Fungal Solid-state Fermentation Research Articles

Response surface optimization of lipase production by <i>Pseudomonas sp.</i> on a low-cost shea-nut cake using solid state fermentation

Shea nut cake (SNC) is the solid residue produced after the shea butter extraction process. The SNC could provide a low-cost substrate for bacterial lipase production. Greater yield are among the benefits Solid State Fermentation (SSF). However, there have been less instances of lipase production by bacteria than fungi in SSF. The current study aimed at improving bacterial lipase production on SNC in SSF using Response Surface Methodology. Lipase-producing bacteria (LPB) were isolated from the shea butter mill effluent pond and screened for lipase production on a tributyrin agar plate. The influence of four independent parameters (temperature, pH, moisture content and biosurfactant) on lipase production was studied using Box Behnken Design (BBD) of RSM. Results showed that isolate AO (Pseudomonas sp.) displayed the highest zone of hydrolysis 2 (53 mm). A quadratic regression with R = 0.931 showed that the model is best fitting, and predicted the optimum conditions of significant (P>0.05) growth parameters, including temperature at 40°C, moisture content at 50%, and biosurfactant at 2 mL, which produced 408.25 U/g of experimental lipase. The findings indicate that the model was useful for predicting optimal growth conditions for incresing lipase production in SSF and the low-cost agro- industrial SNC could be a promising substrate for industrial applications.

Improving Soluble Phenolic Profile and Antioxidant Activity of Grape Pomace Seeds through Fungal Solid-State Fermentation.

Grape pomace seeds contain abundant phenolic compounds, which are also present in both soluble and insoluble forms, similar to many other plant matrices. To further increase the extractable soluble phenolics and their antioxidant activities, grape pomace seeds were fermented with different fungi. Results showed that solid-state fermentation (SSF) with Aspergillus niger, Monascus anka, and Eurotium cristatum at 28 °C and 65% humidity had a significantly positive impact on the release of soluble phenolics in grape pomace seeds. Specifically, SSF with M. anka increased the soluble phenolic contents by 6.42 times (calculated as total phenolic content) and 6.68 times (calculated as total flavonoid content), leading to an overall improvement of antioxidant activities, including DPPH (increased by 2.14 times) and ABTS (increased by 3.64 times) radical scavenging activity. Furthermore, substantial changes were observed in the composition and content of individual phenolic compounds in the soluble fraction, with significantly heightened levels of specific phenolics such as chlorogenic acid, syringic acid, ferulic acid, epicatechin gallate, and resveratrol. Notably, during M. anka SSF, positive correlations were identified between the soluble phenolic content and hydrolase activities. In particular, there is a strong positive correlation between glycosidase and soluble phenols (r = 0.900). The findings present an effective strategy for improving the soluble phenolic profiles and bioactivities of grape pomace seeds through fungal SSF, thereby facilitating the valorization of winemaking by-products.

Bioremediation of Diesel-Contaminated Soil by Fungal Solid-State Fermentation.

To address the poor removal of diesel in soil by indigenous microorganisms, we proposed a fungal solid-state fermentation (SSF) method for bioremediation. We screened Pycnoporus sanguineus 5.815, Trametes versicolor 5.996, and Trametes gibbosa 5.952 for their diesel-degrading abilities, with Trametes versicolor 5.996 showing the most promise. The fungal inoculum was obtained through SSF using wood chips and bran. Trametes versicolor 5.996 was applied to two treatments: natural attenuation (NA, diesel-contaminated soil) and bioremediation (BR, 10% SSF added to diesel-contaminated soil). Over 20 days, NA removed 12.9% of the diesel, while BR achieved a significantly higher 38.3% degradation rate. BR also increased CO2 and CH4 emissions but reduced N2O emissions. High-throughput sequencing indicated SSF significantly enriched known diesel-degrading microorganisms like Ascomycota (83.82%), Proteobacteria (46.10%), Actinobacteria (27.88%), Firmicutes (10.35%), and Bacteroidota (4.66%). This study provides theoretical support for the application of fungal remediation technology for diesel and improves understanding of microbiologically mediated diesel degradation and soil greenhouse gas emissions.

