Solid-state Fermentation System Research Articles

Evaluation of high-value bioproducts production by marine endophytic fungus Arthrinium sp. FAKSA 10 under solid state fermentation using agro-industrial wastes

BackgroundTo address public health challenges, there is increasing interest in bioactive metabolites from unconventional sources for targeted cancer and viral treatments with minimal side effects. Endophytic fungi are promising for these therapies. This study focused on maximizing bioactive agent yields from these fungi using a low-cost medium and optimized fermentation system. ResultsForty-three endophytic fungi isolated from Sinularia polydactyla were cultivated on wheat bran medium and evaluated for bioactive metabolites under solid-state fermentation. Strain FAKSA 10, identified as Arthrinium sp. FAKSA 10, exhibited the highest levels of pharmaceutical metabolites, including L-glutaminase, L-methioninase, L-arginase, L-asparaginase, L-tyrosinase, L-lysine α-oxidase, and ribonuclease, with a 79.12% hepatitis C virus knockdown rate. This strain produced 46 metabolites with anticancer, antioxidant, antiviral, and cytotoxic properties, including major compounds like hexadecanoic acid methyl ester; hexadecanoic acid ethyl ester; 9, 12-octadecadienoic acid (Z, Z), methyl ester; 9-octadecenoic acid (Z)-, methyl ester, and 11, 14-eicosadienoic acid, methyl ester. A cost effective strategy using 11 agro-industrial residues was applied, following by optimization of the solid-state fermentation system. This optimization increased enzyme yields and enhanced antiviral and antioxidant activities. The optimal conditions for solid-state fermentation were a 10 d incubation, 1 mm particle size, 60% initial moisture, 28°C temperature, and 2 × 107 CFU/mL inoculum size. ConclusionsThis study exploited Arthrinium sp. FAKSA 10 metabolites as natural pharmaceuticals against cancer and viral diseases, highlighting their significant antioxidant properties. Among various residues, oil cakes emerged as an effective and cost-efficient medium, capable of significantly enhancing the production of valuable bioactive metabolites.

Solid-State Fermentation Engineering of Traditional Chinese Fermented Food.

Solid-state fermentation (SSF) system involves solid, liquid, and gas phases, characterized by complex mass and heat transfer mechanisms and microbial complex interactions. The SSF processes for traditional Chinese fermented foods, such as vinegar, soy sauce, and baijiu primarily rely on experience, and most of the operations are replaced by auto machine now. However, there is still a lack of engineering in-depth study of the microbial process of SSF for complete process control. To meet the demands of smart manufacturing and green production, this paper emphasizes the engineering analysis of the mechanisms behind SSF. It reviews the progress in the engineering aspects of Chinese traditional SSF, including raw material pretreatment, process parameter detection, mathematical model construction, and equipment innovation. Additionally, it summarizes the challenges faced during intelligent upgrades and the opportunities brought by scientific and technological advancements, proposing future development directions. This review provides an overview of the SSF engineering aspects, offering a reference for the intelligent transformation and sustainable development of the Chinese traditional SSF food industry.

Mining of thermophilic biosurfactant producers for solid-state fermentation

Solid-state fermentation (SSF) is an emerging technology for organic waste valorisation to marketable bio products. SSF scale-up is hindered by heat transfer problems that could be diminished operating under thermophilic conditions. Compost samples collected from food waste composting facility in a primary school in Barcelona and textile industry sample from Faisalabad Pakistan were subjected to isolation of thermophilic biosurfactant producing microorganisms. Altogether 43 isolates were screened through biosurfactant qualitative screening, but 18 isolates dominated with promising results which were further shortlisted under solid-state culture to select the five significant performers belonging to Bacillus sp., Aspergillus sp., and Streptomyces sp. The performance of the five isolates in SSF was assessed in time course studies in 0.5-L packed bed bioreactor using winterization oil cake (WOC), wheat bran and glucose as substrates combined with wheat straw as support material. All five isolates colonized the substrate well in SSF systems and exhibited considerable glucose consumption and adaptation within the SSF setup. The fermentation extracts indicated promising surface-active properties as observed by emulsification and oil displacement analysis. These findings provide an effective enrichment strategy for screening of thermophilic biosurfactant producers and can aid establishment of the scale up for the enhanced metabolite production.

