两株菲降解菌株的特性及其系统发育分析

从石油污染土壤中分离到两株可以菲为唯一碳源的细菌菌株,经形态和生理生化特性分析,脂肪酸含量分析和16S rDNA序列同源性鉴定, 两菌均属鞘氨醇单胞菌属(Sphingomonas),菌株ZX4为少动鞘氨醇单胞菌(Sphingomonas paucimobilis),而菌株EVA17与该属内已知菌的序列同源性在93%~98%之间,推测可能为一新种.两菌株在不同碳源培养基上的生长曲线表明菲对细菌生长有明显的延滞作用.菌株EVA17全细胞蛋白质电泳图谱揭示该细菌在菲诱导下可出现诱导性蛋白,推测可能为一些解毒酶或降解酶.菲降解细菌在以结晶态菲为碳源时生长速率明显低于以粉末态菲为碳源时的生长速率,表明细菌与菲间的接触面积是限制其利用菲的一个重要因素.分离菌株谷胱甘肽S-转移酶(GST酶)具有可与1-氯-2,4-二硝基苯(CDNB)结合的活性.添加表面活性剂吐温-80可促进细菌对菲的利用.     

微囊藻毒素降解菌的分子鉴定和特性研究

在成功筛选出1株能够降解微囊藻毒素(Microcystins,MCs)菌株的基础上,利用16S rDNA的PCR扩增和序列分析对其进行了分子鉴定。由BLAST序列比对及进化树分析可将该菌株定属至鞘氨醇单胞菌USTB-01(Sphingopyxissp.USTB-01),这是我国首次筛选出高效降解MCs的鞘氨醇单胞菌。在鞘氨醇单胞菌USTB-01降解MC-RR和MC-LR活性研究方面发现,温度30℃和pH7.0条件下该菌降解MCs的速率最大,日均降解MC-RR和MC-LR分别达到了23.4mg/L和7.9mg/L,在进一步去除水体中的MCs的基础和应用研究方面都具有非常重要的意义。     

菲降解菌株GY2B的分离鉴定及其降解特性

对一株菲降解菌进行了鉴定并对其降解特性进行了研究.初步鉴定菌株GY2B为鞘氨醇单胞菌(Sphingomonassp.).菌株GY2B有较高的降解效率,当无机盐培养液中菲初始浓度为100mg/L时,48h内对菲降解率达到99.1%.添加100mg/L的葡萄糖和蛋白胨均可以促进菌株的生长和菲的降解,pH值为中性条件时对细胞的生长较为有利.GY2B菌株还能降解1-羟基-2-萘酸、2-萘酚、萘、水杨酸、邻苯二酚和苯酚等多种芳香化合物并利用其为碳源和能源生长繁殖.GY2B菌株对菲的降解可能通过水杨酸途径.     

甲氰菊酯降解菌JQL4-5的分离鉴定及降解特性研究

从农药厂废水处理池的活性污泥中分离到1株能以甲氰菊酯为唯一碳源生长的细菌,命名为JQL4-5.根据其生理生化特征和16S rDNA(GenBank Accession No.DQ177525)序列相似性分析,将该菌株鉴定为鞘氨醇单胞菌属(Sphingomonas sp.).该菌株在24h内对20 mg/L的甲氰菊酯的降解率达到99.8%.降解甲氰菊酯的最适温度为30℃,pH为7.0,降解速率与初始接种量呈正相关.酶的定域试验表明,降解甲氰菊酯的酶为胞内酶.     

DDT降解菌株DB-1的分离、系统发育及降解特性

从农药厂下水道污泥中分离、筛选到1株能够在好氧条件下降解DDT的细菌菌株DB-1,根据表型特征、生理生化特性及16SrDNA序列的系统发育分析,将菌株DB-1初步鉴定为鞘氨醇单胞菌属(Sphingomonassp).该菌株能在含酵母膏(40mg/L)的DDT(40mg/L)无机盐液体培养基中降解DDT,10d降解率达到83.6%,菌株DB-1在25～30℃长势较好,最适生长pH值为8.0.     

