Stereoselective Reduction of Ethyl 4-chloro-3-oxobutanoate by Fungi

The enantioselectivity of ECAA to ECHB by eight fungi of four genus was evaluated. All strains showed (S)-selectivity, and Cylindrocarpon sclerotigenum IFO 31855 gave the highest yield and good optical purity (e.e.; >99%). Cell-free extract and acetone-dried cells of C. sclerotigenum IFO 31855 reduced ECAA to (S)-ECHB in the presence of NADPH (e.e.; >99%) and the e.e. was not decreased by heat treatment of the cell-free extract or the acetone-dried cells. The active fractions shown by two peaks on a DEAE-Toyopearl 650 M column gave preferentially (S)-ECHB (e.e.; >99%).     

全氟辛烷磺酰基化合物对剑尾鱼肝脏抗氧化物酶活性的影响

目的研究全氟辛烷磺酰基化合物(PFOS)暴露对剑尾鱼(Xiphophorus helleri Heckel)抗氧化物酶活性的影响,探讨PFOS对鱼类的致毒机理。方法使用浸润法以3.5、7.0、14.0和28.0 mg/L四个PFOS浓度为剑尾鱼染毒,定量测定了96 h内肝脏组织中的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽过氧化物酶(GSH-PX)活性及丙二醛(MDA)含量的变化。结果 PFOS暴露12 h后,除28.0 mg/L组SOD活性被显著性抑制外,其余各组与对照组均无显著性差异(P>0.05);7.0 mg/L组和14.0 mg/L组在24 h被极显著诱导(P<0.01),并且一直保持至96 h。CAT活性随PFOS浓度的升高而降低,12 h时,除3.5 mg/L组外,其余各组CAT活性被显著或极显著抑制,至24 h时,各组CAT活性有上升趋势,但48 h后,各组呈不断下降趋势持续至96 h,其CAT活性恢复到12 h水平。GSH-PX活性变化与CAT活性变化趋势相似,其中28.0 mg/L组在不同时间均被显著性抑制,并在96 h时抑制率达到最高值64.8%。MDA含量在12 h时呈小幅下降趋势,但随着暴露时间的延长,各处理组MDA含量呈连续上升趋势,并在96 h时达到最高点,诱导率分别为71.2%、70.1%和85.1%。结论结果表明,SOD的高活性是由于机体中超氧阴离子的存在,而高浓度的超氧阴离子能够灭活CAT和GSH-PX活性,因此,CAT和GSH-PX活性始终低于对照组。GSH-PX对PFOS的敏感性高于CAT。MDA含量持续升高反映出细胞组织已经遭受到氧化损伤。剑尾鱼活体的实验表明,PFOS能够诱导肝脏氧化应激反应,氧化损伤是PFOS致毒的主要途径之一。     

Effects of nitrate adaptation by rumen inocula donors and substrate fiber proportion on in vitro nitrate disappearance,methanogenesis, and rumen fermentation acid

A study was conducted to evaluate the main effects of dietary nitrate adaptation by cattle and alfalfa cell wall to starch ratio in in vitro substrates on nitrate disappearance and nitrite and volatile fatty acid (VFA) concentrations, as well as hydrogen (H₂) and methane (CH₄) accumulations. Rumen fluid from steers fed diets containing urea or nitrate was added into in vitro incubations containing sodium nitrate as the sole nitrogen source and 20 cell wall : 80 starch or 80 cell wall : 20 starch as the carbohydrate source. The results showed that during 24 h incubation, rumen fluid inoculums from steers adapted to dietary nitrate resulted in more rapid nitrate disappearance by 6 h of incubation (P < 0.01), no significant effect on nitrite concentration and diminished CH₄ accumulation (P < 0.05). Cell wall to starch ratio did not affect nitrate disappearance, CH₄ accumulation and total VFA concentration. The higher cell wall ratio had the lower total gas production and H₂ concentration (P < 0.05). Ammonia-N (NH₃-N) concentration increased because of adaptation of donors to nitrate feeding (P < 0.05). Nitrate adaptation did not alter total VFA concentration, but increased acetate, and decreased propionate and butyrate molar proportions (P < 0.01).     

