Effects of metabolic inhibitors on the alcoholic fermentation by several yeasts in batch or in immobilized cell systems

Summary In previous papers it was shown that the bacterium Zymomonas mobilis might be an interesting alternative for industrial alcohol production from sugar, compared to Saccharomyces bayanus. Factors that might increase the glucose to ethanol conversion efficiency and which are in favour of the bacterium, are the production of less biomass and less by-products such as glycerol, succinic acid, butanediol, acetoin, and acetic acid. In order to reduce the synthesis of biomass three metabolic inhibitors were now studied: dinitrophenol, azide and arsenate. Their effects on the alcoholic fermentation in batch and in immobilized cell system were investigated, using three yeasts: Saccharomyces bayanus, Schizosacharomyces pombe, and Saccharomyces diastaticus. It was found that dinitrophenol in 0.1 mM concentration was effective in increasing the conversion of glucose to ethanol especially with Saccharomyces bayanus while azide in 0.1 mM concentration was better with Schizosaccharomyces pombe. In immobilized systems high steady state ethanol production from 15% glucose media was obtained by inclusion into the media of dinitrophenol or azide. Arsenate had less effect at the concentration used. Arsenate had less effect at the concentrations used. As a result ethanol productivity in g·l^-1·h^-1 was increased from around 70 in the absence of inhibitor to around 74 in the presence of dinitrophenol with Saccharomyces bayanus. With Schizosaccharomyces pombe the productivity was increased from around 65 in the absence of inhibitor to around 74 in the presence of azide. The specific ethanol productivity expressed as g ethanol formed per hour and per g viable cells was increased from 0.87 to 1.37 for Schizosaccharomyces pombe and from 1.02 to 1.66 for Saccharomyces bayanus.     

Hepatic ethanol metabolism: Respective roles of alcohol dehydrogenase,the microsomal ethanol-oxidizing system,and catalase

The respective role of alcohol dehydrogenase, of the microsomal ethanol-oxidizing system, and of catalase in ethanol metabolism was assessed quantitatively in liver slices using various inhibitors and ethanol at a final concentration of 50 mm. Pyrazole (2 mm) virtually abolished cytosolic alcohol dehydrogenase activity but inhibited ethanol metabolism in liver slices by only 50–60%. The residual pyrazole-insensitive ethanol oxidation in liver slices remained unaffected by in vitro addition of the catalase inhibitor sodium azide (1 mm). At this concentration, sodium azide completely abolished catalatic activity of catalase in liver homogenate as well as peroxidatic activity of catalase in liver slices in the presence of dl-alanine. Similarly, in vivo administration of 3-amino-1,2,4-triazole, a compound which inhibits the activity of catalase but not that of the microsomal ethanol-oxidizing system, failed to decrease both the overall rates of ethanol oxidation and the activity of the pyrazole-insensitive pathway. Finally, butanol, a substrate and inhibitor of the microsomal ethanol-oxidizing system but not of catalase-H₂O₂, significantly decreased the pyrazole-insensitive ethanol metabolism in liver slices. These results indicate that alcohol dehydrogenase is responsible for half or more of ethanol metabolism by liver slices and that the microsomal ethanol-oxidizing system rather than catalase-H₂O₂ accounts for most if not all of the alcohol dehydrogenase-independent pathway.     

Ethanol production by fermentation of sweet-stem sorghum juice using various yeast strains

Ethanol tolerance, osmotolerance and sugar conversion efficiency were used to screen yeasts for potential ethanol production from sweet-stem sorghum juice. Of the ten strains of Saccharomyces sp. that produced ethanol from the sorghum juice or from yeast extract/phosphate/sucrose (YEPS) media, the best sugar conversion efficiencies were greater than 85% for the strains Vin7, SB9, N96 and GSL. Vin7 and SB9 had higher sugar conversion efficiencies for sweet-stem sorghum juice, while strains N96 and GSL gave higher conversions in YEPS.The authors are with the Food and Fermentation Laboratory, Department of Biochemistry, University of Zimbabwe, M.P.167. Mount Pleasant, Harare, Zimbabwe     

Evaluation of ethanol productivity from cellulose by clostridium thermocellum

Clostridium thermocellum, a thermophillic anaerobe, directly converts cellulose to ethanol. To estimate its ethanol production from cellulose, we used a new method based on material balance by which the efficiencies of the enzymes that convert cellulose to ethanol were calculated. Using this method, the maximum efficiency of ethanol production of two strains of C. thermocellum was estimated to be 0.05, with 0.67 as the theoretical maximum.     

