Solid-state Culture Research Articles

Production of Cellulolytic Enzymes by Paecilomyces TP08 Using Grape Stalk and Wheat Bran as Substrates in Solid State Culture

Objective: The objective of this study is to investigate the use of grape stems and wheat bran as substrates in solid state fermentation, for the production of cellulolytic enzymes using the fungus Paecilomyces TP08 sp. Theoretical Framework: In grape production, driven by increased consumption and exports, by-products represent 30% of the total volume of grapes processed for wine, requiring efficient management to ensure sustainability. Method: The methodology adopted for this research involves solid-state cultivation, with a substrate made up of 50% grape stems and 50% wheat bran, with samples taken every 24 hours. Each sample taken went through a filtration process, followed by enzyme activity analysis using the DNS method. Results and Discussion: The results obtained showed that the cultivation with the association of wheat bran and grape stalks presented good total cellulase activity during the process, value of 0.846 U/g in 144h, followed by the activity of 0.663U/g observed in 24h. Research Implications: The practical and theoretical implications of this research are discussed, providing insights into how the results can be applied or influence practices in the field of industrial biotechnology. These implications could cover the food, textile and pharmaceutical industries. Originality/Value: This study contributes to the literature by associating grape stalks with wheat bran for the production of fungal enzymes. The relevance and value of this research are evidenced by the use of an innovative substrate (grape stalks) associated with a conventional substrate (wheat bran) for induction of the microorganism little used in the literature for enzyme production.

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.

Solid-state cultivation of multiple industrial strains of koji mold on different Thai unpolished rice cultivars: biotransformation of phenolic compounds and their effects on antioxidant activity.

Colored rice is abundant in polyphenols, and koji molds have potential for biotransformation. This study aimed to produce Thai-colored rice koji to study its polyphenolic biotransformation. Four industrial koji mold strains: Aspergillus oryzae 6001, A. oryzae 6020, A. sojae 7009, and A. luchuensis 8035, were cultivated on unpolished Thai-colored rice (Riceberry and Sangyod), unpolished Thai white rice (RD43), and polished Japanese white rice (Koshihikari). We discovered that koji molds grew on all the rice varieties. Methanol extracts of all rice kojis exhibited an approximately 2-fold or greater increase in total phenolic content and DPPH antioxidant activity compared to those of steamed rice. Moreover, quercetin, quercetin-3-O-glucoside, isorhamnetin-3-O-glucoside, ferulic acid, caffeic acid, protocatechuic acid, vanillic acid, (+)-catechin, and (-)-epicatechin content increased in Riceberry and Sangyod koji samples. Consequently, Aspergillus solid-state cultivation on unpolished Thai-colored rice exhibited higher functionalization than the cultivation of unpolished Thai white rice and polished Japanese white rice.

Hydrogel-based photobioreactor for Solid-State cultivation of Chlorella vulgaris

Solid-state cultivation is a promising technology for algal biomass production, achieving high productivities without the need for dewatering. However, such systems have suffered from high evaporation, and capital costs. Here is described a hydrogel photobioreactor (hPBR) with the aim of reducing water demand in solid-state cultivations. Two designs are described with “Design A” offering better humidity control overgrowth conditions. A biomass productivity of 2.41gm-2d-1, and 2.87gm-2d-1 when using physically crosslinked poly(vinyl alcohol) (pPVA) and chemically crosslinked PVA (cPVA) respectively were achieved with Chlorella vulgaris with a water demand around 0.44 kg g−1 of biomass. Over the 23 days of growth, the lipid content increased from 18.9 % to 56.6 % and 13.8 % to 43.2 % for pPVA and cPVA respectively, and the chlorophyll content decreased by more than 81 %. However, cell viability stayed high at over 98 % and surface coverage analysis showed good coverage of the gel surface.

