved in a 2G bio-ethanol refinery process. The proposed process relating to pre-treatment operation involves the technique of in-situ adsorption of human urine-derived total dissolved solids (TDS), majorly comprising a consortium of various inorganic salts, followed by a stipulated heating regime. Additionally, prior to subjecting the biomass to the urine-derived salt-based heat treatment, the biomass is subjected to a stage of endolithic fungus-assisted exogenous bio-porosity induction. Subsequently, the biomass is subjected to a whole-mycelial bio-transformation using a fungal strain to perform a controlled depolymerization/reduction in the degree of polymerization of the cellulosic component of the biomass.
The proposed process for pre-treatment includes four-stages four-stage pre-treatment process for lignocellulosic biomass involves an endolithic fungal mycelial proliferation-assisted stage for improving the porosity of the native biomass, thereby paving way for an enhancement in the second stage of controlled cellulose depolymerization. During the first stage, pulverized, waste egg shell-derived paste is applied to the biomass, and it is subjected to a stage of ultrasonication. A process of ultrasonication, in general, is known to cause an improvement in the contact surface area of the biomass, wherein, also, the solid, bulk nature of cellulose is transformed to a disintegrated form, which, in this case, facilitates efficient accessibility of the egg-shell paste into the interior architectural network of the biomass. Usually, an ultrasonic cavitation sequentially proceeds with the formation of bubbles, its successive growth followed by bubble destruction/collapse and formation of a microjet, which forces an entry into the biomass, causes significant distortion in the axis resulting in substantial erosion and axial fissures on the biomass’s surface, thereby weakening the rigidity of its innate cellulosic structure. Furthermore, during the bubble collapse, the radicals of H and OH will cleave the glycosidic linkages of the cellulose molecule, significantly. Owing to the combined action of the sonication and fungal mycelial penetration mentioned above, at the end of the first stage, an alteration of the degree of crystallinity of the biomass occurs. The second stage of the pre-treatment process involves the employment of a fungal strain to perform a short-duration, controlled cellulose depolymerization to result in partial breakdown of the complex cellulosic architecture during the subsequent stages of the process. During this stage, hemicellulose and lignin are degraded to a substantial extent; care has to be taken to limit the duration of the fungus’s cultivation to remain within a stipulated time duration so as to refrain the fungus from completely consuming the cellulosic component towards increasing its mycelial biomass proportion. The ligninolytic activity of the fungi employed in the previous two stages aids in efficiently depolymerizing/degrading the lignin architecture of the biomass, thereby facilitating reduced cellulolytic enzyme complex loading during saccharification. The third stage involves the in-situ adsorption of the overall salt content (estimated as TDS) of human urine onto the biomass in a specially designed customized device called the urine concentrator. The fourth stage involves heat-treating the urine-derived salt-adsorbed biomass in a heating element-based electric oven for 1.5 hours in 15 min cycles with a stipulated quantity of ice cube-addition, at the end of every 15 min. Ultimately, the biomass, thus treated, results in 46.8% lignin degradation, 100% hemicellulose hydrolysis and removal, 40% cellulose depolymerization as soluble oligomers in the pre-treatment hydrolysate, and a Segal crystallinity index of 31.34% of the majorly cellulosic regions post pre-treatment; this implies the occurrence of a higher proportion of amorphous regions in the biomass post pre-treatment. In general, in spite of the several hydroxyl groups, the tightly-packaged cellulose confers it with a highly-impervious nature and recalcitrance. In most cases, only the superficial/surface fibrils and the aggregated between crystallites in cell walls are readily permeated by enzymes/cellulose-solubilizing agents. Subsequent enzymatic saccharification of the pre-treated biomass required 12–15 FPU/g of saccharifying enzyme complex loading, which signifies an ~70% decrease, in comparison to the usually reported (by contemporary research groups) enzyme loading between 33–60 FPU/g of biomass. The amorphous regions of cellulose are initially hydrolysed during enzymatic saccharification in a facile manner, while the crystalline regions are hydrolysed comparatively slower and released into the saccharification hydrolysates as oligomers or monomers. The obtainment of highly amorphous cellulose component of the biomass is an added advantage of the proposed inventive process. While 8.24% (w/v) saccharification yield was recorded for an enzymatic saccharification performed using 100 g of pre-treated biomass, a bio-ethanol yield of 0.372 g/g of β-D-glucose was recorded during a Saccharomyces cerevisiae-based ethanol fermentation. The sugar monomer yield was ~95%+ for the batch of the pre-treatment biomass during saccharification, signifying the efficacy of the described pre-treatment process.
In an embodiment, for pre-preparation of the biomass, after manually removing the visible impurities from the biomass, jets of ambient temperature-bearing municipal water are used to rinse the biomass, followed by an ultra-low temperature freezing at -80oC for 3–4 h and packaging the biomass into heavy-gauge poly-propylene bags capable of being subjected to autoclaving, and tightly sealing the bags.
In an embodiment, for preparing the substrate for inducing exogenous fungal bio-porosity over the biomass, pre-rinsed egg shells are subjected to repeated pulverization until a powder with an average particle size of 2–6 µm (as observed under a microscope) is obtained, followed by adding distilled water to maintain a paste-like consistency of the same. After pre-heating the paste to a temperature of ~80–90oC, it is introduced through the biomass-containing poly-propylene bag and allowed to trickle down so as to permit an even dispersion of the paste in a 4:1 shell powder : biomass loading ratio. While still not completely dry, the shell paste-coated biomass is placed within thick-walled glass beakers and subjected to a stage of ultrasonication (in batches) using a sonicator probe, operated between 20–25 kHz, at an amplitude between 50–60% for a total of ~30 min and, with a default pulse-and-pause duration. After performing an autoclave-based heating at 100oC, at 15 psi, for 24 minutes and draining the excess paste, the biomass is evenly distributed within a solid-state reactor’s rack.
In an embodiment, in order to induce exogenous bio-porosity on the surface of biomass,an endolithic fungus,Aspergillussydowii MTCC 6693, initially grown on 1.5–2.4% (w/v) calcium carbonate agar-containing solid medium is appropriately incubated in a light-deprived environment at ~36–45oC, for ~25 days, followed by the development of hyphal mats and transferring the mats into ~4–5% (w/v) calcium carbonate-containing minimal medium, within a large-bottom Haffkine flask to cultivate large carpets of the growing fungus for ~15–20 days, at a temperature of ~37–45oC, with a very gentle shaking at ~10–15 RPM. The developed mycelial carpets are laid over the surface of the biomass beds, and subjected to ~18–24 day-long incubation, at a temperature of ~45oC.
In an embodiment, in order to perform a fungal strain’s whole-mycelial bio-transformation-assisted controlled depolymerization, a fungal strain Aspergillusniger MTCC 4325, was subjected to primary inoculum preparation, and for the actual inoculum preparation, incubation of flasks, with appropriate spore load, was performed at a temperature of 25–28oC for 6–10 days, at 120 RPM in a shaker. ~46% (v/w) of the primary inoculum is added to the bio-porosity-induced biomass and blended (gently) to ensure spreading of the fungus throughout the biomass. The entire stage of controlled fungal depolymerization of cellulose is performed at a temperature between 25–28oC for a period of ~5–7 days within the human urine-concentrator.
In an embodiment, in order to perform the stage of in-situ adsorption and evaporative concentration of human urine over the biomass, the main vessel of the self-designed and customized urine-concentrator reactor is loaded with the bio-porosity-induced biomass and subjected to pre-heating at ~50–60oC for 2 hours, followed by heating at ~100–112oC. Appropriately collected, pre-processed and stored human urine is fed using a peristaltic pump to the main concentrator vessel containing the biomass until ~27% (w/v) of its (urine’s) TDS content is reached within the biomass sample. Appropriately packed and cooled distillate harvest vessel is used to collect the condensate from the main urine-concentrator vessel.
In an embodiment, in order to perform the concluding stage of heat treatment to the in-situ human urine-derived 27% (w/w) TDS-adsorbed biomass, a pre-calculated/pre-standardized weight of ice flakes are added, followed by a 14-min.-long heating cycle at a temperature between 120–150oC, alternating with a holding cycle at 180oC for 1 min., immediately proceeded by the addition of a stipulated quantity of ice. The entire 15 min cycle is repeated for 1.5 hours. Post the heat treatment’s completion, the biomass is washed with two volumes of water to extract the pre-treatment hydrolysate, comprising the solubilized sugars. Further, after completely rinsing the biomass free of excess urine-derived TDS, it is subjected to in-house enzyme-utilizing enzymatic saccharification and ethanol production using the saccharified hydrolysate.
Figure 3 illustrates a flowchart representing the various stages of the four-stage biomass pre-treatment process along with their critical process variables and set-points during the course of the operation in accordance with an embodiment of the present disclosure.
