Initial Substrate Moisture Content Research Articles

Lignocellulolytic enzyme cocktail produced by plant endophytic Chaetomium globosum exhibits a capacity for high-efficient saccharification of raw rice straw

The aim of this work was to optimize the production of lignocellulolytic enzyme cocktail by a plant endophyte Chaetomium globosum DX-THS3 under solid-state fermentation, and to evaluate the saccharification efficiency of this enzyme cocktail on raw rice straw. The process variables, including substrate type, substrate size, initial moisture content of substrate, culture temperature and fermentative time, were optimized using One-factor at a time approach and Response surface methodology. The results showed that the maximum CMCase, FPase and xylanase production reached 237.04 U/g, 54.8 U/g, and 987.33 U/g, respectively. Meanwhile, the lignocellulolytic enzyme cocktail exhibited good thermal stability between 30 °C and 60 °C, and excellent tolerance to acidic pH values of 3–6. Subsequently, the saccharification of raw rice straw by the enzyme cocktail was performed. After 24 h hydrolysis of raw rice straw, high reducing sugars yield (294.36 mg/g) and hydrolytic rate (47.58 %) were achieved. Furthermore, scanning electron micrograph also indicated that the enzyme cocktail had high hydrolysis performance for raw rice straw. Overall, our result provides a novel class of lignocellulolytic enzyme cocktail with a capacity for high-efficient and eco-friendly saccharification of raw rice straw, and strongly suggests plant endophytic fungi as excellent candidates for the production of lignocellulolytic enzymes.

One step selection strategy for optimization of media to enhance arachidonic acid production under solid state fermentation

Mortierella alpina is the most researched fungus for production of arachidonic acid. Arachidonic acid inclusion in diet is gaining prominence due to its potential role in brain development and age-related disease conditions. The production mainly focuses on submerged fermentation with extensive use of gas chromatography which increases the cost of optimization. The present study focuses on the optimization of arachidonic acid production by M. alpina under solid-state fermentation and developing an effective mycelial staining method as an analytical tool to replace gas chromatography. The optimization method increased the Arachidonic acid yield (41.29 mg/gds) with the supplementation of 3.0% corn gluten, 3.0% flax seed oil and 0.625% of mineral mix-3 in a soybean meal based substrate at an optimum temperature of 25 °C and initial substrate moisture content of 70.0%. For deriving an effective analytical tool, correlation was made between the responses of statistically designed submerged fermentation and solid-state fermentation trials. The comparative regression analysis of the responses i.e., the mycelial Triphenyl tetrazolium chloride staining data and the arachidonic acid values revealed a 98.0% correlation. The submerged fermentation based mycelial staining was thus devised as an effective media optimization strategy for solid state fermentation based arachidonic acid production.

Production of single cell protein by a local Trichoderma reesei in solid state fermentation: effects of process variables

Single cell protein (SCP) has attracted a lot of attention as an alternative protein source to support the increasing demand and to reduce the dependence on fishmeal in the animal feed and poultry industries. The SCP can be produced using lignocellulosic materials which are abundantly generated continuously as agricultural residues by Trichoderma reesei in solid state fermentation system. In this work, we evaluated the effects of various carbon sources (rice straw, corncob, bagasse, and coffee husk) which were combined with various nitrogen sources (Urea, Ammonium Sulphate, NPK) on SCP production. The rice straw and coffee husk gave a higher SCP yield when combined with urea or NPK at C/N ratio of 20:1 and 30:1. The solid substrate which contained rice straw or coffee husk gave a higher SCP yield when combined with urea or NPK at C/N ratio of 20:1 and 30:1. The initial moisture content of the solid substrate gave a significant contribution to the SCP yield, the highest yield of SCP developed in the substrate with the initial water content of 75%. On the other hand, the ratio of inoculum size to the solid substrate of 1:1 (w/v) gave the maximum SCP yield.