2株降解菲的植物内生细菌筛选及其降解特性

为了获得具有菲降解特性的植物内生细菌,通过选择性富集培养,本研究从多环芳烃污染区植物体内分离得到2株能够降解液体培养基中高浓度菲(200 mg·L-1)的植物内生细菌(菌株P1和P3).经形态观察、生理生化特征鉴定和16S rDNA序列同源性分析,分别将菌株P1和P3鉴定为寡养单胞菌属(Stenotrophomonas sp.)和假单胞菌属(Pseudomonas sp.)的细菌.菌株P1和P3均为好氧生长,28℃、150 r·min-1摇床培养7 d,2株菌对无机盐培养基中菲(100 mg·L-1)的降解率均高于90%.条件实验表明,温度20～30℃,pH 6.0～8.0,盐含量0%～4%,装液量10～30 mL(100 mL三角瓶)2菌株生长良好且对菲降解率高于70%.其最适生长和降解温度为30℃,pH为7.0,盐含量≤4%,装液量≤30 mL.综合比较2株菌对菲的降解特性,P1菌株高温耐受性稍强,而P3菌株对环境pH改变和缺氧的耐受性稍强.     

溴氨酸降解菌株的分离鉴定及特性研究

从污泥样品中分离得到一株溴氨酸降解菌,该菌株能够以溴氨酸为唯一碳、氮源及能源生长.通过形态、生理生化特性分析以及对16SrDNA序列进行同源比较,鉴定该菌株属于鞘氨醇单胞菌属,定名为Sphingomonasxenophaga .菌株的最适降解与生长条件为:pH =7 0 ,温度3 0℃,转速15 0r·min- 1 ,接种量8% .在最佳条件下,溴氨酸的降解率可达90 %以上,菌株可耐受的溴氨酸浓度为10 0 0mg·L- 1 .通过分析菌株对水杨酸、邻苯二酚及邻苯二甲酸的利用情况,推测该菌株可能通过邻苯二酚的代谢途径降解溴氨酸.     

混合菌对原油的降解及其降解性能的研究

从污染土壤中分离筛选到四株石油组分降解菌被用于组建降解原油的混合菌体系.石油组分降解菌包括:烷烃降解菌洋葱伯克霍尔德氏菌(Burkholderia cepacia)GS3C、菲降解菌鞘氨醇单胞菌(Sphingomonas sp.)GY2B、芘降解菌GP3(假单胞菌(Pseudomonas sp.)GP3A和伯克菌科的菌株(Pandoraea pnomenusa)GP3B).气相色谱-质谱联用法(GC-MS)用于对原油降解性能的测定,并对原油组分的降解情况进行详细分析.通过对不同菌株的混合培养比较,得到降解原油的最佳组合G8(GS3C+GY2B+GP3B),培养5d后使初始原油浓度为2000mg·mL-1的总去除率达到69.20%,并且对烷烃类和芳烃类化合物都表现出较强的降解能力.混合菌G8对原油的总去除率比单菌提高了近30%,其最适生长条件为:温度为30℃,初始pH值为7,接种量为4%.     

庆大霉素降解菌的筛选及效果验证

抗生素菌渣因残留有抗生素被列为危险固体废物。该研究以庆大霉素菌渣堆肥为菌源,采用浓度梯度递增富集驯化法,筛选出能够降解庆大霉素的微生物菌株。经检测表明,筛选出的降解菌株彼此间无明显拮抗作用,对相对高效的3株降解菌进行16S rDNA序列分析,鉴定其分别属于寡养单胞菌属、鞘氨醇单胞菌属和土壤杆菌属。这3株菌按体积比1∶1∶1组合后,当接种量为5%时,在含1 000 mg/L庆大霉素浓度的液体无机盐基础培养基中,7 d后对庆大霉素的降解率高达71.5%,且外加碳源促进了菌株对庆大霉素的降解率,其中果糖的促进作用最明显。     

南海高效石油降解菌的筛选及降解特性研究

以南海10个采样点采集到的样品为研究材料,以石油降解率为筛选依据,初筛获得52株石油降解菌,从中进一步筛选出6株对石油烃有降解能力的细菌,通过16S rRNA序列分析对筛选得到的6株菌进行初步鉴定,并使用GC-MS内标法测定降解产物,对降解菌的降解特性进行进一步研究.结果表明,采用重量法筛选出来的6株细菌对石油的降解率为20%～55%.与Genbank中的16S rRNA基因序列BLAST对比结果显示,所筛选出的6株菌株中,3株菌株属于芽孢杆菌属(Bacillus),2株菌株属于假交替单胞菌属(Pseudoalteromonas),1株属于交替单胞菌属(Alteromonas).降解特性分析表明,所筛选6株菌的烷烃降解率均在40%以上,多环芳烃降解率均在70%以上,其中,菌株B08500m-3对石油中总烷烃和总芳香烃的降解效果较好,降解率分别为75%和87%.     

