Design of Solid-State Fermentation Systems for Polymer Hydrolytic Extracellular Enzyme Production by Filamentous Fungi

This protocol utilizes wheat bran in a rotary solid-state fermentation system to enhance enzyme production. The substrate, supplemented with inducers such as chitin, supports fungal growth under controlled conditions. Results demonstrate enzyme yields 4-6 times higher compared to submerged fermentation, showcasing the method's adaptability and effectiveness for diverse biotechnological applications.

Solid-state fermentation (SSF) is a bioconversion process that utilizes a solid substrate that does not dissolve in an aqueous medium. Microorganisms grow on the surface of the substrate and penetrate its solid matrix to extract essential nutrients for their development. SSF is characterized by minimal free water, with a substrate moisture content maintained above 70%, and involves three interconnected phases -- gaseous, liquid, and solid. This protocol describes the use of wheat bran, an agro-industrial byproduct, as the base substrate for enzyme production in a rotary system. The substrate is supplemented with an inducer, such as chitin, chitosan, starch, or cellulose, to promote the synthesis of hydrolytic proteins. The system is highly adaptable, allowing the use of different fungal forms, including mycelium, spores, or pellets. In the methodology described, the inducer and substrate are mixed at a ratio of 1:100 (w/w), sterilized via autoclaving, and adjusted to the desired moisture level with sterile water. The fungal inoculum is then added, and the rotary system operates at 10 rpm to ensure adequate mixing and oxygenation. The system is incubated for 6-8 days under optimal growth conditions for mesophilic or thermophilic/thermotolerant fungi, enhancing its versatility. Following incubation, the enzyme is easily extracted using an appropriate cold buffer (e.g., acetate, citrate, or phosphate), depending on the type of enzyme. The extract is clarified through centrifugation and filtration to obtain a cell-free supernatant. The enzyme can then be further concentrated or purified as needed. The results demonstrated a 4-6-fold increase in enzyme activity compared to submerged fermentation (SmF), highlighting the effectiveness of the system. Its adaptability to different substrates, inducers, and fungal species makes it a valuable tool for various biotechnological applications.

Solid-state fermentation (SSF) has emerged as a promising and sustainable bioconversion technology for producing high-value enzymes, bioactive compounds, and secondary metabolites. This technique involves the growth of microorganisms on solid substrates with minimal free water, simulating their natural environment and enabling efficient metabolic activity¹. The primary goal of this protocol is to optimize enzyme production through a rotary SSF system that ensures enhanced substrate utilization, oxygen diffusion, and process scalability. Employing wheat bran, an abundant agro-industrial byproduct, as the base substrate, contributes to the valorization of agricultural residues and promotes circular bioeconomy practices².

SSF has significant advantages over submerged fermentation (SmF), including lower energy and water consumption, higher product concentration, and compatibility with a wide range of inexpensive agricultural residues such as wheat bran, rice husks, and sugarcane bagasse³. Unlike SmF, which requires large volumes of water and expensive nutrient media, SSF systems leverage solid matrices that not only serve as microbial growth surfaces but also provide nutrients essential for microbial activity. Additionally, the limited free water in SSF minimizes contamination risks, making it a more robust option for enzyme production in industrial settings⁴. In addition to its operational advantages, SSF presents significant environmental and economic benefits compared to submerged fermentation (SmF). Studies have reported that SSF reduces water consumption by 50%-70% and lowers energy costs by more than 30% due to the absence of large water volumes requiring constant agitation and aeration. Moreover, the use of agro-industrial residues as substrates minimizes raw material costs and promotes circular economy practices by repurposing agricultural byproducts^2,4.

SSF has been extensively validated for its efficiency and scalability. For example, studies have reported a 4-6-fold increase in enzyme activity using SSF compared to SmF, highlighting the economic and environmental advantages of this technique^2,5. Additionally, the downstream process is simplified, as enzyme extraction typically requires less water and fewer purification steps. This makes SSF particularly attractive for industries aiming to reduce operational costs and environmental impact⁶.

The rotary SSF system described in this protocol offers several improvements over traditional static SSF methods. While static systems often face challenges such as uneven substrate colonization and oxygen limitation, the rotary configuration ensures thorough mixing and aeration, promoting uniform microbial growth^7,8,9. For instance, this system has been successfully employed to produce hydrolytic enzymes such as chitinases, amylases, and proteases using fungal species like Aspergillus and Trichoderma².

