Description:FIELD OF THE INVENTION

The present disclosure relates to the field of biotechnology, specifically to systems and processes for fungal cultivation and enzyme production. More particularly, it pertains to a system and method for prolonged, quasi-continuous hydroponic cultivation of fungi for the production of cellulolytic enzyme complexes and their periodic in-situ harvesting. The invention is relevant to applications in industrial biotechnology, including biofuel production, bioremediation, and other sectors requiring sustainable and efficient production of cellulolytic enzymes.

BACKGROUND OF THE INVENTION

The production of cellulolytic enzyme complexes is a critical and cost-intensive step in second-generation (2G) bio-ethanol production, accounting for approximately 50% of the overall production cost. Existing bio-refinery models face challenges in balancing operational costs and enzyme productivity. Both solid-state fermentation (SSF) and submerged fermentation (SMF) methods have been employed for cellulase production. SSF, which mimics the natural fungal growth process, typically yields higher enzyme activity but poses challenges in maintaining uniform growth, moisture regulation, and prolonging high-yielding processes without over-drying or over-flooding the substrate bed.
Current methods often involve batch-based processes requiring repeated sterilization, adding to operational costs and impacting production efficiency. Hydroponic techniques, traditionally used for plant cultivation and wastewater treatment, offer an innovative solution for enzyme production. The disclosed invention leverages hydroponic principles in a custom-designed reactor to enable quasi-continuous fungal growth on mycelial scaffolds excised from spent mushroom substrates.
Unlike prior art, such as US20150004670A1, which employs a trickle-bed reactor for fungal growth under pyridoxine limitation, or other systems reliant on batch processes with significant manual intervention, the present invention integrates hydroponics for controlled fungal growth, periodic enzyme harvests, and substrate re-feeding. This quasi-continuous process eliminates the need for enzyme recovery via koji harvesting, simplifies operation, and enables scalable enzyme production.
By employing mature mycelial scaffolds from spent mushroom cultivation bags as reusable growth substrates, the invention enhances sustainability and reduces substrate costs. The reactor system incorporates mechanisms for precise temperature and humidity control, nutrient delivery, and in-situ enzyme harvesting. This approach ensures a cost-effective, scalable, and extended enzyme production process, addressing the existing limitations of SSF and SMF methods while meeting the growing demand for cellulases in bio-ethanol and related industries.
In view of the foregoing discussion, it is portrayed that there is a need to have asystem for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest.

SUMMARY OF THE INVENTION

The present disclosure seeks to provide asystem for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest.The present disclosure describes a first-of-its-kind of fungal hydroponic process, performed within a custom-designed reactor, for the quasi-continuous production and periodical in-situ harvest of cellulolytic enzyme complexes in a time-extended and economical procedure, utilizing mycelia-rich spent mushroom substrate as the whole-mycelia-bearing scaffolds. While the mycelial scaffold-bearing trays serve as the cellulase-producing and accumulating region, a tray beneath the scaffold serves as the culture medium-holding tank, which is replenished every 27 days from a master culture medium reservoir with dedicated and strategically designed lines appropriately filling each tank only until the stipulated levels. Since the process is a hydroponic cultivation, jute wicks from the scaffold trays, dipping into the medium tanks, using capillary uptake, provide sufficient medium distribution throughout the mycelial scaffold without overflooding them. The feeding of 25% (v/v) of cellulose-rich substrate is performed using equally-distributing silicone tubing lines introduced into each of the scaffold-holding trays. While the relative humidity (RH) measurement using RH probes and its maintenance (at 70–80%) is performed using timed atomiser units spraying autoclaved water every 1.5-h, the process of incubation is performed at 28oC for a 9-day batch, and is then continued forward with a fresh feeding cycle. Owing to its strategic design, sufficient air transfer happens during the cultivation phase resulting in OUR – 0.14 g/h, OTR – 10–12 g/h and KLa – 60.7/h. Post the completion of each batch, autoclaved water is introduced through dedicated lines into the scaffolds, followed by in-situ enzyme extraction performed using a spring-loaded presser located at the top of the reactor. Enzyme collected in the harvest tanks, through specific lines, get accumulated into a master enzyme-harvest tank. The demonstrative process performed in a prototype of the reactor indicates that the process can be consistently performed for 3 continuous cycles or ~27 days. The disclosure offers a sustainable solution to the requirement of higher cellulase yields for on-site cellulase production in bio-ethanol-producing second-generation bio-refineries.

In an embodiment,a system for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest is disclosed. The system includes a hydroponic reactor configured to provide a controlled environment for cultivation of a fungus.
The system further includes a plurality of scaffold-holding trays horizontally arranged within the reactor, each tray configured to hold a mycelial scaffold, each scaffold comprising an excised portion of a spent mushroom substrate bag, wherein the excised portion is densely populated with mycelia of the mushroom.
The system further includes a culture medium delivery system operatively coupled to the reactor, comprising a master medium reservoirfor bulk storage of culture medium, a plurality of scaffold-specific medium-storage tanksfluidly connected to the master medium reservoir via sequential medium filling lines, wherein each medium-storage tank is positioned to supply culture medium to a respective scaffold-holding tray, each tank associated with a respective scaffold, a plurality of jute wicks extending from each scaffold-holding tray and dipping into a corresponding culture medium-storage tank below, configured for capillary action to transport culture medium from the tank to the mycelial scaffold over approximately 75 minutes, a plurality of fluid conduits configured to deliver culture medium from the master reservoir to the scaffold-specific storage tanks, and a plurality of overflow indication ports associated with each storage tankto indicate maximum fill level and prevent overfilling.
The system further includes a substrate delivery system including a cellulose-loading channel positioned above the scaffold-holding trays configured to deliver exogenous cellulose-rich substrate to the mycelial scaffolds.
The system further includes a temperature control system configured to maintain 28±3oC temperature within the reactor comprising a water-filled jacket disposed at the back surface of the reactor for thermal insulation and temperature regulation, a heated platform supporting the reactorfrom below to provide basal heating, a first temperature sensor positioned within the reactor to monitor internal temperature, and a second temperature sensor positioned within the water-filled jacket to monitor internal temperature.
The system further includes a humidity control system configured to maintain 70–80% humidity within the reactor comprising an atomizer unit disposed within the reactor vessel to introduce autoclaved water as moisture into the reactor, and a plurality of relative humidity sensors positioned within the reactorfor monitoring humidity levels.
The system further includes an enzyme harvest system comprising a plurality of fluid conduits coupled to an autoclaved water reservoir and autoclaved water introduction line configured to deliver autoclaved water to the mycelial scaffolds for enzyme extraction, a plurality of enzyme-harvesting tankspositioned below the scaffold-holding trays to collect enzyme-containing solution, each tank associated with a respective scaffold, a master enzyme-harvesting reservoirfor accumulating harvested enzyme solution from the enzyme-harvesting tanks via an enzyme harvesting line, and a plurality of spring-loaded pressers, each positioned above a respective scaffold-holding tray and actuatable via a spring-loaded press button to gently squeeze the mycelial scaffolds and enhance enzyme extraction.

In another embodiment, a process for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest is disclosed. The process includes preparing mycelium-rich scaffolds from spent mushroom cultivation substrate upon excising mycelia-rich regions from spent mushroom cultivation bags of Pleurotus ostreatus(spawns originally purchased from ICAR-Indian Institute of Horticultural Research).For certain other demonstrations, the mycelium-rich scaffolds were derived from either of the following fungal strains:Aspergillus nigerMTCC 4325; Trichoderma harzianumMTCC 795; Trichoderma reeseiMTCC 3192.
The process further includes aseptically loading the mycelium-rich scaffolds into scaffold-holding trays within a hydroponic reactor, wherein the scaffold-holding trays comprise wicks for capillary uptake of culture medium.
The process further includes supplying a culture medium to the scaffold-holding trays via capillary action from the wicks, wherein the culture medium contains 1.73g/L di-potassium hydrogen phosphate, 0.68g/L potassium di-hydrogen phosphate, 1g/L ammonium nitrate, 0.1g/L magnesium sulfate hepta-hydrate, 0.02g/L calcium chloride di-hydrate, 4g/L sodium chloride, and 0.03g/L ferrous sulfate heptahydrate.
The process further includes supplying a cellulose substrate slurry to the scaffold-holding trays, wherein the cellulose substrate slurry is prepared from pulverized and alkali-pretreated wheat straw, further comprising soaking and autoclaving the pre-treated wheat straw to form the slurry.
The process further includes incubating the mycelium-rich scaffolds in the hydroponic reactor to produce cellulolytic enzyme complexes, wherein during the mycelium-rich scaffolds incubation, 28°C temperature and 70% and 80% relative humidity is maintained within the hydroponic reactor, wherein autoclaved water is introduced by the atomizer approximately every 1.5 hours, wherein the incubation period is approximately 9 to 12 days, wherein humidity is maintained by introducing autoclaved water into the reactor environment using an atomizer at intervals, wherein above 86% dissolved oxygen levels is maintained within the hydroponic reactor.
The process further includes periodically harvesting the cellulolytic enzyme complexes by draining the culture medium from the scaffold-holding trays after an incubation period and washing the mycelium-rich scaffolds with autoclaved water thereby allowing the autoclaved water to soak within the scaffolds for a period of time followed by pressing the mycelium-rich scaffolds to extract the cellulolytic enzyme complexes into an enzyme-harvesting tank; and collecting the extracted cellulolytic enzyme complexes, wherein the step of washing, soaking, and pressing is repeated at least two times, wherein the soaking period with autoclaved water is approximately 1.5 hours.

