CELLULASE PRODUCTION AND OPTIMIZATION IN SOLID STATE FERMENTATION BIOREACTOR

Abstract

Cellulases, constituting the third largest category of industrial enzymes, hold a significant 20% share in the global enzyme market. These versatile enzymes play a crucial role in diverse sectors such as biofuels, food, feed, textiles, detergents, pharmaceuticals, and paper and pulp industries. However, their widespread use faces a major obstacle in the form of high production costs, primarily linked to the current methods that rely on submerged cultivation using costly substrates and genetically modified strains. Addressing this challenge, there is a pressing need to shift towards environmentally friendly and renewable substrates that are abundantly available, ensuring a cost-effective and high-yield production of cellulases. Solid-state fermentation (SSF) emerges as a promising alternative, involving the cultivation of microorganisms in a solid substrate with minimal water. This approach has gained prominence over submerged fermentation due to its effectiveness in utilizing solid organic wastes for producing various valuable products, including enzymes, single-cell proteins, and other secondary metabolites. Despite the advantages of SSF, its industrial potential remains partially untapped due to the absence of a suitable bioreactor system that can operate under stringent aseptic conditions while facilitating efficient heat and mass transfer for enhanced yield and productivity. In this context, preliminary investigations focused on selecting four potent fungal strains and utilizing a range of lignocellulosic substrates, such as sorghum, cottonseed oil cake, pine, kans grass, wheat straw, wheat bran, linseed oil cake etc. The optimization process involved identifying the most effective combination of substrates and environmental parameters using the One-factor-at-a-time approach (OFAT) and advanced methodologies like Response Surface Methodology (RSM). Under optimized conditions, cellulase production showed an enhancement to 21.58 FPU/g-ds, with reduced fermentation time. Similarly, β-glucosidase production under optimized conditions demonstrated a desirability factor of 0.986 with a maximum activity of 5.02 IU/g-ds, confirming the effectiveness of the response model. The optimized parameters were upscaled in a newly designed 6-L SSF bioreactor. In this process, two substrates, namely pine needles and a combination of sorghum and cottonseed oil cake, were individually optimized within the bioreactor using the strain Aspergillus niger and Trichoderma reesei respectively. To explore the influence of varying bed height (BH) on enzyme production, SSF was carried out in 2-L conical flasks with different quantities of untreated pine needles (UPN), resulting in varying BH of 0.5, 1, 2, 3, and 4 cm. Notably, cellulase activity reached its peak on the 4th day (7.15 ± 0.35 FPU/g-ds) at 0.5 cm BH, but subsequent increases in BH led to a significant decline, with a approximately 45% decrease observed at 4 cm BH (p < 0.0001). Similar trends were observed for endoglucanase, β-glucosidase, and xylanase production. The negative impact of higher BH on enzyme activity is attributed to compaction, impeding fungal penetration into the depth of the pine needles. Parallel investigations in the packed bed bioreactor demonstrated enhanced enzyme productivity, with maximum cellulase activity reaching 9.97 ± 0.27 FPU/g-ds at 2 cm BH, surpassing the flask studies (p < 0.05). This improvement is likely attributed to favourable temperature, moisture, and airflow control in the packed bed bioreactor, contributing to enhanced cellulose utilization. Furthermore, analysis of the fermented residue revealed a 53.5% holocellulose consumption, reduced crystallinity index (29.5% from 46%), and increased total lignin content (45.9%). Pellet production from the fermented residue exhibited a higher calorific value (CV) of 19.13 ± 0.69 MJ/kg, surpassing unfermented pine pellets (17.63 ± 0.66 MJ/kg), accompanied by improved compressive strength. Importantly, the sulfur content remained below the permissible limit for SOx emissions in fuels with an ash content below 5% at 0.14%. The optimization strategies for cellulolytic enzyme production from T. reesei, using sorghum and cottonseed oil cake (3:1) consisted of 200 g mixed substrate corresponding to a BH of 3 cm. PBR investigations demonstrated peak enzyme activities at an aeration rate of 0.75 Litres per minute (LPM). Incorporating sand as an inert material at 10% concentration enhanced porosity, and acted as a great moisture reservoir facilitating efficient heat and mass transfer. Mixing regimes, such as initial mixing after a 48-h interval followed by subsequent 24-h intervals, proved beneficial in maintaining optimal moisture content and temperature, promoting higher production levels. Comprehensive assessments included biomass concentration, moisture content, temperature profiles, oxygen uptake rate, and carbon dioxide evolution rate in each case. The optimal mixing regime was further validated by the respiratory activity of T.reesei. Comparative analyses favoured the bioreactor over Tray and SSF-PBR systems, particularly at elevated BH. The maximum biomass productivity obtained in the intermittent mixed bed reactor (19.31 mg glucosamine/g-ds/d) was 2.46-fold as obtained in the Tray bioreactor. After the comprehensive optimization, the maximum cellulase, endoglucanase, ß-glucosidase, and xylanase activity of 28.34 FPU/g-ds, 146.45 IU/ds, 2.89 IU/g-ds, and 25, 175.5 IU/g-ds were achieved at a substrate bed loading (SBL) of 26%. Further validation in a 45-L SSF PBR, maintaining a constant superficial air velocity of 0.0235 cm/s, at an SBL of 26%, showcased promising scaling-up prospects. The crude extract obtained after extraction from the solid substrate underwent concentration using a membrane filtration strategy, with microfiltration and ultrafiltration achieving recovery rates of 94% and 71%, respectively. This yielded a cellulase concentrate with an final activity of 13.54 FPU/mL. It displayed stability across a wide pH and temperature range, with a 24.5-h half-life at 50 °C. The concentrate, meeting rigorous food-grade safety standards, adheres to permissible limits for potential pathogens, heavy metals, mycotoxins, and pesticide residue. It significantly enhanced apple juice clarity (94.37%), reducing turbidity (21%) and viscosity (99%), while elevating total reducing sugar release by 63% compared to untreated juice. In additional applications, the concentrate efficiently extracted eicosapentaenoic acid (EPA)-rich oil from Nannochloropsis sp. biomass, achieving a total fatty acid (TFA) recovery of 369.4 ± 4.6 mg/g dry weight (77% yield) in just 12 h. The enzyme was effectively reused without compromising on the yield yield. Additionally, high protein content of 47% in the defatted biomass showed potential applications in aquafedd ingredient, thus improving the overall process economics. Optimization strategies, including forced aeration, water activity maintenance, and the incorporation of a low-cost inert material, effectively addressed challenges related to temperature and bed compaction. Online monitoring for microbial activity facilitated accurate biomass estimation, while different mixing strategies promoted enhanced cellulase production. The valorization of leftover residues for high-calorific-value fuel pellets added a sustainable dimension to the study. Future studies should concentrate on designing equations for bioreactor configuration, SBL, and operational strategies for scale-up. Continuous SSF, particularly through the valorization of residual cellulose and hemicellulose, holds promise for sustained productivity. Comprehensive techno-economic and life cycle analyses in subsequent studies will provide valuable insights into the overall viability and sustainability of the entire process.