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Item Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and Associated Biofilms on the Crow Reservation, Montana, USA(2018-07) Richards, Crystal L.; Broadaway, Susan C.; Eggers, Margaret J.; Doyle, John T.; Pyle, Barry H.; Camper, Anne K.; Ford, Tim E.Private residences in rural areas with water systems that are not adequately regulated, monitored, and updated could have drinking water that poses a health risk. To investigate water quality on the Crow Reservation in Montana, water and biofilm samples were collected from 57 public buildings and private residences served by either treated municipal or individual groundwater well systems. Bacteriological quality was assessed including detection of fecal coliform bacteria and heterotrophic plate count (HPC) as well as three potentially pathogenic bacterial genera, Mycobacterium, Legionella, and Helicobacter. All three target genera were detected in drinking water systems on the Crow Reservation. Species detected included the opportunistic and frank pathogens Mycobacterium avium, Mycobacterium gordonae, Mycobacterium flavescens, Legionella pneumophila, and Helicobacter pylori. Additionally, there was an association between HPC bacteria and the presence of Mycobacterium and Legionella but not the presence of Helicobacter. This research has shown that groundwater and municipal drinking water systems on the Crow Reservation can harbor potential bacterial pathogens.Item 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.Item 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 FilmTec’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-µmthick stained biofoulant cryo-sections, the accumulation of hydrated biofoulants on RO #1 and #3 were from 0.87 to 1.26µm/day, which was lower than that on RO#2 (2.19µm/day). Biofoulants increased the hydrophobicity of RO #2 to the greatest amount, up to 32°, 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.Item Community-based participatory research in Indian country: Improving health through water quality research and awareness(2010-07) Cummins, C.; Doyle, John T.; Kindness, L.; Lefthand, M. J.; Bear Don't Walk, U. J.; Bends, Ada L.; Broadaway, Susan C.; Camper, Anne K.; Fitch, R.; Ford, Tim E.; Hamner, Steve; Morrison, A. R.; Richards, Crystal L.; Young, Sara L.; Eggers, Margaret J.Water has always been held in high respect by the Apsaalooke (Crow) people of Montana. Tribal members questioned the health of the rivers and well water because of visible water quality deterioration and potential connections to illnesses in the community. Community members initiated collaboration among local organizations, the tribe, and academic partners, resulting in genuine community-based participatory research. The article shares what we have learned as tribal members and researchers about working together to examine surface and groundwater contaminants, assess routes of exposure, and use our data to bring about improved health of our people and our waters.Item Specific and rapid enumeration of viable but non-culturable and viable-culturable gram-negative bacteria using flow cytometry(2010-06) Khan, Mohiuddin M. T.; Pyle, Barry H.; Camper, Anne K.An issue of critical concern in microbiology is the ability to detect viable but non-culturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population and other options (direct viable count and double-staining method using epifluorescence microscopy and inhibitory substance influenced molecular methods) are also biased and time consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella typhimurium after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40 and propidium iodide (PI). The FCM data were compared with specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were non-culturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell-membranes and were therefore theoretically dead. Data obtained using four different gram-negative bacteria exposed to heat and stained with PI also illustrates the usefulness of the approach for the rapid and unbiased detection of dead vs. live organisms.Item 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.Item Comparing the chlorine disinfection of detached biofilm clusters with those of sessile biofilms and planktonic cells in single-and dual-species cultures(2011-10) Behnke, S.; Parker, Albert E.; Woodall, Dawn; Camper, Anne K.Although the detachment of cells from biofilms is of fundamental importance to the dissemination of organisms in both public health and clinical settings, the disinfection efficacies of commonly used biocides on detached biofilm particles have not been investigated. Therefore, the question arises whether cells in detached aggregates can be killed with disinfectant concentrations sufficient to inactivate planktonic cells. Burkholderia cepacia and Pseudomonas aeruginosa were grown in standardized laboratory reactors as single species and in coculture. Cluster size distributions in chemostats and biofilm reactor effluent were measured. Chlorine susceptibility was assessed for planktonic cultures, attached biofilm, and particles and cells detached from the biofilm. Disinfection tolerance generally increased with a higher percentage of larger cell clusters in the chemostat and detached biofilm. Samples with a lower percentage of large clusters were more easily disinfected. Thus, disinfection tolerance depended on the cluster size distribution rather than sample type for chemostat and detached biofilm. Intact biofilms were more tolerant to chlorine independent of species. Homogenization of samples led to significantly increased susceptibility in all biofilm samples as well as detached clusters for single-species B. cepacia, B. cepacia in coculture, and P. aeruginosa in coculture. The disinfection efficacy was also dependent on species composition; coculture was advantageous to the survival of both species when grown as a biofilm or as clusters detached from biofilm but, surprisingly, resulted in a lower disinfection tolerance when they were grown as a mixed planktonic culture.Item Viable real-time PCR in environmental samples: Can all data be interpreted directly?(2011-01) Fittipaldi, M.; Codony, F.; Adrados, B.; Camper, Anne K.; Morato, J.Selective nucleic acid intercalating dyes—ethidium monoazide (EMA) and propidium monoazide (PMA)—represent one of the most successful recent approaches to detect viable cells (as defined by an intact cell membrane) by PCR and have been effectively evaluated in different microorganisms. However, some practical limitations were found, especially in environmental samples. The aim of this work was to show that in the application of viable real-time PCR, there may be significant biases and to propose a strategy for overcoming some of these problems. We present an approach based on the combination of three real-time PCR amplifications for each sample that should provide an improved estimation of the number of viable cells. This approach could be useful especially when it is difficult to determine a priori how to optimize methods using PMA or EMA. Although further studies are required to improve viable real-time PCR methods, the concept as outlined here presents an interesting future research direction.Item Membrane fouling due to dynamic particle size changes in the aerated hybrid PAC–MF system(2011-04) Khan, Mohiuddin M. T.; Takizawa, S.; Lewandowski, Zbigniew; Jones, Warren L.; Camper, Anne K.; Katayama, H.; Ohgaki, S.To quantify the effect of dynamic particle size changes and degradation and accumulation of suspended solids (SS) in influents to reactors on membrane fouling frequency in hybrid powder-activated carbon (PAC)–microfiltration (MF) reactors, we operated a PAC–MF system (hollow-fiber module) for more than five months to purify river water before and after pretreatment by a biofilter. The transmembrane pressure, backwashing pressure, resistance to filtration, and SS accumulation and degradation during these dynamic changes were evaluated. The initial dose of PAC was 40 g/L of the reactor and no additional PAC was added during this continuous operational period. The presence of PAC reduced the membrane resistance to filtration even at the end of filtration period when the number of particles in the smallest range (>1.0–3.6 μm) was the highest measured by the flow cytometer and microscopy image analysis. This resistance was reduced further when the river water was biofiltered prior to membrane filtration. This real-time study demonstrates that over time PAC and other particles coming into the reactors through the influents degrade and/or become smaller because of the turbulence caused by continuous aeration below the MF membrane fibers. The number of particles in the reactors with diameters less than 10 μm increased with time, increasing the fouling frequency; however, the presence of PAC further reduced the particle enhanced fouling. The presence of PAC also increased SS degradation by up to 10%. The increased number of bacteria inside the PAC–MF systems did not contribute to the number of membrane fouling. Even though the particle sizes inside the reactors became smaller with time, the gradual increase in net accumulation of SS was also an important factor controlling the performance of the PAC–MF system.Item 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.