Referring to Figure 3, the process of a fungal strain’s depolymerization-/whole mycelial bio-transformation-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment comprises the following steps:
Firstthe biomass, after manually removing the visible impurities from its surface, is subjected to jets of ambient temperature-bearing municipal water-based rinsing. Prior to being subjected to an ultra-low temperature freezing at -80oC for 3–4 h, the biomass is packed into heavy-gauge poly-propylene bags capable of being subjected to autoclaving, and tightly sealed. Egg shells, after being subjected to water jet washing similar to the regime mentioned above, is subjected to repeated pulverization using a multi-blade setup of a commercially-available mixer/blender until shell powder with an average particle size of 2–6 µm (as observed under a microscope) is obtained. This is followed by the careful addition of autoclaved distilled water to the pulverized shells to maintain a paste-like consistency of the egg shell powder. The temperature of the pasty-mixture is maintained at ~80–90oC until introduction into the frozen biomass. To the frozen biomass, within a laminar air-flow chamber, the paste-like shell mixture is introduced through the top of the opened poly-propylene bag and allowed to trickle down so as to evenly disperse the shell powder’s paste throughout the various sections of the packed biomass. The paste is applied, quite generously, with an application on weight basis as 4:1 shell powder:biomass loading ratio. While still not completely dry, the shell paste-coated biomass is placed within thick-walled glass beakers and subjected to a stage of ultrasonication (in batches) using a sonicator probe, operated between 20–25 kHz, at an amplitude between 50–60% for a total of ~30 min and, with a default pulse-and-pause duration.Post sonication, after manually sealing the poly-propylene bag, the shell material-infused biomass is subjected to an autoclave-based treatment at 100oC, at 15 psi, for 24 minutes. After autoclaving and draining the excess calcium carbonate-rich paste through the bag’s bottom, the biomass is removed from the bag, and within a laminar air-flow chamber, it is evenly distributed on the trays of a solid-state reactor. Aspergillussydowii MTCC 6693, an endolithic fungus maintained in our lab’s repository, is initially grown on fungus growth-specific minimal medium-infused, 1.5–2.4% (w/v) calcium carbonate agar-containing solid medium prepared using sea water as the solvent. A drop culture is made from the preserved glycerol stock to initiate growth of the fungus on the agar plates. The plates are wrapped using aluminium foil to provide a light-deprived environment to the growing bio-porosity-inducing endolithic fungus, and incubated at ~36–45oC, for ~25 days. The developed hyphal mats from the agar plates are carefully introduced as floating layers into ~4–5% (w/v) exogenous calcium carbonate-containing fungus growth-specific minimal medium prepared using sea water, within a large-bottom Haffkine flask. After wrapping the flask using aluminium foil, incubation is performed for ~15–20 days with a very gentle shaking at ~10–15 RPM, at a temperature of ~37–45oC in a temperature-programmable shaker incubator. Throughout the culturing stages of the bio-porosity-inducing fungus, appropriate personal protective equipment must be worn by the experimentalist and the culture’s handling is recommended to be performed within a bio-safety level 2 (BSL-2) facility. After completion of the stage of hyphal carpet growth/formation within the Haffkine flask, inside the laminar air-flow chamber, the carpets are carefully transferred and laid over the surface of the biomass beds already placed within the solid-state reactor. The phase of exogenous, endolithic fungus-facilitated bio-porosity induction lasts for ~18–24 days, and is performed by maintaining the reactor’s temperature at ~45oC, with periodical introduction of autoclaved minimal medium-infused sea water. Post completion of the stipulated period of incubation, the hyphal carpets are removed (under aseptic conditions) and stored in sea water within a Haffkine flask for future use. The biomass, after being subjected to a stage of decontamination within an autoclave, is transferred to a vessel and is washed with jets of volumes of municipal water until the egg shell paste is removed (visually assessed). While the egg shell powder is harvested, dried and stored for future use, the remnant of the biomass is subjected to various analyses. An incognizable/unquantifiable cellulose deconstruction may have also begun to occur as Aspergillus spp. are reported to be prolific cellulase enzyme complex producers as well. The purpose of the fungus-induced exogenous bio-porosity induction was to improve the efficient permeation of human urine (and its total dissolved solid content - TDS) during the subsequent stage of in-situ adsorption and evaporative concentration of human urine over the biomass. Additionally, for the third stage of a fungus-/whole mycelial bio-transformation-assisted cellulose depolymerization, an improved porosity would reduce the time required for hyphal proliferation and controlled enzymatic depolymerization. Together, the induced and enhanced porosity, along with a controlled cellulose depolymerization assists the efficient, concentrated cellulolytic enzyme complex penetration during the stage of enzymatic saccharification to result in maximum cellulose-to-fermentable monomer conversion. Furthermore, during this stage, hemicellulose and lignin are degraded to a substantial extent; care has to be taken to limit the duration of the fungus’s cultivation to remain within a stipulated time duration so as to refrain the fungus from completely consuming the cellulosic component towards increasing its mycelial biomass proportion.
A strain of Aspergillus spp., (AspergillusnigerMTCC 4325) is subjected to the standard primary inoculum preparation steps, such as the introduction of a spore load of 9 x 1012 spores into a volume of 100 mL of fungal growth-specific minimal medium containing 1% (w/v) micro-crystalline cellulose, followed by incubation of the flasks at a temperature of 25–28oC for 6–10 days, at 120 RPM in a temperature-programmable shaker incubator. Akin to the typical preparation stage of a solid-state fermentation (SSF), ~46% (v/w) of the primary inoculum is added to the bio-porosity-induced biomass and blended (gently) using gloved hands so as to ensure the spreading of the fungus throughout the various regions of the biomass; the entire preparation stage is performed within a laminar air-flow chamber belonging to the BSL-1 category. To perform the stage of a fungal strain’s whole mycelial bio-transformation-assisted controlled depolymerization, the inoculum-blended biomass is loaded into the main vessel of the urine concentrator reactor, which provides sufficient oxygen transfer for the growing fungus through an aeration line introduced from the feeding line; the top and bottom flanges are screw-locked and the ball valves for the steam valve is kept open and plugged with cotton to allow ambient air to flow into the reactor. Distilled water is filled into the reactor’s jacket, and it is placed over a hot plate to maintain its temperature between 25–28oC for a period of ~5–7 days as standardized during the demonstrative trials. Extension of the period of incubation beyond the stipulated duration results in complete exhaustion of the biomass’s cellulosic and hemicellulosic content as a result of prolific fungal growth throughout its (biomass’s) surface. The process of controlled depolymerization is based on the exploitation of the thigmotropic effector response of the hyphal network of a fungus to drive it to the interior cellulose/hemicellulose-rich region of the biomass. Accelerating the process is the previously induced bio-porosity, which reduces the time for the fungus to penetrate the otherwise recalcitrant biomass’s architecture/surface. The pre-treatment hydrolysate is extracted post the fungal growth with appropriate safety gear and within a laminar air-flow hood. After removing the cotton plug, aeration lines, screw-locking the flange plates of the biomass-containing urine concentrator vessel, it is subjected to the subsequent stage of in-situ evaporative concentration of urine onto the biomass. It has to be noted that during the stage of controlled depolymerization, the random cleavage of glycosidic bonds throughout the biomass’s cellulosic content would not result in soluble oligomer release into the pre-treatment hydrolysate. However, after the stage of in-situ adsorption of human urine-derived salt and heat treatment, the already delinked glycosidic bonds helps in efficient breakage of the hydrogen bonds and sufficient decrease in the biomass’s crystallinity.
A self-designed and customized urine-concentrator reactor fabricated from stainless steel is used for the process of evaporative concentration and in-situ adsorption of the TDS content of human urine directly onto the biomass. First, the external jacket of the main vessel of the reactor is filled using distilled water until overflow is observed from the designated valve. Post closing the valves, loading a stipulated quantity of the bio-porosity induced biomass (quantity for demonstration was 100 g biomass) into the main reaction vessel, and locking the screws of the top and bottom flange plates, the vessel is subjected to a pre-heating at ~50–60oC for 2 hours on a commercially-available hot plate; the pre-heating is followed by heating at ~100–112oC. The pre-heating process enhances the effective interaction/activation of the cellulase enzyme complex with the biomass’s interior resulting in sufficient microbial depolymerization. The steam release valve of the jacket, the urine-inlet line, and the steam riser line are always maintained in a closed position until temperature attainment in the main reaction vessel. Periodically, throughout the operation, the steam pressure is monitored within the jacket, and if necessary, the steam release valve is opened to reduce the excess pressure within the water jacket. Water is again filled to maintain a uniform temperature within the urine concentrator’s main vessel. The purpose of having a jacketed vessel is to permit the controlled heating of biomass, and a simultaneous evaporative concentration of human urine atop its surface without selectively charring the biomass content when subjecting it to direct heat.
