Photoarsenotrophy is an anoxygenic photosynthesis-dependent arsenite oxidation pathway encoded by the Arx gene cluster and is linked to light-dark cycling of arsenic in freshwater environments.

This dissertation uses techniques from microbiology, molecular genetics, DNA sequencing, and analytical chemistry to characterize photoarsenotrophy in freshwater environments. The hypothesis is that photoarsenotrophy occurs in freshwater environments and is associated with light-dark cycling of arsenic, which may have a different effect on arsenic biogeochemical cycling when compared to Arx-type arsenotrophy. Light-dark arsenic redox cycling is defined herein as variations in arsenic species, arsenite and arsenate, that correlate to light or dark phases.

Since the discovery of photoarsenotrophy in 2008, less than a dozen Arx-dependent arsenotrophs have been isolated, and light-dependent arsenite oxidation has only been detected in three genera (Ectothiorhodospira sp. strains MLW-1, PHS-1, BSL-9, Ect. shaposhnikovii strains DSM 243 and DSM 2001, Halorhodospira halophila SL-1, and now Cereibacter azotoformans str. ORIO). The other studied Arx-dependent arsenotrophs couple arsenite oxidation to anaerobic respiration (i.e., nitrate reduction) instead of anoxygenic photosynthesis (Alkalilimnicola ehrlichii str. MLHE-1, Azoarcus sp. CIB, Sterolibacteraceae strain M52, Desulfotomaculum strain TC-1, and Halomonas sp. ANAO440). In this thesis, I define the light requirement by referring to photosynthesis-dependent arsenite-oxidation as photoarsenotrophy, and photosynthesis-independent Arx-type arsenite-oxidation as Arx-type arsenotrophy. This difference in light requirement is important as it introduces the possibility that light-dark cycling of arsenic could occur in environments containing a photoarsenotroph and arsenate reducer, since Arx-type arsenotrophs are able to oxidize arsenite in the dark so long there is availability of a terminal electron acceptor.

The first question investigated was: Does photoarsenotrophy occur in freshwater environments? This question was studied through the isolation and genetic characterization of a novel photoarsenotroph from freshwater sediments in Owens River, CA, USA. Our results show the photoarsenotrophs are present in freshwater environments and harbor the Arx genes required for photoarsenotrophy.

The second question investigated was: Can arsenic be light-dark cycled in the environment? In other words, do concentrations of different arsenic forms correlate to the light phase or dark phase of a day? To answer this question, we performed anaerobic microcosm studies with sediment collected from Owens River. Arsenic speciation was measured over light-dark cycles and was followed by metagenome sequencing and analysis. The results provide evidence of light-dark cycling in freshwater sediments and the potential genes involved in the cycle.

The third question investigated was: Can we develop a new model organism for studying photoarsenotrophy? We successfully determined that Cereibacter azotoformans str. ORIO is genetically malleable using traditional cloning techniques and can serve as a model for studying the biological mechanism underlying photoarsenotrophy. This was achieved by validating the role of the arxA gene in ORIO and characterizing the physiology surrounding arsenite oxidation.Taken together, these studies show photoarsenotrophy occurs in freshwater environments, ORIO can serve as a model organism for studying photoarsenotrophy, and evidence of light-dark arsenic redox cycling in freshwater sediments.