Combining submerged and solid state fermentation to convert waste bread into protein and pigment using the edible filamentous fungus N. intermedia

Waste streams from ethanol and bread production present inexpensive, abundant and underutilized renewable substrates that are highly available for valorisation into high-value products. A combined submerged to solid state fermentation strategy was studied using the edible filamentous fungus Neurospora intermedia to biotransform ethanol plant residues ‘thin stillage’ and waste bread as substrates for the production of additional ethanol, biomass and a feed product rich in pigment. The fungus was able to degrade the stillage during submerged fermentation, producing 81 kg ethanol and 65 kg fungal biomass per ton dry weight of thin stillage. Concurrently, the second solid state fermentation step increased the protein content in waste bread by 161%. Additionally, 1.2 kg pigment per ton waste bread was obtained at the best conditions (6 days solid state fermentation under light at 95% relative humidity at 35 °C with an initial substrate moisture content of 40% using washed fungal biomass to initiate fermentation). This study presents a means of increasing the value of waste bread while reducing the treatment load on thin stillage in ethanol plants.

Initial Substrate Moisture Content Affects Chemical Properties of Bagged Substrates Containing Controlled Release Fertilizer at Two Different Temperatures

Bagged potting mixes can be stored for weeks or months before being used by consumers. Some bagged potting mixes are amended with controlled release fertilizers (CRFs). The objective of this research was to determine how initial substrate moisture content and storage temperature affect the chemical properties of bagged potting mix with CRF incorporated and stored for up to 180 days. The base substrate composed of 60 sphagnum peat: 30 bark : 10 perlite (by vol.) amended with 5.5 g·L−1 dolomitic limestone and 0.5 g·L−1 granular wetting agent. This base substrate was either not amended with additional fertilizer (control) or amended with 0.59 kg·m−3 N of a CRF (Osmocote 18N–1.3P–5K) that was either ground (CRF-G) or whole prills (CRF-P). Substrates had initial moisture contents (IMCs) of 25%, 45%, or 65% and were stored at temperatures of either 20 or 40 °C. IMC and fertilizer type affected pH, electrical conductivity (EC), and nutrient release. Substrate pH increased with increasing IMC due to greater lime reactivity. About 25% of N from CRF-G treatments was immobilized between 2 and 14 days of storage. Low moisture content of bags, due to low IMC or storage at 40 °C, reduced the rate of N release from CRF-P treatments. Substrate P was rapidly immobilized by microbial communities.

Production of Natural Pigments from Monascus ruber by Solid State Fermentation of Broken Rice and its Application as Colorants of Some Dairy Products

The present study was aimed to maximize the production of natural pigments from Monascus ruber Went AUMC 5705 by solid state fermentation of broken rice to be a safe and healthy substitute of the harmful chemical dyes in the coloring of some flavored dairy products. Maximum pigments productivity was achieved under the following conditions: initial substrate moisture content 39%, substrate particle size 2-2.5 mm, PH 6.5, inoculums size 126×104 spores per 10 g dried rice and incubation period of 15 days at 30°C. The favorable nitrogen source was monosodium glutamate at 2% concentration. The obtained pigments mixture was extracted with 95% ethanol and further separated into red, orange and yellow water soluble pigments. These pigments were applied as coloring agents of flavored yoghurt and milk beverages. On the sensory evaluation, application of these natural colorants in a preparation of flavored dairy products was found to be highly acceptable.

Production of Single Cell Protein from Sugarcane Bagasse by Saccharomyces cerevisiae in Tray Bioreactor

In this study, solid state fermentation (SSF) was carried out to produce single cell protein (SCP) from sugarcane bagasse using Saccharomyces cerevisiae. The SSF experiment were performed in a tray bioreactor. The influence of several parameters including extraction buffer, initial moisture content of substrate, fermentation time, relative humidity in bioreactor, the bioreactor temperature and pretreatment of substrate on SCP production yield was considered. Among the used extraction buffers, carbonate-bicarbonate buffer was the most effective one for protein extraction. The obtained results revealed that the suitable fermentation conditions were initial substrate moisture content of 70%, fermentation time of 72 h, relative humidity of 85%, bioreactor temperature of 35 oC and pretreatment of substrate using 2% NaOH solution; at this optimum condition protein production yield of 13.41% was attained. The amino acid analysis of the produced protein showed that the product contained almost all of the essential amino acid as well as some non-essential amino acids.