An object of the present disclosure is to provide a quasi-continuous system for hydroponic cultivation of fungi for the production and periodical harvest of cellulolytic enzyme complexes, in-situ, with minimal manual intervention.
Another object of the present disclosure is to devise a cost-effective and sustainable method for cellulolytic enzyme production in a hydroponic mode for prolonged operation until as long as ~27 days or 3 operational batches with periodical enzyme harvests at the end of every ~9 days.
Yet another object of the present invention is to deliver an expeditious and cost-effectivemethod for cellulolytic enzyme production in a hydroponic mode for prolonged operation with periodical enzyme harvests.
An object of the invention includes performing a hydroponic SSF for cellulase complex production with appropriate moisture-regulation (using culture medium), time-extended high-yielding operation, and periodical enzyme harvests.
Another object of the invention includes the utilization of the enzyme-secreting, substrate mycelia-rich spent mushroom substrate as whole-cell/whole-mycelial scaffolds for enzyme production within a custom-built hydroponic reactor.
Another object of the invention includes designing a customized hydroponic bio-reactor for a time-extended, quasi-continuous enzyme production and periodical harvests.
Another object of the invention includes, within the reactor, providing uninterrupted culture medium-supplementation using a master medium reservoir (with connecting lines) and mycelial scaffold-specific medium-storage tanks beneath each of the hydroponic trays, which are sequentially filled until a stipulated level, where overflow is indicated by a specific overflow-indication port.
An object of the invention involves, within the reactor, designing dedicated lines for uniformly providing exogenous cellulose-rich substrate, individually, to the hydroponic cultivation scaffolds in each tray.
Another object of the invention involves, at the back of the reactor, providing a water-filling jacket to maintain the temperature of the hydroponic process by placing the entire reactor on a heated platform.
Yet another objective of the invention involves, within the reactor and the water-filled jacket, introducing a thermometer each, to measure and maintain the temperature of the incubation accordingly.
Another object of the invention involves, within the reactor, providing an electrically-powered, timer-based atomiser unit, within which autoclaved water would flow to maintain the relative humidity throughout the inner environment of the reactor.
Another object of the invention involves, within the reactor’s environment and within the scaffold-holding tray, introducing a relative humidity (RH)-sensing probe each to measure the RH of the individual regions.
Another objective of the invention involves the provision of 9 cotton plugs as to facilitate efficient and sufficient air exchange with the growing regions in the scaffold-holding tray.
An object of the invention involves, within the reactor, providing dedicated lines for autoclaved water flow to the hydroponic trays/mycelial scaffolds to wet the scaffolds and draw enzymes in the enzyme-harvesting tanks, and subsequently to a master enzyme-harvesting reservoir.
Yet another objective of the inventio involves, within the reactor, above each of the hydroponic trays, providing a spring-loaded presser to gently squeeze the accumulated enzymes, in situ, to be collected within the enzyme collection tanks.
According to yet another objective of the invention, the harvested enzyme complexes would bear the following enzyme activities as estimated using the standard IUPAC Ghose- and NREL-prescribed cellulolytic enzyme assay protocol for the demonstrative operation: (i) from the P. ostreatus’sharvest: exoglucanase – 5.91±1 IU/g.ds; endoglucanase: 14±2.5 IU/g.ds; beta-glucosidase - 180±30 IU/g.ds. (ii) from the A. niger’sharvest: exoglucanase – 4.5±0.5 IU/g.ds; endoglucanase: 10±1 IU/g.ds; beta-glucosidase - 100±36 IU/g.ds. (iii) fromT. harzianum’sharvest: exoglucanase – 4±0.2 IU/g.ds; endoglucanase: 12±1.5 IU/g.ds; beta-glucosidase - 120±23. (iv) fromTrichoderma sp.’s harvest: exoglucanase – 4.1±1 IU/g.ds; endoglucanase: 15±3 IU/g.ds; beta-glucosidase - 90±27 IU/g.ds. In all the above cases, IU/g.ds. refer to the enzyme activity per gram of the dry solids obtained.
In yet another objective of the invention, the oxygen transfer rate (OTR), oxygen uptake rate (OUR) and mass-transfer co-efficient - KLa for the demonstrative process while using the custom-designed hydroponic reactor are as follows: OUR – 0.14±0.02 g/h; OTR – 10.5 g/h considering the air available in the reactor at each point of time, the density of O2 and its percentage in ambient air; KLa – 60.7/h.

To further clarify the advantages and features of the present disclosure, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are 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 in 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 concerning the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Figure 1 illustrates a block diagram of a system for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest in accordance with an embodiment of the present disclosure;
Figure 2 illustrates a flow chart of a process for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest in accordance with an embodiment of the present disclosure;
Figure 3 illustrates a flow chart with the key variables required to perform the quasi-continuous hydroponic process for the production and periodical harvest of the produced cellulolytic enzyme complex;
Figure 4 illustrates a schematic of the custom-built hydroponic reactor, along with the: (i) mycelial scaffold-loading tray; (ii) culture medium tanks underneath each of the scaffold-holding trays; (iii) spring-loaded presser above each of the scaffold-holding trays; (iv) connecting lines to fill culture medium from its reservoir into each of the medium-holding tanks; (v) connecting lines to deliver autoclaved water from its reservoir into each of the scaffold-holding trays; (vi) individual enzyme harvest ports connected to a master enzyme-harvesting line collecting the enzymes into a reservoir; (vii) thermometers for temperature regulation; (viii) humidity-measurement probes for estimating humidity throughout the course of the operation; (ix) timer-based atomiser lines, within the reactor, to periodically introduce atomised and autoclaved water to maintain the stipulated humidity;
Figure 5 illustrates a schematic of the cellulose-bearing, uniform substrate-loading silicone tubing lines into each of the scaffold-bearing trays;
Figure 6 illustrates an engineering drawing of the outer vessel of the hydroponic reactor along with its hinged-door;
Figure 7 illustrates an engineering drawing of the inner scaffold-bearing tray, medium-holding tank, presser for enzyme extraction; it also presents, the connecting lines/piping for loading the culture medium from its master reservoir into individual culture-medium loading tanks, located beneath each of the scaffold-loading trays;
Figure 8 illustrates the individual cellulose slurry-feeding lines; and
Figure 9 illustrates the connecting lines for the introduction of autoclaved water into each of the scaffold-holding trays.

Further, skilled artisans will appreciate those 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 the benefit of the description herein.

DETAILED DESCRIPTION:

To promote 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 concerning the accompanying drawings.

Referring to Figure 1, a block diagram of a system for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest is illustrated in accordance with an embodiment of the present disclosure. The system (200) includes a hydroponic reactor (202) configured to provide a controlled environment for cultivation of a fungus.

In an embodiment, a plurality of scaffold-holding trays (204) are horizontally arranged within the reactor, each tray configured to hold a mycelial scaffold, each scaffold comprising an excised portion of a spent mushroom substrate bag, wherein the excised portion is densely populated with mycelia of the mushroom.

In an embodiment, a culture medium delivery system (206) is operatively coupled to the reactor, comprising a master medium reservoir, a plurality of scaffold-specific medium-storage tanksfluidly connected to the master medium reservoir via sequential medium filling lines, wherein each medium-storage tank is positioned to supply culture medium to a respective scaffold-holding tray, each tank associated with a respective scaffold, a plurality of jute wicks extending from each scaffold-holding tray and dipping into a corresponding culture medium-storage tank below, configured for capillary action to transport culture medium from the tank to the mycelial scaffold over approximately 75 minutes, a plurality of fluid conduits configured to deliver culture medium from the master reservoir to the scaffold-specific storage tanks, and a plurality of overflow indication ports associated with each storage tankto indicate maximum fill level and prevent overfilling.

In an embodiment, a substrate delivery system (208)including a cellulose-loading channel positioned above the scaffold-holding trays is configured to deliver exogenous cellulose-rich substrate to the mycelial scaffolds.

In an embodiment, a temperature control system (210) is configured to maintain 28±3oC temperature within the reactor comprising a water-filled jacket disposed at the back surface of the reactor for thermal insulation and temperature regulation, a heated platform supporting the reactorfrom below to provide basal heating, a first temperature sensor positioned within the reactor to monitor internal temperature, and a second temperature sensor positioned within the water-filled jacket to monitor internal temperature.

In an embodiment, a humidity control system (212) is configured to maintain 70–80% humidity within the reactor comprising an atomizer unit disposed within the reactor vessel to introduce autoclaved water as moisture into the reactor, and a plurality of relative humidity sensors positioned within the reactorfor monitoring humidity levels.