Oxygen influences spatial heterogeneity and microbial succession dynamics during Baijiu stacking process

The spontaneous solid-state stacking process (SSSP) of Baijiu is an environmentally friendly and cost-effective process for enriching and assembling environmental microorganisms to guarantee the subsequent fermentation efficiency. In this study, how SSSP create spatial heterogeneity of stacking piles were found through spatiotemporal sampling. The degree of difficulty in oxygen exchange categorizes the stacking pile into depleted (≤4%), transitional (4 %–17 %), and enriched (≥17 %) oxygen-defined layers. This results in variation in succession rates (Vdepleted > Vtransitional > Venriched), which accelerates spatial heterogeneity during SSSP. As a dominant species (65 %–99 %) in depleted and transitional layers, Acetilactobacillus jinshanensis can rapidly reduce oxygen disturbance by upregulating poxL and catE, that sustains spatial heterogeneity. The findings demonstrated the value of oxygen control in shaping spatial heterogeneity during SSSP processes, which can create specific functional microbiome. Adding spatial heterogeneity management will help achieve more precise control of such solid-state fermentation systems.

Xylanase production by Penicillium sp. Pn004 and its application for grass hydrolysis: high value subproduct from non-centrifugal sugarcane bagasse and wheat bran

Worldwide more than 7 billion tons of lignocellulosic biomass will be produced by 2025. In Colombia, these residues are mainly disposed of in rivers and open fields, and only 10% is reused and recycled. Non-centrifugal sugarcane bagasse (SCB) is a residue obtained after sugarcane juice extraction during the manufacturing process of panela and is mostly used as fuel for boilers at sugar mills. Wheat bran (WB) is the main waste from wheat flour production. Nonetheless, the chemical composition of these by-products makes them suitable for use as substrates for hemicellulolytic enzyme production from fungi. Here, a whole process for production in a solid-state fermentation system, recovery, and ultrafiltration process for concentration of xylanases by Penicillium sp. Pn004 is presented. The higher productivity (26.7 ± 1.59 U gds-1 day-1) was reached on the fifth day of fermentation with an enzyme activity of 130.0 ± 7.95 U gds-1. The batch ultrafiltration process allowed increasing the xylanase activity up to 19-fold in the retentate, from 66.47 U mL-1 to 1486.83 U mL-1, without reaching a steady state flux through the membrane. Finally, the enzymatic extract achieved a 43% release of sugar from kikuyu grass (Cenchrus clandestinus), showing its potential to be used as an additive for silage or for enzymatic saccharification of lignocellulosic materials for sugar production.

Pediococcus spp.-mediated competition interaction within Daqu microbiota determines the temperature formation and metabolic profiles.

Fermented microbiota is critical to the formation of microenvironment and metabolic profiles in spontaneous fermentation. Microorganisms generate a diverse array of metabolites concurrent with the release of heat energy. In the case of Daqu fermentation, the peak temperature exceeded 60°C, forming a typical high-temperature fermentation system known as high-temperature Daqu. However, microorganisms that cause the quality variation in Daqu and how they affect the functional microbiota and microenvironment in the fermentation process are not yet clear. This study adopted high-throughput sequencing and monitored the dynamic fluctuations of metabolites and environmental factors to identify the pivotal microorganism responsible for the alterations in interaction patterns of functional keystone taxa and quality decline in the fermentation system of different operational areas during the in situ fermentation process that had been mainly attributed to operational taxonomic unit (OTU)_22 (Pediococcus acidilactici). Additionally, we used isothermal microcalorimetry, plate inhibition experiments, and in vitro simulation fermentation experiments to explore the impact of Pediococcus spp. on heat generation, microorganisms, and metabolite profiles. Results showed the heat peak generated by Pediococcus spp. was significantly lower than that of Bacillus spp., filamentous fungi, and yeast. In addition, the preferential growth of P. acidilactici strain AA3 would obviously affect other strains to colonize through competition, and its metabolites made a significant impact on filamentous fungi. The addition of P. acidilactici strain AA3 in simulated fermentation would cause the loss of pyrazines and acids in metabolites. These evidences showed that the overgrowth of Pediococcus spp. greatly influenced the formation of high temperatures and compounds in solid-state fermentation systems. Our work illustrated the vital impact of interaction variability mediated by Pediococcus spp. for microbial assembly and metabolites, as well as in forming temperature. These results emphasized the functional role of Daqu microbiota in metabolites and heat production and the importance of cooperation in improving the fermentation quality.IMPORTANCEThe stable and high-quality saccharifying and fermenting starter in traditional solid-state fermentation was the prerequisite for liquor brewing. An imbalance of microbial homeostasis in fermentation can adversely impact production quality. Identification of such critical microorganisms and verifying their associations with other fermentation parameters pose a challenge in a traditional fermentation environment. To enhance the quality of spontaneous fermented products, strategies such as bioaugmentation or the control of harmful microorganisms would be employed. This work started with the differences in high-temperature Daqu metabolites to explore a series of functional microorganisms that could potentially contribute to product disparities, and found that the differences in interactions facilitated directly or indirectly by Pediococcus spp. seriously affected the development of microbial communities and metabolites, as well as the formation of the microenvironment. This study not only identified functional microbiota in Daqu that affected fermentation quality, but also demonstrated how microorganisms interact to affect the fermentation system, which would provide guidance for microbial supervision in the actual production process. Besides, the application of isothermal microcalorimetry in this study was helpful for us to understand the heat production capacity of microorganisms and their adaptability to the environment. This study presented a commendable framework for improving and controlling the quality of traditional fermentation and inspired further investigations in similar systems.

