DESC:FIELD OF THE INVENTION

The present disclosure relates to a sustainable, green process engineering-incorporated, lignocellulosic biomass system and method for pre-treatment procedure. More specifically, a moderate reaction condition-possessing, lesser inhibitor-generating, self-sustainable biomass pre-treatment procedure, involving a human urine-derived pre-treatment liquid, and the hydrolytic action of a fungi, has been developed.

BACKGROUND OF THE INVENTION

Lignocellulosic biomass, the sought-after, carbohydrate-rich source of second-generation bio-ethanol, comprises three major constituent-polymers — cellulose, hemicellulose, and lignin. While the ß-D-glucose monomer-dense polymer, cellulose, is the polysaccharide of interest for ethanol production using conventional S. cerevisiae-based fermentation, its (cellulose’s) accessibility/usability is hindered by the recalcitrant, phenolic- polymer lignin, and another polysaccharide, hemicellulose.

In bio-refineries, biomasses are subjected to a procedure, termed pre-treatment, where chemical agents are utilized in high-temperature (as high as 300oC) and pressure-driven (as high as 700 psi) operations, which disrupt the innate structure of biomasses, hydrolyse and solubilize hemicelluloses, depolymerize lignin, reduce the overall recalcitrance, and improve accessibility of the inner-most homopolymer: cellulose. Pre-treatment procedures, which incur 40 – 50% of the overall functioning costs of a bio-refinery, usually involve the usage of strong, occupational hazard-possessing concentrations of acids (majorly: sulphuric acid/hydrochloric acid) or alkalis (majorly sodium hydroxide/liquor ammonia/calcium hydroxide) during their operational regimes. Microbe-based/microbial-consortia-based biological pre-treatments, though have been demonstrated to possess selective delignification capabilities, have majorly suffered from prolonged process durations. The pre-treatment efficiencies of chemical, hydrothermal, and biological techniques are on par with each other, recording 70 – 80% lignin degradation, and 85 – 100% hemicellulose solubilization.

In addition to the cost-intensive procurement of chemical agents and heavy electricity/power-requiring equipment’s operations (to perform pre-treatment), the extreme reaction environments, lead to the formation of certain inhibitory compounds, such as furfural, p-coumaric acid, 5-hydroxy-methyl-furfual (5-HMF), acetic acid, pseudo-lignin, etc. While pseudo-lignin has been reported to non-specifically bind to cellulase-enzyme complex during saccharification and decrease monomeric, fermentable sugar yields, furfurals and 5- HMFs have been reported to deter the metabolic activities of S. cerevisiae, and inhibit the key-fermentative enzymes during fermentation of the saccharification-resultant sugars to bio-ethanol.

Hydrotropic pre-treatment of lignocellulosic biomass is currently being practiced by contemporary researchers, owing to its environment-friendliness, process economy, and efficient dissolution and recovery of lignin. Possessing an amphiphilic nature, and using the combinatorial effect of hydrophobic interactions, and multi-layered, lignin-solubilizing micro-environment creation, a hydrotropic agent greatly-promotes the solubility of hydrophobic, water-insoluble, organic compounds such as lignin. While sodium xylene sulphonate (SXS), sodium cumene sulphonate, sodium benzoate, and p-toulenesulfonic acid (p-TsOH), are reported to be effective hydrotropic agents, urea, capable of initiating hydrogen bonding with lignin along with its potential of disrupting its (lignin’s) p-p architecture by forming O- p networks, is currently drawing a lot of interest among bio-fuel researchers.

In the view of the forgoing discussion, it is clearly portrayed that there is a need to have a human urine-based, fungus-assisted, alkali-cum-hydrotropic system and method for pre-treatment of lignocellulosic biomass.