In an embodiment, an enzyme harvest system (214) comprising a plurality of fluid conduits coupled to an autoclaved water reservoir and autoclaved water introduction line configured to deliver autoclaved water to the mycelial scaffolds for enzyme extraction, a plurality of enzyme-harvesting tankspositioned below the scaffold-holding trays to collect enzyme-containing solution, each tank associated with a respective scaffold, a master enzyme-harvesting reservoirfor accumulating harvested enzyme solution from the enzyme-harvesting tanks via an enzyme harvesting line, and a plurality of spring-loaded pressers, each positioned above a respective scaffold-holding tray and actuatable via a spring-loaded press button to gently squeeze the mycelial scaffolds and enhance enzyme extraction.

In another embodiment, the enzyme-secreting fungus is selected from the group consisting of Trichoderma, Aspergillus, and Penicillium, wherein the enzyme harvesting system further comprises a plurality of valves for controlling the flow of water and enzymes, and a pH probe (Figure 2: pH probe) positioned within the reactor for monitoring pH levels.

In a further embodiment, the exogenous cellulose-rich substrate is agricultural waste selected from corn stover, wheat straw, or rice husks, and the jute wicks are configured to release culture medium to the scaffolds providing sustained moisture for approximately 75 minutes via capillary action.

Yet, in another embodiment, the mycelial scaffolds are derived from spent mushroom substrate bags densely colonized by Pleurotus ostreatus mycelium, wherein mycelial scaffold is arranged in trays and supported by a Trays' mounting rod.

Yet, in a further embodiment, the hydroponic reactor (202) further comprising a nutrient delivery system configured to provide nutrients to the mycelia-rich scaffolds, and a gas exchange system including an air inlet and outlet configured to provide oxygen and remove carbon dioxide to maintain aerobic conditions within the reactor.

In one of the above embodiments, the reactor is thermally insulated, wherein the reactor further comprises a water jacket surrounding the reactor for maintaining a constant temperature preferably at 28±3oC, and a transparent window in the reactor wall deployed for visual observation of the mycelial scaffolds.

The system (200) further comprising a first dissolved oxygen (DO) probe (216) disposed within the interior chamber of the reactor vessel, a second DO probe (218) disposed within one of the scaffold-holding trays (204), and a controller (220)operatively connected to the temperature control system and humidity control system, configured to maintain the temperature at 28±3°C and humidity at 70–80% respectively, based on signals from the temperature sensors and relative humidity sensors, and configured to monitor the DO levels measured by the first and second DO probes and to adjust operating parameters of the system to maintain a DO concentration of more than 90% within the reactor vessel, wherein the controller is configured to adjust the aeration rate to maintain the desired DO concentration.

The system (200) further comprising a timer-based controller operatively connected to the atomizer unit, configured to actuate the atomizer to introduce autoclaved water into the reactor vessel at predetermined intervals, approximately every 1.5 hours.

The system (200) further comprising a plurality of feeding lines (222) connected to the reservoir and to each of the scaffold-holding trays (204), and a plurality of valves (224)associated with each of the feeding lines and the cellulose-loading channel for controlling the flow of cellulosic substrate as a slurry-like consistency to each tray, wherein the cellulosic substrate is alkali pre-treated and neutralized pulverized wheat straw having a lignin content of less than 10% w/w.

Figure 2 illustrates a flow chart of a process for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest in accordance with an embodiment of the present disclosure.At step (302), process (300) includespreparing mycelium-rich scaffolds from spent mushroom cultivation substrate upon excising mycelia-rich regions from spent mushroom cultivation bags of Pleurotus ostreatus, wherein the mycelium-rich scaffolds are derived from fungal strains selected from the group consisting of Aspergillus niger, Trichoderma harzianum, and Trichoderma reesei.
At step (304), process (300) includes aseptically loading the mycelium-rich scaffolds into scaffold-holding trays within a hydroponic reactor, wherein the scaffold-holding trays comprise wicks for capillary uptake of culture medium.
At step (306), process (300) includes supplying a culture medium to the scaffold-holding trays via capillary action from the wicks, wherein the culture medium contains 1.73g/L di-potassium hydrogen phosphate, 0.68g/L potassium di-hydrogen phosphate, 1g/L ammonium nitrate, 0.1g/L magnesium sulfate hepta-hydrate, 0.02g/L calcium chloride di-hydrate, 4g/L sodium chloride, and 0.03g/L ferrous sulfate heptahydrate.
At step (308), process (300) includes supplying a cellulose substrate slurry to the scaffold-holding trays, wherein the cellulose substrate slurry is prepared from pulverized and alkali-pretreated wheat straw, further comprising soaking and autoclaving the pre-treated wheat straw to form the slurry.
At step (310), process (300) includes incubating the mycelium-rich scaffolds in the hydroponic reactor to produce cellulolytic enzyme complexes, wherein during the mycelium-rich scaffolds incubation, 28°C temperature and 70% and 80% relative humidity is maintained within the hydroponic reactor, wherein autoclaved water is introduced by the atomizer approximately every 1.5 hours, wherein the incubation period is approximately 9 to 12 days, wherein humidity is maintained by introducing autoclaved water into the reactor environment using an atomizer at intervals, wherein above 86% dissolved oxygen levels is maintained within the hydroponic reactor.
At step (312), process (300) includes periodically harvesting the cellulolytic enzyme complexes by draining the culture medium from the scaffold-holding trays after an incubation period and washing the mycelium-rich scaffolds with autoclaved water thereby allowing the autoclaved water to soak within the scaffolds for a period of time followed by pressing the mycelium-rich scaffolds to extract the cellulolytic enzyme complexes into an enzyme-harvesting tank; and collecting the extracted cellulolytic enzyme complexes, wherein the step of washing, soaking, and pressing is repeated at least two times, wherein the soaking period with autoclaved water is approximately 1.5 hours.

The process for a prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its (enzyme’s) in-situ periodical harvest throughout the cultivation phase, wherein the process comprises:
a. performing a hydroponic SSF for cellulase complex production with appropriate moisture-regulation (using culture medium), time-extended high-yielding operation, and periodical enzyme harvests;
b. the utilization of the enzyme-secreting, substrate mycelia-rich spent mushroom substrate as whole-cell/whole-mycelial scaffolds for enzyme production within a custom-built hydroponic reactor;
c. designing a customized hydroponic bio-reactor for a time-extended, quasi-continuous enzyme production and periodical harvests with minimal manual intervention;
d. within the reactor, providing uninterrupted culture medium-supplementation using a master medium reservoir (with connecting lines) and mycelial scaffold-specific medium-storage tanks beneath each of the hydroponic trays, which are sequentially filled until a stipulated level, where overflow is indicated by a specific overflow-indication port;
e. within the reactor, designing dedicated lines for uniformly providing exogenous cellulose-rich substrate, individually, to the hydroponic cultivation scaffolds in each tray;
f. at the back of the reactor, providing a water-filling jacket to maintain the temperature of the hydroponic process by placing the entire reactor on a heated platform;
g. within the reactor and the water-filled jacket, introducing a thermometer each, to measure and maintain the temperature of the incubation accordingly;
h. within the reactor, providing an electrically-powered, timer-based atomiser unit, within which autoclaved water would flow to maintain the relative humidity throughout the inner environment of the reactor;
i. within the reactor’s environment and within the scaffold-holding tray, introducing a relative humidity (RH)-sensing probe each to measure the RH of the individual regions;
j. within the reactor, providing dedicated lines for autoclaved water flow to the hydroponic trays/mycelial scaffolds to wet the scaffolds and draw enzymes in the enzyme-harvesting tanks, and subsequently to a master enzyme-harvesting reservoir;
k. within the reactor, above each of the hydroponic trays, providing a spring-loaded presser to gently squeeze the accumulated enzymes to be collected, in-situ, within the enzyme-collection tanks.

The preparation of the mycelia-rich scaffold comprises:
a. using a spent mushroom substrate bag to derive the mycelia-rich scaffold. For the demonstrative process, the spent substrate bag of an oyster mushroom, Pleurotus ostreatus(spawns originally purchased from ICAR-Indian Institute of Horticultural Research), which is being cultivated in a commercially operating mushroom farm in Pondicherry, India, was used;
b. utilizing the hanging bag cultivation strategy for mushrooms, which is well-reported in the prior-art. In the demonstrative process, 4 kg of water-washed rice straw was used for mushroom cultivation for a period of 4 months, with 4 fruiting bodies’ (mushroom) harvesting stages. Post completion of the cultivation process, the cellulose content of the straw was negligible and was found to be unsuitable for promoting any further growth as assessed using a biomass’s proximate analyses procedure; the cultivation bag was densely filled with the mushroom’s mycelia, which are, typically, as long as 3–5 m;
c. exploiting an excised portion of the spent substrate bag, packed with dense, cellulase-producing substrate mycelium as whole-mycelia for producing cellulases in a hydroponic cultivation process within a custom-designed reactor;
d. the excision of ~24 cm (L) x 24 cm (B) x 3 cm (W) of mycelia-rich regions from spent mushroom cultivation bags, which will then be serving as whole-mycelia-bearing scaffolds; the aseptic extraction process will have to be performed within a laminar air-flow chamber using gloved hands;
e. placing the excised mycelial scaffolds within the scaffold-holding tray(s) of the hydroponic reactor;
f. prior to commencement of the aseptic transfer, positioning the cotton plugs in their respective positions at the top of the hydroponic reactor and autoclaving. A compositional analysis of the substrate material in the excised region from the mushroom bags showed negligible or complete absence of fungal growth-promoting cellulose or hemicellulose.

