Botanical and geographical origin identification of industrial ethanol by stable isotope analyses of C, H, and O

The isotope ratios of carbon, hydrogen, and oxygen of rectified alcohols were determined to distinguish their botanical and geographical origins by continuous flow-isotope ratio mass spectrometry (CF-IRMS). The (13)C/(12)C and (18)O/(16)O ratios of 27 fermented alcohols with known origins showed clusters derived from each botanical origin, viz. corn, sugarcane, wheat, and tapioca. C3 and C4 plants were easily distinguishable by the (13)C/(12)C ratio. Sugarcane and corn are both C4 plants, and they showed small differences in isotope ratios. The combination plots of the D/H and (18)O/(16)O ratios enabled us to designate the geographical origins of alcohol derived from the same kind of crop, such as Chinese or American corn. The chemically synthetic and fermented alcohols were clearly distinguished by D/H and (18)O/(16)O ratios. Isotope ratios were useful for origin identification of alcohol. We plan to construct a database of alcohol isotope ratios to determine the origins of raw materials in alcohol.     

Improvement of erythromycin production by Saccharopolyspora erythraea in molasses based medium through cultivation medium optimization

In the present work, erythromycin production was carried out in submerged culture using Saccharopolyspora erythraea. Different experiments were conducted to optimize the cultivation medium through the change of carbon and nitrogen sources to cheaper one in order to reduce the cost of medium and to utilize sugar cane molasses as one of major sugar industry by-products in Egypt. It was found that the addition of sugar cane molasses a sole carbon source at a concentration of 60 g/l accompanied by corn steep liquor (as organic N-source) in combination with ammonium sulphate (as inorganic N-source) gave the maximal erythromycin production. The antibiotic production in this medium reached about 600 mg/l which is about 33% higher than the value obtained in glucose based medium. On the other hand, the addition of n-propanol in concentration of 1% (v/v) increased the antibiotic production reaching about 720 mg/l after 144 h. Concluding, the new medium formulation based on cheap carbon source, sugar cane molasses, was a good alternative solution for the production of erythromycin economically.     

The Use of the abundance ratio of 13C and 12C isotopes for characteristic of the origin of ethyl alcohol

During alcohol fermentation, the carbon isotope composition of ethyl alcohol produced depended on the substrate used and was characterized by the value of delta 13C equal to -24.7 +/- 0.8/1000 (wheat grain), -22 +/- 0.1/1000 (rye grain), -22 +/- 0.5/1000 (products of wood hydrolysis), -15.3 +/- 0.3/1000 (maize grain) and -10 +/- 0.1/1000 (sugar cane). The isotope composition of carbon of ethyl alcohol obtained during catalytic hydroxylation of ethylene has a delta 13C of -30.6 +/- 0.3/1000. The possibility of quantitative determination of specific components in mixtures of ethanol samples with various isotope compositions (chemical synthesis and alcohol fermentation of raw material from C3- or C4-plants) was shown.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources

Summary Azotobacter vinelandii strain UWD formed >2 mg/ml poly--hydroxybutyrate (pHB) during exponential growth in media containing ammonium acetate and 1% w/v glucose, fructose, sucrose, or maltose, and >1.5 mg/ml with 1% w/v sodium gluconate or glycerol. After acetate exhaustion, pHB formation accompanied carbohydrate utilization and pHB rapidly accounted for 53%–70% of the cell mass. Strain UWD also formed >2 mg/ml pHB when it was grown with 2% w/v corn syrup, cane molasses, beet molasses, or malt extract. Beet molasses had a growth stimulatory effect which promoted higher yields of pHB/ml and a high ratio of pHB/protein. Malt extract also promoted higher yields of pHB/ml. In this case, pHB formation was no longer subject to acetate repression and the cells contained a higher ratio of pHB/protein. This study shows that unrefined carbon sources support pHB formation in strain UWD and that the yields of pHB were comparable to or better than those obtained with refined carbon sources.     

