Nutrient loading impacts on culturable E. coli and other heterotrophic bacteria fate in simulated stream mesocosms.

Understanding fecal indicator bacteria persistence in aquatic environments is important when making management decisions to improve instream water quality. Routinely, bacteria fate and transport models that rely on published kinetic decay constants are used to inform such decision making but may not adequately represent instream conditions. The objective of this work was to evaluate bacterial responses to applied nutrient amendments and provide additional information regarding bacterial response to applied changes that can be incorporated into future modeling efforts. Re-created stream mesocosms were established in laboratory-based, repurposed algae raceways filled with water and sediment from a small, 3rd order Southeast Texas stream. Mesocosm treatments consisted of low (10x) or high (50x) nutrient doses above ambient water concentrations operated at low (0.032 m/s) or high (0.141 m/s) flow rates. Escherichia coli and heterotrophic bacterial concentrations were quantified in water and sediment over 22 days. No significant differences in kinetic constants were observed among E. coli in water or sediment, and only E. coli in sediment showed any growth response. Heterotrophic plate counts revealed a pronounced growth response in water and sediment within 24 h of nutrient addition but did not differ significantly from control mesocosms. Significant kinetic constant differences between E. coli and heterotrophic bacteria in water were identified (p < 0.01) but did not differ significantly in sediment (p > 0.48). Results indicate that nutrient addition does affect microbial numbers instream, but competition from heterotrophic bacteria may prevent an E. coli growth response.

Impact of enumeration method on diversity of Escherichia coli genotypes isolated from surface water.

There are numerous regulatory-approved Escherichia coli enumeration methods, but it is not known whether differences in media composition and incubation conditions impact the diversity of E. coli populations detected by these methods. A study was conducted to determine if three standard water quality assessments, Colilert® , USEPA Method 1603, (modified mTEC) and USEPA Method 1604 (MI), detect different populations of E. coli. Samples were collected from six watersheds and analysed using the three enumeration approaches followed by E. coli isolation and genotyping. Results indicated that the three methods generally produced similar enumeration data across the sites, although there were some differences on a site-by-site basis. The Colilert® method consistently generated the least diverse collection of E. coli genotypes as compared to modified mTEC and MI, with those two methods being roughly equal to each other. Although the three media assessed in this study were designed to enumerate E. coli, the differences in the media composition, incubation temperature, and growth platform appear to have a strong selective influence on the populations of E. coli isolated. This study suggests that standardized methods of enumeration and isolation may be warranted if researchers intend to obtain individual E. coli isolates for further characterization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized the impact of three USEPA-approved Escherichia coli enumeration methods on observed E. coli population diversity in surface water samples. Results indicated that these methods produced similar E. coli enumeration data but were more variable in the diversity of E. coli genotypes observed. Although the three methods enumerate the same species, differences in media composition, growth platform, and incubation temperature likely contribute to the selection of different cultivable populations of E. coli, and thus caution should be used when implementing these methods interchangeably for downstream applications which require cultivated isolates.

A geographical approach to tracking Escherichia coli and other water quality constituents in a Texas coastal plains watershed.

Diffuse sources of surface water pathogens and nutrients can be difficult to isolate in larger river basins. This study used a geographical or nested approach to isolate diffuse sources of Escherichia coli and other water quality constituents in a 145.7-km(2) river basin in south central Texas, USA. Average numbers of E. coli ranged from 49 to 64,000 colony forming units (CFU) per 100 mL depending upon season and stream flow over the 1-year sampling period. Nitrate-N concentrations ranged from 48 to 14,041 µg L(-1) and orthophosphate-P from 27 to 2,721 µg L(-1). High concentrations of nitrate-N, dissolved organic nitrogen, and orthophosphate-P were observed downstream of waste water treatment plants but E. coli values were higher in a watershed draining an older part of the city. Total urban land use explained between 56 and 72 % of the variance in mean annual E. coli values (p < 0.05) in nine hydrologically disconnected creeks. Of the types of urban land use, commercial land use explained most of the variance in E. coli values in the fall and winter. Surface water sodium, alkalinity, and potassium concentrations in surface water were best described by the proportion of commercial land use in the watershed. Based on our nested approach in examining surface water, city officials are able to direct funding to specific areas of the basin in order to mitigate high surface water E. coli numbers and nutrient concentrations.

Microbial community composition and dynamics in a semi-industrial-scale facility operating under the MixAlco™ bioconversion platform.