Production of bacterial protease via solid-state cultivation with agro-industrial by-products and eco-friendly perspectives of enzyme application

Production of bacterial protease via solid-state cultivation with agro-industrial by-products and eco-friendly perspectives of enzyme application

Mortierella Ramanniana Lipid Fermentation Wastewater as an Innovative Maceration Liquid Medium for Sustainable Solid-State Cultivation of Higher Fungi

Mortierella Ramanniana Lipid Fermentation Wastewater as an Innovative Maceration Liquid Medium for Sustainable Solid-State Cultivation of Higher Fungi

Crecimiento y producción de invertasa por Neurospora crassa en cultivos sumergido y sólido

Filamentous fungi are widely used in industry to produce enzymes, organic acids, and secondary metabolites. Neurospora crassa has gained attention due to its flexibility for easy genetic manipulation, fast growth, and non-pathogenic characteristics. This study evaluated the growth of N. crassa and invertase production in submerged culture (SC) and solid-state culture (SSC) using pine sawdust (SSC-PS) and polyurethane foam (SSC-PUF) as supports. Modified Vogel's medium with initial sucrose concentrations of 1.5%, 3%, and 5% was used. The specific and maximum CO2 production rates were higher in SSC than SC, particularly with SSC-PS. Protein and invertase production were higher in SSC, with SSC-PS demonstrating the highest sucrose concentration as the inducer and carbon source. The water-holding capacity (WHC) of SSC-PUF was approximately 25 times higher than that of SSC-PS, facilitating a more productive process. SSC-PUF enables higher biomass growth, protein, and invertase production than SC. Moreover, using inert supports, such as PUF, allows the correct substrates and product assessment without interferences due to the chemical composition and heterogeneity of conventional agro-industrial by-products, such as PS.

Airlift bioreactor–based strategies for prolonged semi-continuous cultivation of edible Agaricomycetes

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h−1. A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h−1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h−1). However, growth at a D of 0.1 h−1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h−1, a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h−1 and 0.07 h−1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h−1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h−1, while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h−1.Key points• The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h.• Semi-continuous fermentation at D of 0.02 h−1 resulted in 13.4 g/L of fungal biomass.• Semi-continuous fermentation at D of 0.07 h−1 resulted in fungal biomass with 28% of total glucans.

Novel Culture Media Made from Sago Pith Waste Used to Culture the Antagonistic Rhizobacterium Streptomyces angustmyceticus NR8-2

Sag o pith waste (SPW) is an agricultural by-product from the sago starch extraction process. Using SPW supports the bio-circular green economy and sustainable agriculture. Although many bacterial culture media are commercially available, using SPW as a carbon source to develop culture media is considered an alternative way to save costs and limit the import of commercial media. This research aimed to develop culture media from SPW to culture the antagonistic rhizobacterium Streptomyces angustmyceticus NR8-2 and test the antifungal activity of the cell-free culture filtrate (CF) of S. angustmyceticus NR8-2 against Curvularia pseudobrachyspora, the pathogen of brown spot disease in dragon fruit. S. angustmyceticus NR8-2 was cultured on glucose yeast malt agar (GYMA) and glucose yeast malt broth (GYMB) as general media, modified sago pith waste yeast agar (SPYA), and sago pith waste yeast broth (SPYB); then, the bacterial growth was measured. S. angustmyceticus NR8-2 grew on SPYA similarly to on GYMA, and there was no morphological change when it was cultured on SPYA. S. angustmyceticus NR8-2 could also be cultured on both GYMB and modified SPYB, as indicated by spherical pellet development, but the biomass of S. angustmyceticus NR8-2 cultured on SPYB was significantly higher than that on GYMB (p<0.05). The biofilm formation of S. angustmyceticus NR8-2 on the surface of SPYB or GYMB displayed no significant difference in dry or fresh weight (p>0.05). The cell-free CF of S. angustmyceticus NR8-2 was collected and tested against the antifungal ability of C. pseudobrachyspora via agar well diffusion. Cell-free CFs from GYMB and SPYB effectively inhibited the growth of C. pseudobrachyspora with percentage inhibitions of 60.95 and 58.99%, respectively, with no statistical difference (p>0.05). The cell-wall-degrading enzyme activity of chitinase and β-1,3-glucanase showed no significant differences when S. angustmyceticus NR8-2 was cultured on GYMB and SPYB. Furthermore, the solid-state cultivation of S. angustmyceticus NR8-2 on SPW + yeast extract and SPW + dextrose + yeast extract showed bacterial populations of 2.56±1.38 × 108 and 3.38±1.89 × 108 CFU/g, significantly higher than when cultured on SPW alone. The results of our study show that SPW is a novel source for the development of novel media that can be used to culture antagonistic rhizobacterium S. angustmyceticus NR8-2.

