The purpose of this research was to investigate the microorganisms, genetics and biochemistry of anaerobic dechlorination of chlorinated ethanes, which are common groundwater contaminants. Specifically, this project used mixed microbial cultures to study the dechlorination of 1,2-dichloroethane (1,2-DCA), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,1-trichloroethane (1,1,1-TCA). A mixed microbial culture enriched from a contaminated multilayered aquifer in West Louisiana dechlorinated 1,2-DCA, 1,1,2-TCA, tetrachloroethene, trichloroethene, cis-dichloroethene and vinyl chloride (VC) to non-toxic ethene when amended with ethanol as the electron donor. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organisms Dehalobacter and Dehalococcoides spp. Denaturing gradient gel electrophoresis analysis and quantitative PCR (qPCR) with species-specific primers demonstrated that both organisms grew during the dichloroelimination of 1,2-DCA to ethene. Conversely, during the dichloroelimination of 1,1,2-TCA to VC only Dehalobacter grew, while during the reductive dechlorination of VC to ethene only Dehalococcoides grew. Further enrichment with 1,2-DCA, H2 and acetate yielded a co-culture of Dehalobacter and Acetobacterium spp. that did not dechlorinate other chlorinated ethanes or ethenes. Dehalobacter grew in the presence but not in the absence of 1,2-DCA, while Acetobacterium growth was not affected by 1,2-DCA. A novel putative Dehalobacter-associated 1,2-DCA reductive dehalogenase gene was identified and was shown to be transcribed only in the presence of 1,2-DCA. An enrichment microbial culture derived from a 1,1,1-TCA-contaminated site in the northeastern United States was also studied. This culture, referred to as MS, reductively dechlorinated 1,1,1-TCA to 1,1-dichloroethane (1,1-DCA) and then to monochloroethane (CA) when amended with methanol, ethanol, acetate and lactate. 16S rRNA gene sequence analysis revealed the presence of the putative dechlorinating organism Dehalobacter sp., whose growth during 1,1,1-TCA and 1,1-DCA dechlorination was confirmed by qPCR. In the presence of chlorinated ethenes, dechlorination 1,1,1-TCA by the culture MS was slowed, while dechlorination of 1,1-DCA was completely inhibited. Experiments with cell-free extracts and whole cell suspensions of culture MS suggested that chlorinated ethenes have direct inhibitory effects on 1,1,1-TCA reductive dehalogenase(s), while the inhibition of 1,1-DCA dechlorination may be due to effects on non-dehalogenase components of Dehalobacter sp. cells. Additionally, two novel reductive dehalogenase genes associated with 1,1,1-TCA reductive dechlorination were identified.

Microbial activity occurs year-round in Arctic soils, including during the winter when soils are frozen. From 2004 to 2008 I monitored soil microbial and nutrient dynamics in low Arctic wet and dry sedge meadows near Churchill, Manitoba. I documented a consistent annual pattern in which soil microbial biomass (MB) and soil nutrients peak in late winter, and decrease during the early stages of spring thaw, remaining in low abundance during the summer. Based on a series of experiments, resource shortages do not appear to be the cause of the microbial decline, as has been hypothesized. Observations and theoretical considerations regarding soil physical properties indicate that this decrease is driven by the influx of liquid water at thaw that brings about a rapid change in the chemical potential of water, leading to cell lysis. I have used 15N isotope tracing to show that inorganic nitrogen is taken up very quickly at thaw by the roots of the dominant plant, Carex aquatilis. This represents a critical window of opportunity for these plants, as nitrogen remains abundant only for a short time. The described annual pattern was pronounced in wet sedge sites, but some inter-annual variation is evident, for example a post-thaw soil nitrogen pulse in 2006, and low winter MB in 2008. In the dry sedge meadow, fluctuations in MB and nutrients were dampened relative to wet sites, and the annual pattern was variable, particularly after 2006. Over four years, peak winter values of soil MB and nutrient variables declined in both wet and dry sites, and this could be related to a drying trend. This work improves our understanding of the controls on decomposition and primary productivity in a system that is experiencing climate warming and increased precipitation. Changes to hydrology, carbon and nitrogen cycling, and primary productivity will have further effects on vegetation communities and higher trophic levels, including several species of migratory birds.