Human urine from healthy volunteers is collected in sufficient quantities in a container constantly maintained at 4oC. The collected urine, prior to usage, with appropriate personal safety gear is filtered using a motorized 0.22 µm filter cartridge-containing assembly, and subjected to autoclaving. Post autoclave, using a peristaltic pump, an intermittent feeding of autoclaved urine is performed to the main concentrator vessel.
Urine feeding is performed on the basis of a pre-calculated rate of evaporation of the water content from the added quantity of urine, which is ~10–15 min for 200 mL in the demonstrative case. Since the TDS content of the human urine is assessed prior to feeding, the urine is fed until ~27% (w/v) of its TDS content is reached within the biomass sample. The TDS content, majorly comprises a consortium of salts such as sodium chloride, potassium chloride, magnesium sulfate, etc., as evaluated from a sample of human urine in a diagnostic laboratory. Once the first batch of urine is added, the steam riser valve is kept open. The steam rises through the silicone tubing, wrapped around with stainless steel bellows to better transfer the cold temperature from the surrounding frozen gel packs. As the steam travels through the condensation tubing, it gets collected in a urine’s distillate harvest vessel. The distillate harvest vessel contains ice flakes/cubes loaded into its surrounding jacket. The distillate of the urine collected in the ice-cooled harvest vessel can be used for biomass rinsing purposes and microbial culture purposes, thereby signifying a circular bio-economy of the process. The demonstrative process for achieving the in-situ adsorption of 27% (w/w) of the TDS content of the human urine onto the biomass was 2.5–4 h. Since the temperature of the urine concentrator reactor was maintained between ~100–120oC throughout the lengthy duration of the process, the biomass is directly harvested, aseptically, using appropriate PPE, and transferred to the heating element-containing oven/heater for the subsequent stage.
A pre-calculated/pre-standardized weight of ice flakes are addedto 27% (w/v) human urine’s TDS-adsorbed biomass. The weight corresponds to the quantity of water removed/evaporated during every cycle of the heating regimen. The combination of biomass and ice is subjected to a 14-min.-long heating cycle at a temperature between 120–150oC, followed by holding at 180oC for 1 min., immediately followed by the addition of a pre-standardized quantity of ice, after the entire 15 min.-long cycle; the quantity of ice added is calculated on the basis of the quantity of water that evaporates during the heating cycle. The 15 min cycle is repeated for 1.5 hours. During demonstrative trials, the heat treatment was performed in a conventional heating element-containing electric oven. After completion of the treatment, two volumes of water, with respect to the quantity of the biomass, are added and the biomass is manually squeezed (using gloved hands) to extract the pre-treatment hydrolysate. After first two washes using the stipulated quantity of water, a few more washes may be conducted until the wash-resultant water is within its native TDS limits; summarily, it is to be made sure that the biomass is free of the salts adsorbed from the human urine.
Figure 4 illustrates the engineering drawings of the sugar concentrator device in accordance with an embodiment of the present disclosure.
Referring to Figure 4, the human urine- concentrator device is used for performing the in-situ adsorption of human urine on the biomass aided by the simultaneous evaporative concentration of its (urine’s) TDS content. The urine-concentrator device comprises two major vessels fabricated out of SS 316 grade stainless-steel sheets.
The inner vessel of the main, cylindrical urine-concentrator vessel has a diameter of 20 cm and a height of 26 cm. The inner vessel is surrounded by an outer jacket, which has an overall diameter of 24 cm and a height of 28 cm. The distance between the periphery of the inner vessel and the periphery of the outer jacket is 2 cm. On the top of the main vessel, the gap between the jacket and the inner vessel is welded so that the jacket layer is not open to air. The top edge/cylindrical periphery of the outer vessel has slightly broader edges, which functions as the bottom flange with 6 bores to accommodate 6 stainless-steel screws introduced through the top flange plate. A silicone-based gasket (3 mm thickness) is present adhered to the bottom flange. The top flange plate with a diameter of 30.2 cm has 4 screw bores to introduce screws through the top flange and tighten the respective nuts from the bottom. The top flange plate rests on the gasket material present atop the bottom flange’s surface. The stainless-steel inlet and outlet pipes mentioned throughout the description are of the same diameter of 1.5 cm.
At a height of 2.5 cm from the bottom, on the side of the periphery of the jacket, a circular stainless-steel pipe of 1.5 cm diameter with customized screw threads is welded, which functions as the distilled water inlet line to fill the jacket. An appropriate ball valve is attached to the line.
At a height of 4.5 cm from the top, an overflow line of the similar pipe diameter and a similar ball valve setup is welded so as to visualize overflow of distilled water through the pipe and close the jacket’s outlet when necessary.
The water in the jacket would be heated by placing the main vessel on a commercially-available hot plate. A steam release valve is present on the anterior of the vessel, right above the gap between the inner vessel and the jacket, to drive out excess steam from the outer jacket in case of an excessive pressure build up.
Similarly, a jacket’s pressure-monitoring gauge is located on the top plate of the vessel, right above the gap between the inner vessel and the jacket in order to regularly monitor the pressure build up within the jacket.
A human urine inlet line is present on top of the inner vessel. Similarly, the urine-derived steam carrier line/steam outlet line is present on the top of the vessel, to which a ball valve is mounted.
Silicone tubing (1.2 cm dia.) is connected to the inner vessel’s steam outlet valve, which connects to the urine’s distillate collection vessel. Surrounding the steam-harvest silicone tubing, flexible stainless-steel bellows are wrapped so as to enable efficient heat transfer from the surrounding frozen gel packs to the silicone tube. Frozen gel packs are wrapped and fastened around the silicone tube so as to provide a conducive environment for condensation of the water content from the human urine prior to flowing with the effect of gravity into the urine’s distillate collection vessel.
Appropriately autoclaved, stored, and filtered human urine is pumped using a peristaltic pump into the main reaction vessel/urine-concentrator. Silicone tube is used for carrying the human urine to the reaction vessel.
A distillate-collection vessel has an inner diameter of 20 cm, and an inner vessel height of 18 cm. An outer jacket of 20.4 cm diameter and 21 cm height is present outside the inner vessel. The gap between the inner vessel and the outer jacket is 2 cm. Unlike the main reaction vessel, the gap on the top between the inner vessel and the outer jacket of the condensate harvest vessel is not completely sealed. It is welded in selective regions to hold the inner and outer vessels in position, and also to allow loading of ice flakes through the top into the jacket of the harvest vessel.
Similar to the main reaction vessel/urine concentrator, the harvest vessel has a bottom and top flange, capable of being tightened using screws. A 3 mm silicone-based gasket is present between the top and bottom flanges. A distillate collection line is present on top of the inner vessel’s top plate, which is connected to the steam outlet line from the main urine-concentrator device.
Figure 5 illustrates schematic representation of the process of the in-situ adsorption of the human urine-derived salts onto the biomass within a self-designed and fabricated customized human urine concentrator device in accordance with an embodiment of the present disclosure.
The in-situ adsorption of the TDS content of human urine onto biomass followed by its evaporative concentration comprises a reactor with two vessels. The biomass, which has been subjected to the stages of exogenous bio-porosity induction and controlled fungus-assisted depolymerization, is introduced into the main vessel of the urine-concentrator reactor, followed by tightening its (reactor vessel’s) top and bottom flanges using the provided screws. Distilled water is filled into its outer jacket through the bottom inlet port and once the water-filling is assessed by visualizing its overflow through the overflow port, the ball valve is closed. The outer jacket’s steam release valve is kept closed at all times and it is typically used for releasing the steam building in the jacket in case of an excessive pressure increase, as visualized in the pressure monitoring gauge. The sealed, air-tight vessel containing appropriately filtered, autoclaved, and stored human urine batch is connected through a peristaltic pump to the urine inlet line of the main vessel of the urine-concentrator reactor. The main vessel’s steam outlet, which bears steam from the evaporation of urine, is connected to the inlet of the urine’s distillate collection vessel. The stainless-steel bellow-enveloped silicone tubing, which carries the steam from urine to the distillate collection vessel, is wrapped with deep frozen gel packs as to enable efficient condensation of the steam within the tubing and facilitate collection in the distillate collection vessel. The harvest vessel contains a jacket, which is filled with ice flakes. The vessel’s top and bottom flanges are locked using the provided screws.
The evaporation rate of the added volume of human urine is pre-calculated. Based on the rate of evaporation, when the volume of added urine is evaporated, the peristaltic pump is programmed in such a way that a volume of urine, similar to the evaporated volume, is fed to the urine-concentrator device.
The main urine-concentrator vessel is placed over a commercially-available magnetic heating device. After a stipulated pre-heating regime, the temperature on the heating plate is maintained between 120–150oC. The entire urine-feeding and evaporative, in-situ concentration cycle is followed until the required 27% (w/w) of the urine’s TDS content is adsorbed on the biomass sample.