Solid-state fungal fermentation transforms low-quality plant-based foods into products with improved protein quality

To meet global protein demand sustainably in the future, we have to move to alternative, non-animal sources. A problem of many plant-derived foods is their low protein quality compared to animal-based proteins, in particular their deficiency in lysine. To improve the protein quality, plant proteins could be converted to fungal proteins using solid-state fungal fermentation (SSFF). In this project Rhizopus microsporus var. oligosporus and Aspergillus oryzae were used to ferment barley and rice, producing tempeh and koji, respectively. SSFF yielded products with 6–13% (dry weight basis) fungal biomass. Protein quality was defined by the parameters indispensable amino acid index (IAAI) and protein digestibility corrected amino acid score (PDCAAS). SSFF improved both parameters in all samples. Lysine was the limiting amino acid in unfermented barley and rice, with PDCAAS of 0.54 and 0.57, respectively. SSFF increased the PDCAAS of lysine by approximately 30% in barley koji, barley tempeh and rice koji and 10% in rice tempeh. These results demonstrate the use of SSFF to improve the protein quality of staple foods. Thereby, this fermentation method can aid in meeting protein demands sustainably in the future through plant-based diets.

Production of itaconic acid from sweet potato (Ipomoea batata) peel using naturally occurring fungi in solid state fermentation

This study evaluates the potentials of sweet potato peel as a substrate for itaconic acid production in solid state fermentation using naturally occurring fungi. Sweet potato peel was analyzed for proximate composition using standard methods. Fungi were obtained from the peel by solid state fermentation and identified using microscopic and molecular methods. Fermentation for itaconic acid production was done using isolated fungus and Aspergillus niger (ATCC 16888) was used as control. Optimization of fermentation parameters was carried out using standard procedures. Assay for itaconic acid and total titratable acidity (TTA) were analyzed using standard methods. Reducing sugar was obtained using DNSA method. Proximate composition of the peel reveals carbohydrate content to be 79%, protein, 5% and ash, 4%. Fungus isolated was identified as Aspergillus flavus L-2482/2012. Highest itaconic acid production by A. flavus was 7.74±0.00 mg/ml on day 5 at 30 g substrate concentration. Using 2 ml of 1 x 107 spores/ml, A. flavus and A. niger produced 6.97±0.13 and 6.67±0.09 mg/ml respectively. Optimum temperature for itaconic acid production was 30°C for A. niger and A. flavus. TTA ranged between 0.04±0.00 to 0.21±0.01 and 0.05±0.00 to 0.26±0.00 mg/ml for A. niger and A. flavus respectively. The highest reducing sugar, 0.51±0.00 mg/ml was obtained at substrate concentration 40 mg/ml on day 5 by A. flavus while A. niger had reducing sugar value of 0.37±0.00 mg/ml. In conclusion, A. flavus L-2482/2012. has great potentials for itaconic acid production using sweet potato peel in solid state fermentation under optimized conditions.

Follow-up of solid-state fungal wood pretreatment by a novel near-infrared spectroscopy-based lignin calibration model

Lignin removal plays a crucial role in the efficient bioconversion of lignocellulose to fermentable sugars. As a delignification process, fungal pretreatment has gained great interest due to its environmental friendliness and low energy consumption. In our previous study, a positive linear correlation between acid-insoluble lignin degradation and the achievable enzymatic saccharification yield has been found, hereby highlighting the importance of the close follow-up of lignin degradation during the solid-state fungal pretreatment process.However, the standard quantification of lignin, which relies on the two-step acid hydrolysis of the biomass, is highly laborious and time-consuming. Vibrational spectroscopy has been proven as a fast and easy alternative; however, it has not been extensively researched on lignocellulose subjected to solid-state fungal pretreatment. Therefore, the present study examined the suitability of near-infrared spectroscopy (NIR) for the rapid and easy assessment of lignin content in poplar wood pretreated with Phanerochaete chrysosporium. Furthermore, the predictive power of the obtained calibration model and the recently published ATR-FTIR spectroscopy-based model were compared for the first time using the same fungus-treated wood data set.PLSR was used to correlate the NIR spectra to the acid-insoluble lignin contents (19.9%–27.1%) of pretreated wood. After normalization and second derivation, a PLSR model with a good coefficient of determination (RCV2 = 0.89) and a low root mean square error (RMSECV = 0.55%) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. The performance of this PLSR model was comparably good to the one obtained by ATR-FTIR (RCV2 = 0.87) while it required more extensive spectral pre-processing. In conclusion, both methods will be highly useful for the high-throughput and user-friendly monitoring of lignin degradation in a solid-state fungal pretreatment-based biorefinery concept.

