粘质沙雷氏菌对重金属的耐受性和去除作用

微生物修复是治理重金属污染的重要手段之一。本文考察了粘质沙雷氏菌对重金属Cu²⁺、Zn²⁺和Cr⁶⁺耐受性和去除作用。结果表明,粘质沙雷氏菌对重金属Cu²⁺、Zn²⁺、Cr⁶⁺具有良好的耐受性,耐受程度表现为：Zn²⁺> Cu²⁺>Cr⁶⁺。不产灵菌红素时,最大耐受浓度分别为100～150 mg/L、200～250 mg/L、50～100 mg/L;产灵菌红素时能增加菌体对重金属的耐受性,最大耐受浓度分别为150～200 mg/L、>250 mg/L、150～200 mg/L。菌体对重金属的去除效果表现为Zn²⁺>Cr⁶⁺> Cu²⁺,48 h内最大去除率分别为分别为63.3%、44.5%、32.3%。     

培养条件及培养基组分对粘质沙雷氏菌生长及产D-乳酸的影响

通过摇瓶实验,确定粘质沙雷氏菌发酵生产I）-乳酸的培养条件为：温度34℃,pH值6．5,种子液接种量5％,载液量为150mL／500mL,采用前期通气有氧培养至菌体对数生长后期、而后厌氧发酵产酸的两阶段培养工艺。考察培养基各组分对菌体生长及乳酸产量的影响,确定葡萄糖及酵母粉作为碳、氮源,并补充添加适量的无机氮。培养基各组分如下（g·L^-1）：葡萄糖15,酵母粉15,KH2P041．5,K2HP04·3H2O2．0,MgSO4·7H2O1．0,FeSO4·7H2O0．03,MnSO4·H2O0．005,以此培养条件及培养基配方进行摇瓶发酵,D-乳酸产量由（13．5±1．29）g·L^-1提高至（29．0±1．42）g·L^-1,提高一倍以上,光学纯度大于97％。     

粘质沙雷氏菌HB-4吸附重金属镉的机制

从重金属污染土壤中筛选出1株对Cd²⁺具有高耐受能力和高吸附容量的菌株HB-4,经16S rDNA序列分析鉴定为粘质沙雷氏菌（Serratia marcescens）。该菌株能在Cd²⁺浓度为300 mg·L^-1的条件下正常生长;对Cd²⁺的最大吸附量为（154.7±0.9（ mg·g^-1。考察了Cd²⁺初始浓度、pH、盐浓度以及共存离子对HB-4吸附Cd²⁺的影响,结果表明：pH=3.0~8.0时,对吸附效果无影响;NaCl含量为8.0%时,菌株对Cd²⁺的去除率仍可达到49.9%±0.1%;Pb²⁺、Zn²⁺、Cu²⁺与Cd²⁺共存时,几种重金属离子的去除率分别为98.7%±0.2%（Pb²⁺）、44.6%±0.6%（Zn²⁺）、52.7%±0.1%（Cu²⁺）和64.2%±0.3%（Cd²⁺）。解吸实验证明了HB-4对Cd²⁺极强的吸附能力,洗脱液pH=7.0时,解吸率小于2%。检测了细胞内外镉的分布情况,并利用SEM、XPS和FTIR对吸附机理进行了研究,推断HB-4对Cd²⁺的吸附机理为胞外吸附和胞内摄取。     

粘质沙雷氏菌利用蔗糖和柠檬酸铵生产2，3-丁二醇的研究

研究了几种无机氮源对粘质沙雷氏菌发酵生物量和产物2,3-丁二醇形成的影响,在确定柠檬酸铵为无机氮源的基础上,利用响应面法(RSM)对柠檬酸铵和硫酸锰的浓度进行了优化,得出了最佳浓度,并以此最优成分进行了摇瓶发酵和分批补料发酵.在摇瓶发酵中,110 g·L-1的蔗糖最终被转化成44 g·L-1的2,3-丁二醇,转化率为0.4 g·g-1,产率为1.13 g·L-1·h-1;在分批补料发酵中,共有166 g·L-1的蔗糖被消耗,2,3-丁二醇的最高浓度为81.2 g·L-1,乙偶姻的浓度为7.7 g·L-1,2,3-丁二醇转化率达到0.489 g·g-1,产率达到1.7 g·L-1·h-1.     