Characterization and phylogenetic analysis of a phenanthrene-degrading strain isolated from oil-contaminated soil

Bacterium strain EVA17 was isolated from an oil-contaminated soil, and identified as Sphingomonas sp.based on analysis of 16S rDNA sequence, cellular fatty acid composition and physiological-chemical tests. The salicylate hydroxylase and catechol 2, 3-dioxygenase (C23O) were detected in cell-free lysates, suggesting a pathway for phenanthrene catabolism via salicylate and catechol. Alignment showed that both of the C23O and GST genes of the strain EVA17 had high similarity with homologues of strains from genus Sphingomonas. The phylogenetic analysis based on 16S rDNA and C23O gene sequence indicated that EVA17 should be classified into genus Sphingomonas, although the two phylogenetic trees were slightly different from each other. The results of co-amplification and sequence determination indicated that GST gene should be located upstream of the C230 gene.     

布洛芬降解菌降解谱研究

以从西安污水处理厂筛选所得的3株布洛芬降解菌株I2(克雷伯氏菌)、I4(假单胞菌)和I14(不动杆菌)为研究对象,探索其生长特性及对布洛芬同系物降解的广谱性.结果表明,3株菌对7种选定的布洛芬同系物均有降解作用,但降解能力表现出一定的差异.I2、I4菌株的降解能力优于I14菌株,对7种布洛芬同系物的最高耐受浓度均在300 mg·L-1以上.3株降解菌都对布洛芬同系物中的间苯二酚有良好的耐受能力,最高耐受浓度均在1000 mg·L-1以上,I2菌株甚至达到2500 mg·L-1;3株降解菌对于邻苯二酚、对苯二酚的耐受程度最差,其中,I14菌株在对苯二酚达到100 mg·L-1时就会死亡.I2、I4菌株可作为布洛芬及其同系物降解的优良备选菌株,用于进一步研究多重污染治理的方式.     

Degradation of polycyclic aromatic hydrocarbons by Pseudomonas sp. JM2 isolated from active sewage sludge of chemical plant

It is important to screen strains that can decompose polycyclic aromatic hydrocarbons (PAHs) completely and rapidly with good adaptability for bioremediation in a local area. A bacterial strain JM2, which uses phenanthrene as its sole carbon source, was isolated from the active sewage sludge from a chemical plant in Jilin, China and identified as Pseudomonas based on 16S rDNA gene sequence analysis. Although the optimal growth conditions were determined to be pH 6.0 and 37℃, JM2 showed a broad pH and temperature profile. At pH 4.5 and 9.3, JM2 could degrade more than 40% of fluorene and phenanthrene (50 mg/L each) within 4 days. In addition, when the temperature was as low as 4℃, JM2 could degrade up to 24% fluorene and 12% phenanthrene. This showed the potential for JM2 to be applied in bioremediation over winter or in cold regions. Moreover, a nutrient augmentation study showed that adding formate into media could promote PAH degradation, while the supplement of salicylate had an inhibitive effect. Furthermore, in a metabolic pathway study, salicylate, phthalic acid, and 9-fluorenone were detected during the degradation of fluorene or phenanthrene. In conclusion, Pseudomonas sp. JM2 is a high performance strain in the degradation of fluorene and phenanthrene under extreme pH and temperature conditions. It might be useful in the bioremediation of PAHs.     

