We evaluated the molecular diversity of narG gene from Suquía River sediments to assess the impact of the nitrate concentration and water quality on the composition and structure of the nitrate-reducing bacterial community. To this aim, a library of one of the six monitoring stations corresponding to the highest nitrate concentration was constructed and 118 narG clones were screened. Nucleotide sequences were associated to narG gene from alpha-, beta-, delta-, gammaproteobacteria and Thermus thermophilus. Remarkably, 18% of clones contained narG genes with less than 69% similarity to narG sequences available in databases. Thus, indicating the presence of nitrate-reducing bacteria with novel narG genes, which were quantified by real-time PCR. Results show a variable number of narG copies, ranging from less than 1.0 × 102 to 5.0 × 104 copies per ng of DNA, which were associated with a decreased water quality index monitored along the basin at different times.

To reveal the degradation capacity of bacteria in PAH polluted soil and rhizosphere we combined bacterial extradiol ring-cleavage dioxygenase and 16S rRNA analysis in Betula pubescens rhizoremediation. Characterisation of the functional bacterial community by RFLP revealed novel environmental dioxygenases, and their putative hosts were studied by 16S rRNA amplification. Plant rhizosphere and PAH amendment effects were detected by the RFLP/T-RFLP analysis. Functional species richness increased in the birch rhizosphere and PAH amendment impacted the compositional diversity of the dioxygenases and the structural 16S rRNA community. A shift from an Acidobacteria and Verrucomicrobia dominated to an Alpha- and Betaproteobacteria dominated community structure was detected in polluted soil. Clone sequence analysis indicated catabolic significance of Burkholderia in PAH polluted soil. These results advance our understanding of rhizoremediation and unveil the extent of uncharacterized functional bacteria to benefit bioremediation by facilitating the development of the molecular tool box to monitor bacterial populations in biodegradation.

This study investigated antibiotic resistance profiles and tetracycline resistance genes in Enterobacteriaceae family isolates from the Pearl rivers. The Enterobacteriaceae isolates were tested for susceptibility to seven antibiotics ampicillin, chloramphenicol, ciprofloxacin, levofloxacin, sulphamethoxazole/trimethoprim, tetracycline and trimethoprim. In Liuxi reservoir, with an exception to ampicillin resistant strains (11%) no other antibiotic resistance bacterial strains were detected. However, multiple drug resistance in bacterial isolates from the other sites of Pearl rivers was observed which is possibly due to sewage discharge and input from other anthropogenic sources along the rivers. Four tetracycline resistance genes tet A, tet B, tet C and tet D were detected in the isolates from the rivers. The genes tet A and tet B were widely detected with the detection frequencies of 43% and 40% respectively. Ciprofloxacin and levofloxacin resistant enteric bacteria were also isolated from the pig and duck manures which suggest a wider distribution of human specific drugs in the environment. This investigation provided a baseline data on antibiotic resistance profiles and tetracycline resistance genes in the Pearl rivers delta.

A Pb-mine site situated on acidic soil, but comprising of Ca-enriched islands around derelict buildings was used to study the spatial pattern of genetic diversity in Lumbricus rubellus. Two distinct genetic lineages (‘A’ and ‘B’), differentiated at both the mitochondrial (mtDNA COII) and nuclear level (AFLPs) were revealed with a mean inter-lineage mtDNA sequence divergence of approximately 13%, indicative of a cryptic species complex. AFLP analysis indicates that lineage A individuals within one central ‘ecological island’ site are uniquely clustered, with little genetic overlap with lineage A individuals at the two peripheral sites. FTIR microspectroscopy of Pb-sequestering chloragocytes revealed different phosphate profiles in residents of adjacent acidic and calcareous islands. Bioinformatics found over-representation of Ca pathway genes in ESTPb libraries. Subsequent sequencing of a Ca-transport gene, SERCA, revealed mutations in the protein's cytosolic domain. We recommend the mandatory genotyping of all individuals prior to field-based ecotoxicological assays, particularly those using discriminating genomic technologies. Landscapes punctuated by Pb-polluted islands have engendered local genetic differentiation in resident earthworms.