Changes in chemical properties and microbial communities' composition of a forest litter-based biofertilizer produced through aerated solid-state culture under different oxygen conditions.

Fermented forest litter (FFL) is a bioproduct used as biofertilizer for several decades in Eastern Asia and Latin America. It is locally handcrafted by farmers in anaerobic conditions by fermenting forest litter added with agricultural by-products such as whey, cereal bran, and molasses. The aim of this study was to characterize the FFL process and product through gas and liquid chromatography analyses. It also provides some highlights on the influence of O2 on this solid-state culture. Under anoxic condition, a maximum CO2 production rate (CDPR) of 0.41mL/h∙g dry matter (dm) was reached after 8days. The main volatile organic compounds (VOCs) were ethanol and ethyl acetate, with a production rate profile similar to CDPR. After 21days of culture, no residual sucrose nor lactose was detected. Lactic and acetic acids reached 58.8mg/g dm and 10.2mg/g dm, respectively, ensuring the acidification of the matrix to a final pH of 4.72. A metabarcoding analysis revealed that heterolactic acid bacteria (Lentilactobacillus, Leuconostoc), homolactic acid bacteria (Lactococcus), and yeasts (Saccharomyces, Clavispora) were predominant. Predicted genes in the microbiome confirmed the potential link between detected bacteria and acids and VOCs produced. When O2 was fed to the cultures, final pH reached values up to 8.5. No significant amounts of lactic nor acetic acid were found. In addition, a strong shift in microbial communities was observed, with a predominance of Proteobacteria and molds, among which are potential pathogens like Fusarium species. This suggests that particular care must be brought to maintain anoxic conditions throughout the process.

A formulation of combined Poria cocos and Cordyceps militaris rice ameliorates depressive-like effects by downregulating p38 MAPK signaling pathways

Background and aimPoria cocos (PC) is traditional Chinese medicine with sedative, diuretic, and tonic properties. Cordyceps militaris rice (CMR) is a matrix used for the solid-state culture of C. militaris, which has been used for anti-angiogenetic and anti-inflammation effects. To investigate a formulation of combined Poria cocos and Cordyceps militaris rice (PC-CMR) alleviates anxiety behaviours and depressive-like effects for the first time. Experimental procedureAfter five-week of stress, rats were induced by unpredictable chronic mild stress (UCMS) treated with PC-CMR formulation. Then, rats underwent various behavior tests, testing serum inflammatory mediators by enzyme-linked immunosorbent assays (ELISA), evaluated monoamines by high-performance liquid chromatography (HPLC) analysis, and protein expression by western blotting. ResultsTreatment with doses of formulated PC-CMR reversed sucrose preference and increased amount of time spent on the open arms in rat behavioral tests. The formulated PC-CMR significantly reduced glutamate content and turnover rates of serotonin and dopamine in the prefrontal cortex of rats. In the inflammation-related pathway, PC-CMR decreased interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels in serum and downregulated the expression of p38 mitogen-activated protein kinase (p38 MAPK), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in the prefrontal cortex and signal transducer and activator of transcription 3 (STAT3), and indoleamine 2,3-dioxygenase (IDO) in the amygdala. ConclusionOur findings suggest that the formulated PC-CMR could have antidepressive and anxiolytic effects by modulating neurotransmitter levels and reducing inflammation.

Leaching Approach for β-Glucosidase Extraction from Fermented Rice Husk in Solid State Cultivation by Aspergillus protuberus.

