Julia R. de Lipthay

In Situ Exposure to Low Herbicide Concentrations Affects Microbial Population Composition and Catabolic Gene Frequency in an Aerobic Shallow Aquifer

ABSTRACT The aim of this study was to evaluate how the in situ exposure of a Danish subsurface aquifer to phenoxy acid herbicides at low concentrations (<40 μg l−1) changes the microbial community composition. Sediment and groundwater samples were collected inside and outside the herbicide-exposed area and were analyzed for the presence of general microbial populations, Pseudomonas bacteria, and specific phenoxy acid degraders. Both culture-dependent and culture-independent methods were applied. The abundance of microbial phenoxy acid degraders (100 to 104 g−1 sediment) was determined by most probable number assays, and their presence was only detected in herbicide-exposed sediments. Similarly, PCR analysis showed that the 2,4-dichlorophenoxyacetic acid degradation pathway genes tfdA and tfdB (102 to 103 gene copies g−1 sediment) were only detected in sediments from contaminated areas of the aquifer. PCR-restriction fragment length polymorphism measurements demonstrated the presence of different populations of tfd genes, suggesting that the in situ herbicide degradation was caused by the activity of a heterogeneous population of phenoxy acid degraders. The number of Pseudomonas bacteria measured by either PCR or plating on selective agar media was higher in sediments subjected to high levels of phenoxy acid. Furthermore, high numbers of CFU compared to direct counting of 4′,6-diamidino-2-phenylindole-stained cells in the microscope suggested an increased culturability of the indigenous microbial communities from acclimated sediments. The findings of this study demonstrate that continuous exposure to low herbicide concentrations can markedly change the bacterial community composition of a subsurface aquifer.

Bacterial diversity and community structure of a sub‐surface aquifer exposed to realistic low herbicide concentrations

An increasing number of herbicides are found in our groundwater environments. This underlines the need for examining the effects of herbicide exposure on the indigenous groundwater microbial communities, as microbial degradation is the major process responsible for the complete removal of most contaminants. We examined the effect of in situ exposure to realistic low concentrations of herbicides on the microbial diversity and community structure of sub-surface sediments from a shallow aquifer near Vejen (Denmark). Three different community analyses were performed: colony morphology typing, sole-carbon source utilisation in Biolog EcoPlates, and denaturing gradient gel electrophoresis. Cluster analysis demonstrated that the microbial communities of those aquifer sediments that acclimated to the herbicide exposure also had similar community structure. This observation was concurrent for all three community analyses. In contrast, no significant effect was found on the bacterial diversity, except for the culturable fraction where a significantly increased richness and Shannon index was found in the herbicide acclimated sediments. The results of this study show that in situ exposure of sub-surface aquifers to realistic low concentrations of herbicides may alter the overall structure of a natural bacterial community, although significant effects on the genetic diversity and carbon substrate usage cannot be detected. The observed impact was probably due to indirect effects. In future investigations, the inclusion of methods that specifically detect relevant microbial sub-populations and functional genes is therefore recommended.

Quantification of the atrazine-degrading Pseudomonas sp. strain ADP in aquifer sediment by quantitative competitive polymerase chain reaction

The widely used herbicide atrazine and some of its degradation products are among the most commonly found xenobiotics in groundwater in Europe as well as in the USA. The bacterium Pseudomonas sp. strain ADP (P. ADP) possesses genes encoding atrazine mineralization on the self-transmissible plasmid pADP-1. In the present study, this ability of the strain to mineralize atrazine in aquifer sediment under both aerobic and denitrifying conditions at 10 degrees C was studied. P. ADP was able to mineralize more than 50% of 2.8 muM atrazine within 14 days under both growth conditions. Counts of degraders as colony forming units (CFU) on atrazine plates and counts of atzA gene copies as determined by quantitative competitive polymerase chain reaction (cPCR) were performed. The atzA gene encodes the enzyme which catalyzes the first step of atrazine mineralization by the strain. Quantification of the atzA gene gave rise to higher numbers than did counts of CFU. High nitrate concentrations inhibited atrazine mineralization and culturability on agar plates, but atzA copy numbers remained stable throughout the experiment. The results show a potential for bioaugmentation using P. ADP at both aerobic and denitrifying conditions and the use of cPCR as a tool for monitoring the bacteria independent of culturability.

Acclimation of subsurface microbial communities to mercury

We studied the acclimation to mercury of bacterial communities of different depths from contaminated and noncontaminated floodplain soils. The level of mercury tolerance of the bacterial communities from the contaminated site was higher than those of the reference site. Furthermore, the level of mercury tolerance and functional versatility of bacterial communities in contaminated soils initially were higher for surface soil, compared with the deeper soils. However, following new mercury exposure, no differences between bacterial communities were observed, which indicates a high adaptive potential of the subsurface communities, possibly due to differences in the availability of mercury. IncP-1 trfA genes were detected in extracted community DNA from all soil depths of the contaminated site, and this finding was correlated to the isolation of four different mercury-resistance plasmids, all belonging to the IncP-1beta group. The abundance of merA and IncP-1 plasmid carrying populations increased, after new mercury exposure, which could be the result of selection as well as horizontal gene exchange. The data in this study suggest a role for IncP-1 plasmids in the acclimation to mercury of surface as well as subsurface soil microbial communities.

Microbial Diversity and PAH Catabolic Genes Tracking Spatial Heterogeneity of PAH Concentrations

We analyzed the within-site spatial heterogeneity of microbial community diversity, polyaromatic hydrocarbon (PAH) catabolic genotypes, and physiochemical soil properties at a creosote contaminated site. Genetic diversity and community structure were evaluated from an analysis of denaturant gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified sequences of 16S rRNA gene. The potential PAH degradation capability was determined from PCR amplification of a suit of aromatic dioxygenase genes. Microbial diversity, evenness, and PAH genotypes were patchily distributed, and hot and cold spots of their distribution coincided with hot and cold spots of the PAH distribution. The analyses revealed a positive covariation between microbial diversity, biomass, evenness, and PAH concentration, implying that the creosote contamination at this site promotes diversity and abundance. Three patchily distributed PAH-degrading genotypes, NAH, phnA, and pdo1, were identified, and their abundances were positively correlated with the PAH concentration and the fraction of soil organic carbon. The covariation of the PAH concentration with the number and spatial distribution of catabolic genotypes suggests that a field site capacity to degrade PAHs may vary with the extent of contamination.