A key feature of this SSF system is its adaptability. The use of wheat bran as a base substrate demonstrates the potential of agro-industrial residues for cost-effective bioconversion³. Moreover, the supplementation of the substrate with inducers such as chitin, chitosan, and starch further enhances enzyme synthesis by stimulating specific metabolic pathways^2,10. The system is also compatible with different fungal forms, including spores, mycelium, and pellets, allowing users to tailor the process to their specific requirements².

SSF offers broad potential for application in various fields such as food biotechnology, biofuel production, and environmental remediation¹¹. Its integration of cost-effective substrates, exceptional enzyme yields, and high process flexibility establishes SSF as an essential approach for industrial-scale biotechnological innovations.

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Substrate preparation

NOTE: Use a commercial brand of wheat bran to minimize significant variations in substrate characteristics. Each batch of wheat bran varies due to multiple factors, making it a heterogeneous material that is difficult to standardize, leading to fluctuations in its constituent content. If a standardized material is required, choose an alternative matrix or perform a proximate chemical analysis of each batch of wheat bran to adjust it according to the needs.

1. Wash the wheat bran three times with sterile distilled water to remove organic matter residues, debris, and dust. This also removes simple sugars that could interfere with fermentation.
2. Spread the washed bran on an aluminum tray and dry it in an oven at 60 °C for 24 h.
3. Once dried, place the wheat bran into a sterile 50 mL conical tube.

2. Preparation of inoculum

NOTE: This protocol describes three methods for inoculum preparation: spore suspension, direct inoculation with mycelium disks, and cellular suspension. Establish the initial inoculum concentration and quantify protein levels for accurate yield calculations.

1. Preparation of spore suspension
   1. Transfer a 5 mm diameter agar disk saturated with mycelium onto a fresh potato dextrose agar plate. Incubate the plate at 28 °C for 5-7 days, or until the mycelium saturates the medium. Some fungi may require a longer incubation time.
   2. Add 5 mL of sterile distilled water to the plate and mechanically detach the spores using a sterile loop.
   3. Prepare a 1:100 dilution of the spore suspension. Place 10 µL in the center of a Neubauer chamber and count spores under a microscope. Calculate the spore concentration (spores/mL) based on the chamber's factor and dilution.
2. Cultivation of mycelium in liquid medium
   1. Transfer a 5 mm agar disk saturated with mycelium onto a fresh potato-dextrose-agar plate and incubate at 28 °C until saturation.
   2. Prepare 25 mL of potato-dextrose broth in a sterile 125 mL flask and autoclave.
   3. Transfer a 5 mm mycelium disk from the saturated plate into the sterile broth.
   4. Incubate the flask on a shaker at 125 rpm for 24-48 h, depending on the fungal strain. Extend the incubation time for slow-growing fungi.
   5. Collect 2 mL for the inoculum and 2 mL for dry weight determination.
3. Direct inoculation of mycelium disks
   1. Place a 5 mm agar disk saturated with mycelium on a fresh potato-dextrose-agar plate. Incubate at 28 °C until saturation.
   2. Use one mycelium disk as inoculum and another to determine the dry weight.

3. Preparation of the SSF system

NOTE: Inducers can be natural or commercial. Purified commercial inducers are preferred to minimize impurities that could alter fermentation efficiency. Adjust water additions to maintain a relative humidity of at least 90%.

