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.     

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.     

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.     

Assessing microbial communities for a metabolic profile similar to activated sludge.

To search for reliable testing inocula alternatives to activated sludge cultures, several model microbial consortia were compared with activated sludge populations for their functional diversity. The evaluation of the metabolic potential of these mixed inocula was performed using the Biolog EcoPlates and GN and GP MicroPlates (Biolog, Inc., Hayward, California). The community-level physiological profiles (CLPPs) obtained for model communities and activated sludge samples were analyzed by principal component analysis and hierarchic clustering methods, to evaluate the ability of Biolog plates to distinguish among the different microbial communities. The effect of different inocula preparation methodologies on the community structure was also studied. The CLPPs obtained with EcoPlates and GN MicroPlates showed that EcoPlates are suitable to screen communities with a metabolic profile similar to activated sludge. New, well-defined, standardized, and safe inocula presenting the same metabolic community profile as activated sludge were selected and can be tested as surrogate cultures in activated-sludge-based bioassays.     

Assessment of nematode community structure as a bioindicator in river monitoring

Nematode communities from river water and sediments were assessed for the abundance, feeding types, maturity indices and nematode channel ratio (NCR). The sampling sites studied included different levels of pollution and contamination from agricultural, industrial and sewage sources. The nematode abundance found in the sediment samples was more than that in the water samples. The lowest nematode abundance in sediment samples and the lowest NCR in water samples were both found at the industrial pollution site. Water samples showed positive correlation between the NCR and river pollution index (RPI). Mean maturity indices in sediment samples were inversely correlated with RPI. The pollutant source determined the relationship between NCR and pollution level, while maturity index always showed negative correlation with pollutant level regardless of the pollutant sources. The nematode abundance and its community structure were both reliable bioindicators for monitoring long-term river pollution in both qualitative and quantitative aspects.     

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.     

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。     

Detecting changes in insect herbivore communities along a pollution gradient

The forests surrounding the urban areas of the Los Angeles basin are impacted by ozone and nitrogen pollutants arising from urban areas. We examined changes in the herbivore communities of three prominent plant species (ponderosa pine, California black oak and bracken fern) at six sites along an air pollution gradient. Insects were extracted from foliage samples collected in spring, as foliage reached full expansion. Community differences were evaluated using total herbivore abundance, richness, Shannon-Weiner diversity, and discriminant function analysis. Even without conspicuous changes in total numbers, diversity or richness of herbivores, herbivore groups showed patterns of change that followed the air pollution gradient that were apparent through discriminant function analysis. For bracken fern and oak, chewing insects were more dominant at high pollution sites. Oak herbivore communities showed the strongest effect. These changes in herbivore communities may affect nutrient cycling in forest systems.     

Influence of ciprofloxacin on the microbial catabolic diversity in soil

In this study, the effect of ciprofloxacin (CIP) on the catabolic diversity of soil microbial communities was evaluated. Soil samples were spiked with ciprofloxacin (0, 1, 5 and 50 mg?kg^?1) and were incubated for 1, 3, 9, 22 and 40 days. Untreated controls received only water. The functional diversity of the microbial community studied was characterized using a catabolic response profile (CRP). Six substrate groups were tested: carbohydrates, amino acids, carboxylic acids, aromatic chemicals, alcohols and polymers. After 40 days, the CIP concentrations in the soil samples ranged from 25% to 58% of the initial concentrations. Soil respiratory responses to the individual substrates D-glucose, lactose, D-mannose, L-glutamic, Na-citrate, malic acid and inosine were inhibited at the high CIP concentrations (5 and 50 mg·kg^?1) in the soils and were increased at the lowest CIP concentration (1 mg·kg^?1). Soil respiration was inhibited at all of the CIP concentrations after the addition of D-galactose and glycerol. The CIP concentration and incubation time explained 45.3% of the variance of the catabolic responses. The CRP analysis clearly discriminated among the different CIP concentrations. The results suggest that CIP strongly affects the catabolic diversities of soil microbial communities and that its effect is more significant than that of incubation time.     

Development and Integration of Quantitative Real-Time PCR Methods for Detection of Mitochondrial DNA and Methanobrevibacter smithii nifH Gene as Novel Microbial Source Tracking Tools

A novel microbial source tracking (MST) method based on the detection of human and non-human markers was developed and applied to track the origin of fecal pollution in water systems. Mitochondrial DNA sequences were used to develop new quantitative real-time polymerase chain reaction (qPCR) assays for dog, poultry, and gull. The targets were included as part of a toolbox including human, cow, pig, and sheep assays. A primer and probe set for the detection of the human-specific nifH gene of Methanobrevibacter smithii was also designed as an indicator of human fecal contamination. The assays were tested for specificity and applied to fecal-spiked surface waters and environmental samples collected from two river catchments impacted by sources of human and non-human fecal contamination. The MST methods described were applicable to both spiked waters and environmental samples, and using the two approaches the origin of fecal pollution could be successfully determined in mixed source fecally polluted waters.     