Steam–air fluidized bed gasification of distillers grains: Effects of steam to biomass ratio,equivalence ratio and gasification temperature

In this study, thermochemical biomass gasification was performed on a bench-scale fluidized-bed gasifier with steam and air as fluidizing and oxidizing agents. Distillers grains, a non-fermentable byproduct of ethanol production, were used as the biomass feedstock for the gasification. The goal was to investigate the effects of furnace temperature, steam to biomass ratio and equivalence ratio on gas composition, carbon conversion efficiency and energy conversion efficiency of the product gas. The experiments were conducted using a 3 × 3 × 3 full factorial design with temperatures of 650, 750 and 850 °C, steam to biomass ratios of 0, 7.30 and 14.29 and equivalence ratios of 0.07, 0.15 and 0.29. Gasification temperature was found to be the most influential factor. Increasing the temperature resulted in increases in hydrogen and methane contents, carbon conversion and energy efficiencies. Increasing equivalence ratio decreased the hydrogen content but increased carbon conversion and energy efficiencies. The steam to biomass ratio was optimal in the intermediate levels for maximal carbon conversion and energy efficiencies.     

The Feasibility of Producing Adequate Feedstock for Year-Round Cellulosic Ethanol Production in an Intensive Agricultural Fuelshed

To date, cellulosic ethanol production has not been commercialized in the United States. However, government mandates aimed at increasing second-generation biofuel production could spur exploratory development in the cellulosic ethanol industry. We conducted an in-depth analysis of the fuelshed surrounding a starch-based ethanol plant near York, Nebraska that has the potential for cellulosic ethanol production. To assess the feasibility of supplying adequate biomass for year-round cellulosic ethanol production from residual maize (Zea mays) stover and bioenergy switchgrass (Panicum virgatum) within a 40-km road network service area of the existing ethanol plant, we identified ～14,000 ha of marginally productive cropland within the service area suitable for conversion from annual rowcrops to switchgrass and ～132,000 ha of maize-enrolled cropland from which maize stover could be collected. Annual maize stover and switchgrass biomass supplies within the 40-km service area could range between 429,000 and 752,000 metric tons (mT). Approximately 140–250 million liters (l) of cellulosic ethanol could be produced, rivaling the current 208 million l annual starch-based ethanol production capacity of the plant. We conclude that sufficient quantities of biomass could be produced from maize stover and switchgrass near the plant to support year-round cellulosic ethanol production at current feedstock yields, sustainable removal rates and bioconversion efficiencies. Modifying existing starch-based ethanol plants in intensive agricultural fuelsheds could increase ethanol output, return marginally productive cropland to perennial vegetation, and remove maize stover from productive cropland to meet feedstock demand.     

Full-scale On-farm Pretreatment of Perennial Grasses with Dilute Acid for Fuel Ethanol Production

Biorefineries that rely on lignocellulosic feedstocks require dependable and safe methods for storing biomass. Storing biomass wet in the presence of sulfuric acid and the absence of oxygen has been shown to preserve carbohydrates and enhance cellulose conversion but has not been demonstrated at farm-scale. To that end, switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated with 18?N sulfuric acid with two methods: during bagging (on-line) and thoroughly mixed in a commercial feed mixer (mixed) and both stored for 90 days. The two methods, applied at rates from 28 to 54 g(kg DM)^?1 not only helped to preserve biomass substrates under on-farm conditions (anaerobic, ambient temperature and pressure) through inhibition of microbial activity but also enhanced conversion of cellulose to ethanol by simultaneous saccharification and fermentation (SSF) using Saccharomyces cerevisiae. Acid-pretreated substrate yielded 19 and 7 percentage points higher ethanol conversion efficiencies than fresh reed canarygrass and switchgrass, respectively. The on-line method of pretreatment out-yielded the mixed method both as a preservative and as an agent for enhanced cell wall degradation. This result was thought to be an outcome of more uniform acid application as indicated by the on-line method’s more consistent pH profile and decreased fermentation products, as compared to the mixed method. Although significant levels of acetate and lactate were present in the biomass following storage, concentrations were not sufficient to inhibit S. cerevisiae in SSFs with a 10% solids loading.     

Studies on the transport mechanism of 5-methyltetrahydrofolic acid in freshly isolated hepatocytes: effect of ethanol.