BIOPROCESS DEVELOPMENTS FOR CELLULASE PRODUCTION BY Aspergillus oryzae CULTIVATED UNDER SOLID-STATE FERMENTATION

Bioprocess development studies concerning the production of cellulases are of crucial importance due to the significant impact of these enzymes on the economics of biomass conversion into fuels and chemicals. This work evaluates the effects of solid-state fermentation (SSF) operational conditions on cellulase production by a novel strain of Aspergillus oryzae using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The use of SSF cultivation under controlled conditions substantially improved cellulase production. Highest production of FPase (0.40 IU g-1), endoglucanase (123.64 IU g-1), and β-glucosidase (18.32 IU g-1) was achieved at 28 °C, using an initial substrate moisture content of 70%, with an inlet air humidity of 80% and an airflow rate of 20 mL min-1. Further studies of kinetic profiles and respirometric analyses were performed. The results showed that these data could be very useful for bioprocess development of cellulase production and scale-up.

Optimization of Phytase Production from Escherichia coli by Altering Solid-State Fermentation Conditions

Cultivation of Escherichia coli on wheat-bran substrate under various Solid-State Fermentation (SSF) conditions was evaluated for phytase yield along with the enzyme activity profile as a potential, low-cost alternative to submerged-liquid fermentation. The maximum phytase activity achieved by E. coli was 350 ± 50 SPU of phytase activity per gram of bran, incubated for 96 h with a substrate bed moisture content of 70% (w/v) at 37 °C with a relative air humidity of 90%, and supplemented with 10% (w/w bran) Luria-Bertani broth powder which translates into a 300% increase in phytase activity compared with an un-supplemented culture. The greatest improvements in phytase yield were associated with nutrient supplementation and the optimization of initial substrate moisture content. E. coli production of phytase utilizing solid-state fermentation technology was shown to be feasible utilizing the low-cost agro-residue wheat bran as substrate. Furthermore, the effect of pH and temperature on phytase activity was monitored from pH 2.5 to pH 7.5, and for temperatures ranging from 20 °C to 70 °C. Optimal phytase activity was at pH 5.5 and 50 °C when produced under the SSF optimized conditions.

Bioprocess development to add value to canola cake used as substrate for proteolytic enzyme production

Microbial proteases are one of the largest groups of industrial enzymes. This important market has led to a need for technically and economically efficient bioprocesses for protease production that could be exploited commercially. The aim here was to develop a complete bioprocess for protease production, from microbial fermentation up to dried product formulation. Evaluation was made of the effects of operational conditions on the production of protease by a selected strain of Aspergillus oryzae, cultivated under solid-state fermentation (SSF) with canola cake as a sole carbon source in an instrumented lab-scale bioreactor. Statistical experimental design revealed that the air flow rate, inlet air relative humidity, and initial substrate moisture content had significant effects on the efficiency of protease production. The highest protease production by A. oryzae was achieved at a fixed air flow rate of 12mL/min, with inlet air relative humidity within the range 66–94% and initial substrate moisture content between 30% and 40%. The enzymatic extract produced under the selected conditions was spray dried using different concentrations of additives (glucose, maltodextrin, and CMC). The stability of the dried enzymatic powder during shelf storage was evaluated over a period of 90 days. There was a positive effect of CMC and a negative effect of glucose on protease activity and stability, while the influence of maltodextrin was negative in enzyme recovery at time zero, but it was positive on protease stability over a longer period. The spray dried proteolytic enzymatic formulation obtained from SSF of canola cake using A. oryzae has potential for applications in the industrial sector.

