ORP对酿酒酵母在木质纤维素水解液抑制物中发酵的影响

木质纤维素水解液中的毒性副产物对酿酒细胞具有抑制作用,采用氧化还原电位(oxidoreduction potential, ORP)调控,能够提高细胞对抑制物的耐受性。考察了代表性抑制物乙酸、糠醛及苯酚对酵母细胞生长和代谢的影响,并通过添加氧化剂赤血盐(K₃[Fe(CN)₆])和还原剂二硫苏糖醇(DTT)调节发酵液ORP初始值在(305±5)mV、(157±8)mV及(-150±5)mV,研究不同ORP条件下细胞对抑制物的耐受性影响。结果表明,氧化态能提高生物量并缩短发酵时间。除苯酚外,氧化态也利于提高乙醇的收率。对细胞保护剂甘油合成的方面,氧化态和还原态都会促进甘油合成。在水解液脱毒方面,氧化态表现比还原态要好,因为氧化态促进生物量积累,有利于通过细胞自身代谢脱毒。     

Biochemistry of Citric Acid Production from Rapeseed Oil by <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> Yeast

The growth of wild type strain Yarrowia lipolytica VKM Y-2373 and its mutant Y. lipolytica NG40/UV7 as well as the biosynthesis of citric and isocitric acid on rapeseed oil were studied. It was indicated that the initial step of assimilation of rapeseed oil in the yeast Y. lipolytica is its hydrolysis by extracellular lipases with the formation of glycerol and fatty acids, which appear in the medium in the phase of active growth. The concentrations of these metabolites were changed insignificantly upon further cultivation. Lipase and the key enzymes of glycerol metabolism (glycerol kinase) and the glyoxylate cycle responsible for the metabolism of fatty acids (isocitrate lyase and malate synthase) are induced just at the beginning of the growth phase and remain active in the course of further cultivation. These results, taken together, suggest that glycerol and fatty acids available in the medium do not suppress the metabolism of each other. Citric acid production and a ratio between citric and isocitric acids depended on the strain used. It was revealed that the wild strain produced almost equal amounts of citric and isocitric acids while the mutant produced only citric acid (175 g/L) with a yield of 1.5 g of CA per g of oil.     

酒精-沼气双发酵耦联工艺中丙酸对酿酒酵母酒精发酵的影响(英文)

Propanoic acid accumulated in an ethanol-methane coupled fermentation process affects the ethanol fermentation by Saccharomyces cerevisiae. The effects of propanoic acid on ethanol production were examined in cassava mash under different pH conditions. Final ethanol concentrations increased when undissociated propanoic acid was <30.0 mmol·L-1 . Propanoic acid, however, stimulated ethanol production, as much as 7.6% under proper conditions, but ethanol fermentation was completely inhibited when undissociated acid was >53.2 mmol·L-1 . Therefore, the potential inhibitory effect of propanoic acid on ethanol fermentation may be avoided by controlling the undissociated acid concentrations through elevated medium pH. Biomass and glycerol production decreased with propanoic acid in the medium, partly contributing to increased ethanol concentration.     

Optimization of the synthesis of lower glycerides rich in unsaturated fatty acid residues obtained via enzymatic ethanolysis of sesame oil

The lipases Novozym 435, Lipozyme TL IM and Lipozyme RM IM were employed in the production of lower acylglycerols (LG), i.e. mono‐ (MAG) and diacylglycerols (DAG), rich in unsaturated fatty acids from sesame oil in batch reactors. The effect of the molar ratio of ethanol to fatty acids on the reusability of these immobilized lipases was studied in detail. The effects of pretreatment on lipase activity for ethanolysis were investigated. Glycerol had a strong product inhibition effect on the ethanolysis reaction, and a relatively large excess of ethanol was necessary to remove the glycerol adsorbed on these biocatalysts. The enzymatic activity was drastically reduced by addition of water to the reaction medium. The presence of organic solvents (hexane and acetone) did not favor the production of LG. For the Novozym 435‐catalyzed reaction, optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 15 wt‐% lipase and 50 °C. For Lipozyme TL IM, the optimum conditions were a molar ratio of ethanol to fatty acid residues of 5 : 1, 20 wt‐% biocatalyst, and 30 °C. Novozym 435 and Lipozyme TL IM produced LG with molar ratios of unsaturated to saturated fatty acids of 20.4 in 1 h and 25.3 in 5 h, respectively. In the original oil, this ratio was 5. For trials conducted under optimum conditions, the products from the Novozym 435 trials contained 21.8 wt‐% triacylglycerols (TAG), 24 wt‐% DAG and 54.2 wt‐% MAG. The products of the Lipozyme TL IM trials consisted of 12.9 wt‐% DAG and 87.1 wt‐% MAG. No TAG species were detected.     

