Effects of Pellet Characteristics on L-Lactic Acid Fermentation by R. oryzae: Pellet Morphology,Diameter, Density,and Interior Structure

The effects of pellet morphology, diameter, density, and interior structure on L-lactic acid fermentation by Rhizopus oryzae were characterized for different inoculum sizes and concentrations of peptone and CaCO₃. Inoculum size was the most important factor determining pellet formation and diameter. The diameter decreased with increasing inoculum size, and larger pellets were observed for lower inoculum sizes. Peptone concentration had the greatest effect on pellet density, which increased with increasing peptone concentration. L-lactic acid production depended heavily on pellet density but not on pellet diameter. Low-density pellets formed easily under conditions of low peptone concentration and often had a relatively hollow structure, with a thin condensed layer surrounding the pellet and an extraordinarily loose biomass or hollow center. As expected, this structure greatly decreased production. The production of L-lactic acid increased until the density reached a certain level (50–60 kg/m³), in which the compact part was distributed homogeneously in the thick outer layer of the pellet and loose in the central layer. Homogeneously structured, denser pellets had limited mass transfer, causing a lower overall turnover rate. However, the interior structure remained nearly constant throughout all fermentation phases for pellets with the same density. CaCO₃ concentration only had a slight influence on pellet diameter and density, probably because it increases spore germination and filamentous hypha extension. This work also provides a new analysis method to quantify the interior structure of pellets, thus giving insight into pellet structure and its relationship with productivity.     

Fumaric Acid Production from Alkali-Pretreated Corncob by Fed-Batch Simultaneous Saccharification and Fermentation Combined with Separated Hydrolysis and Fermentation at High Solids Loading

Production of fumaric acid from alkali-pretreated corncob (APC) at high solids loading was investigated using a combination of separated hydrolysis and fermentation (SHF) and fed-batch simultaneous saccharification and fermentation (SSF) by Rhizopus oryzae. Four different fermentation modes were tested to maximize fumaric acid concentration at high solids loading. The highest concentration of 41.32 g/L fumaric acid was obtained from 20 % (w/v) APC at 38 °C in the combined SHF and fed-batch SSF process, compared with 19.13 g/L fumaric acid in batch SSF alone. The results indicated that a combination of SHF and fed-batch SSF significantly improved production of fumaric acid from lignocellulose by R. oryzae than that achieved with batch SSF at high solids loading.     

Mycelial pellet formation by Rhizopus oryzae ATCC 20344

Factors in a cultivation medium affecting fungal growth morphology and funmaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted, the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.     

Optimization of Lactic Acid Production by Pellet-Form Rhizopus oryzae in 3-L Airlift Bioreactor Using Response Surface Methodology

The influence of two key environmental factors, pH and oxygen transfer coefficient (k _La), was evaluated on the lactic acid production as the main answer and, on the size of cell pellets of the fungal strain Rhizopus oryzae KPS106, as second dependant answer by response surface methodology using a central composite design. The results of the analysis of variance and modeling demonstrated that pH and k _La had a significant effect on lactic acid production by this strain. However, no interaction was observed between these two experimental factors. pH and k _La had no significant influence on the pellet size. Optimal pH and k _La of the fermentation medium for lactic acid production from response surface analysis was 5.85 and of 3.6 h^?1, respectively. The predicted and experimental lactic acid maximal values were 75.4 and 72.0 g/l, respectively, with pellets of an average of 2.54?±?0.41 mm. Five repeated batches in series were conducted with a mean lactic acid production of 77.54 g/l. The productivity was increased from 0.75 in the first batch to 0.99 g/l h in the last fifth batch.     

