Bacterial diversity in Fez tanneries and Morocco’s Binlamdoune River, using 16S RNA gene based fingerprinting

The response of bacteria to various carbohydrates in the deep-sea sediments and the Antarctic soils was investigated using cellulose, chitin, and olive oil. It was found that the carbohydrates significantly increased the corresponding specific ectoenzyme activity (β- glucosidase, β-N-acetylglucosaminidase, lipase) in the samples from deep-sea sediments. In the case of Antarctic soil samples, the cellulose or olive oil amendments had minor or no effect on β-glucosidase or lipase activity, except the chitin which stimulated β- N-acetylglucosaminidase production. The responses of the bacteria in the deep-sea sediment sample WP02-3 and the Antarctic soil sample CC-TY2 towards the chitin amendment were further analyzed. Chitin amendments were shown to stimulate the ectoenzyme activity in all the tested sediments and the soils. The bacterial response before and after the carbohydrates amendments were compared by denaturing gradient gel electrophoresis and quantitative competitive polymerase chain reaction. Significant changes were found in the structure and density of the bacterial community in the deep sea sediments as compared to the Antarctic soil sample, where the effects were relatively lower. There was no change in the bacterial population in both studied samples in response to carbohydrates amendments. These data indicate that the bacterial communities in the oligotrophic deep-sea sediments are more dynamic than that in the Antarctic soils as they respond to the nutrient sources efficiently by regulation of ectoenzyme activity and/or changing community structure.     

Impacts of wastewater sludge amendments in restoring nitrogen cycle in p-nitrophenol contaminated soil

The possible impacts on nitrogen-cycle in a p-nitrophenol (PNP) polluted soil and the e ectiveness of wastewater sludge amendments in restoring nitrification potential and urease activity were evaluated by an incubation study. The results indicated that PNP at 250 mg/kg soil inhibited urease activity, nitrification potential, arginine ammonification rate and heterotrophic bacteria counts to some extents. After exposure to PNP, the nitrification potential of the tested soil was dramatically reduced to zero over a period of 30 days. Based on the findings, nitrification potential was postulated as a simple biochemical indicator for PNP pollution in soils. Nitrogen-cycling processes in soils responded positively to the applications of wastewater sludges. A sludge application rate of 200 tons/ha was su cient for successful biostimulation of these nitrogen processes. The microbial activities in sludge-amended, heavy PNP-polluted soils seemed to recover after 30–45 days, indicating the e ectiveness of sludge as a useful soil amendment.     

Characterization of contamination, source and degradation of petroleum between upland and paddy fields based on geochemical characteristics and phospholipid fatty acids

To evaluate contamination caused by petroleum, surface soil samples were collected from both upland and paddy fields along the irrigation canals in the Hunpu wastewater irrigation region in northeast China. N-alkanes, terpanes, steranes, and phospholipid fatty acids (PLFA) in the surface soil samples were analyzed. The aliphatic hydrocarbon concentration was highest in the samples obtained from the upland field near an operational oil well; it was lowest at I-3P where wastewater irrigation promoted the downward movement of hydrocarbons. The Hunpu region was found contaminated by heavy petroleum from oxic lacustrine fresh water or marine deltaic source rocks. Geochemical parameters also indicated significantly heavier contamination and degradation in the upland fields compared with the paddy fields. Principal component analysis based on PLFA showed various microbial communities between petroleum contaminated upland and paddy fields. Gram-negative bacteria indicated by 15:0, 3OH 12:0, and 16:1(9) were significantly higher in the paddy fields, whereas Gram-positive bacteria indicated by i16:0 and 18:1(9)c were significantly higher in the upland fields (p < 0.05). These PLFAs were related to petroleum contamination. Poly-unsaturated PLFA (18:2ωup6, 9; indicative of hydrocarbon-degrading bacteria and fungi) was also significantly elevated in the upland fields. This paper recommends more sensitive indicators of contamination and degradation of petroleum in soil. The results also provide guidelines on soil pollution control and remediation in the Hunpu region and other similar regions.     