In forest ecosystems, substantial part of carbon enters soil in the form of plant litter. The decomposition of litter and soil organic matter represents an important process affecting nutrient cycling and carbon balance in soils. Fungi are considered the primary decomposers in terrestrial ecosystems due to the production of wide range of extracellular enzymes that allow them to attack the lignocellulose matrix in litter. Even if fungi represent key players in organic matter decomposition, the information about the structure and diversity of their communities is still limited and the roles of individual fungal taxa in forest soils remain unclear. This Ph.D. thesis focused on the characterization of fungal communities in forest soils and their potential to decompose plant litter. The method for in-depth analysis of complex microbial communities from environmental samples was established and used. In addition, single eukaryotic functional gene was analysed in soil for the first time at a depth that allowed reliable estimation of diversity. It was demonstrated that microbial community composition differs among horizons of forest soil profile. Despite similar diversity, significant differences in microbial community composition were observed between the DNA and RNA. Several microbial groups highly...

Dead plant biomass is a key pool of carbon in terrestrial ecosystems. Its decomposition in soil environments is thus an essential process of the carbon cycle. Fungi are considered to be the primary decomposers in soil ecosystems because of their physiological adaptations and enzymatic apparatus composed from highly effective oxidative and hydrolytic enzymes. Many recent works show that in addition to fungi, bacteria may also play a significant role in lignocellulose decomposition and among bacteria, the members of the phylum Actinobacteria are often regarded to significantly contribute to cellulose and lignocellulose decomposition. This thesis is focused on the evaluation of the role that fungi and Actinobacteria play in dead plant biomass degradation. First, it explored mechanisms involved in degradation, in particular the enzymatic breakdown of major lignocellulose components as cellulose, hemicelluloses and lignin. Enzymatic apparatus of the saprotrophic fungus Fomes fomentarius was explored both in vitro as well as in vivo. Several Actinobacteria were isolated from soil and comparative experiments, investigating production of hydrolytic enzymes, were carried out to track the transformation of polysaccharides and lignin by these strains. To explain the roles of lignocellulose decomposers in...

Vegetation of post-mining sites determines soil microbial community structure and soil processes Mgr. Michaela Urbanová Abstract The aim of this thesis, which consists of four published articles, was to investigate the effect of vegetation on soil microbial communities and processes in de novo developing soil substrate on the brown-coal spoil heaps in the surrounding of city Sokolov. Spoil material - soil clayey substrate, which had been gradually mined from the opencast brown coal mine, stratified onto spoil heaps and reclaimed by assisted afforestation with selected tree species or left for spontaneous plant succession, changes its biotic and abiotic characteristic in the course of time and particularly under the influence of plants. Changes of spoil substrate characteristics are related to the growth of plant roots and particularly also to the production of plant biomass, which is decomposed gradually and takes part of soil, where participates to soil organic matter. The process of plant dead materials decomposition and transformation is the function of the activity of soil organisms and among them notably soil microorganisms. Moreover, the presence of many of them is closely related to the presence of vegetation, whose symbionts or pathogens are. The exact mechanisms of the plant-microbes interactions...