Optimization of l -(+)-lactic acid production using pelletized filamentous Rhizopus oryzae NRRL 395

Lactic acid is used as a food additive for flavor and preservation and a precursor in the development of poly-lactic acid, a product used to make biodegradable plastics and textiles. Rhizopus oryzae NRRL 395 is known to be a strain that produces optically pure l-(+)-lactic acid. The morphology of Rhizopus cultures is complex, forming filamentous, clumps, and pellet mycelia. Different morphology growth has significant effects on lactic acid production. In bioreactors, the filamentous or clump mycelia increase the viscosity of the medium, wrap around impellers, and block the nutrient transportation, leading to a decrease in production efficiency and bioreactor performance. Growing fungi in pellet form can significantly improve these problems. In this study, factors that affect lactic acid production in pelletized flask cultures using R. oryzae NRRL 395 were investigated in detail. Completely randomized designs were used to determine the influence of culture temperature, time, concentration of glucose, and inoculum size. Lactic acid fermentation using clump and pellet morphologies were performed in a 5 L fermentor at the optimal values obtained from flask culture. Finally, fed-batch culture was used to enhance the lactate concentration in broth. The final lactate concentration of fed-batch culture reached 92 g/L. The data presented in the article can provide useful information on optimizing lactic acid production using alternative source materials.     

Mixed Food Waste as Renewable Feedstock in Succinic Acid Fermentation

Mixed food waste, which was directly collected from restaurants without pretreatments, was used as a valuable feedstock in succinic acid (SA) fermentation in the present study. Commercial enzymes and crude enzymes produced from Aspergillus awamori and Aspergillus oryzae were separately used in hydrolysis of food waste, and their resultant hydrolysates were evaluated. For hydrolysis using the fungal mixture comprising A. awamori and A. oryzae, a nutrient-complete food waste hydrolysate was generated, which contained 31.9 g L^?1 glucose and 280 mg L^?1 free amino nitrogen. Approximately 80–90 % of the solid food waste was also diminished. In a 2.5 L fermentor, 29.9 g L^?1 SA was produced with an overall yield of 0.224 g g^?1 substrate using food waste hydrolysate and recombinant Escherichia coli. This is comparable to many similar studies using various wastes or by-products as substrates. Results of this study demonstrated the enormous potential of food waste as renewable resource in the production of bio-based chemicals and materials via microbial bioconversion.     

Production of fumaric acid with immobilized biocatalysts

Immobilization ofRhizopus arrhizus mycelium improved fumaric acid production. The optimum conditions for fumaric acid production with immobilized cells were investigated using a statistical experimental design. Substrate concentration, carbon:nitrogen ratio, and residence time were chosen as independent variables. In the repeated batch shake flask fermentation, the fumaric acid yield from xylose was as much as 3.5 times higher with immobilized mycelium than with free mycelium. Polyurethane foam cubes, in this case, gave better results than nylon net cubes as a carrier.     

Phytase Production by Aspergillus oryzae in Solid-State Fermentation and its Applicability in Dephytinization of Wheat Ban

Aspergillus oryzae SBS50 secreted a high titre of phytase in solid-state fermentation (SSF) using wheat bran at 30 °C after 96 h at the initial substrate to moisture ratio of 1:2 and a water activity of 0.95. The production of phytase increased when wheat bran was supplemented with sucrose and beef extract. Further enhancement in enzyme production was recorded when the substrate was supplemented with the surfactant Triton X-100 (145 U/g of DMB). An overall 29-fold improvement in phytase production was achieved owing to optimization. Under optimized conditions, the mould secreted 9.3-fold higher phytase in SSF as compared to submerged fermentation (SmF). The mesophilic mould also secreted amylase, cellulase (CMCase), pectinase and xylanase along with phytase in SSF. Scanning electron microscopy revealed luxuriant growth of A. oryzae on wheat bran with abundant spores. The enzyme dephytinized wheat bran with concomitant liberation of inorganic phosphate.     

Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production

Using pelletized fungal biomass can effectively improve the fermentation performance for most of fugal strains. This article studied the effects of inoculum and medium compositions such as potato dextrose broth (PDB) as carbon source, soybean peptone, calcium carbonate, and metal ions on pellet formation of Rhizopus oryzae. It has been found that metal ions had significantly negative effects on pellet formation whereas soybean peptone had positive effects. In addition PDB and calcium carbonate were beneficial to R. oryzae for growing small smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9) spores/L had no significant influence on pellet formation. Thus, a new approach to form pellets has been developed using only PDB, soybean peptone, and calcium carbonate. Meanwhile, palletized fungal fermentation significantly enhanced organic acid production. Lactic acid concentration reached 65.0 g/L in 30 h using pelletized R. oryzae NRRL 395, and fumeric acid concentration reached 31.0 g/L in 96 h using pelletized R. oryzae ATCC 20344.     

