环境样品中DNA提取方法的研究进展

利用微生物降解污染物是废水处理中的常用方法,研究废水处理工艺中微生物的多样性和动态性对于优化废水处理工艺的性能、提高其处理效率具有重要的指导意义.分子生物学方法是研究微生物多样性和动态性的有效方法.由于大多数分子生物学方法都是以提取研究对象的基因组DNA为前提,因此建立一种高效的环境样品DNA的提取方法具有重要作用.综述了从环境样品中提取DNA的主要方法及其研究进展,并提出了一种有效的从活性污泥中提取DNA的具体方法.     

应用于PCR-DGGE分析的活性污泥微生物总DNA提取方法比较

探讨适用于PCR-DGGE分析研究的活性污泥细菌和真菌的DNA提取方法。采用5种方法提取活性污泥微生物基因组DNA,以DNA纯度、含量、片段大小及DGGE条带多样性作为考察指标评价提取方法的优劣,以确定最佳实验方案。紫外吸收法和琼脂糖凝胶电泳结果显示,试剂盒法提取的DNA含量最低,其余4种方法获得的DNA含量无显著差异,就DNA纯度而言,试剂盒法最优;除高温裂解法对真菌细胞壁裂解效果较差外,其他4种方法均能不同程度地裂解细菌和真菌细胞;DGGE结果表明,高温裂解法获得的细菌条带最多,基于SDS的细胞裂解法得到的真菌条带最多。综合分析,高温裂解法更适合于活性污泥中细菌的PCR-DGGE分析,基于SDS的细胞裂解法则更适合于污泥中真菌的PCR-DGGE分析。     

剩余污泥中微生物絮凝剂的提取方法比较

分别采用低浓度和高浓度剩余污泥对剩余污泥中微生物絮凝剂(MBF)的多种提取方法(超声法、树脂法、超声-树脂法、树脂-超声法、超声-树脂-超声法和树脂-超声-树脂法等)进行了比较研究.结果表明,各种复合形式的提取效果均优于超声法或树脂法单独使用的提取效果.在各种复合形式中,以树脂-超声法所提取MBF的含量最高且絮凝效果最好.此外,所提取的MBF浓度与污泥浓度正相关.从剩余污泥中直接提取MBF,在降低MBF生产成本的同时实现了污泥的资源化利用.     

巢湖入湖河流沉积物中有机磷的形态分级研究

为识别巢湖流域污染物的特征、来源及其沉积物有机磷各形态分布与富营养化的关系,测定了7条巢湖入湖河流沉积物中有机磷各形态的含量,分析不同污染类型人湖河流沉积物中有机磷各形态分布的差异及与其他因素间的相关性。研究发现,不同污染类型人湖河流沉积物中水土保持控制型河流沉积物中有机磷各组分的相对含量顺序为残渣态Po〉富里酸-Po〉HCl-Po〉胡敏酸-Po〉NaHCO3-Po,平均的相对比例为7．5：3．1：1．9：1．5：1．0,而城市污染控制型和面源污染控制型河流沉积物中有机磷各组分的相对含量顺序恰好相同,面源污染控制型河流沉积物Po各形态含量低于城市污染控制型和水土保持控制型河流。中活性P。和OM、TP、Pi、Po、TN、NaHCO3-Pi、NaOH—Pi呈正相关,非活性Po与Po、NaOH-Pi呈显著正相关关系,反映了中活性Po很容易转化为生物可利用磷和非活性Po,且非活性Po仍然具有潜在的生物活性。     

废水处理系统中活性污泥总DNA的提取

对废水脱氮除磷厌氧-缺氧-好氧系统中的活性污泥样品经过或未经预处理,比较了超声波法、玻璃珠震荡法和冻融法3种细胞裂解方法对DNA提取效果的影响。吸光度和琼脂糖凝胶电泳检测结果表明:活性污泥经TENP和PBS预处理后采用冻融法加2%SDS裂解细胞的DNA提取操作,可以得到数量多、纯度高的活性污泥DNA样品,可不经纯化直接进行PCR扩增反应。     

本源微生物菌剂修复城市污染河流

通过丰湖河投菌过程中和总投菌后两周的各水质指标分析,对本源微生物菌剂作用城市重污染河流过程进行了探讨和研究。结果表明,在2010年11月20日全河布菌后,以COD下降后上升的情况作为补菌时间的判断依据,实现菌种效力最大化。继2010年11月25日、27日和30日3次补菌后上游至下游硐桥、久泰桥和益民水闸处COD的去除率分别达到52.7%、68.8%和61.6%,氨氮和总磷有所去除。水色由深灰绿色转变为深绿色,浮油减少,臭味明显消除。表明此法在冬季水温较低的天然河流条件下,不需大型设备便可较快修复水质,具有较好的经济环境效益。     