Valorization of Caribbean Sargassum biomass as a source of alginate and sugars for de novo biodiesel production

Since 2011, a massive influx of pelagic brown algae Sargassum has invaded coastlines causing environmental and economic disaster. Valorizing this plentiful macroalgae can present much needed economic relief to the areas affected. Here the production of biodiesel and a high-value alginate stream using Sargassum biomass collected from the coast of Quintana Roo, Mexico is reported. Biomass was pretreated via AEA (Alginate Extraction Autohydrolysis) and enzymatic saccharification via fungal Solid State Fermentation, releasing 7 g/L total sugars. The sugar mixture was fermented using engineered Yarrowia lipolytica resulting in 0.35 g/L total lipid titer at the lab tube scale. Additionally, the capability of extracting 0.3875 g/g DW of a high-value, purified alginate stream from this material is demonstrated. The findings presented here are promising and suggest an opportunity for the optimization and scale up of a biodiesel production biorefinery for utilization of Sargassum seaweeds during seasons of high invasion.

Rapid lignin quantification for fungal wood pretreatment by ATR-FTIR spectroscopy

Lignin determination in lignocellulose with the conventional two-step acid hydrolysis method is highly laborious and time-consuming. However, its quantification is crucial to monitor fungal pretreatment of wood, as the increase of acid-insoluble lignin (AIL) degradation linearly correlates with the achievable enzymatic saccharification yield. Therefore, in this study, a new attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy method was developed to track fungal delignification in an easy and rapid manner.Partial least square regression (PLSR) with cross-validation (CV) was applied to correlate the ATR-FTIR spectra with the AIL content (19.9 %–27.1 %). After variable selection and normalization, a PLSR model with a high coefficient of determination (RCV2 = 0.87) and a low root mean square (RMSECV = 0.60 %) were obtained despite the heterogeneous nature of the fungal solid-state fermentation. These results show that ATR-FTIR can reliably predict the AIL content in fungus-treated wood while being a high-throughput method. This novel method can facilitate the transition to the wood-based economy.

Solid-state fermentation of Pleurotus ostreatus to improve the nutritional profile of mechanically-fractionated canola meal

Canola meal is an abundant and economic source of energy and nutrients for production animals, but it contains more antinutrients, less protein, and has a lower nutrient digestibility compared to soybean meal. In this study, mechanical fractionation and solid-state fungal fermentation were conducted to improve nutrient profile and reduce antinutrient content of canola meal. Mechanical processing, including milling, sieving, and air classification, was used to fractionate canola meal and redistribute its components in different fractions. Then these fractions were inoculated for fungal fermentation. The results showed that 12-day solid-state fermentation with Pleurotus ostreatus increased the protein content in all fractions by 11–18 %, decreased sinapine, glucosinolates, and phytate up to 99.8 %, 98.8 % and 75.8 %, respectively, and increased the in vitro digestibility of selective fractions. Overall, Pleurotus ostreatus-based solid-state fermentation has the potential to be an effective treatment to improve the nutritional profile of canola meal.

