Quantification of depth-dependent microbial growth in encapsulated systems.

Encapsulated systems have been widely used in environmental applications to selectively retain and protect microorganisms. The permeable matrix used for encapsulation, however, limits the accessibility of existing analytical methods to study the behaviour of the encapsulated microorganisms. Here, we present a novel method that overcomes these limitations and enables direct observation and enumeration of encapsulated microbial colonies over a range of spatial and temporal scales. The method involves embedding, cross-sectioning, and analysing the system via fluorescence in situ hybridization and retains the structure of encapsulants and the morphology of encapsulated colonies. The major novelty of this method lies in its ability to distinguish between, and subsequently analyse, multiple microorganisms within a single encapsulation matrix across depth. Our results demonstrated the applicability and repeatability of this method with alginate-encapsulated pure (Nitrosomonas europaea) and enrichment cultures (anammox enrichment). The use of this method can potentially reveal interactions between encapsulated microorganisms and their surrounding matrix, as well as quantitatively validate predictions from mathematical models, thereby advancing our understanding of microbial ecology in encapsulated or even biofilm systems and facilitating the optimization of these systems.

The potential for bacteria from carbon-limited deep terrestrial environments to participate in chlorine cycling.

Bacteria capable of dehalogenation via reductive or hydrolytic pathways are ubiquitous. Little is known, however, about the prevalence of bacterial dechlorination in deep terrestrial environments with a limited carbon supply. In this study we analyzed published genomes from three deep terrestrial subsurface sites: a deep aquifer in Western Siberia, the Sanford Underground Research Facility in South Dakota, USA, and the Soudan Underground Iron Mine (SUIM) in Minnesota, USA to determine if there was evidence to suggest that microbial dehalogenation was possible in these environments. Diverse dehalogenase genes were present in all analyzed metagenomes, with reductive dehalogenase and haloalkane dehalogenase genes the most common. Taxonomic analysis of both hydrolytic and reductive dehalogenase genes was performed to explore their affiliation; this analysis indicated that at the SUIM site, hydrolytic dehalogenase genes were taxonomically affiliated with Marinobacter species. Because of this affiliation, experiments were also performed with Marinobacter subterrani strain JG233 ('JG233'), an organism containing three predicted hydrolytic dehalogenase genes and isolated from the SUIM site, to determine whether hydrolytic dehalogenation was an active process and involved in growth on a chlorocarboxylic acid. Presence of these genes in genome appears to be functional, as JG233 was capable of chloroacetate dechlorination with simultaneous chloride release. Stable isotope experiments combined with confocal Raman microspectroscopy demonstrated that JG233 incorporated carbon from 13C-chloroacetate into its biomass. These experiments suggest that organisms present in these extreme and often low-carbon environments are capable of reductive and hydrolytic dechlorination and, based on laboratory experiments, may use this capability as a competitive advantage by utilizing chlorinated organic compounds for growth, either directly or after dechlorination.

Unraveling encapsulated growth of Nitrosomonas europaea in alginate: An experimental and modeling study.

Encapsulation is a promising technology to retain and protect autotrophs for biological nitrogen removal. One-dimensional biofilm models have been used to describe encapsulated systems; they do not, however, incorporate chemical sorption to the encapsulant nor do they adequately describe cell growth and distribution within the encapsulant. In this research we developed a new model to describe encapsulated growth and activity of Nitrosomonas europaea, incorporating ammonium sorption to the alginate encapsulant. Batch and continuous flow reactors were used to verify the simulation results. Quantitative PCR and cross-section fluorescence in situ hybridization were used to analyze the growth and spatial distribution of the encapsulated cells within alginate. Preferential growth of Nitrosomonas near the surface of the encapsulant was predicted by the model and confirmed by experiments. The modeling and experimental results also suggested that smaller encapsulants with a larger surface area to volume ratio would improve ammonia oxidation. Excessive aeration caused the breakage of the encapsulant, resulting in unpredicted microbial release and washout. Overall, our modeling approach is flexible and can be used to engineer and optimize encapsulated systems for enhanced biological nitrogen removal. Similar modeling approaches can be used to incorporate sorption of additional species within an encapsulant, additional nitrogen-converting microorganisms, and the use of other encapsulation materials.

