Microbial transformation of validamycin A to valienamine by immobilized cells

Immobilized Pseudomonas sp. HZ519 cells have been used for transformation of validamycin A to valienamine and the degradation pathway of validamycin A by Pseudomonas sp. HZ519 has also been studied. Substrate inhibition in immobilized cell system was avoided. An average of 8.6 g L^?1 valienamine concentration was obtained when concentration of validamycin A was increased up to 120 g L^?1. Through a treatment of the immobilized cells with 0.3 mol L^?1 substrate, the activity of the immobilized cells was increased distinctly. Compared with free cells, the productivity of valienamine by CA-immobilized cells was improved about three times. The reusability of the immobilized cells was evaluated with repeated–batch degradation experiments. The Tiele modulus was obtained from the experimental effectiveness factor. The result showed that the degradation process in the immobilized system was governed by intraparticle diffusion and chemical reaction.     

焦化厂地肤根内解芘细菌的筛选及促生潜力

从多环芳烃耐受植物根内分离具多环芳烃降解功能的内生细菌并研究其促生特性,为内生菌协同宿主植物修复多环芳烃污染土壤提供基础.以长期受多环芳烃污染的焦化厂区生长的地肤为材料,从其根内分离出以芘和1-氨基环丙烷-1-羧酸(ACC)为唯一碳源和氮源的内生细菌8株.通过芘降解试验,筛选得到3株高效芘降解内生细菌KSE4、KSE7和KSE8,经鉴定分别为芽孢杆菌属、假单胞菌属和鞘氨醇菌属.通过液体培养试验,研究了3株菌在芘胁迫下产ACC脱氨酶的能力和对地肤种子萌发的影响.结果表明: 随着芘浓度(0~15 mg·L^-1)的升高,ACC脱氨酶活性降低,其中KSE7的效果最好,在芘浓度为15 mg·L^-1时,地肤发芽率和芽长分别比对照提高了44.8%和61.1%,在地肤-微生物修复焦化厂污染土壤的修复中具有一定的应用潜力.     

Isolation and identification of a novel valienamine-producing bacterium

AIMS: To isolate, identify and assess valienamine production by a soil bacterial isolate from a wheat field in Hangzhou, China. METHODS AND RESULTS: A validamycin A-degrading bacterial strain, numbered ZJB-041, was isolated and identified as Stenotrophomonas maltophilia, based on morphology, physiological tests, ATB system (ID32 GN), and 16S rDNA analysis. The strain was capable of producing valienamine by decomposing validamycin A. After fermentation in shaking flasks at 30 degrees C for 7 days, 96.0% of 34.49 mmol l(-1) of validamycin A was degraded and 2.65 mmol l(-1) of valienamine was obtained. The resting cells of this strain also produced valienamine by degrading validamycin A. After 72 h of incubation in 0.2 mol l(-1) of phosphate buffer (pH 7.5), 90.2% of 17.16 mmol l(-1) of validamycin A was degraded, and 1.77 mmol l(-1) of valienamine was obtained. CONCLUSIONS: Our data suggested that S. maltophilia ZJB-041, a bacterial isolate, has the potential for validamycin A degradation and valienamine production. SIGNIFICANCE AND IMPACT OF THE STUDY: The validamycin A-degrading bacterium could potentially be utilized in the disposal of validamycin residues and in the production of valienamine.     

Immobilized Cyclodextrin Glycosyl Transferase for the Continuous Production of Cyclodextrins

Cyclodextrin glycosyl transferase (E.C: 2.4.1.19) from Bacillus, macerans and from a Bacillus sp. isolate was immobilized by two methods, viz. to epoxy-activated Sepharose and to alkylamine silica treated with glutaraldehyde. Because of the ready availability, low cost ($0.01/g), good surface area (30 M²/g) and ease of operation of a continuous cylindrical reactor, the high silica fabric was chosen. The immobilized enzyme had a pH optimum shifted to the alkaline side (from 6.5 to 7.5) and had a reduced temperature optimum (from 60°C to 50-55°C). Reuse efficiency showed 65% reduction in the overall activity of the immobilized enzyme after 10 cycles of 48 h each. Continuous operation at 55°C of a cylindrical reactor of 141 ml capacity, using the immobilized enzyme (80 g of high - silica fabric containing 114 mg of purified enzyme) gave a maximum productivity of 10.2 g of cyclodextrins L^-1 h,^-1, at a dilution rate of 0.32 h^-1 and a substrate concentration of 20 g L^-1. The half life of the biocatalyst was found to be 22 days, which could be further improved by using a lower operating temperature. Over the useful life time of the immobilized biocatalyst (22 days), the total Cyclodextrin produced was of the order of 88 Kg.     

