几种生物学指标与生物强化系统降解效率的关系

针对炼油废水生物强化处理系统中有时存在的不稳定造成排水超标的问题,研究了SBR反应器中采用生物强化技术,其细菌数量、脱氢酶、邻苯二酚1,2-双加氧酶(C12O)和邻苯二酚2,3-双加氧酶(C23O)等关键生物学指标与污染物去除效率的综合对应关系,并运用ERIC-PCR技术研究了运行前后处理系统内微生物群落的变化.结果表明,投加菌剂的生物强化处理系统提高了细菌数量和酶活,使处理效率明显提高.生物强化系统中细菌数量及C23O酶活与污染物去除效率呈正相关关系,且微生物群落结构十分稳定.C12O为一种诱导酶,其酶     

多环芳烃降解菌的分离鉴定及其生理特性研究

从兰州石化污水处理厂的污泥中分离出一株能分别以萘、蒽为唯一碳源生长的细菌(BDP01). 通过菌株形态观察及16S rDNA序列比对, 确定该菌株属于假单胞菌属的绿脓杆菌(Pseudomonas aeruginosa). 菌株在LB培养基中48 h内呈对数生长, 48 h到72 h进入生长稳定期, 72 h后开始进入衰亡期;其最佳培养温度为30 ℃, 最适生长pH为中性或弱碱性, 该菌能在萘和蒽的质量浓度分别为100 mg·L^-1和50 mg·L^-1的无机盐培养基中良好生长, 与已报道的其他微生物比较, BDP01在较短时间内具有较强的降解多环芳烃能力, 1%的接种量15 d内可降解89.64%的萘和77.21%的蒽. 采用PCR和琼脂糖凝胶电泳技术检测到菌株基因组中有邻苯二酚2,3-双加氧酶 (C23O)基因. 测序结果与NCBI数据库发布的C23O基因序列相似度为92%. 该酶在20~65 ℃以邻苯二酚为底物时, 细胞裂解液中酶活力变化范围为13.10~540.86 U.     

邻苯二酚双加氧酶传感器的研究

洋葱伯克霍尔德氏菌L68的细胞裂解液经硫酸铵分级沉淀后,依次经DEAE-SepharoseFastFlow柱层析,Hydroxyapatite柱层析和SephadexG-150凝胶过滤分离,提取到邻苯二酚双加氧酶.经SDS-PAGE检测,达到电泳纯.将酶反应与氧电极偶联,研究了检测水体中邻苯二酚的邻苯二酚双加氧酶传感器.以100mmol/L磷酸钠标准缓冲液(pH7.24)作为该酶传感器的的工作介质,在25℃条件下测得传感器对邻苯二酚标准液的工作曲线线性范围为0.2～3mmol/L,检测限为0.05mmol/L,响应时间为1min,平均回收率达到1.03%.     

菲降解菌的特性及其降解酶纯化研究

研究考察了一株伯克霍尔德氏菌(Burkholderia sp.AFF)的基本特性,并对降解过程中的邻苯二酚2,3-双加氧酶进行了初步分离纯化。菌株AFF对链霉素和四环素具有抗性;当培养基中NaCl的浓度>1.5%时,菌株的生长降解能力受到抑制。菌株AFF生长与降解菲的最佳条件:温度30℃,pH=7.0,摇床转速为150r/min,接种量10%。菌株AFF的细胞粗提物具有邻苯二酚2,3-双加氧酶活性,用阴离子层析交换柱对其进行纯化,对收集到的具有酶活的组分进行SDS-PAGE分析,结果表明目的蛋白分子量在31kDa和43kDa之间。经过一步纯化后,比酶活提高33%,蛋白的回收率为27.6%。     

苯酚降解菌红球菌PNAN5菌株(Rhodococcus sp.strain PNAN5)的分离鉴定、降解特性及其开环双加氧酶性质研究

分离到一株能以苯酚、苯甲酸、对甲酚、萘为唯一碳源和能源生长、具有同时降解单环和双环芳烃能力的细菌菌株,经生理生化、16SrRNA基因序列分析等鉴定为红球菌PNAN5菌株(Rhodococcussp.strainPNAN5).在实验条件下和在温度为20～40℃、pH7 0～9 0范围内菌株PNAN5降解苯酚的效率保持在80%～100%之间,苯酚浓度在2～10mmol·L-1范围内变化对降解效率没有明显的影响.该菌株通过邻苯二酚1,2 双加氧酶催化的开环途径降解芳烃,不同于已知的浑浊红球菌(R.opacus)是通过邻苯二酚2,3 双加氧酶催化芳烃降解.以细胞裂解液测定该酶的酶促反应动力学常数Km值为35 94μmol·L-1,Vmax为0 84μmol·L-1·min-1·mg-1.     

