Enzyme Production In Solid-state Fermentation Research Articles

Enzymatic One-Pot Hydrolysis of Extracted Sugar Beet Press Pulp after Solid-State Fermentation with an Engineered Aspergillus niger Strain

Extracted sugar beet press pulp (SBPP) is a promising agricultural residue for saccharification and further bioconversion. Combining solid-state fermentation of SBPP with engineered Aspergillus niger for enzyme production followed by hydrolysis of additionally added SBPP in the same bioreactor was studied to produce a sugar solution (hydrolysate) in a one-pot process. The initial aerobic solid-state fermentations were carried out in duplicate on non-milled, wet SBPP (moisture content of 72% (w/v)) with an A. niger strain engineered for constitutive pectinase production for 96 h, and this resulted in polygalacturonase activities of up to 256 U mL−1 in the wet media. Afterwards, water was added to the bioreactor, and the remaining solids were suspended by stirring to dissolve the hydrolytic enzymes. Metabolic activities of A. niger were inactivated by a N2-atmosphere and by increasing the temperature to 50 °C. High solid loads of milled SBPP were added to the stirred-tank reactor with a delay of 24 h to enable sugar yield calculations based on the compositional analysis of the SBPP used. The resulting final sugar concentrations of the hydrolysate after 166 h were 17 g L−1 d-glucose, 18.8 g L−1 l-arabinose, and 12.5 g L−1 d-galacturonic acid, corresponding to sugar yields of 98% d-glucose, 86% l-arabinose, and 50% d-galacturonic acid, respectively. Including the other sugars released during enzymatic hydrolysis in the one-pot process (d-xylose, d-mannose, d-galactose), a total sugar concentration of 54.8 g L−1 was achieved in the hydrolysate. The one-pot process combining hydrolytic enzyme production in solid-state fermentation with high solid loads during enzymatic hydrolysis of the milled SBPP reduces hydrolytic process costs by replacing chemical pre-treatments, enabling the in situ production of SBPP-adapted hydrolytic enzymes, as well as avoiding intermediate enzyme extraction and preparation steps.

Coconut waste valorization to produce biochar catalyst and its application in cellulose-degrading enzymes production via SSF

Solid waste management and waste valorization are key concerns and challenges around the globe. Solid wastes generated by food industries are found in a diverse variety, are key sources of enormously valuable compounds, and can be effectively transformed into useful products for broad industrial applications. Biomass-based catalysts, industrial enzymes, and biofuels are some of the very prominent and sustainable products that are developed using these solid wastes. The aims of the current study are therefore centered on the multiple valorizations of coconut waste (CWs) to develop biochar as a catalyst and its application in fungal enzyme production in solid-state fermentation (SSF). Biochar as a catalyst using CWs has been prepared via a calcination process lasting 1 h at 500 °C and characterized through X-ray diffraction, Fourier-transformed infrared spectroscopy, and scanning electron microscope techniques. The produced biochar has been implemented for boosting enzyme production through SSF. In addition, studies have been performed on enzyme production with varying time and temperature, and it is found that the maximum 92 IU/gds BGL enzyme could be produced at a 2.5 mg concentration of biochar-catalyst at 40 °C in 72 h.

Solid-state fermentation increases secretome complexity in Aspergillus brasiliensis

Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF. Although differences related to lower catabolite repression and substrate inhibition, as well as higher extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying such differences are still unknown. To explain some differences among SSF and SmF, the secretome of Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most abundant and upregulated protein found in SSF was the transaldolase, which could perform a moonlighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins, and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion. All these differences can be related to the fact that molds are more specialized to growth in solid materials because they mimic their natural habitat.