Sorbitol and Gluconate Production in a Hollow Fibre Membrane Reactor by Immobilized Zymomonas Mobilis

This study describes the results of a hollow fibre membrane reactor with immobilized treated cells of Zymomonas mobilis which produced sorbitol and gluconic acid continuously from fructose and glucose respectively. A productivity of 10-20 g sorbitol · L^-1 · h^-1 and 10-20 gluconate · L^-1 · h^-1 (based on total bioreactor volume) from a feed of 100 g · L^-1 each of glucose and fructose was possible at high dilution rates. Kinetic parameters describing the reaction rate of treated cells in batch reactors were used to analyse the performance of the hollow fibre membrane reactor employing significant convective mass transfer. No significant mass transfer limitation was apparent.     

横岗水库富营养化特征分析

于2005年5月和11月调查了横岗水库的营养盐、叶绿素a(Chl.a)和浮游植物,对水库富营养化特征进行了分析。5月的总氮(TN)平均浓度为3.810mg·L^-1,总磷(TP)平均浓度为0.172mg·L^-1,Chl.a平均浓度为17.888mg·m^-3,综合营养状态指数(TSI_M)为73.3,该水库已达重度富营养化;11月份TSI_M下降到55.6,处于轻度富营养化状态,TN和TP平均浓度分别下降到1.302mg·L^-1和0.096mg·L^-1,Chl.a平均浓度却上升到26.935mg·m^-3,Chl.a浓度的上升与氨氮(NH₃-N)和正磷(PO₄-P)浓度上升以及浮游植物群落组成改变有关。从5月到11月,水库由蓝藻型富营养化水体变为绿藻型富营养化水体。5月浮游植物平均细胞密度高达2.75×10⁸cells·L^-1,优势种为美丽平裂藻和银灰平裂藻,其中平裂藻细胞数约占总细胞数的90%。11月浮游植物平均细胞密度降低到1.27×10⁷cells·L^-1,平裂藻的优势度下降,只占总细胞数的14.89%,绿藻成为主优势类群,其中栅藻是优势种之一,占总细胞数的11.52%。     

椭圆背角无齿蚌胃的组织学及铜对其结构的影响

硫酸铜常用于水产养殖的疾病防治,但当水体中的铜离子浓度超过一定范围时便对软体动物产生一定的毒害作用.研究选取椭圆背角无齿蚌(Anodonta woodiana elliptica)作为研究对象,采用苏木精-伊红染色方法进行组织石蜡切片制作,从组织学角度研究了浓度为0.01 mg·L^-mg·L^-1、0.1 mg·L^-1、1 mg·L^-1和10 mg·L^-1的硫酸铜作用不同时间后椭圆背角无齿蚌的胃结构的变化.结果表明,0.01 mg·L^-1硫酸铜处理7d和14d后,仅胃绒毛变短,随着硫酸铜浓度的增大和处理时间的延长,胃绒毛逐渐变短,肌层变薄,腺细胞数量减少直至空化;低于10mg·L^-1的硫酸铜中毒7d和14d后,胃晶杆头直径和胃楯长度出现增大的现象.     

Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^-1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1-3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^-1, as any increase in concentration (0.75 and 1.0 g L^-1) precipitated the precursor, resulting in no further degradation.     

镉胁迫对绢毛委陵菜-丛枝菌根真菌共生体根系结构和生理的影响

为从生理水平上揭示绢毛委陵菜（Potentilla sericea L.）-丛枝菌根真菌共生体对镉胁迫的应答机制,以绢毛委陵菜多年生植株为试验材料,分别接种根内根生囊霉（Rhizophagus intraradices）和摩西球囊霉（Funneliformis mosseae）,80 d后以不同浓度的镉胁迫处理并测定菌根侵染率,研究侵染率较高菌种的共生体植株的生理指标变化和结构变化。结果表明：摩西球囊霉较根内根生囊霉对绢毛委陵菜菌根侵染率高,达100%,而根内根生囊霉侵染率为89.33%;接种摩西球囊霉真菌的植物,当镉质量浓度达10 mg?L^-1时,细胞壁小范围破裂、液泡出现变小的趋势,高浓度镉（20 mg?L^-1）处理时,根系细胞壁破裂,细胞无法维持原有形态;镉胁迫下,Cd（5 mg?L^-1）~ Cd（10 mg?L^-1）MDA含量显著上升（P<0.05）,Cd（0 mg?L^-1）~Cd（5 mg?L^-1）SOD活性和脯氨酸含量变化显著（P<0.05）,可溶性蛋白含量和叶绿素含量显著减少（P<0.05）,Cd（10 mg?L^-1）~Cd（15 mg?L^-1）可溶性蛋白含量和叶绿素含量变化不显著（P>0.05）,植物可以抵抗一定浓度范围的镉胁迫。综合来看,接种摩西球囊菌可以增强绢毛委陵菜耐镉胁迫能力,为利用绢毛委陵菜修复镉污染土壤提供了理论依据。     

