RAPD marker and substrate utilization pattern applied to study microbial community diversity in the soil affected by agricultural chemicals

Present analyses of random amplified polymorphic DNA (RAPD) and Biolog GN substrate utilization pattern are combined to further study the diversity of microbial communities in four soils affected by agricultural chemicals. The results showed that the four soil microbial communities were apparently distinguishable in the diversity at RAPD level in terms of the richness and modified richness in the summer, which supports our previous report using the same soils in winter. A significant difference for the average well color development (AWCD) at 72 h incubation was found among the soils in winter using Biolog GN substrate utilization pattern, but this difference was not found among the soils in summer. However, Shannon-Weaver indices for microbial communities in the summer soils polluted by agricultural chemicals were significantly higher than those in winter at metabolic level; in contrast, no significant difference existed between the two seasons for microbial communities in the soil without chemical pollution. Present results suggest that the combined approach using RAPD and substrate utilization pattern could be used to effectively quantify microbial community diversity and its changes among the seasons in the soils affected by agricultural chemicals, simultaneously at molecular and physiological levels.     

RAPD Marker and Substrate Utilization Pattern Applied to Study Microbial Community Diversity in the Soil Affected by Agricultural Chemicals

Abstract

Present analyses of random amplified polymorphic DNA (RAPD) and Biolog GN substrate utilization pattern are combined to further study the diversity of microbial communities in four soils affected by agricultural chemicals. The results showed that the four soil microbial communities were apparently distinguishable in the diversity at RAPD level in terms of the richness and modified richness in the summer, which supports our previous report using the same soils in winter. A significant difference for the average well color development (AWCD) at 72 h incubation was found among the soils in winter using Biolog GN substrate utilization pattern, but this difference was not found among the soils in summer. However, Shannon-Weaver indices for microbial communities in the summer soils polluted by agricultural chemicals were significantly higher than those in winter at metabolic level; in contrast, no significant difference existed between the two seasons for microbial communities in the soil without chemical pollution. Present results suggest that the combined approach using RAPD and substrate utilization pattern could be used to effectively quantify microbial community diversity and its changes among the seasons in the soils affected by agricultural chemicals, simultaneously at molecular and physiological levels.     

Ni2＋对活性污泥活性及群落多样性的影响

通过检测活性污泥的电子传递体系活性以及生物多样性,研究Ni2＋对活性污泥微生物活性及群落多样性的影响。结果表明：与对照系统相比,5mg／L的Ni2＋对2,3,5-lriphenylteItrazoliumchloride（TTC．ETS）活性未产生显著的影响;但当Ni2＋的浓度进一步增大到10、20和40mg／L后,其对序批式反应器内活性污泥TTC—ETS活性的抑制率分别达到（36．794-11．14）％、（55．88±13．90）％和（70．97±6．78）％。低浓度Ni2＋．能增强活性污泥微生物对碳源的利用,但高于10mg／L的Ni2＋则显著抑制了活性污泥微生物对碳源的利用。各个SBR系统中微生物群落最常见的物种相近,物种丰富度和均一性则均有所不同,其中群落物种丰富度随着Ni2＋浓度的增加而逐渐减小。TTC—ETS活性、平均每孔颜色变化率、Shan—liOn指数和Simpson指数,与Ni2＋的胁迫浓度之间的显著相关性表明,它们均可有效地表征Ni2＋胁迫对活性污泥微生物活性及群落多样性的影响程度。     

Functional biodiversity of microbial communities in aerobic selector slaughterhouse wastewater.

The biodiversity of the microbial communities of an aerobic selector and a conventional system treating slaughterhouse wastewater were evaluated using the Biolog technique (Biolog Inc., Hayward, California). Principal components analysis of patterns and level of microbial activity indicate that microbial communities statistically differentiate between the selector and conventional system. Biolog data indicated that there was seasonal variation in the structure and function of the microbial community in conventional samples, which may be a useful indicator of wastewater community disturbance and unsteadiness, while, for the selector system, there were no recognizable statistical differences between winter and summer data communities, Biodiversity indices indicated that the selector system maintained a high functional diversity (Shannon-Weaver diversity index [H'] from 3.8 to 4.6) than the conventional system (H' from 1.8 to 2.8). The high values in the selector system were a reflection of the fact that most of the carbon sources were used, contributing to the very high Shannon indices. In addition, the high substrate equitability values (J) obtained for the selector samples indicated that the microbial communities between the summer and winter samples used the carbon sources in the same proportion. In contrast, differences in the equitability of the microbial communities within the conventional system were observed. This indicated a pattern representing unevenness.     