In vitro–in vivo study on the effects of plant compounds on rumen fermentation,microbial abundances and methane emissions in goats

Two in vitro and one in vivo experiments were conducted to investigate the effects of a selection of plant compounds on rumen fermentation, microbial concentration and methane emissions in goats. Treatments were: control (no additive), carvacrol (CAR), cinnamaldehyde (CIN), eugenol (EUG), propyl propane thiosulfinate (PTS), propyl propane thiosulfonate (PTSO), diallyl disulfide (DDS), a mixture (40 : 60) of PTS and PTSO (PTS+PTSO), and bromochloromethane (BCM) as positive control with proven antimethanogenic effectiveness. Four doses (40, 80, 160 and 320 µl/l) of the different compounds were incubated in vitro for 24 h in diluted rumen fluid from goats using two diets differing in starch and protein source within the concentrate (Experiment 1).The total gas production was linearly decreased (P<0.012) by all compounds, with the exception of EUG and PTS+PTSO (P⩾0.366). Total volatile fatty-acid (VFA) concentration decreased (P⩽0.018) only with PTS, PTSO and CAR, whereas the acetate:propionate ratio decreased (P⩽0.002) with PTS, PTSO and BCM, and a tendency (P=0.064) was observed for DDS. On the basis of results from Experiment 1, two doses of PTS, CAR, CIN, BCM (160 and 320 µl/l), PTSO (40 and 160 µl/l) and DDS (80 and 320 µl/l) were further tested in vitro for 72 h (Experiment 2). The gas production kinetics were affected (P⩽0.045) by all compounds, and digested NDF (DNDF) after 72 h of incubation was only linearly decreased (P⩽0.004) by CAR and PTS. The addition of all compounds linearly decreased (P⩽0.009) methane production, although the greatest reductions were observed for PTS (up to 96%), DDS (62%) and BCM (95%). No diet–dose interaction was observed. To further test the results obtained in vitro, two groups of 16 adult non-pregnant goats were used to study in vivo the effect of adding PTS (50, 100 and 200 mg/l rumen content per day) and BCM (50, 100 and 160 mg/l rumen content per day) during the 9 days on methane emissions (Experiment 3). The addition of PTS and BCM resulted in linear reductions (33% and 64%, respectively, P⩽0.002) of methane production per unit of dry matter intake, which were lower than the maximum inhibition observed in vitro (87% and 96%, respectively). We conclude that applying the same doses in vivo as in vitro resulted in a proportional lower extent of methane decrease, and that PTS at 200 mg/l rumen content per day has the potential to reduce methane emissions in goats. Whether the reduction in methane emission observed in vivo persists over longer periods of treatments and improves feed conversion efficiency requires further research.     

Effects of different tannin-rich extracts and rapeseed tannin monomers on methane formation and microbial protein synthesis in vitro

Tannins, polyphenolic compounds found in plants, are known to complex with proteins of feed and rumen bacteria. This group of substances has the potential to reduce methane production either with or without negative effects on digestibility and microbial yield. In the first step of this study, 10 tannin-rich extracts from chestnut, mimosa, myrabolan, quebracho, sumach, tara, valonea, oak, cocoa and grape seed, and four rapeseed tannin monomers (pelargonidin, catechin, cyanidin and sinapinic acid) were used in a series of in vitro trials using the Hohenheim gas test, with grass silage as substrate. The objective was to screen the potential of various tannin-rich extracts to reduce methane production without a significant effect on total gas production (GP). Supplementation with pelargonidin and cyanidin did not reduce methane production; however, catechin and sinapinic acid reduced methane production without altering GP. All tannin-rich extracts, except for tara extract, significantly reduced methane production by 8% to 28% without altering GP. On the basis of these results, five tannin-rich extracts were selected and further investigated in a second step using a Rusitec system. Each tannin-rich extract (1.5 g) was supplemented to grass silage (15 g). In this experiment, nutrient degradation, microbial protein synthesis and volatile fatty acid production were used as additional response criteria. Chestnut extract caused the greatest reduction in methane production followed by valonea, grape seed and sumach, whereas myrabolan extract did not reduce methane production. Whereas chestnut extract reduced acetate production by 19%, supplementation with grape seed or myrabolan extract increased acetate production. However, degradation of fibre fractions was reduced in all tannin treatments. Degradation of dry matter and organic matter was also reduced by tannin supplementation, and no differences were found between the tannin-rich extracts. CP degradation and ammonia-N accumulation in the Rusitec were reduced by tannin treatment. The amount and efficiency of microbial protein synthesis were not significantly affected by tannin supplementation. The results of this study indicated that some tannin-rich extracts are able to reduce methane production without altering microbial protein synthesis. We hypothesized that chestnut and valonea extract have the greatest potential to reduce methane production without negative side effects.     