一株重金属镉耐受小球藻的分离鉴定及其生长条件优化

以龙江河镉污染水体预留样本为实验材料,从中筛选得到一株重金属镉耐受藻株,经形态和分子生物学鉴定为小球藻属,命名为Chlorella sp.LQQ-1。实验从温度、pH、Cd²⁺、有机碳源及有机氮源等方面对该藻株生长条件进行优化,并考察其在最佳生长条件下对水体中Cd²⁺的去除效果。结果表明:该小球藻最适宜生长温度为30℃,最适宜pH为7.0左右。Cd²⁺在低浓度下能促进小球藻的生长,当Cd²⁺浓度超过30μmol·L^-1时,小球藻的生长受到抑制。适宜该小球藻生长的最佳葡萄糖添加浓度为10g·L^-1,最佳尿素添加浓度为1g·L^-1。在最佳生长条件下,该小球藻对水体中Cd²⁺的去除率达89.7%。     

不同土壤水分条件下CO2浓度对禾谷缢管蚜种群的影响

利用开顶式熏气室研究了不同土壤水分条件下不同CO₂浓度对禾谷缢管蚜种群的影响,以期对未来大气CO₂浓度升高条件下不同降雨地区的小麦蚜虫关系发展趋势做出初步预测.结果表明,随CO₂浓度升高,禾谷缢管蚜种群持续增长,但以CO₂浓度从350μl·L^-1上升到550μl·L^-1时增长最快;禾谷缢管蚜种群大小与土壤水分密切相关,各CO₂浓度下均以60%土壤水分的最大;当CO₂浓度从350μl·L^-1上升到550μl·L^-1时,60%土壤水分下的种群增长最快;当CO₂浓度从550μl·L^-1上升到700μl·L^-1时,60%和40%土壤水分下的种群增长相近,且高于80%土壤水分下的增长.据此可以认为,随大气CO₂浓度升高,禾谷缢管蚜种群会持续增长,从目前至下世纪中叶的时间内可能是蚜虫种群增长最快的阶段,特别在干旱、半干旱地区禾谷缢管蚜种群增长幅度较大、小麦受害较重.     

不同比例盐生小球藻-枯草芽孢杆菌共生体对砷酸盐耐性及富集差异

采用盐生小球藻与枯草芽孢杆菌为供试材料,构建不同藻菌比(1∶0、1∶1、1∶2、1∶3、1∶4)的共生体,在不同浓度砷酸盐[As(Ⅴ)]处理7 d后,测定藻菌共生体生长及其对砷的富集、吸附、吸收和形态转化。结果表明: 在As(Ⅴ)处理下,随着枯草芽孢杆菌比例的增加,藻菌共生体叶绿素含量、干重、比增长率显著提高,在750 μg·L^-1As(Ⅴ)处理时,1∶4的藻菌共生体分别达到1.81 mg·L^-1、125.0 mg、0.28 mg·L^-1·d^-1。藻菌比例由1∶0变为1∶4时,藻菌共生体对砷的富集和吸收呈下降趋势;砷的富集方式随砷浓度的增加而变化,在75～150 μg·L^-1As(Ⅴ)处理下以吸收为主,在300～750 μg·L^-1 As(Ⅴ)处理下以吸附为主。藻菌共生体内存在As(Ⅴ)和As(Ⅲ)两种形态,且随枯草芽孢杆菌比例的上升,As(Ⅴ)还原率增大(最高达到12.6%)。综上,枯草芽孢杆菌的添加提升了藻菌共生体对As(Ⅴ)的耐性和还原,但减少了对As(Ⅴ)的富集。     

巢湖春夏季节浮游植物的动态变化

2009年3月至8月,利用原位围隔实验和野外调查,研究了巢湖春夏季节浮游植物的动态变化.结果显示:围隔中浮游植物密度和生物量的变动范围分别是(2.95～102.43)×10⁵ cells·L^-1和0.0-7.39 mg·L^-1,优势种类为鱼腥藻属(Anabaena)、梅尼小环藻(Cyclotella meneghiniana)和圆筒锥囊藻(Dinobryon cylindricum),其最大生物量分别为0.82 mg·L^-1、0.66mg·L^-1和2.98 mg·L^-1,均出现在3月或4月.在巢湖湖水中,春季(3～5月)浮游植物平均生物量为5.43mg·L^-1,其中绿藻占47.59%、硅藻占40.81%、蓝藻占10.18%,优势种类为盘星藻属(Pediastrum)和梅尼小环藻;而夏季(6～7月)浮游植物平均生物量为7.89mg·L^-1,其中蓝藻占58.7%、绿藻占2.77%、硅藻占11.4%,优势种类为微囊藻属(Microcystis).磷和枝角类滤食对巢湖春夏季节浮游植物的生物量和群落结构有重要影响.     