The individual steps of the disclosed fungal strain’s controlled depolymerization-assisted, in-situ human urine’s adsorption-facilitated efficient biomass deconstruction procedure are explained below in detail.
Step 1: Preparation of biomass, pre-inoculum and inoculum, and induction of exogenous fungal bio-porosity
Rice straw was used as the model biomass for the entire biomass pre-treatment procedure described in the disclosure. After manually removing the visible impurities from the biomass, jets of ambient temperature-bearing municipal water are used to rinse the biomass. Prior to being subjected to an ultra-low temperature freezing at -80oC for 3–4 h, the biomass is packed into heavy-gauge poly-propylene bags capable of being subjected to autoclaving, and tightly sealed. Egg shells, after being subjected to water jet washing similar to the regime mentioned above, is subjected to repeated pulverization using a multi-blade setup of a commercially-available mixer/blender until shell powder with an average particle size of 2–6 µm (as observed under a microscope) is obtained. This is followed by the careful addition of autoclaved distilled water to the pulverized shells to maintain a paste-like consistency of the egg shell powder. The temperature of the pasty-mixture is maintained at ~80–90oC until introduction into the frozen biomass. To the frozen biomass, within a laminar air-flow chamber, the paste-like shell mixture is introduced through the top of the opened poly-propylene bag and allowed to trickle down so as to evenly disperse the shell powder’s paste throughout the various sections of the packed biomass. The paste is applied, quite generously, with an application on weight basis as 4:1 shell powder : biomass loading ratio. While still not completely dry, the shell paste-coated biomass (along with the bag) is placed within thick-walled glass beakers and subjected to a stage of ultrasonication (in batches) using a sonicator probe, operated between 20–25 kHz, at an amplitude between 50–60% for a total of ~30 min and, with a default pulse-and-pause duration. Post sonication, after manually sealing the poly-propylene bag, the shell material-infused biomass is subjected to an autoclave-based treatment at 100oC, at 15 psi, for 24 minutes. After autoclaving and draining the excess calcium carbonate-rich paste through the bag’s bottom, the biomass is removed from the bag, and within a laminar air-flow chamber, it is evenly distributed on the trays of a solid-state reactor.
Aspergillussydowii MTCC 6693, an endolithic fungus maintained in the lab’s repository, is initially grown on fungus growth-specific minimal medium-infused, 1.5–2.4% (w/v) calcium carbonate agar-containing solid medium prepared using sea water as the solvent. A drop culture is made from the preserved glycerol stock to initiate growth of the fungus on the agar plates. The plates are wrapped using aluminium foil to provide a light-deprived environment to the growing bio-porosity-inducing endolithic fungus, and incubated at ~36–45oC, for ~25 days. The developed hyphal mats from the agar plates are carefully introduced as floating layers into ~4–5% (w/v) exogenous calcium carbonate-containing fungus growth-specific minimal medium prepared using sea water, within a large-bottom Haffkine flask. After wrapping the flask using aluminium foil, incubation is performed for ~15–20 days with a very gentle shaking at ~10–15 RPM, at a temperature of ~37–45oC in a temperature-programmable shaker incubator. Throughout the culturing stages of the bio-porosity-inducing fungus, appropriate personal protective equipment must be worn by the experimentalist and the culture’s handling is recommended to be performed within a bio-safety level 2 (BSL-2) facility. After completion of the stage of hyphal carpet growth/formation within the Haffkine flask, inside the laminar air-flow chamber, the carpets are carefully transferred and laid over the surface of the biomass beds already placed within the solid-state reactor. The phase of exogenous, endolithic fungus-facilitated bio-porosity induction lasts for ~18–24 days, and is performed by maintaining the reactor’s temperature at ~45oC, with periodical introduction of autoclaved minimal medium-infused sea water. Post completion of the stipulated period of incubation, the hyphal carpets are removed (under aseptic conditions) and stored in sea water within a Haffkine flask for future use. The biomass, after being subjected to a stage of decontamination within an autoclave, is transferred to a vessel and is washed with jets of volumes of municipal water until the egg shell paste is removed (visually assessed). While the egg shell powder is harvested, dried and stored for future use, the remnant of the biomass is subjected to scanning electron microscopy (SEM) observations, X-ray diffraction (XRD) study, and Fourier-transform infrared (FT-IR) spectroscopy analysis to visualize the changes in the features of the biomass prior to and after the stage of bio-porosity induction. Unlike the native biomass, SEM observations of the bio-porosity-induced biomass indicated the presence of an uneven surface with several perceivable crevices, which were created by the thigmotropic response-driven mycelia of the porosity-inducing fungus. Such invasive penetration by the mycelia could be attributed to the stage of acoustic cavitation, wherein, microbubbles evolving from the egg-shell paste grow and explode so as to cause a dramatic erosion in the axial direction of the biomass’s surface, thereby facilitating enhanced permeation of the egg-shell paste, and thus the mycelia, to consume and degrade the imbibed shell paste. Furthermore, during the autoclave-based heat treatment post the stage of mycelial penetration, the biomass’s external surface area is greatly reduced, owing to substantial peeling of the external lignin and hemicellulose networks. During empirical estimation using the retention of water as an evaluation factor, an 42% increase in the porosity of the biomass was observed in comparison to its innate porosity of its native state. A proximate compositional analysis of the biomass after the phase of bio-porosity induction revealed an 60% decrease in the content of hemicelluloses, and a 35.46% decrease in the lignin content. The above observation is substantiated by the fact of a mild reduction in the absorption peaks between the FT-IR spectroscopy’s finger-print region in the range 3200–3600 cm-1, corresponding to the O–H stretching vibrations, qualitatively indicating lignin content’s reduction. Additionally, the absorption intensities between 1364–1367 cm-1,is also attributed to the qualitative assessment of O–H phenolic group, indicative of lignin’s presence. A reduction in the absorbances’ intensity between the region 1710–1028 cm-1 is attributed to a reduction in the hemicellulose content of the biomass. As a consequence of the removal of the innately amorphous lignin and hemicellulose components of the biomass during the stage of exogenous bio-porosity induction, the crystallinity index of the biomass measured and found to be increased to 34.26% in comparison to a crystallinity index of 27.33% for the native biomass. Prominent X-ray diffraction peaks at 2Ɵ of 14.5o, 16.5o, and 22.5o corresponding to the Miller indices 110, 11 ̅0 and 200, respectively, indicated a higher content of cellulose I in the native, untreated biomass (rice straw). Additionally, confirming the presence of cellulose I, with respect to interpretation of FT-IR spectrum, prominent peaks were observed between the range 1800–400 cm-1, near 893 cm-1 and between the region 1430 and 556 cm-1 forthe native biomass. In comparison to the untreated biomass, the exogenous bio-porosity-induced biomass, apart from displaying the above-mentioned prominent peaks in the diffractogram, exhibited inconspicuous diffraction peaks at 2Ɵ of 12o, 20o, 22o, thereby revealing that a portion of the native biomass was gradually being converted to cellulose II, a comparatively amorphous and thermodynamically stable form of cellulose. The native biomass, typically, possess the cellulose polymorph, Cellulose I; more specifically, owing to the presence of absorbances in the FT-IR spectral peak regions 3270 cm-1, 710 cm-1 and 3320 cm-1, the presence of the cellulose allomorph, Cellulose Iβ in the native biomass can be ascertained. During the bio-porosity induction, a disruption of the cellulose architecture, the Van der waals forces and the inter- and intra-molecular hydrogen bonding within the cellulose polymer leads to a marginal decrease in the intrinsic crystallinity of the cellulose. For a consolidated overview of the stage-wise variation of the composition of the rice straw during each stage of the pre-treatment, the Table 1, provided at the end of this section can be referred. Aspergillus spp. are known producers of lignin-degrading enzymes, such as lignin peroxidases, manganese peroxidases, and laccases. Though the preliminary objective of this stage is the introduction of exogenous bio-porosity in the biomass, thigmotropic response-driven enzyme secretion patterns may have resulted in degradation/depolymerization of the lignin content of the biomass. An incognizable/unquantifiable cellulose deconstruction may have also begun to occur as Aspergillus spp. are reported to be prolific cellulase enzyme complex producers as well. The purpose of the fungus-induced exogenous bio-porosity induction was to improve the efficient permeation of human urine (and its total dissolved solid content - TDS) during the subsequent stage of in-situ adsorption and evaporative concentration of human urine over the biomass. Additionally, for the third stage of a fungal strain-assisted controlled cellulose depolymerization, an improved porosity would reduce the time required for hyphal proliferation and controlled enzymatic depolymerization. Together, the induced and enhanced porosity, along with a controlled cellulose depolymerization assists the efficient, concentrated cellulolytic enzyme complex penetration during the stage of enzymatic saccharification to result in maximum cellulose-to-fermentable monomer conversion.
