Mark D. Burr

Genotypic Microbial Community Profiling: A Critical Technical Review

Microbial ecology has undergone a profound change in the last two decades with regard to methods employed for the analysis of natural communities. Emphasis has shifted from culturing to the analysis of signature molecules including molecular DNA-based approaches that rely either on direct cloning and sequencing of DNA fragments (shotgun cloning) or often rely on prior amplification of target sequences by use of the polymerase chain reaction (PCR). The pool of PCR products can again be either cloned and sequenced or can be subjected to an increasing variety of genetic profiling methods, including amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. In this document, we present and critically compare these methods commonly used for the study of microbial diversity.

Use of Propidium Monoazide for Live/Dead Distinction in Microbial Ecology

ABSTRACT One of the prerequisites of making ecological conclusions derived from genetic fingerprints is that bacterial community profiles reflect the live portion of the sample of interest. Propidium monoazide is a membrane-impermeant dye that selectively penetrates cells with compromised membranes, which can be considered dead. Once inside the cells, PMA intercalates into the DNA and can be covalently cross-linked to it, which strongly inhibits PCR amplification. By using PCR after PMA treatment, the analysis of bacterial communities can theoretically be limited to cells with intact cell membranes. Four experiments were performed to study the usefulness of PMA treatment of mixed bacterial communities comprising both intact and compromised cells in combination with end-point PCR by generating community profiles from the following samples: (i) defined mixtures of live and isopropanol-killed cells from pure cultures of random environmental isolates, (ii) wastewater treatment plant influent spiked with defined ratios of live and dead cells, (iii) selected environmental communities, and (iv) a water sediment sample exposed to increasing heat stress. Regions of 16S rRNA genes were PCR amplified from extracted genomic DNA, and PCR products were analyzed by using denaturing gradient gel electrophoresis (DGGE). Results from the first two experiments show that PMA treatment can be of value with end-point PCR by suppressing amplification of DNA from killed cells. The last two experiments suggest that PMA treatment can affect banding patterns in DGGE community profiles and their intensities, although the intrinsic limitations of end-point PCR have to be taken into consideration.

Development and structure of drinking water biofilms and techniques for their study

Drinking water systems are known to harbour biofilms, even though these environments are oligotrophic and often contain a disinfectant. Control of these biofilms is important for aesthetic and regulatory reasons. Study of full‐scale systems has pointed to several factors controlling biofilm growth, but cause‐and‐effect relationships can only be established in controlled reactors. Using laboratory and pilot distribution systems, along with a variety of bacterial detection techniques, insights have been gained on the structure and behaviour of biofilms in these environments. Chlorinated biofilms differ in structure from non‐chlorinated biofilms, but often the number of cells is similar. The number and level of cellular activity is dependent on the predominant carbon source. There is an interaction between carbon sources, the biofilm and the type of pipe material, which complicates the ability to predict biofilm growth. Humic substances, which are known to sorb to surfaces, appear to be a usable carbon source for biofilms. The finding offers an explanation for many of the puzzling observations in full scale and laboratory studies on oligotrophic biofilm growth. Pathogens can persist in these environments as well. Detection requires methods that do not require culturing.

Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA

ABSTRACT We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing Bacteria and Eukarya taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and Bacteria-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the Firmicutes and the α-Proteobacteria. Unexpectedly, there were no Archaea clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and Archaea-biased primers and unmodified RNA demonstrated the presence of novel Archaea in the soil. Experiments with pure cultures of Sulfolobus solfataricus and Halobacterium halobium revealed that some Archaea rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.

Influence of Season and Plant Species on the Abundance and Diversity of Sulfate Reducing Bacteria and Ammonia Oxidizing Bacteria in Constructed Wetland Microcosms