克雷伯氏杆菌发酵生产1,3-丙二醇的代谢通量优化分析

以克雷伯氏杆菌为研究对象,系统研究了1,3-丙二醇厌氧和微氧代谢途径.采用代谢通量分析法建立了代谢通量平衡模型,通过线性规划对1,3-丙二醇厌氧和微氧发酵进行了优化分析,得到其最优代谢通量分布和最大理论得率,并分析了比生长速率、底物浓度以及乙醇生成速率、氧气消耗速率、呼吸商对1,3-丙二醇得率的影响.     

克鲁斯假丝酵母分批发酵生产甘油的代谢流分布

采用Candida krusei分批发酵生产甘油，在碳元素平衡基础上作代谢流分析得知，典型生长阶段（6－12h)形成菌体流量较大，占50％以上，且有12％的乙醇流量，甘油流量较小：典型生产阶段（48－54h)甘油流量增大为42%，菌体和乙醇流量为0，有11％的有机酸流量。     

Effect of temperature on fermentation kinetics of waste sulphite liquor by Saccharomyces cerevisiae

The optimum operating temperatures for the maximum production of ethanol and the maximum utilization of substrate in batch fermentations of a waste sulphite liquor (WSL) as well as a synthetic medium using Saccharomyces cerevisiae were determined. The fastest consumption of substrate resulting in the shortest fermentation times of 13 h and 45 h was achieved at 35°C and 30°C for the synthetic medium and the WSL, respectively. The concentrations of ethanol in the two media were also maximum under these conditions: 11.6g dm^?3 and 9.4 g dm^?3 for the synthetic medium and the WSL, respectively. The productivities of biomass and ethanol increased with the increase of temperature and reached maximum values of 0.89 g dm^?3 h^?1 and 0.21 g dm^?3 h^?1 in the synthetic medium and the WSL, respectively. The inhibiting agents in the waste sulphite liquor affected the metabolic rates of microbial activities and prolonged the overall fermentation time while decreasing the productivities of biomass and ethanol. From analysis of the fermentation kinetics a mathematical model based on the Monod model was developed to describe the cellular growth and ethanol production. The model included inhibition terms for ethanol and the inhibiting agents in the waste liquor. The temperature dependence of the model parameters followed the Arrhenius law for temperatures between 15°C and 35°C. The activation energies (E) and the frequency constants (A) of these parameters were also determined.     

不同类型载气对乙醇气提发酵的影响

研究了在乙醇气提发酵过程中,分别使用空气、二氧化碳、氮气和高纯氮作为气提载气对细胞生理及乙醇发酵的影响. 结果表明,空气能维持细胞生长和活力,但发酵效果最差,二氧化碳使细胞发酵能力波动不稳定且抑制副产物甘油合成;而以浓度为99.5%～99.8%的氮气为气提载气取得了较好的效果,乙醇和甘油的生产速率比不气提批式发酵分别提高2倍和1.9倍. 对以高纯氮(99.999%)为气提载气的研究发现,发酵进行到47 h开始出现菌种大量死亡现象,此时适当通氧和补充生长所需营养物质,细胞数量回升,活性恢复,最终乙醇生产速率比不气提时提高1.3倍.     