Metabolic diversity of the heterotrophic microorganisms and potential link to pollution of the Rouge River

The heterotrophic microbial communities of the Rouge River were tracked using Biolog Ecoplates to understand the metabolic diversity at different temporal and spatial scales, and potential link to river pollution. Site less impacted by anthrophogenic sources (site 1), showed markedly lower metabolic diversity. The only substrates that were utilized in the water samples were carbohydrates. Sites more impacted by anthrophogenic sources (sites 8 and 9) showed higher metabolic diversity. Higher functional diversity was linked to the physico-chemical and biological properties of the water samples (i.e. higher concentrations of DO, DOC, chlorophyll, and bacterial density). Biolog analysis was found to be useful in differentiating metabolic diversity between microbial communities; in determining factors that most influence the separation of communities; and in identifying which substrates were most utilized by the communities. It can also be used as an effective ecological indicator of changes in river function attributable to urbanization and pollution.     

Inocula from activated sludge for ready biodegradability testing: homogenization by preconditioning

The use of activated sludge as inoculum source in ready biodegradability tests (RBT) suffers from several drawbacks related to the heterogeneity of these communities. In this work, the ability of a 7-day aeration period in a mineral medium to homogenize the characteristics of various activated sludges, as suggested by some RBT, was studied. The biodegradation potential of three activated sludge supernatants obtained from different wastewater treatment plants was assessed in terms of cultivable cell density, dehydrogenasic activity and a profile of hydrolytic enzymes. After the preconditioning, the homogenization of these characteristics in the supernatants was observed, as well as a decrease. When preconditioned inocula were used in acetate RBT, the biodegradation kinetics were homogenized. However, some preconditioned supernatants lost their ability to degrade an easily-assimilable xenobiotic compound (aniline) during the observation period, showing the effect of inoculum preconditioning on the behavior of complex bacterial communities, specialist populations (e.g. aniline degraders) being more sensitive than generalist populations (e.g. acetate degraders). These results show that preconditioning cannot be an optional inoculum pretreatment in RBT, and emphasize the importance of further studies focusing on inoculum homogenization.     

Exploring the potential of applying proteomics for tracking bisphenol A and nonylphenol degradation in activated sludge

A significant percentage of bisphenol A and nonylphenol removal in municipal wastewater treatment plants relies on biodegradation. Nonetheless, incomplete information is available concerning their degradation pathways performed by microbial communities in activated sludge systems. Hydroquinone dioxygenase (HQDO) is a specific degradation marker enzyme, involved in bisphenol A and nonylphenol biodegradation, and it can be produced by axenic cultures of the bacterium Sphingomonas sp. strain TTNP3. Proteomics, a technique based on the analysis of microbial community proteins, was applied to this strain. The bacterium proteome map was obtained and a HQDO subunit was successfully identified. Additionally, the reliability of the applied proteomics protocol was evaluated in activated sludge samples. Proteins belonging to Sphingomonas were searched at decreasing biomass ratios, i.e. serially diluting the bacterium in activated sludge. The protein patterns were compared and Sphingomonas proteins were discriminated against the ones from sludge itself on 2D-gels. The detection limit of the applied protocol was defined as 10^?3 g TTNP3 g^?1 total suspended solids (TSSs). The results proved that proteomics can be a promising methodology to assess the presence of specific enzymes in activated sludge samples, however improvements of its sensitivity are still needed.     

污泥好氧颗粒化过程中微生物群落结构的演变与分析

为了揭示颗粒污泥形成过程中微生物群落结构多样性的演变过程,以人工配水为进水,在SBR中采用厌氧/好氧循环的手段成功培育出具有聚磷特性的颗粒污泥,利用基于16S rDNA的PCR-DGGE技术获得了微生物群落的DNA特征指纹图谱,对条带进行了统计分析和切胶测序,并建立了系统发育树。结果表明,污泥沉降性能的改善要先于颗粒污...     