Production of α-amylase with Aspergillus oryzae on spent brewing grain by solid substrate fermentation

Ten Aspergillus oryzae strains were screened in solid substrate fermentation for α-amylase production on spent brewing grain (SBG) and on corn fiber. SBG proved to be a better substrate for enzyme production than corn fiber. A Plackett-Burman experimental design was used to optimize the medium composition for the best strain. Solid substrate fermentation on optimized medium with A. oryzae NRRL 1808 (=ATCC 12892) strain in stationary 500-mL Erlenmeyer flask culture yielded 4519 U of α-amylase/g of dry matter substrate in 3 d. The whole solid substrate fermentation material (crude enzyme, in situ enzyme) may be considered a cheap biocatalytic material for animal feed rations and for bioalcohol production from starchy materials.     

Food Waste Fermentation to Fumaric Acid by <Emphasis Type="Italic">Rhizopus arrhizus RH7-13</Emphasis>

Fumaric acid as a four-carbon unsaturated dicarboxylic acid is widely used in the food and chemical industries. Food waste (FW), rich in carbohydrates and protein, is a promising potential feedstock for renewable bio-based chemicals. In this research, we investigated the capability of Rhizopus arrhizus RH7-13 in producing fumaric acid from FW. The liquid fraction of the FW (L-FW) was proven to be the best seed culture medium in our research. When it was however used to be fermentation medium, the yield of fumaric acid reached 32.68 g/L, at a volumetric production of 0.34 g/L h. The solid fraction of FW mixed with water (S-FW) could also be used as fermentation medium when a certain amount of glucose was added, and the yield of fumaric acid reached 31.26 g/L. The results indicated that both fractions of FW could be well utilized in fermentation process and it could replace a part of common carbon, nitrogen, and nutrient. The process has an application potential since reducing the costs of raw materials.     

Pythium irregulare Fermentation to Produce Arachidonic Acid (ARA) and Eicosapentaenoic Acid (EPA) Using Soybean Processing Co-products as Substrates

Arachidonic acid (ARA) and eicosapentaenoic acid (EPA) were produced by Pythium irregulare fungus using soybean cotyledon fiber and soy skim, two co-products from soybean aqueous processing, as substrates in different fermentation systems. Parameters such as moisture content, substrate glucose addition, incubation time, and vegetable oil supplementation were found to be important in solid-state fermentation (SSF) of soybean fiber, which is to be used as animal feed with enriched long-chain polyunsaturated fatty acids (PUFA). Soybean fiber with 8 % (dwb) glucose supplementation for a 7-day SSF produced 1.3 mg of ARA and 1.6 mg of EPA in 1 g of dried substrate. When soy skim was used as substrate for submerged fermentation, total ARA yield of 125.7 mg/L and EPA yield of 92.4 mg/L were achieved with the supplementation of 7 % (w/v) soybean oil. This study demonstrates that the values of soybean fiber and soy skim co-products could be enhanced through the long-chain PUFA production by fermentation.     

Animal Bone Char Solubilization with Itaconic Acid Produced by Free and Immobilized Aspergillus terreus Grown on Glycerol-Based Medium

Cells of Aspergillus terreus, free and immobilized in polyurethane foam, were employed in itaconic acid fermentation processes on glycerol-based media. The purpose was to assess their suitability for animal bone char solubilization and the development of a biotechnological alternative to P fertilizers chemically produced from rock phosphate. Animal bones constitute a renewable source of P that can replace the traditionally used finite, nonrenewable rock phosphate as a P source. Glycerol was an excellent substrate for growth (10.2 g biomass L^?1) and itaconic acid production (26.9 g?L^?1) by free fungal cells after 120-h fermentation. Simultaneously, A. terreus solubilized the insoluble phosphate to a yield of 23 to 50 %, depending on the particle size and concentration. Polyurethane foam cut into cubes of 0.5–0.6 cm per side, with 0.3 mm pore size and applied at 2.0 g?L^?1 proved to be an excellent cell carrier. In repeated batch fermentation, the immobilized mycelium showed a high capacity to solubilize animal bone char, which resulted on average in 168.8 mg?L^–1 soluble phosphate per 48-h cycle and 59.4 % yield (percent of total phosphate) registered in the fourth batch.     

