Paige J. Novak

Effect of protein, polysaccharide, and oxygen concentration profiles on biofilm cohesiveness

ABSTRACT It is important to control biofilm cohesiveness to optimize process performance. In this study, a membrane-aerated biofilm reactor inoculated with activated sludge was used to grow mixed-culture biofilms of different ages and thicknesses. The cohesions, or cohesive energy levels per unit volume of biofilm, based on a reproducible method using atomic force microscopy (F. Ahimou, M. J. Semmens, P. J. Novak, and G. Haugstad, Appl. Environ. Microbiol. 73:2897-2904, 2007), were determined at different locations within the depths of the biofilms. In addition, the protein and polysaccharide concentrations within the biofilm depths, as well as the dissolved oxygen (DO) concentration profiles within the biofilms, were measured. It was found that biofilm cohesion increased with depth but not with age. Level of biofilm cohesive energy per unit volume was strongly correlated with biofilm polysaccharide concentration, which increased with depth in the membrane-aerated biofilm. In a 12-day-old biofilm, DO also increased with depth and may therefore be linked to polysaccharide production. In contrast, protein concentration was relatively constant within the biofilm and did not appear to influence cohesion.

A novel in situ technology for the treatment of nitrate contaminated groundwater.

A novel in situ membrane technology was developed to remove nitrate (NO3-) from groundwater. Membrane-fed hydrogen gas (H2) was used as an electron donor to stimulate denitrification. A flow-through reactor fit with six hollow-fiber membranes (surface area = 93 cm2) was designed to simulate groundwater flowing through an aquifer with a velocity of 0.3 m/day. This membrane technology supported excellent NO3- and nitrite (NO2-) removal once H2 and carbon limitations were corrected. The membrane module achieved a maximum H2 flux of 1.79 x 10(-2) mg H2/m2 s, which was sufficient to completely remove 16.4 mg/L NO3(-)-N from a synthetic groundwater with no NO2- accumulation. In addition, this model in situ treatment process produced a high quality water containing <0.5 mg/L total organic carbon.

Biofilm cohesiveness measurement using a novel atomic force microscopy methodology

ABSTRACT Biofilms can be undesirable, as in those covering medical implants, and beneficial, such as when they are used for waste treatment. Because cohesive strength is a primary factor affecting the balance between growth and detachment, its quantification is essential in understanding, predicting, and modeling biofilm development. In this study, we developed a novel atomic force microscopy (AFM) method for reproducibly measuring, in situ, the cohesive energy levels of moist 1-day biofilms. The biofilm was grown from an undefined mixed culture taken from activated sludge. The volume of biofilm displaced and the corresponding frictional energy dissipated were determined as a function of biofilm depth, resulting in the calculation of the cohesive energy. Our results showed that cohesive energy increased with biofilm depth, from 0.10 ± 0.07 nJ/μm3 to 2.05 ± 0.62 nJ/μm3. This observation was reproducible, with four different biofilms showing the same behavior. Cohesive energy also increased from 0.10 ± 0.07 nJ/μm3 to 1.98 ± 0.34 nJ/μm3 when calcium (10 mM) was added to the reactor during biofilm cultivation. These results agree with previous reports on calcium increasing the cohesiveness of biofilms. This AFM-based technique can be performed with available off-the-shelf instrumentation. It could therefore be widely used to examine biofilm cohesion under a variety of conditions.

Natural niche for organohalide-respiring Chloroflexi.

ABSTRACT The phylum Chloroflexi contains several isolated bacteria that have been found to respire a diverse array of halogenated anthropogenic chemicals. The distribution and role of these Chloroflexi in uncontaminated terrestrial environments, where abundant natural organohalogens could function as potential electron acceptors, have not been studied. Soil samples (116 total, including 6 sectioned cores) from a range of uncontaminated sites were analyzed for the number of Dehalococcoides-like Chloroflexi 16S rRNA genes present. Dehalococcoides-like Chloroflexi populations were detected in all but 13 samples. The concentrations of organochlorine ([organochlorine]), inorganic chloride, and total organic carbon (TOC) were obtained for 67 soil core sections. The number of Dehalococcoides-like Chloroflexi 16S rRNA genes positively correlated with [organochlorine]/TOC while the number of Bacteria 16S rRNA genes did not. Dehalococcoides-like Chloroflexi were also observed to increase in number with a concomitant accumulation of chloride when cultured with an enzymatically produced mixture of organochlorines. This research provides evidence that organohalide-respiring Chloroflexi are widely distributed as part of uncontaminated terrestrial ecosystems, they are correlated with the fraction of TOC present as organochlorines, and they increase in abundance while dechlorinating organochlorines. These findings suggest that organohalide-respiring Chloroflexi may play an integral role in the biogeochemical chlorine cycle.