Table 1: Proximate analysis of the biomass composition throughout the course of the 4-stages of fungal depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic pre-treatment
Proximate analysis of the native rice straw(weight of biomass - 100 g)
S. No. Total saccharide content of the biomass (% w/w) Lignin – 14.1% (w/w) Others – 1.7% (w/w)
Cellulose – 39% (w/w) Hemicellulose – 33.8% (w/w)
1. Glucosec(g) Xyloseh(g) Arabinoseh(g) Mannoseh(g)
Galactoseh(g)
Acid Soluble Lignin (g) Klason Lignin (g) Protein (g)
Ash (g)
43±1 24±1.4 12±0.8 2±0.3 0.4 3±0.6 11.1±1.1 0.5±0.1 1.2±0.3
2. Proximate analysis of the rice straw after the stage of fungal bio-porosity induction – Stage 1 (weight of biomass – 76.8 g)
Total saccharide content of the biomass (% w/w) Lignin – 11.8% (w/w) Others – 0.3% (w/w)
Cellulose – 60% (w/w) Hemicellulose – 17.7% (w/w)
Glucosec(g) Xyloseh(g) Arabinoseh(g) Mannoseh(g) Galactoseh(g) Acid Soluble Lignin (g) Klason Lignin (g) Protein (g)
Ash (g)
51±0.6 14±0.3 0.3±0.1 1±0.2 0.2±0.1 1±0.1 8.1±0.2 0.1 0.1

Proximate analysis of the rice straw after the stage of the fungal strain-based controlled depolymerization – Stage 2
(Weight of biomass – 60 g)
Total saccharide content of the biomass (% w/w) Lignin – 13.1% (w/w) Others
3. Cellulose – 68% (w/w) Hemicellulose – 10% (w/w)
Glucosec(g) Xyloseh(g) Arabinoseh(g) Mannoseh(g)
Galactoseh(g)
Acid Soluble Lignin (g) Klason Lignin (g) Protein (g)
Ash (g)
45.3±0.8 6±0.1 0.1 0.6±0.1 0.1 Negligible 7.81±0.6 Negligible negligible
4. Proximate analysis of the rice straw after the in-situ urine’s TDS adsorption and heat treatment – Stage 3 and 4
Quantity of solubilized cellulose released into the pre-treatment hydrolysate – 40% (w/w); (Weight of remnant biomass – 34.6 g)
Total saccharide content of the biomass (% w/w) Lignin – 21.6% (w/w) Others
Cellulose– 70.5%(w/w) Hemicellulose – negligibly low
Glucosec – (g) Xyloseh (g) Arabinoseh(g) Mannoseh(g) Galactoseh(g)
Acid Soluble Lignin (g) Klason Lignin (g) Protein (g)
Ash (g)
27.1±1 0 0 0 0 negligible 7.5±0.2 Negligible negligible
5. Saccharification using in-house cellulolytic enzyme complex – expressed in (w/v)
Glucosec Xyloseh Arabinoseh Mannoseh Galactoseh
Cellobiosec Cellodextrins

4.12±0.3% 1±0.8% 0.03% 0.08±0.01% 0.02% 0.25±0.1% 0.23±0.15%
6. Bio-ethanol fermentation using the pre-treated solid biomass – expressed in (w/v)
Bio-ethanol Glucosec Arabinoseh Xyloseh Mannose Cellobiose Cellodextrin
4.2±0.3 3.7±0.1 0.001 0.001 0.002 0.12±0.01 0.06±0.02
GlucoseC: glucose expressed as cellulose equivalents using an anhydro correction factor of 0.90.
Xyloseh and arabinoseh: xylose and arabinose expressed as hemicellulose equivalents using an anhydro correction factor of 0.88.
Step 2: A fungal strain-assisted, controlled depolymerization of the biomass’s architecture
A strain of Aspergillusniger, was isolated from degrading twigs picked from the mangrove areas of the sub-urban region of Pondicherry, India. After complete morpho-molecular analysis and a phylogenetic tree construction, the fungus has been identified to be a novel strain of Aspergillusniger;however, owing to restrictions in deposition of the strain due to non-acceptance of Trichoderma spp., Aspergillus spp. and Penicillium spp. by collection repositories of India, the experimental trials were repeated using a strain of Aspergillusniger MTCC 4325. The fungus has been found to possess a shorter time for increasing its population, and additionally, its enhanced cellulose depolymerization activity has been exploited for the present work. Initially, the cultures are preserved in 1% (w/v) micro-crystalline cellulose-infused minimal medium-containing solid agar plates, which are prepared using ~50% (v/v) sea water to mimic the fungus’s natural environment in laboratory conditions. The standard primary inoculum preparation steps for the cellulose-depolymerizing fungus includes the introduction of a spore load of 9 x 1012 spores into a volume of 100 mL of fungal growth-specific minimal medium containing 1% (w/v) micro-crystalline cellulose, followed by incubation of the flasks at a temperature of 25–28oC for 6–10 days, at 120 RPM in a temperature-programmable shaker incubator. Akin to the typical preparation stage of a solid-state fermentation (SSF), ~46% (v/w) of the primary inoculum is added to the bio-porosity-induced biomass and blended (gently) using gloved hands so as to ensure the spreading of the fungus throughout the various regions of the biomass; the entire preparation stage is performed within a laminar air-flow chamber belonging to the BSL-1 category. To perform the stage of a fungus-assisted controlled depolymerization, the inoculum-blended biomass is loaded into the main vessel of the urine concentrator reactor, which provides sufficient oxygen transfer for the growing fungus through an aeration line introduced from the feeding line; the top and bottom flanges are screw-locked and the ball valves for the steam valve is kept open and plugged with cotton to allow ambient air to flow into the reactor. Distilled water is filled into the reactor’s jacket, and it is placed over a hot plate to maintain its temperature between 25–28oC for a period of ~5–7 days as standardized during the demonstrative trials. Extension of the period of incubation beyond the stipulated duration results in complete exhaustion of the biomass’s cellulosic and hemicellulosic content as a result of prolific fungal growth throughout its (biomass’s) surface. The process of controlled depolymerization is based on the exploitation of the thigmotropic effector response of the hyphal network of a fungus to drive it to the interior cellulose/hemicellulose-rich region of the biomass. Accelerating the process is the previously induced bio-porosity, which reduces the time for the fungus to penetrate the otherwise recalcitrant biomass’s architecture/surface. Another additional feature of the process, is a ~44.7% degradation of the biomass’s lignin content during this stage; furthermore, while subjecting a portion of the biomass to a stage of the NREL-prescribed proximate analysis procedure, it signified an ~82.3% degradation and removal of hemicellulose. ~6% (w/w) cellulose is consumed by the growing fungus. The pre-treatment hydrolysate was extracted post the fungal growth with appropriate safety gear and within a laminar air-flow hood. It has to be noted that during the stage of controlled depolymerization, the random cleavage of glycosidic bonds throughout the biomass’s cellulosic content would not result in soluble oligomer release into the pre-treatment hydrolysate. However, after the stage of in-situ adsorption of human urine-derived salt and heat treatment, the already delinked glycosidic bonds help in efficient breakage of the hydrogen bonds and sufficient decrease in the biomass’s crystallinity. After removing the cotton plug, aeration lines, screw-locking the flange plates of the biomass-containing urine concentrator vessel, it is subjected to the subsequent stage of in-situ evaporative concentration of urine onto the biomass.
During the demonstrative phase, in order to specifically analyse the changes after the stage of controlled microbial depolymerization, the biomass, which was subjected to the heating phase (incubation) of cellulose depolymerization, was carefully removed from the urine-concentrator device within a laminar air-flow hood. The biomass along with the excess minimal medium, if any, is subjected to autoclaving to decontaminate the same. While the pre-treatment hydrolysate is subjected to a stage of DNSA-based sugar analysis to estimate the components released after microbial depolymerization, the rest of the biomass is subjected to characterization using SEM, XRD, and FT-IR spectroscopy. However, during real-time performance of the 4-stage pre-treatment, following controlled depolymerization, the in-situ adsorption of human urine and immediate heat treatment is carried out; in summary the controlled depolymerization step, the urine adsorption and heat treatment are all performed as a single process.