In this report, the toxic effect of TCE (trichloroethylene), PCE (tetrachloroethylene), and potassium dichromate on P. subcapitata was investigated. The test was conducted at different concentrations of pollutants, starting from the European Community limit values defined for each analysed contaminant. Mixtures of pollutants were also tested to verify the combined effect of algae cells. Results suggest that both TCE and PCE were able to reduce P. subcapitata growth and metabolism starting from 0.05 and 0.02 mg L⁻¹ of contaminant, respectively. PCE seems to be substantially more toxic than TCE. Chromium produces a clear effect on algae growth and esterase activity only starting from 1 mg L⁻¹ of potassium dichromate; this result confirms the suitability of EU limit value. AFLP analysis showed that all tested pollutants produce DNA mutations probably due to oxygen radicals. Generally, chromium, at high concentrations, is more toxic and genotoxic that TCE or PCE. Test performed with a mixture of pollutants showed a synergic effect of chromium and organic compounds suggesting that the membrane damage induced from organic substances should increase the chromium cellular access.

The evolution of microbial communities with increasing carbon tetrachloride concentrations was studied in two anaerobic columns containing sand and two different clay soils, one of which contained high levels of iron. Microbial communities were characterized through analysis of column effluents with denaturing gradient gel electrophoresis and quantitative polymerase chain reaction for archaea and eubacteria as inlet carbon tetrachloride concentrations were increased from 0.8 to 29 μM. Inhibition of microbial activity was observed in both columns, and was associated with the accumulation of chloroform at concentrations of 0.2 to 0.4 μM as inlet CT concentrations were increased to 2.4-3.0 μM in the low-iron clay column and approximately 16 μM in the iron rich clay column. Inhibition was indicated by decreasing rates of methanol and carbon tetrachloride degradation, decreases in effluent levels of DNA, and shifts in microbial communities of the columns. Even with the inhibition observed, in the iron-rich clay column CT degradation continued to the end of the study with inlet CT concentrations of 29 μM, in contrast to the low-iron clay column in which minimal CT degradation occurred once CT inlet concentrations exceeded 3 μM. The greater capacity for CT degradation in the column containing the iron-rich clay was hypothesized to be the result of reaction with biogenic ferrous iron produced by biological dissimilatory iron reduction.

Applying an integrated approach using the Comet, micronucleus (MN), and random amplified polymorphic DNA (RAPD) assays, occurrence of erythrocytic nuclear abnormalities (ENAs) and the liver activity of antioxidants enzymes (catalase and glutathione-S-transferase (GST)) was carried out to evaluate the effects of acute (6, 24, and 96 h) and subchronic (15 days) exposures to aluminum on fish Prochilodus lineatus. The Comet assay showed that fish erythrocytes exhibited significantly higher DNA damage after 6 and 96 h of Al exposure. MN frequencies were very low and did not increase significantly after Al exposures, while ENAs frequency increased significantly after all exposure periods. RAPD profiles obtained with DNA from fish fins collected before the toxicity tests were compared to the profiles with DNA from gills and liver of the same fish sampled after Al exposures. Alterations in RAPD profiles, including appearance and disappearance of bands, after 6 h, 24 h, and 15 days of Al exposure were detected. Fish exposed to Al for 6 and 24 h also showed significant increases in GST and catalase activities. These results indicated that Al exposure was genotoxic to P. lineatus, inducing DNA damage in peripheral erythrocytes. The induction of antioxidant enzymes might be an indication that Al causes oxidative damage to DNA, while the very low frequency of MN suggests that Al does not produce clastogenic or aneugenic effects. Genotoxic effects after 15 days of Al exposure was revealed only by RAPD, showing that this assay represents a sensitive method to detect genotoxic damage, occasionally not detected by other genotoxic tests used in toxicological genetics studies.

The remediation of groundwater contaminated with benzene, toluene, ethylbenzene, and the xylenes (BTEX) typically involves in situ biodegradation. Although the mechanisms of aerobic BTEX biodegradation in laboratory cultures have been well studied, less is known about the microorganisms responsible in mixed culture samples or at contaminated sites. In this study, the microorganisms responsible for in situ degradation within mixed culture samples were investigated using the molecular method stable isotope probing (SIP). For this, m-xylene was utilized as a model BTEX contaminant. Specifically, DNA-based SIP was utilized to identify active m-xylene degraders in microcosms constructed with soil from three sources (a gasoline-contaminated site and two agricultural sites). Replicate microcosms were amended with either labeled (¹³C) or unlabeled m-xylene, and the extracted DNA samples were ultracentrifuged, fractioned, and subjected to terminal restriction fragment length polymorphism (TRFLP). The dominant m-xylene degraders (responsible for ¹³C uptake) were determined by comparing relative abundance of TRFLP phylotypes in heavy fractions of labeled m-xylene (¹³C) amended samples to the controls (from unlabeled m-xylene amended samples). Four phylotypes were identified as the dominant m-xylene degrading species, falling within either the β Proteobacteria or the Bacilli. Of these, two 16S rRNA gene sequences were highly novel, displaying very limited similarity (94% and 90%) to any previously reported 16S rRNA gene sequence. Further, three of these phylotypes fell within genera with limited or no previous links to BTEX degradation, suggesting much information is still to be gained concerning the identity of microorganisms responsible for degradation within mixed culture samples.

