Browsing by Author "Camper, Anne K."

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Analysis of bacterial spatial patterns at the initial stage of biofilm formation
(1995) Hamilton, Martin A.; Johnson, K. R.; Camper, Anne K.; Stoodley, Paul; Harkin, Gary; Gillis, Richard J.; Shope, Paul A.
Apparent surface associated lag time in growth of primary biofilm cells
(2000-07) Rice, A. R.; Hamilton, Martin A.; Camper, Anne K.
The ability of microorganisms to form biofilms has been well documented. Bacterial cells make a transition from a planktonic state to a sessile state, replicate, and subsequently populate a surface. In this study, organisms that initially colonize a ``clean'' surface are referred to as ``primary'' biofilm cells. The progeny of the first generation of sessile cells are known as ``secondary'' biofilm cells. This study examined the growth of planktonic, primary, and secondary biofilm cells of a green fluorescent protein producing (GFP+) Pseudomonas aeruginosa PA01. Biofilm experiments were performed in a parallel plate flow cell reactor with a glass substratum. Individual cells were tracked over time using a confocal scanning laser microscope (CSLM). Primary cells experience a lag in their growth that may be attributed to adapting to a sessile environment or undergoing a phenotypic change. This is referred to as a surface associated lag time. Planktonic and secondary biofilm cells both grew at a faster rate than the primary biofilm cells under the same nutrient conditions.
Archaeal ammonium oxidation coupled with bacterial nitrite oxidation in a simulated drinking water premise plumbing system
(2016-01) Santillana, G. E.; Smith, Heidi J.; Burr, Mary; Camper, Anne K.
Simulated copper and PVC premise plumbing reactors modeling chloramine decay were monitored for complete nitrification of 0.71 mg NH4-N L−1 ammonium to nitrate with no nitrite detected. PCR, qPCR, fluorescent in situ hybridization (FISH) and DNA sequencing were used to investigate the microbial community responsible for nitrification in the reactors' influent and biofilm on copper and PVC surfaces. No bacterial ammonium oxidizers were detected by directly targeting the bacterial amoA gene or 16S rRNA gene amplicons. FISH images indicated an archaeal population on both surfaces. Archaeal 16S rRNA and amoA gene sequences showed 98.6% and 87.6% similarity to the known archaeal ammonium oxidizer, Candidatus Nitrosotenuis uzonenis. Copy numbers of the archaeal 16S rRNA gene and archaeal amoA approximated a 1:1 ratio, suggesting that any archaea in the systems are likely to be ammonium oxidizers. Further, there was evidence for the presence of bacterial nitrite oxidizers. Copper surfaces supported fewer archaea as detected using the archaeal 16S rRNA and amoA genes. The results provide strong evidence for biofilms in a drinking water premise plumbing system composed of archaeal ammonium oxidizers and bacterial nitrite oxidizers, capable of complete oxidation of ammonium to nitrate. Since no bacterial ammonium oxidizers were found, this study adds to the growing body of research indicating an important role for archaeal ammonium oxidizers in freshwater/drinking water environments in the conversion of ammonium to nitrite.
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.
Assessing technician effects when extracting quantities from microscope images
(2003-04) Webb, D.; Hamilton, Martin A.; Harkin, Gary; Lawrence, S.; Camper, Anne K.; Lewandowski, Zbigniew
Consider an experiment where the response is based on an image; e.g., an image captured to a computer file by a digital camera mounted on a microscope. Suppose relevant quantitative measures are extracted from the images so that results can be analyzed by conventional statistical methods. The steps involved in extracting the measures may require that the technicians, who are processing the images, perform some subjective manipulations. In this case, it is important to determine the bias and variability, if any, attributable to the technicians' decisions. This paper describes the experimental design and statistical analyses that are useful for those determinations. The design and analysis are illustrated by application to two biofilm research projects that involved quantitative image analysis. In one investigation, the technician was required to choose a threshold level, then the image analysis program automatically extracted relevant measures from the resulting black and white image. In the other investigation, the technician was required to choose fiducial points in each of two images collected on different microscopes; then the image analysis program registered the images by stretching, rotating, and overlaying them, so that their quantitative features could be correlated. These investigations elucidated the effects of the technicians' decisions, thereby helping us to assess properly the statistical uncertainties in the conclusions for the primary experiments.
Assessment of the ability of the bioelectric effect to eliminate mixed-species biofilms
(2005-10) Shirtliff, Mark E.; Bargmeyer, Alex Martin; Camper, Anne K.
Microbes have been able to persist in water distribution systems through the development of multicellular communities known as biofilms. This study evaluated the usefulness of the bioelectric effect for the elimination of water distribution system biofilms from annular reactors. The bioelectric effect did not have any bactericidal action either alone or when coupled with free chlorine.
Attachment and fate of carbon fines in simulated drinking water distribution system biofilms
(1997-03) Morin, P.; Camper, Anne K.