Biological nitrogen removal (BNR) has been applied for more than a century in the interests of preserving and enhancing public health and the environment. But only during the last few decades has the development of molecular techniques using biomolecules such as nucleic acids (DNA and RNA) and proteins allowed the accurate description and characterization of the phylogenetic and functional diversity of microbial communities. Moreover, thanks to recent advances in genomics and next-generation sequencing technologies, microbial community analyses have initiated a new era of microbial ecology. Notwithstanding the fact that the efficiency and robustness of a wastewater treatment mainly depend on the composition and activity of BNR communities, research on the structural and functional microbial ecology of the engineered BNR process remains rare with respect to next-generation sequencing and bioinformatics. This dissertation aims to bridge high-priority knowledge gaps in determining and applying knowledge of microbial structure (who is there and how many?) and function (what are they doing? what else can they do?) to the practice of BNR processes, and to opening up the ‘black-box’ of energy and resource efficient engineered BNR processes using a systems biology approach. Specific objectives were to (1) selectively enrich Nitrospira spp. from a mixed environmental microbial consortium (such as activated sludge) in a continuously operated bioreactor and characterize the microbial ecology during the course of enrichment, determine key kinetic parameters of enriched Nitrospira spp., (2) examine the inhibitory effects of nitrogenous intermediates (such as hydroxylamine, presented herein) on the physiological and molecular responses of Nitrospira spp. in terms of both catabolism and anabolism, (3) characterize bacterial community composition and their dynamics by 16S rRNA gene amplicon sequencing under varying reactor operational conditions from full-scale WWTPs and identify process parameters that most significantly correlate with those dynamics, (4) interpret metagenomic (DNA-based) and metatranscriptomic (RNA-based) derived structure, metabolic function and activity of the full-scale BNR microbial communities, and (5) describe gene expression in the same full-scale BNR communities in response to alternating anoxic-aerobic conditions using a metatranscriptomic approach. First, planktonic Nitrospira spp. were successfully enriched from activated sludge in a sequencing batch reactor by maintaining sustained limiting extant nitrite and dissolved oxygen concentrations for a half year. The determined parameters collectively reflected not just higher affinities of this enrichment for nitrite and oxygen, respectively, but also a higher biomass yield and energy transfer efficiency relative to other NOB such as Nitrobacter spp. Used in combination, these kinetic and thermodynamic parameters can help toward the development and application of energy-efficient biological nutrient removal processes through effective Nitrospira out-selection. Second, using quantitative activity measurements (respirometrc rates) with functional gene expression profiles, this study demonstrated that N-intermediates such as hydroxylamine (NH¬2OH) can strongly inhibit the activity and expression of key anabolic (energy synthesis) and catabolic (biomass synthesis) pathways of Nitrospira spp. A strategy that relies upon the transient accumulation and consumption of such intermediates (such as transient aeration) could provide the platform for successful suppression of Nitrospira spp. in the next generation of energy efficient engineered BNR processes. Third, 16S rRNA gene amplicon sequencing revealed that microbial community structure and their dynamics significantly varied depending on seven differing wastewater treatment processes. The findings showed that five process parameters of wastewater influenced the dynamics of BNR communities; water temperature was correlated most strongly to the variance of bacterial communities, followed by effluent NH3, effluent NO3-, removed N, and effluent NO2-. The results provided insights into the underlying ecological pattern of community compositions and dynamics in full-scale WWTPs; and correlation with process parameters brought about distinct communities that enable different microbial activities. However, one of the greatest challenges was to elucidate the relationship between microbial structure and their “active” functions, which are related to reactor performance (This challenge continued into fourth study chapter summarized below). Fourth, continuing from the previous study, combined metagenomics and metatranscriptomics revealed far superior richness of information of not just microbial structure, but also potential (through metagenomics) and expressed function (through metatranscriptimics) within the complex activated sludge processes. Via independent analysis of whole-DNA and whole-RNA, the entire microbial community and its in situ active members, involved in nitrificaiton and denitrification, were compared. Active nitrifiers and denitrifiers obtained by RNA analysis exhibited relatively high abundances in DNA-derived communities. Further gene expression annotation on nitrogen removal revealed that the expressions of denitrification-related genes except nos were increased under anoxic conditions relative to aerobic conditions, while the expressions of nitrifying genes were decreased. Our findings led to an improved understanding of metabolic activities and roles of BNR microbial communities, and offer the first metatranscriptional insights on engineered nutrient removal in anoxic conditions relative to aerobic conditions in full-scale wastewater systems. In sum, next-generation sequencing as well as traditional molecular techniques shed light on microbial diversity and different functional genes in varying engineered BNR systems. Furthermore, this dissertation provides a wealth of knowledge on systematic explorations of the linkage between structure and function of BNR communities, and offers engineering applications to BNR processes including energy and resource efficient engineered systems. It is expected that the implementation and further expansion of this work will improve the design and operation of engineered BNR processes, eventually producing benefits for the global population and the environment.

