Approaches for Producing Fungal Cellulases Through Submerged Fermentation.

Fungal cellulases are the most sought-after biological molecules produced from microbial sources in the last four decades. Owing to their emerging applications in the bioenergy industry for hydrolyzing cellulose, for which they are the most abundant source on this planet, research trends are shifting heavily toward adapting to submerged fermentation. However, filamentous fungal species, which are efficient cellulase producers, are well-adapted to low-moisture solid support as the substrate, such as in nature. Therefore, various fermentation strategies are currently being investigated to adapt them to submerged fermentation for large and high-quality production of cellulases. Emerging research trends, such as the use of inexpensive feedstocks, nutrient and/or culture optimization, innovative bioreactor designs, microparticle-assisted fungal growth, and innovative genetic engineering approaches, are some of the recent efforts by researchers to exploit the full potential of these biological molecules. This review discusses some of these strategies and their success rates in various research conditions. In addition, specific focus was provided to both increasing the market value of cellulases and the innovative strategies required to enhance their production on an industrial scale.

High hydrostatic pressure-assisted extraction of lipids from Lipomyces starkeyi biomass.

The purpose of this study is to evaluate the effect of high hydrostatic pressure (HHP) as a novel approach for yeast cell disruption and lipid extraction from Lipomyces starkeyi DSM 70295 grown in glucose medium (40 g/L and C/N:55/1) at initial pH of 5.0, 25°C, and 130 rpm for 8 days. HHP extraction conditions including pressure, time, and temperature were optimized by response surface methodology. The high speed homogenizer-assisted extraction (HSH) was also used for comparison. The biomass subjected to HHP was examined under scanning electron microscopy and light microscope. A maximal lipid yield of 45.8 ± 2.1% in dry cell basis (w/w) was achieved at 200 MPa, 40°C, and 15 min, while a minimum yield of 15.2 ± 0.9% was observed at 300 MPa, 40°C, and 10 min (p < 0.05). The lipid yield decreased with increasing pressure. It was demonstrated that low pressure (200 MPa) collapsed the cells, while high pressure (400 MPa) created protrusions on the cell wall and cell fragments spread in the environment. This study favors HHP as a promising method for Lipomyces oil extraction. PRACTICAL APPLICATION: Single-cell oils are considered future alternatives to plant-based oils as food additives and dietary supplements. Oleaginous microorganisms accumulate oils in their cell plasma, which makes extraction essential. One of the main obstacles with existing methods is the utilization of strong acids to destroy cell walls. This study aims to demonstrate high hydrostatic pressure as a rapid method for lipid extraction from oleaginous yeast Lipomyces starkeyi.

Salt and nitrogen amendment and optimization for cellulase and xylanase production using dilute acid hydrolysate of distillers' dried grains with solubles (DDGS) as the feedstock.

Distillers' dried grains with solubles (DDGS) is a by-product of dry-mill corn ethanol production comprising a high nutritional value due to residual fiber, protein, and lipid contents. The fiber content of DDGS is high enough to be considered a valuable source for the production of hydrolytic enzymes, such as cellulase and xylanases, which can be used for hydrolysis of lignocellulosic feedstock during ethanol production. The DDGS-based medium prepared after acid hydrolysis provides adequate sugars for enzyme production, while additional macronutrients, such as salts and nitrogen sources, can enhance the enzyme production. Therefore, this study was undertaken to evaluate the effect of salts (KH2PO4, CaCl2·2H2O, MgSO4·7H2O, FeSO4·7H2O, CoCl2·6H2O, and MnSO4·H2O), peptone, and yeast extract on enzyme secretion by four different Aspergillus niger strains and to optimize the nitrogen source for maximum enzyme production. Yeast extract improved the cellulase production (0.38 IU/ml) for A. niger (NRRL 1956) as compared to peptone (0.29 IU/ml). However, maximum cellulase productions of 0.42 IU/ml and 0.45 IU/ml were obtained by A. niger (NRRL 330) and A. niger (NRRL 567), respectively, in presence of ammonium sulfate. The optimized nitrogen amounts resulted in a significant increase in the cellulase production from 0.174 to 0.63 IU/ml on day 9 of the fermentation with A. niger (NRRL 330). The composite model improved both cellulase and xylanase production. In conclusion, the optimization of all three nitrogen sources improved both cellulase and xylanase production in the DDGS-based media.

Distillers' dried grains with solubles (DDGS) and its potential as fermentation feedstock.

