Browsing by Author "Burr, Mark D."

Now showing 1 - 8 of 8

Are they alive? Detection of viable organisms and functional gene expression using molecular techniques
(2010) Rochelle, Paul A.; Camper, Anne K.; Nocker, Andreas; Burr, Mark D.
The ultimate measure of microbial viability and biological activity is growth in some form of culture system. Unfortunately, due to many limitations, growth is usually not the most sensitive or rapid detection method. This chapter describes many of the molecular-based tools for assessing viability and functional gene expression, and their applications for specific microbes in environmental samples. Methods include fluorescent nucleic acid binding dyes, enzymatic conversion of substrates to fluorescent compounds (often in conjunction with nucleic acid-based methods), various techniques based on amplification and detection of nucleic acids, nucleic acid amplification linked to biosensors and microarray detection platforms, detection and characterization of proteins, and molecular detection coupled with culturing. Principles supporting each of these techniques are discussed along with applications to bacteria, protozoa, and viruses, focusing primarily on microbes of concern to the drinking water and wastewater industries.
Assessing biofouling on polyamide reverse osmosis (RO) membrane surfaces in a laboratory system
(2010-04) Khan, Mohiuddin M. T.; Stewart, Philip S.; Moll, D. J.; Mickols, W. E.; Burr, Mark D.; Nelson, Sara E.; Camper, Anne K.
Biofouling of reverse osmosis (RO) membranes is a major impediment in both wastewater reuse and desalination of sea/brackish waters. A benefit to the industry would be a simple screening approach to evaluate biofouling resistant RO membranes for their propensity to biofoulants. To observe the relationship between initial membrane productivity and control of biofilm formation governed by surface modification to the aromatic polyamide thin-film composite RO membranes, three different RO membranes developed by the FilmTec Corporation including FilmTecâ€™s commercial membrane BW30 (RO#1) and two experimental membranes (RO #2 and #3) were used. RO #2 and RO #3 were modified with a proprietary aliphatic group and with an extra proprietary aromatic group, respectively. Membrane swatches were fixed on coupons in rotating disk reactor systems without filtration and exposed to water with indigenous organisms supplemented with 1.5 mg/L organic carbon under continuous flow. After biofouling had developed, the membranes were sacrificed and subjected to several analyses. Staining and epifluorescence microscopy revealed more cells on RO #2 and #3 compared to RO #1. Based on image analysis of 5-Âµmthick stained biofoulant cryo-sections, the accumulation of hydrated biofoulants on RO #1 and #3 were from 0.87 to 1.26Âµm/day, which was lower than that on RO#2 (2.19Âµm/day). Biofoulants increased the hydrophobicity of RO #2 to the greatest amount, up to 32Â°, as determined by contact angle. In addition, a wide range of changes of the chemical elements of the RO surfaces was observed with X-ray photoelectron spectroscopy analysis. RO #2 with the highest initial membrane productivity showed the poorest biofouling resistance. A combination of these novel approaches showed good agreement and suggested that membrane productivity, heterogeneity of anti-biofouling agents on membrane surface, stability of surface chemical elements and the role of virgin RO surface hydrophobicity should be jointly considered during the development of anti-biofouling polyamide thin-film RO surfaces.
Denaturing gradient gel electrophoresis (DGGE) can rapidly display the bacterial diversity contained in 16S rDNA clone libraries
(2006-04) Burr, Mark D.; Clark, Stewart James; Camper, Anne K.
Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.
Development and structure of drinking water biofilms and techniques for their study
(1999-12) Camper, Anne K.; Burr, Mark D.; Ellis, B. D.; Butterfield, Phillip W.; Abernathy, Calvin G.
Floating treatment wetlands for domestic wastewater treatment
(2011-11) Faulwetter, J. L.; Burr, Mark D.; Cunningham, Alfred B.; Stewart, Frank M.; Camper, Anne K.; Stein, Otto R.
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 NO3-N. Complete removal of NO3-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.
Influence of season and plant species on the abundance and diversity of sulfate reducing bacteria and ammonia oxidizing bacteria in constructed wetland microcosms
(2013-01) Faulwetter, J. L.; Burr, Mark D.; Parker, Albert E.; Stein, Otto R.; Camper, Anne K.
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.
Temperature, plant species and residence time effects on nitrogen removal in model treatment wetlands
(2013-12) Allen, Christopher Robert; Stein, Otto R.; Hook, Paul B.; Burr, Mark D.; Parker, Albert E.; Hafla, Erin C.
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 Â°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 (40â€“95% versus 20â€“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.
Variability in presence-absence scoring of AP PCR fingerprints affects computer matching of bacterial isolates
(1997-04) Burr, Mark D.; Pepper, I. L.
Sources of variation in scoring bands in arbitrarily primed PCR (AP PCR) fingerprints of bacterial isolates were identified, and their effect on computer matching of fingerprints was determined. E. coli and five Salmonella serotypes were fingerprinted. PCR reactions and gel electrophoresis analysis of PCR products were replicated, including comparisons in the same gel and in different gels. Bands in the images were assigned by two different people on a presence–absence basis. Variations in scoring the images occurred at all levels, and prevented correct identification of isolates. E. coli was distinguished from Salmonella, but discrimination among different Salmonella serotypes and between two isolates of the same serotype was poor. Our results suggest that computer analysis of AP PCR fingerprints scored on a presence–absence basis may not correctly match isolates. Side-by-side visual comparison of isolates is recommended.