Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "May 2002." Typescript. Vita. Includes bibliographical references (leaves 237-245).

Despite the environmental significance of microbial manganese reduction, the molecular mechanism of microbial manganese respiration remains poorly understood. Soluble Mn(III) has been recently found to be a dominant soluble species in aquatic systems, yet little is known about the identity of microbial populations catalyzing Mn(III) reduction in the environment nor the molecular mechanism of Mn(III) respiration. In this research, a suite of Mn(III) reduction-deficient mutant strains were isolated, including Mn(III) reduction-deficient mutant strain Mn3-1 that also displayed the ability to reduce soluble organic-Fe(III), but not solid Fe(III) oxides, demonstrating for the first time that the reduction of soluble organic-Fe(III) and solid Fe(III) oxides proceed through electron transport pathways with at least one distinct component. This work also shows that the electron transport pathway for Mn(III) reduction in S. oneidensis shares many of the electron transport components of Fe(III) and Mn(IV) reduction pathways and that Mn(IV) reduction to Mn(II) proceeds step-wise through two one-electron transfer reactions with Mn(III) as a transient intermediate. Finally, sediment incubations were carried out to enrich for NH4+ oxidizing- Mn(III) reducing consortia. The Mn(III) reducing consortium was found to be dominated by an electrogenic Ochrobactrum sp. and a Shewanella sp. The isolated Shewanella strain is able to oxidize acetate with Mn(III) as electron acceptor, an activity never observed before in a metal-reducing member of the Shewanella genus.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Anaerobic digestion of agricultural wastes provides an opportunity for renewable energy production while reducing emissions of greenhouse gasses such as carbon dioxide and methane from crop and livestock production. While anaerobic digestion is possible under a wide range of temperatures and reactor configurations, it does require a stable methanogenic community composed of hydrolytic and fermentative bacteria and methanogenic archaea in order to maintain robust methane production. Research focused on characterizing and optimizing the microbial community during anaerobic digestion is increasingly exploiting DNA-based methods. In addition to providing an in-depth phylogenetic survey, these techniques permit examination of dynamic changes in α- and β-diversity during the digestion process and in response to perturbations in the system. This study used universal target amplification, next generation sequencing, and quantitative PCR to characterize the Bacteria and Archaea in digestate from thermophilic batch anaerobic digesters processing different combinations wheat ethanol stillage waste and cattle manure. The results indicated that the bacterial community was composed primarily of Firmicutes, with Proteobacteria and Bacteroidetes also numerically abundant. While less phylogenetically diverse, the archaeal community showed robust populations of both hydrogenotrophic and acetoclastic methanogens. A core microbiome present across all reactors was identified and differences in the relative abundances of the bacteria within the core community suggested significant niche overlap and metabolic redundancy in the reactors. A time-course study correlating the abundances of individual Bacteria and Archaea to methane production and volatile fatty acid catabolization identified several microorganisms hypothesized to be critical to both hydrogenotrophic and acetoclastic methanogenesis. Individual Bacteria most closely related to Clostridium spp. and Acetivibrio spp. were 10-1000-fold less abundant in reactors suffering from volatile fatty acid accumulation and inhibition of methanogenesis. Additionally, failing reactors were devoid of robust populations of acetoclastic methanogens. Microorganisms identified as critical during the time-course study were targeted for isolation in vitro and a robust methanogenic consortium consisting of at least 9 bacteria and both a hydrogenotrophic and an acetoclastic methanogen was stably propagated. Addition of this bioaugmentation consortium to digesters experiencing classic symptoms of acid crisis resulted in reduced acetate accumulation and initiation of methanogenesis. One acetoclastic methanogen, most likely a novel species from the genus Methanosarcina, showed particularly robust growth in the recovered bioaugmented reactors, increasing 100-fold in the first 7 days post-treatment. A combination of Illumina shotgun and Roche 454 paired-end sequencing chemistry was used to generate a high quality draft genome for this organism. Analysis of the annotated genome revealed diverse metabolic potential with a full complement of genes for acetoclastic, hydrogenotrophic and methylotrophic methanogenesis pathways represented. Taken as a whole, this thesis provides the foundation for using microbial community characterization to inform anaerobic digester design and operation. By identifying organisms of interest, correlating their abundance to specific biochemical functions and confirming their hypothesized functions in situ, microorganisms critical for robust methane production were acquired. The logical extension of this work is to establish monitoring tools for microorganisms identified as critical to specific performance parameters, to enumerate them in real-time, and to use that data to improve reactor operation.