Fungal solid-state fermentation of food waste for biohydrogen production by dark fermentation

The dark fermentation process was evaluated for biohydrogen production from food waste through fungal solid-state fermentation (SSF). Three fungal cultures (one strain of Aspergillus tubingensis and two strains of Meyerozyma caribbica) were compared, being A. tubingensis the best hydrolyser culture for releasing soluble carbohydrates. The biochemical hydrogen potential of food waste hydrolysate (FWH) at different substrate-inoculum ratios obtained a lower hydrogen yield than untreated food waste (RFW). The highest hydrogen yield value corresponded to treatments RFW-20 and RFW-30 with 77.0 ± 2.6 and 76.9 ± 1.4 mL H2 normalized by per gram volatile solid added (NmL H2/gVSadded), respectively. The microbial community of food waste was analysed, being detected lactic-acid bacteria genera as Latilactobacillus and Leuconostoc. The presence of actively growing bacteria during the SSF could explain the lowest hydrogen yield (20.1–36.0 NmL H2/gVSadded) in the FWH treatment due to the substrate competition between lactic-acid bacteria and hydrogen-producing bacteria, where the lactic-acid bacteria were favoured by their faster growth rate.

Nutritional and antioxidant changes in lentils and quinoa through fungal solid-state fermentation with Pleurotus ostreatus

Solid-state fermentation (SSF) may be a suitable bioprocess to produce protein-vegetal ingredients with increased nutritional and functional value. This study assessed changes in phenol content, antinutrient content, biomass production and protein production resulting from the metabolic activity of Pleurotus ostreatus, an edible fungus, in lentils and quinoa over 14 days of SSF. The impact of particle size on these parameters was also assessed because the process was conducted in both seeds and flours. Fungus biomass increased during fermentation, reaching 30.0 ± 1.4 mg/g dry basis and 32 ± 3 mg/g dry basis in lentil grain and flour and 52.01 ± 1.08 mg/g dry basis and 45 ± 2 mg/g dry basis in quinoa seeds and flour after 14 days of SSF. Total protein content also increased by 20% to 25% during fermentation, in all cases except lentil flour. However, the soluble protein fraction remained constant. Regarding phytic acid, SSF had a positive impact, with a progressive decrease being higher in flours than in seeds. Regarding antioxidant properties, autoclaving of the substrates promoted the release of polyphenols, together with antioxidant activity (ABTS, DPPH and FRAP), in all substrates. However, these parameters drastically decreased as fermentation progressed. These results provide scientific knowledge for producing lentil- or quinoa-based ingredients with low antinutrient content enriched with protein fungal biomass.Graphical

Insights on fungal solid-state fermentation for waste valorization: Conidia and chitinase production in different reactor configurations

Different reactor configurations are paired with a wide variety of agro-industrial wastes of different biodegradability to produce fungal conidia by solid-state fermentation.This work presents a preliminary comparative study between packed-bed and tray reactor configurations to produce Beauveria bassiana and Trichoderma harzianum conidia using two different substrates in terms of biodegradability: rice husk or beer draff complemented with wood chips. Conidia production, mean temperature and respiration indexes have been analysed in most of the presented reactor configurations. Both strains showed higher conidia production when using beer draff complemented with wood chips as substrate due to the use of a mixture as substrate. When working with beer draff, chitinase analyses obtained similar profiles in both strains but higher overall values using TH. Conidia and chitinase production maximums were not achieved at the same time, having 2–3 days of difference depending on the strain. No significant differences in mean temperature were shown between most of the performed fermentations. As a result of the present work, further scaling of both packed bed and tray configurations using beer draff and wood chips to produce BB or TH conidia would be advisable. More experiments should be performed to optimize both conidia and chitinase productions to enhance the quality of the final product.

Applicability and information value of biocalorimetry for the monitoring of fungal solid-state fermentation of lignocellulosic agricultural by-products

The applicability of biocalorimetry for monitoring fungal conversion of lignocellulosic agricultural by-products during solid-state fermentation (SSF) was substantiated through linking the non-invasive measurement of metabolic heat fluxes to conventional invasive determination of fungal activity (growth, substrate degradation, enzyme activity) parameters. For this, the fast-growing, cellulose-utilising ascomycete Stachybotrys chlorohalonata and the comparatively slow-growing litter-decay basidiomycete Stropharia rugosoannulata were investigated as model organisms during growth on solid wheat straw. Both biocalorimetric and non-calorimetric data may suggest R (ruderal)- and C (combative)-selected life history strategies in S. chlorohalonata and S. rugosoannulata, respectively. For both species, a strong linear correlation of the released metabolic heat with the corresponding fungal biomass was observed. Species-specific YQ/X values (metabolic heat released per fungal biomass unit) were obtained, which potentially enable use of biocalorimetric signals for the quantification of fungal biomass during single-species SSF processes. Moreover, YQ/X values may also indicate different fungal life history strategies and therefore be considered as useful parameters aiding fungal ecology research.