The pre-preparation of the custom-designed hydroponic bio-reactor comprises:
a. subjecting the master reservoirs of the: typical fungal growth-specific minimal medium, autoclaved water, cellulosic substrate-feeding tank, and enzyme-collection tank to autoclaving after appropriately sealing and closing their valves;
b. subjecting the hydroponic reactor, with all its valves and line-openings appropriately sealed, to a stage of autoclaving. Post the stage of autoclaving, within a laminar air-flow hood, the appropriate reservoir lines are connected to the hydroponic bio-reactor.
and
c. preparation of the minimal medium used to promote fungal growth during demonstrative trials, which comprises in (g/L): di-potassium hydrogen phosphate – 1.73, potassium di-hydrogen phosphate – 0.68, ammonium nitrate – 1, magnesium sulfate hepta-hydrate – 0.1, calcium chloride di-hydrate – 0.02, sodium chloride – 4, ferrous sulfate heptahydrate – 0.03;
d. preparation of the cellulose-rich slurry using pulverized and alkali-pre-treated wheat straw; the pre-treatment can be performed using any of the process parameters as known and established well in the prior-art; the average particle size of the fine, pulverized biomass was kept as low as 300 µm. Post pre-treatment, neutralization and drying, a mixture containing biomass and distilled water in the ratio (1:2) was soaked for 3-h at a temperature of 60oC and subjected to a stage of autoclave to create the feed slurry for the demonstrative process.

The strategically designed culture medium-filling process comprises:
a. uniform culture medium supplementation throughout the scaffold-holding region, wherein, each of the scaffold-holding trays has 6–7 cm-long jute wicks to perform the capillary-uptake of medium from the medium-holding tanks;
b. designing each of the culture medium-holding trays to be roughly able to accommodate ~1.7 L of the minimal medium;
c. designing the sides and bottom of the scaffold-holding trays with meshes, which permit sufficient oxygen transfer to the growing scaffolds from all sides;
d. filling up the water-jacket located at the back surface of the reactor is with water until it flows out of its water-inlet valve indicating complete filling; post loading the scaffolds into their respective trays, the water in the jacket would serve to maintain the temperature uniform throughout the reactor during the hydroponic cultivation process.

The process for setting up the hydroponic reactor prior to each quasi-steady-state operation comprises:
a. opening the culture medium reservoir’s master valve, and filling up, first, the top-most tray until it reaches the exit/overflow valve of tray 1; next, the second tray is filled with the overflow of tray 1, followed by a similar mechanism to fill up the tray 3. After filling to its optimum in tray 3, medium flows out of the master overflow port located outside the enclosure of the reactor, indicating complete filling of all the medium trays; the similar construction can be extended to any number of medium trays, where gravity functions to fill the trays until the stipulated levels. The master reservoir and the conclusive overflow valves are closed after the phase of medium filling completes.

The commencement of substrate feeding and the quasi-continuous hydroponic cultivation process comprises:
a. feeding each of the scaffold-holding trays, the cellulose-rich substrate slurry’s master reservoir is opened, and based on its flow rate, calculated manually, an equal volume of the slurry is allowed to fill up each of the scaffold-holding trays to a final feed volume of ~25% (v/v) within the scaffold-holding tray; then, the valves of the individual feeding lines are then closed.
b. Shifting the reactor to be placed atop a hot-plate/heated platform to maintain the reactor’s temperature at 28oC throughout the ~9–12 days of cultivation; the shifting is performed after a visual inspection of the trays, the entire reactor, within the laminar air-flow chamber;
and
c. maintenance of the stipulated humidity, when the incubation begins, using the electrically-powered, programmed, timer-based atomiser is as it introduces autoclaved water every 1.5 hours into the reactor’s environment;
d. maintaining the DO using two DO probes, one within the bio-reactor’s environment and the other within the scaffold aid to appropriately monitor and maintain the DO% concentration in the environment, at a value of more than 86%;
e. calibrating the pH probes using any of the methods as well-reported in prior-art.

The process of optimum moisture-maintenance throughout the scaffold region using culture medium comprises:
a. uptake of culture medium from the jute wicks through capillary action, wherein, as the mycelia and the scaffold material dry up, the medium is transferred to the scaffold from the jute wicks, fresh medium from the tank is up-taken by the jute wicks. Such a wick-based transfer allows for appropriate moisture maintenance without overflooding the scaffold region.
b. replenishment of the culture-medium in the medium tanks with fresh medium from the master reservoir, every 3 cycles or ~27 days, as standardized during the demonstrative run;
c. maintenance of relative humidity (RH) within the reactor and within the scaffold between 70–80%, and monitoring it using two RH-sensing probes provided within the reactor throughout the duration of cultivation.
d. periodically inspecting the scaffold trays through the transparent window on the door of the reactor, for the stalks of the mushroom bud or developing fruiting bodies, which have to be manually removed by moving the reactor to the laminar air-flow chamber.
e. The 9 cotton plug provisions on top of the reactor facilitate sufficient and efficient air transfer to the interior of the reactor and scaffold, resulting in an OTR of ~10–12 g/h, OUR of 0.14 g/h, and KLa of 60.7/h.

The process of periodical enzyme harvest(s) during the quasi-continuous hydroponic cultivation comprises:
a. opening the culture medium tanks’ outlets, after completion of the incubation time of ~9–12 days; the medium flows into the excess flow collection reservoir, and the reservoir is emptied using a peristaltic pump. The optimum period of incubation is deduced on the basis of the yield coefficient (Yx/s) of the mushroom cultivation phase recorded during a preliminary trial performed using a day-to-day analysis of the mycelial scaffold, which is not possible during actual experimental trials within the hydroponic reactor.
b. Emptying the medium-holding tanks by opening their exit valves. Subsequently, the autoclaved water’s reservoir line is opened carefully, and roughly ~900 mL of the water is allowed to fill into the scaffold region (until the first drop drips at the bottom) and it is allowed to stay for 1.5 h in the scaffold region;
c. depressing the spring-loaded presser on the top of the reactor, gently, for a few times, after the completion of the 1.5-h of water-standing, taking caution to not damage the mycelia in each of the trays;
d. depressing the presser, gently, as it squeezes out the enzymes, in-situ, from each of the scaffold-bearing trays. At this stage, the culture medium-holding tank beneath each of the scaffold-holding tray becomes the master enzyme-harvesting tank, and when each of the lines are opened, the enzyme fills up into the enzyme-harvesting master reservoir;
e. repeating the autoclaved water-filling, pressing, in-situ enzyme extraction steps for 2 more times for each of the extraction stages. The collected enzyme is aseptically collected and appropriately stored.

The commencement of the subsequent/consecutive cycle of feeding to take forward the quasi-continuous hydroponic fungal cultivation process comprises:
a. filling culture medium into their respective trays, cellulosic slurry feeding to the scaffold trays, topping-up the water-filling jacket and respective reservoirs. In the demonstrative trial, we were able to operate the reactor for ~27 days or 3 consecutive cycles until the enzyme activity reduced as the mycelia started ageing.

and

b. Assessing the suitability of the hydroponic cultivation process for the quasi-continuous growth and periodical harvest(s) of the cellulolytic enzyme complexes, was performed using three fungal strains from the culture bank of our fungal biotechnology laboratory. The strains are: Aspergillus nigerMTCC 4325; Trichoderma harzianumMTCC 795; Trichoderma reeseiMTCC 3192.Initially, the fungi are grown in a solid-state fermentation model using rice straw as the substrate in a large-bottom cultivation flask until the growth-supporting cellulose/hemi-cellulose content of the substrate exhausts as assessed using a compositional analysis procedure. The cellulose-exhausted substrate along with the dense mycelia are then aseptically transferred to the scaffold-holding trays and the rest of the process of the hydroponic cultivation is continued.