Detection of Norovirus Recombinant GII.2[P16] Strains in Oysters in Thailand

Human norovirus causes sporadic and epidemic acute gastroenteritis worldwide, and the predominant strains are genotype GII.4 variants. Recently, a novel GII.17[P17] and a recombinant GII.2[P16] strain have been reported as the causes of gastroenteritis outbreaks. Outbreaks of norovirus are frequently associated with foodborne illness. In this study, each of 75 oyster samples processed by a proteinase K extraction method and an adsorption-elution method were examined for noroviruses using RT-nested PCR with capsid primers. Thirteen (17.3%) samples processed by either method tested positive for norovirus genogroup II (GII). PCR amplicons were characterized by DNA sequencing and phylogenetic analysis as GII.2 (n?=?6), GII.4 (n?=?1), GII.17 (n?=?3), and GII.unclassified (n?=?3). Norovirus-positive samples were further amplified by semi-nested RT-PCR targeting the polymerase-capsid genes. One nucleotide sequence revealed GII.17[P17] Kawasaki strain. Five nucleotide sequences were identified as belonging to the recombinant GII.2[P16] strains by recombination analysis. The collected oyster samples were quantified for norovirus GII genome copy number by RT-quantitative PCR. Using the proteinase K method, GII was found in 13/75 (17.3%) of samples with a range of 8.83–1.85?×?10⁴ genome copies/g of oyster. One sample (1/75, 1.3%) processed by the adsorption-elution method was positive for GII at 5.00?×?10¹ genome copies/g. These findings indicate the circulation of a new variant GII.17 Kawasaki strain and the recombinant GII.2[P16] in oyster samples corresponding to the circulating strains reported at a global scale during the same period of time. The detection of the recombinant strains in oysters emphasizes the need for continuing systematic surveillance for control and prevention of norovirus gastroenteritis.

     

耐冷腐殖酸吸附态PAHs降解菌筛选及其降解特性

为了寻找自然环境中高效PAHs降解菌,并应用于北方寒冷地区PAHs污染土壤修复,从沈抚灌渠冻融土壤中筛选出一株以腐殖酸(HA)吸附态PAHs为碳源和能源且生长良好的耐冷菌株,结合其生理生化特征和16S rDNA序列比对,鉴定此菌株为假单胞菌(Pseudomonas sp.SDR4),并研究了其对冻融土壤中HA吸附态PAHs的降解性能.结果表明:Pseudomonas sp.SDR4对冻融土壤中Phe、Pyr和Ba P均具有一定的降解能力,42 d降解率分别为73.88%、64.88%和49.39%.加入HA能够提高其对Phe、Pyr和Ba P的降解率,42 d降解率分别提高15.9%、13.8%和8.6%;在降解初期加入HA可显著提高Phe、Pyr和Ba P的降解速率,第1周,降解速率分别提高37.8%、28.4%和39.1%,但在后期促进效果减弱.若要在寒冷地区选择Pseudomonas sp.SDR4作为固定化菌株,可添加HA提高其修复效果.本研究为北方寒冷地区PAHs污染土壤修复提供了新的固定化菌种.     

纯菌株与混合菌株在MFC中降解喹啉及产电性能的研究

微生物燃料电池(microbial fuel cell,MFC)阳极微生物菌群组成与MFC产电性能有重要关系.从稳定运行了210 d以上,以200 mg.L-1喹啉为燃料的MFC阳极室分离提纯出4株兼性厌氧菌Q1、b、c和d,分别代表原MFC中所有4类不同菌落形态的可培养菌.16S rDNA序列分析结果表明,菌株Q1、c和d属于假单胞菌属(Pseudomonas sp.),菌株b属于伯克霍尔德菌属(Burkholderia sp.).通过构建双室MFC,以200 mg.L-1喹啉和300 mg.L-1葡萄糖为混合燃料,以铁氰化钾为电子受体测定各菌株产电能力,结果表明菌株b、c和d均为非产电菌.产电菌Q1与非产电菌b、c、d复合产电电荷量依次为3.00、3.57和5.13C,库仑效率依次为3.85%、4.59%和6.58%,产电菌与非产电菌对燃料的降解利用存在竞争关系,使得复合菌产电能力比产电菌Q1单独时的产电能力差.在MFC中,非产电菌与产电菌复合产电时24h内对喹啉的去除率均可以达到100%,降解喹啉效果优于4株菌单独构建的MFC,即混合菌更有利于利用复杂碳源.GC/MS的测定结果表明,产电菌株Q1构建的纯菌MFC和原混合菌MFC周期结束时出水中存在的喹啉代谢产物均为2-羟基喹啉和苯酚.     

