1,2-Diazole and 2,2,2-Trifluoroethanol and Their Regulatory Effects on Ethanol and Lactic Acid Formation in the Living Culture of Rhizopus oryzae

In heterofermentation of Rhizopus oryzae, ethanol is the major byproduct which reduces the production of a desired product, an optically pure l-lactic acid. To improve lactic acid production, regulating the alcohol fermentative pathway to limit ethanol production has been done by various techniques. In vitro study on alcohol dehydrogenase (ADH) inhibition in several organisms showed that 1,2-diazole and 2,2,2-trifluoroethanol were competitively bound at the active sites that eventually limited ethanol production. In this study, 1,2-diazole and 2,2,2-trifluoroethanol were present during fermentation of R. oryzae. It was found that both 1,2-diazole and 2,2,2-trifluoroethanol not only strongly affected ethanol formation but they also indirectly regulated lactate production as observed by the decreasing affinity for glucose flux toward lactate and ethanol production. The increase in both ethanol and lactate formation rates revealed 1,2-diazole and 2,2,2-trifluoroethanol not only regulated the reversible redox reaction by ADH, but they also caused the dynamic change in the conversion of all metabolites in the living R. oryzae in order to maintain the balanced flux for cellular growth and maintenance.     

In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in <Emphasis Type="Italic">Rhizopus Oryzae</Emphasis> to Improve <Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid Fermentation

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.     

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.     

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l ?(+)?lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure l-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine l-lactate dehydrogenase (l-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in lowcost medium, cane juice-based medium was used in fermentation with neutralizing conditions. l-lactate production reached 122 g/L, with 61% of sugar being transformed into l-lactate finally. The optical purity of this l-lactate, that affects the physical characteristics of poly-l-lactic acid, was extremely high, 99.9% or over. These two authors contributed equally to this work.     

Construction and characterization of fermentative lactate dehydrogenase Escherichia coli mutant and its potential for bacterial hydrogen production

In Escherichia coli, classified as a mixed-acid producer in fermentation, D-lactate is one of the final metabolites from pyruvate. In order to achieve a high efficiency of bacterial hydrogen production from glucose, we have constructed an E. coli strain deficient in fermentative lactate dehydrogenase (LDH-A) by P1 transduction. The mutant, designated as MC13-4, entirely lost LDH-A activity while retaining whole formate hydrogenlyase activity. This mutation resulted in an increase in hydrogen production based on glucose consumed. The effect of uptake hydrogenases on the hydrogen production was also discussed.     

Metabolic engineering of Saccharomyces cerevisiae for efficient production of pure l−(+)−lactic acid

We developed a metabolically engineered Saccharomyces cerevisiae, which produces optically pure L-lactic acid efficiently using cane juice-based medium. In this recombinant, the coding region of pyruvate decarboxylase (PDC)1 was completely deleted, and six copies of the bovine L-lactate dehydrogenase (L-LDH) genes were introduced on the genome under the control of the PDC1 promoter. To confirm optically pure lactate production in low cost medium, cane juice-based medium was used in fermentation with neutralizing conditions. L-lactate production reached 122 g/L, with 61% of sugar being transformed into L-lactate finally. The optical purity of this L-lactate, that affects the physical characteristics of poly-L-lactic acid, was extremely high, 99.9% or over.     

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.     

Effect of water extracts of aloe and some herbs in decreasing blood ethanol concentration in rats. II

Oral administration of ethanol to rats at a dose of 3 g/kg decreased alcohol dehydrogenase (ADH) activity and metabolism of lactate to pyruvate in the liver. The effects of water extracts of Aloe and some other herbs on blood ethanol concentration and on ADH activity in liver cytosol were examined. The water extracts of these herbs caused a faster elimination of ethanol from blood of normal rats when administered orally 30 min before oral administration of ethanol. The rapid elimination of ethanol seems to be due to a protection of ADH activity and the supply of nicotinamide dinucleotide, both of which are reduced by high ethanol concentration. The effects of ethanol in decreasing the enzyme activities relating to its own metabolism occur when high concentrations of ethanol pass through the liver, and thus may primarily appear during the absorption of alcohol from the gastrointestinal tract, when portal concentration of ethanol are very high.     

Development of Industrial Brewing Yeast with Low Acetaldehyde Production and Improved Flavor Stability

Higher acetaldehyde concentration in beer is one of the main concerns of current beer industry in China. Acetaldehyde is always synthesized during beer brewing by the metabolism of yeast. Here, using ethanol as the sole carbon source and 4-methylpyrazole as the selection marker, we constructed a new mutant strain with lower acetaldehyde production and improved ethanol tolerance via traditional mutagenesis strategy. European Brewery Convention tube fermentation tests comparing the fermentation broths of mutant strain and industrial brewing strain showed that the acetaldehyde concentration of mutant strain was 81.67 % lower, whereas its resistant staling value was 1.0-fold higher. Owing to the mutation, the alcohol dehydrogenase activity of the mutant strain decreased to about 30 % of the wild-type strain. In the meantime, the fermentation performance of the newly screened strain has little difference compared with the wild-type strain, and there are no safety problems regarding the industrial usage of the mutant strain. Therefore, we suggest that the newly screened strain could be directly applied to brewing industry.     