1株对叔丁基邻苯二酚降解菌的筛选鉴定及响应面法优化其降解

从某化工厂污水处理车间活性污泥中分离、筛选到1株能以对叔丁基邻苯二酚(p-tert-Butylcatechol,TBC)为唯一碳源和能源生长的菌株YH1.经形态特征、生理生化、BIOLOG细菌自动鉴定系统和16S r DNA序列分析,鉴定菌株YH1为皱纹假单胞菌(Pseudomonas corrugate).在温度为24~36℃,p H为7.0~10.0的条件下,菌株YH1可使浓度低于500 mg·L-1的TBC降解率达到82%以上.运用单因素实验初步确定TBC降解的最适外加碳源和氮源分别为蔗糖和胰蛋白胨,最适温度为30℃,最适初始p H为7.0,最适接种量为2%.为了提高降解率,首先利用Plackett-Burman实验设计评估并筛选出影响TBC降解的3个关键因素:蔗糖、胰蛋白胨、初始p H.用最陡爬坡实验逼近以上3个因子的最大响应区域,采用Box-Behnken实验设计及响应面法分析,确定其最优降解条件为蔗糖浓度3%(ρ)、胰蛋白胨浓度1.44%(ρ)、TBC浓度400 mg·L-1、初始p H值8.12、接种量2.97%(φ)、温度30℃、培养时间96 h,在此条件下TBC降解率可达98.21%.TBC降解酶活性及酶定域实验表明,菌株YH1相关降解酶为胞内酶,且TBC可诱导邻苯二酚1,2双加氧酶(C12O)的合成.通过降解酶特异性引物从菌株YH1扩增得到C12O基因片段,经质粒检测和消除实验发现菌株YH1相关降解基因位于质粒上.此外,菌株YH1能耐受高浓度Na Cl和多种重金属离子,对多种抗生素具有抗性.研究结果为有效处理复杂工业废水提供了理论基础.     

菲降解菌株的分离鉴定及特性研究

从某石化公司污水处理厂的活性污泥中分离到1株对菲具有降解活性的菌株AFF,该菌株能够以菲为唯一碳源和能源生长.根据形态特征及16S rRNA基因序列分析,该菌株初步鉴定为Burkholderia sp..在以菲为唯一碳源的培养基中,84 h内菌株对100 mg·L-1菲的降解率可达74.1%.通过液相-质谱联用及紫外可见分光光度法分析,推测该菌株代谢菲的途径为水杨酸途径.测定了降解途径中相关酶的活性,表明该菌株降解酶系中具有邻苯二酚2,3-双加氧酶活性,并证明该酶为诱导酶;确定了粗酶降解邻苯二酚的动力学常数:米氏常数Km为10.93μmol·L-1,最大反应速率Vmax为106.38μmol·min-1,说明酶与邻苯二酚的亲和力较大,邻苯二酚是该酶的良好底物.     

苯酚降解菌TX1的分离鉴定及其降解特性

从受酚类污染的土壤中分离筛选出1株能够高效降解苯酚的菌株。通过形态学、生理生化以及26S核糖体（rDNA）测序等手段对其进行初步鉴定,确定该菌株为丝孢酵母菌属（Trichosporon sp．）,命名为TX1。初步研究结果表明,该菌株对苯酚的代谢途径为将苯酚先转化为邻苯二酚的苯酚羟化酶定位于细胞膜和细胞质;进而通过邻苯二酚1,2-双加氧酶（C120）邻位开环裂解,C120为诱导型胞内酶。通过分批培养得到菌株TX1降解苯酚的生长动力学模型可用霍尔丹（Haldane）方程表述,相关参数μmax=0．667h^-1,Ks=51．14mg／L,Ki=271．7mg／L。     