Contaminants of Emerging Concern in the Lower Volta River, Ghana, West Africa: The Agriculture, Aquaculture, and Urban Development Nexus.

Contaminants of emerging concern (CECs) are ubiquitous in aquatic environments across all continents and are relatively well known in the developed world. However, few studies have investigated their presence and biological effects in low- and middle-income countries. We provide a survey of CEC presence in the Volta River, Ghana, and examine the microbial consequences of anthropogenic activities along this economically and ecologically important African river. Water and sediment samples were taken by boat or from shore at 14 sites spanning 118 km of river course from the Volta estuary to the Akosombo dam. Sample extracts were prepared for targeted analysis of antimicrobial CECs, N,N-diethyl-meta-toluamide, and per- and polyfluoroalkyl substances (PFAS; water only). Concurrent samples were extracted to characterize the microbial community and antibiotic-resistant genes (ARGs). Antibiotics and PFAS (PFAS, 2-20 ng/L) were found in all water samples; however, their concentrations were usually in the low nanograms per liter range and lower than reported for other African, European, and North American studies. N,N-Diethyl-meta-toluamide was present in all samples. The number of different genes detected (between one and 10) and total ARG concentrations varied in both water (9.1 × 10-6 to 8.2 × 10-3 ) and sediment (2.2 × 10-4 to 5.3 × 10-2 ), with increases in gene variety at sites linked to urban development, sand mining, agriculture, and shellfish processing. Total ARG concentration spikes in sediment samples were associated with agriculture. No correlations between water quality parameters, CEC presence, and/or ARGs were noted. The presence of CECs in the lower Volta River highlights their global reach. The overall low concentrations of CECs detected is encouraging and, coupled with mitigation measures, can stymie future CEC pollution in the Volta River. Environ Toxicol Chem 2022;41:369-381. © 2021 SETAC.

Dissolved oxygen concentrations affect the function but not the relative abundance of nitrifying bacterial populations in full-scale municipal wastewater treatment bioreactors during cold weather.

This study aimed to understand the effect of different dissolved oxygen (DO) concentrations on the abundance and performance of nitrifying bacteria in full-scale wastewater treatment bioreactors, particularly during the winter when nitrifying bacterial activity is often negligible. Biomass samples were collected from three parallel full-scale bioreactors with low DO concentrations (<1.3 mg/ L) and from two full-scale bioreactors with higher DO concentrations (~4.0 and ~2.3 mg/ L). The relative abundance of nitrifying bacteria was determined by sequencing of PCR-amplified 16S rRNA gene fragments. In the three bioreactors with low DO concentrations, effluent ammonia concentrations sharply increased with a decline in temperature below approximately 17 °C, while the bioreactors with high DO concentrations showed stable nitrification regardless of temperature. Even with the decline in nitrification during the winter in the three low DO bioreactors, the relative abundance of ammonia oxidizing bacteria (mostly Nitrosomonas spp.) was curiously maintained. The relative abundance of nitrite oxidizing bacteria was similarly maintained, although there were substantial seasonal fluctuations in the relative abundance values of Nitrospira spp. versus Nitrotoga spp. This research suggests that nitrification activity can be controlled during the winter via DO to produce better effluent quality with high DO concentrations or to reduce aeration costs with a concomitant decline in nitrification activity.

Diverse dechlorinators and dechlorination genes enriched through amendment of chlorinated natural organic matter fractions.