Daily Development of Nutritional Composition of Canola Sprouts Followed by Solid-state Fungal Fermentation

Daily Development of Nutritional Composition of Canola Sprouts Followed by Solid-state Fungal Fermentation

Effect of feeding of 10 % prefermented feed on fatty acid profile and oxidation changes in chicken breast meat

Abstract The aim of this work was to evaluate the effect of addition of 10 % fermented feed (FF) into commercial broiler feed on fatty acid profile, chemical composition and oxidative stability of chicken breast meat fat. FF was prepared by the process of fungal solid-state fermentation. Application of FF into the commercial feed mixtures resulted in increased content of gamma-linolenic and oleic acid in broiler breast meat (P < 0.05). Oxidative stability of meat fat was significantly higher in experimental broilers group fed diet with the addition of 10% FF (P < 0,05) after storage period of samples for seven days at temperature of 4 °C.

Closing the loop of cereal waste and residues with sustainable technologies: An overview of enzyme production via fungal solid-state fermentation

Abstract The circular economy is one of the leading sustainable development strategies that can help utilize waste to produce new products. Cereal food waste and residues represent a significant resource of million tonnes globally and a feasible feedstock for enzyme production via environmentally-friendly solid-state fermentation (SSF). This study presents a targeted review of 487 examples of quantified enzyme production from 209 studies reported on Science Direct. The collection of data enabled the identification of the key substrates and enzymes for potential production pathways. The main substrates were wheat (53 experiments), rice (22 experiments), and corn (22 experiments), while the most common enzymes reported were xylanase, CMCase, FPase with 92, 83, and 61 examples, respectively. Their activities showed a wide range with xylanase activities 7-10 000 U/gds, CMCase activities 1.4-950 U/gds, and FPase activities 1.7-438 U/gds. Other enzymes reported were β-glucosidase, Laccase, β-xylosidase. Both single substrates and mixed substrates were included, and several mixtures showed a synergistic effect for higher enzyme activities. Xylanase from wheat bran, rice straw, and their combination showed great potential for reaching high activity and promising revenues. Scale-up experiments were reported and demonstrated increased production of enzymes as a crucial step towards industrial application. Identified improvements in transparency and knowledge sharing for cross-disciplinary research are: i) harmonized enzyme names ii) expression of enzyme activity per mass unit specifying moisture content as found in 52% of studies. Only the results using the same unit (or enabling unit conversion) can be compared and used to identify the most promising production.

Valorisation of fungal hydrolysates of exhausted sugar beet pulp for lactic acid production.

Exhausted sugar beet pulp pellets (ESBPP) were used as raw material for lactic acid (LA) fermentation. The enzymatic hydrolysis of ESBPP was performed with the solid obtained after the fungal solid-state fermentation of ESBPP as a source of hydrolytic enzymes. Subsequently, a medium rich in glucose and arabinose was obtained, which was used to produce LA by fermentation. For LA production, two Lactobacillus strains were assayed and the effects of the supplementation of the hydrolysate with a nitrogen source and the mode of pH regulation of the fermentation were investigated. Moreover, a kinetic model for LA fermentation by Lactobacillus plantarum of ESBPP hydrolysates was developed. L. plantarum produced a LA concentration 34% higher than that produced by L. casei. The highest LA concentration (30g L-1 ) was obtained with L. plantarum when the hydrolysate was supplemented with 5g L-1 yeast extract and the pH was controlled with CaCO3 . The concentration of acetic acid differed depending on the concentration of CaCO3 added, producing its maximum value with 27g L-1 CaCO3 . The proposed kinetic model was able to predict the evolution of substrates and products depending on the variation of the pH in the hydrolysate, according to the amount of CaCO3 added. ESBPP can be revalorised to produce LA. A pure LA stream or a mixture of LA and acetic acid, depending on the pH control method of the fermentation, can be produced. Thus, this control is of great interest depending on the destination of the effluent. © 2020 Society of Chemical Industry.