Metabolism is a key attribute of life on Earth at multiple spatial and temporal scales, involved in processes ranging from cellular reproduction to biogeochemical cycles. While metabolic network modeling approaches have enabled significant progress at the cellular-scale, extending these techniques to address questions at both the ecosystem and planetary-scales remains highly unexplored. In this thesis, I integrate various multi-scale metabolic network modeling approaches to address key questions with regard to both the long-term evolution of metabolism in the biosphere and the metabolic processes that take place in complex microbial communities. The first portion of my thesis work, focused on the evolution of ancient metabolic networks, attempts to model the emergence of ecosystem-level metabolism from simple geochemical precursors. By integrating network-based algorithms, physiochemical constraints, and geochemical estimates of ancient Earth, I explored whether a complex metabolic network could have emerged without phosphate, a key molecular component in modern-day living systems, known to be poorly available at the onset of life. We found that phosphate may have not been essential in early living systems, and that thioesters may have been the primitive energy currency in ancient metabolic networks. By generalizing this approach to explore the scope of geochemical scenarios that could have given rise to living systems, I found that other key biomolecules, including fixed nitrogen, may have not been required at the earliest stages in biochemical evolution. The second portion of my thesis deals with a different aspect of ecosystem-level metabolism, namely the role of metabolism in shaping the structure of microbial communities. I studied the relationship between metabolism and microbial community assembly using microbial communities grown in synthetic laboratory environments. We found that a generalized statistical consumer-resource model recapitulates the emergent phenomena observed in these experiments. Future work could seek to better clarify the connection between the fundamental rules that led to life’s emergence over 4 billion years ago and the laws that shape microbial ecosystems today. An ecosystems-level metabolic perspective may aid in our understanding of both the emergence and maintenance of the biosphere.

Knowledge about whether/how natural water chemistry influences the fate, dissolution, and toxicity of silver nanoparticles (AgNPs) should contribute to ecological risk assessment and informed decision making. The effects of three critical water chemistry parameters – dissolved organic carbon (DOC), pH, and hardness – were investigated on the colloidal stability, dissolution dynamics, and antimicrobial activity of citrate-functionalized AgNPs (citrate–AgNPs) against Escherichia coli. Toxicities of citrate–AgNPs and AgNO3 were also determined in the river water samples collected across three seasons (for seven months). Detectable changes in hydrodynamic diameter, surface charge, and plasmonic resonance revealed the modulating effects of the water chemistry parameters on the colloidal stability of citrate–AgNPs. Although, overall Ag release from citrate–AgNPs was low (0.33–3.62%), it increased with increasing DOC concentrations (0–20 mg L−1) but decreased with increasing pH (5–7.5) or hardness (150–280 mg L−1). Citrate–AgNP toxicity was 3–44 fold lower than of AgNO3 (Ag mass basis). Notably, higher DOC or pH conferred protection to E. coli against citrate–AgNPs or AgNO3; increasing solution hardness tended to enhance toxicity, however. Citrate–AgNPs or AgNO3 toxicity in the river water matrix revealed no seasonality. Generalized linear models developed, by parameterizing particle properties, could fairly predict empirically-derived nanotoxicity. Our results show that particle size, surface properties, ion release kinetics, and toxicity of citrate–AgNPs can be modified upon release into aquatic environments, suggesting potential implications to ecosystem health and functions.

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