Extracellular L-asparaginase production in solid-state fermentation by using sugarcane bagasse as support material

L-asparaginase is an important enzyme used in the pharmaceutical and food industry, which can be produced by different microorganisms using low cost feedstocks. In this work, sugarcane bagasse (SCB) was used as support for enzyme production in solid-state fermentation (SSF) by A. terreus. Initially, the influence of the variables carbon and nitrogen sources on the enzyme production was studied following an experimental design carried out in Erlenmeyer flasks. Statistical analysis indicated the use of 0.54% of starch, 0% of maltose, 0.44% of asparagine, and 1.14% of glutamine in the medium, resulting in enzyme activity per volume of produced extract of 120.723 U/L. Then, these conditions were applied in a horizontal column reactor filled with SCB, producing 105.3 U/L of enzyme activity. Therefore, the potential of extracellular L-asparaginase enzyme production in the column reactor using sugarcane bagasse as support was demonstrated and it represents a system that can favor large scale production.

Aproveitamento de resíduos da agroindústria do óleo de dendê para a produção de lipase por Aspergillus Níger

RESUMO: Neste trabalho, foi avaliado o desempenho de duas linhagens de Aspergillus niger (mutante 11T53A14 e selvagem C) previamente selecionadas como promissoras para a produção de lipases, utilizando meios de cultivo formulados a partir da torta de dendê (palmiste) e da borra alcalina do refino do óleo de dendê (borra de dendê), resíduos provenientes da agroindústria do óleo de dendê (palma) por fermentação em estado sólido (FES). Os experimentos de produção da enzima em FES foram conduzidos em colunas aeradas, incubadas em banho-maria a 32ºC com entrada controlada de ar não umedecido de 1,0vvm. Os melhores resultados foram obtidos com a cepa mutante A. niger 11T53A14 em meio contendo torta de dendê umedecida com solução sulfato de amônio (1,2%) e com a adição de 3% da borra de dendê. O valor máximo da atividade da lipase neste meio foi de 72,57U gss-1 em 48 horas. Esse valor foi 47,5% superior ao obtido no meio sem a borra de dendê. A comparação do desempenho da cepa mutante com a cepa selvagem mostrou que o meio composto por torta de dendê adicionado com sulfato de amônio e borra de dendê induziu ambas as linhagens a produzir lipases com bons níveis de atividade, além de reduzir o tempo de processo de fermentação.

Lignocellulolytic Enzyme Production in Solid-State Fermentation of Corn Stalk with Ammoniation Pretreatment by Lentinus edodes L-8

This study investigated the effect of ammoniation pretreatment of corn stalk (CS) with different temperatures, ammonia proportions, and processing times on lignocellulolytic enzyme production in solid-state fermentation (SSF) by Lentinus edodes L-8. The total N content and lignocellulose contents of ammoniated corn stalk were determined for analysis of the effect of ammoniation pretreatment on lignocellulose structure. The variation patterns of enzyme activity were analyzed according to the enzyme data determined every 2 days during the fermentation. A 4% w/w high-temperature ammoniation pretreatment had a significant effect on cellulase production, and the highest enzyme activity reached almost triple that of the control group. The results also showed that ammoniation pretreatment inhibited the generation of ligninases, such that ligninases appeared later and at lower activities in experimental groups compared to the control group.

Comparative characterization of proteins secreted by Neurospora sitophila in solid-state and submerged fermentation

Although submerged fermentation (SmF) accounts for most of current enzyme industries, it has been reported that solid-state fermentation (SSF) can produce higher enzyme yields in laboratory scale. In order to understand the reasons contributing to high enzyme production in SSF, this study compared the cellulase activities and secretomes of Neurospora sitophila cultured in SSF and SmF using steam exploded wheat straw as carbon source and enzyme inducer. The total amounts of protein and biomass (glucosamine content) in SSF were respectively 30 and 2.8 times of those inSmF. The CMCase, FPA and β-glucoside activities in SSF were 53-181 times of those in SmF. Both in SSF and SmF,N.sitophila secreted the most critical cellulases and hemicellulases known for Trichoderma reesei, although a β-xylosidasewas exclusively identified in SSF. Six endoglucanases were identified in N.sitophila secretion with the high CMCase activity. The non-enzyme proteins in SSF were involved in fungal mycelia growth and conidiation; while those in SmF were more related to glycometabolism and stress tolerance. This revealed that SSF more likely serves as a natural habitat for filamentous fungi to facilitate the enzyme secretion.