湿地植物-沉积物微生物燃料电池产电及河流底泥修复

以受污染的城市河涌底泥为底质,湿地植物选用风车草（Clinopodium Urticifolium）或短叶茳芏（Cyperus Malaccensis）,构建了湿地植物-沉积物微生物燃料电池（P-SMFC）及无植物的沉积物微生物燃料电池（SMFC）共3个电极处理组,研究了P-SMFC与SMFC的产电特性,并探讨了它们与对照组中底泥及上覆水中氮磷的迁移转化规律。结果表明,产电方面,在系统启动运行的7个月内,PSM1、SM和PSM2三个电极处理组均能维持较稳定的产电,输出电压在整个运行阶段总体稳定在0.30~0.50 V,且植物的引入提升了系统的产电性能。底泥修复方面,设置的5个处理组对底泥中有机质均有一定的降解作用,表现出PSM1、SM和PSM2处理组有机质的降解要显著高于PS1和PS2处理组,P-SMFC系统对底泥有机物的去除有显著的促进作用;系统中系统运行前2个月,2个P-SMFC处理组氨氮含量显著低于其他3个处理组,之后随着运行时间的延长,各处理组之间的变化差异性不大,5个处理组底泥中氨氮去除率均达到80%以上;电极的引入对底泥中硝氮的去除没有产生显著影响;各处理组底泥中总磷去除率不同,分别为PSM1处理组8.67%、SM处理组8.89%、PSM2处理组7.33%、PS1处理组12.45%、PS2处理组8.89%,产电过程抑制了磷的迁移,有助于底泥中磷的稳定。     

人工河流沉积物中磷的分布特征及其形态研究：以肇兰新河为例

以黑龙江省肇兰新河流域为研究对象,在调查区域水质和沉积物基本理化性质的基础上,采用SMT化学提取方法分析流域内表层、柱状沉积物中磷形态及含量,明确了人工河流沉积物各形态磷的空间分布特征。结合上覆水与沉积物理化性质,探讨了影响人工河流沉积物磷形态及释放因素。结果表明,肇兰新河流域内水体TP均值为2.61 mg·L⁻¹,属于劣V类水体。沉积物的TP为692.91~2 197.87 mg·kg⁻¹,整体处于中度污染水平。沉积物中Fe/Al-P含量占比较高。通过相关性分析发现,上覆水中ORP是影响OP、Fe/Al-P迁移的重要环境因子,沉积物中TC、OM、Fe含量是影响OP含量的重要因素。作为一条典型人工河流,肇兰新河流域磷污染负荷严重,相比自然河流形态结构单一,缓冲能力和纳污能力较弱。本研究可为了解人工河流沉积物磷形态及其含量,并进行流域污染防控提供参考。     

深圳市河流沉积物重金属污染特征及评价

分析了深圳市的深圳河、布吉河、龙岗河和茅洲河等4条河流沉积物中重金属的含量和富集状况,并对重金属污染的潜在生态危害进行了评价。结果表明：（1）1996-2003年,深圳河、布吉河、龙岗河和茅洲河沉积物中重金属的含量都有上升的趋势;布吉河和龙岗河沉积物中的重金属含量较高,特别是Cu、Zn和Cr的含量比其他两条河流高出较多,深圳河居中,茅洲河较小;这4条河流都以Cd、Cu、Cr和Zn的富集为主。（2）在研究时段内,这4条河流沉积物重金属污染的潜在生态危害指数（RI）都有上升的趋势;其中深圳河,布吉河和龙岗河的RI在多数年份都达到强或很强的程度,Cd、Hg、Cu为主要贡献元素;4条河流中,茅洲河受重金属污染最小,其RI为轻微或中等,只有Cd和Hg的生态危害系数为中等或强。针对深圳市河流沉积物中重金属的累积机理,建议通过改善区域水文条件、控制污染物的排放等措施加以控制和治理。     

Degradation of nonylphenol by anaerobic microorganisms from river sediment

We investigated the degradation of nonylphenol monoethoxylate (NP1EO) and nonylphenol (NP) by anaerobic microbes in sediment samples collected at four sites along the Erren River in southern Taiwan. Anaerobic degradation rate constants (k1) and half-lives (t1/2) for NP (2 microg/g) ranged from 0.010 to 0.015 1/day and 46.2 to 69.3 days respectively. For NP1EO (2 microg/g), the ranges were 0.009-0.014 1/day and 49.5-77.0 days respectively. Degradation rates for NP and NP1EO were enhanced by increasing temperature and inhibited by the addition of acetate, pyruvate, lactate, manganese dioxide, ferric chloride, sodium chloride, heavy metals, and phthalic acid esters. Degradation was also measured under three anaerobic conditions. Results show the high-to-low order of degradation rates to be sulfate-reducing conditions > methanogenic conditions > nitrate-reducing conditions. The results show that sulfate-reducing bacteria, methanogen, and eubacteria are involved in the degradation of NP and NP1EO, with sulfate-reducing bacteria being a major component of the river sediment.     