木薯和玉米原料丁醇发酵中丁醇丙酮质量比的图论理论计算及其验证

在丁醇发酵产溶剂阶段,乙酸和丁酸的生成途径、消耗途径同时存在,各自形成一个闭环路径。本研究利用图论对丁醇发酵中丁醇丙酮质量比进行了理论计算,并对以木薯和玉米为原料的丁醇发酵进行了模拟计算,结果表明:丁酸闭环路径(L2环)的代谢强度是影响丁醇丙酮质量比的主要因素,并且L2环的代谢强度越弱,丁醇丙酮质量比越高;与玉米原料丁醇发酵相比,木薯原料发酵的m(丁醇)/m(丙酮)提高了16.7%。实验结果证实了以上计算结果:在传统发酵、油醇萃取发酵和生物柴油萃取发酵中,以木薯(适时添加酵母浸粉)为原料的发酵批次与以玉米为原料的发酵批次相比,由于其丁酸闭环路径代谢强度较弱,相应发酵方式下丁醇丙酮质量比分别提高了12.9%、61.4%和6.7%,而且两种原料相应发酵方式的丁醇总产量和生产效率基本持平。另外,高丁醇丙酮质量比的木薯发酵所得改良型生物柴油中丁醇浓度与玉米发酵的相比提高了16%,性能得到进一步提高。     

The use of tamarind waste to improve ethanol production from cane molasses

Tamarind wastes such as tamarind husk, pulp, seeds, fruit and the effluent generated during tartaric acid extraction were used as supplements to evaluate their effects on alcohol production from cane molasses using yeast cultures. Small amounts of these additives enhanced the rate of ethanol production in batch fermentations. Tamarind fruit increased ethanol production (9.7%, w/v) from 22.5% reducing sugars of molasses as compared to 6.5% (w/v) in control experiments lacking supplements after 72 h of fermentation. In general, the addition of tamarind supplements to the fermentation medium showed more than 40% improvement in ethanol production using higher cane molasses sugar concentrations. The direct fermentation of aqueous tamarind effluent also yielded 3.25% (w/v) ethanol, suggesting its possible use as a diluent in molasses fermentations. This is the first report, to our knowledge, in which tamarind-based waste products were used in ethanol production. Received 2 April 1998/ Accepted in revised form 13 November 1998     

The use of13C/12C isotope abundance ratio for characterization of the origin of ethyl alcohol

The carbon isotope composition of ethyl alcohol produced during alcohol fermentation depended on the substrate used and was characterized by the value of δ¹³C equal to −24.7 ± 0.8%. (wheat grain), −22 ± 0.1%. (rye grain), −22 ± 0.5%. (products of wood hydrolysis), −15.3 ±0.3%. (maize grain) and −10 ± 0.1%. (sugar cane). The isotope composition of carbon of ethyl alcohol obtained during catalytic hydroxylation of ethylene has a δ¹³C of −30.6 ± 0.3%.. The possibility of quantitative determination of specific components in mixtures of ethanol samples with various isotope compositions (chemical synthesis and alcohol fermentation of raw material from C₃ or C₄ plants) was shown.     

Effect of gamma-ray irradiation on alcohol production from corn

Cracked corn was irradiated with gamma rays at 0-100 Mrad and the effects of the irradiation on sugar yield, susceptibility to enzymatic hydrolysis of starch, yeast growth, and alcohol production were studied. Gamma irradiation at 50 Mrad or greater produced a considerable amount of reducing sugar but little glucose. At lower dosages, gamma irradiation significantly increased the susceptibility of corn starch to enzymatic hydrolysis, but dosages of 50 Mrad or greater decomposed the starch molecules as indicated by the reduction in iodine uptake. About 12.5% reducing sugar was produced by amylase treatment of uncooked, irradiated corn. This amount exceeded the level of sugar produced from cooked (gelatinized) corn by the same enzyme treatment. The yeast numbers in submerged cultivation were lower on a corn substrate that was irradiated at 50 Mrad or greater compared to that on an unirradiated control. About the same level of alcohol was produced on uncooked, irradiated (10(5)-10(6) rad) corn as from cooked (121 degrees C for 30 min) corn. Therefore, the conventional cooking process for gelatinization of starch prior to its saccharification can be eliminated by irradiation. Irradiation also eliminated the necessity of sterilization of the medium and reduced the viscosity of high levels of substrate in the fermentation broth.     

Comparative hydrolysis and fermentation of sugarcane and agave bagasse

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treatment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Celluclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.     

Production of PHB by a Bacillus megaterium strain using sugarcane molasses and corn steep liquor as sole carbon and nitrogen sources

Poly(hydroxybutyric acid) (PHB) and other biodegradable polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of cane molasses and corn steep liquor, two of the cheapest substrates available in Egypt, may help to reduce the cost of producing such biopolyesters. In this work, the effect of different carbon sources was studied. Maximum production of PHB was obtained with cane molasses and glucose as sole carbon sources (40.8, 39.9 per mg cell dry matter, respectively). The best growth was obtained with 3% molasses, while maximum yield of PHB (46.2% per mg cell dry matter) was obtained with 2% molasses. Corn steep liquor was the best nitrogen source for PHB synthesis (32.7 mg per cell dry matter), on the other hand, best growth was observed when ammonium chloride, ammonium sulphate, ammonium oxalate or ammonium phosphate were used as nitrogen sources.     