The effect of ethanol on the transport of 5-methyltetrahydrofolate in freshly isolated hepatocytes in vitro resulted in about a 30% increase in accumulation of substrate. It was shown that this was not due to differences in metabolism, nor to an inhibition of efflux. Preincubation with 40 mm ethanol for 45 min resulted in a significantly increased rate of entry of 5-methyltetrahydrofolate into the cells. The stimulatory effect was specific to 5-methyltetrahydrofolate since ethanol inhibited uptake of folate and methotrexate. The increased uptake was due to metabolism of ethanol as shown by studies with pyrazole. Also, the n-alkanols, propanol through pentanol, and sorbitol but not methanol were stimulatory. Anaerobiosis and sodium azide stimulated uptake of 5-methyl-tetrahydrofolate but were inhibitory to methotrexate uptake. These data, taken together, suggest that the ethanol effect is due to increased entry of 5-CH₃-H₄PteGlu into the cells possibly as the result of an increased cellular $\text{NADH}\text{NAD}$ ratio.     

The effects of Mn2+ on ethanol production by Kluyveromyces marxianus IMB3 during growth on lactose-containing media at 45°C

Summary It has previously been demonstrated that the thermotolerant yeast strain, K. marxianus IMB3 is capable of growth and ethanol production on lactose containing media at 45°C. Although the organism is capable of producing a -galactosidase, that enzyme has been shown to be extremely thermolabile at 45°C and this has been reflected in reduced efficiencies with respect to conversion of lactose to ethanol. In this paper we demonstrate that addition of Mn²⁺ ions to enzyme preparations contributes significantly to enzyme stability at 45°C. We also demonstrate that addition of Mn²⁺ to fermentations results in increased efficiency of conversion of lactose to ethanol at this temperature.     

Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of Zymomonas mobilis

A laboratory process was established for ethanol production by fermentation of sugar beet molasses with the bacterium Zymomonas mobilis. Sucrose in the molasses was hydrolyzed enzymatically to prevent levan formation. A continuous system was adopted to reduce sorbitol formation and a two-stage fermentor was used to enhance sugar conversion and the final ethanol concentration. This two-stage fermentor operated stably for as long as 18 d. An ethanol concentration of 59.9 g/l was obtained at 97% sugar conversion and at high ethanol yield (0.48 g/g, 94% of theoretical). The volumetric ethanol productivity (3.0 g/l·h) was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the thanol concentration was increased to a level acceptable for economical recovery. The process proposed in this paper is the first report of successful fermentation of sugar beet molasses in the continuous mode using the bacterium Z. mobilis.     

The utilization of metabolic inhibitors for shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis

Summary Xylose, glucose and xylose/glucose mixtures were fermented with Candida tropicalis ATCC 32113 under aerobic, oxygen limited and anaerobic conditions. Ethanol yields were highest under oxygen limited conditions with xylose and xylose/glucose. Anaerobic conditions were best for glucose fermentations.The effect of four metabolic inhibitors (azide, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), oligomycin A and valinomycin-K⁺) were then studied under oxygen limited conditions. Only azide had a significant influence on ethanol production. At 2¢10^-4 M concentrations, ethanol yield increased up to two times and xylitol levels were repressed by 90% for xylose and glucose/xylose fermentations. 4.2×10^-3 M azide gave highest ethanol yields in glucose fermentations. At this concentration of azide, however, cell growth was inhibited, which seemed to prevent ethanol production in xylose fermentations. The effect of azide is discussed in terms of fine-tuning the respiratory activity necessary for metabolism.     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.     

The direct oxidation of ethanol by a catalase- and alcohol dehydrogenase-free reconstituted system containing cytochrome P-4501.

Ethanol oxidation activity has been reconstituted in a system composed of NADPH-cytochrome c reductase, synthetic dilauroylglycerol-3-phosphorylcholine and cytochrome P-450 purified from liver microsomes of phenobarbital-treated rats. This system is free of alcohol dehydrogenase and catalase activities. Furthermore, sodium azide (1 mm), a catalase inhibitor, is without effect on ethanol metabolism. There is a requirement for both NADPH-cytochrome c reductase and cytochrome P-450 and a partial requirement for phospholipid for ethanol oxidation by the reconstituted system. In addition, both NADPH and O₂ are required for catalysis. Under optimal reaction conditions, the rate of acetaldehyde formation if 25 to 50 nmol/min/nmol of cytochrome P-450. Cytochrome P-450 from other sources, including the homogeneous P-450LM₂ from phenobarbital-treated rabbits, have also been found to catalyze ethanol oxidation in reconstituted systems. Antibody prepared against cytochrome P-450 inhibits ethanol metabolism in the reconstituted system consistent with a cytochrome P-450-mediated reaction. Furthermore, cumene hydroperoxide can replace both NADPH and NADPH-cytochrome c reductase in ethanol oxidation and catalysis can be demonstrated in a system composed of only cytochrome P-450, lipid, ethanol, and cumene hydroperoxide. These data implicate cytochrome P-450 in the direct oxidation of ethanol by this system.     