Constructed wetlands offer an effective means for treatment of wastewater from a variety of sources. An understanding of the microbial ecology controlling nitrogen, carbon and sulfur cycles in constructed wetlands has been identified as the greatest gap for optimizing performance of these promising treatment systems. It is suspected that operational factors such as plant types and hydraulic operation influence the subsurface wetland environment, especially redox, and that the observed variation in effluent quality is due to shifts in the microbial populations and/or their activity. This study investigated the biofilm associated sulfate reducing bacteria and ammonia oxidizing bacteria (using the dsrB and amoA genes, respectively) by examining a variety of surfaces within a model wetland (gravel, thick roots, fine roots, effluent), and the changes in activity (gene abundance) of these functional groups as influenced by plant species and season. Molecular techniques were used including quantitative PCR and denaturing gradient gel electrophoresis (DGGE), both with and without propidium monoazide (PMA) treatment. PMA treatment is a method for excluding from further analysis those cells with compromised membranes. Rigorous statistical analysis showed an interaction between the abundance of these two functional groups with the type of plant and season (p < 0.05). The richness of the sulfate reducing bacterial community, as indicated by DGGE profiles, increased in planted vs. unplanted microcosms. For ammonia oxidizing bacteria, season had the greatest impact on gene abundance and diversity (higher in summer than in winter). Overall, the primary influence of plant presence is believed to be related to root oxygen loss and its effect on rhizosphere redox.

Floating treatment wetlands for domestic wastewater treatment

Floating islands are a form of treatment wetland characterized by a mat of synthetic matrix at the water surface into which macrophytes can be planted and through which water passes. We evaluated two matrix materials for treating domestic wastewater, recycled plastic and recycled carpet fibers, for chemical oxygen demand (COD) and nitrogen removal. These materials were compared to pea gravel or open water (control). Experiments were conducted in laboratory scale columns fed with synthetic wastewater containing COD, organic and inorganic nitrogen, and mineral salts. Columns were unplanted, naturally inoculated, and operated in batch mode with continuous recirculation and aeration. COD was efficiently removed in all systems examined (>90% removal). Ammonia was efficiently removed by nitrification. Removal of total dissolved N was ∼50% by day 28, by which time most remaining nitrogen was present as NO(3)-N. Complete removal of NO(3)-N by denitrification was accomplished by dosing columns with molasses. Microbial communities of interest were visualized with denaturing gradient gel electrophoresis (DGGE) by targeting specific functional genes. Shifts in the denitrifying community were observed post-molasses addition, when nitrate levels decreased. The conditioning time for reliable nitrification was determined to be approximately three months. These results suggest that floating treatment wetlands are a viable alternative for domestic wastewater treatment.

Temperature, plant species and residence time effects on nitrogen removal in model treatment wetlands

Total nitrogen (TN) removal in treatment wetlands (TWs) is challenging due to nitrogen cycle complexity and the variation of influent nitrogen species. Plant species, season, temperature and hydraulic loading most likely influence root zone oxygenation and appurtenant nitrogen removal, especially for ammonium-rich wastewater. Nitrogen data were collected from two experiments utilizing batch-loaded (3-, 6-, 9- and 20-day residence times), sub-surface TWs monitored for at least one year during which temperature was varied between 4 and 24 °C. Synthetic wastewater containing 17 mg/l N as NH4 and 27 mg/l amino-N, 450 mg/l chemical oxygen demand (COD), and 13 mg/l SO4-S was applied to four replicates of Carex utriculata, Schoenoplectus acutus and Typha latifolia and unplanted controls. Plant presence and species had a greater effect on TN removal than temperature or residence time. Planted columns achieved approximately twice the nitrogen removal of unplanted controls (40-95% versus 20-50% removal) regardless of season and temperature. TWs planted with Carex outperformed both Typha and Schoenoplectus and demonstrated less temperature dependency. TN removal with Carex was excellent at all temperatures and residence times; Schoenoplectus and Typha TN removal improved at longer residence times. Reductions in TN were not accompanied by increases in NO3, which was consistently below 1 mg/l N.

Pathogens in Water and Biofilms

Abstract Biofilms can play a major role for waterborne pathogens. Apart from being a ‘meeting place’ allowing for interaction with other microorganisms (bacteria, protozoa, viruses) and exchange of genetic material, the incorporation of pathogens into biofilms can pose a couple of distinct advantages compared with a planktonic lifestyle. This chapter aims to give an overview of current knowledge in this area and includes information about the efficiencies of different pathogens to form biofilms or to associate with existing biofilms and the consequences of being in this sheltered environment in regard to susceptibility to stress and disinfection, the pathogen’s phenotype and survival tendencies, and last but not least, for pathogenicity and infectivity.