Environmental problems are caused by the disposal of agrowastes in developing countries. It is imperative to convert such wastes into useful products, which require enzymes such as β-glucosidase. β-Glucosidase has variety of applications in biotechnology including food, textile, detergents, pulp and paper, pharmaceutical and biofuel industries. β-Glucosidase production was performed using the locally isolated Aspergillus protuberus using best growth circumstances on rice husk in solid-state fermentation (SSF). Leaching of β-glucosidase from fermented rice husk with number of solvents to evaluate their extraction efficacy. Among the different solvents examined, acetate buffer (0.02M, pH 5.0) proved to be the best solvent. The subsequent parameters were optimized with acetate buffer. Two washes with acetate buffer each by shaking (30min) in a ratio of 1g of rice husk: 5ml of acetate buffer together attained maximum recovery of β-glucosidase with 41.95 U/g of rice husk.

Spray-drying of xylanases produced by Myceliophthora thermophila under solid-state cultivation

Spray-drying of xylanases produced by Myceliophthora thermophila under solid-state cultivation

The hidden treasures in endophytic fungi: a comprehensive review on the diversity of fungal bioactive metabolites, usual analytical methodologies, and applications.

This review provides a comprehensive overview of the key aspects of the natural metabolite production by endophytic fungi, which has attracted significant attention due to its diverse biological activities and wide range of applications. Synthesized by various fungal species, these metabolites encompass compounds with therapeutic, agricultural, and commercial significance. We delved into strategies and advancements aimed at optimizing fungal metabolite production. Fungal cultivation, especially by Aspergillus, Penicillium, and Fusarium, plays a pivotal role in metabolite biosynthesis, and researchers have explored both submerged and solid-state cultivation processes to harness the full potential of fungal species. Nutrient optimization, pH, and temperature control are critical factors in ensuring high yields of the targeted bioactive metabolites especially for scaling up processes. Analytical methods that includes High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-Mass Spectrometry (LC-MS), Gas Chromatography-Mass Spectrometry (GC-MS), Nuclear Magnetic Resonance (NMR), and Mass Spectrometry (MS), are indispensable for the identification and quantification of the compounds. Moreover, genetic engineering and metabolic pathway manipulation have emerged as powerful tools to enhance metabolite production and develop novel fungal strains with increased yields. Regulation and control mechanisms at the genetic, epigenetic, and metabolic levels are explored to fine-tune the biosynthesis of fungal metabolites. Ongoing research aims to overcome the complexity of the steps involved to ensure the efficient production and utilization of fungal metabolites.

Supercritical CO2 extraction of solid-state cultivation fungus producing azaphilone polyketides

Supercritical Fluid Extraction (SFE) methods dedicated to microorganisms specialized metabolites are very scarce in the literature and limited to liquid cultivation. We proposed here a new sample preparation method to achieve SFE of specialized metabolites from solid-state cultivation. SFE parameters, including CO2 pressure, temperature of extraction cell and percentage of co-solvent, were optimized in the case of solid-state cultures of Penicillium sclerotiorum SNB-CN111, a filamentous fungus producing azaphilone pigments. The metabolic composition of the extracts was then analyzed by reverse-phase liquid chromatography coupled to electrospray ionization and tandem mass spectrometry in data dependent acquisition mode. The resulting molecular networks generated by MetGem software allowed the annotation of the extracted metabolites in the different conditions, confirming the enrichment of fractions according to the polarity of azaphilone subfamilies. First, the 100 % CO2 fraction a yield ten times higher than hexane maceration The optimization of SFE method led to an extraction yield twice as high as ethyl acetate maceration when mixing CO2 with ethanol and, indicating that CO2/ethanol SFE is more environmentally-friendly and efficient than standard maceration methods for the extraction of azaphilones from solid-state fermentation.

Designing sustainable circular bioeconomy solutions for the pulse industry: The case of crude pea starch as a substrate for single cell protein production