Production of Acid-Stable and High-Maltose-Forming α-Amylase of Bacillus acidicola by Solid-State Fermentation and Immobilized Cells and Its Applicability in Baking

Among matrices used for immobilizing Bacillus acidicola cells [calcium alginate, chitosan + alginate, scotch brite, and polyurethane foam (PUF)], α-amylase production was highest by PUF-immobilized cells (9.1U ml(-1)), which is higher than free cells (7.2U ml(-1)). The PUF-immobilized cells could be reused over seven cycles with sustained α-amylase production. When three variables (moisture, starch, and ammonium sulfate), which significantly affected enzyme production in solid-state fermentation (SSF), were optimized using response surface methodology, 5.6-fold enhancement in enzyme production was attained. The enzyme production in SSF is 3.8-fold higher than that in submerged fermentation. The bread made by supplementing dough with α-amylase of B. acidicola scored better than those with the xylanase of Bacillus halodurans and thermostable α-amylase of Geobacillus thermoleovorans.

Optimization of Xylanase Production byThermomyces lanuginosusin Solid State Fermentation

Extracellular xylanase production by the thermophilic fungus Thermomyces lanuginosus 195 in solid state fermentation (SSF) was found to be significantly affected by fermentation temperature, duration, and inoculum volume (p < or = 0.001). Optimization of these parameters corresponded to a 21.7% increase in xylanase yield. Maximum activity (2,335 U/g of wheat bran) was obtained when 10 g of wheat bran was inoculated with 10 ml of liquid culture and cultivated at 45 degrees C for 40 h. The influence of supplemental carbon and nitrogen sources (3% w/v) on xylanase production was also assessed. Wheat bran, supplemented with glucose and cellulose, facilitated 10% and 7% increases in relative activity respectively. Ammonium based salts, nitrates, and a number of organic nitrogen sources served only to reduce xylanase production (p < or = 0.005) significantly. The enhanced xylanase titers achieved in the present study emphasize the need for optimizing growth conditions for maximum enzyme production in SSF.

Biotransformation of rice straw by Phanerachaete chrysosporium and the related ligninolytic enzymes

Biotransformation of lignin is related to the biomass utilisation of agricultural wastes. Ligninolytic enzyme from Phanerochaete chrysosporium plays an important role in biodegrading lignin and is specially concerned by researchers. The aim of the study is to optimise the conditions for producing ligninolytic enzymes in solid-state fermentation, and to biotransform the rice straw for the biomass utilisation. The effects of five factors on enzyme activity in state-solid medium were studied using orthogonal experiments, which were expected to offer reference for condition design of enzyme production in solid-state fermentation. The results indicated that the optimal condition for enzyme production by P. chrysosporium during solid-state fermentation could be realised when fungi were domesticated for 8 days, 0.8% fungi suspension (weight ratio) inoculated, temperature kept at 37?C, water content of the solid substrate maintained by 85% and 0.3% Tween80 added, respectively. Also, it was found that the temperature and water content were the foremost influencing factors for the ligninolytic enzyme production of P. chrysosporium during solid-state fermentation. The lignocellulose of rice straw was reduced significantly. The structures of the untreated straw and the straw degraded under the optimal fermentation condition were analysed through ultraviolet spectroscopy and infrared spectroscopy. It could be observed that long-bond and difficult-to-biodegrade hydrocarbons of giant molecule were degraded into short-bond hydrocarbons of small molecule, which could be easily biodegraded.