Canola Cake as a Potential Substrate for Proteolytic Enzymes Production by a Selected Strain of Aspergillus oryzae: Selection of Process Conditions and Product Characterization.

Oil cakes have excellent nutritional value and offer considerable potential for use in biotechnological processes that employ solid-state fermentation (SSF) for the production of high value products. This work evaluates the feasibility of using canola cake as a substrate for protease production by a selected strain of Aspergillus oryzae cultivated under SSF. The influences of the following process parameters were considered: initial substrate moisture content, incubation temperature, inoculum size, and pH of the buffer used for protease extraction and activity analysis. Maximum protease activity was obtained after cultivating Aspergillus oryzae CCBP 001 at 20°C, using an inoculum size of 107 spores/g in canola cake medium moistened with 40 mL of water to 100 g of cake. Cultivation and extraction under selected conditions increased protease activity 5.8-fold, compared to the initial conditions. Zymogram analysis of the enzymatic extract showed that the protease molecular weights varied between 31 and 200 kDa. The concentrated protease extract induced clotting of casein in 5 min. The results demonstrate the potential application of canola cake for protease production under SSF and contribute to the technological advances needed to increase the efficiency of processes designed to add value to agroindustrial wastes.

Scale-Up Production of Bacillus Thuringiensis by Box-Type Solid-State Fermentation Equipment

The Bacillus thuringiensis (Bt) which is considered as the most effective bio-pesticide is widely used in the world. There are mainly two modes for the production of this microbial pesticides: submerged fermentation and solid-state fermentation. In this paper, The Scale-up of Kg-class Bt production was studied on wheat bran based media in the Box-type solid-state fermentation equipment made by our lab. The fermentation conditions were optimized and the result show that, Bt production yielded a significant increase in spore count and toxin content at the following conditions: actiactivation of culture for 8h, initial moisture content of solid substrate 55%, pH value 7.5, keeping the thick of inoculated media about 4cm and fermenting for 45 h, Maintaining the temperature within a range of 25-30°C, The bioassay efficacy of the product could be above 13000IU·mg-1, The production of Bt kilograms class by solid-state fermentation was successfully represented as well as providing a strong technical support to the large-scale industrial production of Bt bio-pesticide.

Optimization of L-asparaginase production by Serratia marcescens (NCIM 2919) under solid state fermentation using coconut oil cake

Abstract Background The present study focused on utilization of agrowaste byproducts generated from oil mill for L-asparaginase enzyme production using Serratia marcescens under solid state fermentation. Classical and statistical methods were employed to optimize the process variables and the results were compared. Results The classical one factor at a time (OFAT) and response surface methodology (RSM) are employed to optimize the fermentation process. When used as the sole carbon source in SSF, coconut oil cake (COC) showed maximum enzyme production. The optimal values of substrate amount, initial moisture content, pH and temperature were found to be 6 g, 40%, 6 and 35°C respectively under classical optimization method with maximum enzyme activity of 3.87 (U gds-1). Maximum enzyme activity of 5.86 U gds-1 was obtained at the predicted optimal conditions of substrate amount 7.6 g of COC, initial moisture content of substrate 50%, temperature 35.5°C and pH 7.4. Validation results proved that a good relation existed between the experimental and the predicted model. Conclusions RSM optimization approach enhances the enzyme production to 33% when compared to classical method. Utilization of coconut oil cake as a low cost substrate in SSF for L-asparaginase production makes the process economical and also reduces the environmental pollution by converting the oil mill solid waste into a useful bioproduct.