Kinetics and Thermodynamics of Ethanol Production by Saccharomyces cerevisiae MLD10 Using Molasses

In this study, we have used ultraviolet (UV) and γ-ray induction to get a catabolite repression resistant and thermotolerant mutant with enhanced ethanol production along with optimization of sugar concentration and temperature of fermentation. Classical mutagenesis in two consecutive cycles of UV- and γ-ray-induced mutations evolved one best catabolite-resistant and thermotolerant mutant Saccharomyces cerevisiae MLD10 which showed improved ethanol yield (0.48?±?0.02 g g^?1), theoretical yield (93?±?3 %), and extracellular invertase productivity (1,430?±?50 IU l^?1 h^?1), respectively, when fermenting 180 g sugars l^?1 in molasses medium at 43 °C in 300 m³ working volume fermenter. Ethanol production was highly dependent on invertase production. Enthalpy (ΔH*) (32.27 kJ M^?1) and entropy (ΔS*) (?202.88 J M^?1 K^?1) values at 43 °C by the mutant MLD10 were significantly lower than those of β-glucosidase production by a thermophilic mutant derivative of Thermomyces lanuginosus. These results confirmed the enhanced production of ethanol and invertase by this mutant derivative. These studies proved that mutant was significantly improved for ethanol production and was thermostable in nature. Lower fermentation time for ethanol production and maintenance of ethanol production rates (3.1 g l^?1 h^?1) at higher temperature (43 °C) by this mutant could decrease the overall cost of fermentation process and increase the quality of ethanol production.     

Enhanced Fumaric Acid Production from Brewery Wastewater and Insight into the Morphology of Rhizopus oryzae 1526

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5 % (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465?±?0.04 mm), the highest concentration of fumaric acid achieved was 31.3?±?2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.     

Production and application of amylases of Rhizopus oryzae and Rhizopus microsporus var. oligosporus from industrial waste in acquisition of glucose

Amylases from Rhizopus oryzae and Rhizopus microsporus var. oligosporus were obtained using agro-industrial wastes as substrates in submerged batch cultures. The enzymatic complex was partially characterised for use in the production of glucose syrup. Type II wheat flour proved better than cassava bagasse as sole carbon source for amylase production. The optimum fermentation condition for both microorganisms was 96 hours at 30°C and the amylase thus produced was used for starch hydrolysis. The product of the enzymatic hydrolysis indicated that the enzyme obtained was glucoamylase, only glucose as final product was attained for both microorganisms. R. oligosporus was of greater interest than R. oryzae for amylase production, taking into account enzyme activity, cultivation time, thermal stability and pH range. Glucose syrup was produced using concentrated enzyme and 100 g L^?1 starch in a 4 hours reaction at 50°C. The bioprocess studied can contribute to fungus glucoamylase production and application.     

Metabolic Engineering of Escherichia coli Cells for Ethanol Production under Aerobic Conditions

The ethanol production by recombinant Escherichia coli introducing of pyruvate decarboxylase (pdc) and alcohol dehydrogenase (adhB) from Zymomonas mobilis was investigated under aerobic conditions. In aerobic culture (K_La = 1.5 min^-1), the cells expressing pdc and adhB produced 0.4 g l^-1 ethanol when cultured for 18 h. This value was improved in BW25113Δpta/pHfdh/pTadhB-pdc following 4 g l^-1 formate feeding at 0.8 g l^-1 ethanol. In higher oxygenation level (K_La = 6.1 min^-1), the production of ethanol was further enhanced at 1.79 g l^-1 ± 0.37 g l^-1 after 24 h cultivation. Formate was found not detectable at the end of culture, indicating complete degradation this organic acid to regenerate NADH from NAD⁺. The culture strategy was effective to inactivate lactate dehydrogenase, which is major competitor for ethanol production in utilizing NADH.     

Selective n-Butanol Production by Clostridium sp. MTButOH1365 During Continuous Synthesis Gas Fermentation Due to Expression of Synthetic Thiolase, 3-Hydroxy Butyryl-CoA Dehydrogenase, Crotonase, Butyryl-CoA Dehydrogenase, Butyraldehyde Dehydrogenase, and NAD-Dependent Butanol Dehydrogenase

Acetogen Clostridum sp. MT1962 produced 287 mM acetate (p?<?0.005) and 293 mM ethanol (p?<?0.005) fermenting synthesis gas blend 60 % CO and 40 %?H₂ in single-stage continuous fermentation. This strain was metabolically engineered to the biocatalyst Clostridium sp. MTButOH1365. The engineered biocatalyst lost production of ethanol and acetate while initiated the production of 297 mM of n-butanol (p?<?0.005). The metabolic engineering comprised Cre-lox66/lox71-based elimination of phosphotransacetylase and acetaldehyde dehydrogenase along with integration to chromosome synthetic thiolase, 3-hydroxy butyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, and NAD-dependent butanol dehydrogenase. This is the first report on elimination of acetate and ethanol production genes and expression of synthetic gene cluster encoding n-butanol biosynthesis pathway in acetogen biocatalyst for selective fuel n-butanol production with no antibiotic support for the introduced genes.     