SUMMARY OF THE INVENTION

The present disclosure seeks to provide a human urine-based, fungus-assisted, alkali-cum-hydrotropic system and method for pre-treatment of lignocellulosic biomass. A green, self-sustainable, cost-effective, waste-stream-based, moderate operational-regime possessing, lesser inhibitor-generating, efficient pre-treatment procedure for lignocellulosic biomass. The disclosure presents a sequential, single-pot, combinatorial-procedure, where, within a solid-state bio-reactor, a human urine- derived, 0.3% (w/v) urea-containing solution is allowed to percolate into pre-processed biomass’s interiors, which is then inoculated with 60% (v/w) Aspergillus niger VVS-15 NKR. The fungus, pre-accustomed to a urea-rich environment, follows a solid-state growth regime and accumulates a mycelial biomass concentration of ~12 - 14 g/L during the dual-staged, urea pulse-fed incubation, each lasting 120 h. Once fungal growth stabilizes, post the 240-h-long growth phase, an excess of urine-derived 6.66% (w/v) urea-containing solution is added to the reactor, incubation temperature is risen to 42o C, and the accumulated fungal urease catalyses an in-situ hydrolysis of the fed urea. The hydrolysis is allowed to progress until 288 h when the continuously-monitored pH plateaus. Subsequently, the reactor is incubated at -80oC (minus 80o C), for 48 h, to facilitate the stable interaction of ammonia and the lignocellulosic biomass. Following the 48-h ultra-low temperature incubation, the bio-reactor, along with its contents, is heat-treated at 100 o C, for 1 h, at 5 – 6 psi in an autoclave; during this phase, as the ammonia-imbibed, and remnant, unreacted urea-containing biomass is subjected to heat treatment, a simultaneous alkali pre-treatment and hydrotropic pre-treatment is carried out. As a demonstrative process, while subjecting waste, weedy biomass from rice fields to the 336-h-long, fungus-assisted, alkali-cum-hydrotropic pre-treatment procedure, 93% hemicellulose degradation, 75% lignin degradation, and 25% reduction in biomass crystallinity has been recorded. For the pre-treated weedy biomass, saccharified with a reduced cellulase loading of ~8 – 10 FPU/g of biomass, the percentage of enzymatic-saccharification is 62.8%, the bio-ethanol yield 0.44 g/g of glucose, indicating a cellulose bio-conversion of 69.54%.

The present disclosure seeks to provide a self-sustainable, cost-efficient, milder operational regime-possessing, green process engineered-pre-treatment, which simultaneously integrates the aspects of alkali pre-treatment and hydrotropic pre-treatment of lignocellulosic biomass. The process comprises various stages, such as: (i) continuous-distillation of human urine to obtain a concentrated urea-containing solution; (ii) pre-processing water-soaked lignocellulosic biomass by freezing it at - 80oC for 24 h, followed by rapid autoclaving to facilitate structural damage, and to increase biomass’s water absorptivity; (iii) soaking the pre-processed biomass with human urine-derive urea solution, and inoculation using Aspergillus niger VVS-15 NKR and incubation for 240 h, at 28oC, in two phases, each lasting 120 h, within a solid-state bio-reactor; (iv) addition of remaining, urine-derived urea solution, rising the temperature regime to 42oC, for 48 h, to facilitate urease-based, in-situ hydrolysis of remnant urea in the biomass; (v) incubating the reactor at – 80oC for 48 h, to permit the interaction of ammonia and the biomass; (vi) heat-treating the reactor at 100oC for 1 hour, at 5 – 6 psi, within an autoclave to result in a combined alkali-cum-hydrotropic pre-treatment of the biomass.

In an embodiment, a human urine-based, fungus-assisted, alkali-cum-hydrotropic system for pre-treatment of lignocellulosic biomass is disclosed. The system includes a urine collection apparatus for collecting human urine. The system further includes a distillation chamber for continuously distilling the collected urine to concentrate a crude urea content while water from urine is collected as a distillate. The system further includes a pre-processing module connected to the distillation chamber for pre-processing a biomass by soaking the biomass in water, and freezing the water-soaked biomass. The system further includes a solid-state bio-reactor connected to the pre-processing module for accepting frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, followed by inoculation and incubation of the soaked biomass using Aspergillus niger to facilitate an in-situ ureolysis, and subsequently a stable-interaction of ammonia with the biomass. The system further includes a heat-treatment apparatus attached to the bio-reactor for heating the biomass at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

In another embodiment, a human urine-based, fungus-assisted, alkali-cum-hydrotropic method for pre-treatment of lignocellulosic biomass is disclosed. The method includes collecting by a urine collection apparatus 102, human urine. The method further includes distilling by a distillation chamber 104, the collected urine to concentrate a crude urea content while water from urine is collected as a distillate. The method further includes pre-processing by a pre-processing module 106, a biomass by soaking the biomass in water, and freezing the water-soaked biomass. The method further includes accepting by a solid-state bio-reactor 108 frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, followed by inoculation and incubation of the soaked biomass using a ureolytic fungus to facilitate an in-situ ureolysis, and subsequently to promote the stable-interaction of ammonia with the biomass. The method further includes heating by a heat-treatment apparatus 110 the biomass at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

An object of the present disclosure is to provide a self-sustainable, green process-engineered, lesser inhibitor-generating, and economical pre-treatment procedure for lignocellulosic biomass.