Concern over the release of colonized carbon fines from filters has increased with the interest in operating granular activated carbon filters optimized for biological activity. These fines may transport bacteria to the distribution system and become entrained in biofilms. The capture and release of carbon fines of two size ranges (1.2–50 μm and 1.2–8 μm) in biofilms and their impact on the biomass was studied using a bench-scale simulated drinking water system. Size of the particles was important, with large carbon fines (1.2–50 μm) remaining in the biofilms while the small ones (1.2–8 μm) were entrained and then detached. The presence of carbon fines did not influence the number or activity of bacteria present in the biofilms. Disinfection assays on biofilms containing particles were performed with chlorine (0.5 mg/L) and monochloramine (1 mg/L). Even in the presence of substantial numbers of carbon fines, the effect of the disinfectants on the biofilms as determined by destructive analysis and direct microscopic observation was limited. Carbon fines were released from the biofilms during disinfection.
Bacteria associated with granular activated carbon particles in drinking water
(1986-09) Camper, Anne K.; LeChevallier, Mark W.; Broadaway, Susan C.; McFeters, Gordon A.
A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barries and enter potable water supplies.
Bacterial colonization of surfaces in flowing systems: methods and analysis
(1994) Camper, Anne K.; Hamilton, Martin A.; Johnson, K. R.; Stoodley, Paul; Harkin, Gary; Daly, Don Simone
Bacteriology of granular activated carbon
(2002) Camper, Anne K.
The goal of drinking water treatment has always been to provide microbiology safe water to the consumer. To accomplish this, especially when surface water is being treated, gravity filters containing sand and/or anthracite are typically used to remove particular matter. When filtration is followed by disinfection, pathogens should be removed and the microbial load of the water reduced to an acceptable level. As water quality regulations have become more restrictive, granular activated carbon (GAC) has been used to a greater extent as a filter medium because of its ability to adsorb regulated synthetic organic carbon compounds, materials that contribute to taste and odor in finished water, and organic matter that leads to the formation of disinfection by-products. GAC is known to become colonized with bacteria, and this trend has been exploited in treatment by using the organisms on biologically active carbon (BAC) to metabolize a fraction of the organic compound found in the water. However, there has been concern that indicator organisms and bacteria of public health interests may colonize and proliferate on GAC and BAC filters. For these bacteria to be found in finished water they must successfully colonize and compete with the indigenous organisms, be released from the filter and penetrate the disinfection barrier. This review describes these issues and the beneficial aspects of BAC filtration in water treatment.
Biofilm barriers to contain and degrade dissolved trichloroethylene
(2004-04) Komlos, John; Cunningham, Alfred B.; Camper, Anne K.; Sharp, Robert R.
Biologically produced subsurface barriers (i.e., biofilm barriers) are a viable technology for controlling contaminant migration from hazardous waste sites. Biofilm barriers are created through the injection of bacteria and selective growth medium into a series of wells downstream of a contaminant plume. Adequate substrate addition enables the bacteria to grow and form thick biofilms capable of uniform plugging of the subsurface. This technology has been successful in significantly reducing porous media permeability in bench-scale and field-scale applications. The research presented herein expands on current biofilm. barrier technology by examining the feasibility of using a biofilm barrier to not only control contaminant migration through permeability reduction, but also facilitate contaminant biodegradation. ne experimental scenario involved the creation of a dual-species biofilm matrix: one organism to reduce porous media permeability through thick biofilm formation and another organism to degrade a contaminant, in this case trichloroethylene (TCE). Porous medium column experiments demonstrated that a dual-species biofilm barrier can be created and that growth medium concentration was a very important variable in controlling simultaneous TCE degradation and permeability reduction.
Biofilms growing on gas permeable membranes
(1994) Rothemund, C.; Camper, Anne K.; Wilderer, P. A.
Biofilms in drinking water treatment and distribution
(2000) Camper, Anne K.
Challenges and Opportunities for Tribal Waters: Addressing Disparities in Safe Public Drinking Water on the Crow Reservation in Montana, US
(2018-03) Doyle, John T.; Kindness, L.; Real Bird, James; Eggers, Margaret J.; Camper, Anne K.
Disparities in access to safe public drinking water are increasingly being recognized as contributing to health disparities and environmental injustice for vulnerable communities in the United States. As the Co-Directors of the Apsaálooke Water and Wastewater Authority (AWWWA) for the Crow Tribe, with our academic partners, we present here the multiple and complex challenges we have addressed in improving and maintaining tribal water and wastewater infrastructure, including the identification of diverse funding sources for infrastructure construction, the need for many kinds of specialized expertise and long-term stability of project personnel, ratepayer difficulty in paying for services, an ongoing legacy of inadequate infrastructure planning, and lack of water quality research capacity. As a tribal entity, the AWWWA faces additional challenges, including the complex jurisdictional issues affecting all phases of our work, lack of authority to create water districts, and additional legal and regulatory gaps—especially with regards to environmental protection. Despite these obstacles, the AWWWA and Crow Tribe have successfully upgraded much of the local water and wastewater infrastructure. We find that ensuring safe public drinking water for tribal and other disadvantaged U.S. communities will require comprehensive, community-engaged approaches across a broad range of stakeholders to successfully address these complex legal, regulatory, policy, community capacity, and financial challenges.