Dechlorination of polychlorinated biphenyl congeners by anaerobic microorganisms from river sediment.

The microbial dechlorination of seven kinds of polychlorinated biphenyls (PCBs) by anaerobic microorganisms from river sediment was investigated. Dechlorination rates were found to be affected by the chlorine level of PCB congeners; dechlorination rates decreased as chlorine levels increased. Dechlorination rates were fastest under methanogenic conditions and slowest under nitrate-reducing conditions. The addition of individual electron donors (acetate, pyruvate, and lactate) enhanced the dechlorination of PCB congeners under methanogenic and sulfate-reducing conditions but delayed the dechlorination of PCB congeners under nitrate-reducing conditions. PCB congener dechlorination also was delayed by the addition of various polycyclic aromatic hydrocarbons (PAHs) under three reducing conditions and by surfactants, such as brij30, triton SN70, and triton N101. The results suggest that methanogen, sulfate-reducing bacteria, and nitrate-reducing bacteria all are involved in the dechlorination of PCB congeners.     

Biodegradation of nonylphenol in river sediment

We investigated the biodegradation of nonylphenol monoethoxylate (NP1EO) and nonylphenol (NP) by aerobic microbes in sediment samples collected at four sites along the Erren River in southern Taiwan. Aerobic degradation rate constants (k1) and half-lives (t1/2) for NP (2 microg g(-1)) ranged from 0.007 to 0.051 day(-1) and 13.6 to 99.0 days, respectively; for NP1EO (2 microg g(-1)) the ranges were 0.006 to 0.010 day(-1) and 69.3 to 115.5 days. Aerobic degradation rates for NP and NP1EO were enhanced by shaking and increased temperature, and delayed by the addition of Pb, Cd, Cu, Zn, phthalic acid esters (PAEs), and NaCl, as well as by reduced levels of ammonium, phosphate, and sulfate. Of the microorganism strains isolated from the sediment samples, we found that strain JC1 (identified as Pseudomonas sp.) expressed the best biodegrading ability. Also noted was the presence of 4'-amino-acetophenone, an intermediate product resulting from the aerobic degradation of NP by Pseudomonas sp.     

Biodegradation of phenanthrene in river sediment

The aerobic biodegradation potential of phenanthrene (a polycyclic aromatic hydrocarbon [PAH]) in river sediment was investigated in the laboratory. Biodegradation rate constants (k1) and half-lives (t1/2) for phenanthrene (5 microg/g) in sediment samples collected at five sites along the Keelung River in densely populated northern Taiwan ranged from 0.12 to 1.13 l/day and 0.61 to 5.78 day, respectively. Higher biodegradation rate constants were noted in the absence of sediment. Two of the sediment samples were capable of biodegrading phenanthrene at initial concentrations 5-100 microg/g; lower biodegradation rates occurred at higher concentrations. Optimal biodegradation conditions were determined as 30 degreesC and pH 7.0. Biodegradation was not significantly influenced by the addition of such carbon sources as acetate, pyruvate, and yeast extract, but was significantly influenced by the addition of ammonium, sulfate, and phosphate. Results show that anthracene, fluorene, and pyrene biodegradation was enhanced by the presence of phenanthrene, but that phenanthrene treatment did not induce benzo[a]pyrene biodegradation during a 12-day incubation period.     

Comparative study of extraction methods for the determination of PAHs from contaminated soils and sediments

The following four methods were compared on the extraction efficiency of 16 EPA (US Environmental Protection Agency) polycyclic aromatic hydrocarbons (PAHs): German method of the Verband Deutscher Landwirtschaftlicher Untersuchungs und Forschungsanstalten (VDLUFA), two methods of the International Organization for Standardization using shaking (ISO A) and Soxhlet extraction (ISO B) and an ultrasonic method. Recovery rates of 16 PAHs were determined in two soils. Extraction efficiency was evaluated in five soils and three sediments. Effect of drying soils and sediments on extraction efficiency was tested using the VDLUFA and the ultrasonic methods. Our study shows that the number of aromatic rings, rather than extraction procedures, significantly influenced recovery rates of individual PAHs. No significant differences in extraction efficiency of the four methods were observed for less polluted samples. For highly polluted soils, extraction efficiency decreased in the following order: VDLUFA method > ISO A > ultrasonic method > ISO B. Influence of soil moisture on extraction efficiency depended to some extent on both solvent used and content of PAHs in samples. A mixture of dichloromethane/acetone (5:1) is recommended for PAH extraction from moist samples when the ultrasonic method is used.     