Improvement of the production and quality of Cordyceps javanica conidia for the control of Diaphorina citri adults.

This study aimed to evaluate the use of palm kernel meal (PKM) in the traditional solid-state fermentation system to improve the production and quality of Cordyceps javanica conidia. The impact of PKM was determined by measuring conidia yield, viability, hydrophobicity, shelf life, and conidia pathogenicity against Diaphorina citri adults. By supplementing rice grains with 5% palm kernel meal increased the conidial yield by up to 40%, without compromising conidia viability and hydrophobicity. In addition, conidia caused higher levels of mortality by mycosis against D. citri adults (90%), relative to conidia harvested from rice (52%). The conidia recovered from rice/palm kernel meal mixtures also retained viability greater than 90% after storage for 10months at 4°C, while the conidia produced on rice reached 80%. Thus, conidia produced in the presence of palm kernel meal can be consumed immediately or in the medium term. Some process advantages of the palm kernel meal as co-substrate in the traditional production system of C. javanica are also mentioned. These results are attractive for improving the mycoinsecticide production process, with excellent cost-benefit and minimal changes in infrastructure and process.

Agricultural Bioinputs Obtained by Solid-State Fermentation: From Production in Biorefineries to Sustainable Agriculture

Agriculture plays a major role on society, especially in developing countries which rely on commodity exportation markets. To maintain high crop productivity, the use of agrochemicals was once employed as the main strategy, which in turn affected soil, water, and human health. In order to aid this issue, identifying some alternatives, such as the implementation of biofertilizers and inoculants as bioinputs in modern agriculture, are imperative to improve ecosystem quality. Among these bioinputs, a few bioproducts have shown good performances, such as phytohormones (e.g., auxins and giberellins), biosurfactants, and other enzymes; thus, it is extremely important to assure the quality and feasibility of their production in biorefinery scenarios. These bioproducts can be synthesized through fermentation processes through utilizing plant biomasses and agricultural byproducts as carbon sources. In this sense, to increase the tecno-economical availability of these processes, the implementation of solid-state fermentation (SSF) has shown great potential due to its ease of operation and cost-attractiveness. Therefore, this study aims to describe the main substrates used in SSF systems for the production of potential bioinputs; their associated operation hurdles, parameters, and conditions selection; the most suitable microorganisms; and the underlying mechanisms of these molecules in soil dynamics. Within this context, this study is expected to contribute to the development of new processes in modern biorefineries and to the mitigation of environmental impacts.

Production of Xylanase by Trichoderma Species Growing on Olive Mill Pomace and Barley Bran in a Packed-Bed Bioreactor.