1. Combine the following components in a sterile 50 mL conical tube: 5 g of dry wheat bran; 0.2 g of the inducer (e.g., commercial chitin); 5.5 mL of water (adjust based on the inducer's water absorption capacity); 5 mL of a sterile salt solution containing 16 g/L monobasic potassium phosphate, 4 g/L sodium sulfate, 2 g/L potassium chloride, 1 g/L calcium chloride, 400 mg/L zinc chloride, 60 mg/L boric acid, 40 mg/L sodium molybdate, 150 mg/L magnesium chloride, 100 mg/L ferric chloride, and 400 mg/L copper sulfate.
2. Measure the relative humidity using an electrode-based hygrometer, ensuring a minimum of 90% humidity. Insert the electrode probe directly into the reactor at varying depths to obtain a representative measurement of moisture distribution.
   1. Follow the steps to adjust the humidity if below 90%:
      1. Gradually add sterile distilled water in 1 mL increments per 10 g of substrate. After each addition, mix thoroughly to ensure uniform distribution of moisture.
      2. Allow the substrate to equilibrate for 10-15 min. Remeasure the humidity level.
      3. Repeat the above steps until the target humidity of 90% is reached. Avoid over-wetting the substrate throughout the process.
   2. Follow the steps to adjust the humidity if above 90%:
      1. Spread the substrate thinly in a sterile environment. Remove excess moisture by (1) exposing the substrate to laminar airflow, or (2) placing it in a drying chamber at 30 °C for 10-15 min.
      2. Alternatively, gently mix the substrate to promote even moisture redistribution. After treatment, reassess the humidity level.
      3. Repeat the drying or mixing step as needed until the humidity reaches 90%. Proceed with fermentation only once the target humidity is achieved.
3. Autoclave the tube at 15 psi for 15 min.
4. After cooling, inoculate the substrate with one of the following: 1 mL of spore suspension (1 x 10⁶-1 x 10⁷ spores/mL), 2 mL of cellular suspension, or one 5 mm mycelium disk.

4. Solid-state fermentation (SSF) procedure

NOTE: For kinetic studies or parameter evaluations at different times, prepare separate tubes for each time point to ensure representativity.

1. Avoid substrate clumping by vortexing the tubes at maximum speed for 5 min in 1-min cycles.
2. Place the tubes in a rotary mixer with a horizontal axis. Ensure the substrate moves freely inside the tubes. Set the mixer to operate at 10 rpm.
3. Incubate the mixer in an incubator at the microorganism's optimal growth temperature. Maintain the temperature reported for optimal enzymatic activity when using heat-sensitive inducers.

5. Extraction of enzymes

NOTE: The extraction fundamentals are based on the solubility and pH-maximum activity of the extracellular enzyme. As SSF avoids the water medium, the extracellular enzyme is involved in the water surrounding the solid matrix, which means the concentration is higher than in SmF. In this context, the selection of the best extraction buffer depends on the knowledge of the desired activity. The optimization of the extractions depends on the final enzyme concentration and the type of extraction buffer used.

1. After the desired fermentation period, resuspend the substrate in 20 mL of pre-chilled buffer. Examples include: 0.1 M acetate buffer, pH 5.6, for chitinase extraction; 0.02 M phosphate buffer, pH 6.9, for amylase extraction.
2. Vortex the tubes in cycles: 1 min at maximum speed, followed by 1 min on ice. Repeat 10 times.
3. Filter the suspension using paper filters and mechanically extract the supernatant by pressing.
4. Clarify the supernatant by centrifuging at 3000 x g for 15 min at 4 °C.
5. Use the crude extract directly or further purify the enzyme via column chromatography or centrifugal filters. Kinetic studies are also recommended to determine the Michaelis constant (Km) and the maximum rate of enzymatic transformation (Vmax)¹².

6. Optimization process

NOTE: Optimize this protocol by evaluating and adjusting the quality and concentration of inducers, as well as the type and concentration of the inoculum.

1. Determine the ideal fermentation time and refine the extraction steps to improve efficiency.
2. Control and fine-tune environmental conditions, including temperature, pH, and aeration.
3. Test various buffers and extraction conditions to enhance enzyme yield and stability.
4. Perform statistical analyses, such as response surface methodology, to identify the most influential variables and achieve optimal enzyme production.

Figure 1A presents the schematic representation of the rotary mixer used in this system, which has a capacity for six conical tubes of 50 mL. Figure 2B illustrates the changes that occur in the wheat bran during conditioning before entering the solid-state fermentation process. As seen, no significant structural changes were observed.

Figure 2 shows the saturation of wheat bran after 6 days of solid-state fermentation for chitinase production by the Trichoderma harzianum fungus in this system, using commercial chitin as an inducer. Figure 2A shows the original material before the fermentation process. The micrographs confirm the substrate's utilization by the fungus, which is also reflected in the modifications it undergoes, losing its fractal-like structure due to interaction with the fungus.