published_or_final_version / Civil and Structural Engineering / Master / Master of Philosophy

The anaerobic oxidation of ammonium (anammox) with nitrite as electron acceptor is a microbial process that generates nitrogen gas as main final product. After being discovered in the Netherlands in the 1990s, anammox has been applied in state-of-the-art biotechnologies for the removal of N pollution from ammonium rich wastewaters. The anammox process offers significant advantages over traditional nitrification-denitrification based processes. Since anammox does not need elemental oxygen, it allows for important savings in aeration. Furthermore, due to the autotrophic nature of the bacteria, anammox does not require external addition of electron donor, often needed in systems with post-denitrification. Although the anammox bacteria have high specific activity, they are slow growing, with doubling times that can range from 10 to 25 d. Therefore, in case of a toxic event causing the death of the biomass, a long recovery period will be required to reestablish full treatment capacity. The purpose of this work is to investigate the inhibition of anammox bacteria by compounds commonly found in wastewaters, including substrates, intermediates and products of the anammox reaction. Among common wastewater constituents, sulfide was shown to be especially harmful, causing complete inhibition of anammox activity at concentrations as low as 11 mg H₂S L⁻¹. Dissolved oxygen was moderately toxic with a 50% inhibiting concentration of 2.3 and 3.8 mg L⁻¹ to granular and suspended anammox cultures, respectively. Among the various compounds involved in the anammox reaction, special attention was paid to nitrite. Numerous literature reports have indicated inhibition of anammox bacteria by its terminal electron acceptor. However to date, there is no consensus explanation as to the mechanism of nitrite inhibition nor on how the inhibition is impacted by variations in the physiological status of anammox cells. The mechanisms of anammox inhibition by nitrite were thoroughly investigated in batch and continuous experiments of this dissertation. The results of this work demonstrate that conditions hindering generation of metabolic energy have a detrimental effect on the tolerance of anammox cells to toxic levels of nitrite. The absence of ammonium during events of nitrite exposure was shown to exacerbate its toxic effect. As a result of nitrite inhibition, nitric oxide, an intermediate of the anammox reaction, accumulated in the head space of the batch experiments. Moreover, nitrite inhibition was enhanced at the lowest range of pH tested (6.4-7.2), while same nitrite concentrations caused no inhibition under mildly alkaline conditions (7.5-7.8). Although other authors have relied on the classic concept that undissociated nitrous acid is the species responsible for the inhibition, the results in this work indicate that the pH affects the inhibitory effect of nitrite, irrespective of the free nitrous acid concentration. Nitrite stress triggered an active response of the anammox bacteria, which temporarily increased their ATP content to mitigate the inhibition. Additionally, starvation of anammox microorganisms, caused during storage or by sustained underloading of bioreactors, was found to limit the capacity of the bacteria to tolerate exposure to nitrite. The results of this dissertation indicate that the tolerance of anammox bacteria to NO₂⁻ inhibition relies on limiting its accumulation in sensitive regions of the cell. Active metabolism in presence of NH₄⁺ allows for active consumption of NO₂⁻, avoiding accumulation of toxic intracellular NO₂⁻ concentrations. Furthermore, secondary active transport proteins may be used by anammox bacteria to translocate nitrite to non-sensitive compartments. Nitrite active transport relies on a proton motive force. Therefore, conditions such as low pH (below 7.4) or absence of energy sources, which may disturb the maintenance of the intracellular proton gradient, will increase the sensitivity of anammox cells to NO₂⁻ inhibition. Strategies for the operation and control of anammox bioreactors must be designed to avoid exposure of the biomass to nitrite under the absence of ammonium, low pH or after periods of starvation.