Selective effects of fatty acids upon cell growth and metabolic regulation

Positional isomers ofcis-methyleneoctadecanoic acid differed greatly in their efficiency for growth of an unsaturated fatty acid auxotroph ofEscherichia coli upon glucose as a carbon source. The 8, 9, and 11 isomers were more efficient in producing cells (60–70 cells/fmole) than the others (0–7 cells/fmole), although all isomers were found esterified to a similar extent into cellular lipid. WithSaccharomyces cerevisiae mutants, all isomers between 6 and 12 supported some growth of the eukaryotic cells, and the 7 and 9 isomers were slightly more efficient than the 8-isomer. WhenE. coli were grown with glycerol, all isomers from 5 to 14 supported growth, and those with the substituent near the center of the acyl chain had the greatest efficiency (70 cells/fmole). With the glycerol medium, the pattern of efficiencies for the variouscis-methylene acyl chains resembled the broad selectivity reported earlier for thecis-ethylenic isomers in glucose medium, which agreed closely with predictions based upon the physical property of their phospholipid derivatives. Thus, metabolism of glycerol appeared to allow the cyclopropane acyl chains to support cell functions to the limits expected for bulk phase chain-chain fluidity considerations. This broad specificity was also obtained when cells were grown on glucose with cyclic AMP added to the culture. Therefore, the selective inadequacies of the 5, 6, 7, 10, 12 and 13 isomers in supporting cell growth on glucose may occur through an interaction modified by cAMP and dependent upon reduced cellular levels of cyclic AMP. The highly selective pattern of efficiency of thecis-methylene acids forE. coli growth on glucose resembles that with the acetylenic acids, but was shifted one carbon atom toward the methyl terminus. This observed selectivity pattern seems due to interactions of the individual acyl chains with cellular protein(s) rather than to chain-chain interactions in a bulk phase. The ability of certain positional isomers to support cell function equally well in both nutrient conditions suggests that the role of those acyl chain isomers may be independent of metabolite flux or cyclic nucleotide contents of the cell, whereas the actions of other isomeric fatty acids seem closely related to the metabolic status of the cell. A highly selective role for different fatty acids in modulating cellular function seems possible on the basis of the current evidence.     

Kinetics of Ultrasound-Assisted Polyphenol Extraction from Spent Filter Coffee Using Aqueous Glycerol

Culinary filter coffee residues were utilized as raw material, with the aim of recovering functional polyphenolic components. The extraction was performed under ultrasonication using aqueous glycerol, and for this reason, the process was initially optimized with regard to glycerol concentration (C_gl) and extraction time (t), by implementing response surface methodology via a central composite (Box-Behnken) experimental design. The optimized conditions determined were C_gl = 3.6% (w/v) and t = 175 min. The comparative assessment, based on a kinetic assay, illustrated that the progression of the extraction was slower in aqueous glycerol than in water, yet aqueous glycerol was proven a more efficient extraction medium, providing 7.4% higher total polyphenol yield. Liquid chromatography-diode array-mass spectrometry analysis revealed that the major antioxidant phytochemicals in the aqueous glycerol extract were chlorogenic acids (caffeic acid derivatives), accompanied by some other polyphenolic metabolites.     

Enzymatic production and physicochemical characterization of uncommon wax esters and monoglycerides

Wax esters from fatty alcohols and uncommon fatty acids were synthesized in yields up to 90% when commercially available microbial lipases fromRhizomucor miehei (Lipozyme^™) andCandida antarctica (SP 435) were used with limited water content in nonpolar solvents under mild conditions. The corresponding fatty acids were prepared by chemical conversion of naturally occurring resources (agricultural surpluses). Also, when phenylboronic acid was added as solubilizing agent in a nonpolar solvent, the direct enzymatic monoacylation of glycerol with uncommon fatty acids was successful. The measurement of π/A-isotherms by means of a Langmuir film balance indicated medium film pressures, medium or large molecular areas, and interesting phase behavior. The monolayer of a wax ester at the air/water interface could be directly visualized by Brewster angle microscopy.     

Microbial mediated dimerization of fattyacids of sunflower oil: An effective role of lipase and biosurfactant

This study emphasizes microbial mediated transformation of sunflower oil to an adhesive product and characterization in detail. Marine bacterial isolates Bacillus (MTCC 5514), when grown in mineral medium, releases both hydrolytic enzymes and surface‐active components during the log phase of growth. When this species was grown in the presence of sunflower oil at an optimized concentration of 5% (w/v) under room temperature, enzymatic hydrolysis of oil proceeds with the release of fatty acids and glycerol. Further, on increasing the incubation period, the presence of surface‐active components, lipase and glycerol, influence the dimerization of the fatty acids, which further, transformed to a polymerized product sunflower oil‐based adhesive product with adhesive nature. Liquid chromatography‐mass spectrometry (electrospray ionization) analysis further authenticates the presence of dimeracids. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40555.     