Another object of the present disclosure is to concentrate human urine by continuous distillation, and obtain a crude solution of urea, which would be used as a hydrotropic biomass pre-treatment agent.

Another object of the present disclosure is to utilize the water, obtained by distillation of human urine, for biomass’s soaking and washing purposes.

Another object of the present disclosure is to incubate the hydrolyzed urea-containing biomass at – 80oC, for 48 h, to allow the stable interaction of ammonia with the biomass.

Yet another object of the present invention is to deliver an expeditious and cost-effective human urine-based, fungus-assisted, alkali-cum-hydrotropic system.

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 block diagram of a human urine-based, fungus-assisted, alkali-cum-hydrotropic system for pre-treatment of lignocellulosic biomass in accordance with an embodiment of the present disclosure;
Figure 2 illustrates a flow chart of a human urine-based, fungus-assisted, alkali-cum-hydrotropic method for pre-treatment of lignocellulosic biomass in accordance with an embodiment of the present disclosure;
Figure 3 illustrates a block diagram of a green process-engineered, self-sustainable, fungus assisted, alkali-cum-hydrotropic pre-treatment of lignocellulosic biomass in accordance with an embodiment of the present disclosure; and
Figure 4 illustrates a schematic representation of the solid-state bio-reactor used to perform the proposed pre-treatment procedure 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 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.

Referring to Figure 1, a block diagram of a human urine-based, fungus-assisted, alkali-cum-hydrotropic system for pre-treatment of lignocellulosic biomass is illustrated in accordance with an embodiment of the present disclosure. The system 100 includes a urine collection apparatus 102 for collecting human urine.

In an embodiment, a distillation chamber 104 is used for continuously distilling the collected urine to concentrate a crude urea content while water from urine is collected as a distillate.

In an embodiment, a pre-processing module 106 is attached to the distillation chamber 104 for pre-processing a biomass by soaking the biomass in water, and freezing the water-soaked biomass.

In an embodiment, a solid-state bio-reactor 108 is attached to the pre-processing module 106 for accepting frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, inoculating and incubating the soaked biomass to facilitate the stable-interaction of ammonia with the biomass.

In an embodiment, a heat-treatment apparatus 110 is connected to the bio-reactor 108 for heating the biomass at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

In another embodiment, the collected urine is stored at a temperature of 3-5oC.

In another embodiment, 85% (v/v) water from human urine is collected as the distillate while the 15% (v/v) residue is a crude solution of urea, along with other components of human urine, wherein post distillation, the urea content of the moderately-viscous residue is 1% (w/v) as quantified using high-performance liquid chromatography.

In another embodiment, the soaked biomass is stored at – 80oC, for 24 hours, followed by rapid autoclaving of the frozen, water-soaked biomass.

In another embodiment, the incubation is followed by a temperature-up-shift, to catalyse the in-situ, enzymatic-hydrolysis of urea into ammonia.

In another embodiment, the bio-reactor 108 consists of sieved trays, over which the urea-imbibed biomass is loaded, and inoculated with a uratolytic fungus.

Figure 2 illustrates a flow chart of a human urine-based, fungus-assisted, alkali-cum-hydrotropic method for pre-treatment of lignocellulosic biomass in accordance with an embodiment of the present disclosure. At step 202, the method 200 includes collecting by a urine collection apparatus 102, human urine.

At step 204, the method 200 includes distilling by a distillation chamber 104, the collected urine to concentrate a crude urea content while water from urine is collected as a distillate.
At step 206, the method 200 includes pre-processing by a pre-processing module 106, a biomass by soaking the biomass in water, and freezing the water-soaked biomass.

At step 208, the method 200 includes accepting by a solid-state bio-reactor 108 frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, followed by inoculation and incubation of the soaked biomass using a ureolytic fungus Aspergillus niger to facilitate an in-situ ureolysis, and subsequently a stable-interaction of ammonia with the biomass.