In comparison to the bio-porosity-induced biomass, the SEM image of the biomass subjected to microbial depolymerization exhibits almost complete peeling of its top-surface, revealing the interior cellulose-dense architecture. Akin to the previous stage, the fungal strain readily propagates its mycelia throughout the interior of the biomass to catalyse cellulose depolymerization/enzymatic reduction of the degree of polymerization. During the course of the process, owing to its innate capability of secreting lignin-degrading enzymes, the lignin and hemicellulose-rich outer-most regions of the biomass are degraded. Proximate analysis of the microbially-depolymerized biomass exhibited an ~82.3% decrease in the content of hemicelluloses, and a ~44.7% decrease in the lignin content. The above observation is substantiated by the fact of a reduction of the absorption peaks between the FT-IR spectroscopy’s finger-print region between the ranges 3200–3600 cm-1, corresponding to the O–H stretching vibrations, and 1364–1367 cm-1, attributed to the O–H phenolic group, indicating significant lignin degradation. Considerable reduction of the absorbance peaks between the range 1710–1028 cm-1 is attributed to a reduction in the hemicellulose content of the biomass. Owing to the removal of a significant quantity of the hemicellulose and lignin components of the biomass, the crystallinity index of the biomass as estimated from X-ray diffractogram using the Segal method was found to be 46.6%. With respect to the determination of cellulose polymorph, the characteristic regions, as mentioned in the earlier discussions, indicated a gradual yet considerable transformation of the Cellulose I to its Cellulose II polymorph.
Step 3: In-situ evaporative concentration of human urine onto biomass in a specially-designed urine concentrator reactor/device
The self-designed and customized urine-concentrator reactor fabricated from stainless steel is used for the process of evaporative concentration and in-situ adsorption of the TDS content of human urine directly onto the biomass. First, the external jacket of the main vessel of the reactor is filled using distilled water until overflow is observed from the designated valve. Post-closing the valves, the main reaction vessel is loaded with the bio-porosity-induced biomass, resulting from the previous stage, to 1/4th of the reactor’s volume; after locking the screws of the top and bottom flange plates, the vessel is subjected to a pre-heating at ~50–60oC for 2 hours on a commercially-available hot plate; the pre-heating is followed by heating at ~100–112oC. The pre-heating process enhances the effective interaction/activation of the cellulase enzyme complex with the biomass’s interior resulting in sufficient microbial depolymerization. The steam release valve of the jacket, the urine-inlet line, and the steam riser line are always maintained in a closed position until temperature attainment in the main reaction vessel. Periodically, throughout the operation, the steam pressure is monitored within the jacket, and if necessary, the steam release valve is opened to reduce the excess pressure within the water jacket. Water is again filled to maintain a uniform temperature within the urine concentrator’s main vessel. The purpose of having a jacketed vessel is to permit the controlled heating of biomass, and a simultaneous evaporative concentration of human urine atop its surface without selectively charring the biomass content when subjecting it to direct heat.
Human urine from healthy volunteers is collected in sufficient quantities in a container constantly maintained at 4oC. The TDS content of human urine was found to be 5.4% (w/v) during the demonstrative trial. The collected urine, prior to usage, with appropriate personal safety gear is filtered using a motorized 0.22 µm filter cartridge-containing assembly, and subjected to autoclaving. Post autoclave, as described in the flow-schema in Figure5, using a peristaltic pump, an intermittent feeding of autoclaved urine is performed to the main concentrator vessel.
Urine feeding is performed on the basis of a pre-calculated rate of evaporation of the water content from the added quantity of urine, which is ~10–15 min for 200 mL in the demonstrative case. Since the TDS content of the human urine is assessed prior to feeding, the urine is fed until ~27% (w/v) of its TDS content is reached within the biomass sample. The TDS content, majorly comprises a consortium of salts such as sodium chloride, potassium chloride, magnesium sulfate, etc., as evaluated from a sample of human urine in a diagnostic laboratory. Once the first batch of urine is added, the steam riser valve is kept open. The steam rises through the silicone tubing, wrapped around with stainless steel bellows to better transfer the cold temperature from the surrounding frozen gel packs. As the steam travels through the condensation tubing, it gets collected in a urine’s distillate harvest vessel. The distillate harvest vessel contains ice flakes/cubes loaded into its surrounding jacket. The distillate of the urine collected in the ice-cooled harvest vessel can be used for biomass rinsing purposes and microbial culture purposes, thereby signifying a circular bio-economy of the process. The demonstrative process for achieving the in-situ adsorption of 27% (w/w) of the TDS content of the human urine onto the biomass was 2.5–4 hours. Since the temperature of the urine concentrator reactor was maintained between ~100–120oC throughout the lengthy duration of the process, the biomass is directly harvested, aseptically, using appropriate PPE, and transferred to the heating element-containing oven/heater for the subsequent stage 4.
Step 4: A heating element-facilitated treatment of the urine-derived salt-adsorbed biomass
To 27% (w/v) human urine’s TDS-adsorbed biomass, a pre-calculated/pre-standardized weight of ice flakes are added. The weight corresponds to the quantity of water removed/evaporated during every cycle of the heating regimen. The biomass + ice is subjected to a 14-min.-long heating cycle at a temperature between 120–150oC, followed by holding at 180oC for 1 min., immediately followed by the addition of the pre-standardized quantity of ice, after the entire 15 min.-long cycle. The 15 min cycle is repeated for 1.5 hours. During demonstrative trials, the heat treatment was performed in a conventional heating element-containing electric oven. After completion of the treatment, two volumes of water, with respect to the quantity of the biomass, are added and the biomass is manually squeezed (using gloved hands) to extract the pre-treatment hydrolysate. After first two washes using the stipulated quantity of water, a few more washes may be conducted until the wash-resultant water is within its native TDS limits; summarily, it is to be made sure that the biomass is free of the adsorbed salts/TDS. While a proximate analysis of the remnant biomass showed ~46.8% lignin degradation and ~100% hemicellulose solubilization/removal, a ~40% decrease in the cellulose content indicated the removal of depolymerized cellulose as soluble cellodextrins/oligomers in the pre-treatment hydrolysate.
The heating duration of the final stage of the pre-treatment is advised to not be prolonged beyond the optimum 1.5 h. At the optimum heating duration, considerable cellulose depolymerization and de-crystallization is observed; further extension of the heating duration beyond the standardized 1.5 h led to an increase in the crystallinity of the cellulose, which is unfavoured and unintended. It could be attributed to the fact that the complete degradation of the amorphous cellulose of the biomass leads to re-ordering of the cellulose linkages towards a crystalline arrangement, in turn resulting in an increased crystallinity. It was observed that during the sequence of pre-treatment, in the final heat-treatment stage, while using the microbially depolymerized and concentrated urine’s TDS-adsorbed biomass, the rate of production of % (w/w) soluble β-D-glucose oligomers was higher; it could be attributed to the fact that the partially depolymerized (random and controlled fungal depolymerized) biomass has the potential to be subjected to cleavage of glycosidic linkage, thereby decreasing crystallinity, resulting in enhanced glucose monomer release into the pre-treatment liquid.
SEM observations of the remnant insolubilized fraction of the biomass indicated that the biomass had undergone a thorough degradation from its top surface until its inner core. The released oligomers and the short-chain cellulosic fibres further undergo enhanced hydrolysis to release reducing sugars during interaction with hydrogen radicals. Sodium chloride, a major component of the total dissolved solids in concentrated human urine, initially swells the biomass, effectively attenuates the intra- and inter-molecular hydrogen bonding, hampers the mobility of the hydrogen linkages, which interact with all ions, thereby perturbing and altering the crystallinity of the native cellulose I and the converted cellulose II. SEM images revealed hollow interiors of the biomass and highly-disordered fibril structures unlike the native biomass. In the present stage, the remnant lignin and hemicellulose after the previous stage of the pre-treatment process were further reduced to a major extent as visualized by variations in the characteristic FT-IR spectral regions (% transmittances) 1605, 1513, and 1426 cm-1 relating to the aromatic skeletal vibrations pertaining to the degradation of lignin. A similar trend is observed for the peak regions 1225 and 1125 cm-1, which relate to C-C, C-O, and C=O and ether linkages, respectively. The FT-IR spectral intensity decrease between 1710–1028 cm-1 explicates complete hydrolysis and removal of hemicelluloses as also evidenced by the proximate compositional analysis of the remnant biomass post pre-treatment. The 27.1% (w/w) cellulose content in the remnant biomass showed a 31.34% crystallinity index as measured using Segal’s method, which is a 15.26% decrease, in comparison to the biomass crystallinity from the previous stage, thereby indicating a large reduction in crystallinity and conversion of the cellulose to a more amorphous and amenable form. Insignificant X-ray diffraction peaks at 2Ɵ of 14.5o, 16.5o, and 22.5o corresponding to the Miller indices 110, 11 ̅0 and 200, respectively, indicate a major reduction in the cellulose I content of the native biomass. The characteristic FT-IR spectral peaks (1800–400 cm-1, 893 cm-1, and 1430–556 cm-1) corresponding to Cellulose I had significantly reduced. The X-ray diffractogram exhibited prominent peaks at 2Ɵ of 12o, 20o, 22o, elucidating a major conversion of the Cellulose I to the amorphous cellulose II polymorph after the entire sequence of the pre-treatment.