活性污泥总DNA提取方法的比较

从处理某制药废水的MBR反应器中采集活性污泥,评价不同DNA提取方法对其总DNA提取效率的影响。DNA提取分细胞裂解和DNA纯化2步,对细胞裂解比较了珠磨匀浆法、反复冻融法、十二烷基磺酸钠（sodium dodecyl sulfate,SDS）裂解法等7种方法;对DNA纯化比较了酚/氯仿纯化法和胶回收纯化法。结果表明,SDS裂解法、酚氯仿纯化法最优。通过条件优化实验,确定SDS裂解酚/氯仿纯化法在污泥量1.1 g,10 000 r/min离心5 min的操作条件下,获得的DNA产量（10 774 μg/g泥重）和纯度（OD₂₆₀∶OD₂₈₀=1.84）等综合指标最好。     

河流重金属污染底泥的稳定化实验研究

以实现河道疏浚底泥中富含的重金属Cu、Zn、Ni的稳定化为目标,选用EDTA、DTCR、Na2S、Na2S2O3、膨润土、水泥及自主研发的特殊胶凝材料作为稳定剂,对底泥中的重金属进行固化稳定,通过分析稳定后重金属浸出液浓度及赋存形态变化,探讨各药剂稳定效果,并寻求稳定剂的最佳投加量.研究表明,7种稳定剂对3种重金属的综合稳定化效果由好到差依次为:水泥> 胶凝材料> 膨润土> DTCR> 硫化钠> 硫代硫酸钠> EDTA;对Cu、Zn、Ni单种重金属而言,稳定效果最好的药剂分别为膨润土、胶凝材料及DTCR,而EDTA则使重金属浸出液浓度升高.除EDTA外,其他药剂对重金属Cu和Zn的稳定效果要明显优于Ni.根据稳定后重金属赋存形态变化结果,EDTA可使重金属可交换态比例升高,而膨润土对重金属4种形态分布基本无影响,其他药剂使重金属可交换态比例降低.     

PAHs biodegradation in intertidal surface sediment by indigenous microorganisms

In this study, the 30-day aerobic microorganism-mediated biodegradation of polycyclic aromatic hydrocarbons (PAHs) was investigated in four size fractions (i.e., <0.002, 0.002–0.031, 0.031–0.063 and >0.063 mm) of sand-dominated sediment S1 and mud-dominated S2 collected from intertidal zones in Bohai Bay (China). Prior to biodegradation, the total quantity of phenanthrene, fluoranthene and pyrene comprised more than 80 % of the total quantity of 16 EPA-priority PAHs in each size fraction, with the exception of 70.33 % found in the >0.063 mm fraction of sediment S1. Among the three dominant compounds, the intermediate size fraction (0.031–0.063 mm) showed higher levels of biodegradation than other size fractions in sediment S1 and S2. After pooling data from sediment S1 and S2 for joint analysis, it was observed that the biodegraded portion of the three dominant compounds showed negative correlations with both total organic carbon (TOC) and humic coverage index (HCI) in the size fractions. The observed negative correlation with TOC was in agreement with findings in many other studies, but the negative correlation with HCI had not been observed in early studies, which only investigated aged sediment/soil samples. The findings in this study indicated that the greatest bioavailability of PAHs in intertidal surface sediment may be present in sediment particles of intermediate size and mobility, and that intertidal sediment particles are less likely to experience sufficient ageing given periodical tidal disturbance. These findings have important implications for the assessment of the environmental fate of PAHs in intertidal regions.     

Aerobic degradation of tetrabromobisphenol-A by microbes in river sediment

This study investigated the aerobic degradation of tetrabromobisphenol-A (TBBPA) and changes in the microbial community in river sediment from southern Taiwan. Aerobic degradation rate constants (k₁) and half-lives (t_1/2) for TBBPA (50 μg g⁻¹) ranged from 0.053 to 0.077 d⁻¹ and 9.0 to 13.1 d, respectively. The degradation of TBBPA (50 μg g⁻¹) was enhanced by adding yeast extract (5 mg L⁻¹), sodium chloride (10 ppt), cellulose (0.96 mg L⁻¹), humic acid (0.5 g L⁻¹), brij 30 (55 μM), brij 35 (91 μM), rhamnolipid (130 mg L⁻¹), or surfactin (43 mg L⁻¹), with rhamnolipid yielding a higher TBBPA degradation than the other additives. For different toxic chemicals in the sediment, the results showed the high-to-low order of degradation rates were bisphenol-A (BPA) (50 μg g⁻¹) > nonylphenol (NP) (50 μg g⁻¹) > 4,4′-dibrominated diphenyl ether (BDE-15) (50 μg g⁻¹) > TBBPA (50 μg g⁻¹) > 2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether (BDE-209) (50 μg g⁻¹). The addition of various treatments changed the microbial community in river sediments. The results also showed that Bacillus pumilus and Rhodococcus ruber were the dominant bacteria in the process of TBBPA degradation in the river sediments.