Background, aim, and scope Soil contamination with heavy metals occurs as a result of both anthropogenic and natural activities. Heavy metals could have long-term hazardous impacts on the health of soil ecosystems and adverse influences on soil biological processes. Soil enzymatic activities are recognized as sensors towards any natural and anthropogenic disturbance occurring in the soil ecosystem. Similarly, microbial biomass carbon (MBC) is also considered as one of the important soil biological activities frequently influenced by heavy metal contamination. The polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) has recently been used to investigate changes in soil microbial community composition in response to environmental stresses. Soil microbial community structure and activities are difficult to elucidate using single monitoring approach; therefore, for a better insight and complete depiction of the soil microbial situation, different approaches need to be used. This study was conducted in a greenhouse for a period of 12 weeks to evaluate the changes in indigenous microbial community structure and activities in the soil amended with different application rates of Cd, Pb, and Cd/Pb mix. In a field environment, soil is contaminated with single or mixed heavy metals; so that, in this research, we used the selected metals in both single and mixed forms at different application rates and investigated their toxic effects on microbial community structure and activities, using soil enzyme assays, plate counting, and advanced molecular DGGE technique. Soil microbial activities, including acid phosphatase (ACP), urease (URE), and MBC, and microbial community structure were studied. Materials and methods A soil sample (0-20 cm) with an unknown history of heavy metal contamination was collected and amended with Cd, Pb, and Cd/Pb mix using the CdSO₄ and Pb(NO₃)₂ solutions at different application rates. The amended soils were incubated in the greenhouse at 25 ± 4°C and 60% water-holding capacity for 12 weeks. During the incubation period, samples were collected from each pot at 0, 2, 9, and 12 weeks for enzyme assays, MBC, numeration of microbes, and DNA extraction. Fumigation-extraction method was used to measure the MBC, while plate counting techniques were used to numerate viable heterotrophic bacteria, fungi, and actinomycetes. Soil DNAs were extracted from the samples and used for DGGE analysis. Results ACP, URE, and MBC activities of microbial community were significantly lower (p < 0.05) in the metal-amended samples than those in the control. The enzyme inhibition extent was obvious between different incubation periods and varied as the incubation proceeded, and the highest rate was detected in the samples after 2 weeks. However, the lowest values of ACP and URE activities (35.6% and 36.6% of the control, respectively) were found in the Cd₃/Pb₃-treated sample after 2 weeks. Similarly, MBC was strongly decreased in both Cd/Pb-amended samples and highest reduction (52.4%) was detected for Cd₃/Pb₃ treatment. The number of bacteria and actinomycetes were significantly decreased in the heavy metal-amended samples compared to the control, while fungal cells were not significantly different (from 2.3% to 23.87%). In this study, the DGGE profile indicated that the high dose of metal amendment caused a greater change in the number of bands. DGGE banding patterns confirmed that the addition of metals had a significant impact on microbial community structure. Discussion In soil ecosystem, heavy metals exhibit toxicological effects on soil microbes which may lead to the decrease of their numbers and activities. This study demonstrated that toxicological effects of heavy metals on soil microbial community structure and activities depend largely on the type and concentration of metal and incubation time. The inhibition extent varied widely among different incubation periods for these enzymes. Furthermore, the rapid inhibition in microbial activities such as ACP, URE, and MBC were observed in the 2 weeks, which should be related to the fact that the microbes were suddenly exposed to heavy metals. The increased inhibition of soil microbial activities is likely to be related to tolerance and adaptation of the microbial community, concentration of pollutants, and mechanisms of heavy metals. The DGGE profile has shown that the structure of the bacterial community changed in amended heavy metal samples. In this research, the microbial community structure was highly affected, consistent with the lower microbial activities in different levels of heavy metals. Furthermore, a great community change in this study, particularly at a high level of contamination, was probably a result of metal toxicity and also unavailability of nutrients because no nutrients were supplied during the whole incubation period. Conclusions The added concentrations of heavy metals have changed the soil microbial community structure and activities. The highest inhibitory effects on soil microbial activities were observed at 2 weeks of incubation. The bacteria were more sensitive than actinomycetes and fungi. The DGGE profile indicated that bacterial community structure was changed in the Cd/Pb-amended samples, particularly at high concentrations. Recommendations and perspectives The investigation of soil microbial community structure and activities together could give more reliable and accurate information about the toxic effects of heavy metals on soil health.