Biochemical and microbial soil functioning after application of the insecticide imidacloprid

Imidacloprid is one of the most commonly used insecticides in agricultural practice, and its application poses a potential risk for soil microorganisms. The objective of this study was to assess whether changes in the structure of the soil microbial community after imidacloprid application at the field rate(FR, 1 mg/kg soil) and 10 times the FR(10 × FR, 10 mg/kg soil)may also have an impact on biochemical and microbial soil functioning. The obtained data showed a negative effect by imidacloprid applied at the FR dosage for substrate-induced respiration(SIR), the number of total bacteria, dehydrogenase(DHA), both phosphatases(PHOS-H and PHOS-OH), and urease(URE) at the beginning of the experiment. In 10 × FR treated soil, decreased activity of SIR, DHA, PHOS-OH and PHOS-H was observed over the experimental period. Nitrifying and N2-fixing bacteria were the most sensitive to imidacloprid. The concentration of NO3-decreased in both imidacloprid-treated soils,whereas the concentration of NH4+in soil with 10 × FR was higher than in the control.Analysis of the bacterial growth strategy revealed that imidacloprid affected the r- or K-type bacterial classes as indicated also by the decreased eco-physiological(EP) index.Imidacloprid affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time for growth. Principal component analysis showed that imidacloprid application significantly shifted the measured parameters, and the application of imidacloprid may pose a potential risk to the biochemical and microbial activity of soils.     

Bacterial diversity in soils around a lead and zinc mine

Five samples of soil collected from a lead and zinc mine were used to assess the effect of combined contamination of heavy metals on soil bacterial communities using a polyphasic approach including characterization of isolates by culture method, community level catabolic profiling in BIOLOG GN microplates, and genetic community fingerprinting by denaturing gradient gel electrophoresis of 16S rDNA fragments amplified by PCR from community DNA （PCR-DGGE）. The structure of the bacterial community was affected to a certain extent by heavy metals. The PCR-DGGE analysis of 16S rRNA genes showed that there were significant differences in the structure of the microbial community among the soil samples, which were related to the contamination levels. The number of bacteria and the number of denaturing gradient gel electrophoresis （DGGE） bands in the soils increased with increasing distance from the lead and zinc mine tailing, whereas the concentration of lead （Pb） and cadmium （Cd） was decreased. Heavily polluted soils could be characterized by a community that differs from those of lightly polluted soils in richness and structure of dominating bacterial populations. The clustering analysis of the DGGE profiles showed that the bacteria in all the five samples of soil belonged to three clusters. The data from the BIOLOG analysis also showed the same result. This study showed that heavy metal contamination decreased both the biomass and diversity of the bacterial community in soil.     

Biochemical and microbial soil functioning after application of the insecticide imidacloprid

Imidacloprid is one of the most commonly used insecticides in agricultural practice, and its application poses a potential risk for soil microorganisms. The objective of this study was to assess whether changes in the structure of the soil microbial community after imidacloprid application at the field rate (FR, 1 mg/kg soil) and 10 times the FR (10× FR, 10 mg/kg soil) may also have an impact on biochemical and microbial soil functioning. The obtained data showed a negative effect by imidacloprid applied at the FR dosage for substrate-induced respiration (SIR), the number of total bacteria, dehydrogenase (DHA), both phosphatases (PHOS-H and PHOS-OH), and urease (URE) at the beginning of the experiment. In 10× FR treated soil, decreased activity of SIR, DHA, PHOS-OH and PHOS-H was observed over the experimental period. Nitrifying and N₂-fixing bacteria were the most sensitive to imidacloprid. The concentration of NO₃^- decreased in both imidacloprid-treated soils, whereas the concentration of NH₄⁺ in soil with 10× FR was higher than in the control. Analysis of the bacterial growth strategy revealed that imidacloprid affected the r- or K-type bacterial classes as indicated also by the decreased eco-physiological (EP) index. Imidacloprid affected the physiological state of culturable bacteria and caused a reduction in the rate of colony formation as well as a prolonged time for growth. Principal component analysis showed that imidacloprid application significantly shifted the measured parameters, and the application of imidacloprid may pose a potential risk to the biochemical and microbial activity of soils.     