The results in Figure 3 show a significant increase in chitinase activity (Figure 3A) and amylase activity (Figure 3B) obtained from T. harzianum and Aspergillus lentulus, respectively, when comparing the solid-state fermentation (SSF) and submerged fermentation (SmF) systems. The data were validated by a one-way ANOVA and Tukey test with p < 0.05 using SigmaPlot software. In this context, it is observed that this system is applicable to different enzymatic activities and fungi. A. lentulus was characterized as a thermotolerant fungus, incubated at 40 °C, showing a significant increase in its amylase activity. The chitinase activity results from T. harzianum in both liquid and solid fermentation have been previously reported separately by our research group^2,5, with this work confirming and comparing a significant increase in SSF in comparison to SmF, similar to the amylase activity. Our group has worked with more than 50 fungal strains for the production of proteases and amylases under mesophilic and thermophilic conditions, and the results are consistent.

The results confirm the success of the protocol by demonstrating significant increases in enzyme activity, with solid-state fermentation yielding higher outputs compared to submerged fermentation. This indicates the effectiveness of SSF in enhancing both chitinase and amylase production. Previously published figures have been reused with proper reprint permission.

Figure 1: Overview of the rotary solid-state fermentation (SSF) system and wheat bran pretreatment process. (A) Schematic representation of the rotary SSF system. (B) Modifications in the wheat bran pretreatment process: (I) Initial raw material, (II) Moist wheat bran following the washing step, (III) Dried wheat bran. Please click here to view a larger version of this figure.

Figure 2: Substrate saturation by Trichoderma harzianum. (A) Substrate before SSF. (B,C) Substrate saturated with fungal mycelium at different magnifications. Scale bars: (A), 50 µm; (B), 200 µm; (C), 100 µm. Please click here to view a larger version of this figure.

Figure 3: Enzymatic activity in SSF and submerged fermentation (SmF) by T. harzianum and A. lentulus. Comparison of (A) Glucosamine formation through the hydrolysis of chitosan by enzymes produced by Trichoderma harzianum in SSF and SmF, and (B) Amylase activity of Aspergillus lentulus obtained in SSF and SmF. Error bars represent the standard deviation of three replicates. Asterisks (*) indicate a statistically significant difference between the data. Note that the statistical variation is specific to each type of enzyme and does not apply to comparisons between (A) and (B). Please click here to view a larger version of this figure.

This study outlines a relevant protocol for optimizing enzyme production through solid-state fermentation (SSF) systems, specifically designed for filamentous fungi. Below, critical aspects of the methodology are discussed, alongside its significance, limitations, and potential applications.

The success of the protocol is highly dependent on key steps such as the preparation of the substrate and inoculum. Proper washing and drying of the wheat bran are essential for eliminating impurities that may interfere with fungal growth or enzyme production. Additionally, the careful adjustment of substrate moisture to maintain relative humidity above 90% ensures optimal fungal colonization and activity¹³. The rotary system's operation at 10 rpm is another crucial parameter, as it promotes even mixing and oxygenation, preventing substrate clumping and ensuring uniform fungal growth¹⁴.

The adaptability of this protocol lies in its compatibility with diverse fungal species and inducers. For instance, while commercial chitin and starch were used as inducers in this study, other substrates, such as lignin, cellulose, or chitosan, could be substituted depending on the target enzyme. Troubleshooting common challenges, such as uneven substrate colonization, involves refining the mixing parameters or adjusting the inoculum concentration². Furthermore, ensuring sterility during substrate preparation and inoculation is critical to prevent contamination, especially in industrial-scale applications¹⁵.

While SSF offers several advantages over submerged fermentation (SmF), it is not without limitations¹⁶. One major challenge is scaling up the rotary SSF system without compromising substrate mixing, oxygen diffusion, or accurate biomass determination, another common problem in SSF^17,18. Additionally, the protocol's reliance on agro-industrial residues such as wheat bran may introduce variability in results due to differences in substrate composition across batches. These limitations highlight the need for further optimization when transitioning to large-scale production^19,20.

The described rotary SSF system demonstrates significant advantages over traditional static SSF and SmF methods. Compared to static SSF, the rotary system ensures more uniform microbial growth, reducing issues related to oxygen limitations. Additionally, the system's adaptability allows for its application to various fungal forms and enzyme types, making it highly versatile¹⁸. SSF systems can have various configurations to enhance metabolite productivity. Each type of system has advantages and disadvantages that must be analyzed in depth to determine the best configuration. The rotary SSF system offers several advantages over traditional static SSF and SmF; however, it faces challenges when compared to other SSF systems, such as tray and packed-bed bioreactors. Tray bioreactors, commonly used for large-scale SSF, offer simplicity and low energy consumption but face challenges related to limited oxygen transfer and moisture distribution, leading to uneven microbial growth and reduced enzyme yields. Packed-bed bioreactors, on the other hand, improve aeration through forced airflow but may encounter issues with pressure drops and uneven temperature distribution, particularly in tall columns. In contrast, the rotary SSF system promotes continuous mixing and homogeneous conditions, reducing anaerobic zones and enhancing enzyme productivity. Nonetheless, energy consumption and mechanical wear due to continuous rotation may increase operational costs²¹.