Large Deformation of Helix F during the Photoreaction Cycle of Pharaonis Halorhodopsin in Complex with Azide

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl⁻ and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.     

An experimental study of the oxygen consumption,growth, and metabolism of the COD (Gadus Morhua L.)

The rate of oxygen consumption of cod in sea water at 12 °C containing MS222 (25 mg/l) can be expressed as: Q_o2 = 0.245 W^0,82(mg/h), where W is the lived weight of the fish (g). The maximum efficiency of conversion of assimilated food into growth was 24% during the feeding experiment. Digestion efficiencies were estimated at over 98% using fillets of plaice as food. The effect of increasing the rate of food intake was to increase liver weight and condition factor. The relative proportions of protein and lipid in the body did not change over the range of feeding levels used. The conversion efficiency had a maximum value at an intermediate feeding rate.     

Scale-up of ethanol production from sugarcane usingZymomonas mobilis

Summary TheZymomonas fermentation for industrial ethanol production has been successfully scaled up. Pilot plant experiments at 100 and 1,000 litre fermentation capacity gave 91–95% conversion efficiencies and up to 10% (v/v) ethanol yields within 17–20 hours using sugar cane syrup, A-, B-, and C-molasses with the addition of sucrose or syrup to a final 15% total sugar concentration.     

Ethanol production from wheat flour by Zymomonas mobilis

Starch from wheat flour was enzymatically hydrolyzed and used for ethanol production by Zymmonas mobilis. The addition of a nitrogen source like ammonium sulfate was sufficient to obtain a complete fermentation of the hdyrolyzed strach. In batch culture a glucose concentration as high as 223 g/l could be fermented (conversion 99.5%) to 105 g/l of ethanol in 70 h with an ethanol yield of 0.47 g/g (92% of theoretical). In continuous culture the use of a flocculent strain and a fermentor with an internal settler resulted (D=1,4 h⁻¹) in a high ethanol productivity of 70.7 g/l·h with: ethanol concentration 49.5 g/l, ethanol yield 0.50 g/g (98% of theoretical and substrate conversion 99%.     

A new dawn for industrial photosynthesis

Several emerging technologies are aiming to meet renewable fuel standards, mitigate greenhouse gas emissions, and provide viable alternatives to fossil fuels. Direct conversion of solar energy into fungible liquid fuel is a particularly attractive option, though conversion of that energy on an industrial scale depends on the efficiency of its capture and conversion. Large-scale programs have been undertaken in the recent past that used solar energy to grow innately oil-producing algae for biomass processing to biodiesel fuel. These efforts were ultimately deemed to be uneconomical because the costs of culturing, harvesting, and processing of algal biomass were not balanced by the process efficiencies for solar photon capture and conversion. This analysis addresses solar capture and conversion efficiencies and introduces a unique systems approach, enabled by advances in strain engineering, photobioreactor design, and a process that contradicts prejudicial opinions about the viability of industrial photosynthesis. We calculate efficiencies for this direct, continuous solar process based on common boundary conditions, empirical measurements and validated assumptions wherein genetically engineered cyanobacteria convert industrially sourced, high-concentration CO₂ into secreted, fungible hydrocarbon products in a continuous process. These innovations are projected to operate at areal productivities far exceeding those based on accumulation and refining of plant or algal biomass or on prior assumptions of photosynthetic productivity. This concept, currently enabled for production of ethanol and alkane diesel fuel molecules, and operating at pilot scale, establishes a new paradigm for high productivity manufacturing of nonfossil-derived fuels and chemicals.     

Unsaturated fatty acid but not ergosterol is essential for high ethanol production in Saccharomyces

Summary The membrane lipid composition of Saccharomyces was manipulated by growing cells anaerobically with or without ergosterol and unsaturated fatty acid. Cells low in ergosterol but enriched in unsaturated fatty acid residues on membrane phospholipids produced high concentrations, 13–15.5% w/v, of ethanol at substrate conversion efficiencies of around 90%.