Microbial activity related to N cycling in the rhizosphere of maize stressed by heavy metals

A greenhouse experiment was carried out to compare differences in potential activities of ammonification, nitrification and denitrification in rhizosphere and bulk soil in a heavy-metal-stressed system. Exchangeable fractions of Cd, Cu and Cr were all higher in the rhizosphere of maize than in bulk soil. Results showed that the mineralization of N in soil was stimulated by low concentration of Cd.Addition of Cd at low levels stimulated the ammonifying and nitrifying activity in soil, while inhibitory influences were shown at high levels.Nitrifying bacteria was proved to be the most sensitive one, whilst the effect on denitrifying bacteria was very limited. Comparing Cd, Cu and Cr(Ⅵ) at 20 mg/kg soil, Cd was the most effective inhibitor of ammonification and denitrification, while Cr(Ⅵ) had the strongest influence on nitrifying activity. Root exudates played important roles on the different exchangeable metal fractions and bacterial activities between rhizosphere and non-rhizosphere. Nitrate was the main form of mineral N in soil, as well as the main form of N absorbed by plants, but the formation and relative absorption of ammonium were promoted in response to high Cd exposure.     

Diversity surveys of soil bacterial community by cultivation--based methods and molecular fingerprinting techniques

By combining the cultivation methods with molecular fingerprinting techniques, the diversity surveys of soil bacterial community in 13 areas of China were carded out. The cultivable heterotrophic diversity was investigated by colony morphology on solid LB medium. Genetic diversity was measured as bands on denaturing gradient gel electrophoresis(DGGE) by the extraction and purification of the total soil DNA, and amplification of bacterial 16S rDNA fragments by polymerase chain reaction(PCR). The Shannon-Wiener indices of diversity(H), richness(S) and evenness(EH) were employed to estimate the diversity of soil bacterial community. The results showed that there was an obvious diversification existed in soil from the different areas. However, the genetic diverslty estimated by PCR-DGGE can provide more comprehensive information on bacterial community than the cultivation-based methods. Therefore, it is suggested to combine the traditional methods with genetic fingerprinting techniques to survey and estimate soil bacterial diversity.     

Soil enzymatic activities and microbial community structure with di erent application rates of Cd and Pb

This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd andor Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd andor Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction-denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.     

Responses of soil ammonia oxidizers to a short-term severe mercury stress

The responses of soil ammonia-oxidizing archaea(AOA) and ammonia-oxidizing bacteria(AOB) to mercury(Hg) stress were investigated through a short-term incubation experiment.Treated with four different concentrations of Hg(CK,Hg25,Hg50,and Hg100,denoting 0,25,50,and 100 mg Hg/kg dry soil,respectively),samples were harvested after 3,7,and 28 day incubation.Results showed that the soil potential nitrification rate(PNR) was significantly inhibited by Hg stress during the incubation.However,lower abundances of AOA(the highest in CK: 9.20 × 10~7 copies/g dry soil; the lowest in Hg50: 2.68 × 10~7 copies/g dry soil) and AOB(the highest in CK: 2.68 × 10~7 copies/g dry soil; the lowest in Hg50:7.49 × 10~6 copies/g dry soil) were observed only at day 28 of incubation(P 0.05).Moreover,only the community structure of soil AOB obviously shifted under Hg stress as seen through DGGE profiles,which revealed that 2–3 distinct AOB bands emerged in the Hg treatments at day 28.In summary,soil PNR might be a very useful parameter to assess acute Hg stress on soil ecosystems,and the community structure of soil AOB might be a realistic biological indicator for the assessment of heavy metal stress on soil ecosystems in the future.     