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。     

A+OSA污泥减量工艺的微生态特性

采用16S rDNA序列与PCR-DGGE(polymerase chain reaction-denaturing gradient gel electrophoresis)分析技术相结合的方法,研究了A+OSA(the anoxic+oxic-settling-anaerobic)污泥减量工艺在不同工况下的减量效果及其微生态特性。结果显示,在自然条件下,A+OSA工艺可有效减少剩余污泥27%左右。分子生物研究表明,解耦联池的插入可以明显改变系统微生物的群落结构,且随着解耦联池水力停留时间的延长,系统中部分微生物被"淘洗",微生物丰富度和多样性指数均有所降低。相似性分析表明,参照系统和A+OSA工艺分属于2个不同的集群,但在A+OSA工艺内部各反应池样品间具有较高的相似性,且各反应池在HRT为5.16 h和7.14 h时,表现为显著相似。通过上述研究可为该工艺优化及调控提供理论指导。     

Spatial variability in the degradation rate of isoproturon in soil

Thirty samples of soil were taken at 50-m intersections on a grid pattern over an area of 250 x 200 m within a single field with nominally uniform soil characteristics. Incubations of isoproturon (3-(4-isopropylphenyl)-1,1-dimethylurea) under standard conditions (15 degrees C; -33 kPa soil water potential) indicated considerable variation in degradation rate of the herbicide, with the time to 50% loss (DT50) varying from 6.5 to 30 days. The kinetics of degradation also varied between the sub-samples of soil. In many of them, there was an exponential decline in isoproturon residues; in others, exponential loss was followed by more rapid rates of decline; in a few soil samples, rapid rates of loss began shortly after the start of the incubations. In more detailed studies with soils from a smaller number of sub-sites (20), measurements were again made of isoproturon degradation rate, and the soils were analysed for organic matter content, pH, and nutrient status (N, P, K). Measurements were also made of isoproturon adsorption by the soils and of soil microbial biomass. Patterns of microbial metabolism were assessed using 95 substrates in Biolog GN plates. Soils showing rapid biodegradation were generally of higher pH and contained more available potassium than those showing slower degradation rates. They also had a larger microbial biomass and greater microbial metabolic diversity as determined by substrate utilisation on Biolog GN plates. The implications of the results for the efficacy and environmental behaviour of isoproturon are discussed.     

Effect of activated sludge properties and membrane operation conditions on fouling characteristics in membrane bioreactors

Biofouling control is considered to be a major challenge in operating membrane bioreactors (MBRs) for the treatment of wastewater. This study examined the impact of biological, chemical, and physical properties of activated sludge on membrane filtration performance in laboratory-scale MBRs. Sludges with different microbial communities were produced using pseudo-continuous stirred-tank reactors and pseudo-plug flow reactors treating a synthetic paper mill wastewater. Various filtration resistances were used to investigate membrane fouling characteristics, and molecular biology tools targeting 16S ribosomal DNA gene sequences were used to identify predominant bacterial populations in the sludges or attached to the fouled membranes. Filtration experiments using axenic cultures of Escherichia coli, Acinetobacter calcoaceticus, and Gordonia amarae were also performed to better understand the initiation and development of biofouling. The results showed that the tendency of membranes to biofoul depended upon membrane operating conditions as well as the properties of the activated sludge in the MBR systems. Specific bacterial populations, which were not dominant in the activated sludges, were selectively accumulated on the membrane surface leading to the development of irreversible biofouling.     

生物降解实验中的接种物标准化研究

针对降解实验中接种物采集困难、活性差异大、实验可重复性及结果可比性差的问题展开接种物标准化研究。对多来源的接种物在实验室可控条件下进行驯养,驯养后生长状态良好的活性污泥分别固定于灭菌麸皮和藻酸钙凝胶等2种微生物载体上,形成2种固定化接种物(分别简称为Bran及Ball)。对上述固定化接种物进行微生物活性、适应性、降解能力以及稳定性研究:通过2种固定化接种物与商品化接种物Polyseed对葡萄糖-谷氨酸(GGA)五日生化需氧量(BOD5)的结果与活菌细胞数的关系,确认3种接种物在降解实验中的用量可控制在相同数量级;比较不同批次及保存时间的2种固定化接种物、相同批次不同保存时间的Polyseed以及污水处理厂曝气池污泥为接种物对苯胺及二甘醇的快速生物降解实验(OECD301F)结果,发现Ball及Bran较Polyseed及新鲜采集活性污泥的微生物适应性及降解能力更佳。此外,在较长保存期内2种固定化接种物活性相对稳定。2种固定化接种物初具成为标准化接种物的必要条件。     

Microbial metabolic activities in soils of old-field communities following eleven years of nutrient enrichment

Effects of 11 years (1978-1988) of nutrient enrichment (fertilizer or sludge) on microbial metabolic activity in soil samples collected from contrasting types of old-field communities were studied during September 1989. During the 1989 growing season, subplots were manipulated by tilling and/or liming to evaluate mechanisms of ecosystem recovery or were left undisturbed. Metabolic activities of soil microorganisms were determined by measuring dehydrogenase activity within soil samples collected from these subplots. The amounts of 2,3,5-triphenyltetrazolium formazan formed during incubation by the reduction of 2,3,5-triphenyltetrazolium chloride were used to evaluate dehydrogenase activity. Plots that had received long-term applications of sludge or fertilizer had significantly lower rates of microbial activity (P<0.05) than did control plots. Fertilizer and sludge plots treated with lime had significantly higher microbial metabolic activity (P<0.05) than those not receiving lime. Whereas liming stimulated microbial activity to near control levels, tilling had no significant treatment effect.     