While analysing the extracted pre-treatment hydrolysate using a thin-layer chromatographic technique on 20 cm-long silica gel-based commercially-available TLC plates, it showed the prominent hydrolysis of the xylan backbone and the release of xylose (majorly), as identified with a proximate position of the separated bands in the molecular weight range of ~150–200 g/mol., and certain other unknown oligomers between the range of ~180–350 g/mol; precisely, the unknown oligomer spots lay below glucose (180.1 g/mol) and in the molecular weight near the ranges of agar (336.3 g/mol) and cellobiose (342.3 g/mol) standards. The observation was further strengthened during a closer examination of the peaks from an electro-spray ionization mass spectrometry (ESI-MS) data conducted for the human urine-derived salt-adsorbed and heat-treated biomass sample. The ESI-MS of the hydrolysate revealed the presence of a prominent peak in the molecular weight range of ~340 g/mol, elucidating the release of soluble cellobiose (homo-dimer of β-D-glucose) units. Peaks corresponding to the presence of the hydrolysed portions of the xylan backbone were also visible in the spectra. The above observations confirm the pre-treatment’s efficiency in depolymerizing and hydrolysing the biomass’s cellulosic content. For the TLC, the mobile phase consists of acetonitrile : water in the volume-ratio (85:15). P-anisaldehyde-based staining followed by a brief heating regimen using a heat gun was used to develop the chromatograms for visualization. Usually, residual pre-treatment agent/ionic liquid of the pre-treatment operation, which is the TDS content of urine in this case, hamper cellulolytic enzymes’ saccharifying ability. It is, hence, recommended to perform the separation of the hydrolysed sugar entities in the pre-treatment hydrolysate from the TDS content of the urine using a process of electrodialysis. The remnant solid biomass, when subjected to enzymatic saccharification using 12–15 FPU/g of biomass loading of in-house cellulases, showed 98±1.2% saccharification efficiency, and while the saccharification hydrolysate was subjected to an ethanol fermentation process, it resulted in a bio-ethanol yield of 0.37 g/g of glucose indicating an 74% fermentation yield. Fermentation was performed using a 10% (w/v) inoculum cream of a strain of Saccharomyces cerevisiae, for 36–50 hours, at 28–30oC, in a micro-aerophilic environment, with ~15% (w/v) β-D-glucose loading. Analysis of biomass samples without the bio-porosity induction or/and microbial depolymerization resulted in at least ~70% decreased efficiency of the urine-concentration and heat treatment alone; this elucidates the significance of the bio-porosity induction and microbial depolymerization stages prior to the in-situ adsorption of human urine and the heat treatment. Furthermore, biomass samples, not subjected to the stages of the first two fungal treatments, did not exhibit cognizable lignin degradation.
The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts necessarily need to be performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.
Benefit s, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.
, Claims:1. A process for fungal-strain-catalyzed controlled-depolymerization-assisted, in-situ human urine’s adsorption-based lignocellulosic biomass pre-treatment for 2G-bio-ethanol production, comprising:
a) pre-preparing biomass and applying eggshell paste to the pre-prepared biomass thereby preparing a pre-inoculum and inoculum followed by inoculating the biomass with the pre-inoculum and inoculum to induce exogenous bio-porosity;
b) reducing a degree of polymerization of a cellulose architecture of the biomass by performing a fungal strain-assisted controlled biomass depolymerization process using a whole-mycelial bio-transformation technique;
c) performing an in-situ adsorption of human urine onto depolymerized biomass and evaporatively concentrating adsorbed human urine in a human urine concentrator reactor; and
d) treating salt-adsorbed biomass by subjecting the salt-adsorbed biomass to a heating element for enhancing cellulose solubilization, resulting in a reduction in a degree of polymerization of a cellulose thereby positively reducing cellulase enzyme complex-loading during saccharification and subsequently serving as a significant breakthrough in reduction of a final production cost of 2G bio-ethanol.

2. The process as claimed in claim 1, wherein the biomass preparation claimed in step a comprises:
manually removing visible impurities from the biomass and rinsing the biomass with jets of ambient temperature-bearing municipal water;
packing the biomass into heavy-gauge polypropylene bags capable of autoclaving; and
tightly sealing the bags and freezing the biomass at an ultra-low temperature of -80°C for 3–4 hours.

3. The process as claimed in claim 1, wherein the egg shell application onto the biomass comprises:
rinsing egg shells using a water jet washing method;
pulverizing the egg shells using a multi-blade mixer/blender to produce powder with an average particle size of 2–6 µm;
mixing the pulverized egg shell powder with autoclaved distilled water to form a paste, maintaining a temperature between 80–90°C;
introducing the shell paste into the frozen biomass within a laminar air-flow chamber through a top of an opened poly-propylene bag and allowing it to trickle down so as to evenly disperse the shell powder’s paste throughout various sections of the packed biomass;
applying a shell powder to biomass weight ratio of 4:1;
subjecting the paste-coated biomass, placed within thick-walled glass beakers, to ultrasonication in batches using a sonicator probe, operating between 20–25 kHz at an amplitude of 50–60%, for a duration of ~30 minutes;
sealing the polypropylene bag containing the shell-infused biomass;
subjecting the biomass to autoclave treatment at a temperature of 100°C, at 15 psi, for 24 minutes;
draining excess calcium carbonate-rich paste through a bottom of the polypropylene bag, and removing the biomass from the bag within a laminar air-flow chamber; and
evenly distributing the biomass on trays within a solid-state reactor for further processing.

4. The process as claimed in claim 1, wherein the preparation of pre-inoculum of the bio-porosity-inducing fungus comprises:
culturing Aspergillussydowii MTCC 6693, an endolithic fungus by preparing a solid medium by infusing a fungus growth-specific minimal medium with 1.5–2.4% (w/v) calcium carbonate and using seawater as solvent;
introducing a drop starter culture of Aspergillussydowii MTCC 6693 from a preserved glycerol stock onto the prepared calcium carbonate agar plates;
wrapping agar plates with aluminum foil to create a light-deprived environment for fungus; and
incubating wrapped plates at a temperature range of ~36–45°C for approximately 25 days to promote fungal growth.

5. The process as claimed in claim 1, wherein the preparation of the inoculum/inoculum carpets of the exogenous bio-porosity-inducing fungus comprises:
harvesting developed hyphal mats from calcium carbonate agar plates grown under a light-deprived environment at ~36–45°C for approximately 25 days;
preparing a fungus growth-specific minimal medium containing 4–5% (w/v) exogenous calcium carbonate, using seawater as the solvent;
introducing harvested hyphal mats as floating layers into prepared medium within a large-bottom Haffkine flask, ensuring that the mats do not sink to the bottom to maintain a floating condition;
wrapping the Haffkine flask with aluminum foil to create a light-deprived environment to further support a growth of the fungus;
incubating the wrapped flask in a temperature-programmable shaker incubator for approximately 15–20 days at a controlled temperature of ~37–45°C;
setting a shaker to perform gentle shaking at a speed of ~10–15 RPM to maintain homogeneity of the culture medium while preventing shear stress on the hyphal mats;
wearing appropriate personal protective equipment, including gloves, lab coat, and face mask, to ensure safety during handling of the culture; and
conducting all handling and culturing activities within a bio-safety level 2 (BSL-2) facility to minimize contamination and ensure safety of experimentalist.

6. The process as claimed in claim 1, wherein the endolithic fungus-assisted exogenous bio-porosity induction comprises:
transferring developed hyphal carpets, previously cultured in a Haffkine flask, into a laminar air-flow chamber to ensure an aseptic environment;
laying the hyphal carpets over surface of biomass beds placed within a solid-state reactor;
maintaining the reactor at a temperature of approximately 45°C during the bio-porosity induction phase for a duration of 18–24 days;
periodically introducing autoclaved minimal medium-infused seawater into the reactor to provide essential nutrients and maintain moisture content throughout induction phase, wherein the biomass is subjected to a decontamination process within an autoclave prior to washing, wherein the biomass is washed with jets of water until the egg shell paste is visually assessed to be completely removed;
monitoring environmental conditions within the reactor to ensure optimal growth conditions for the endolithic fungus;
after completion of an incubation period, removing the hyphal carpets under aseptic conditions to prevent contamination; and
storing the removed hyphal carpets in seawater within a Haffkine flask for future use in further experiments or applications.