Static solid-state fermentation systems, such as tray bioreactors, are constrained by limited heat and mass transfer, often leading to internal temperature gradients of over 30 °C, resulting in suboptimal microbial performance. These systems usually operate at small volumes (0.15-0.25 m³) and suffer from heterogeneous microbial colonization, with studies indicating that only about 34% of substrate pores are effectively utilized. In contrast, rotating bioreactors offer mechanical agitation that enhances oxygen distribution and substrate homogeneity, while supporting larger operational volumes of up to 13 m³ and substrate loads reaching 40% (w/v). A notable example involves cellulase production by Thermoascus aurantiacus, where a rotating SSF reactor maintained at 49 °C and ventilated at 5 L/min·kg yielded 14,098 IU/g of enzyme activity-over three times higher than the 4,212 IU/g obtained under static conditions²².

Scaling up solid-state fermentation requires a delicate balance between maintaining microbial kinetics and ensuring effective mass and heat transfer in progressively larger systems. The traditional stepwise approach includes laboratory (5-20 kg), pilot (50-5,000 kg), and industrial (25-1,000 tons) stages. One of the main challenges during scale-up is the dissipation of metabolic heat, which can reach up to 3,200 kcal/kg of dry material, which is particularly problematic in static or poorly ventilated systems. To address this, scale-up strategies often rely on the control of dimensionless design parameters and the application of mathematical modeling approaches, including mass and energy balance equations, to preserve key variables such as oxygen availability and substrate moisture retention across different scales. Pilot-scale systems (e.g., 150 L and 6 m³) have successfully incorporated engineering features such as water jackets, curved stirring blades, and controlled humidity to improve process reproducibility and ensure consistent product yields^18,21,22.

The protocol presented demonstrated effectiveness for enzyme production on a small laboratory scale. However, scaling up the process for industrial applications poses significant challenges, primarily related to maintaining efficient mixing and aeration in larger volumes. One promising approach to address these limitations is the use of a continuous-screw reactor, which mimics the function of a vertical mixer by promoting continuous mixing and improved oxygenation throughout the solid substrate. This design reduces the formation of anaerobic zones and enhances mass transfer, which are critical factors for the success of solid-state fermentation on an industrial scale^2,15. Nevertheless, potential challenges include maintaining the structural integrity of the solid matrix and preventing the formation of temperature gradients along the reactor. Further studies should focus on optimizing operational parameters and validating enzyme yields under scaled-up conditions to ensure the feasibility and efficiency of the process.

The potential applications of this protocol extend to multiple sectors, including food biotechnology, bioenergy, and environmental remediation. For example, the enhanced production of hydrolytic enzymes like chitinases and amylases can support bioconversion processes in agriculture and industry. Furthermore, the use of agro-industrial byproducts such as wheat bran aligns with sustainable practices, promoting the valorization of waste and contributing to a circular bioeconomy²⁰.

The authors declare that they have no conflicts of interest.

This work was supported by the Secretaría de Investigación y Posgrado of the Instituto Politécnico Nacional (SIP-IPN) through grant/project numbers 20220487, 20230676, 20240793, and 20251269 awarded to GGS, and 20220492, 20230427, 20240335, and 20251139 awarded to DROH. The authors would like to express their gratitude to ENCB-IPN, the Secretaría de Ciencia, Humanidades, Tecnología e Innovación de México (Secihti), formerly known as the Consejo Nacional de Ciencia, Humanidades y Tecnología (CONAHCyT), and BEIFI-program as well as Centro de Nanociencias y Micro y Nanotecnologías of Instituto Politécnico Nacional for their invaluable support. López-García acknowledges Secihti (previously CONAHCyT) for the master's fellowship, as well as IPN for the SIP-BEIFI fellowship. Legorreta-Castañeda is a recipient of a postdoctoral fellowship from the "Estancias Posdoctorales por México" program of Secihti, previously known as CONAHCyT.