陕北石油污染土壤微生物种群变化及影响因素

采集陕北地区10个产油区县的石油污染土壤和洁净土壤,利用化学分析法和分子生物学技术对土壤理化性质和细菌群落结构进行测定,采用Pearson相关性分析和冗余分析研究石油污染对土壤理化性质、微生物种群分布和多样性的影响.结果表明,与未受污染土壤相比,石油污染土壤的NO₃^--N、总有机碳、全碳、全硫含量和碳氮比增加,pH值减小;土壤细菌群落均匀度明显降低,丰富度无明显变化.不同地区土壤受到石油污染后,一些优势细菌的相对丰度变化明显.变形菌门（Proteobacteria）和绿弯菌门（Chloroflexi）的相对丰度变化与石油烃含量呈显著正相关,Candidatus Saccharibacteria的相对丰度变化与石油烃含量呈极显著负相关.定边、靖边、子长、绥德、吴起、安塞和富县石油污染土壤中假单胞菌属（Pseudomonas）、诺卡氏菌属（Nocardioides）、节杆菌属（Arthrobacter）、不动杆菌属（Acinetobacter）、Alkanindiges、Gaiella和Gp4相对丰度显著增加;定边、靖边、子长和绥德石油污染土壤中Ohtaekwangia、假诺卡氏菌属（Pseudonocardia）、Ramlibacter、Sacchariacteria_genera_incertae_sedis相对丰度显著降低.假单胞菌属（Pseudomonas）、诺卡氏菌属（Nocardioides）、节杆菌属（Arthrobacter）和Alkanindiges为陕北地区石油污染土壤中的主要“嗜油菌”.土壤pH值、含水率、总石油烃和总有机碳等理化性质是影响陕北油田区石油污染土壤微生物种群结构的关键因素.     

棉秆还田对咸水滴灌棉田土壤酶活性和细菌群落结构多样性的影响

咸水长期灌溉会增加土壤盐分,对土壤理化性质产生不利影响,改变土壤细菌的多样性.秸秆还田可以改善土壤微环境,进而影响土壤酶活性和细菌群落结构多样性.试验设置淡水(FW,0.35 dS·m-1)和咸水(SW,8.04 dS·m-1)两种灌溉水盐度,在每个灌溉水盐度下秸秆用量分别为0 kg·hm-2和6 000 kg·hm-...     

Influence of oxytetracycline on the structure and activity of microbial community in wheat rhizosphere soil

The microbial community composition in wheat rhizosphere was analyzed by detecting colony forming units (CFUs) in agar plates. The total CFUs in rhizosphere were 1.04 109/g soil with 9.0 108/g bacteria, 1.37 108/g actinomyces and 3.6 106/g fungi. The 10 dominant bacteria were isolated from wheat rhizosphere and were grouped into genus Bacillus according to their full length 16S rRNA gene sequences. Although belonging to the same genus, the isolated strains exhibited di erent sensitivities to oxytetracycline. When a series of the rhizosphere soil was exposed under various concentrations of oxytetracycline, the microbial community structure was highly a ected with significant decline of CFUs of bacteria and actinomyces (22.2% and 31.7% at 10 mg/kg antibiotic, respectively). This inhibition was clearly enhanced with the increase exposure dosage of antibiotic and could not be eliminated during 30 d incubation. There was no obvious influence of this treatment on fungi population. Among the four soil enzymes (alkaline phosphatase, acidic phosphatase, dehydrogenase and urease), only alkaline phosphatase was sensitive to oxytetracycline exposure with 41.3% decline of the enzyme activity at 10 mg/kg antibiotic and further decrease of 64.3%–80.8% when the dosage over 30 mg/kg.     