Optimization of lignocellulolytic enzyme production by the white-rot fungus Trametes trogii in solid-state fermentation using response surface methodology

The white-rot basidiomicete Trametes trogii (MYA 28-11) is an outstanding producer of laccase. A Doehlert experimental design was applied to optimize its lignocellulolytic enzyme production in solid-state fermentation. The impact on enzyme production of three quantitative variables, namely pH, copper and nitrogen concentrations, was investigated by using a wood-based solid medium supplemented with malt extract. Optimization was aimed at simultaneously minimizing cellulase activity and maximizing ligninolytic enzyme production. Such conditions were: pH 4.5, peptone 12.5 g l −1, and CuSO 4 11 mM. Highest activities achieved were: laccase 901 and Mn-peroxidase 20 U g −1. There was not a direct correlation between the levels of enzymatic activities and the extent of losses of wood weight or components. The factorial approach also allowed us to quantify the enzyme activity in any part of the experimental domain, consequently, it was also possible to determine a culture medium to obtain crude extracellular extracts with high laccase (510 U g −1) and endoxylanase (780 U g −1) yields. These enzymes have gained renewed interest mainly due to their applications in paper industries for pulp treatment, improving the effectiveness of conventional bleaching. Considering the results obtained, T. trogii could be an attractive source of both enzymes.

Xylanase Production by the Thermophilic Mold &lt;I&gt;Humicola lanuginosa&lt;/I&gt; in Solid-State Fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate--to--moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 x 106 spores/10 g of wheat bran at a water activity (aw) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.

Advantages of fungal enzyme production in solid state over liquid fermentation systems

The present paper attempts to explain why enzyme production in solid-state fermentation (SSF) is higher than in submerged fermentation (SmF). Recent work done in our laboratory [Biotechnol. Lett. 22 (2000) 1255; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 271; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 296] related to the production of invertase, pectinases and tannases, by Aspergillus niger grown by SSF and SmF is reviewed. To do such a comparative study, logistic and Luedeking–Piret equations are used in order to estimate the values of the following coefficients: maximal specific growth rate ( μ M), maximal biomass level ( X M), enzyme/biomass yield ( Y P / X ) and secondary rate of production, or breakdown ( k). It is shown that enzyme productivity is proportional to group, μ M Y P/ X X M, corrected by a function of ν= k/ Y P/ X μ M. In all three cases of enzyme production studied, productivity using a SSF system was higher than in SmF. Studies with invertase resulted in higher values of μ M X M. Studies with pectinases resulted in higher values of Y P/ X X M. Studies with tannases resulted in higher Y P / X and less negative values of k. Finally, a reaction–diffusion model is presented to try to explain such differences based on micrographic measurements of mycelial aggregates for each kind of fermentation system.

Hydrolytic enzyme production in solid-state fermentation by Aspergillus niger 3T5B8

A mixture containing polygalacturonase, cellulase, xylanase and protease enzymes was produced using Aspergillus niger 3T5B8 on different agroindustrial residues by solid-state fermentation and tested for vegetable oil extraction. The enzymic activities were evaluated using second-order empirical models from experimental data as a function of fermentation time and cellobiose concentration in the fermentation medium. The use of wheat bran as substrate without cellobiose addition and 42 h of fermentation were the most favourable conditions for producing the mixture of hydrolytic enzymes (polygalacturonase 30.75 U/ml, xylanase 30.62 U/ml, protease 5.27 U/ml). Another favourable condition was obtained when mango peel was used as substrate in the presence of 0.2% cellobiose and 24 h of fermentation (FPCAse 3.5 U/ml, xylanase 20.33 U/ml). Enzymic formulations with suitable polygalacturonase and FPCAse activities were favourable to extract oil from the pulp of tropical fruits and oleaginous seeds.

Effect of particle size of substrate of enzyme production in solid-state fermentation

Abstract Asperigillus niger (RRL isolate) produced about 900 units of glucoamylase/g dry substrate (gds) containing fungal biomass in solid-state fermentation using wheat bran mixed with corn flour at 10% w/w ratio as substrate of particle size 425–500 μm and 500–600 μm. However, substrates with mixed particle sizes containing particles smaller than 180 μm to bigger than 1·4 mm gave comparable yields.