Antibiotic resistance genes in an urban river as impacted by bacterial community and physicochemical parameters

Antibiotic resistance genes (ARGs) in urban rivers are a serious public health concern in regions with poorly planned, rapid development. To gain insights into the predominant factors affecting the fate of ARGs in a highly polluted urban river in eastern China, a total of 285 ARGs, microbial communities, and 20 physicochemical parameters were analyzed for 17 sites. A total of 258 unique ARGs were detected using high-throughput qPCR, and the absolute abundance of total ARGs was positively correlated with total organic carbon and total dissolved nitrogen concentrations (P < 0.01). ARG abundance and diversity were greatly altered by microbial community structure. Variation partitioning analysis showed that the combined effects of multiple factors contributed to the profile and dissemination of ARGs, and variation of microbial communities was the major factor affecting the distribution of ARGs. The disparate distribution of some bacteria, including Bacteroides from mammalian gastrointestinal flora, Burkholderia from zoonotic infectious diseases, and Zoogloea from wastewater treatment, indicates that the urban river was strongly influenced by point-source pollution. Results imply that microbial community shifts caused by changes in water quality may lead to the spread of ARGs, and point-source pollution in urban rivers requires greater attention to control the transfer of ARGs between environmental bacteria and pathogens.

     

Spatial distribution and source apportionment of water pollution in different administrative zones of Wen-Rui-Tang (WRT) river watershed, China

Water quality degradation in river systems has caused great concerns all over the world. Identifying the spatial distribution and sources of water pollutants is the very first step for efficient water quality management. A set of water samples collected bimonthly at 12 monitoring sites in 2009 and 2010 were analyzed to determine the spatial distribution of critical parameters and to apportion the sources of pollutants in Wen-Rui-Tang (WRT) river watershed, near the East China Sea. The 12 monitoring sites were divided into three administrative zones of urban, suburban, and rural zones considering differences in land use and population density. Multivariate statistical methods [one-way analysis of variance, principal component analysis (PCA), and absolute principal component score—multiple linear regression (APCS-MLR) methods] were used to investigate the spatial distribution of water quality and to apportion the pollution sources. Results showed that most water quality parameters had no significant difference between the urban and suburban zones, whereas these two zones showed worse water quality than the rural zone. Based on PCA and APCS-MLR analysis, urban domestic sewage and commercial/service pollution, suburban domestic sewage along with fluorine point source pollution, and agricultural nonpoint source pollution with rural domestic sewage pollution were identified to the main pollution sources in urban, suburban, and rural zones, respectively. Understanding the water pollution characteristics of different administrative zones could put insights into effective water management policy-making especially in the area across various administrative zones.     

Macroinvertebrate community response to acid mine drainage in rivers of the High Andes (Bolivia)

Several High Andes Rivers are characterized by inorganic water pollution known as acid mine drainage (AMD). The aim of this study was to assess the relationship between metal concentrations in the sediments and the macroinvertebrate communities in two river basins affected by AMD. In general, the taxon diversity of the macroinvertebrate community at the family level was low. The concentrations of Cd, Cu, Zn, Pb and Ni at mining sites were higher than at unpolluted sites. The pH of the water was alkaline (7.0-8.5) in unpolluted sites, whereas it dropped to very low values (<3) at mining sites. Redundancy Analysis (RDA) showed that pH was the best predictor of macroinvertebrate community richness. The number of macroinvertebrate families decreased gradually with increasing acidity, both in pools and riffles, though it is suggested that riffle communities were more affected because they are in closer contact with the acid water.     

Response of the fish populations of the River Don in South Yorkshire to water quality and habitat improvements

The River Don in Yorkshire, UK, has suffered a legacy of pollution and land contamination that dates back to the Industrial Revolution. The river continues to be impacted by discharges from industry, mining and sewage sources, resulting in poor water quality and considerable damage to the fish stocks. Some modest reductions in ammonia, biochemical oxygen demand (BOD) and metal levels have been achieved through improvements to sewage treatment works and the shutting down of some industries. The decline in the steel industry has generally led to sustained low and acceptable levels of iron and nickel in the river, although some locations are impacted by ochre from abandoned mines. As a consequence, recreational fishing in the catchment remains restricted at many locations. Fish stocking and enhancement programmes have so far been ineffective due to setbacks from ad hoc pollution incidents and degraded habitats. It is suggested that the water quality and habitat degradation problems be addressed prior to embarking on any large-scale fish stock enhancement programmes. This would require substantial financial investment and it is recommended that funding be actively sought from both internal and external agencies to achieve this objective.     