油污土微生态环境非生物因子与微生物活性关系

油污土微生态环境的非生物因子通过改变降解优势菌的含量及活性最终影响石油烃的生物降解速率及模式.本研究通过对我国北方部分油田和石油化工区的取样分析,揭示油污土微生态环境的非生物因子与微生物活性的关系,研究影响微生物活性和石油烃降解效率的环境及污染物等非生物因子.结果表明,我国北方油田区及石油化工区土壤受到了不同程度的石油烃污染,石油烃含量最高可达34 000mg/kg干土.柱层析分析结果表明,污染物中烷烃、芳烃等轻质组分含量超过50%,有利于微生物活性的提高.油田区土壤的pH值一般在7.8以上,不利于石油烃降解微生物生长代谢;油污土的营养水平普遍较低,如可被生物利用的速效氮含量低于30mg/kg干土,速效磷含量低于10mg/kg干土,仅占土壤总氮、总磷的5%左右,远远达不到石油烃生物降解所需的营养水平(C:N:P=100:10:1).毫无疑问,调节土壤pH值,增加对营养水平等非生物因子的调控对于提高微生物活性,加快石油烃的降解有重要的意义.     

邻苯二甲酸酯复合污染对土壤微生态的影响

在实验室模拟条件下,通过测定土壤呼吸,w(PAEs)和微生物RAPD条带数,研究了邻苯二甲酸二甲酯(DMP)、邻苯二甲酸二乙酯(DEP)和邻苯二甲酸二辛酯(DOP)3种PAEs复合污染对农田土壤基础呼吸、微生物多样性的影响. 结果表明,PAEs污染初期的土壤基础呼吸被激活,但这种激活作用随着培养时间的延长而减弱,400 mg/kg组土壤基础呼吸最高;3种PAEs含量均有一定程度的下降,其中w(DEP)显著降低;试验终止时各组多样性条带的比例较预培养后有所增加(平均增加13%);不同处理组土壤微生物群落DNA序列Shannon-Weaver指数顺序为0 mg/kg>50 mg/kg>100 mg/kg>200 mg/kg>400 mg/kg. 可见,PAEs复合污染提高了土壤基础呼吸,但降低了土壤微生物多样性.      

Changes in enzymes activity, substrate utilization pattern and diversity of soil microbial communities under cadmium pollution

Heavy metal pollution has received increasing attention in recent years mainly because of the public awareness of environmental issues. In this study we have evaluated the effect of cadmium （Cd） on enzymes activity, substrate utilization pattern and diversity of microbial communities in soil spiked with 0, 20, 40, 60, 80, and 100 mg/kg Cd, during 60 d of incubation at 25℃. Enzyme activities determined at 0, 15, 30, 45, and 60 d after heavy metal application（DAA） showed marked declines for various Cd treatments, and up to 60 DAA, 100 mg/kg Cd resulted in 50.1%, 47.4%, and 39.8% decreases in soil urease, acid phosphatase and dehydrogenase activities, respectively to control. At 60 DAA, substrate utilization pattern of soil microbial communities determined by inoculating Biolog ECO plates indicated that Cd addition had markedly inhibited the functional activity of soil microbial communities and multivariate analysis of sole carbon source utilization showed significantly different utilization patterns for 80 and 100 mg/kg Cd treatments. The structural diversity of soil microbial communities assessed by PCR-DGGE method at 60 DAA, illustrated that DGGE patterns in soil simplified with increasing Cd concentration, and clustering of DGGE profiles for various Cd treatments revealed that they had more than 50% difference with that of control.     

改良剂对土壤铜镉有效性和微生物群落结构的影响

以一次性添加木炭、石灰和磷灰石稳定化修复4年后的土壤为研究对象,探讨了土壤中铜镉有效性和土壤微生物群落结构的变化.结果表明,3种改良剂的添加提高了土壤pH值,降低了土壤交换性酸和交换性铝的含量,使铜镉由活性态向非活性态和潜在活性态转化,其中磷灰石和石灰的处理效果优于木炭处理; Biolog测试发现,培养192h,木炭、石灰和磷灰石处理土壤AWCD值(0.61、0.76和0.70)分别是对照的1.33、1.65和1.52倍;且Shannon和McIntosh指数均较对照有所提高,表现为:石灰 > 磷灰石 > 木炭,表明改良剂处理后增加了土壤微生物对碳源的利用能力,提高了其功能多样性.PCR-DGGE分析结果显示,改良剂处理后土壤细菌优势群的数量显著增加,木炭、石灰和磷灰石处理Shannon指数分别比对照提高了0.22、0.39和0.24.相关性分析表明,土壤酸度和重金属有效性是影响稳定化修复重金属污染土壤细菌结构多样性差异的主要因素.     