At step 210, the method 200 includes heating by a heat-treatment apparatus 110 the biomass at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

In another embodiment, 0.3% (w/v) urea-containing solution is added to the autoclaved biomass, within a laminar air-flow chamber, wherein a soil-inhabiting, ureolytic, microbial isolate, is prepared in a standard, fungal growth-specific basal medium, containing 1% (w/v) glucose as a carbon source.

In another embodiment, 6.66% (w/v) urea-containing solution is evenly-dispersed throughout the biomass bed (substrate in the bio-reactor) and volume of the solution is pre-calculated on the basis of the maximum water-absorption capacity of a substrate bed.

In another embodiment, the biomass is pre-heated to 100oC, the frozen biomass is loaded evenly onto the sieved trays of a solid-state bio-reactor, and subjected to autoclave.

Figure 3 illustrates a block diagram of a green process-engineered, self-sustainable, fungus assisted, alkali-cum-hydrotropic pre-treatment of lignocellulosic biomass in accordance with an embodiment of the present disclosure. The system includes a 1 st stage of collection of human urine in sterile, capped-plastic containers, and storing them at 4oC. The collected urine is then continuously-distilled to concentrate its crude urea content while the water from urine is collected as the distillate.

In an embodiment, a 2nd stage involves the pre-processing of biomass by soaking it using water distilled out of human urine, and freezing the water-soaked biomass at – 80oC, for 24 h, followed by rapid autoclaving of the frozen, water-soaked biomass.

In an embodiment, a 3rd stage process involves the transfer of frozen, autoclaved, dried biomass to a solid-state reactor, addition of dilute, urine-derived urea solution to soak the biomass, inoculation of the urea-imbibed biomass using Aspergillus niger VVS-15 NKR, and incubation within a solid-state bio-reactor.

In an embodiment, a 4th stage involves the addition of an excess of urine-derived urea solution, obtained by distillation as described in stage 1, to the biomass being incubated in the bio-reactor. An incubation, followed by a temperature-up-shift, catalyses the in-situ, enzymatic-hydrolysis of urea into ammonia.

In an embodiment, a 5 th stage involves the incubation of the bio-reactor at – 80oC, to facilitate the stable-interaction of ammonia with the interior, fibrillar architecture of the biomass.