Enhancement of Fumaric Acid Production by Rhizopus oryzae Using a Two-stage Dissolved Oxygen Control Strategy

Batch fermentative production of fumaric acid by Rhizopus oryzae ME-F12 was investigated in a 7-l stirred tank fermentor under different dissolved oxygen (DO) concentrations. High fumaric acid yield on glucose (0.56 g/g) was achieved under high DO concentration (80%), but the glucose consumption rate and fumaric acid productivity were rather low (0.91 and 0.51 g/l/h). Fumaric acid productivity was enhanced under low DO concentration (30%), but the fuamric acid yield on glucose decreased to 0.52 g/g. In order to achieve the high fumaric acid yield and productivity simultaneously, a two-stage dissolved oxygen control strategy was proposed, in which the DO concentration was controlled at 80% in the first 18 h and then switched to 30%. This was experimentally proven to be successful. Relatively high fumaric acid production (56.2 g/l), high fumaric acid yield on glucose (0.54 g/g), and high glucose consumption rate (1.3 g/l/h) were achieved by applying this strategy. The productivity (0.7 g/l/h) was improved by 37%, 21%, and 9%, respectively, compared with fermentations in which DO concentrations were kept constant at 80%, 60%, and 30%.     

<Emphasis Type="Italic">Arcopilus aureus</Emphasis>, a Resveratrol-Producing Endophyte from <Emphasis Type="Italic">Vitis vinifera</Emphasis>

Due to great interest on producing bioactive compounds for functional foods and biopharmaceuticals, it is important to explore the microbial degradation of potential sources of target biomolecules. Gallotannins are polyphenols present in nature, an example of them is tannic acid which is susceptible to enzymatic hydrolysis. This hydrolysis is performed by tannase or tannin acyl hydrolase, releasing in this way, biomolecules with high-added value. In the present study, chemical profiles obtained after fungal degradation of tannic acid under two bioprocesses (submerged fermentation (SmF) and solid state fermentation (SSF)) were determined. In both fermentation systems (SmF and SSF), Aspergillus niger GH1 strain and tannic acid as a sole carbon source and inducer were used (the presence of tannic acid promotes production of enzyme tannase). In case of SSF, polyurethane foam (PUF) was used like as support of fermentation; culture medium only was used in case of submerged fermentation. Fermentation processes were monitored during 72 h; samples were taken kinetically every 8 h; and all extracts obtained were partially purified to obtain polyphenolic fraction and then were analyzed by liquid chromatography-mass spectrometry (LC-MS). Molecules like gallic acid and n-galloyl glucose were identified as intermediates in degradation of tannic acid; during SSF was identified ellagic acid production. The results obtained in this study will contribute to biotechnological production of ellagic acid.     

Bioconversion potential of Trichoderma viride HN1 cellulase for a lignocellulosic biomass Saccharum spontaneum

The industrialisation of lignocellulose conversion is impeded by expensive cellulase enzymes required for saccharification in bioethanol production. Current research undertakes cellulase production from pretreated Saccharum spontaneum through Trichoderma viride HN1 under submerged fermentation conditions. Pretreatment of substrate with 2% NaOH resulted in 88% delignification. Maximum cellulase production (2603 ± 16.39 U/mL/min carboxymethyl cellulase and 1393 ± 25.55 U/mL/min FPase) was achieved at 6% substrate at pH 5.0, with 5% inoculum, incubated at 35°C for 120 h of fermentation period. Addition of surfactant, Tween 80 and metal ion Mn⁺², significantly enhanced cellulase yield. This study accounts proficient cellulase yield through process optimisation by exploiting cheaper substrate to escalate their commercial endeavour.     