Xylanases are hydrolytic enzymes that have tremendous applications in different sectors of life, but the high cost of their production has limited their use. One solution to reduce costs and enhance xylanase production is the use of agro-wastes as a substrate in fungal cultures. In this study, olive mill pomace (OMP) and barley bran (BB) were used as carbon sources and possible inducers of xylanase production by three species of Trichoderma (atroviride, harzianum, and longibrachiatum), one major xylanase producer. The experiments were conducted under a solid-state fermentation system (SSF) in flask cultures and a packed-bed bioreactor. Cultures of OMP and BB were optimized by examining different ratios of OMP and BB, varied particle sizes, and inoculum size for the three species of Trichoderma. The ratio of 8:2 OMP and BB yielded the highest xylanase activity, with a particle size of 1 mm at 29 °C and an inoculum size of 1 × 107 spores/mL. Studying the time profile of the process revealed that xylanase activity was highest after seven days of incubation in flask SSF cultures (1.779 U/mL) and after three days in a packed-bed bioreactor (1.828 U/mL). The maximum percentage of OMP degradation recorded was about 15% in the cultures of T. harzianum flask SSF cultures, compared to about 11% in T. longibrachiatum bioreactor cultures. Ammonium sulfate precipitation and dialysis experiments showed that Xylane enzyme activity ranged from 0.274 U/mL in T. harzianum to 0.837 U/mL in T. atroviride when crude extract was used, with the highest activity (0.628 U/mL) at 60% saturation. Xylose was the main sugar released in all purified fractions, with the G-50 and G-75 fractions showing the maximum units of xylanase.

Novel Bioformulations with Trichoderma lixii to Improve the Growth Dynamics and Biocontrol of the Cowpea Damping-Off Disease

Because agricultural wastes are abundant in biologically active substances, they can be used as a substitute source to produce highly valuable products while lowering pollution levels in the environment. Therefore, we aimed at determining the best agricultural wastes to increase the biomass production rate and the effectiveness of the biocontrol strain Trichoderma lixii SARS 111 in a solid-state fermentation system. The potential for its use in enhancing growth dynamics and controlling the Fusarium oxysporum NCAIM-F-00779-caused damping-off disease of cowpea plants grown in greenhouse conditions was also studied. Using a one-factor-at-a-time experiment, five cheap agricultural waste substrates (faba bean, cowpea, sweet potato, pumpkin, and cassava) were studied using the Plackett–Burman design (PBD) and the central composite design (CCD) to optimize the nutritional and growth conditions to maximize the production of Trichoderma conidia. The findings demonstrated that increasing Candida production quantitatively required the use of 3 g of sweet potato, 3 g of cassava, pH 6, 25 °C, and pre-treatment with dH2O. The shelf life and viability of T. lixii strain were measured as log10 CFU g−1 per substrate at room temperature (RT, 25 °C) at the beginning of month 0 and subsequently at 2-month intervals for 12 months. Data showed that the fungal counts increased with the use of 4 g of sweet potato + 2 g of cassava up to 7 months and then sharply decreased, lasting up to 12 months. Additionally, this bioformulation was applied to cowpea plants in a greenhouse experiment, where a significantly higher level of plant growth traits, photosynthetic pigments, antioxidant enzymes, and chemical content in the leaves, as well as lower incidence of the damping-off disease, were noted. Accordingly, it is possible to suggest 4 g of sweet potato and 2 g of cassava as a suitable bioformulation for the industrial-scale production of the T. lixii strain, which may be a potential biocontrol agent for preventing the cowpea damping-off disease caused by F. oxysporum and improving the growth dynamics.

Dynamic analysis Caffeic acid production driven by the key physicochemical factor and microbial community succession in Baijiu Daqu: A multi-microorganism fermentation of solid-state fermentation system

Daqu is a saccharifying fermenting agent in the solid-state fermentation process of Strong aromatic Baijiu, providing important raw materials, microorganisms and enzymes for the fermentation of Baijiu, however, a little information about organic acids production in Daqu until now. This work analyzed the correlation among microbial community succession, physicochemical factors and caffeic acid-producing metabolic pathways in key fermentation time-point of strong aromatic Baijiu Daqu, and the results showed that the main functional communities of caffeic acid metabolism were Staphylococcus, Klebsiella, Lactobacillus and Pichia, respectively. The correlation results showed that in the fermentation stage of the Daqu, caffeic acid was mainly influenced by miosture and reducing sugar. Co-occurrence network analysis showed that microbial interactions were stronger in days 0–8 than in days 10–27. The PICRUSt2 marker gene sequence was used to predict information on the function and abundance of enzymes related to different fermentation stages of Daqu, and combined with the related enzymes involve in caffeic acid metabolism, the results indicated that the microbial community in the pre-fermentation 0–8 days had the potential to produce caffeic acid. The results provide a theoretical basis for the analysis of the formation mechanism of phenolic acids in the multi-bacterial co-fermentation system of Daqu fermentation.