In an embodiment, a 6 th stage involves heat-treatment of the bio-reactor at 100oC, for 1 h, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre- treatments.
Figure 4 illustrates a schematic representation of the solid-state bio-reactor used to perform the proposed pre-treatment procedure in accordance with an embodiment of the present disclosure. The bio-reactor consists of sieved trays, manufactured out of stainless-steel 316L-grade, over which the urea-imbibed biomass is loaded, and inoculated with the ureolytic fungus. The reactor maintains a sterile, internal environment. Provisions for pH probes, dissolved oxygen probes, addition of fluid samples, and aeration lines are depicted in the figure.
The individual steps of the proposed pre-treatment procedure are explained below in detail.
Step 1: Collection and distillation of human urine to obtain a concentrated, crude urea solution
In this step, human urine, collected in sterile, capped-plastic containers, and stored at 4oC, is subjected to a stage of continuous-distillation in a temperature-programmable distillation unit, operated at 60 – 70oC. 85% (v/v) water from human urine is collected as the distillate while the 15% (v/v) residue is a crude solution of urea, along with other components of human urine. On an average, post distillation, the urea content of the moderately-viscous residue is 1% (w/v) as quantified using high-performance liquid chromatography. The urea-containing residue from the distillation apparatus is stored at – 20oC.
Step 2: Pre-processing of biomass by freezing-and-rapid-autoclaving
In this step, lignocellulosic biomass, with an initial water-absorption capacity of 100% (w/w) is soaked in water until it attains its complete water-absorption capacity (calculated using Equation 1). Packed into sterile, autoclavable, heavy-gauge, poly-propylene bags, the water-soaked biomass is frozen at – 80oC for 24 h. In an autoclave, pre-heated to 100o C, the frozen biomass, loaded evenly onto the sieved trays of a solid-state bio-reactor, is placed and subjected to autoclave following the standard operational procedure. Excess water, post autoclave, is drained through the bottom harvest port of the bio-reactor.
Water-absorption capacity = [((W_2-W_1))/((W_1 ) )]x 100 … Eq. 1
where,
W 1 = initial weight of the biomass (g)
W 2 = final weight of the water-absorbed biomass (g)
Post water-soaking, the cells of the biomass remain swollen, and owing to their increased cell-volume, they are partially disrupted during freezing. When frozen biomass is autoclaved, the rapid thermal expansion disrupts the orientation of its structural pores and opens up the tightly-networked lignocellulosic architecture, moderately-deconstructs the highly-crystalline cellulose, thereby enhancing better accessibility to its interior cellulosic fibers. In the demonstrative process, performed using weedy biomass, the resultant structural damage is visible in the scanning electron micrographs, observed prior to and after the freezing-rapid-autoclave-based pre-processing. A proximate analysis of biomass performed using the NREL-recommended protocol, revealed 30% hemicellulose hydrolysis and solubilization, and 1.2% lignin degradation, and additionally substantiated by variations in characteristic peak intensities of lignin and hemicellulose in the FT-IR spectra. As confirmed using an X-ray diffraction, the percentage of crystallinity had decreased by 10% (calculated using Equation 2).
Percentage of crystallinity = [((I_Cr-I_am))/((I_cr ) )]x 100 … Eq. 2
where,
ICr = spectral peak intensity at 2O - 22.5
Iam = spectral peak intensity at 2O - 18.5
Step 3: Solid-state fermentation for culturing the ureolytic fungi over urea-imbibed biomass
In this step, to the autoclaved biomass within a laminar air-flow chamber, 0.3% (w/v) urea-containing solution is added to the biomass’s complete water-absorption capacity. In the demonstrative process, the weedy biomass’s water-absorption capacity after pre-processing had increased to 150% (w/w). Aspergillus niger VVS-15 NKR, a soil-inhabiting, ureolytic, microbial isolate, is prepared in a standard, fungal growth-specific basal medium, containing 1% (w/v) glucose as the carbon source. The primary culture of A. niger is incubated at 28oC, for 144 h, at a pH of 5 – 5.5, and at 100 – 120 rpm shaking in an orbital-shaker. During the 144-h growth period, post 72 h of incubation and complete exhaustion of glucose, 0.25% (w/v) urea is added to the culture-flask and incubation is prolonged until 144 h. The enzyme activity of urease produced during the primary culture phase is ~12 IU/mL. 60% (v/w) of the starter culture is used to inoculate the urea-imbibed biomass. Following inoculation, pre-calibrated dissolved-oxygen probes and pH probes are introduced into their provisions in the bio-reactor, the aeration lines are set to operate at 1 – 0.5 vvm, and the reactor is incubated at 28oC, for 120 h, until the moisture content of the fermentation bed reduces to minimum. In the demonstrative process, the first 120 h of growth results in a urease activity of 18 IU/g. dry substrates, and the second 120-h growth phase resulted in 22.5 IU/g. dry substrates. The moisture content is periodically assessed visually, and by measuring the overall weight of the bio-reactor. Following complete drying of the substrate bed, a feed solution containing 0.3% (w/v) urea is added to the bed until the biomass’s complete water-absorption capacity is reached. Incubation, following the above-mentioned regime, is continued until 240 h. Dense growth of A. niger over the substrate bed is visually assessed, and the DO probes and aeration lines are removed.
Step 4: Addition of excess human urine-derived urea solution, and in-situ fungal urease-assisted hydrolysis of urea