Optimization of novel hyperthermostable β amylase production by Bacillus subtilis DJ5 using solid agroresidual substrates

Eight different low cost starchy agroresidues namely Barley (B), Wheat bran (WB), Sattu (S), Rice powder (RP), Corn flour (CF), Rice husk (RH), Yellow peas split (YPS) and arrowroot (A) were used for solid culturing of Bacillus subtilis DJ5 for production of novel hyperthermostable β amylase. Various process parameters like initial moisture content, inoculum load, medium pH and incubation temperature affecting enzyme production were optimized to ensure maximum enzyme yield. Only 10 % inoculums load and medium pH of 6.9 was found sufficient to achieve maximum enzyme production in all substrates in a decreasing order, B > WB > S > RP > CF > RH > YPS > A. Optimum β amylase production was highly dependent on initial moisture content of substrate as observed from varying requirement of moisture for different substrates. Only 50 % moisture was sufficient for maximum enzyme production of 84.29 U/gdm in CF. For B, RH, YPS, and A 60 % initial moisture resulted in higher production of 120.34, 35.19, 26.59, and 21.58 U/gdm, respectively, at 37 °C. However, for S and RP higher (70 %) moisture content allowed 113.4 and 85.56 U/gdm enzyme production, respectively. Under optimized conditions, maximum β amylase production was observed after 25 h for A, YPS, RH, RP; 41 h for B, WB, CF, and 45 h for S.

Enhancing xylanases production by a new Amazon Forest strain of Aspergillus oryzae using solid-state fermentation under controlled operation conditions

Bioprocess studies towards improving the production of xylanases are of great value due to the importance of these enzymes on several industrial applications. This work evaluates the effects of operational conditions on xylanase production by a new strain of Aspergillus oryzae (P6B2) isolated from the Amazon Rain Forest. The fungus was cultivated under solid-state fermentation (SSF) using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of initial substrate moisture content and temperature on xylanase production were evaluated and compared to static conditions. It was outstanding the positive effect on xylanase production by SSF cultivations under controlled conditions of forced over static aeration conditions. The conditions selected for further characterization resulted in the highest production of xylanase (2830.7IU/g), which was achieved using an initial substrate moisture content of 80%, at 28°C with an air flow rate of 20mL/min and an inlet air humidity of 80%. Results of respirometric analysis showed good agreement with enzyme production, suggesting that these data could be very useful for optimization and scale-up studies.

Production of Biomass-Degrading Multienzyme Complexes under Solid-State Fermentation of Soybean Meal Using a Bioreactor

Biomass-degrading enzymes are one of the most costly inputs affecting the economic viability of the biochemical route for biomass conversion into biofuels. This work evaluates the effects of operational conditions on biomass-degrading multienzyme production by a selected strain of Aspergillus niger. The fungus was cultivated under solid-state fermentation (SSF) of soybean meal, using an instrumented lab-scale bioreactor equipped with an on-line automated monitoring and control system. The effects of air flow rate, inlet air relative humidity, and initial substrate moisture content on multienzyme (FPase, endoglucanase, and xylanase) production were evaluated using a statistical design methodology. Highest production of FPase (0.55 IU/g), endoglucanase (35.1 IU/g), and xylanase (47.7 IU/g) was achieved using an initial substrate moisture content of 84%, an inlet air humidity of 70%, and a flow rate of 24 mL/min. The enzymatic complex was then used to hydrolyze a lignocellulosic biomass, releasing 4.4 g/L of glucose after 36 hours of saccharification of 50 g/L pretreated sugar cane bagasse. These results demonstrate the potential application of enzymes produced under SSF, thus contributing to generate the necessary technological advances to increase the efficiency of the use of biomass as a renewable energy source.

Integrated Strategies to Enhance Cellulolytic Enzyme Production Using an Instrumented Bioreactor for Solid-State Fermentation of Sugarcane Bagasse