典型气田土壤铁还原活性与微生物群落关系研究

由微生物驱动的土壤铁还原过程在铁的生物地球化学循环中起到重要作用,该过程还可与土壤重金属的转化及石油烃类有机污染物的降解等过程相偶联.油气田土壤常具有潜在有机污染物风险,本研究以重庆涪陵页岩气田的土壤（潜在烃类有机物污染风险）为对象,测定土壤铁还原活性（Iron Reducing Potential,IRP）,并利用Illumina Miseq测序解析其中的铁还原微生物类群,进而探讨IRP、土壤基本性质及微生物群落之间的关系.结果表明,该区域土壤铁还原菌群的优势菌门为厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）和拟杆菌门（Bacteroidetes）.与低IRP样品相比,高IRP样品中Pseudomonas、norank Peptococcaceae及Lentimicrobium等菌属具有较高的相对丰度.基于各样品OTU （Operational Taxonomic Unit）组成的PCoA （Principal Co-ordinates Analysis）分析表明,高、中及低IRP样品中铁还原菌群落结构差异显著（R²=0.25,p<0.01）,且一些分别属于Acetoanaerobium、Proteiniphilum、Petrimonas、Tessaracoccus及Exiguobacterium菌属中的OTUs在高IRP样品中显著上调.结构方程模型表明,铁还原微生物的群落结构是直接决定土壤IRP的主要因子,土壤氨氮及有效磷均可通过影响微生物群落结构来间接影响IRP,且氨氮还可通过直接影响有效磷来间接影响土壤IRP.本研究揭示了影响典型页岩气田土壤铁还原活性的关键因子及微生物机制,为进一步深入研究铁还原条件下土壤有机污染物去除的微生物机制打下基础.     

典型油田区油污土壤微生物群落区域性分布研究

石油污染改变土壤微生态环境,驱动了土壤微生物群落结构的演替与进化.为了深入探究油田区油污土壤中微生物群落分布特征,揭示区域性的土壤微生物群落结构成因,采用Miseq平台的16S rDNA扩增子测序技术分析了辽河油田和大庆油田区6组共计18个土壤样品的微生物群落多样性及结构组成,并结合土壤环境因子指标剖析了群落结构成因,进而预测了具有石油代谢能力的功能菌属.结果表明,石油含量随着距井口距离增加而减少,石油的空间分布特征是影响微生物群落结构变化的关键因子,6组土壤样品的OTU分属于49门、131纲、169目、328科和564属,微生物种群多样性随着污油浓度的增加而减小;两油田共有5个相同优势菌门,2种优势菌属;辽河油田区独特优势菌门为Saccharibacteria门,优势菌属为微枝形杆菌属（Microvirga）、分支杆菌属（Mycobacterium）和Defluviicoccus属;大庆油田独特优势菌门为拟杆菌门（Bacteroidetes）,独特优势菌属包括盐单胞菌属（Halomonas）、食烷菌属（Alcanivorax）和海杆菌属（Marinobacter）等.冗余分析（RDA）结果表明污油组成是微生物群落差异性分布的决定性因素,胶质的高含量与强毒性诱导辽河油田区微生物群落获得较强的胁迫抗性;同时区域生态环境背景差异也是影响微生物群落整体胁迫抗性的重要因子.结合PICRUSt分析预测,共发现2种辽河油田区和5种大庆油田区石油功能降解优势菌属,为石油降解功能菌剂的种质资源的高效开发提供目标菌株.     

Effects of humic acid on phytodegradation of petroleum hydrocarbons in soil simultaneously contaminated with heavy metals

Effects of humic acid on phytoremediation of petroleum hydrocarbons in soil simultaneously contaminated with heavy metals were evaluated.