In uncontaminated environments, chlorinated natural organic matter (Cl-NOM) can act as an electron acceptor for organohalide-respiring bacteria. It is unknown, however, whether different types of Cl-NOM are preferentially dechlorinated or whether enrichment with Cl-NOM affects the ability of bacteria to dechlorinate contaminants. In this research NOM was extracted from sediment, fractionated based on hydrophobicity, and either amended to polychlorinated biphenyl-contaminated soil directly or chlorinated and then amended to soil. Amendments of the least hydrophobic Cl-NOM fraction were dechlorinated most rapidly, followed by the moderately hydrophobic Cl-NOM fraction. Soil that had been enriched on the moderately hydrophobic fraction of Cl-NOM was also capable of faster dechlorination of the contaminants trichloroethene and tetrachlorobenzene. Community analysis of the soil during enrichment showed that some known organohalide-respiring bacteria were present and may have played a role in dechlorination; nevertheless, many bacteria appeared to be enriched during both Cl-NOM and contaminant dechlorination. In addition, the quantities of two haloalkane dehalogenase genes increased during enrichment on Cl-NOM. These results show for the first time that Cl-NOM can prime contaminant dechlorination and also suggest that hydrolytic dechlorination processes were involved in both Cl-NOM and contaminant dechlorination.

Aqueous film forming foam and associated perfluoroalkyl substances inhibit methane production and Co-contaminant degradation in an anaerobic microbial community.

Aqueous film forming foams (AFFF) can contain gram per liter concentrations of per- and polyfluoroalkyl substances (PFAS) and are often released in large quantities directly to the environment as they are used to fight fires. AFFF composition is complex and contains many unknown PFAS in addition to ingredients such as hydrocarbons, solvents, and corrosion inhibitors. While biological effects of single PFAS have been studied, the effects of PFAS-containing mixtures, such as AFFF, are unknown. The effect of PFAS on microorganisms is also not well understood; nevertheless, we rely on microorganisms in locations containing elevated PFAS concentrations to perform certain functions, such as carbon cycling and co-contaminant degradation. This study focused on determining the functional consequences of AFFF and PFAS exposure in a microbial community in both the presence and the absence of a co-contaminant. AFFF, select PFAS, and a PFAS mixture were tested to determine the effect of AFFF on an anaerobic microbial community and the characteristics of the PFAS that drive toxicity in such mixtures. To study this, anaerobic digester communities were exposed to PFAS and a co-contaminant (2,4-dichlorophenol, DCP); methane production, as an indicator of toxicity and the community's ability to cycle carbon, and co-contaminant degradation were monitored. Results showed that PFAS and AFFF can alter the toxicity of DCP, inhibit DCP degradation, decrease the number of methanogens present, and change the microbial community structure. DCP was also able to decrease the toxicity of the PFAS perfluorooctane sulfonate (PFOS), possibly by changing the sorption of PFOS to the microorganisms present. Additionally, it was determined that while PFOS was responsible for AFFF toxicity, no single PFAS or simple PFAS mixture accurately accounted for the inhibition of DCP degradation caused by AFFF exposure.

Modeling alginate encapsulation system for biological hydrogen production.

Wastewater treatment using encapsulated biomass is a promising approach for high-rate resource recovery. Encapsulation matrices can be customized to achieve desired biomass retention and mass transport performance. This, in turn, facilitates treatment of different waste streams. In this study, a model was developed to describe calcium-alginate beads encapsulating hydrogen-producing biomass, with the goal of enabling appropriate a priori customization of the system. The model was based on a classic diffusion-reaction model, but also included the growth of encapsulated biomass and product inhibition. Experimental data were used to verify the model, which accurately described the effect of hydraulic retention time, bead size, and feed concentration on resource (hydrogen) recovery from brewery wastewater. Sensitivity analyses revealed that the hydrogen production rate was insensitive to substrate diffusivity and bead size, but sensitive to the substrate partition coefficient, initial encapsulated biomass concentration, and the total volume of beads in the reactor, demonstrating that this system was growth-limited rather than diffusion-limited under the tested conditions. Because the model quantifies the relationship between the hydrogen production rate and various input and operating parameters, it should be possible to extend the model to determine the most cost-effective system for optimal performance with a given waste stream.