The custom-designed prototype of the lab-scale hydroponic reactor comprises:
fabrication of the entire reactor out of 1 mm-thick SS 316-grade stainless-steel sheets;
an outer casing of 24 cm (L) x 24 cm (B) x 4.2 cm (W) dimensions (fig. 6). The closure has a screw-lockable, silicone-gasketed door of ~39 cm (L) x 24.6 cm (B) x 0.1 cm (W) dimensions (fig. 7). A polycarbonate-based transparent window is present on the door of the reactor to facilitate visual examination of the hydroponic region.
location, at the back of the reactor, a hollow jacket of dimensions 45 cm (L) x 30 cm (B) x 5 cm (W) through which water is to be filled until it reaches overflow (fig. 6). The entire reactor, when placed over a heating platform, facilitates the maintenance of the incubation temperature of ~28oC, within the reactor’s environment, throughout the entire period of ~9-days of batch cultivation.
6 provisions at the top of the reactor, wherein, cotton plugs are made so as to facilitate sufficient gaseous exchange with ambient air (fig. 6).
scaffold-holding tray(s), bearing the dimensions of (24.6 cm x 24.6 cm x 4.2 cm) (fig. 7). The sides and bottom of the scaffold-holding trays have stainless-steel meshes (of ~1 mm mesh sizing) to permit the circulation of air throughout the mycelial scaffold layers;
within each of the scaffold-holding tray, a silicone-based cellulose-introduction line (~0.3 cm diameter), which has 6 outlets within the mycelial scaffold to evenly distribute the feed within the scaffold layers (fig. 5).
connecting the silicone layers to a master cellulose slurry reservoir;
location of an autoclaved water-introducing line at a distance of ~2.1 cm above the base of the tray (fig. 9); autoclaved water’s flow begins when its (water’s) master reservoir is opened.
Provision of jute-based wicks, manually tied to the meshes, at the bottom of each of the meshes of the scaffold-holding trays. The diameter of the jute wicks is ~0.3 cm. The length of the jute wicks immersed into the medium is 6–7 cm.
Spacing the wicks in such a manner that the entire scaffold has medium transfer through the capillary action of the wicks in ~55 minutes (as calculated from demonstrative trials). Accordingly, the wicks are evenly spaced at a distance of ~3 cm from each other.
Construction of each of the culture medium-holding tanks with a dimension of 24.6 cm (L) x 4.2 cm (H) x 24.6 cm (B) (fig. 7). The sequential medium-filling line’s opening of 3 cm diameter is located at 0.6 cm above the base of the medium-holding tank (fig. 7). When a medium-holding master reservoir’s valve is opened, the medium-filling lines fill the culture medium, sequentially, from the top medium-holding tank until the bottom medium-holding tank until a stipulated level through the strategic location of the lines and by the influence of gravity. The lines are so placed that the overflow from the top tank fills up the middle tank and then the middle tank’s overflow fills the bottom-most tank and an overflow from the bottom-most tank located ~3-cm above the floor of the base tank indicates that all the tanks have been filled until their stipulated levels (fig. 7).
On the side opposite to the overflow lines, individual enzyme-harvest lines connecting to a master enzyme-harvesting reservoir (fig. 7);
a presser of dimension 19.5 cm (L) x 19.5 cm (B) x 4.2 cm (W), bearing an ~3 cm of thick silicone-based cushioning stuck to its bottom using a heat-resistant adhesive (fig. 7);
two stainless-steel rods of diameter 0.6 cm, spaced at a distance of ~9. 75 cm, and welded to the floor of the hydroponic reactor;
welding the scaffold trays and the culture medium-holding tanks to the mounting rods (fig. 7). The gap between a scaffold-holding tray and a culture medium-holding tray is ~0.15 cm.
Welding a third mounting-rod to a spring attached to the hydroponic reactor’s platform. The presser is welded in such a way that a presser exists on top of each of the scaffold-holding tray at a height of ~0.15-cm from a completely filled scaffold tray.

Figure 3 illustrates a flow chart with the key variables required to perform the quasi-continuous hydroponic process for the production and periodical harvest of the produced cellulolytic enzyme complex.
Referring to figure 3, which explains the entire quasi-cultivation hydroponic fungal cultivation process 100 for cellulolytic enzyme complex production and their periodical harvests involves:
At step 102, the process begins with the aseptic excision of 24 cm x 24 cm x 3 cm mycelia-rich regions from spent mushroom cultivation bags, which will then be serving as whole-mycelia-bearing scaffolds; the aseptic extraction process will have to be performed within a laminar air-flow chamber. The excised mycelial scaffold is placed within the scaffold-holding tray of the hydroponic reactor; prior to commencement of the aseptic transfer, the cotton plugs are placed in their respective positions at the top of the hydroponic reactor and autoclaved. A composition analysis of the substrate material in the excised region from the mushroom bags showed negligible or complete absence of fungal growth-promoting cellulose or hemicellulose. Each of the scaffold-holding trays has 6–7 cm-long jute wicks to perform the capillary-uptake-based uniform culture medium supplementation throughout the scaffold region. The culture medium is constituted by the typical minimal medium composition mentioned in this disclosure. The sides and bottom of the scaffold-holding trays are meshed, which permit sufficient oxygen transfer to the growing scaffolds from all sides; prior to using the hydroponic reactor and connecting its respective lines, the reactor is placed within an autoclave and subjected to the conventional process of steam-based sterilization.
At step 104, after autoclaving and within a laminar air-flow chamber, post loading the scaffolds into their respective trays, the water-jacket located at the back surface of the reactor is filled with water to maintain the stipulated temperature during the hydroponic cultivation process.
At step 104, the temperature within the reactor and the water jacket is maintained at 28±3oC, which is monitored using two thermometers, placed within the reactor and its water-filling jacket. The prototype of the reactor was designed in such a way that the reactor is placed over a heating platform to maintain temperature within the water-filled jacket.
At step 106, the culture medium reservoir’s master valve is opened and as the medium begins to flow out of the overflow-indication port after filling each of the culture medium holding tanks sequentially, the reservoir’s master valve is closed.
At step 108, Alkali pre-treated and neutralized pulverized wheat straw (with lignin content less than 10% w/w) is the cellulosic substrate used for the demonstrative process. After autoclaving the material and mixing it with autoclaved distilled water to a slurry-like consistency, it is loaded into the cellulose-feeding reservoir and introduced to 25% (v/v) within the scaffold-holding tray; the valves of the feeding lines are then closed.
At step 110, after loading the cellulose-based substrate into the scaffold trays, the electrically-powered, programmed, timer-based atomiser is turned on to maintain humidity, as it introduces autoclaved water every 1.5 hours into the reactor’s environment.
At step 112, two DO probes, one within the bio-reactor’s environment and the other within the scaffold aid in appropriately monitor and maintain the DO% concentration in the environment, at a value of more than 90%.
At step 114, The hydroponic cultivation is allowed to operate for ~9–12 days. As the mycelia and the scaffold material dry up, they uptake culture medium from the jute wicks through capillary action, and as the medium is transferred to the scaffold from the jute wicks, fresh medium from the tank is up-taken by the jute wicks. Such a wick-based transfer allows for appropriate moisture maintenance without overflooding the scaffold region. The culture medium in the tank beneath the trays, as observed during the demonstrative trials, has to be replenished with fresh medium from reservoir every 3 cycles or ~27 days.
At step 116, Throughout the duration of incubation, the relative humidity (RH) within the reactor and within the scaffold is maintained between 70–80%, and monitored using two RH-sensing probes provided within the reactor. During the period of mushroom cultivation, if the stalks of the mushroom bud or develop into fruiting bodies, they have to be manually removed by moving the reactor to the laminar air-flow chamber.
AT step 118, after completion of the incubation time of ~9–12 days, the culture medium tanks’ outlets are opened and the medium flows into the excess flow collection reservoir, and the reservoir is emptied using a peristaltic pump. The optimum period of incubation is deduced on the basis of the yield coefficient (Yx/s) of the mushroom cultivation phase recorded during a preliminary trial performed using a day-to-day analysis of the mycelial scaffold, which is not possible during actual experimental trials within the hydroponic reactor.
At step 118, post the completion of the incubation period, the culture medium-holding tanks are emptied by opening their exit valves. Subsequently, the autoclaved water’s reservoir line is opened carefully, and roughly ~900 mL of the water is allowed to fill into the scaffold region (until the first drop drips at the bottom) and it is allowed to stay for 1.5-h in the scaffold region. After the stipulated 1.5-h, the spring-loaded presser on the top of the reactor is gently pressed a few times, taking caution to not damage the mycelia in each of the trays. As the presser is depressed, it gently squeezes out the enzymes, in situ, from each of the scaffold-bearing trays. At this stage, the culture medium-holding tank beneath each of the scaffold-holding tray becomes the enzyme harvesting tank, and when each of the lines are opened, the enzyme fills up into the enzyme-harvesting master reservoir. The autoclaved water-filling, pressing, enzyme extraction steps are repeated 2 more times for each of the extraction stages.
At step 120, the cellulose-feeding process can be commenced for the subsequent/consecutive hydroponic cultivation and enzyme production-cum-harvest phases.

Figure 4 illustrates a schematic of the custom-built hydroponic reactor, along with the: (i) mycelial scaffold-loading tray; (ii) culture medium tanks underneath each of the scaffold-holding trays; (iii) spring-loaded presser above each of the scaffold-holding trays; (iv) connecting lines to fill culture medium from its reservoir into each of the medium-holding tanks; (v) connecting lines to deliver autoclaved water from its reservoir into each of the scaffold-holding trays; (vi) individual enzyme harvest ports connected to a master enzyme-harvesting line collecting the enzymes into a reservoir; (vii) thermometers for temperature regulation; (viii) humidity-measurement probes for estimating humidity throughout the course of the operation; (ix) timer-based atomiser lines, within the reactor, to periodically introduce atomised and autoclaved water to maintain the stipulated humidity.