The impacts of triclosan on anaerobic community structures, function, and antimicrobial resistance.

Triclosan is a widespread antimicrobial agent that accumulates in anaerobic digesters used to treat the residual solids generated at municipal wastewater treatment plants; there is very little information, however, about how triclosan impacts microbial communities in anaerobic digesters. We investigated how triclosan impacts the community structure, function and antimicrobial resistance genes in lab-scale anaerobic digesters. Previously exposed (to triclosan) communities were amended with 5, 50, and 500 mg/kg of triclosan, corresponding to the median, 95th percentile, and 4-fold higher than maximum triclosan concentration that has been detected in U.S. biosolids. Triclosan amendment caused all of the Bacteria and Archaea communities to structurally diverge from that of the control cultures (based on ARISA). At the end of the experiment, all triclosan-amended Archaea communities had diverged from the control communities, regardless of the triclosan concentration added. In contrast, over time the Bacteria communities that were amended with lower concentrations of triclosan (5 mg/kg and 50 mg/kg) initially diverged and then reconverged with the control community structure. Methane production at 500 mg/kg was nearly half the methane production in control cultures. At 50 mg/kg, a large variability in methane production was observed, suggesting that 50 mg/kg may be a tipping point where function begins to fail in some communities. When previously unexposed communities were exposed to 500 mg triclosan/kg, function was maintained, but the abundance of a gene encoding for triclosan resistance (mexB) increased. This research suggests that triclosan could inhibit methane production in anaerobic digesters if concentrations were to increase and may also select for resistant Bacteria. In both cases, microbial community composition and exposure history alter the influence of triclosan.

Sources and transport of contaminants of emerging concern: A two-year study of occurrence and spatiotemporal variation in a mixed land use watershed

The occurrence and spatiotemporal variation of 26 contaminants of emerging concern (CECs) were evaluated in 68 water samples in 2011-2012 in the Zumbro River watershed, Minnesota, U.S.A. Samples were collected across a range of seasonal/hydrological conditions from four stream sites that varied in associated land use and presence of an upstream wastewater treatment plant (WWTP). Selected CECs included human/veterinary pharmaceuticals, personal care products, pesticides, phytoestrogens, and commercial/industrial compounds. Detection frequencies and concentrations varied, with atrazine, metolachlor, acetaminophen, caffeine, DEET, and trimethoprim detected in more than 70% of samples, acetochlor, mecoprop, carbamazepine, and daidzein detected in 30%-50% of samples, and 4-nonylphenol, cotinine, sulfamethoxazole, erythromycin, tylosin, and carbaryl detected in 10%-30% of samples. The remaining target CECs were not detected in water samples. Three land use-associated trends were observed for the detected CECs. Carbamazepine, 4-nonylphenol, erythromycin, sulfamethoxazole, tylosin, and carbaryl profiles were WWTP-dominated, as demonstrated by more consistent loading and significantly greater concentrations downstream of the WWTP and during low-flow seasons. In contrast, acetaminophen, trimethoprim, DEET, caffeine, cotinine, and mecoprop patterns demonstrated both seasonally-variable non-WWTP-associated and continual WWTP-associated influences. Surface water studies of CECs often target areas near WWTPs. This study suggests that several CECs often characterized as effluent-associated have additional important sources such as septic systems or land-applied biosolids. Finally, agricultural herbicide (atrazine, acetochlor, and metolachlor) profiles were strongly influenced by agricultural land use and seasonal application-runoff, evident by significantly greater concentrations and loadings at upstream sites and in early summer when application and precipitation rates are greatest. Our results indicate that CEC monitoring studies should consider a range of land uses, seasonality, and transport pathways in relation to concentrations and loadings. This knowledge can augment CEC monitoring programs to result in more accurate source, occurrence, and ecological risk characterizations, more precisely targeted mitigation initiatives, and ultimately, enhanced environmental decision-making.

Contaminants of Emerging Concern: Mass Balance and Comparison of Wastewater Effluent and Upstream Sources in a Mixed-Use Watershed

Understanding the sources, transport, and spatiotemporal variability of contaminants of emerging concern (CECs) is important for understanding risks and developing monitoring and mitigation strategies. This study used mass balances to compare wastewater treatment plant (WWTP) and upstream sources of 16 CECs to a mixed-use watershed in Minnesota, under different seasonal and hydrological conditions. Three distinct CEC groups emerged with respect to their source proportionality and instream behavior. Agricultural herbicides and daidzein inputs were primarily via upstream routes with the greatest loadings and concentrations during high flows. Trimethoprim, mecoprop, nonprescription pharmaceuticals, and personal care products entered the system via balanced/mixed pathways with peak loadings and concentrations in high flows. Carbaryl, 4-nonylphenol, and the remaining prescription pharmaceuticals entered the system via WWTP effluent with relatively stable loadings across sampling events. Mass balance analysis based on multiple sampling events and sites facilitated CEC source comparisons and may therefore prove to be a powerful tool for apportioning sources and exploring mitigation strategies.