In this step, a 6.66% (w/v) urea-containing solution is evenly-dispersed throughout the biomass bed (substrate in the bio-reactor). The volume of the solution is pre-calculated on the basis of the maximum water-absorption capacity of the substrate bed. The pH within the bio-reactor is ~6.9 – 7.2 prior to the start of enzyme-assisted ureolysis. The temperature is set to 42oC, which is the optimum temperature for maximum hydrolytic action of urease. The in-situ hydrolysis process is continued for 48 h until the increasing-pH reaches constancy. In the demonstrative process using weedy biomass, the pH reached 8.7 - 9, and remained constant. Post in-situ hydrolysis, the initial resultant products are NH3 and CH2NO2. As CH2 NO2 rapidly-hydrolyses to yield NH3 and H2CO3, they contribute to a steadily-increasing pH. However, towards 48 h, the observed constancy in pH is due to the buffering-action between NH4+, NH3, and HCO3. Additionally, a reduction in the available proportion of free enzyme, catalysing ureolysis, reduces with time, and as it encounters increasing substrate concentration, the reaction rate hits a plateau. This phase marks the end of the in-situ enzymatic-hydrolysis.
Step 5: Ultra-low temperature incubation to facilitate the percolation of ammonia
Post the end-point of in-situ ureolysis, during this step, the reactor is incubated at -80oC for 48 h. Gaseous ammonia, being readily soluble in water, owing to its hydrogen bonding with water molecules, results in a basic solution. A proportion of dissolved ammonia results in the formation of ammonium hydroxide. The solid-state bio-reactor, which maintains sterility throughout the process, is air-tight and the accumulated ammonia is retained within the system. In order to allow the biomass to be steadily-imbued with ammonia to initiate structural changes as in stage 1, and to arrest the on-going enzymatic-ureolysis, the ultra-low temperature incubation is performed.
Step 6: Autoclave-based alkali-cum-hydrotropic pre-treatment of biomass
In this step, post the ultra-low temperature incubation, the reactor is subjected to a heat-treatment at 100oC, for 1 h, at 5 – 6 psi, performed within an autoclave. Portions of the biomass imbued with ammonia, and portions of the biomass, which retain unhydrolyzed urea, together undergo the heat-based pre-treatment, which integrates the operational conditions of ammonia-based alkali pre-treatment and urea-based hydrotropic pre-treatment. The maintenance of temperature at 100oC is to facilitate efficient, urea-based hydrotropic solubilization. Furthermore, since urea thermally-decomposes at ~120 – 130oC, the process is carried out at 100 oC so as to avert the formation of ammelide, cyanuric acid, and isocyanic acid, which are associated with occupational hazards, and several adverse health conditions, such as asphyxiation, topical and ocular trauma, azotemia, injury to the respiratory system, etc. Post the 1-h pre-treatment, the biomass is washed thrice using an equal volume of water, distilled out from urine. The stage of autoclave-based heat treatment doubles as a disinfection procedure as the accumulated mycelia of Aspergillus niger are thermally disinfected during this stage. In the demonstrative process, performed using weedy biomass, a proximate analysis, carried out after the complete, integrated process starting from freezing-rapid-autoclaving to in-situ ureolysis to alkali-cum-hydrotropic pre-treatment revealed: 75% lignin degradation/solubilization; 93% hemicellulose hydrolysis/solubilization; 25% reduction in cellulose-crystallinity. It was observed that undetectable/inconspicuous quantities of process inhibitors are generated. The observations are further substantiated by variations in peak intensities between the characteristic regions of the FT-IR spectra. The weedy biomass had undergone extreme structural distortion, and disruption of its innate fibrillar arrangement, as visualized using SEM.
In order to evaluate the efficiency of the process, the weedy biomass, subjected to alkali-cum-hydrotropic pre-treatment, is saccharified and subsequently fermented to bio-ethanol. Performed with a cellulolytic enzyme-loading of 8 – 10 FPU/g biomass, at a temperature of 50oC, for 48 h, the enzymatic hydrolysis process resulted in 62.8% saccharification (calculated using Equation 3). Fermentation of the saccharified hydrolysate for 30 – 36 h, at 30 - 32oC, using a sugar concentration of 15% (w/v), and 12% (w/v) S. cerevisiae, resulted in a bio-ethanol yield of 0.44 g/g of glucose, elucidating a cellulose bio-conversion of 69.54% (calculated using Equation 4).
Percentage of saccharification = [((sugar content in the hydrolysate)(g)x 0.9)/((quantity of biomass) (g))]x 100
Equation 3
Cellulose bio-conversion = [(Ethanol in the fermentation sample)(g)/(0.51 x (fraction of cellulose(g)x total biomass (g)) x 1.111)]x 100 Equation 4
In an embodiment, the proposed, green, human urine-based, fungus-assisted, alkali-cum-hydrotropic pre-treatment is found to be on par with chemical pre-treatment methods in terms of its biomass pre-treatment efficiency, and it is suitable for wide-applicability in bio- fuel-production plants. The process is readily usable for other sources of lignocellulosic biomass as well. The process’s independence on external sources of expensive chemical agents, and its capability of utilizing water, which is distilled from urine, makes it an attractive, self-sustainable procedure.