Effect of Solid-state Fermentation Parameters on Growth of Interest and Environmental Enzyme Production with Aspergillus niger

Using residual biomass in biological processes is increasingly promoted to alleviate environmental impacts. However, the focus is mainly based on emerging technologies and limited resources, and the conditions of the technologies remain unclear. The objective of this research is to establish the conditions of a solid-state fermentation system (SSF) for the growth of fungal biomass and the production of ligninolytic enzymes of environmental interest using Aspergillus niger. In a screening step, the biomass and lipase enzyme expression of three A. niger strains obtained from different screening sites will be extended. In the second stage, the effect of packing density (ρs) and airflow (vvm) was evaluated through a 23, on the growth of fungal biomass, fiber degradation, and CO2 generation in a lipid-contaminated SSF system. The generation of biomass and the concentration of specific enzymatic activity (U/L) of lipase present a correlation for all the strains evaluated. It was estimated that the A. niger strain AN19bc isolated from sugarcane bagasse presents the highest accumulation of biomass and concentration of specific enzyme activity of lipase (0.027 U/L) after nine days. The most appropriate conditions for the production of fungal biomass of the AN19bc strain in an SSF system are presented with an airflow of 33.33 vvm and packing density ρs = 360 kg Mss/m3, levels with which Yx/s= 0.91 is reached. After the process, the solid support used in the SSF presents a change in composition, with the fiber being the component that suffers a considerable degradation of 78.70%.

Bacillus subtilis SPB1 lipopeptide biosurfactant: antibacterial efficiency against the phytopathogenic bacteria Agrobacterium tumefaciens and compared production in submerged and solid state fermentation systems.

Bacillus subtilis SPB1 derived biosurfactants (BioS) proved its bio-control activity against Agrobacterium tumefaciens using tomato plant. Almost 83% of disease symptoms triggered by Agrobacterium tumefaciens were reduced. Aiming potential application, we studied lipopeptide cost-effective production in both fermentations systems, namely the submerged fermentation (SmF) and the solid-state fermentation (SSF) as well as the use of Aleppo pine waste and confectionery effluent as cheap substrates. Optimization studies using Box-Behnken (BB) design followed by the analysis with response surface methodology were applied. When using an effluent/sea water ratio of 1, Aleppo pine waste of 14.08g/L and an inoculum size of 0.2, a best production yield of 17.16 ± 0.91mg/g was obtained for the SmF. While for the SSF, the best production yield of 27.59 ± 1.63mg/g was achieved when the value of Aleppo pine waste, moisture, and inoculum size were, respectively, equal to 25g, 75%, and 0.2. Hence, this work demonstrated the superiority of SSF over SmF.

Studies on red-pigment production by Talaromyces atroroseus TRP-NRC mutant II from wheat bran via solid-state fermentation