粘质沙雷氏菌产灵菌红素的碳源分析、菌种诱变及动力学

使用流式细胞仪研究了不同碳源对粘质沙雷氏菌ZSG新陈代谢的影响,发现不同碳源导致ZSG的DNA含量、细胞内部颗粒密度、表面粗糙度和细胞大小在发酵过程中呈现有差异的变化。对ZSG进行复合诱变筛得一株稳定突变菌株ZSG7,在250 ml摇瓶和5 L发酵罐中进行发酵,其灵菌红素（PG）产量比出发菌株分别提高了62.5%和269%。对ZSG7进行发酵培养基优化后PG产量比优化前提高了100%。对ZSG7发酵进行溶氧分段控制模式调控后PG产量比DO调控前提高了30.9%。对ZSG7发酵进行恒定pH调控后比pH调控前提高了35.9%。对ZSG7发酵进行补料组分优化后比补料前提高了47.6%。基于Logistic方程和Luedeking-Piret方程建立了恒定pH7分批发酵和补料分批发酵的菌体生长模型和PG合成模型。拟合模型参数后,模型可以合理地描述恒定pH7分批发酵和补料分批发酵的过程。     

粘质沙雷氏菌G1利用玉米浆干粉和磷酸氢二铵生产2,3-丁二醇的研究

研究了几种工业氮源对粘质沙雷氏菌G1发酵生产2,3-丁二醇的影响,在确定玉米浆干粉为氮源的基础上,利用Plackett-Burman(PB)实验和响应面法(RSM)实验对玉米浆干粉和磷酸氢二铵[(NH4)2HPO4]的浓度进行了优化,确定优化培养基(g·L-1)为:蔗糖90,玉米浆干粉20.32,(NH4)2HPO47.21,NaAc 4,柠檬酸钠14,MgSO40.5,Fe-SO40.02,MnSO40.01。并以此优化培养基进行了摇瓶和分批补料发酵,结果表明,摇瓶发酵中,90g·L-1的蔗糖最终被转化成43.06g·L-1的2,3-丁二醇;分批补料发酵中,2,3-丁二醇浓度为128.28g·L-1,产率为2.67g·L-1·h-1,转化率为0.48g·g-1蔗糖。以玉米浆干粉和(NH4)2HPO4为氮源,2,3-丁二醇浓度较高,培养基的成本大幅降低,为工业化生产奠定了基础。     

铜绿假单胞菌TBPY与黏质沙雷氏菌SMA协同降解三溴苯酚的特性

三溴苯酚是一类难降解、对环境危害大的物质.在本课题组前期的研究基础上,本研究以自有的铜绿假单胞菌株(Pseudomonas aeruginosa,TBPY)和黏质沙雷氏菌株(Serratia marcescens AB 90027,SMA)为出发菌种,研究两菌降解TBP的协同性,同时考察相关因素对该协同降解的影响.研究结果表明,这两种微生物具有良好的协同性,两菌协同降解TBP的效果明显优于单菌.在初始pH值为6.5,接入稳定期的TBPY,TBP先经TBPY降解两天后再接入稳定期的SMA,两菌接种量比为2:1时,TBP降解效果最好,100 mg/L的TBP 5天内降解率可达到97%.本研究不仅为三溴苯酚的微生物降解提供了一个新的有效途径,也为卤代芳香化合物,乃至环境污染的生物降解提供一种新的思路.     

恒定pH值对粘质沙雷氏菌产灵菌红素的影响

探讨了恒定pH值对粘质沙雷氏菌ZSG7发酵产灵菌红素的影响。结果表明,当发酵系统pH值恒定为7时,粘质沙雷氏菌生长速度最快,灵菌红素产生速度最快且产量最高(0.3033g·L-1),比不调pH值的灵菌红素最高产量0.2296g·L-1高出32.1%。     

粘质沙雷氏菌产灵菌红素的碳源分析、菌种诱变及动力学

使用流式细胞仪研究了不同碳源对粘质沙雷氏菌ZSG新陈代谢的影响,发现不同碳源导致ZSG的DNA含量、细胞内部颗粒密度、表面粗糙度和细胞大小在发酵过程中呈现有差异的变化。对ZSG进行复合诱变筛得一株稳定突变菌株ZSG7,在250 ml摇瓶和5 L发酵罐中进行发酵,其灵菌红素(PG)产量比出发菌株分别提高了62.5%和269%。对ZSG7进行发酵培养基优化后PG产量比优化前提高了100%。对ZSG7发酵进行溶氧分段控制模式调控后PG产量比DO调控前提高了30.9%。对ZSG7发酵进行恒定pH调控后比pH调控前提高了35.9%。对ZSG7发酵进行补料组分优化后比补料前提高了47.6%。基于Logistic方程和Luedeking-Piret方程建立了恒定pH7分批发酵和补料分批发酵的菌体生长模型和PG合成模型。拟合模型参数后,模型可以合理地描述恒定pH7分批发酵和补料分批发酵的过程。     

羟基和氨基苯类化合物的生物氧化与代谢

引言 芳香类污染物是自然界分布广泛的一类化合物,主要来源于石油工业、造纸业、塑料加工、纤维制造以及农药生产等[1].其中大多数酚(羟基苯)和芳香胺(氨基苯)具有很强的毒性和致癌性,因此被很多国家列为首要污染物[2-3].由于物理处理法和化学处理法成本高且可能引入其他有害物质[4],人们日益关注成本低廉而处理彻底的生物法[3].     