Valorization of crude pea starch has become a key focus in the pea industry's sustainability pursuit. This study aimed to explore the circularity potential of crude pea starch as a nutrient-dense substrate for the solid-state cultivation of yeast (Saccharomyces cerevisiae) Single Cell Protein (SCP). Following the ISO 2006:14040/44 standard, a life cycle assessment (LCA) was performed to ascertain the environmental performance and operational dynamics of baseline and scenario pea starch-based yeast SCP process designs and identify optimal design considerations. Results demonstrated a higher relative contribution to the toxicity categories, with a relatively less contribution to global warming and land use. The distribution and media enrichment processes were identified as the hotspots, contributing about 32–55 % and 40–56 % to global warming and land use, respectively. Generally, train and air freight were more sustainable than lorry freight, respective of mileage and mass. Regarding system alteration, eliminating the media enrichment process could offset about 26 % of land footprint, with a similar trend for most impact categories. Process benchmarking showed up to a 3-fold reduction in global warming impacts relative to soybean meal, and about 71 % offset relative to fishmeal. Consequential LCA showed a general sustainability preference for substituting the aquacultural feeds with pea starch-based SCP, with a stronger emphasis on fishmeal substitution. Overall, these findings highlight the potential of the proposed SCP design as a sustainable upcycling solution with substitutionary potentials for conventional food and feeds, recommending further exploration in value and wealth creation.

Copper ion (Cu2+) is involved in the transcription of the tyrosinase-encoding melB gene of Aspergillus oryzae in solid-state culture.

In Aspergillus oryzae, the tyrosinase-encoding gene melB causes undesirable browning of sake and sake lees. This gene is known to be expressed specifically in solid-state culture; however, its expression mechanisms remain unknown. Here, we evaluated the possible factors affecting the transcription of melB and found that the copper ion (Cu2+) significantly enhanced the transcription level of melB in solid-state culture.

Solid state culture conditions for improved conidial production of the mycoparasitic fungus Escovopsis weberi

ABSTRACT We evaluated culture conditions (temperature, substrate moisture content, agitation and light conditions) for optimising conidia production of Escovopsis weberi Ew1 using solid state culture. In addition, several substrates such as agricultural waste and food industry by-products were evaluated with the aim of reducing spore production costs in a strategic way towards circular bioeconomy. Finally, we selected substrate combinations in order to achieve a suitable system for the production of conidia.

Expression of heterochromatin protein 1 affects citric acid production in Aspergillus luchuensis mut. kawachii

A putative methyltransferase, LaeA, controls citric acid production through epigenetic regulation of the citrate exporter gene, cexA, in the white koji fungus Aspergillus luchuensis mut. kawachii. In this study, we investigated the role of another epigenetic regulator, heterochromatin protein 1, HepA, in citric acid production. The ΔhepA strain exhibited reduced citric acid production in liquid culture, although to a lesser extent compared to the ΔlaeA strain. In addition, the ΔlaeA ΔhepA strain showed citric acid production similar to the ΔlaeA strain, indicating that HepA plays a role in citric acid production, albeit with a less-significant regulatory effect than LaeA. RNA-seq analysis revealed that the transcriptomic profiles of the ΔhepA and ΔlaeA strains were similar, and the expression level of cexA was reduced in both strains. These findings suggest that the genes regulated by HepA are similar to those regulated by LaeA in A.luchuensis mut. kawachii. However, the reductions in citric acid production and cexA expression observed in the disruptants were mitigated in rice koji, a solid-state culture. Thus, the mechanism by which citric acid production is regulated differs between liquid and solid cultivation. Further investigation is thus needed to understand the regulatory mechanism in koji.

PRODUCTION, VIRULENCE, AND VIABILITY OF Beauveria bassiana (Bals.) Vuill. CONIDIA OBTAINED BY FERMENTATION OF AMARANTH STUBBLE

Entomopathogenic fungi biological formulations require viable concentrations of infective units (conidia) with high virulence. These are mainly produced by solid-state cultivation of agro-industrial residues with high C/N ratios as substrates, such as amaranth stubble (Amaranthus hypochondriacus L.), which is discarded in the fields due to its lack of postharvest utility and thus becomes a source of contamination. Therefore, its use can reduce production costs when compared to common substrates such as rice (Oryza sativa). The objective of this work was to compare the effect of amaranth stubble on the production, virulence, and viability of Beauveria bassiana (Bals.) Vuill. (Hypocreales: Cordycipitaceae) conidia produced by solid-state cultivation to those produced using rice as a substrate. The results showed that the yield of B. bassiana conidia produced with rice was 15 higher than that produced with amaranth stubble. However, there were no significant differences in the virulence and viability of conidia produced by both substrates. Therefore, the use of amaranth stubble is an economical alternative to produce B. bassiana conidia compared to common substrates like rice.