Effects of ethanol-based fuel contamination: microbial community changes, production of regulated compounds, and methane generation.

Ethanol-based fuels are becoming more heavily used, increasing the likelihood of ethanol-based fuel spills during transportation and storage. Although ethanol is well-known to be readily biodegradable, very little is known about the effects that such a spill might have on an indigenous microbial community. Of particular concern is that ethanol contamination could stimulate the growth of organisms that can generate regulated compounds and/or produce explosive quantities of methane gas. A column-based study was performed to elucidate the potential impacts of ethanol-based fuel (E85) on the indigenous microbial community during a simulated fuel spill. A continuous dilute supply of E85 resulted in profound shifts in both the bacterial and archaeal communities. The shift was accompanied by the production of high concentrations of volatile fatty acids and butanol, a compound that is regulated in groundwater by some states. Results also indicated that a continuous feed of dilute E85 generated explosive levels of methane within one month of column operation. Quantitative PCR data showed a statistically significant increase in methanogenic populations when compared to a control column. The elevated population numbers correlated to areas of the column receiving a sustained carbon load. Toxicity data indicated that microbial growth was completely inhibited (as evidenced by absence of ethanol breakdown products) at ethanol levels above 6% (v/v). These data suggest that ethanol from ethanol-based fuel can be readily degraded, but can also produce metabolic products that are regulated as well as explosive levels of methane. The core of an E85 spill may serve as a long-term source of contamination as it cannot be degraded until significant dilution has occurred.

Passive dissolution of hydrogen gas into groundwater using hollow-fiber membranes

A new hollow-fiber membrane remediation system has recently been developed to passively supply groundwater with dissolved hydrogen (H2) to stimulate the biodegradation of chlorinated solvents. Understanding the mass transfer behavior of membranes under conditions of creeping flow is critical for the design of such systems. Therefore, the objectives of this research were to evaluate the gas transfer behavior of hollow-fiber membranes under conditions typical of groundwater flow and to assess the effect of membrane configuration on gas transfer performance. Membrane gas transfer was evaluated using laboratory-scale glass columns operated at low flow velocities (8.6-12,973 cm/d). H2 was supplied to the inside of the membrane fibers while water flowed on the outside and normal to the fibers (i.e. cross-flow). Membrane configuration (single fiber and fabric) and membrane spacing for the fabric modules did not affect gas transfer performance. Therefore, the results from all of the experiments were combined to obtain the following dimensionless Sherwood number (Sh) correlation expressed as a function of Reynolds number (Re) and Schmidt number (Sc): Sh = 0.824Re(0.39)Sc(0.33) (0.0004<Re<0.6). This correlation is useful for predicting the rate of transfer of any gas from clean membranes to flowing water at low Re. This correlation provides a basis for estimating the membrane surface area requirements for groundwater remediation as illustrated by a simple example.

The role of biodegradation in limiting the accumulation of petroleum hydrocarbons in raingarden soils

Previous studies have indicated that raingardens are effective at removing petroleum hydrocarbons from stormwater. There are concerns, however, that petroleum hydrocarbons could accumulate in raingarden soil, potentially resulting in liability for the site owner. In this work, 75 soil samples were collected from 58 raingardens and 4 upland (i.e., control) sites in the Minneapolis, Minnesota area, representing a range of raingarden ages and catchment land uses. Total petroleum hydrocarbon (TPH) concentrations in the samples were quantified, as were 16S rRNA genes for Bacteria and two functional genes that encode for enzymes used in the degradation of petroleum hydrocarbons. TPH levels in all of the raingarden soil samples were low (<3 μg/kg) and not significantly different from one another. The TPH concentration in raingarden soil samples was, however, significantly greater (p ≤ 0.002) than TPH levels in upland sites. In addition, the number of copies of Bacteria 16S rRNA genes and functional genes were greater in the raingardens planted with deeply-rooted natives and cultivars than in raingardens containing simply turf grass or mulch (p < 0.036), suggesting that planted raingardens may be better able to assimilate TPH inputs. The ability of microorganisms present in the soil samples to degrade a representative petroleum hydrocarbon (naphthalene) was also investigated in batch experiments. A sub-set of the field sites was selected for re-sampling, and all soil samples tested (n = 8) were able to mineralize naphthalene. In these experiments the initial mineralization rate correlated with the number of copies of Bacteria 16S rRNA genes present.