耐受高浓度氨氮异养硝化菌的筛选及其脱氮条件优化

研究了异养硝化菌对高浓度氨氮的耐受能力和去除能力.采用多点取样、高浓度氨氮废水强行驯化、驯化液连续梯度稀释、颜色指示剂快速硝化效果检测、平板划线分离等步骤,筛选能耐受高浓度氨氮废水的异养硝化菌株,以各菌株16S rDNA序列的系统发育分析来鉴定其种属,考察了菌株的脱氮特性,并通过提高C/N比和优化菌株配伍的方式对其脱氮能力进行了优化.结果共筛出8株高效的异养硝化菌株,并将其命名为N1～N8.系统发育分析表明8株菌分属丛毛单胞菌属（Comamonassp.）、红球菌属（Rhodococcus sp.）、假单胞菌属（Pseudomonas sp.）、节杆菌属（Arthrobacter sp.）、副球菌属（Paracoccus sp.）,其对起始氨氮浓度为256.9 mg.L-1、C/N=5.5的人工废水,72 h后氨氮去除率约在65%～80%之间,其中最高为N4的80.2%.若将上述废水的C/N比提高至8.0,则各菌株的氨氮去除率相应提高至约80%～90%.部分菌株配伍后脱氨氮效果优于任一单菌株,其中N4＋N5＋N6对起始浓度为261.1 mg.L-1的氨氮、在C/N=5.5的条件下,48 h去除率为88.2%.将N4＋N5＋N6组合驯化菌液,则能将该氨氮去除率提高至99.8%;在将起始氨氮浓度提高至446.9 mg.L-1、C/N比降为3.2后,52h后氨氮去除率亦可达99.9%,且最终几乎无亚硝酸盐氮和硝酸盐氮的积累,总氮去除率为66.5%,菌株同化的氮仅占损失氨氮的33%.可见驯化菌液中一些未能分离的菌株对分离出的菌株的脱氨氮效果有显著的协同作用.     

一株新颖的Providencia sp.菌降解吲哚及合成靛蓝的特性研究

吲哚是一种典型的氮杂环芳烃污染物,在焦化废水和畜牧废水中大量存在.本研究从近海泥沙中分离纯化得到一株高效吲哚降解菌DCX,经16S rRNA基因序列比对分析,鉴定其为普罗维登斯菌属(Providencia sp.).该菌株能够以吲哚为唯一碳源,在28 h内将100 mg·L~(-1)吲哚完全降解.液相色谱/飞行时间-质谱联用(LC/TOF-MS)的结果表明,靛蓝、靛红、靛红酸及邻氨基苯甲酸是菌株DCX降解吲哚过程中的中间产物.此外,本研究中发现,外加营养物质可以促进菌株DCX降解吲哚,特别是加入酵母浸粉后,反应体系中会产生大量靛蓝.利用表面响应法确定菌株DCX转化吲哚合成靛蓝的最优条件为:吲哚207.49 mg·L~(-1),酵母浸粉2.9 g·L-1,接菌量4.23%(V/V),并且在最优条件下,靛蓝产率达到最高(9.90%),比初始条件提高了4.38倍.     

降解多环芳烃的菌株Gordonia sp. He4的分离鉴定及其在菲污染土壤修复过程中的动态变化

从石油污染土壤中分离得到1株降解石油烃类污染物的He4菌株.该菌株能够以正十六烷、苯、萘、蒽、菲和芘作为唯一的碳源生长.经过对其形态特征、生理生化、以及16S rRNA基因序列分析,该菌株初步鉴定为Gordoniasp..通过分析其16S rRNA基因序列,设计引物并构建了竞争性模板.通过竞争性定量PCR（quantitative competitive-PCR）分析了该菌株在含有菲的污染土壤中数量的变化.结果表明,部分菌株He4在土壤中转变为不可培养状态,采用传统的稀释涂布菌落计数法（CFU）无法对其进行定量,而通过QC-PCR能够较准确地测定土壤中微生物的动态变化.