Background, aim, and scope Current Australian legislation permits the beneficial application of grease trap waste (GTW) to agricultural soil, viewing it as a beneficial source of organic matter and soil conditioner containing no/low amounts of metals or pathogenic organisms. However, little is known about the influence of GTW on soil bacterial community. A field experiment was established at Menangle in south western Sydney in Australia to quantitatively assess the impacts of different types (GTW CO and GTW CL) and amounts of GTW application on the soil bacterial community and diversity. Furthermore, a municipal solid waste (MSW) compost was simultaneously examined to compare against the other organic wastes. Knowledge about the shifts in microbial community structure and diversity following the applications of organic wastes could help to evaluate the ecological consequences on the soil and thus to develop sound regulatory guidelines for the beneficial reuse of organic wastes in agricultural lands. Materials and methods Soil samples were collected from recycled organics plots treated with different types and quantity of organic wastes. The field experimental treatments included control (CK, without application of any organic wastes), low amount of GTW CO (COL), GTW CL (CLL), and MSW (ML), and high amounts of GTW CO (COH), GTW CL (CLH), and MSW compost (MH). Microbial DNA was extracted from soil samples and the 16S rRNA genes were polymerase chain reaction (PCR)-amplified. The PCR products were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. The bacterial community structures and diversity were assessed using the DGGE profiles and clone libraries constructed from the excised DGGE bands. Results DGGE-based analyses showed that application of the GTW CO, regardless of the amount applied, had significant negative effects on soil bacterial genotypic diversity and community structure compared with the control, while the applications of other organic wastes including the GTW CL and MSW had no clear effects. The effects of the rate of organic waste application on soil bacterial community characteristics varied with the types of organic wastes applied. Sequence-based analyses of 126 clones indicated that Proteobacteria (53.2%) was the dominant taxa at the experimental site, followed by Actinobacteria (9.5%), Bacteroidetes (7.9%), Firmicutes (7.9%), Gemmatimonadetes (5.6%), Chloroflexi (2.4%), Acidobacteria (1.6%) and the unclassified group (11.9%). In the COH treatment, Acidobacteria, Bacteroidetes, and Gemmatimonadetes were not detected; the percentages of Firmicutes, Proteobacteria, and Actinobacteria in the COH treatment were significantly different from those in CK. There is a significant positive correlation (r = 0.71, p = 0.002) between the C/N ratio of organic wastes and the bacterial genotypic communities. Discussion Both the type and the amount of GTW applied affected soil bacterial genotypic diversity and community structure. The different effects of various types of organic wastes on soil bacterial characteristics may be predicted by the differences in specific properties of organic wastes such as C/N ratio, as evidenced by the strong and significant positive relationship between the bacterial community distance and the environmental distance of C/N ratio. This also indicates that the C/N ratio of GTW applied can be a major driver for the shift in the soil bacterial community. Conclusions Our results revealed that the effects of organic wastes on soil bacterial communities varied with the types of organic wastes, and depending on the rate of application. Application of the GTW CO led to significant shifts in soil bacterial community diversity and structure. The effects of different types of organic wastes on the soil bacterial characteristics can be predicted by the differences of specific properties of organic wastes, such as the C/N ratio. Sequence-based analyses of 126 clones indicated that Proteobacteria was the dominant taxa at the experimental site. Recommendations and perspectives Our results have important implications for developing sound regulatory guidelines for the beneficial reuse of organic wastes, indicating that GTW CO and similar organic waste treatments may not be suitable for application in agricultural soils due to its significant negative effect on soil bacterial community.