Production of polyols and waterborne polyurethane dispersions from biodiesel‐derived crude glycerol

This study investigated the preparation of polyols and waterborne polyurethane dispersions (CG‐WPUDs) from biodiesel‐derived crude glycerol. The polyols were produced from biodiesel‐derived crude glycerol via a thermochemical conversion process, which converted crude glycerol components such as glycerol, free fatty acids, and methyl esters of fatty acids (FAMEs) into polyols under optimized reaction conditions. CG‐WPUDs with different hard segments (41.0% to 63.2 wt %) were prepared from the crude glycerol‐based polyols produced. PU coating films cast from CG‐WPUDs showed increasing glass transition temperatures (T_g) from 63°C to 81°C when hard segment content increased from 41.0% to 63.2% and had good thermal stability up to 240°C. CG‐WPUD‐based coatings showed excellent adhesion to steel panel surfaces, pencil hardness as high as F, but relatively low flexibility. This study demonstrated the potential of biodiesel‐derived crude glycerol for the production of bio‐based polyols and WPUDs. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41425.     

Effects of changes in the major carbon source on the fatty acids ofEuglena gracilis

Euglena gracilis was cultured under both heterotrophic and phototrophic growth conditions using ethanol, glucose or CO₂ as the major carbon source. Total fatty acid analyses indicated that ethanol produced more highly unsaturated acids than did glucose under both growth conditions. Growth in the light on CO₂ yielded a very high content of 18∶3, 16∶3 and 16∶4 (33%), compared to ethanol (11%) or glucose (10%). These two preformed carbon sources enhanced the content of the C₂₀ and C₂₂ polyenes compared to CO₂, and growth in the dark on to CO₂, and growth in the dark on ethanol caused a further increase in these polyenes. Growth in the dark on glucose caused only a slight increase of the C₂₀ and C₂₂ polyenes compared to growth in the light on this carbon source. When the fatty acid patterns of the two dark-grown heterotrophs were compared, two observations were quite evident. First, there was a two-fold increase in the saturated acids in the cells grown on glucose. This was largely due to myristic acid. Second, the C₂₀ and C₂₂ polyenes were almost twice as concentrated in the cells grown on ethanol.     

甘油连续生物歧化过程的过渡行为及其数学模拟

用肺炎杆菌将甘油转化为１,３－丙二醇过程对稀释速率和底物进料浓度变化的过渡行为反映了细胞生长和胞内物质代谢在不同条件下存在着显著的差异。体系的过渡响应取决于操作条件变化前后系统所处的状态,当底物浓度由限制状态向剩余状态过渡时,甘油的残余浓度不断上升,而乙醇的浓度则逐渐下降,同时生物量、１,３－丙二醇和乙酸的浓度呈现先增后降的趋势。本文用“甘油浓度变化速率”的概念修正了菌体生长模型,并对甘油歧化过程     

Improvement of ethanol production in Saccharomyces cerevisiae by hetero‐expression of GAPN and FPS1 deletion

BACKGROUND: During anaerobic bioethanol fermentation of Saccharomyces cerevisiae, the main byproduct glycerol is essential to regulate redox balance (reoxidize NADH to NAD⁺), which is necessary to maintain cell growth and fermentation. Hetero‐expression of a NADP⁺‐dependent glyceraldehydes‐3‐phosphate dehydrogenase (GAPN) [EC.1.2.1.9] in S. cerevisiae could redirect the carbon flux from glycerol to ethanol involving a net oxidation of NADH. The present study investigates whether combination of GAPN hetero‐expression and glycerol exporter Fps1p disruption would result in less glycerol and more ethanol production without affecting growth rate during anaerobic fermentations. RESULTS: The results of anaerobic fermentations showed that the fps1Δ mutant with GAPN (named 4FG) produced 21.47% less glycerol and 9.18% more ethanol compared with a parental strain with a control plasmid, while the rates of growth and fermentation were not changed. Moreover, the engineered strain 4FG yielded less glycerol and acetic acid, and more ethanol than the control, fps1Δ mutant or with GAPN only. CONCLUSIONS: During anaerobic fermentations, hetero‐expression of GAPN restored the reduced grow rate of the fps1Δ mutant, and led to less byproducts and more ethanol production. This combination strategy could be used to modulate glycerol metabolism and optimize the anaerobic fermentation of S. cerevisiae. Copyright © 2011 Society of Chemical Industry     

Biohydrogen and Bioethanol Production from Biodiesel-Based Glycerol by Enterobacter aerogenes in a Continuous Stir Tank Reactor