Presence, Diversity, and Enrichment of Respiratory Reductive Dehalogenase and Non-respiratory Hydrolytic and Oxidative Dehalogenase Genes in Terrestrial Environments.

Organohalide-respiring bacteria have been linked to the cycling and possible respiration of chlorinated natural organic matter (Cl-NOM) in uncontaminated soils and sediments. The importance of non-respiratory hydrolytic/oxidative dechlorination processes in the cycling of Cl-NOM in terrestrial soil and sediment, however, is still not understood. This research analyzes the dechlorination potential of terrestrial systems through analysis of the metagenomes of urban lake sediments and cultures enriched with Cl-NOM. Even with the variability in sample type and enrichment conditions, the potential to dechlorinate was universal, with reductive dehalogenase genes and hydrolytic or oxidative dehalogenase genes found in all samples analyzed. The reductive dehalogenase genes detected grouped taxonomically with those from organohalide-respiring bacteria with broad metabolic capabilities, as opposed to those that obligately respire organohalides. Furthermore, reductive dehalogenase genes and two haloacid dehalogenase genes increased in abundance when sediment was enriched with high concentrations of Cl-NOM. Our data suggests that both respiratory and non-respiratory dechlorination processes are important for Cl-NOM cycling, and that non-obligate organohalide-respiring bacteria are most likely involved in these processes.

Partitioning and Accumulation of Perfluoroalkyl Substances in Model Lipid Bilayers and Bacteria.

Perfluoroalkyl substances (PFAS) are ubiquitous and persistent environmental contaminants, yet knowledge of their biological effects and mechanisms of action is limited. The highest aqueous PFAS concentrations are found in areas where bacteria are relied upon for functions such as nutrient cycling and contaminant degradation, including fire-training areas, wastewater treatment plants, and landfill leachates. This research sought to elucidate one of the mechanisms of action of PFAS by studying their uptake by bacteria and partitioning into model phospholipid bilayer membranes. PFAS partitioned into bacteria as well as model membranes (phospholipid liposomes and bilayers). The extent of incorporation into model membranes and bacteria was positively correlated to the number of fluorinated carbons. Furthermore, incorporation was greater for perfluorinated sulfonates than for perfluorinated carboxylates. Changes in zeta potential were observed in liposomes but not bacteria, consistent with PFAS being incorporated into the phospholipid bilayer membrane. Complementary to these results, PFAS were also found to alter the gel-to-fluid phase transition temperature of phospholipid bilayers, demonstrating that PFAS affected lateral phospholipid interactions. This investigation compliments other studies showing that sulfonated PFAS and PFAS with more than seven fluorinated carbons have a higher potential to accumulate within biota than carboxylated and shorter-chain PFAS.

Novel Microbial Assemblages Dominate Weathered Sulfide-Bearing Rock from Copper-Nickel Deposits in the Duluth Complex, Minnesota, USA.