A chemometric approach to the evaluation of atmospheric and fluvial pollutant inputs in aquatic systems: the Guadalquivir River estuary as a case study

To establish the quality of waters it is necessary to identify both point and non-point pollution sources. In this work, we propose the combination of clean analytical methodologies and chemometric tools to study discrete and diffuse pollution caused in a river by tributaries and precipitations, respectively.During a two-year period, water samples were taken in the Guadalquivir river (selected as a case study) and its main tributaries before and after precipitations. Samples were characterized by analysing nutrients, pH, dissolved oxygen, total and volatile suspended solids, carbon species, and heavy metals. Results were used to estimate fluvial and atmospheric inputs and as tracers for anthropic activities.Multivariate analysis was used to estimate the background pollution, and to identify pollution inputs. Principal Component Analysis and Cluster Analysis were used as data exploratory tools, while box-whiskers plots and Linear Discriminant Analysis were used to analyse and distinguish the different types of water samples.     

京杭大运河(杭州段)三氮变化及影响因素浅析

根据已建立的ＮＨ_３－Ｎ、ＮＯ_２~－－Ｎ、ＮＯ_３~－－Ｎ基本水质数据库,对京杭大运河（杭州段）三氮的水质现状和趋势作了分析,结果表明,工业废水与生活污水是河水中ＮＨ_３－Ｎ升高的主要原因,ＮＯ_２~－－Ｎ和ＮＯ_３~－－Ｎ与硝化反应有关,不同水期对三氮都有影响。     

Patterns of insect communities along a stress gradient following decommissioning of a Cu-Ni smelter

The diversity, estimated richness and abundance of terrestrial insect communities were examined along a stress gradient of past pollution in the region of Sudbury, Ontario, Canada. This gradient represents the natural recovery and lingering effects of a decommissioned copper-nickel smelting complex. Ant genera and sixteen higher taxonomic groups (family and order) had the highest abundance at the sites with intermediate stress. Eight families increased in abundance with distance from the decommissioned source of pollution and eleven families decreased reflecting a complex response of diversity to pollution. Carabid beetles show an increase in diversity further from the smelter; however, examination of the species composition reveals a distinct carabid community closest to the smelter, emphasizing the unique habitat created by severe pollution. Although almost forty years since decomissioning of the smelter complex, the terrestrial insect community in the vicinity remains significantly impacted suggesting slow recovery.     

光合细菌改善Cd、Pb及呋喃丹污染土壤的微生物群落DNA序列多样性的研究

应用DNA随机扩增多态性(RAPD)分子标记技术研究了光合细菌(PSB)对Cd、Pb及呋喃丹污染土壤的微生物群落DNA序列多样性的影响.结果表明,Cd、Pb及呋喃丹单一污染或3者复合污染土壤的微生物群落DNA序列的丰富度相对对照土样(S0)都有不同程度的增加,受Cd、Pb或呋哺丹污染,可能会引起土壤微生物群落DNA序列...     

Studies on the restoration succession of PFU microbial communities in a pilot-scale microcosm

In order to imitate the restoration succession process of natural water ecosystem, a laboratory microcosm system of constant-flow-restoration was designed and established. A eutrophycation lake, Lake Donghu, was selected as the subject investigated. Six sampling stations were set on the lake, among which the water of station IV was natural clean water, and others were polluted with different degrees. Polyurethane foam unit microbial communities, which had colonized in the stations for a month, were collected from these stations and placed in their respective microcosms, using clean water of station IV to gradually replace the water of these microcosms. In this process, the healthy community in clean water continuously replaced the damaged communities in polluted water, the restoration succession of the damaged communities was characterized by weekly determination of several functional and structural community parameters, including species number (S), diversity index (DI), community pollution value (CPV), heterotrophy index (HI), and similarity coefficient. Cluster analysis based on similarity coefficient was used to compare the succession discrepancies of these microbial communities from different stations. The ecological succession of microbial communities during restoration was investigated by the variable patterns of these parameters, and based on which, the restoration standards of these polluted stations were suggested in an ecological sense. That was, while being restored, the water of station 0 (supereutrophycation) should be substituted with natural clean water by 95%; station I (eutrophycation), more than 90%; station II (eutrophycation), more than 85%; station III (eutrophycation), about 85%; station V (mesoeutrophycation), less than 50%. The effects of the structural and functional parameters in monitoring and assessing ecological restoration are analyzed and compared.     

Organic pollutants in the Odra river ecosystem

The paper presents the results of the analysis of water and bottom sediment samples from different locations along the Odra river, collected during eight campaigns in the years 1997-2000. All the basic organic pollutants were determined. Pollution of water with PCBs was negligible while with PAHs was not critical. Concentrations of volatile organochlorine compounds in water were the highest in Brzeg Dolny and its surroundings. Concentrations of tetrachloroethene were exceptionally high in the Szczecin Lagoon. The Odra sediments were non-polluted or only slightly polluted with PCBs, pesticides and different classes of volatile organic pollutants. However, PAHs were present in rather large concentrations, especially in the upper course of the Odra river. The data obtained contributed to the evaluation of pollutants discharges to the Baltic sea via Szczecin Lagoon as well as to the characterization of pollution sources (point sources and area sources) in the Odra catchment area.