Effect of textile auxiliaries on the biodegradation of dyehouse effluent in activated sludge

The textile industry is confronted with serious environmental problems associated with its immense wastewater discharge, substantial pollution load, extremely high salinity, and alkaline, heavily coloured effluent. Particular sources of recalcitrance and toxicity in dyehouse effluent are two frequently used textile auxiliaries; i.e. dye carriers and biocidal finishing agents. The present experimental work reports the observation of scientific and practical significance related with the effect of two commercially important textile dye carriers and two biocidal finishing agents on biological activated sludge treatment at a textile preparation, dyeing and finishing plant in Istanbul. Respirometric measurements of the dyehouse effluent spiked with the selected textile chemicals were carried out for the assessment of the "readily biodegradable COD fraction" of the wastewater. The respirometric data obtained to visualize the effect of the selected textile auxiliaries on biomass activity was evaluated by an adopted activated sludge model. Results have indicated that the tested biocides did not exert any significant inhibitory effect on the treatment performance of the activated sludge reactor at the concentrations usually encountered in the final, total dyehouse effluent. The situation with the dye carriers was inherently different; one dye carrier appeared to be highly toxic and caused serious inhibition of the microbial respirometric activity, whereas the other dye carrier, also known as the more ecological alternative, i.e. the "Eco-Carrier", appeared to be biodegradable. Finally, the respirometric profile obtained for the Eco-Carrier was described by a simplified respirometric model.     

Evaluation of TCDD biodegradability under different redox conditions

Polychlorinated dibenzo-p-dioxins have been generated as unwanted by-products in many industrial processes. Although their widespread distribution in different environmental compartments has been recognized, little is known about their fate in the ultimate environment sinks. The highly stable dioxin isomer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been called the most toxic compound known to man. In this laboratory microcosm study, TCDD bioavailability was evaluated under five reduction/oxidation (redox) conditions including aerobic biodegradation, aerobic cometabolism, methanogenesis, iron reduction, and reductive dechlorination. Activated sludge and aquifer sediments from a TCDD and a pentachlorophenol (PCP) contaminated site were used as the inocula. Acetate, sludge cake, and cane molasses were used as the primary substrates (carbon sources) in cometabolism and reductive dechlorination microcosms. After a 90-day incubation period, microcosms constructed under reductive dechlorination conditions were the only treatment showing promising remediation results. The highest TCDD degradation rate [up to 86% of TCDD removal (with an initial concentration of 96 microg/kg of soil)] was observed in the microcosms with anaerobic activated sludge as the microbial inocula and sludge cakes as the primary substrates. Except for reductive dechlorination microcosms, no significant TCDD removal was observed in the microcosms prepared under other conditions. Thus, application of an effective primary substrate to enhance the reductive dechlorination process is a feasible method for TCDD bioremediation. Bioremediation expense can be significantly reduced by the supplement of some less expensive alternative substrates (e.g., sludge cakes, cane molasses). Results would be useful in designing a scale-up in situ or on-site bioremediation system such as bioslurry reactor for field application.     

Standard inocula preparations reduce the bacterial diversity and reliability of regulatory biodegradation tests

OECD ready biodegradability tests have been central to understanding the biodegradation of chemicals from a regulatory perspective for many decades. They are not fit for contemporary prioritisation of chemicals based on persistence, however, due to the low concentration of inocula used, short duration and high variability between tests. Two OECD standard inoculum pretreatment methods (settlement and filtration) were investigated to observe their effect on the probability of biodegradation and associated changes in bacterial community structure and diversity of inocula sourced from the activated sludge process of wastewater treatment plants. Both settlement and filtration were shown to dramatically and significantly reduce the probability and increase the variability of biodegradation of 4-nitrophenol compared to the use of unprocessed inocula. These differences were associated with a significant hundred-fold reduction in cell numbers and solids content and a significant shift in bacterial community structure that was sometimes accompanied by significant reductions in detectable operational taxonomic unit richness and evenness. The natural variation (between different environments) and variation due to differential selection of bacterial communities (by different pretreatment methods) is offered as an explanation for the historical high variability in standard OECD ready biodegradability tests.     