Elevated polysaccharide production by mutants of the fungus Aureobasidium pullulans

Abstract Two mutants of the fungus Aureobasidium pullulans ATCC 42023 were isolated that exhibited elevated polysaccharide production. Both mutants were isolated using a combination of chemical mutagenesis and resistance to growth inhibitors. It was found that both mutants elaborated higher polysaccharide levels after 7 days of growth on corn syrup or sucrose, respectively, compared to ATCC 42023. The dry weights of the mutant cells were found not to differ greatly from those of the parent cells whether corn syrup or sucrose served as the carbon source. The pullulan content of the polysaccharide synthesized by the mutants or parent cells on sucrose was consistently lower than polysaccharide synthesized on corn syrup. Using corn syrup as a carbon source, the pullulan content of the polysaccharide elaborated by the parent was higher than either mutant. The inverse was found to occur with respect to pullulan content when the strains were grown on sucrose as a carbon source.     

Production of Aspergillus terreus alpha-L-rhamnosidase by solid state fermentation

AIMS: The study of production of Aspergillus terreus CECT 2663 alpha-L-rhamnosidase in solid state fermentation using wheat bran, washed sugar cane bagasse and polyurethane foam as substrates or supports for the enzyme production. METHODS AND RESULTS: Cultures were carried out in Petri dishes under controlled temperature and humidity. Naringin or rhamnose were the enzyme inducers and carbon sources. The enzyme activity to inducer ratio was appreciably greater when using sugar cane bagasse or polyurethane foam than wheat bran. The influence of inoculum size, inducer, airflow, humidity and temperature were determined. Under optimum conditions, about four units of enzyme per ml nutrient solution were obtained after 4-6 d. CONCLUSIONS: The activity to inducer ratio was higher, and the cultivation time was shorter in solid state fermentation than those observed in submerged cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Solid cultures, using naringin as inducer, can be appropriate alpha-L-rhamnosidase production.     

Isolation of a high-specific-growth-rate mutant of Cellulomonas flavigena on sugar cane bagasse

By treatment of a wild-type strain of Cellulomonas flavigena with N-methyl-N'-nitro-N-nitrosoguanidine at 150 g/ml, mutants PN-7 and PN-10 were obtained, which produce 1.38 and 1.5 times more carboxymethylcellulase than the wild strain when cultured in a batch system with sugar cane bagasse as the sole carbon source. These mutants also exhibited higher specific growth rates compared to the wild strain. From a second mutagenesis of mutant PN-10, mutant PN-120 was obtained in continuous culture. This mutant was able to use a larger portion of sugar cane bagasse than did the wild-type and therefore its biomass yield was also higher. The mutant showed a specific growth rate on sugar cane bagasse threefold higher than the wild strain.     

Microbial synthesis of 3-dehydroshikimic acid: a comparative analysis of D-xylose, L-arabinose, and D-glucose carbon sources.

3-Dehydroshikimic acid is a hydroaromatic precursor to chemicals ranging from L-phenylalanine to adipic acid. The concentration and yield of 3-dehydroshikimic acid microbially synthesized from various carbon sources has been examined under fed-batch fermentor conditions. Examined carbon sources included D-xylose, L-arabinose, and D-glucose. A mixture consisting of a 3:3:2 molar ratio of glucose/xylose/arabinose was also evaluated as a carbon source to model the composition of pentose streams potentially resulting from the hydrolysis of corn fiber. Escherichia coli KL3/pKL4.79B, which overexpresses feedback-insensitive DAHP synthase, synthesizes higher concentrations and yields of 3-dehydroshikimic acid when either xylose, arabinose, or the glucose/xylose/arabinose mixture is used as a carbon source relative to when glucose alone is used as a carbon source. E. coli KL3/pKL4.124A, which overexpresses transketolase and feedback-insensitive DAHP synthase, synthesizes higher concentrations and yields of 3-dehydroshikimic acid when the glucose/xylose/arabinose mixture is used as the carbon source relative to when either xylose or glucose is used as a carbon source. Observed high-titer, high-yielding synthesis of 3-dehydroshikimic acid from the glucose/xylose/arabinose mixture carries significant ramifications relevant to the employment of corn fiber in the microbial synthesis of value-added chemicals.     

