Identifying fecal sources in a selected catchment reach using multiple source-tracking tools

Given known limitations of current microbial source-tracking (MST) tools, emphasis on small, simple study areas may enhance interpretations of fecal contamination sources in streams. In this study, three MST tools-Escherichia coli repetitive element polymerase chain reaction (rep-PCR), coliphage typing, and Bacteroidales 16S rDNA host-associated markers-were evaluated in a selected reach of Plum Creek in south-central Nebraska. Water-quality samples were collected from six sites. One reach was selected for MST evaluation based on observed patterns of E. coli contamination. Despite high E. coli concentrations, coliphages were detected only once among water samples, precluding their use as a MST tool in this setting. Rep-PCR classification of E. coli isolates from both water and sediment samples supported the hypothesis that cattle and wildlife were dominant sources of fecal contamination, with minor contributions by horses and humans. Conversely, neither ruminant nor human sources were detected by Bacteroidales markers in most water samples. In bed sediment, ruminant- and human-associated Bacteroidales markers were detected throughout the interval from 0 to 0.3 m, with detections independent of E. coli concentrations in the sediment. Although results by E. coli-based and Bacteroidales-based MST methods led to similar interpretations, detection of Bacteroidales markers in sediment more commonly than in water indicates that different tools to track fecal contamination (in this case, tools based on Bacteroidales DNA and E. coli isolates) may have varying relevance to the more specific goal of tracking the sources of E. coli in watersheds. This is the first report of simultaneous, toolbox approach application of a library-based and marker-based MST analyses to flowing surface water.     

Effect of Changing VOC Influent Composition on the Microbial Community Structure of TBABs

Microbial communities in trickle bed air biofilters (TBABs) were evaluated under conditions of interchanging the feed volatile organic compounds (VOCs) and VOC mixtures. Three independent TBABs (Biofilter “A,” “B,” and “C”) were run under interchanging VOCs conditions with different initial VOCs. Two aromatic compounds (toluene and styrene) and two oxygenated compounds (methyl ethyl ketone (MEK) and methyl isobutyl ketone (MIBK)) were interchanged as single solutes. Two other TBABs “D” and “E” were run for two VOC mixtures. Biofilter “D” had a VOC mixture with equal molar ratio of the four components and Biofilter “E” received a VOC mixture with its composition based on EPA 2003 emission report. Denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA genes was used to assess the microbial richness in TBABs for treating the VOC mixtures and the impact of interchanging VOCs on the bacterial community structure in the biofilters. The results from DGGE indicated that the microbial community structure in the biofilter was different after each interchange of VOCs. Some bands of microbial species faded and some bands were strengthened. For the two TBABs treating VOC mixtures, the microbial species did not show significant difference, but the richness among these species was different from each other.     

16S ribosomal RNA tools identify an unexpected predominance of Paenibacillus-like bacteria in an industrial activated sludge system suffering from poor biosolids separation.

Molecular biology tools targeting 16S ribosomal RNA (16S rRNA) were used to identify a predominant bacterial population in a full-scale dairy wastewater activated sludge system suffering from poor biosolids separation. Gram and acridine orange staining indicated that viable, Gram-positive microorganisms were present in samples removed from the influent waste stream and represented approximately 50% of total cell counts in samples removed from the mixed liquor. Subsequently, the "full-cycle 16S rRNA approach" showed that phylogenetic relatives of Paenibacillus spp., a low guanine-plus-cytosine percent DNA-content, Gram-positive microorganism, represented up to 30% of total 4,6-diamidino-2-phenylindole (DAPI)-stained cell counts in samples of mixed liquor. Although fluorescent in situ hybridizations with 16S rRNA-targeted oligonucleotide hybridization probes identified Paenibacillus-like spp. in samples removed from the influent waste stream, their abundance was less than 10% of total stained cell counts. Results of this study suggest that Paenibacillus-like spp. were present in low abundance in the influent waste stream, increased in relative abundance within the treatment system, and should be examined further as a candidate bacterial population responsible for poor biosolids separation. This study demonstrates that the full-cycle 16S rRNA approach can be used to identify candidate bacterial populations that may be responsible for operational upsets in full-scale activated sludge systems without prior information from cultivation or microscopic analyses.     

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.