Figure 5 illustrates a schematic of the cellulose-bearing, uniform substrate-loading silicone tubing lines into each of the scaffold-bearing trays.

Figure 6 illustrates an engineering drawing of the outer vessel of the hydroponic reactor along with its hinged-door.
The custom-designed prototype of the lab-scale hydroponic reactor includes the following components with the below-mentioned specifications:
Referring to Figure 6, the entire reactor is fabricated out of 1 mm-thick SS 316-grade stainless-steel sheets.
The reactor has an outer casing of 45 cm (H) x 30 cm (B) x 30 cm (W) dimensions (fig. 6). The closure has a screw-lockable, silicone-gasketed door of 39 cm (L) x 24.6 cm (B) x 0.1 cm (W) dimensions. At the back of the reactor, there exists a hollow jacket of thickness of 5 cm through which water is to be filled until it reaches overflow. The entire reactor, when placed over a heating platform, facilitates the maintenance of the incubation temperature of ~28oC, within the reactor’s environment, throughout the entire period of ~9-days of batch cultivation. The top of the reactor has 6 provisions where cotton plugs are made so as to facilitate sufficient gaseous exchange with ambient air (fig. 6).

Figure 7 illustrates an engineering drawing of the inner scaffold-bearing tray, medium-holding tank, presser for enzyme extraction; it also presents, the connecting lines/piping for loading the culture medium from its master reservoir into individual culture-medium loading tanks, located beneath each of the scaffold-loading trays.
The scaffold-holding tray bears the dimensions of 24.6 cm x 24. 6 cm x 4.2 cm (fig. 7). The sides and bottom of the scaffold-holding trays have stainless-steel meshes (of ~1 mm mesh sizing) to permit the circulation of air throughout the mycelial scaffold layers.

Within each of the scaffold-holding trays, a silicone-based cellulose-introduction line (~0.3 cm diameter) is present, which has 6 outlets within the mycelial scaffold to evenly distribute the feed within the scaffold layers (fig. 5). The silicone layers are connected to a master cellulose slurry reservoir. The scaffold-holding trays have an autoclaved water-introducing line located at ~2.1 cm above the base of the tray (fig. 9); autoclaved water’s flow begins when its (water’s) master reservoir is opened. The bottom of each of the trays has jute-based wicks manually tied to the meshes at the bottom. The diameter of the jute wicks is ~0.3 cm. The length of the jute wicks immersed into the medium is 3–4 cm. The wicks are spaced in such a manner that the entire scaffold has a complete and a uniform medium transfer through the capillary action of the wicks in ~75 minutes (as calculated from demonstrative trials). Accordingly, the wicks are evenly spaced at a distance of ~3 cm from each other.
Each culture medium-holding tank has a dimension of 4.2 cm (H) x 24.6 cm (L) x 24.6 cm (B) (fig. 7). The sequential medium-filling line’s opening of ~1.5 cm diameter is located at ~0.6 cm above the base of the medium-holding tank (fig. 7). When a medium-holding master reservoir’s valve is opened, the medium-filling lines fill the culture medium, sequentially, from the top medium-holding tank until the bottom medium-holding tank until a stipulated level through the strategic location of the lines and by the influence of gravity. The lines are so placed that the overflow from the top tank fills up the middle tank and then the middle tank’s overflow fills the bottom-most tank and an overflow from the bottom-most tank located ~3 cm above the floor of the base tank indicates that all the tanks have been filled until their stipulated levels (fig. 7). On the side opposite to the overflow lines, the individual enzyme harvest lines connecting to a master enzyme-harvesting reservoir are present (fig. 7).
A presser has a ~3 cm of thick silicone-based cushioning stuck to its bottom using a heat-resistant adhesive (fig. 7).
Two stainless-steel rods of diameter 0.6 cm are spaced at a distance of 19.5 cm and welded to the floor of the hydroponic reactor.
The scaffold trays and the culture medium-holding tanks are welded to the mounting rods (fig. 7). The gap between a scaffold-holding tray and a culture medium-holding tray is ~0.15 cm.
A third mounting-rod is welded to a spring attached to the hydroponic reactor’s platform. The presser is welded in such a way that a presser exists on top of each of the scaffold-holding tray at a height of ~0.15-cm from a completely filled scaffold tray.

The individual steps of the disclosed hydroponic bio-process employing mushroom mycelial scaffolds for a quasi-continuous production of cellulolytic enzyme complexes are as described hereunder:
Step 1: Pre-preparation of the hydroponic reactor and loading the mycelium-rich scaffolds into the designated trays
The master reservoirs of the: typical fungal growth-specific minimal medium, autoclaved water, cellulosic substrate-feeding tank, and enzyme-collection tank are subjected to autoclaving after appropriately sealing and closing their valves. The hydroponic reactor, with all its valves and line-openings appropriately sealed, is also subjected to stage of autoclaving. Post the stage of autoclaving, within a laminar air-flow hood, the appropriate reservoir lines are connected to the hydroponic bio-reactor.
The minimal medium used to promote fungal growth during demonstrative trials comprised in (g/L): di-potassium hydrogen phosphate – 1.73, potassium di-hydrogen phosphate – 0.68, ammonium nitrate – 1, magnesium sulfate hepta-hydrate – 0.1, calcium chloride di-hydrate – 0.02, sodium chloride – 4, ferrous sulfate heptahydrate – 0.03. The cellulose-rich slurry was prepared using pulverized and alkali-pre-treated wheat straw; the pre-treatment can be performed using any of the process parameters as known and established well in the prior-art; the average particle size of the fine, pulverized biomass was kept as low as 300 µm. Post pre-treatment, neutralization and drying, a mixture containing biomass and distilled water in the ratio (1:2) was soaked for 3-h at a temperature of 60oC and subjected to a stage of autoclave to create the feed slurry for the demonstrative process.
The demonstrative trials performed for the hydroponic bio-process involved the usage of the spent cultivation bags of the mushroom, Pleurotus ostreatus (spawns originally purchased from ICAR-Indian Institute of Horticultural Research), which is being cultivated in a commercially operating mushroom farm in Pondicherry, India. The hanging bag cultivation strategy for mushrooms are well-reported in the prior-art. In our process, 4 kg of water-washed rice straw was used for mushroom cultivation for a period of 4 months, with 4 fruiting bodies’ (mushroom) harvesting stages. Post completion of the cultivation process, the cellulose content of the straw was negligible and was found to be unsuitable for promoting any further growth as assessed using a biomass’s proximate analyses procedure. The cultivation bag was densely filled with the mushroom’s mycelia, which are, typically, as long as ~3–5 m. In the present disclosure, the spent substrate bag, packed with dense, cellulase-producing substrate mycelium are exploited as whole-mycelia for producing cellulases in a hydroponic cultivation process within a custom-designed reactor.
The excision of ~24 cm x 24 cm x 3 cm of mycelia-rich regions from spent mushroom cultivation bags is performed, which will then be serving as whole-mycelia-bearing scaffolds; the aseptic extraction process will have to be performed within a laminar air-flow chamber. The excised mycelial scaffold is placed within the scaffold-holding tray(s) of the hydroponic reactor; prior to commencement of the aseptic transfer, the cotton plugs are placed in their respective positions at the top of the hydroponic reactor and autoclaved. A compositional analysis of the substrate material in the excised region from the mushroom bags showed negligible or complete absence of fungal growth-promoting cellulose or hemicellulose. Each of the scaffold-holding trays has 6–7 cm-long jute wicks to perform the capillary-uptake-based uniform culture medium supplementation throughout the scaffold region. Each of the culture medium-holding trays can roughly accommodate ~1.7 L of the minimal medium. The sides and bottom of the scaffold-holding trays are meshed, which permit sufficient oxygen transfer to the growing scaffolds from all sides. Post loading the scaffolds into their respective trays, the water-jacket located at the back surface of the reactor is filled with water until it flows out of its water-inlet valve indicating complete filling. The water in the jacket would serve to maintain the temperature uniform throughout the reactor during the hydroponic cultivation process.
Step 2: Setting up the hydroponic cultivation reactor for uninterrupted, prolonged operational cycles
The culture medium reservoir’s master valve is opened. The medium first fills up the top-most tray until it reaches the exit/overflow valve of tray 1; next, the second tray is filled with the overflow of tray 1, followed by a similar mechanism to fill up the tray 3. After filling to its optimum in tray 3, medium flows out of the master overflow port located outside the enclosure of the reactor, indicating complete filling of all the medium trays. The similar construction can be extended to any number of medium trays, where gravity functions to fill the trays until the stipulated levels. The master reservoir and the conclusive overflow valves are closed.
The cellulose-rich substrate slurry’s master reservoir is opened, and based on its flow rate, calculated manually, an equal volume of the slurry is allowed to fill up each of the scaffold-holding trays to a final feed volume of ~25% (v/v) within the scaffold-holding tray. The valves of the individual feeding lines are then closed. After a visual inspection of the trays, the entire reactor, from within the laminar air-flow chamber, the entire reactor is shifted to be placed atop a hot-plate/heated platform to maintain the reactor’s temperature at 28oC throughout the 9–12 days of cultivation.
When the incubation begins, the electrically-powered, programmed, timer-based atomiser is turned on to maintain humidity, as it introduces autoclaved water every 1.5 hours into the reactor’s environment. Two DO probes, one within the bio-reactor’s environment and the other within the scaffold aid in appropriately monitor and maintain the DO% concentration in the environment, at a value of more than 86%. The pH probes are calibrated using any of the methods as well-reported in prior-art.
As the mycelia and the scaffold material dry up, they uptake culture medium from the jute wicks through capillary action, and as the medium is transferred to the scaffold from the jute wicks, fresh medium from the tank is up-taken by the jute wicks. Such a wick-based transfer allows for appropriate moisture maintenance without overflooding the scaffold region. The culture medium in the tank beneath the trays, as observed during the demonstrative trials, has to be replenished with fresh medium from reservoir every 3 cycles or ~27 days. Throughout the duration of incubation, the relative humidity (RH) within the reactor and within the scaffold is maintained between 70–80%, and monitored using two RH-sensing probes provided within the reactor. During the period of mushroom cultivation, if the stalks of the mushroom bud or develop into fruiting bodies, they have to be manually removed by moving the reactor to the laminar air-flow chamber. The inspection has to be manually performed through the transparent polycarbonate window of the hydroponic reactor every 5–6 days throughout the cultivation process.
Step 3: Periodical harvest of the enzyme in situ, and commencement of the consecutive feeding and incubation cycles
After completion of the incubation time of ~9–12 days, the culture medium tanks’ outlets are opened and the medium flows into the excess flow collection reservoir, and the reservoir is emptied using a peristaltic pump. The optimum period of incubation is deduced on the basis of the yield coefficient (Yx/s) of the mushroom cultivation phase recorded during a preliminary trial performed using a day-to-day analysis of the mycelial scaffold, which is not possible during actual experimental trials within the hydroponic reactor. Post completion of the incubation period, the culture medium-holding tanks are emptied by opening their exit valves. Subsequently, the autoclaved water’s reservoir line is opened carefully, and roughly ~900 mL of the water is allowed to fill into the scaffold region (until the first drop drips at the bottom) and it is allowed to stay for 1.5 h in the scaffold region. After the stipulated 1.5-h, the spring-loaded presser on the top of the reactor is gently pressed a few times, taking caution to not damage the mycelia in each of the trays. As the presser is depressed, it gently squeezes out the enzymes from each of the scaffold-bearing trays. At this stage, the culture medium-holding tank beneath each of the scaffold-holding tray becomes the in situ enzyme harvesting tank, and when each of the lines are opened, the enzyme fills up into the enzyme-harvesting master reservoir. The autoclaved water-filling, pressing, enzyme extraction steps are repeated 2 more times for each of the extraction stages. The collected enzyme is aseptically collected and appropriately stored. The next cycle of incubation begins with culture medium filling into the respective trays, cellulosic slurry feeding to the scaffold trays, topping-up the water-filling jacket and respective reservoirs. In the demonstrative trial, we were able to operate the reactor for ~27 days or 3 consecutive cycles.
In order to assess the suitability of the hydroponic cultivation process for the quasi-continuous growth and periodical harvest(s) of the cellulolytic enzyme complexes, three fungal strainswere employed. The strains are: Aspergillus nigerMTCC 4325; Trichoderma harzianumMTCC 795; Trichoderma reeseiMTCC 3192. Initially, the fungi are grown in a solid-state fermentation model using rice straw as the substrate in a large-bottom cultivation flask until the growth-supporting cellulose/hemi-cellulose content of the substrate exhausts as assessed using a compositional analysis procedure. The cellulose-exhausted substrate along with the dense mycelia are then aseptically transferred to the scaffold-holding trays and the rest of the process of the hydroponic cultivation is continued.