Evaluation of high butanol/acetone ratios in ABE fermentations with cassava by graph theory and NADH regeneration analysis

Higher butanol/acetone ratio is always desirable in ABE fermentation, and this ratio is closely associated with the complicated patterns of metabolic reactions and NADH generation rate. The patterns of acetate/butyrate formation and re-assimilation in multiple closed reaction loops, as well as NADH regeneration in ABE fermentation using different substrates varies. In this study, we evaluated butanol/acetone ratio in ABE fermentations utilizing cassava and corn based media by graph theory and NADH regeneration analysis. The theoretical calculations and experimental data revealed that a lower metabolic strength in butyrate loop and enhanced NADH generation rate were responsible for the achievement of higher butanol/acetone ratio when fermenting cassava based substrate. In traditional fermentations and extractive fermentations with oleyl alcohol/bio-diesel as the extractants when using cassava based substrate, butanol/acetone ratios reached 2.24, 2.84, and 2.19 with the increasing increments of 14.9, 61.4, and 6.8% respectively, while butanol productivities stayed at comparably high levels as compared with those of the fermentations when cultivating on corn based substrate.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> as a biocatalyst for biofuel production

Microbial fermentation of sugars from plant biomass to alcohols represents an alternative to petroleum-based fuels. The optimal biocatalyst for such fermentations needs to overcome hurdles such as high concentrations of alcohols and toxic compounds. Lactic acid bacteria, especially lactobacilli, have high innate alcohol tolerance and are remarkably adaptive to harsh environments. This study assessed the potential of five Lactobacillus casei strains as biocatalysts for alcohol production. L. casei 12A was selected based upon its innate alcohol tolerance, high transformation efficiency and ability to utilize plant-derived carbohydrates. A 12A derivative engineered to produce ethanol (L. casei E1) was compared to two other bacterial biocatalysts. Maximal growth rate, maximal optical density and ethanol production were determined under conditions similar to those present during alcohol production from lignocellulosic feedstocks. L. casei E1 exhibited higher innate alcohol tolerance, better growth in the presence of corn stover hydrolysate stressors, and resulted in higher ethanol yields.     

Genotypic Variations in Non-Structural Carbohydrate and Cell-Wall Components of the Stem in Rice,Sorghum, and Sugar Vane

We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.     

Genotypic variations in non-structural carbohydrate and cell-wall components of the stem in rice, sorghum, and sugar vane

We evaluated genetic variations in the non-structural carbohydrate (NSC) and the cell-wall components of stem in rice, sorghum, and sugar cane to assess the potential suitability of these gramineous crops for bioethanol production. For NSC, the maximum soluble sugar concentration was highest in sugar cane, followed by sorghum with sucrose. The major NSC in rice was starch, but there were wide variations in the starch to soluble sugar ratios among the cultivars. The total concentration of cell-wall components was negatively correlated with the NSC concentration, indicating competition for carbon sources. Among the cell-wall components, lignin was relatively stable within each group. The major sugar species composing hemicellulose was xylose in all crop groups, but there were differences in composition, with a higher fraction of arabinose and glucose in rice as compared to the other crops. In rice, there was less lignin than in sorghum or sugar cane; this might be advantageous for the efficient saccharification of cellulose.     

Impact of pre-harvest burning versus trash conservation on soil carbon and nitrogen stocks on a sugarcane plantation in the Brazilian Atlantic forest region

Owing to the increased demand for ethanol biofuel from sugar cane, the area planted to this crop in Brazil has increased from 4.8 to 9.5 Mha since 2000. At the same time there has been pressure from environmental groups and others to cease the pre-harvest burning of cane, and today over 40% of the crop is harvested without burning, thus conserving the trash on the soil surface. While most trash decomposes during the year, it is generally assumed that this transition from burning to trash conservation will have benefits for cane productivity and increase soil carbon stocks. To investigate the possible benefits of this change of practice an experiment was carried out in the state of Espírito Santo, south-eastern Brazil, to investigate the long-term effects of the practice of pre-harvested burning compared to trash conservation on soil fertility and soil C and N stocks. The results showed that over a 14-year period, trash conservation marginally decreased soil acidity and significantly increased soil C and N stocks in 0–10 cm depth interval. Although the trash conservation treatment accumulated 13 Mg C ha^?1 more than the burned treatment, this difference was not statistically different. However, the stocks of N to 100 cm depth were 900 kg ha^?1 higher under the trash conservation treatment and this difference was statistically significant. The ¹³C abundance data suggested that where trash was conserved, more soil C was derived from the sugar cane than from the original native vegetation.