Background Recently, the need of finding eco-friendly and less-hazardous pigments focused on an important alternative to harmful synthetic dyes. High productivity of various pigments from microorganisms, their rapid growth throughout the year, stability, and solubility of their pigments provide them advantages more than pigments produced from other natural sources.Objective The objective of this study is to improve red-pigment production from local isolated fungus Talaromyces atroroseus TRP-NRC on an inexpensive substrate (wheat bran) under solid-state fermentation system by using different mutants. Then, comparing between pigment released from fungi after mutation by different mutants, comparing the efficiency of different solvents for the extraction of red biopigments under different conditions, and then extraction of pigment and studying its structure.Materials and methods A novel locally non-mycotoxin-producing fungus T. atroroseus TRP-NRC was treated with γ-ray radiation followed by subjecting to ultraviolet rays and grown on wheat bran as a complete medium via solid-state fermentation technique. Different solvents, including water, ethanol, methanol, and acetone, were applied to extract pigment from dried fermented wheat bran. The effect of pH, temperature, and contact time on yield of pigment extraction was studied. Stability of extracted pigment to heat, autoclaving, and ultraviolet rays was studied. Antimicrobial activity of extracted pigment was studied. The extracted sample was subjected to high-performance liquid-chromatography analysis and gas chromatography–mass spectrometry analysis. Statistical analyses were performed using SPSS program at P value less than 0.05.Results and conclusion The mutant fungus (I) by gamma radiation achieved 30% increase in red pigment compared with the wild type. The mutant fungus (I) was subjected to ultraviolet rays, mutant (II) added 22% increase in pigment production compared with mutant obtained by gamma radiation. About 70% v/v of methanol, ethanol, and acetone were more efficient for extracting pigment with an advantage of 70% v/v acetone. The yield of pigment extraction was affected by pH, temperature, and contact time, and was at pH 6.5 at 50°C after 16 h. The produced pigment appeared to be heat-stable when subjected to heat from 30 to 80°C for 6 h. The pigment was also stable when autoclaved at 121°C for 15 min. The pigment was stable when subjected to ultraviolet rays for 6 h. The extracted pigment showed antibacterial activity against Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative). Gas chromatography–mass spectrometry analysis revealed that eighteen compounds were identified in the acetone extract of pigment. In general, the prevailing two compounds of fermented wheat bran by T. atroroseus TRP-NRC mutant-II extract were 9, octadenoic acid (43.72) and 1,1′-bicyclopropyl-2-octanoic acid, 2′-hexyl-, methyl ester 43.72%.

A novel method to assess heat transfer and impact of relevant physicochemical parameters for the scaling up of solid state fermentation systems

Heat production during fermentation is undesirable. It can affect the growth of biomass, sporulation, products formation and the scaling-up. Physico-chemical properties of substrates influence heat and mass transfer in Solid State Fermentation. Heat is chemically produced into substrates without micro-organism to allow better reproducibility. A 24–1 fractional factorial design is chosen to study the influence of four physicochemical parameters on heat transfer: Granulometry, Bulk Density, Carr Index (compressibility index) and Water Absorption Capacity. Results show that the two main physicochemical parameters which influence heat transfer are Granulometry and Carr Index. High Granulometry and low Carr Index have influence on maximum temperature reached during the test, warm-up speed and cooling speed. These two parameters allow efficient air flow through the substrate bed with large interparticle spaces enhancing exchange surface between air and particles. A substrate with these characteristics facilitates heat transfers in forced-aerated reactors.

Trichoderma harzianum sensu lato TSM39: A wheat microbiome fungus that mitigates spot blotch disease of wheat (Triticum turgidum L. subsp. durum) caused by Bipolaris sorokiniana

Bipolaris sorokiniana - the causal agent of wheat spot blotch - is an emerging phytopathogenic fungus in the Yaqui Valley, Sonora, Mexico. At present, its control relies on the use of harmful chemicals, partly due to the lack of effective alternatives. Thus, this work aimed to identify and characterize indigenous wheat-associated fungal strains with biocontrol capacity against B. sorokiniana that can be integrated into control strategies for spot blotch management. Out of 59 fungal strains evaluated, we identified 5 strains with antagonistic activity against B. sorokiniana by dual confrontation assay, and these were taxonomically assigned as Trichoderma harzianum sensu lato (strains TSM39 and TSO39), Aspergillus terreus (strains TRQ89 and TRQ93), and Aspergillus nidulans (strain TRQ83) based on phylogenetic analysis using the ITS region of the rRNA cistron. Trichoderma harzianum TSM39 showed interesting biological control traits (i.e., strong antagonistic activity, high growth rate, non-cytotoxicity) and its application to wheat plants - under highly conducive conditions for spot blotch outbreak- resulted in a significant reduction of disease severity. In vitro experiments revealed that the chitinolytic system of strain TSM39 responds to B. sorokiniana lysates (signals), which could have potential biotechnological applications to improve its biocontrol activity. Additionally, a solid-state fermentation system was successfully established for spore production of strain TSM39. Taken together, Trichoderma harzianum sensu lato TSM39 is a biocontrol agent against B. sorokiniana with the potential to be integrated into spot blotch management strategies and contribute to the sustainability of the region.