AIMS: To monitor microbial community dynamics in a semi-industrial-scale lignocellulosic biofuel reactor system and to improve our understanding of the microbial communities involved in the MixAlco™ biomass conversion process. METHODS AND RESULTS: Reactor microbial communities were characterized at six time points over the course of an 80-day, mesophilic, semi-industrial-scale fermentation using community qPCR and 16S rRNA tag-pyrosequencing. We found the communities to be dynamic, bacterially dominated consortia capable of changing quickly in response to reactor conditions. Clostridia- and Bacteroidetes-like organisms dominated the reactor communities, but ultimately the communities established consortia containing complementary functional capacities for the degradation of lignocellulosic materials. Eighteen operational taxonomic units were found to share strong correlations with reactor acid concentration and may represent taxa integral to fermentor performance. CONCLUSIONS: The results of this study indicate that the emergence of complementary functional classes within the fermentor consortia may be a trait that is consistent across scales, and they suggest that there may be flexibility with respect to the specific identities of the organisms involved in the fermentor's degradation and fermentation processes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information regarding the composition, dynamics and potential flexibility of the microbial communities associated with the MixAlco™ process and is likely to inform the improvement of this and other applications that employ mixed microbial communities.

Microarray applications in microbial ecology research.

Microarray technology has the unparalleled potential to simultaneously determine the dynamics and/or activities of most, if not all, of the microbial populations in complex environments such as soils and sediments. Researchers have developed several types of arrays that characterize the microbial populations in these samples based on their phylogenetic relatedness or functional genomic content. Several recent studies have used these microarrays to investigate ecological issues; however, most have only analyzed a limited number of samples with relatively few experiments utilizing the full high-throughput potential of microarray analysis. This is due in part to the unique analytical challenges that these samples present with regard to sensitivity, specificity, quantitation, and data analysis. This review discusses specific applications of microarrays to microbial ecology research along with some of the latest studies addressing the difficulties encountered during analysis of complex microbial communities within environmental samples. With continued development, microarray technology may ultimately achieve its potential for comprehensive, high-throughput characterization of microbial populations in near real time.

Changes in bacterial community structure correlate with initial operating conditions of a field-scale denitrifying fluidized bed reactor.

High levels of nitrate are present in groundwater migrating from the former waste disposal ponds at the Y-12 National Security Complex in Oak Ridge, TN. A field-scale denitrifying fluidized bed reactor (FBR) was designed, constructed, and operated with ethanol as an electron donor for the removal of nitrate. After inoculation, biofilms developed on the granular activated carbon particles. Changes in the bacterial community of the FBR were evaluated with clone libraries (n = 500 partial sequences) of the small-subunit rRNA gene for samples taken over a 4-month start-up period. Early phases of start-up operation were characterized by a period of selection, followed by low diversity and predominance by Azoarcus-like sequences. Possible explanations were high pH and nutrient limitations. After amelioration of these conditions, diversification increased rapidly, with the appearance of Dechloromonas, Pseudomonas, and Hydrogenophaga sequences. Changes in NO3, SO4, and pH also likely contributed to shifts in community composition. The detection of sulfate-reducing-bacteria-like sequences closely related to Desulfovibrio and Desulfuromonas in the FBR have important implications for downstream applications at the field site.

Chlorobenzoate-degrading bacteria in similar pristine soils exhibit different community structures and population dynamics in response to anthropogenic 2-, 3-, and 4-chlorobenzoate levels.

A study was conducted to determine the diversity of 2-, 3-, and 4-chlorobenzoate (CB) degraders in two pristine soils with similar physical and chemical characteristics. Surface soils were collected from forested sites and amended with 500 microg of 2-, 3-, or 4-CB g(-1) soil. The CB levels and degrader numbers were monitored throughout the study. Degraders were isolated, grouped by DNA fingerprints, identified via 16S rDNA sequences, and screened for plasmids. The CB genes in selected degraders were isolated and/or sequenced. In the Madera soil, 2-CB and 4-CB degraded within 11 and 42 d, respectively, but 3-CB did not degrade. In contrast, 3-CB and 4-CB degraded in the Oversite soil within 14 and 28 d, respectively, while 2-CB did not degrade. Approximately 10(7) CFU g(-1) of degraders were detected in the Madera soil with 2-CB, and the Oversite soil with 3- and 4-CB. No degraders were detected in the Madera soil with 4-CB even though the 4-CB degraded. Nearly all of the 2-CB degraders isolated from the Madera soil were identified as a Burkholderia sp. containing chromosomally encoded degradative genes. In contrast, several different 3- and 4-CB degraders were isolated from the Oversite soil, and their populations changed as CB degradation progressed. Most of these 3-CB degraders were identified as Burkholderia spp. while the majority of 4-CB degraders were identified as Bradyrhizobium spp. Several of the 3-CB degraders contained the degradative genes on large plasmids, and there was variation between the plasmids in different isolates. When a fresh sample of Madera soil was amended with 50, 100, or 200 microg 3-CB g(-1), 3-CB degradation occurred, suggesting that 500 microg 3-CB g(-1) was toxic to the degraders. Also, different 3-CB degraders were isolated from the Madera soil at each of the three lower levels of 3-CB. No 2-CB degradation was detected in the Oversite soil even at lower 2-CB levels. These results indicate that the development of 2-, 3-, and 4-CB degrader populations is site-specific and that 2-, 3-, and 4-CB are degraded by different bacterial populations in pristine soils. These results also imply that the microbial ecology of two soils that develop under similar biotic and abiotic environments can be quite different.