Distillers' dried grain with solubles (DDGS) is a byproduct of bioethanol fermentation, which uses the dry milling technology for starch-rich grains such as corn, wheat, and barley. The current interest in bioethanol is increasing due to the need for renewable liquid fuels specifically in the transportation sector. Since DDGS is rich in crude protein, fat, fiber, vitamins, and minerals, it is currently used as aquaculture, livestock, and poultry feeds. In recent years, DDGS has been used as feedstock in the production of value-added products via microbial fermentation. Numerous studies reported the production organic acids, methane, biohydrogen, and hydrolytic enzymes using DDGS. While DDGS contains remarkable amounts of macronutrients, pre-treatment of DDGS is required for release of the fermentable sugars. The pre-treatment methods such as chemical, physical, and biological origin are either solely used or combined to obtain maximal yields for different applications. Therefore, this review summarizes some of the most prominent pre-treatment processes generating high fermentable sugar yields for the productions of value-added products in the last 5 years. A special focus has been given to the effect of the variability of DDGS on the final product. Integration of hydrolytic enzyme production with the traditional bioethanol production facilities has been discussed for further improvement of bioethanol, methane, and biohydrogen using DDGS as fermentation feedstock.Key points• Distillers' dried grain with solubles (DDGS) has high nutritional value, but the nutritional profile is variable.• DDGS can be used for microbial fermentation feedstock to produce value-added products.• A review of the microbial products using DDGS is given for the last 5 years.• DDGS has the potential to replace expensive feedstocks of value-added products.

Optimization of dilute sulfuric acid, aqueous ammonia, and steam explosion as the pretreatments steps for distillers' dried grains with solubles as a potential fermentation feedstock.

Distillers' dried grains with solubles (DDGS) is the by-product of bioethanol production from starch-rich grains through dry-mill fermentation. In this study, dilute sulfuric acid hydrolysis, aqueous ammonia, and steam explosion as the pre-treatment methods were optimized. The central composite response surface methodology (RSM) design was used for optimization of dilute acid pretreatment, aqueous ammonia pretreatment. The steam explosion trials were evaluated. The results show that the dilute acid pretreatment at 121 °C is the most effective way of obtaining simple fermentable sugars (0.382 g/g DDGS). The levels of furfural and HMF was also 5.2 mg/g DDGS) and 1.6 mg/g DDGS, respectively, in the dilute sulfuric acid pretreated DDGS. On the other hand, maximum sugar yield for ammonia pretreatment was 0.129 g/g DDGS and 0.055 g/g DDGS for the steam pretreatment, while no significant amounts of furfural and HMF were observed for these two pretreatment methods.

Assessment and optimization of xylanase production using co-cultures of Bacillus subtilis and Kluyveromyces marxianus.

Co-cultures of Bacillus subtilis and Kluyveromyces marxianus were investigated in submerged fermentation for xylanase production at shake-flask scale. Xylanase production markedly increased when arabinose, xylose, or hazelnut shells were used as the single carbon source. Maximal xylanase of 49.5 IU/mL was achieved with 4% B. subtilis, 4% K. marxianus, 40% solid load of hazelnut shells, and pH 7.0. Overall, xylanase was enhanced by 4.4-fold compared to initial un-optimized monoculture production and 2.8-fold compared to initial un-optimized co-cultured production, after optimization by response surface method.

Evaluation of heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05 on vinasse and its scale up for biodiesel production.

Direct disposal of vinasse, a by-product of molasses fermentation plants, threatens environmental health. This study investigated the usage of vinasse as a nutrient source for the heterotrophic and mixotrophic cultivation of novel Micractinium sp. ME05. The 500-mL flask experiments resulted in higher biomass productivities under mixotrophic conditions (0.16 ±â€¯0.01 g L-1 day1) than the heterotrophic conditions (0.13 ±â€¯0.01 g L-1 day1). A 1.7-fold increase in biomass productivity was achieved by scaling up from 500-mL flasks (0.16 ±â€¯0.01 g L-1 day1) to 2-L flasks (0.27 ±â€¯0.019 g L-1 day1). The 5-L bioreactor experiments resulted in a biomass productivity of 0.32 ±â€¯0.2 g L-1 day1 and lipid productivity of 3.4 ±â€¯0.20 g L-1 day-1. This study demonstrated that Micractinium sp. ME05 can be cultivated with vinasse to produce large amounts of biomass. The FAME profile of mixotrophic Micractinium sp. ME05 cells was promising for further biodiesel production. This study highlights the feasibility of industrial by- product-vinasse as the nutrient source for biomass and lipid productions using the novel Micractinium sp. ME05 cells.

Kinetic modeling of enzymatic hydrolysis of pretreated kitchen wastes for enhancing bioethanol production.