Figure 8 illustrates the individual cellulose slurry-feeding lines.

Figure 9 illustrates the connecting lines for the introduction of autoclaved water into each of the scaffold-holding trays.

The present disclosure relates to a first-of-its-kind of hydroponic fungal cultivation process, performed using mushroom mycelial scaffolds within a quasi-continuously operated custom-designed hydroponic reactor, with periodical cellulase enzyme harvests. Typically, after a process of hanging-bag mushroom cultivation is performed using any of the conventional techniques known in the prior art, the final phase of fruiting body harvest is completed and the spent mushroom substrate bags are discarded. However, the bags contain a dense mycelial network (as long as 5 m) of the mushrooms, and the substrates are devoid of any growth promoting nutrients such as cellulose/hemicellulose etc. So, the nutrient-depleted substrate material functions as a scaffold to hold the dense mycelium in place. It is a known fact that, while the fruiting bodies are aerial mycelium of the mushroom enabling oxygen transfer, the dense mycelial network within the bag constitute the substrate mycelia, which derive nutrients from the substrate material by secreting enzymes (majorly cellulase complexes) during the growth phase of the mushroom. Hence, in the present disclosure, the dense mycelial network from the spent substrate bag is exploited as the whole-mycelial mass enabling enzyme production within the hydroponic reactor, while provided with exogenous cellulose-rich substrates. The custom-designed hydroponic reactor facilitates controlled mycelial growth, substrate feeding, humidity regulation, aeration, and temperature control, thereby prolonging cellulase yields with reduced operational costs. As is the case with hydroponic cultivation, medium supplementation happens through a capillary uptake-based operation from a culture medium-filled reservoir beneath the mycelia-containing tray. Within the reactor, a spring-loaded presser is manually used to squeeze the mycelial scaffold to collect the accumulated enzyme in the tank beneath the scaffold region, followed by the in-situ enzyme fraction collection in the overall enzyme-harvest reservoir. Such periodic enzyme harvests ensure consistent yields over multiple cycles, making the process economically viable and sustainable.