Maximizing Hydrogen Production and Substrate Consumption by Escherichia coli WDHL in Cheese Whey Fermentation

Fermentative hydrogen production is strongly affected by pH. In order to maximize hydrogen production and substrate consumption in Escherichia coli ΔhycA, ΔlacI (WDHL) cheese whey fermentation, the influence of pH control at values of 5.5, 6, and 6.5 was studied in batch stirred-tank bioreactors. From the conditions evaluated, pH 6.5 was the best condition, at which the highest cumulative hydrogen production and yield (1.78 mol H₂/mol lactose) were obtained. Moreover, at this pH, all carbohydrates from the cheese whey were consumed, and a mix of ethanol and organic acids, mainly lactate, were produced from glucose, whereas galactose yielded acetate, ethanol, and succinate. Operating the reactor at pH 5.5 resulted in the highest maximum specific production rate, but smaller hydrogen yield because only glucose was metabolized and galactose was accumulated. At pH 6, not all cheese whey carbohydrates were consumed, and it was not favorable for hydrogen production. Lactose consumption and growth kinetics were not affected by the pH. The results show the importance of controlling pH to maximize hydrogen production and substrate consumption using cheese whey as substrate.     

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.     

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.     

Corn steep liquor as a cost-effective nutrition adjunct in high-performanceZymomonas ethanol fermentations

The economics of large-scale production of fuel ethanol from biomass and wastes requires the efficient utilization of all the sugars derived from the hydrolysis of the heteropolymeric hemicellulose component of lignocellulosic feedstocks. Glucuronic and 4-0-methyl-glucuronic acids are major side chains in xylans of the grasses and hardwoods that have been targeted as potential feedstocks for the production of cellulosic ethanol. The amount of these acids is similar to that of arabinose, which is now being viewed as another potential substrate in the production of biomass-derived ethanol. This study compared the end-product distribution associated with the fermentation of D-glucose (Glc) and D-glucuronic acid (GlcUA) (as sole carbon and energy sources) byEscherichia coli B (ATCC 11303) and two different ethanologenic recombinants—a strain in whichpet expression was via a multicopy plasmid (pLOI297) and a chromosomally integrated construct, strain KO11. pH-stat batch fermentations were conducted using a modified LB medium with 2% (w/v) Glc or GlcUA with the set-point for pH control at either 6.3 or 7.0. The nontransformed host culture produced only lactic acid from glucose, but fermentation of GlcUA yielded a mixture of ethanol, acetic, and lactic acids, with acetic acid being the predominant end-product. The ethanol yield associated with GlcUA fermentation by both recombinants was similar, but acetic acid was a significant by-product. Increasing the pH from 6.3 to 7.0 increased the rate of glucuronate fermentation, but it also decreased the ethanol mass yield from 0.22 to 0.19 g/g primarily because of an increase in acetic acid production. In all fermentations there was good closure of the carbon mass balance, the exception being the recombinant bearing plasmid pLOI297 that produced an unidentified product from GlcUA. The metabolism of GlcUA by this metabolically engineered construct remains unresolved. The results offered insights into metabolic fluxes and the regulation of pyruvate catabolism in the wild-type and engineered strains. End-product distribution for metabolism of glucuronic acid by the nontransformed, wild-typeE. coli B and recombinant strain KO11 suggests that the enzyme pyruvate-formate lyase is not solely responsible for the production of acetylCoA from pyruvate and that derepressed pyruvate dehydrogenase may play a significant role in the metabolism of GlcUA.     

A novel fermentation pathway in anEscherichia coli mutant producing succinic acid,acetic acid,and ethanol

Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E.coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.     

Efficient Production of Ethanol from Empty Palm Fruit Bunch Fibers by Fed-Batch Simultaneous Saccharification and Fermentation Using Saccharomyces cerevisiae

The concentration of ethanol produced from lignocellulosic biomass should be at least 40 g l^?1 [about 5 % (v/v)] to minimize the cost of distillation process. In this study, the conditions for the simultaneous saccharification and fermentation (SSF) at fed-batch mode for the production of ethanol from alkali-pretreated empty palm fruit bunch fibers (EFB) were investigated. Optimal conditions for the production of ethanol were identified as temperature, 30 °C; enzyme loading, 15 filter paper unit g^?1 biomass; and yeast (Saccharomyces cerevisiae) loading, 5 g l^?1 of dry cell weight. Under these conditions, an economical ethanol concentration was achieved within 17 h, which further increased up to 62.5 g l^?1 after 95 h with 70.6 % of the theoretical yield. To our knowledge, this is the first report to evaluate the economic ethanol production from alkali-pretreated EFB in fed-batch SSF using S. cerevisiae.