好氧颗粒污泥处理畜禽养殖沼液污染物的特性

采用序批式反应器,研究了好氧颗粒污泥处理畜禽养殖沼液中传统和新兴污染物的去除特性,以及系统的微生物群落结构演变情况.结果表明,畜禽沼液所含高浓度污染物不会对好氧颗粒污泥结构产生显著毒性胁迫,好氧颗粒污泥系统可实现对沼液所含有机物和氨氮的稳定去除.系统出水平均化学需氧量和氨氮浓度分别为(267±81) mg·L~(-1)和(62±12) mg·L~(-1),去除效率分别为73%±8%和91%±2%.同时,也可实现对沼液所含四环素类和磺胺类抗生素的有效去除,去除效率分别为65%±16%和98%±2%.但对磷素的去除效率较低,约为16%±2%.好氧颗粒污泥系统处理沼液过程中微生物群落结构稳定,但其中功能微生物菌群丰度会受到水质的作用影响,从毛单胞菌科Comamonadaceae(相对丰度约为16. 66%)为污泥中的主导微生物群落.     

膜曝气-生物膜反应器生物强化处理阿特拉津废水运行性能

在疏水SPG(shirasu porous glass)膜表面形成基因工程菌生物膜,构建SPG膜曝气-生物膜反应器(MABR)生物强化处理阿特拉津废水,考察MABR反应器稳定运行过程中污染物去除性能及其影响因素.结果表明,增大SPG膜孔径和曝气压力,能够提高曝气供氧能力,改善COD和阿特拉津生物强化去除效能.1.5μm疏水SPG膜在70 k Pa曝气压力下的最大供氧能力约为22.4 g·(m~2·d)~(-1).曝气压力为70 k Pa、水力停留时间(HRT)为1.5 h时,1.5μm膜MABR反应器COD平均去除率为80.1%,平均去除负荷为1.86 kg·(m~3·d)~(-1);阿特拉津平均去除率为62.5%,平均去除负荷为0.18 kg·(m~3·d)~(-1).进一步缩短HRT、增加进水负荷后,MABR反应器DO浓度显著下降,COD和阿特拉津去除效率大幅降低.DO浓度对阿特拉津去除的影响更为显著.随着MABR反应器的稳定运行,SPG膜表面单一基因工程菌生物膜逐渐演化为复杂微生物群落,但基因工程菌可以较好地存在于生物膜内,从而保持阿特拉津生物强化去除能力.     

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.     

荧光定量PCR检测生物强化SBR系统中苯胺降解菌的数量变化

生物强化SBR系统中苯胺降解菌Pseudomonas otitidis strain JY9在降解苯胺废水过程中发挥重要作用.本研究根据该菌株的邻苯二酚1,2双加氧酶(C12O)的基因序列,设计特异引物,扩增特异片段并克隆到PGM-T载体中,以此重组质粒为参照,系统活性污泥中提取的总DNA为模板,应用实时荧光定量PCR方法定量检测该菌在苯胺废水生物强化SBR系统中的丰度变化.同时应用高效液相色谱法检测系统中苯胺的残留.结果表明,苯胺初始浓度在200~500 mg·L-1范围内,苯胺降解率均达到96%以上.并且SBR反应系统活性污泥中每单位(mg)MLVSS的C12O基因拷贝数随苯胺浓度的升高而显著上升,最高拷贝数达到2.7×109个,表明该菌的数量随苯胺浓度的升高而显著上升.结果显示,在苯胺去除过程中Pseudomonas otitidis strain JY9的投加能够稳定活性污泥中的MLVSS,当含有高浓度苯胺废水系统运行稳定时,该降解菌逐渐成为活性污泥当中的优势菌.     

Promotion mechanism of self-transmissible degradative plasmid transfer in maize rhizosphere and its application in naphthalene degradation in soil

Rhizospheres can promote self-transmissible plasmid transfer, however, the corresponding mechanism has not received much attention. Plant-microbe remediation is an effective way to promote pollutant biodegradation; however, some pollutants, such as naphthalene, are harmful to plants and result in inefficient plant-microbe remediation. In this study, transfer of a TOL-like plasmid, a self-transmissible plasmid loaded with genetic determinants for pollutant degradation, among different bacteria was examined in bulk and rhizosphere soils as well as addition of maize root exudate and its artificial root exudate (ARE). The results showed that the numbers of transconjugants and recipients as well as bacterial metabolic activities, such as xylE mRNA expression levels and catechol 2,3-dioxygenase (C23O) activities of bacteria, remained high in rhizosphere soils, when compared with bulk soils. The number of transconjugants and bacterial metabolic activities increased with the increasing exudate and ARE concentrations, whereas the populations of donor and recipient bacteria were substantially unaltered at all concentrations. All the experiments consistently showed that a certain number of bacteria is required for self-transmissible plasmid transfer, and that the increased plasmid transfer might predominantly be owing to bacterial metabolic activity stimulated by root exudates and ARE. Furthermore, ARE addition increased naphthalene degradation by transconjugants in both culture medium and soil. Thus, the combined action of a wide variety of components in ARE might contribute to the increased plasmid transfer and naphthalene degradation. These findings suggest that ARE could be an effectively alternative for plant-microbe remediation of pollutants in environments where plants cannot survive.     