海洋浮游古菌MGII的研究进展

海洋浮游古菌MGⅡ是海洋表层水体中最丰富的古菌类群。自1992年被发现以来,如今依然没有被成功分离纯化。前人基于16S rRNA基因的研究认为MGⅡ可以被分为MGⅡa、MGⅡb和MGⅡc三个亚类。近年来,对大量的宏基因组测序数据的分析表明,MGⅡ在分类学上属于广古菌门热源体纲下的一个目,包含MGⅡa和MGⅡb两个科。以前通过16S rRNA基因高通量测序结果得出的少量MGⅡc,在宏基因组测序的数据中并没有找到,因此最近两年的研究认为MGⅡ主要由MGⅡa和MGⅡb组成。本文综述了海洋浮游古菌MGⅡ的丰度和多样性分布特征、潜在的生态功能、生态关系以及培养等方面的研究进展,比较了MGⅡa和MGⅡb的异同点,并对当前的研究热点和趋势进行了讨论和展望。     

典型煤层水微生物产甲烷潜力及其群落结构研究

内蒙古自治区二连盆地、海拉尔盆地是我国重要的煤层气产区,其中生物成因煤层气是煤层气的重要来源,但复杂物质转化产甲烷相关微生物群落结构及功能尚不清楚。【目的】研究煤层水中的微生物代谢挥发性脂肪酸产甲烷的生理特征及群落特征。【方法】以内蒙古自治区二连盆地和海拉尔盆地的四口煤层气井水作为接种物,分别添加乙酸钠、丙酸钠和丁酸钠厌氧培养;定期监测挥发性脂肪酸降解过程中甲烷和底物的变化趋势,应用高通量测序技术,分析原始煤层气井水及稳定期产甲烷菌液的微生物群落结构。【结果】除海拉尔盆地H303煤层气井微生物不能代谢丙酸外,其他样品均具备代谢乙酸、丙酸和丁酸产生甲烷的能力,其生理生态参数存在显著差异,产甲烷延滞期依次是乙酸丁酸丙酸;最大比产甲烷速率和底物转化效率依次是丙酸乙酸丁酸。富集培养后,古菌群落结构与煤层气井水的来源显著相关,二连盆地优势古菌为氢营养型产甲烷古菌Methanocalculus (相对丰度13.5%–63.4%)和复合营养型产甲烷古菌Methanosarcina (7.9%–51.3%),海拉尔盆地的优势古菌为氢营养型产甲烷古菌Methanobacterium(24.3%–57.4%)和复合营养型产甲烷古菌Methanosarcina(29.6%–66.5%);细菌群落则与底物类型显著相关,硫酸盐还原菌Desulfovibrio(12.0%–41.0%)、互营丙酸氧化菌Syntrophobacter(39.6%–75.5%)和互营丁酸菌Syntrophomonas(8.5%–21.9%)分别在乙酸钠、丙酸钠和丁酸钠处理组显著富集。【结论】煤层气井水微生物可降解挥发性脂肪酸(乙酸、丙酸和丁酸)并具有产甲烷潜力;乙酸可能被古菌直接代谢产甲烷,而丙酸和丁酸通过互营细菌和产甲烷古菌代谢产甲烷。Desulfovibrio、Syntrophobacter和Syntrophomonas分别在乙酸、丙酸和丁酸代谢过程中发挥了重要作用。这些结果为煤层气生物强化开采提供了一定的微生物资源基础。     

基因组编辑对酿酒酵母DNA的损伤作用及修复响应

【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。     

Effects of nitrogen fertilisation rate and maturity of grass silage on methane emission by lactating dairy cows