Serratia marcescens非特异性核酸内切酶的原核表达及其应用

目的在大肠杆菌中表达Serratia marcescens非特异性核酸内切酶(SMNE),并进行纯化、活性检测及应用。方法合成smne基因,应用PCR技术在基因的5′端引入6个组氨酸标签序列,将其插入分泌表达载体pET-20b(+)中,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达。表达产物经镍离子螯合琼脂糖凝胶一步纯化后,检测其活性并计算比活。将纯化的SMNE用于重组腺病毒的制备,对外源性核酸进行降解,并采用Southern blot对外源性DNA残留量进行测定。结果重组表达质粒pET-20b-smne经PCR、双酶切和测序证明构建正确。重组蛋白的表达量为8.0 mg/L,纯化后纯度达95%,比活达1.1×106 U/mg。在重组腺病毒制备过程中使用后,成品中的外源性DNA残留量≤10 ng/5.0×1011 VP。结论已成功地在大肠杆菌中表达了SMNE,纯化的SMNE活性高,有望应用于重组生物制品制备过程中外源性核酸的去除。     

Ultrafiltration of Coagulation-Pretreated Serratia marcescens Fermentation Broth: Flux Characteristics and Prodigiosin Recovery

The dead-end ultrafiltration (UF) of coagulation-pretreated fermentation broth of Serratia marcescens SMΔR for prodigiosin recovery was studied. Experiments were performed using different types (regenerated cellulose, YM; polyethersulfone, PES) and molecular weight cut-offs (MWCOs, 1–10 kDa) of the membranes, feed concentrations of prodigiosin (300–1000 mg/L), applied pressures (68.9–206.8 kPa), and stirring speeds (200–400 rpm). With the same MWCO, the YM membrane had a higher retention of prodigiosin and a lower flux than the PES membrane. A two-fold concentration of prodigiosin was observed in the retentate using a 1-kDa YM membrane compared to the concentration in the permeate using a 10-kDa YM membrane. In addition, the extent of membrane fouling was quantitatively analyzed in terms of the modified fouling index. Flux decline in the present batch UF process was mainly due to cake layer formation and partly due to pore blocking. A two-stage UF process was proposed for this purpose, with 81% recovery yield and four-fold concentration.     

Production of chitinolytic enzymes by Serratia marcescens QMB1466 using various chitinous substrates

The chitinolytic activity of submerged cultures of Serratia marcescens QMB1466 in media containing four forms of chitin as the main substrate was investigated via a full factorial design experiment with pH, temperature and substrate concentration as the main parameters. At the optimum conditions (pH 7.0, 32.5 °C and 1.0% (w/v) substrate), bioprocessed chitin (BP), isolated by lactic acid fermentation of prawn shell (Nephrops sp), induced a higher level of enzyme activity than untreated prawn shell and colloidal chitin but not that of a chemically isolated chitin (CP). The optimal conditions of pH and temperature were then applied in a bench‐top bioreactor and the chitinolytic activity monitored temporally under the influence of higher concentrations of BP and CP. Increasing the concentration of substrate in the bioreactor (>1.0% w/v) was found to inhibit the enzyme activity of the bacteria. The enzyme mixtures in selected 120‐h culture supernatants were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and the main proteins characterised by molecular weight. The electrophoretic patterns obtained from cultures from different experiments and by the different chitin substrates showed marked similarity and the main proteins isolated were largely homologous to well‐documented chitinases found in the literature. BP chitin was found to be an efficient elicitor of chitinolytic activity from this bacterium and hence is a suitable substrate to employ in an integrated biotechnological process, whereby several commercially applicable products can be obtained from a waste product of the fishing industry. Copyright © 2004 Society of Chemical Industry     

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole ( 5 a , resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole ( 6 , resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.     

活性黄铒染料的制备及其光谱性能

以活性黄和氧化铒为原料,制备了活性黄铒染料,用该染料对真丝绸进行了染色,分别测定了活性黄、活性黄铒的紫外可见光吸收光谱以及染色后真丝绸的红外光谱。在200～800nm范围内,活性黄和活性黄铒的最大吸收峰的波长分别为383nm和380nm。活性黄与铒离子是以配位键相结合,活性黄铒在保持活性黄的上染基团的基础上增加了与真丝绸结合的基团。     

驱蚊蚊帐的制备和表征

采用氨基丙基三乙氧基硅烷偶联剂处理涤纶蚊帐,然后将处理后的蚊帐浸泡在添加溴氰菊酯悬浮液的聚氨酯胶粘剂乳液中,室温固化得到表面含有溴氰菊酯的蚊帐,该蚊帐具有驱蚊效果。利用紫外可见吸收光谱可以非常简便地测定蚊帐中溴氰菊酯含量。