Production of a novel milk-clotting enzyme from solid-substrate Mucor spp. culture.

Calf rennet has been traditionally used for cheese making all over the world since ancient times. It is primarily a type of aspartic protease. Calf rennet, also known as chymosin, is considered the best milk coagulant in cheese manufacturing. Its usage and demand are increasing day by day in the food industry; however, some ethical issues are also related since it is naturally present in the calf's stomach and obtained by the slaughtering of young animals. Therefore, researchers are trying to introduce some new and better alternatives for chymosin in the food industry. Mucor racemosus f. racemosus CBS 381, Mucor racemosus DSM 62760, and Aspergillus oryzae were cultivated by solid substrate fermentation using the design of experiment (DoE) (MODDE; Umetrics, Sweden) to optimize and analyze the various combinations of different factors and responses (milk-clotting activity, proteolytic activity, specific activity). Based on the analysis of the screening and optimization results, Mucor racemosus CBS 381 was found to be the potential strain in terms of high production of aspartic protease, as well as had high milk-clotting activity under a solid-state fermentation system. However, molasses and casein were determined to be significant carbon and nitrogen sources, respectively, under conditions such as 70% moisture content and 25°C temperature. The molecular weight of the enzyme (Mucor CBS 381) is ∼30 KDa and it exhibits maximum activity at pH 4.8 at 45°C. The investigated enzyme was pronounced as thermal-sensitive and lost activity completely after 10 min incubation at 55°C. The remarkable qualities of the studied enzyme, such as cost-effective production, milk-clotting and proteolytic activity make Mucor racemosus CBS 381 a promising alternate to calf chymosin in the cheese-making industry. PRACTICAL APPLICATION: The milk-clotting enzyme (aspartic protease) produced by the Mucor racemosus is the alternative to calf chymosin. It can be used to produce cheese on the industrial level with some desired properties such as good taste and texture that includes gumminess. Nowadays, consumers prefer products that do not involve any animal cruelty whereas a huge group of consumers oppose the use of genetically modified enzymes. Therefore, the enzyme by Mucor racemosus would produce the cheese that is going to meet the demands of various types of cheese consumers.

Efficient biodegradation of straw and persistent organic pollutants by a novel strategy using recombinant Trichoderma reesei

Efficient biodegradation of lignocellulosic biomass needs a battery of enzymes targeting cellulose, hemicellulose, and lignin. In this study, recombinant Trichoderma reesei ZJ-09 with Pycnoporus sanguineus laccase gene was used to degrade rice straw by in situ production of laccase, xylanase, and cellulases under solid-state fermentation (SSF). Effects of parameters on key enzymes (cellulase, xylanase, and laccase) in biodegradation during SSF were investigated. Under the optimized SSF conditions, the FPA, xylanase activity, and laccase activity reached 110.47 FPU/g, 5787.59 IU/g, and 24.45 IU/g, respectively, on day 12. The obtained recombinant T. reesei SSF system achieved efficient degradation of rice straw with the final mass loss up to 51.16% which was 1.4-fold higher than the host strain. Further, bioconversion of rice straw into a novel laccase-enriched koji for persistent organic pollutants bioremediation (LKPB) was conducted by the optimized SSF system. LKPB was found to degrade persistent organic pollutants (POPs) effectively without the addition of mediators. 4-h removal rates of three POPs mediated by LKPB (87.21% for 2,4,5-trichlorophenol, 92.45% for nonylphenol, and 90.73% for oxytetracycline) were comparable to those achieved by laccase-co-mediator system. The newly established recombinant T. reesei SSF system could be potential to effectively degrade lignocellulosic wastes as well as organic pollutants.Graphical abstract

Safe production of Aspergillus terreus xylanase from Ricinus communis: gene identification, molecular docking, characterization, production of xylooligosaccharides, and its biological activities