Grass silage is typically fed to dairy cows in temperate regions. However, in vivo information on methane (CH₄) emission from grass silage of varying quality is limited. We evaluated the effect of two rates of nitrogen (N) fertilisation of grassland (low fertilisation (LF), 65 kg of N/ha; and high fertilisation (HF), 150 kg of N/ha) and of three stages of maturity of grass at cutting: early maturity (EM; 28 days of regrowth), mid maturity (MM; 41 days of regrowth) and late maturity (LM; 62 days of regrowth) on CH₄ production by lactating dairy cows. In a randomised block design, 54 lactating Holstein–Friesian dairy cows (168±11 days in milk; mean±standard error of mean) received grass silage (mainly ryegrass) and compound feed at 80 : 20 on dry matter basis. Cows were adapted to the diet for 12 days and CH₄ production was measured in climate respiration chambers for 5 days. Dry matter intake (DMI; 14.9±0.56 kg/day) decreased with increasing N fertilisation and grass maturity. Production of fat- and protein-corrected milk (FPCM; 24.0±1.57 kg/day) decreased with advancing grass maturity but was not affected by N fertilisation. Apparent total-tract feed digestibility decreased with advancing grass maturity but was unaffected by N fertilisation except for an increase and decrease in N and fat digestibility with increasing N fertilisation, respectively. Total CH₄ production per cow (347±13.6 g/day) decreased with increasing N fertilisation by 4% and grass maturity by 6%. The smaller CH₄ production with advancing grass maturity was offset by a smaller FPCM and lower feed digestibility. As a result, with advancing grass maturity CH₄ emission intensity increased per units of FPCM (15.0±1.00 g CH₄/kg) by 31% and digestible organic matter intake (33.1±0.78 g CH₄/kg) by 15%. In addition, emission intensity increased per units of DMI (23.5±0.43 g CH₄/kg) by 7% and gross energy intake (7.0±0.14% CH₄) by 9%, implying an increased loss of dietary energy with advancing grass maturity. Rate of N fertilisation had no effect on CH₄ emissions per units of FPCM, DMI and gross energy intake. These results suggest that despite a lower absolute daily CH₄ production with a higher N fertilisation rate, CH₄ emission intensity remains unchanged. A significant reduction of CH₄ emission intensity can be achieved by feeding dairy cows silage of grass harvested at an earlier stage of maturity.     

Prediction of enteric methane emissions from Holstein dairy cows fed various forage sources

Milk fatty acid (FA) profile has been previously used as a predictor of enteric CH₄output in dairy cows fed diets supplemented with plant oils, which can potentially impact ruminal fermentation. The objective of this study was to investigate the relationships between milk FA and enteric CH₄ emissions in lactating dairy cows fed different types of forages in the context of commonly fed diets. A total of 81 observations from three separate 3×3 Latin square design (32-day periods) experiments including a total of 27 lactating cows (96±27 days in milk; mean±SD) were used. Dietary forages were included at 60% of ration dry matter and were as follows: (1) 100% corn silage, (2) 100% alfalfa silage, (3) 100% barley silage, (4) 100% timothy silage, (5) 50 : 50 mix of corn and alfalfa silages, (6) 50 : 50 mix of barley and corn silages and (7) 50 : 50 mix of timothy and alfalfa silages. Enteric CH₄output was measured using respiration chambers during 3 consecutive days. Milk was sampled during the last 7 days of each period and analyzed for components and FA profile. Test variables included dry matter intake (DMI; kg/day), NDF (%), ether extract (%), milk yield (kg/day), milk components (%) and individual milk FA (% of total FA). Candidate multivariate models were obtained using the Least Absolute Shrinkage and Selection Operator and Least-Angle Regression methods based on the Schwarz Bayesian Criterion. Data were then fitted into a random regression using the MIXED procedure including the random effects of cow, period and study. A positive correlation was observed between CH₄ and DMI (r=0.59,P<0.001), whereas negative associations were observed between CH₄ and cis9-17:1 (r=−0.58, P<0.001), and trans8, cis13-18:2 (r=−0.51,P<0.001). Three different candidate models were selected and the best fit candidate model predicted CH₄ with a coefficient of determination of 0.84 after correction for cow, period and study effects and was: CH₄ (g/day)=319.7−57.4×15:0−13.8×cis9-17:1−39.5×trans10-18:1−59.9×cis11-18:1−253.1×trans8, cis12-18:2−642.7×trans8, cis13-18:2−195.7×trans11, cis15-18:2+16.5×DMI. Overall and linear prediction biases of all models were not significant (P>0.19). Milk FA profile and DMI can be used to predict CH₄emissions in dairy cows across a wide range of dietary forage sources