It is well known that use of low cost and abundant waste materials in microbial fermentations can reduce product costs. Kitchen wastes disposed of in large amounts from cafeterias, restaurants, dining halls, food processing plants, and household kitchens contain high amounts of carbohydrate components such as glucose, starch, and cellulose. Efficient utilization of these sugars is another opportunity to reduce ethanol costs. In this study, the effect of pretreatment methods (hot water, acid solutions, and a control) on enzymatic hydrolysis of kitchen wastes was evaluated using a kinetic modeling approach. Fermentation experiments conducted with and without traditional fermentation nutrients were assessed at constant conditions of pH 4.5 and temperature of 30°C for 48h using commercial dry baker's yeast, Saccharomyces cerevisiae. The control, which involved no treatment, and hot water treated samples gave close glucose concentrations after 6h. The highest and lowest rates of glucose production were found as 0.644 and 0.128 (h(-1)) for the control (or no-pretreated (NPT)) and 1% acid solutions, respectively. The fermentation results indicated that final ethanol concentrations are not significantly improved by adding nutrients (17.2-23.3g/L). Thus, it was concluded that product cost can be lowered to a large extent if (1) kitchen wastes are used as a substrate, (2) no fermentation nutrient is used, and (3) hydrolysis time is applied for about 6h. Further optimization study is needed to increase the yield to higher levels.

Optimization of culture conditions for Aspergillus sojae expressing an Aspergillus fumigatus α-galactosidase.

Using Response Surface Methodology, carbon and nitrogen sources and agitation speed for cultivation of Aspergillus sojae expressing the α-galactosidase gene, aglB of Aspergillus fumigatus IMI 385708 were optimized. Compared to cultivation in modified YpSs medium, cultivation in 250-mL Erlenmeyer flasks agitated at 276 rpm and containing 100 mL of optimized medium consisting of 10.5% molasses (w/v) and 1.3% NH(4)NO(3) (w/v), 0.1% K(2)HPO(4), and 0.005% MgSO(4)·7H(2)O achieved a 4-fold increase in α-galactosidase production (10.4 U/mL). These results suggest the feasibility of industrial large scale production of an α-galactosidase known to be valuable in galactomannan modification.

Cost-effective approach to ethanol production and optimization by response surface methodology.

Food wastes disposed from residential and industrial kitchens have gained attention as a substrate in microbial fermentations to reduce product costs. In this study, the potential of simultaneously hydrolyzing and subsequently fermenting the mixed carbohydrate components of kitchen wastes were assessed and the effects of solid load, inoculum volume of baker's yeast, and fermentation time on ethanol production were evaluated by response surface methodology (RSM). The enzymatic hydrolysis process was complete within 6h. Fermentation experiments were conducted at pH 4.5, a temperature of 30°C, and agitated at 150 rpm without adding the traditional fermentation nutrients. The statistical analysis of the model developed by RSM suggested that linear effects of solid load, inoculum volume, and fermentation time and the quadratic effects of inoculum volume and fermentation time were significant (P<0.05). The verification experiments indicated that the developed model could be successfully used to predict ethanol concentration at >90% accuracy. An optimum ethanol concentration of 32.2g/l giving a yield of 0.40g/g, comparable to yields reported to date, was suggested by the model with 20% solid load, 8.9% inoculum volume, and 58.8h of fermentation. The results indicated that the production costs can be lowered to a large extent by using kitchen wastes having multiple carbohydrate components and eliminating the use of traditional fermentation nutrients from the recipe.

Feedstock optimization of in-vessel food waste composting systems for inactivation of pathogenic microorganisms.

An optimum composting recipe was investigated to reduce pathogenic microorganisms in a forced-aerated in-vessel system (55 liters). The feedstocks used for in-vessel composting were food waste, cow manure, and bulking materials (wood shavings and mulch hay). A statistical extreme vertices mixture design method was used to design the composting experiments and analyze the collected data. Each mixture (nine total) was replicated randomly three times. Temperature was monitored as an indicator of the efficiency of the composting experiments. The maximum temperature values of the mixtures were used as a response for both extreme vertices mixture design and statistical analyses. Chemical changes (moisture content, carbon/nitrogen ratio, volatile solids, and pH) and reductions of indicator (fecal coliforms and fecal streptococci) and pathogenic microorganisms (Salmonella and Escherichia coli O157:H7) were measured by the most-probable-number method before and after a 12-day composting period. Maximum temperatures for the tested compost mixtures were in the range of 37.0 to 54.7 degrees C. Extreme vertices mixture design analysis of the surface plot suggested an optimum mixture containing 50% food waste, 40% manure, and 10% bulking agents. This optimum mixture achieved maximum temperatures of 54.7 to 56.6 degrees C for about 3.3 days. The total reduction of Salmonella and E. coli O157:H7 were 92.3%, whereas fecal coliforms and fecal streptococci reductions were lower (59.3 and 27.1%, respectively). Future study is neededto evaluate the extreme vertices mixture design method for optimization of large-scale composting.