BackgroundThe production of industrial enzymes such as xylanase using sufficient cost-effective substrates from potent microorganisms is considered economically feasible. Studies have reported castor cake (Ricinus communis) as the most potent and inexpensive alternative carbon source for production of xylanase C by using Aspergillus terreus (A. terreus). ResultsA. terreus strain RGS Eg-NRC, a local isolate from agro-wastes, was first identified by sequencing the internal transcribed spacer region of a nuclear DNA encoding gene cluster deposited in GenBank (accession number MW282328). Before optimization of xylanase production, A. terreus produced 20.23 U/g of xylanase after 7 days using castor cake as a substrate in a solid-state fermentation (SSF) system that was employed to achieve ricin detoxification and stimulate xylanase production. Physicochemical parameters for the production of xylanase were optimized by using a one-variable-at-a-time approach and two statistical methods (two-level Plackett–Burman design and central composite design, CCD). The maximum xylanase yield after optimization was increased by 12.1-fold (245 U/g). A 60–70% saturation of ammonium sulfate resulted in partially purified xylanase with a specific activity of 3.9 IU/mg protein. At 60 °C and pH 6, the partially purified xylanase had the highest activity, and the activation energy (Ea) was 23.919 kJmol. Subsequently, antioxidant capacity and cytotoxicity tests in normal Ehrlich ascites carcinoma human cells demonstrated xylooligosaccharides produced by the xylanase degradation of xylan as a potent antioxidant and moderate antitumor agent. Further investigations with sodium dodecyl sulfate polyacrylamide gel electrophoresis then determined the molecular weight of partially purified xylanase C to be 36 kDa. Based on the conserved regions, observations revealed that xylanase C belonged to the glycosyl hydrolase family 10. Next, the xylanase-encoding gene (xynC), which has an open reading frame of 981 bp and encodes a protein with 326 amino acids, was isolated, sequenced, and submitted to the NCBI GenBank database (accession number LC595779.1). Molecular docking analysis finally revealed that Glu156, Glu262, and Lys75 residues were involved in the substrate-binding and protein-ligand interaction site of modeled xylanase, with a binding affinity of −8.7 kcal. mol−1. ConclusionThe high production of safe and efficient xylanase could be achieved using economical materials such as Ricinus communis.

Fungal Interactions Strengthen the Diversity-Functioning Relationship of Solid-State Fermentation Systems.

ABSTRACTTraditional fermentation processes are driven by complex fungal microbiomes. However, the exact means by which fungal diversity affects fermentation remains unclear. In this study, we systematically investigated the diversity of a fungal community and its functions during the multibatch Baijiu fermentation process. Metabolomics analysis showed that the metabolic profiles of the Baijiu were enhanced with an increase in the fermentation time, as determined from the characteristic volatile flavors. High-throughput sequencing technology revealed that the major fungal species involved in sauce-flavor Baijiu fermentation are Pichia sp. (41.75%, average relative abundance), Saccharomyces sp. (13.07%), thermophilic species (9.16%), Monascus sp. (6.80%), Aspergillus sp. (4.69%), Schizosaccharomyces sp. (3.76%), Thermomyces sp. (3.74%), and Zygosaccharomyces sp. (1.41%). In addition, the fungal diversity increased as the number of fermentation batches increased. Moreover, the increased fungal diversity contributed to the modularity of the fungal communities, wherein Pichia sp., Torulaspora sp., and Saccharomyces sp. maintained the stability of the fungal community. In addition, metatranscriptomics sequencing technologies were used to reconstruct the key metabolic pathways during fermentation, and it was found that the increased microbial diversity significantly promoted glucose-mediated carbon metabolism. Finally, functional gene analysis showed that functional microorganisms, such as Zygosaccharomyces and Pichia, can enhance fermentation as a result of the high expression of pyruvate decarboxylase and propanol-preferring alcohol dehydrogenase during the metabolism of pyruvate. These results indicate that fungal biodiversity can be exploited to enhance fermentation-based processes via network interactions and metabolism during multiple-batch fermentation.IMPORTANCE Biodiversity and network interactions act simultaneously on the microbial community structure in the Baijiu fermentation process, thereby rendering the microbiome dynamics challenging to manage and predict. Understanding the complex fermentation community and its relationship to community functions is therefore important in the context of developing improved fermentation biotechnology systems. Our work demonstrates that multiple-batch fermentation steps increase microbial diversity and promote community stability. Crucially, the enhanced modularity in the microbial network increases the metabolism of flavor compounds and ethanol. This study highlights the power of biodiversity and network interactions in regulating the function of the microbiome in food fermentation ecosystems.