The conversion of agro-industrial residues, such as sugarcane bagasse, into high-value products and renewable energy, within the biorefinery concept, is a potential alternative towards the sustainable management of these resources. This work evaluates the production of cellulolytic enzymes by a selected strain of Aspergillus niger cultivated in sugarcane bagasse under solid-state fermentation using an instrumented lab-scale bioreactor. The effects of environmental factors including the type of substrate and medium composition, as well as the operational conditions (air flow rate, inlet air relative humidity, and initial substrate moisture content) on the production of the enzymatic complex were evaluated using statistical design tools. Significant increases in FPase, endoglucanase, and xylanase activities were achieved under the optimized conditions predicted by the models, with values of 0.88, 21.77, and 143.85 IU/g of dry solid substrate, respectively, representing around ten-, four-, and twofold increases compared to the activities obtained under the initial growth conditions. This demonstrates the importance of evaluating environmental and operational criteria in order to achieve efficient enzyme production. The crude enzymatic extract obtained under optimized conditions was employed for enzymatic hydrolysis of pretreated sugarcane bagasse. Approximately 13 % of total reducing sugars, and a glucose concentration of 2.54 g/L, were obtained after 22 h of hydrolysis of steam exploded sugarcane bagasse, indicating that the enzymatic cocktail produced has good potential for use in the conversion of biomass.

Solid substrate fermentation for cellulase production using palm kernel cake as a renewable lignocellulosic source in packed-bed bioreactor

Palm kernel cake (PKC), is an agro-industrial residue created in the palm oil industry, and large quantities of PKC are produced in Malaysia. Sustainable development of the palm oil industry in Malaysia demands an economical technology for the environmentally friendly utilization of PKC in industrial utility systems. This research was carried out to evaluate the use of PKC in the production of cellulase by the cultivation of Aspergillus niger FTCC 5003 in a laboratory packed-bed bioreactor for seven days. A central composite design was used to perform eighteen trials of solid substrate fermentation under selected conditions of incubation temperature, initial moisture content of substrate, and airflow rate. Experimental results showed that a cellulase yield of 244.53 U/g of dry PKC was obtained when 100 g of PKC was hydrolyzed at an incubation temperature of 32.5°C, an initial moisture level of 60%, and an aeration rate of 1.5 L/min/g PKC. An empirical second-order polynomial model was adjusted to the experimental data to evaluate the effects of the studied operating variables on cellulase production. The statistical model revealed that the quadratic term for initial moisture content had a significant effect on the production of cellulase (P < 0.01). The regression model also indicated that the quadratic terms for incubation temperature and interaction effects between initial moisture content and aeration rate significantly influenced cellulase production (P < 0.05). The empirical model determined that the optimum conditions for cellulase production were an incubation temperature of 31.0°C, an initial moisture content of 59.0% and an airflow rate of 1.55 L/min/g PKC.

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of beta-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on beta-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of beta-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5 degrees C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of beta-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of beta-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of beta-mannanase were incubation temperature of 32 degrees C, initial moisture level of 59% and aeration rate of 0.5 l/min. A beta-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.

Xylanase Production by Aspergillus niger FTCC 5003 Using Palm Kernel Cake in Fermentative Bioprocess

The production of xylanase from palm kernel cake as a substrate was studied in solid substrate fermentation. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and air flow rate on xylanase production were evaluated by response surface methodology using central composite face centered design. A total of 18 experiments were carried out in which Aspergillus niger FTCC 5003 was cultivated on palm kernel cake in a column bioreactor for 7 days under incubation temperature, moisture level and aeration rate determined. Test results showed that the highest xylanase activity of 174.88 U g(-1) was produced at incubation temperature, initial moisture level and aeration rate of 25 degrees C, 60% and 1.5 L min(-1), respectively. The statistical analysis of the experimental results revealed that the linear effect of incubation temperature and quadratic term of initial moisture content had highly significant effects on xylanase production (p<0.01). Statistical results also showed that interaction effect between incubation temperature and initial moisture content as well as interaction effect between moisture level and aeration rate influenced the yield ofxylanase at probability levels of 95%. Optimum conditions determined by statistical model for attaining maximum xylanase production were incubation temperature of 25 degrees C, initial moisture level of 63% and aeration rate of 1.76 L min(-1). The xylanase activity of 192.50 U g(-1) was obtained when solid substrate fermentation was performed under the optimal circumstances.