In an embodiment, the proposed-process offers the benefit of being economical in terms of the requirement of minimal equipment, investment, labour, and electricity, when the process is being performed with a standard operating procedure in commercial-scale operations. The pre-treatment conditions are optimally-balanced in the procedure so as to circumvent occupational hazards, owing to generation of unwarranted chemical agents, and to avert the formation of saccharification and fermentation inhibitors. A prolonged process duration of ~360 h, distillation of large volumes of human urine to obtain the required-titers of crude urea-containing solution, and variation in process parameters, based on nature, composition, and source of the biomass are aspects, which provide scope for process improvements.
A self-sustainable, green process engineering-incorporated, fungus-assisted pre-treatment procedure, which integrates the aspects of alkali and hydrotropic pre-treatments in a single-pot process, which comprises:
a first-stage process for distilling and concentrating human urine, previously collected and stored at 4o C, to obtain a solution of crude urea.
a second-stage process of soaking lignocellulosic biomass in water, distilled from human urine, and freezing-and-rapid-autoclaving-based pre-processing of biomass to impart structural damage, porosity, and prepare it for uptake of the human-derived pre-treatment agent, urea, during subsequent stages of the procedure.
a third-stage process, wherein after the addition of crude urea solution to the pre-processed biomass, a stipulated percentage of a urea-adapted primary culture for performing a solid-state cultivation of Aspergillus niger VVS-15 NKR is used to inoculate the biomass; attainment of high mycelial density in the solid-state reactor is carried out in two growth phases, possessing identical operational regimes and duration, and in-flow of stipulated urea-concentration-bearing pulse feeds. The primary culture of Aspergillus niger is performed ahead of the biomass-inoculation stage, wherein after attaining a high mycelial density in the submerged culture flask, the fungus is adapted to growth in urea-containing medium.
A fourth-stage process, wherein after completion of the dual-staged growth phase, a stipulated quantity of excess crude urea-bearing solution is introduced into the solid-state cultivation bed, so as to facilitate in-situ, enzymatic ureolysis of the fed urea solution by the fungal urease accumulated within the solid-state cultivation bed.
A fifth-stage process, wherein after completion of in-situ ureolysis, the reactor is incubated at an ultra-low temperature to facilitate the stable-permeation of ureolysis-derived alkali and the interiors of the biomass.
A sixth-stage process, wherein, within an autoclave, the alkali-imbued biomass is heat-treated at 100oC using stipulated process parameters to facilitate an alkali-based, and remnant-urea-based alkali-cum-hydrotropic pre-treatment for efficient de-construction and disintegration of its innate architecture to be amenable to subsequent bio-refinery stages.
The human urine is initially collected in sterile, capped-plastic containers, and stored at 4oC, prior to being subjected to a process of continuous-distillation at 60 – 70oC to separate its 85% (v/v) water content as the distillate, and collect a 15% (v/v) solution as residue, which contains ~1 - 1.2% (w/v) urea.
The lignocellulosic biomass is subjected to a stage of pre-processing by soaking it in water, allowing it to absorb water to its total absorption capacity, transferring it to a solid-state cultivation bio-reactor, incubating
the water-soaked biomass at - 80oC for 24 h, and subjecting it to rapid autoclave in an autoclaving-equipment, pre-heated to 100oC.
Appropriately-pre-processed biomass is washed thrice using equal volumes of urine-derived distilled water, treated with 0.3% (w/v) urine-derived, urea-containing pre-treatment solution, inoculated with 60% (v/w) of primary inoculum of Aspergillus niger VVS-15 NKR, and incubated at 28oC for 120 h. A second sub-stage of the process is performed for another 120-h using identical process conditions and duration with the addition of another 0.3% (w/v) urea-bearing solution to build mycelial density.
The primary culture of Aspergillus niger VVS-15 NKR is prepared in conical flasks using a spore suspension of 3.2 x 10 12 spores/mL in a basal media, containing 1% (w/v) glucose as the carbon source, incubated at 28o C, for 72 h; after complete exhaustion of glucose, 0.25% urea is added, and the incubation is carried out until 150 h to accustom the fungus to a urea-containing environment.
A 6.66% (w/v) urine-derived, urea-containing solution is introduced into the biomass, allowed to be imbibed to the biomass’s complete absorption-capacity, to facilitate in-situ, fungal enzyme-catalyzed ureolysis at 42oC, for 48 h, until the increasing trend of pH attains constancy, signifying the end-point of ureolysis.
Post in-situ ureolysis, the bio-reactor is incubated at – 80oC, for 48 h, to facilitate the steady-permeation of the in-situ ureolysis-resultant ammonia to the biomass’s interior architecture.
Post the ultra-low temperature incubation, the bio-reactor, containing the biomass, is incubated at 100oC for 1 h, at 5 – 6 psi, within an autoclave, to simultaneously facilitate ammonia-based alkali pre-treatment and remnant, unhydrolyzed urea-based hydrotropic pre-treatment.
In a demonstrative operation using weedy biomass, performed using the fungus-assisted, human urine-based, alkali-cum-hydrotropic pre-treatment process as claimed in claim 1, a 62.8% saccharification, and bio-ethanol yield of 0.44 g/g glucose, indicating a cellulose bio-conversion of 69.54%, are recorded.
The self-sustainable and economical process, employs a waste-stream-derived pre-treatment agent, moderate operational regimes, with a maximum temperature of 121oC, thereby averting the possibility of the formation of bio-refinery process inhibitors, and the generation of toxic, occupational hazard-posing by-products.
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.