7. The process as claimed in claim 1, wherein the fungal strain-/whole mycelial bio-transformation-assisted controlled cellulose depolymerization/reduction in the cellulose’s degree of polymerizationclaimed in step b comprises:
preserving the fungal culture on solid agar plates infused with 1% (w/v) micro-crystalline cellulose in a minimal medium, prepared using approximately 50% (v/v) seawater, to mimic fungus's natural habitat;
preparing a primary inoculum by introducing a spore load of 9 x 10¹² spores into 100 mL of fungal growth-specific minimal medium containing 1% (w/v) micro-crystalline cellulose;
incubating the inoculum at a temperature of 25–28°C for 6–10 days with shaking at 120 RPM in a temperature-programmable shaker incubator;
adding approximately 46% (w/v) of the prepared primary inoculum to the bio-porosity-induced biomass and gently blending the mixture with gloved hands within a BSL-1 category laminar air-flow chamber to ensure even distribution of the fungus throughout the biomass;
transferring the inoculum-blended biomass into the main vessel of a urine concentrator reactor, equipped with an aeration line for oxygen transfer, and securing the top and bottom flanges with screw-locks while keeping the steam valve open and plugged with cotton to facilitate ambient air flow;
filling the reactor jacket with distilled water and placing the reactor on a hot plate to maintain a temperature range of 25–28°C for a standardized period of 5–7 days;
monitoring the biomass to prevent over-incubation, which may lead to complete exhaustion of its cellulosic and hemicellulosic content due to excessive fungal growth;
utilizing the thigmotropic effector response of the fungal hyphal network to promote penetration into the interior cellulose/hemicellulose-rich regions of the biomass, enhanced by previously induced bio-porosity;
achieving approximately 44.7% degradation of the biomass's lignin content, with an observed degradation of approximately 82.3% hemicellulose and approximately 6% (w/w) cellulose consumption by the fungus during the controlled depolymerization phase;
extracting the pre-treatment hydrolysate post-fungal growth while wearing appropriate safety gear and working within a laminar air-flow hood, ensuring that random cleavage of glycosidic bonds does not result in soluble oligomer release into the pre-treatment hydrolysate;
subjecting the biomass to in-situ adsorption of human urine-derived salt followed by heat treatment to facilitate the efficient breakage of hydrogen bonds due to previously delinked glycosidic bonds and reduction of biomass crystallinity; and
removing the cotton plug and aeration lines from the reactor, screw-locking the flange plates, and proceeding to the subsequent stage of in-situ evaporative concentration of urine onto the biomass.

8. The process as claimed in claim 1, wherein the in-situ evaporative concentration of human urine onto biomass comprises:
fabricating a stainless steel urine concentrator reactor comprising a main vessel with an external jacket for controlled heating and a steam release valve;
filling the external jacket of the reactor with distilled water until overflow is observed from designated valve, followed by closing the valves to maintain water levels;
loading the main reaction vessel with bio-porosity-induced biomass to a volume of approximately 1/4th of the reactor’s total volume;
securing the top and bottom flange plates of the reactor with screws to prevent leaks;
subjecting the loaded reactor to pre-heating at a temperature range of approximately 50–60°C for 2 hours on a commercially available hot plate to enhance the interaction and activation of the cellulase enzyme complex with the biomass, thereby promoting microbial depolymerization;
heating the reactor to a temperature range of approximately 100–112°C, while keeping the steam release valve, urine-inlet line, and steam riser line closed until the target temperature is attained in the main reaction vessel;
monitoring steam pressure within the jacket throughout the operation, opening the steam release valve as needed to relieve excess pressure, and maintaining uniform water levels within the jacket by periodically adding distilled water;
collecting human urine from healthy volunteers in a container stored at 4°C, wherein the total dissolved solids (TDS) content is determined to be 5.4% (w/v) during a demonstrative trial;
filtering the collected urine using a motorized 0.22 µm filter cartridge assembly to remove particulate matter while wearing appropriate personal protective equipment (PPE);
subjecting the filtered urine to autoclaving to ensure sterilization before use in the evaporation process;
utilizing a peristaltic pump to intermittently feed the autoclaved urine into the main concentrator vessel at a pre-calculated rate based on the observed evaporation rate, approximately every 10–15 minutes for an initial 200 mL quantity;
feeding the urine until the TDS content reaches approximately 27% (w/v) in the biomass, wherein the TDS is predominantly composed of salts such as sodium chloride, potassium chloride, and magnesium sulfate as confirmed by diagnostic laboratory analysis;
keeping the steam riser valve open after the first batch of urine is added, allowing steam to rise through silicone tubing wrapped with stainless steel bellows to maintain cold temperatures via surrounding frozen gel packs;
collecting condensed steam in a distillate harvest vessel equipped with an ice-cooled jacket, allowing the distillate to be utilized for biomass rinsing and microbial culture purposes, thereby creating a circular bio-economy;
performing the in-situ adsorption of approximately 27% (w/w) TDS content from human urine onto the biomass over a period of approximately 2.5–4 hours while maintaining reactor temperatures between 100–120°C; and
aseptically harvesting the treated biomass post-process using appropriate PPE and transferring it to a heating element-containing oven or heater for subsequent processing.

9. The process as claimed in claim 1, wherein the urine-derived salt-adsorbed biomass treatment comprises:
utilizing a biomass pre-treated through a process that achieves in-situ adsorption of total dissolved solids (TDS) from human urine, resulting in a biomass that contains approximately 27% (w/v) of human urine's TDS;
measuring and adding a pre-calculated weight of ice flakes to the TDS-adsorbed biomass, wherein the weight of the ice corresponds to the quantity of water that has been evaporated during the previous concentration cycles;
subjecting the biomass-ice mixture to a heating regimen in a conventional electric oven and implementing a first heating cycle at a temperature range of 120–150°C for 14 minutes, followed immediately by a holding phase at 180°C for 1 minute;
repeating heating cycle for a total duration of 1.5 hours, ensuring that a total time for heating does not exceed this limit to prevent excessive crystallization of cellulose;
adding two volumes of water relative to the biomass quantity, and manually squeezing the biomass with gloved hands to extract a hydrolysate containing soluble sugars and other byproducts;
conducting subsequent washing steps, ensuring that the wash water is devoid of salts and within native TDS limits;
performing proximate analysis on remaining biomass, confirming approximately 46.8% lignin degradation, complete removal of hemicellulose, and a 40% reduction in cellulose content;
measuring and reporting a crystallinity index of remnant biomass using a Segal's method, confirming a decrease to approximately 31.34% crystallinity;
subjecting the remnant biomass to enzymatic saccharification using 12–15 FPU/g of biomass loading of in-house cellulases, achieving a saccharification efficiency of 98±1.2%; and
fermenting the resultant saccharified hydrolysate using a 10% (w/v) inoculum of Saccharomyces cerevisiae under micro-aerophilic conditions, at temperatures of 28–30°C, in a micro-aerophilic environment, with ~15% (w/v) β-D-glucose loading, to yield a bio-ethanol output of 0.37 g/g of glucose, achieving a fermentation yield of approximately 74%.

10. A human urine concentrator reactor as claimed in claim 1, comprising:
an inner vessel, cylindrical in shape, designed to hold human urine;
an outer jacket surrounding the inner vessel, creating a user-defined gap between peripheries of the inner vessel and outer jacket, wherein the gap between the jacket and the inner vessel is welded at a top to prevent air exposure;
a bottom flange with a cylindrical periphery, featuring four screw bores for securing with stainless-steel screws, allowing a top flange plate to be affixed, wherein the top flange plate containing four bores for introducing screws, and rests silicone gasket adhered to the bottom flange;
a stainless-steel inlet pipe for human urine, located at a top of the inner vessel;
a steam outlet line mounted with a ball valve at the top of the inner vessel, facilitating removal of urine-derived steam;
an outlet line positioned at a height of user-defined distance from a bottom of the jacket, equipped with a circular stainless-steel pipe and customized screw threads, allowing distilled water to enter the jacket;
an overflow line at a user-defined height from a top of the jacket, similar in diameter to the inlet pipe, and equipped with a ball valve for monitoring distilled water levels;
a steam release valve located on an anterior of the vessel above the gap between the inner vessel and the jacket, permitting excess steam to escape during pressure build-up;
a pressure-monitoring gauge on the top plate of the jacket, positioned above a user-defined gap to measure internal pressure;
a silicone tubing connected to a steam outlet valve of the inner vessel, extending to a urine distillate collection vessel;
a plurality of flexible stainless-steel bellows wrapped around the silicone tubing to enhance heat transfer from frozen gel packs surrounding the tube;
one or more frozen gel packs fastened around the silicone tube, ensuring an optimal temperature for a condensation of water vapor from the human urine;
a distillate collection vessel, surrounded by an outer jacket, featuring a user-defined gap between the vessels;
a non-fully sealed gap at a top of a condensate collection vessel, allowing loading of ice flakes while maintaining structural integrity through selective welding;
wherein a harvest vessel includes a bottom and top flange capable of being tightened using screws, with a 3 mm silicone-based gasket positioned between the flanges;
a peristaltic pump for transfer of appropriately autoclaved, stored, and filtered human urine into the inner vessel, utilizing silicone tubing for transport; and
a distillate collection line present at the top plate of the inner vessel, directly connecting the steam outlet line from the main urine concentrator device for effective collection of distilled water.