Thesis Statement: Managed aquifer recharge (MAR) is a set of tools and protocols that purposefully collects water to replenish underground aquifers. Types of MAR vary in their capacity to recharge, the price to implement them, and the water quality of infiltrating water. Distributed stormwater collection MAR uses hillslopes and gravity to direct stormwater into collection basins where it then infiltrates back down into the aquifer. This dissertation uses techniques from microbiology, water analysis, carbon characterization, and metagenomic sequences to elucidate how the design of a distributed stormwater collection MAR basin impacts the microbial community, thus impacting the geochemical cycling and water quality. The addition of a carbon-rich permeable reactive barrier (PRB) layered on top of a collection basin promotes the removal of nitrates from agricultural stormwater. The removal of nitrates, along with most of the geochemical cycling in the subsurface soil, is primarily done by the native microbial community. However, prior to this research, it was unclear how the microbial was being impacted by either infiltration or infiltration with a carbon-rich PRB. There was also little research into how different PRB materials could have different results in both water quality and microbial community composition. This research’s overarching hypothesis was that the addition of a carbon-rich PRB would provide carbon to the native subsurface microbes and power their denitrification. We also hypothesized this carbon shift would provide a trophic benefit to a common consort of microbes and the type of carbon being provided would have different outcomes. This research gives first insights into the soil-microbe-water interactions that occur during MAR and helps provide a framework for a predictive understanding of the carbon and nutrient cycling occurring during MAR infiltration. We first looked at how the soil and water characteristics influence the microbial communities of three pilot-scale field experiments simulating shallow MAR infiltration. We found there was a common shift towards Proteobacteria in sites that had coarser soil profiles (mainly sand) after infiltration, while sites with finer soil profiles (more clay and silt) did not exhibit significant changes in microbial community composition. This soil characteristic trend continued when we compared soil communities below a carbon-rich PRB of woodchips to communities below a plot with no PRB. The finer soil did not show major differences in genera with the addition of a PRB, but the coarse soils had similar increases in bacteria capable of complex carbon degradation and decreases in genera capable of nitrification. The next question we asked was: how is the PRB influencing the metabolism of the underlying microbial soil community? To answer this, we took the leachate from two PRB materials: almond shells and woodchips, and created anaerobic microcosms with soil to mimic fully saturated MAR conditions. We observed the almond shell leachate removed nitrate as quickly as the positive control microcosm amended with lactate, while woodchip leachate was a little slower but faster than the unamended microcosm. This corresponded to the metagenomic analysis where we saw an increase in denitrifying genes such as napA and nosZ in the after samples. Overall the division of genes into different subcategories of metabolism remains unchanged, although the assigned taxonomy of the samples varied greatly between pre- and post- incubation. The final question we asked was: How can we characterize the carbon being leached off the different PRB materials? In order to answer this, we used a biodegradable organic carbon assay, an assimilable organic carbon assay, ultraviolet-visible absorption, and nuclear magnetic resonance to characterize the leachates. Our results show that microbes are able to use the carbon leached off of the almond shells better than the woodchips. The woodchips were composed of more complex carbon which could provide a long-term solution for a MAR basin that is experiencing wet-dry/ oxic/ anoxic cycling. These results taken together give a greater understanding of how the design of a MAR system is impacting the native communities in the subsurface soil. It also provides the foundation of important predictive factors for future models and analyses of MAR infiltration.

Thesis statement:

Anoxygenic photosynthetic arsenite oxidation encoded by arxA is a bacterial arsenic metabolism that contributes to the biogeochemical cycle of arsenic in extreme environments.

Abstract:

This dissertation provides molecular genetics and environmental insight into the poorly-understood phenomenon of a photosynthetic microbial metabolism fueled by arsenic. The hypothesis is that arxA is critical for photosynthetic arsenite oxidation and actively found in the environment, which has an impact on the arsenic cycle in euphotic arsenite rich extreme environments.

The discovery of light dependent arsenic metabolisms that allows bacteria to conserve energy by oxidizing arsenite to arsenate in the absence of oxygen was described in 2008. This light dependent (photo) arsenic fueled (arsenotrophy) metabolism is referred to as “photoarsenotrophy” or more specifically anoxygenic photosynthetic arsenite oxidation. These earlier findings have yielded interesting molecular genetics and environmental ecology questions that are address in this dissertation. The first question is that of a molecular genetic nature, namely: Is the arsenite oxidase, ArxA, essential to the photoarsenotrophy mechanism? This question was validated after a genetic system was developed in Ectothiorhodospira sp. strain BSL-9, since there were no genetic models to study photoarsenotrophy.