The Duluth Complex in northeastern Minnesota hosts economically significant deposits of copper, nickel, and platinum group elements (PGEs). The primary sulfide mineralogy of these deposits includes the minerals pyrrhotite, chalcopyrite, pentlandite, and cubanite, and weathering experiments show that most sulfide-bearing rock from the Duluth Complex generates moderately acidic leachate (pH 4 to 6). Microorganisms are important catalysts for metal sulfide oxidation and could influence the quality of water from mines in the Duluth Complex. Nevertheless, compared with that of extremely acidic environments, much less is known about the microbial ecology of moderately acidic sulfide-bearing mine waste, and so existing information may have little relevance to those microorganisms catalyzing oxidation reactions in the Duluth Complex. Here, we characterized the microbial communities in decade-long weathering experiments (kinetic tests) conducted on crushed rock and tailings from the Duluth Complex. Analyses of 16S rRNA genes and transcripts showed that differences among microbial communities correspond to pH, rock type, and experimental treatment. Moreover, microbial communities from the weathered Duluth Complex rock were dominated by taxa that are not typically associated with acidic mine waste. The most abundant operational taxonomic units (OTUs) were from the genera Meiothermus and Sulfuriferula, as well as from diverse clades of uncultivated Chloroflexi, Acidobacteria, and Betaproteobacteria Specific taxa, including putative sulfur-oxidizing Sulfuriferula spp., appeared to be primarily associated with Duluth Complex rock, but not pyrite-bearing rocks subjected to the same experimental treatment. We discuss the implications of these results for the microbial ecology of moderately acidic mine waste with low sulfide content, as well as for kinetic testing of mine waste.IMPORTANCE Economic sulfide mineral deposits in the Duluth Complex may represent the largest undeveloped source of copper and nickel on Earth. Microorganisms are important catalysts for sulfide mineral oxidation, and research on extreme acidophiles has improved our ability to manage and remediate mine wastes. We found that the microbial assemblages associated with weathered rock from the Duluth Complex are dominated by organisms not widely associated with mine waste or mining-impacted environments, and we describe geochemical and experimental influences on community composition. This report will be a useful foundation for understanding the microbial biogeochemistry of moderately acidic mine waste from these and similar deposits.

Rapid Enrichment of Dehalococcoides-Like Bacteria by Partial Hydrophobic Separation.

Organohalide-respiring bacteria can be difficult to enrich and isolate, which can limit research on these important organisms. The goal of this research was to develop a method to rapidly (minutes to days) enrich these organisms from a mixed community. The method presented is based on the hypothesis that organohalide-respiring bacteria would be more hydrophobic than other bacteria as they dehalogenate hydrophobic compounds. The method developed tests this hypothesis by separating a portion of putative organohalide-respiring bacteria, those phylogenetically related to Dehalococcoides mccartyi, at the interface between a hydrophobic organic solvent and an aqueous medium. This novel partial separation technique was tested with a polychlorinated biphenyl-enriched sediment-free culture, a tetrachloroethene-enriched digester sludge culture, and uncontaminated lake sediment. Significantly higher fractions, up to 20.4 times higher, of putative organohalide-respiring bacteria were enriched at the interface between the medium and either hexadecane or trichloroethene. The selective partial separation of these putative organohalide-respiring bacteria occurred after 20 min, strongly suggesting that the separation was a result of physical-chemical interactions between the cell surface and hydrophobic solvent. Dechlorination activity postseparation was verified by the production of cis-dichloroethene when amended with tetrachloroethene. A longer incubation time of 6 days prior to separation with trichloroethene increased the total number of putative organohalide-respiring bacteria. This method provides a way to quickly separate some of the putative organohalide-respiring bacteria from other bacteria, thereby improving our ability to study multiple and different bacteria of potential interest and improving knowledge of these bacteria.IMPORTANCE Organohalide-respiring bacteria, bacteria capable of respiring chlorinated contaminants, can be difficult to enrich, which can limit their predictable use for the bioremediation of contaminated sites. This paper describes a method to quickly separate Dehalococcoides-like bacteria, a group of organisms containing organohalide-respiring bacteria, from other bacteria in a mixed community. From this work, Dehalococcoides-like bacteria appear to have a hydrophobic cell surface, facilitating a rapid (20 min) partial separation from a mixed culture at the surface of a hydrophobic liquid. This method was verified in a polychlorinated biphenyl-enriched sediment-free culture, an anaerobic digester sludge, and uncontaminated sediment. The method described can drastically reduce the amount of time required to partially separate Dehalococcoides-like bacteria from a complex mixed culture, improving researchers' ability to study these important bacteria.

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.