Enhanced production of valienamine by Stenotrophomonas maltrophilia with fed-batch culture in a stirred tank bioreactor

Valienamine is an important medicinal intermediate with broad use in the synthesis of some stronger α-glucosidase inhibitors. In order to improve valienamine concentration in the fermentation broth and make the downstream treatment easy, a fed-batch process for the enhanced production of valienamine by Stenotrophomonas maltrophilia in a stirred tank bioreactor was developed. Results showed that supplementation of validamycin A in the process of cultivation could increase the valienamine concentration. One-pulse feeding was observed to be the best strategy. The maximum valienamine concentration of 2.35 g L⁻¹ was obtained at 156 h when 86.4 g of validamycin A was added to a 15-L bioreactor containing 8 L fermentation medium with one-pulse feeding. The maximum valienamine concentration had a great improvement and was increased above 100% compared to batch fermentation in the stirred tank bioreactor. The pH-controlled experiments showed that controlling the pH in the process of one-pulse feeding fermentation had not obvious effect on the production of valienamine.     

水生植物修复铁污染水体的效果及作用机制

为解决铁离子污染导致水体发黄问题,采用水培试验,分析了水禾、粉绿狐尾藻、圆币草、黄花水龙、大薸和圆叶节节菜等6种水生植物对铁离子污染水体的修复能力,研究了铁离子浓度、pH和植物生物量对水禾修复效果的影响,并探讨了曝气对水禾除铁的强化作用。结果表明: 6种水生植物均不同程度地促进了水中二价铁和全铁的去除,不同植物对铁的去除效果差异显著;试验24 h,水禾和圆币草处理二价铁浓度分别由5.0 mg·L^-1降至0.23和0.26 mg·L^-1,满足《地表水环境质量标准》(GB 3838—2002)限值要求(二价铁浓度≤0.3 mg·L^-1),全铁浓度降至0.84和1.21 mg·L^-1,去除率达83.2%和75.8%。pH在5、6、7、8时,各pH处理组二价铁和全铁浓度无显著差异,二价铁和全铁去除率分别为95.4%～98.4%和92.2%～94.6%。二价铁初始浓度≤5.0 mg·L^-1时,二价铁和全铁去除率随二价铁浓度增加而增大;高浓度二价铁(10.0 mg·L^-1)对水禾生长有一定的抑制作用,试验期间全铁去除不稳定,试验结束时全铁去除率较对照仅提高7.0%,远低于其他浓度处理。生物量≥300 g时,处理24 h,二价铁浓度从5.0 mg·L^-1降至0.3 mg·L^-1以下,且各生物量处理去除效果差异不显著。间歇曝气和连续曝气均强化水禾对铁的去除,连续曝气更利于稳定去除全铁。     

壬基酚对三角褐指藻的毒性效应及其机理

为了探讨壬基酚(Nonylphenol,NP)对海洋微藻的生态毒性效应,实验选择三角褐指藻(Phaeodactylum triconutum Bohlin)作为受试对象,设置7 个NP 质量浓度梯度(0、0.2、0.4、0.6、0.8、1.0 和2.0 mg·L^-1),测定了不同浓度处理下三角褐指藻的生长情况、光合色素含量、可溶性蛋白含量、丙二醛(MDA)含量及最大光能转化效率(Fv/Fm)以及24 h,48 h,72 h,96 hEC₅₀ 等指标。结果表明,NP 对三角褐指藻的96 h EC50 为0.84 mg·L^-1;当NP 暴露浓度在0.4 mg·L^-1 以上时对三角褐指藻生长表现抑制效应,而且随着NP 质量浓度的增加,生长抑制效应加强,当暴露浓度≥1.0mg·L^-1 时即可造成藻细胞大量死亡,当NP暴露浓度≥2.0mg·L^-1 时,藻细胞基本不能生长。三角褐指藻的细胞密度、可溶性蛋白含量、光合色素含量以及Fv/Fm 等指标随NP 暴露浓度质量浓度的增加而下降的幅度更加显著,暴露处理24 h,MDA 含量随NP 质量浓度的增加而上升,表明较高质量浓度的NP 胁迫,使藻细胞膜脂过氧化程度增强,可能会导致细胞膜结构的破坏和功能的丧失。NP 对三角褐指藻的生长具有明显的毒性效应,并可以造成抗氧化酶系统和光合系统的损伤。     