In an embodiment, the primary objective is to devise a cost-effective and sustainable method for cellulolytic enzyme production in a hydroponic mode for prolonged operation until as long as ~27 days or 3 operational batches with periodical enzyme harvests at the end of every ~9 days.
In an embodiment the process involves performing a hydroponic SSF for cellulase complex production with appropriate moisture-regulation (using culture medium), time-extended high-yielding operation, and periodical enzyme harvests.
In an embodiment the overall process includes the utilization of the enzyme-secreting, substrate mycelia-rich spent mushroom substrate as whole-cell/whole-mycelial scaffolds for enzyme production within a custom-built hydroponic reactor.
In another embodiment, the process includes designing a customized hydroponic bio-reactor for a time-extended, quasi-continuous enzyme production and periodical harvests.
In an embodiment, within the reactor, the process flow involves providing uninterrupted culture medium-supplementation using a master medium reservoir (with connecting lines) and mycelial scaffold-specific medium-storage tanks beneath each of the hydroponic trays, which are sequentially filled until a stipulated level, where overflow is indicated by a specific overflow-indication port.
In yet another embodiment, within the reactor, the process involves designing dedicated lines for uniformly providing exogenous cellulose-rich substrate, individually, to the hydroponic cultivation scaffolds in each tray.
In another embodiment, at the back of the reactor, the process involves providing a water-filling jacket to maintain the temperature of the hydroponic process by placing the entire reactor on a heated platform.
In an embodiment, within the reactor and the water-filled jacket, the process involves introducing a thermometer each, to measure and maintain the temperature of the incubation at ~28oC.
In an embodiment, within the reactor, the process involves providing an electrically-powered, timer-based atomiser unit, within which autoclaved water would flow to maintain the relative humidity throughout the inner environment of the reactor.
In another embodiment, within the reactor’s environment and within the scaffold-holding tray, the process involves introducing a relative humidity (RH)-sensing probe each to measure the RH of the individual regions, wherein the specified RH is between 70–80%.
In yet another embodiment, within the reactor, the process involves providing dedicated lines for autoclaved water flow to the hydroponic trays/mycelial scaffolds to wet the scaffolds and draw enzymes in the enzyme-harvesting tanks, and subsequently to a master enzyme-harvesting reservoir.
In another embodiment, the process involves the provision of 9 cotton plug provisions atop the reactor’s enclosure to allow sufficient air exchange to the scaffold region, akin to fungi’s natural mode of growth.
In an embodiment, within the reactor, the process involves, above each of the hydroponic trays, providing a spring-loaded presser to gently squeeze the accumulated enzymes to be collected, in situ, within the enzyme collection tanks.
In an embodiment, the harvested enzyme complexes would bear the following enzyme activities as estimated using the standard IUPAC Ghose- and NREL-prescribed cellulolytic enzyme assay protocol for the demonstrative operation: (i) from the P. ostreatus’sharvest: exoglucanase – 5.91±1 IU/g.ds; endoglucanase: 14±2.5 IU/g.ds; beta-glucosidase - 180±30 IU/g.ds. (ii) from A. niger’sharvest: exoglucanase – 4.5±0.5 IU/g.ds; endoglucanase: 10±1 IU/g.ds; beta-glucosidase - 100±36 IU/g.ds. (iii) fromT. harznianum’sharvest: exoglucanase – 4±0.2 IU/g.ds; endoglucanase: 12±1.5 IU/g.ds; beta-glucosidase - 120±23. (iv) from Trichoderma sp.’s harvest: exoglucanase – 4.1±1 IU/g.ds; endoglucanase: 15±3 IU/g.ds; beta-glucosidase - 90±27 IU/g.ds. In all the above cases, IU/g.ds. refer to the enzyme activity per gram of the dry solids obtained.
In yet another embodiment, the oxygen transfer rate (OTR), oxygen uptake rate (OUR) and mass-transfer co-efficient - KLa for the demonstrative process of P. ostreatus’s hydroponic cultivation while using the custom-designed hydroponic reactor are as follows: OUR – 0.14±0.02 g/h; OTR – 10.5 g/h considering the air available in the reactor at each point of time, the density of O2 and its percentage in ambient air; KLa – 60.7/h.
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 about 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 system for prolonged, quasi-continuous hydroponic cultivation of fungus for cellulolytic enzyme complex production and its in-situ periodical harvest, comprising:
a hydroponic reactor configured to provide a controlled environment for cultivation of a fungus;
a plurality of scaffold-holding trays horizontally arrangedwithin the reactor, each tray configured to hold a mycelial scaffold, each scaffold comprising an excised portion of a spent mushroom substrate bag, wherein the excised portion is densely populated with mycelia of the mushroom;
a culture medium delivery system operatively coupled to the reactor, comprising:
a master medium reservoirfor bulk storage of culture medium,
a plurality of scaffold-specific medium-storage tanksfluidly connected to the master medium reservoir via sequential medium filling lines, wherein each medium-storage tank is positioned to supply culture medium to a respective scaffold-holding tray, each tank associated with a respective scaffold,
a plurality of jute wicks extending from each scaffold-holding tray and dipping into a corresponding culture medium-storage tank below, configured for capillary action to transport culture medium from the tank to the mycelial scaffold over approximately 75 minutes,
a plurality of fluid conduits configured to deliver culture medium from the master reservoir to the scaffold-specific storage tanks, and
a plurality of overflow indication ports associated with each storage tankto indicate maximum fill level and prevent overfilling;
a substrate delivery system including a cellulose-loading channel positioned above the scaffold-holding trays configured to deliver exogenous cellulose-rich substrate to the mycelial scaffolds;
a temperature control system configured to maintain 28±3oC temperature within the reactor, the temperature control systemcomprising:
a water-filled jacket disposed at the back surface of the reactor for thermal insulation and temperature regulation,
a heated platform supporting the reactorfrom below to provide heating from the base,
a first temperature sensor positioned within the reactor to monitor internal temperature, and
a second temperature sensor positioned within the water-filled jacketto monitor internal temperature;
a humidity control system configured to maintain 70–80% humidity within the reactor, the humidity control systemcomprising:
an atomizer unit positioned within the reactor vessel to introduceautoclaved water as moisture into the reactor, and
a plurality of relative humidity sensors positioned within the reactorfor monitoring humidity levels;
an enzyme harvest system comprising:
a plurality of fluid conduits coupled to an autoclaved water reservoir and autoclaved water introduction line configured to deliver autoclaved water to the mycelial scaffolds for enzyme extraction,
a plurality of enzyme-harvesting tankspositioned below the scaffold-holding trays to collect enzyme-containing solution, each tank associated with a respective scaffold,
a master enzyme-harvesting reservoirfor accumulating harvested enzyme solution from the enzyme-harvesting tanks via an enzyme harvesting line, and
a plurality of spring-loaded pressers, each positioned above a respective scaffold-holding tray and actuatable via a spring-loaded press button to gently squeeze the mycelial scaffolds and enhance enzyme extraction.

2. The system as claimed in claim 1, wherein the enzyme-secreting fungus is selected from the group consisting of Trichoderma, Aspergillus, and Penicillium, wherein the enzyme harvesting system further comprises a plurality of valves for controlling the flow of water and enzymes, and a pH probe (Figure 2: pH probe) positioned within the reactor for monitoring pH levels.

3. The system as claimed in claim 1, wherein the exogenous cellulose-rich substrate is agricultural waste selected from corn stover, wheat straw, or rice husks, and the jute wicks are configured to release culture medium to the scaffolds providing sustained moisture for approximately 75 minutes via capillary action.

4. The system as claimed in claim 1, wherein the mycelial scaffolds are derived from spent mushroom substrate bags densely colonized by Pleurotus ostreatus mycelium, wherein mycelial scaffold is arranged in trays and supported by a Trays' mounting rod.

5. The system as claimed in claim 1, wherein the hydroponic reactor further comprising:
a nutrient delivery system configured to provide nutrients to the mycelia-rich scaffolds; and
a gas exchange system including an air inlet and outlet configured to provide oxygen and remove carbon dioxide to maintain aerobic conditions within the reactor.

6. The system as claimed in claim 1, wherein the reactor is thermally insulated, wherein the reactor further comprises:
a water jacket surrounding the reactor for maintaining a constant temperature preferably at 28±3oC; and
a transparent window in the reactor wall deployed for visual observation of the mycelial scaffolds.

7. The system as claimed in claim 1, further comprising:
a first dissolved oxygen (DO) probe disposed within the interior chamber of the reactor vessel;
a second DO probe disposed within one of the scaffold-holding trays; and
a controller operatively connected to the temperature control system and humidity control system, configured to maintain the temperature at 28±3°C and humidity at 70–80% respectively, based on signals from the temperature sensors and relative humidity sensors, and configured to monitor the DO levels measured by the first and second DO probes and to adjust operating parameters of the system to maintain a DO concentration of more than 90% within the reactor vessel, wherein the controller is configured to adjust the aeration rate to maintain the desired DO concentration.

8. The system as claimed in claim 1, further comprising:
a timer-based controller operatively connected to the atomizer unit, configured to actuate the atomizer to introduce autoclaved water into the reactor vessel at predetermined intervals, approximately every 1.5 hours.

9. The system as claimed in claim 1, further comprising:
a plurality of feeding lines connected to the reservoir and to each of the scaffold-holding trays; and
a plurality of valves associated with each of the feeding lines and the cellulose-loading channel for controlling the flow of cellulosic substrate as a slurry-like consistency to each tray, wherein the cellulosic substrate is alkali pre-treated and neutralized pulverized wheat straw having a lignin content of less than 10% w/w.

10. A process for producing cellulolytic enzyme complexes, comprising:
a) preparing mycelium-rich scaffolds from spent mushroom cultivation substrate upon excising mycelia-rich regions from spent mushroom cultivation bags of Pleurotus ostreatus, and in second demonstration using the mycelium-rich scaffolds derived from fungal strains:Aspergillus niger, Trichoderma harzianum, and Trichoderma reesei;
b) aseptically loading the mycelium-rich scaffolds into scaffold-holding trays within a hydroponic reactor, wherein the scaffold-holding trays comprise wicks for capillary uptake of culture medium;
c) supplying a culture medium to the scaffold-holding trays via capillary action from the wicks, wherein the culture medium contains 1.73g/L di-potassium hydrogen phosphate, 0.68g/L potassium di-hydrogen phosphate, 1g/L ammonium nitrate, 0.1g/L magnesium sulfate hepta-hydrate, 0.02g/L calcium chloride di-hydrate, 4g/L sodium chloride, and 0.03g/L ferrous sulfate heptahydrate;
d) supplying a cellulose substrate slurry to the scaffold-holding trays, wherein the cellulose substrate slurry is prepared from pulverized and alkali-pretreated wheat straw, further comprising soaking and autoclaving the pre-treated wheat straw to form the slurry;
e) incubating the mycelium-rich scaffolds in the hydroponic reactor to produce cellulolytic enzyme complexes, wherein during the mycelium-rich scaffolds incubation, 28°C temperature and 70% and 80% relative humidity is maintained within the hydroponic reactor, wherein autoclaved water is introduced by the atomizer approximately every 1.5 hours, wherein the incubation period is approximately 9 to 12 days;
wherein humidity is maintained by introducing autoclaved water into the reactor environment using an atomizer at intervals, wherein above 86% dissolved oxygen levels is maintained within the hydroponic reactor; and
f) periodically harvesting the cellulolytic enzyme complexes by draining the culture medium from the scaffold-holding trays after an incubation period and washing the mycelium-rich scaffolds with autoclaved water thereby allowing the autoclaved water to soak within the scaffolds for a period of time followed by pressing the mycelium-rich scaffolds to extract the cellulolytic enzyme complexes into an enzyme-harvesting tank; and collecting the extracted cellulolytic enzyme complexes, wherein the step of washing, soaking, and pressing is repeated at least two times, wherein the soaking period with autoclaved water is approximately 1.5 hours.