Soil microbial population dynamics following bioaugmentation with a 3-chlorobenzoate-degrading bacterial culture. Bioaugmentation effects on soil microorganisms.

Changes in microbial populations were evaluated following inoculation of contaminated soil with a 3-chlorobenzoate degrader. Madera sandy loam was amended with 0, 500, or 1,000 microg 3-chlorobenzoate g(-1) dry soil. Selected microcosms were inoculated with the degrader Comamonas testosteroni BR60. Culturable bacterial degraders were enumerated on minimal salts media containing 3-chlorobenzoate. Culturable heterotrophic bacteria were enumerated on R2A. Isolated degraders were grouped by enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction fingerprints and identified based on 16S ribosomal-DNA sequences. Bioaugmentation increased the rate of degradation at both levels of 3-chlorobenzoate. In both the 500 and 1,000 microg 3-chlorobenzoate g(-1) dry soil inoculated microcosms, degraders increased from the initial inoculum and decreased following degradation of 3-CB. Inoculation delayed the development of indigenous 3-chlorobenzoate degrading populations. It is unclear if inoculation altered the composition of indigenous degrader populations. In the uninoculated soil, degraders increased from undetectable levels to 6.6 x 10(7) colony-forming-units g(-1) dry soil in the 500 microg 3-chlorobenzoate g(-1) dry soil microcosms, but none were detected in the 1,000 microg 3-chlorobenzoate g(-1) dry soil microcosms. Degraders isolated from uninoculated soil were identified as one of two distinct Burkholderia species. In the uninoculated soil, numbers of culturable heterotrophic bacteria initially decreased following addition of 1,000 microg 3-chlorobenzoate g(-1) dry soil. Inoculation with C. testosteroni reduced this negative impact on culturable bacterial numbers. The results indicate that bioaugmentation may not only increase the rate of 3-chlorobenzoate degradation but also reduce the deleterious effects of 3-chlorbenzoate on indigenous soil microbial populations.

Comparison of 2,4-dichlorophenoxyacetic acid degradation and plasmid transfer in soil resulting from bioaugmentation with two different pJP4 donors.

A pilot field study was conducted to assess the impact of bioaugmentation with two plasmid pJP4-bearing microorganisms: the natural host, Ralstonia eutropha JMP134, and a laboratory-generated strain amenable to donor counterselection, Escherichia coli D11. The R. eutropha strain contained chromosomal genes necessary for mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D), while the E. coli strain did not. The soil system was contaminated with 2,4-D alone or was cocontaminated with 2,4-D and Cd. Plasmid transfer to indigenous populations, plasmid persistence in soil, and degradation of 2,4-D were monitored over a 63-day period in the bioreactors. To assess the impact of contaminant reexposure, aliquots of bioreactor soil were reamended with additional 2,4-D. Both introduced donors remained culturable and transferred plasmid pJP4 to indigenous recipients, although to different extents. Isolated transconjugants were members of the Burkholderia and Ralstonia genera, suggesting multiple, if not successive, plasmid transfers. Upon a second exposure to 2,4-D, enhanced degradation was observed for all treatments, suggesting microbial adaptation to 2,4-D. Upon reexposure, degradation was most rapid for the E. coli D11-inoculated treatments. Cd did not significantly impact 2,4-D degradation or transconjugant formation. This study demonstrated that the choice of donor microorganism might be a key factor to consider for bioaugmentation efforts. In addition, the establishment of an array of stable indigenous plasmid hosts at sites with potential for reexposure or long-term contamination may be particularly useful.