Animal Fat as a Substrate for Production of n-6 Fatty Acids by Fungal Solid-State Fermentation.

The method of solid-state fermentation (SSF) represents a powerful technology for the fortification of animal-based by-products. Oleaginous Zygomycetes fungi are efficient microbial cell factories used in SSF to valorize a wide range of waste and rest cereal materials. The application of this fermentation technique for utilization and biotransformation of animal-based materials represents a distinguished step in their treatment. In this study, for the first time, the strain Umbelopsis isabellina CCF2412 was used for the bioconversion of animal fat by-products to the fermented bioproducts enriched with n-6 polyunsaturated fatty acids, mainly γ-linolenic acid (GLA). Bioconversion of both cereals and the animal fat by-product resulted in the production of fermented bioproducts enriched with not just GLA (maximal yield was 6.4 mg GLA/g of fermented bioproduct), but also with high yields of glucosamine. Moreover, the fermentation on the cornmeal matrix led to obtaining bioproduct enriched with β-carotene. An increased amount of β-carotene content improved the antioxidant stability of obtained fermented bioproducts. Furthermore, the application of Fourier-transform infrared spectroscopy for rapid analysis and characterization of the biochemical profile of obtained SSF bioproducts was also studied.

Conversion of Exhausted Sugar Beet Pulp into Fermentable Sugars from a Biorefinery Approach

In this study, the production of a hydrolysate rich in fermentable sugars, which could be used as a generic microbial culture medium, was carried out by using exhausted sugar beet pulp pellets (ESBPPs) as raw material. For this purpose, the hydrolysis was performed through the direct addition of the fermented ESBPPs obtained by fungal solid-state fermentation (SSF) as an enzyme source. By directly using this fermented solid, the stages for enzyme extraction and purification were avoided. The effects of temperature, fermented to fresh solid ratio, supplementation of fermented ESBPP with commercial cellulase, and the use of high-solid fed-batch enzymatic hydrolysis were studied to obtain the maximum reducing sugar (RS) concentration and productivity. The highest RS concentration and productivity, 127.3 g·L−1 and 24.3 g·L−1·h−1 respectively, were obtained at 50 °C and with an initial supplementation of 2.17 U of Celluclast® per gram of dried solid in fed-batch mode. This process was carried out with a liquid to solid ratio of 4.3 mL·g−1 solid, by adding 15 g of fermented solid and 13.75 g of fresh solid at the beginning of the hydrolysis, and then the same amount of fresh solid 3 times every 2.5 h. By this procedure, ESBPP can be used to produce a generic microbial feedstock, which contains a high concentration of monosaccharides.

Enhanced antioxidant activity of okara through solid state fermentation of GRAS Fungi

Abstract The enhancement of antioxidant activity of fermented okara was investigated using fungal solid-state fermentation. The initial fungal concentration and water activity were found to influence the enhanced antioxidant capacity of fermented okara. Extracellular enzymes, especially proteases and carbohydrases might facilitate the hydrolysis of okara protein that yielded antioxidative amino acids and peptides, as well as the release of isoflavones-derived components. Fermentation of okara with R. oligosporus with 1.04×10-3 gDCW/gokara as initial concentration, water activity of 0.931 was found to yield the highest antioxidant strength. Whereas for A. Oryzae, 1.6×10-3 gDCW/gokara was optimum as the initial concentration with water activity of 0.913. With these optimum conditions, solid-state fermentation of okara could enhance the antioxidant activity up to 2.6- and 5.5-fold, respectively for R. oligosporus and A. oryzae. Besides having an enhanced antioxidant activity, the fermented okara also had higher protein content, amino nitrogen content, and protein digestibility.