Crude glycerol from the biodiesel manufacturing process is being produced in increasing quantities due to the expanding number of biodiesel plants. It has been previously shown that, in batch mode, semi-anaerobic fermentation of crude glycerol by Enterobacter aerogenes can produce biohydrogen and bioethanol simultaneously. The present study demonstrated the possible scaling-up of this process from small batches performed in small bottles to a 3.6-L continuous stir tank reactor (CSTR). Fresh feed rate, liquid recycling, pH, mixing speed, glycerol concentration, and waste recycling were optimized for biohydrogen and bioethanol production. Results confirmed that E. aerogenes uses small amounts of oxygen under semi-anaerobic conditions for growth before using oxygen from decomposable salts, mainly NH₄NO₃, under anaerobic condition to produce hydrogen and ethanol. The optimal conditions were determined to be 500 rpm, pH 6.4, 18.5 g/L crude glycerol (15 g/L glycerol) and 33% liquid recycling for a fresh feed rate of 0.44 mL/min. Using these optimized conditions, the process ran at a lower media cost than previous studies, was stable after 7 days without further inoculation and resulted in yields of 0.86 mol H₂/mol glycerol and 0.75 mol ethanol/mole glycerol.     

Influence of culture pH and aeration on ethanol production and pullulan molecular weight by Aureobasidium pullulans

The formation of ethanol by the polymorphic fungus Aureobasidium pullulans was examined under a range of culture pH values and aeration conditions. Although culture pH had a profound effect on fungal morphology, with a greatly increased proportion of the biomass in the unicellular (yeast-like) form at pH 6·5, there appeared to be no direct link between morphological form and ethanol formation. The levels of ethanol noted may have influenced the morphology. Cessation of aeration rapidly led to a halt in growth and exopolysaccharide synthesis, while ethanol synthesis proceeded rapidly. The ethanol formed could be re-oxidised under conditions where the available carbon source was depleted. Two distinct exopolysaccharides of different molecular weights were recovered from the culture fluids. Overall, the molecular weight of both declined with process time.     

Trans-monoenoic and polyunsaturated fatty acids in phospholipids of aVibrio species of bacterium in relation to growth conditions

AVibrio species of bacterium known to contain the polyunsaturated fatty acid 20∶5n−3 was grown in both freshwater and seawater media at 5 and 20°C and examined for adaptive changes in lipid composition. Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), together with a smaller proportion of nonesterified fatty acids (NEFA), comprised almost all the lipid under all growth conditions examined. Temperature had a more pronounced effect than the salinity of the medium on lipid composition. The proportion of PE in total lipid was always higher at 5 than at 20°C. Conversely, the proportion of NEFA was lower at 5 than 20°C whereas that of PG was not altered. The levels of saturated fatty acids in total lipid, PE and PG were all decreased by growth at 5°C. No differences were observed with respect to growth temperature in the levels ofcis 16∶1n−7, the principal monoenoic fatty acid in both PE and PG.Trans 16∶1n−7 was found to comprise 12.8–15.2% of fatty acids in PE and PG of bacteria grown at 5°C but only 4.4–8.5% of phospholipid fatty acids in bacteria cultured at 20°C. Regardless of medium composition, a reduction in growth temperature from 20 to 5°C also caused the proportions of 20∶5n−3 to increase from around 0.8 to 4.4% in PE and from around 4 to 20% in PG. The simultaneous occurrence oftrans 16∶1n−7 and 20∶5n−3 is unique to thisVibrio species of bacterium. The increased proportions of both these fatty acids with decreasing temperature suggest that they have a role in retailoring biomembrane phospholipids during temperature acclimation of the bacterium.     

Purification of crude glycerol derived from waste used-oil methyl ester plant

The purification of crude glycerol from a biodiesel plant using waste used-oil as a raw material was carried out on a laboratory scale by using the combined chemical and physical treatments based upon repeated cycles of acidification to the desired pH within the range of 1–6 using 1.19 M H₂SO₄, allowing phase separation and harvesting of the glycerol-rich middle phase followed by neutralization of the harvested glycerol phase with 12.5M NaOH. Subsequently, the glycerol-enriched fraction was extracted by ethanol. The results indicated that increasing the pH of the acidification step led to an increased yield of the glycerol-rich layer and decreased amount of inorganic salt and free fatty acids phase. Under strong acid conditions, large quantities of fatty acid and salt in the glycerol-enriched fraction were eliminated and, at pH 1, high purity glycerol (∼93.34%) with relatively low contaminant levels (0.00045% (w/w) ash and 5.16% (w/w) MONG) was obtained.