同步硝化反硝化（SND）过程污泥聚集状态对N2O释放的影响

为实现SND高效生物脱氮及N2O减量化释放的双重目标,采用气升环流生物反应器,在实现SND高效脱氮的基础上研究了污泥的聚集状态与N2O释放特征之间的关系.采用关键酶酶促反应速率法评估了不同聚集状态污泥的硝化/反硝化活性,在相关理论分析的基础上,进行了污泥聚集状态的优化选择.结果表明,SND体系内活性污泥的聚集状态不同会...     

生物强化方式对生物转盘处理养殖废水效果及生物多样性的影响

畜禽养殖废水污染物浓度高、生物毒性大,导致传统生物处理工艺难以满足达标排放要求.为探究高效的畜禽养殖废水处理工艺,采用生物转盘作为主体工艺,利用异养硝化-好氧反硝化菌（简称"HN-AD菌"）为生物强化菌剂,对比了强化污泥挂膜和菌剂挂膜两种不同生物强化方式下该工艺在启动时间、碳耗、能耗、真实废水处理效果及微生物多样性方面的差异,以确定最佳生物强化方式.结果表明:①模拟废水试验中,菌剂挂膜反应器启动时间（19 d）明显短于强化污泥挂膜（33 d）,参数优化后发现,在相同处理效果下,前者碳耗、能耗较后者分别低48.22%、33.33%.②真实废水处理试验中,菌剂挂膜反应器的COD_Cr、NH₄+-N、TN平均去除率较后者分别高7.11%、26.97%、29.14%.③在微生物多样性方面,菌剂挂膜体系中Comamonas（丛毛单胞菌属）相对丰度是强化污泥挂膜体系的10倍左右,推测Comamonas可能是在异养硝化好氧反硝化过程中发挥关键作用的菌属.④SEM观察发现菌剂挂膜生物转盘盘片上的生物膜更薄,但HN-AD优势菌富集程度更高.研究显示,菌剂挂膜反应器对模拟废水、真实废水的处理效果均优于强化污泥挂膜反应器,且效果更稳定.      

Simultaneous biodegradation of nitrogen-containing aromatic compounds in a sequencing batch bioreactor

Many nitrogen-containing aromatic compounds (NACs), such as nitrobenzene (NB), 4-nitrophenol (4-NP), aniline (AN), and 2,4-dinitrophenol (2,4-DNP), are environmentally hazardous, and their removal from contaminated water is one of the main challenges facing wastewater treatment plants. In this study, synthetic wastewater containing NB, 4-NP, 2,4-DNP, and AN at concentrations ranging from 50 to 180 mg/L was fed into a sequencing batch reactor (SBR). Analyses of the SBR system indicated that it simultaneously removed more than 99% of the NACs at loading rates of 0.36 kg NB/(m3·d), 0.3 kg 4-NP/(m3·d), 0.25 kg AN/(m3·d), and 0.1 kg 2,4-DNP/(m3·d). Bacterial groups of Bacteriodetes, Candidate division TM7, α-Proteobacteria, and β-Proteobacteria were dominant in the clone libraries of 16S rRNA genes retrieved from the microbial communities in the SBR system. "Cycle tests" designed to alter feeding and aeration parameters of the SBR system demonstrated that the resident microbial biome of the SBR system responded rapidly to changing conditions. Consumption of O2 was concomitant with the apparent mineralization of NACs. Aromatic ring-cleaving dioxygenase activities suggested that (1) AN and NB were degraded via catechol 2,3-dioxygenase; (2) 4-NP was degraded via 1,2,4-benzentriol 1,2-dioxygenase; and (3) 2,4-DNP was degraded via an unresolved pathway.