甲烷及三氯乙烯驯化对垃圾填埋场覆盖土细菌群落结构的影响

对典型垃圾填埋覆盖土进行CH₄原位富集和三氯乙烯(TCE)驯化,研究了其生物氧化能力和微生物群落结构变化.覆盖土CH₄氧化速率为0.20～0.87 μmol·g^-1 soil·h^-1,TCE降解速率为0.009～0.013 mg·L^-1·h^-1,其中山东垃圾填埋场覆盖土土样甲烷氧化活性高于广东、上海和重庆地区土样.通过Illumina MiSeq测序技术分析了α多样性和驯化前后微生物菌群结构变化规律.结果表明: 在所有被注释的操作分类单元聚类结果中,细菌OTUs分配为39个门,85个纲,562个属,富集驯化后变形杆菌门、拟杆菌门、绿弯菌门和酸杆菌门仍为各土样的优势菌群,所占比例之和高于77.4%;γ-变形杆菌纲、β--变形杆菌纲、α-变形杆菌纲、放线菌纲和酸杆菌纲所占比例之和高于26.5%.嗜甲基菌属、厌氧绳菌属、节杆菌属和假单胞菌属经TCE驯化后,其相对丰度呈增加趋势.表明在覆盖土氯代烃生物降解过程中,除了被广泛认可的甲烷氧化菌异养共代谢机制以外,还存在非甲烷共代谢机制和氯代烃自养降解机制.     

獐ISSR-PCR反应体系的建立与优化及引物筛选

利用正交试验L₁₆(4⁵)对影响獐(Hydropotes inermis)ISSR-PCR反应的Taq DNA聚合酶浓度、dNTP浓度、引物浓度、Mg²⁺浓度及模板DNA浓度5个因素在4个水平上进行优化,同时对退火温度进行梯度PCR反应,以建立适合于獐ISSR-PCR反应的最佳体系.最终确定獐25μL ISSR-PCR反应体系为:Taq酶1.25 U·25μL^-1、Mg²⁺浓度2.5 mmol·L^-1、引物浓度0.3μmol·L^-1、DNA模板量350 ng·25μL^-1、dNTP浓度0.15 mmol·L^-1.在此基础上,利用优化的反应体系成功筛选出10条用于獐相关研究的ISSR引物并确定了各自的最佳退火温度,为今后利用ISSR技术进行獐的物种鉴定与分类、亲缘关系、系统发育和生理病理学研究奠定了技术基础,也为开展其它大型资源动物如黑麂的保护遗传学研究提供理论基础.     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

嗜麦芽寡养单胞菌PX1的降解芘特性及定殖效能

为丰富多环芳烃降解菌菌种库、降低农作物的污染风险,本研究对一株可高效降解多环芳烃(PAHs)的植物内生菌进行筛选鉴定,并初步探究其降解途径以及定殖效能。结果表明: 菌株PX1为嗜麦芽寡养单胞菌。该菌株对多环芳烃的降解具有广谱性,7 d几乎可彻底降解PAH无机盐培养基中的萘,在分别含有50.0 mg·L^-1菲、20.0 mg·L^-1芘、20.0 mg·L^-1荧蒽和10.0 mg·L^-1苯并[a]芘的培养体系中,对菲、芘、荧蒽、苯并[a]芘的降解率分别为72.6%、50.7%、31.9%和12.9%。选取芘作为PAHs模型研究菌株PX1的降解特性。酶活性试验表明,芘可诱导菌株PX1体内邻苯二甲酸双加氧酶、邻苯二酚-1,2-双加氧酶和邻苯二酚-2,3-双加氧酶的活性。在芘降解过程中检测到4,5-环氧化芘、4,5-二羟基芘、龙胆酸/原茶儿酸、水杨酸、顺-己二烯二酸/2-羟粘糠酸半醛、顺-2′-羧基苯丙酮酸、1-羟基-2-萘甲酸、水杨醛等中间产物。浸种定殖试验表明,菌株PX1可高效定殖到空心菜和小麦体内,显著促进空心菜和小麦生长,并能够将空心菜、小麦体内及其生长基质中的芘浓度分别降低29.8%～50.7%、52.4%～67.1%和8.0%～15.3%。表明菌株PX1主要通过“水杨酸途径”和“邻苯二甲酸途径”降解芘,且可以定殖到植物体内,促进植物生长。