The second question that was investigated was: Do anoxygenic photosynthetic arsenite oxidizing bacteria transform arsenite to arsenate in arsenic rich environments? We surveyed Paoha Island Mono Lake in California and Big Soda Lake, NV, two extreme environments rich in arsenic known to have ideal physicochemical conditions, photosynthetic purple sulfur bacterial and the arsenite oxidase arxA gene. The results provide evidence of anoxygenic photosynthetic arsenite oxidation activity by demonstrating in situ arxA gene expression within the hot spring pool biofilms of Paoha Island, and also provide evidence for a microbial arsenic cycle that was light dependent within anoxic water column samples that were collected from Big Soda Lake, Nevada. The environmental-functional gene ecology study demonstrated that Big Soda Lake contains microbial populations that can perform anoxygenic photosynthesis coupled to arsenite oxidation and arsenate reduction. These ecological studies further elucidate the biogeochemical cycle of photoarsenotrophy in extreme environments, lay the foundation for future fundamental molecular genetics and raising many questions that have yet to be addressed.

Microorganisms play a significant role in environmental arsenic cycling. The most recent discovery to the ever growing collection of known arsenic metabolisms is photosynthesis-linked arsenite oxidation (photoarsenotrophy). However, it is poorly understood and has only been identified in thermal springs on Paoha Island of Mono Lake, CA. The arsenite oxidase ArxA is thought to be responsible for the oxidation of arsenite in photoarsenotrophy. However, the first and only isolated photoarsenotroph, Ectothiorhodospira sp PHS-1, has not proven amenable to genetic manipulations, making genetic confirmation impossible. Water, sediment, and tufa collected from Big Soda Lake and Mono County were used for enrichment culturing and functional gene analyses. arxA-specific primers successfully amplified products from 15 out of 18 environmental samples, and cloned products show high homology to existing arxA-like sequences. Strains containing arxA-like sequences and capable of arsenite oxidation belonging to the genera Ectothiorhodospira and Halomonas were isolated. This work demonstrates that arxA sequences and photoarsenotrophs can also be found in Big Soda Lake, NV, and presents two novel arxA-containing, arsenite-oxidizing strains: the chemoautotroph Halomonas sp. BSL-1 and the photoarsenotroph Ectothiorhodospira sp. BSL-9.

Arsenic is a naturally occurring ubiquitous metalloid that is usually associated with Iron, sulfur and other compounds in the earth’s crust. In some places around the world the bio-geochemical conditions can cause the mineral bound form of arsenic (arsenate) to be reduced to a more water-soluble form (arsenite). In its reduced state, arsenic can seep from the soil down into ground water aquifers and contaminate drinking water supplies. The effects of drinking arsenic tainted water are devastating, however the current methods of detecting arsenic contamination are difficult to implement in most communities that rely on individual tube wells for drinking water.

Dissimilatory arsenic reducing microbes can release terminal electrons, from their electron transport chain, onto arsenic in the nearby environment. These electrons then cause arsenate to be reduced to arsenite leading to eventual arsenic pollution of drinking water. However, arsenic reduction only occurs when more favorable terminal electron acceptors, like oxygen or nitrate, are depleted. An understanding of the genetic and biochemical triggers that activate dissimilatory arsenic reduction will help future detection and bioremediation efforts. This dissertation is a compilation of several systems level studies I performed in order to investigate the molecular mechanisms regulating arsenic reduction in Shewanella sp. ANA-3. The techniques include transcriptomics, molecular biology and comparative genomics. Using systems biology techniques I identified important regulatory parameters in activating respiratory arsenic reduction in three forms:

• Transcriptomics of ANA-3 during arsenic reduction vs. growth on oxygen or fumarate

• Characterization of the role of cAMP as a global regulator of cellular respiration in ANA-3

• Isolation of a novel arsenic respiring microbe from an arsenic rich hot spring

Systems biological approaches in Shewanella represent a new frontier that considers the sum of biological processes that allow this genre of microbes to thrive in some of the most hostile environments on earth. My findings contribute to both a broad understanding of cellular respiration in Shewanella and the mechanistic criteria for arsenic reduction.