Effect of green manure amendment on herbicide pendimethalin on soil

Manure amendment in agricultural practice can have a large effect on herbicide dissipation because the period of manure plowing is close to the period of herbicide application. In addition, manure amendment is among the frequently encountered options in ameliorating pesticide pollution. In this research, the dissipation of the herbicide pendimethalin was examined after amendment with two common green manures, Lupinus luteus (L) or Cosmos bipinnatus (C), for 110 days in pH 5.2 and 7.7 soils (Sankengtzu [Sk] and Erhlin [Eh] soil, respectively). The microbial activity and ecology changes were examined by using Biolog EcoPlate and denaturing gradient gel electrophoresis (DGGE). In Sk soil, the half-lives of pendimethalin with L, C, and blank treatment were 49.0, 54.9, and 62.2 days, respectively, whereas that in Eh soil they were 46.3, 52.6, and 34.8 days, respectively. Pendimethalin dissipated quickly in more neutral soil (Eh soil), but the addition of manure can only increase the dissipation rate in acidic soil (Sk soil), indicating that the amendment of manures exerted different effect in pendimethalin dissipation rates in different pH soils. The application of pendimethalin and/or manure altered the microbial community activity after 24 h of incubation. After 110 days, the microbial community activities in green manure–amended soil were more similar to that with blank than pendimethalin treatment in both types of soils. In comparison with treatment C, microbial communities were more similar between treatment L and blank, indicating the superior effect over pendimethalin on microbial communities when applying Lupinus luteus. The research showed that the application of herbicide pendimethalin changed soil microbial community, and the amendment of manures exerted different effect in pendimethalin dissipation rates in different pH soils. It is assumed that the change in dissipation rates was originated from the microbial community change after different manure amendment.     

Stoichiometric and molecular evidence for the enrichment of anaerobic ammonium oxidizing bacteria from wastewater treatment plant sludge samples

Anammox enrichments were readily developed from seven municipal wastewater treatment plants (WWTPs) sludge, but not with methanogenic granular sludge from two agro-industrial WWTPs. Only 50 d was required for the first evidence of anammox activity from a return activated sludge obtained from a WWTP operated for nutrient removal. The molar ratios of nitrite and ammonium consumption of approximately 1.32 as well as nitrate and dinitrogen gas product ratios of approximately 0.095 provided evidence of the anammox reaction. The presence of anammox was confirmed by polymerase chain reaction (PCR) using primer sets (PLA46F and AMX820R) specific for anammox bacteria. The 16S rRNA gene fragment of anammox bacteria was detected in seven enrichment cultures (ECs) with demonstrated anammox activity but not in the original inocula from which the ECs were derived and also not in the two methanogenic sludge samples, which indicates the PCR predicted the anammox activity. Two genera, Brocadia and Kuenenia, were successfully identified as the Planctomycetes occurring in the clone libraries of successful anammox enrichments. Brocadia dominated in cultures that were respiked extensively; whereas Kuenenia predominated in cultures that were less aggressively respiked. These findings indicate that respiking management may play an important role on selecting the genus of anammox bacteria. The batch enrichment results clearly illustrate that anammox can be readily enriched from municipal sludge from a wide variety of process operations at WWTPs.     

Carbendazim dissipation in the biomixture of on-farm biopurification systems and its effect on microbial communities

The impact of repeated carbendazim (CARB) applications on the extent of CARB dissipation, the microbial diversity, the community level physiological profile (CLPP), and the enzymatic activity within the biomixture of an on-farm biopurification system was evaluated. After three successive CARB applications, the CARB dissipation efficiency was high; the efficiency of dissipation was 87%, 94% and 96% after each application, respectively. Although microbial enzymatic activity was affected significantly by CARB application, it could recover after each CARB pulse. Likewise, the numbers of cultivable bacteria, fungi and actinomycetes (as measured in CFUs) were slightly affected by the addition of CARB, but the inhibitory effect of the pesticide application was temporary. Denaturing gradient gel electrophoresis (DGGE) and Biolog Ecoplate assays demonstrated that the microbial populations remained relatively stable over time when compared to the control. The results obtained herein therefore demonstrate the high dissipation capacity of this biomixture and highlight the microbiological robustness of this biological system.