Organic and inorganic carbon can enter inland waters in different ways, and often a considerable amount of this carbon is coming from terrestrial input. Once this terrestrial carbon enters a lake, the carbon may be degraded, mineralized or eventually buried in the sediment. Below the oxic zone of the sediment carbon may be used by archaea to produce methane (CH4). The CH4 can then diffuse up in the sediment and escape to the bottom waters, or the CH4 can be oxidized by bacteria using oxygen as an oxidant. There is also an anoxic process to oxidize CH4 (anaerobic oxidation of methane: AOM), using sulfate (SO4) and by recent findings also ferric iron (Fe(III)) as electron acceptors. In this study the main questions of interest were if CH4 is produced in deep (i.e. old) lake sediments and if CH4 is oxidized anaerobically using Fe(III). Two Swedish boreal forest lakes were studied, sediment profiles of CH4 were conducted in the field (down to 60 cm). Collected sediments were sliced anoxically at different depths and then analyzed for ferrous iron (Fe(II)), Fe(III) and SO4. Sediment from different depths was also incubated anoxic in order to test if CH4 production depends on sediment age. The results show that methanogenic activity occurs by degrading old carbon in deep boreal forest lake sediments, and that a certain part of this might then be oxidized anaerobically. However, all cores exposed a general trend of increasing CH4 concentrations with sediment depth, indicating that CH4 production in old sediment layers is greater than AOM. AOM could therefore only act as a partial sink for CH4 in anoxic deep sediments.

Bacteriophytochromes (Bphs) comprise a family of protein photoreceptors that help bacteria sense changes in light. Bphs contain a chromophore that, upon absorption of red or far-red light, undergoes a cis-trans isomerization that leads to a conformational change in the holoprotein (photoconversion). In the active conformation, Bphs act as a kinase and regulate gene expression through phosphorylation of target proteins. Two putative Bph orfs (rpa0122 and rpa0990) in the Rhodopseudomonas palustris genome encode Bph-like proteins that have a conserved chromophore-binding cysteine residue. The hypothesis is that one or both of these unique Bph-like genes encode proteins that are capable of binding a chromophore and functioning to modulate the cell’s phenotype. I expressed and purified His-tagged RPA0990 in R. palustris, because proteolytic degradation was observed during overexpression in an E coli. expression system. The results show that RPA0990 contains a chromophore and is capable of photoconversion. The wavelengths of light absorbed by the Pr/Pfr forms of RPA0990, predicted to be active and inactive forms respectively, were determined to be 695 nm and 755 nm. Investigation into the phenotype of the bph mutants rpa0122 and rpa0990 revealed that both of these Bphs may have a small effect on light-harvesting complexes. Also, it was observed that the absence of O₂ does not inhibit the normal function of Bphs, although O₂ was thought to be needed to make a linear tetrapyrrole cofactor, by cleaving heme using heme oxygenase. I suggest that a linear tetrapyrrole can be made anaerobically, either through anaerobic heme cleavage by a novel enzyme, or directly from the heme precursor hydroxymethylbilane without ring cleavage. The activity of a divergent promoter region between the rpa1490 (bph3) and rpa1491 (pucBe) genes was evaluated by using the E. coli lacZ gene as a reporter. The results indicated that the pucBe promoter has much higher activity than the bph3 promoter. It was also found that double knockout of the regulatory genes ppsR1ˉ2ˉ led to an increase in bph3::lacZ expression and a decrease in pucBe::lacZ expression.

The human colon is an anaerobic environment densely populated with bacterial species, creating what is known as the human intestinal microbiome; an ecosystem imperative to physiological function with regards to metabolism of non-digestible residues, growth of cells and immune protection from invading organisms. As such, quantifying, and subsequently developing an understanding of the behaviour of this microbial population can be of great value. Unfortunately, because of the physical inaccessibility of many parts of the gastro-intestinal (GI) tract, routine experimentation with this environment is not practical. However, theoretical modelling techniques including in vitro and in silico simulation/experimental platforms provide a means by which further studying of intestinal microflora can be approached. Perfecting these theoretical models is an important step in further understanding colon microbiota. An existing in silico model of carbohydrate digestion in the colon, developed by Munoz-Tamayo et al. (2010) has been used as a platform for experimentation with the intention of of discovering features which may be removed and/or added to improve the performance and reliability of the design. The model is an adaptation of the waste-water engineering based mathematical model ADM1 (Anaerobic Digestion Model 1), developed to incorporate biochemical and environmental specifications as well as physical structures particular to the human colon. The model is then a system of 102-ordinary differential equation with 66 parameters.Simulations with the default model configuration as well as variations of input variables, namely dietary fiber consumption and system flow rate, were completed to study the effect on average biomass concentration, demonstrating significant sensitivity to input variables and an unexpected linearity based on the non-linearity of the original complex system. Simulations and further study suggest that advancements in in silico modelling of the colon rely on the development of a metric or scheme that can effectively compare mathematically generated data with that collected through traditional experimentation. Also, experimenting with various reactor configurations as a basis for mathematical modelling may prove simpler configurations capable of generating comparable data to more complicated designs which may then also be applicable to existing in vitro representations of the colon.