Benefits, 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 human urine-based, fungus-assisted, alkali-cum-hydrotropic pre-treatment of lignocellulosic biomass, wherein a system (100) comprises of:
a urine collection apparatus (102) for collecting human urine;
a distillation chamber (104) for continuously distilling the collected urine to concentrate a crude urea content while water from urine is collected as a distillate;
a pre-processing module (106) connected to the distillation chamber (104) for pre-processing a biomass by soaking the biomass in water, and freezing the water-soaked biomass;
a solid-state bio-reactor (108) attached to the pre-processing module (106) for accepting frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, followed by inoculation and incubation of the soaked biomass with a ureolytic fungus to later facilitate the stable-interaction of ammonia with the biomass; and
a heat-treatment apparatus (110) connected to the bio-reactor (108) for heating the biomass at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

2. The system as claimed in claim 1, wherein the collected urine is stored at a temperature of 3-5oC.

3. The system as claimed in claim 1, wherein 85% (v/v) water from human urine is collected as the distillate while the 15% (v/v) residue is a crude solution of urea, along with other components of human urine, wherein post distillation, the urea content of the moderately-viscous residue is 1% (w/v).

4. The system as claimed in claim 1, wherein the soaked biomass is stored at – 80oC, for 24 hours, followed by rapid autoclaving of the frozen, water-soaked biomass.

5. The system as claimed in claim 1, wherein the incubation is followed by a temperature-up-shift, which catalyses the in-situ, enzymatic-hydrolysis of urea into ammonia.

6. The system as claimed in claim 1, wherein the bio-reactor (108) consists of sieved trays, over which the urea-imbibed biomass is loaded, and inoculated with a ureolytic fungus.

7. A human urine-based, fungus-assisted, alkali-cum-hydrotropic pre-treatment of lignocellulosic biomass, wherein a method (200) comprises of:
collecting by a urine collection apparatus (102), distillation of human urine in a distillation chamber (104), the collected urine is subjected to concentrate its crude urea content while water from urine is collected as a distillate;
pre-processing by a pre-processing module (106), wherein the biomass after soaking in water, is subjected to deep freezing;
introduction into a solid-state bio-reactor (108), the frozen, autoclaved, dried biomass, wherein the dilute, urine-derived urea solution is added to soak the biomass, followed by inoculation and incubation of the soaked biomass using a ureolytic fungus to later facilitate the stable-interaction of ammonia with the biomass; and
heating the biomass using a heat-treatment apparatus (110) at a defined temperature of 80-120oC, for 0.5-1.5 hours, at 5 – 6 psi, in an autoclave, to simultaneously carry out alkali and hydrotropic pre-treatments.

8. The method as claimed in claim 7, wherein a 0.3% (w/v) urea-containing solution is added to the autoclaved biomass within a laminar air-flow chamber, wherein a soil-inhabiting, ureolytic, microbial isolate, is prepared in a standard, fungal growth-specific basal medium, containing 1% (w/v) glucose as a carbon source.

9. The method as claimed in claim 7, wherein a 6.66% (w/v) urea-containing solution is evenly-dispersed throughout the biomass bed (substrate in the bio-reactor) and the volume of the solution is pre-calculated on the basis of the maximum water-absorption capacity of the substrate bed.

10. The method as claimed in claim 7, wherein during an initial stage, the frozen biomass is dispersed onto the sieved trays` of the bio-reactor, loaded into a pre-heated (100oC) autoclave and subjected to autoclave.