High Level Production of β-Galactosidase Exhibiting Excellent Milk-Lactose Degradation Ability from Aspergillus oryzae by Codon and Fermentation Optimization

A β-galactosidase gene from Aspergillus oryzae was engineered utilizing codon usage optimization to be constitutively and highly expressed in the Pichia pastoris SMD1168H strain in a high-cell-density fermentation. After fermentation for 96 h in a 50-L fermentor using glucose and glycerol as combined carbon sources, the recombinant enzyme in the culture supernatant had an activity of 4,239.07 U mL^?1 with o-nitrophenyl-β-d-galactopyranoside as the substrate, and produced a total of extracellular protein content of 7.267 g L^?1 in which the target protein (6.24 g L^?1) occupied approximately 86 %. The recombinant β-galactosidase exhibited an excellent lactose hydrolysis ability. With 1,000 U of the enzyme in 100 mL milk, 92.44 % lactose was degraded within 24 h at 60 °C, and the enzyme could also accomplish the hydrolysis at low temperatures of 37, 25, and 10 °C. Thus, this engineered strain had significantly higher fermentation level of A. oryzae lactase than that before optimization and the β-galactosidase may have a good application potential in whey and milk industries.     

Production of Fumaric Acid by Bioconversion of Corncob Hydrolytes Using an Improved <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Strain

The use of microorganism fermentation for production of fumaric acid (FA), which is widely used in food, medicine, and other fields, can provide technical support for the FA industry. In this study, we aimed to increase the titer of FA production by using an improved Rhizopus oryzae WHT5, which was domesticated to obtain a furfural-resistant strain in corncob hydrolytes. The metabolic pathways and metabolic network of this strain were investigated, and the related enzymes and metabolic flux were analyzed. Metabolic pathway analysis showed that the R. oryzae WHT5 strain produced FA mainly through two pathways. One occurred in the cytoplasm and the other was a mitochondrial pathway. The key parameters of the fermentation process were analyzed. The FA titer was 49.05 g/L from corncob hydrolytes using R. oryzae WHT5 in a 7-L bioreactor. The use of a furfural-resistant strain developed through domestication effectively increased the titer of FA. This capacity of the microorganisms to produce high amounts of FA by bioconverting corncob hydrolyte can be further applied for industrial production of FA.     

Enzymatic Hydrolysis and Succinic Acid Fermentation from Steam-Exploded Corn Stalk at High Solid Concentration by Recombinant Escherichia coli

Steam-exploded corn stalk biomass was used as the substrate for succinic acid production via lignocellulose enzymatic hydrolysis and fermentation. Succinic acid fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). For the washed steam-exploded corn stalk at 30 % substrate concentration, i.e., 30 % water-insoluble solids (WIS), enzymatic hydrolysis yielded 97.5 g/l glucose solution and a cellulose conversion of 73.6 %, thus a high succinic acid level up to 38.6 g/l. With the unwashed steam-exploded corn stalk, though a cellulose conversion of 71.2 % was obtained in hydrolysis at 30 % solid concentration (27.9 % WIS), its hydrolysate did not ferment at all, and the hydrolysate of 25 % solid loading containing 3.8 g/l acetic acid and 168.2 mg/l furfural exerted a strong inhibition on succinic acid production.     

Endoglucanase and Total Cellulase from Newly Isolated Rhizopus oryzae and Trichoderma reesei: Production,Characterization, and Thermal Stability

A multienzymatic complex production was evaluated, as well as endoglucanase and total cellulase characterization, during solid-state fermentation of rice industry wastes with Rhizopus oryzae CCT 7560 (newly isolated microorganism) and Trichoderma reesei QM 9414 (control). R. oryzae produced enzymes with higher activity at 15 h of fermentation (5.1 and 2.3 U g^?1 to endoglucanase and total cellulase), while T. reesei produced them at 55 h (15.3 and 2.8 U g^?1 to endoglucanase and total cellulase). The optimum temperature for total cellulase and endoglucanase was 60 °C. For Trichoderma and Rhizopus, the optimum pH was 5.0 and 6.0 for total cellulase and 6.0 and 5.0 for endoglucanase, respectively. The enzymes produced by Rhizopus presented higher stability at the temperature range evaluated (25–100 °C); the endoglucanase K _M value was 20 times lower than the one found for Trichoderma. The characterization of the cellulolytic enzymes from the fungal species native of rice husk revealed that they can be more efficient than the genetically modified enzymes when rice husk and rice bran are used as substrates.