Characterization and effect of biofouling on polyamide reverse osmosis and nanofiltration membrane surfaces
(2011-01) Khan, Mohiuddin M. T.; Stewart, Philip S.; Moll, D. J.; Mickols, W. E.; Nelson, Sara E.; Camper, Anne K.
Biofouling is a major reason for flux decline in the performance of membrane-based water and wastewater treatment plants. Initial biochemical characterization of biofilm formation potential and biofouling on two commercially available membrane surfaces from FilmTec Corporation were investigated without filtration in laboratory rotating disc reactor systems. These surfaces were polyamide aromatic thin-film reverse osmosis (RO) (BW30) and semi-aromatic nanofiltration (NF270) membranes. Membrane swatches were fixed on removable coupons and exposed to water with indigenous microorganisms supplemented with 1.5 mg l−1 organic carbon under continuous flow. After biofilms formed, the membrane swatches were removed for analyses. Staining and epifluorescence microscopy revealed more cells on the RO than on the NF surface. Based on image analyses of 5-μm thick cryo-sections, the accumulation of hydrated biofoulants on the RO and NF surfaces exceeded 0.74 and 0.64 μm day−1, respectively. As determined by contact angle the biofoulants increased the hydrophobicity up to 30° for RO and 4° for NF surfaces. The initial difference between virgin RO and NO hydrophobicities was ∼5°, which increased up to 25° after biofoulant formation. The initial roughness of RO and NF virgin surfaces (75.3 nm and 8.2 nm, respectively) increased to 48 nm and 39 nm after fouling. A wide range of changes of the chemical element mass percentages on membrane surfaces was observed with X-ray photoelectron spectroscopy. The initial chemical signature on the NF surface was better restored after cleaning than the RO membrane. All the data suggest that the semi-aromatic NF surface was more biofilm resistant than the aromatic RO surface. The morphology of the biofilm and the location of active and dead cell zones could be related to the membrane surface properties and general biofouling accumulation was associated with changes in the surface chemistry of the membranes, suggesting the validity of the combination of these novel approaches for initial assessment of membrane performance.
Characterization of phenotypic changes in pseudomonas putida in response to surface-associated growth
(2001-11) Sauer, K.; Camper, Anne K.
The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P. putida and pure C12-HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.
Chlorination of model drinking water biofilm: implications for growth and organic carbon removal
(2002-10) Butterfield, Phillip W.; Camper, Anne K.; Ellis, B. D.; Jones, Warren L.
The influence of chlorine on biofilm in low organic carbon environments typical of drinking water or industrial process water was examined by comparing biomass and kinetic parameters for biofilm growth in a chlorinated reactor to those in a non-chlorinated control. Mixed-population heterotrophic biofilms were developed in rotating annular reactors under low concentration, carbon-limited conditions (<2 mg/L as carbon) using three substrate groups (amino acids, carbohydrates and humic substances). Reactors were operated in parallel under identical conditions with the exception that chlorine was added to one reactor at a dose sufficient to maintain a free chlorine residual of 0.09¯0.15 mg/L in the effluent. The presence of free chlorine resulted in development of less biofilm biomass compared to the control for all substrates investigated. However, specific growth and organic carbon removal rates were on the average five times greater for chlorinated biofilm compared to the control. Observed yield values were less for chlorinated biofilm. Although chlorinated biofilm's specific organic carbon removal rate was high, the low observed yield indicated organic carbon was being utilized for purposes other than creating new cell biomass. The impacts of free chlorine on mixed-population biofilms in low-nutrient environments were different depending upon the available substrate. Biofilms grown using amino acids exhibited the least difference between control and chlorinated kinetic parameters; biofilm grown using carbohydrates had the greatest differences. These findings are particularly relevant to the fundamental kinetic parameters used in models of biofilm growth in piping systems that distribute chlorinated, low-carbon-concentration water.
Chlorine dioxide disinfection of single and dual species biofilms, detached biofilm and planktonic cells
(2012-07) Behnke, S.; Camper, Anne K.
Disinfection efficacy testing is usually done with planktonic cells or more recently, biofilms. While disinfectants are much less effective against biofilms compared to planktonic cells, questions regarding the disinfection tolerance of detached biofilm clusters remain largely unanswered. Burkholderia cepacia and Pseudomonas aeruginosa were grown in chemostats and biofilm tubing reactors, with the tubing reactor serving as a source of detached biofilm clusters. Chlorine dioxide susceptibility was assessed for B. cepacia and P. aeruginosa in these three sample types as monocultures and binary cultures. Similar doses of chlorine dioxide inactivated samples of chemostat and tubing reactor effluent and no statistically significant difference between the log10 reductions was found. This contrasts with chlorine, shown previously to be generally less effective against detached biofilm particles. Biofilms were more tolerant and required chlorine dioxide doses ten times higher than chemostat and tubing reactor effluent samples. A second species was advantageous in all sample types and resulted in lower log10 reductions when compared to the single species cultures, suggesting a beneficial interaction of the species.
Coliform regrowth and biofilm accumulation in drinking water systems: a review
(1993) Camper, Anne K.