The carboxylate biofuels platform (CBP) involves the conversion of cellulosic biomass into carboxylate salts by a mixed microbial community. Chemical engineering approaches to convert these salts to a variety of fuels (diesel, gasoline, jet fuel) are well established. However, prior to initiation of this project, little was known about the influence of inoculum source on platform performance. The studies in this dissertation test the hypothesis that microbial communities from particular environments in nature (e.g. saline and/or thermal sediments) are pre-adapted to similar industrial process conditions and, therefore, exhibit superior performances. We screened an extensive collection of sediment samples from extreme environments across a wide geographic range to identify and characterize microbial communities with superior performances in the CBP. I sought to identify aspects of soil chemistry associated with superior CBP fermentation performance. We showed that CBP productivity was influenced by both fermentation conditions and inocula, thus is clearly reasonable to expect both can be optimized to target desired outcomes. Also, we learned that fermentation performance is not as simple as finding one soil parameter that leads to increases in all performance parameters. Rather, there are complex multivariate relationships that are likely indicative of trade-offs associated within the microbial communities. An analysis of targeted locus pyrosequence data for communities with superior performances in the fermentations provides clear associations between particular bacterial taxa and particular performance parameters. Further, I compared microbial community compositions across three different process screen technologies employed in research to understand and optimize CBP fermentations. Finally, we assembled and characterized an isolate library generated from a systematic culture approach. Based on partial 16S rRNA gene sequencing, I estimated operational taxonomic units (OTUs), and inferred a phylogeny of the OTUs. This isolate library will serve as a tool for future studies of assembled communities and bacterial adaptations useful within the CBP fermentations. Taken together the tools and results developed in this dissertation provide for refined hypotheses for optimizing inoculum identification, community composition, and process conditions for this important second generation biofuel platform.

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Geologic carbon storage (GCS) is a crucial part of a proposed mitigation strategy to reduce the anthropogenic CO2 emissions to the atmosphere. During this process, CO2 is injected as super critical carbon dioxide (SC-CO2) in confined deep subsurface storage units, such as saline aquifers and depleted oil reservoirs. The deposition of vast amounts of CO2 in subsurface geologic formations may ultimately lead to CO2 leakage into overlying freshwater aquifers. Introduction of CO2 into these subsurface environments will greatly increase the CO2 concentration and will create CO2 concentration gradients that drive changes in the microbial communities present. While it is expected that altered microbial communities will impact the biogeochemistry of the subsurface, there is no information available on how CO2 gradients will impact these communities. The overarching goal of this dissertation is to understand how CO2 exposure will impact subsurface microbial communities at temperature and pressure that are relevant to GCS and CO2 leakage scenarios. To meet this goal, unfiltered, aqueous samples from a deep saline aquifer, a depleted oil reservoir, and a fresh water aquifer were exposed to varied concentrations of CO2 at reservoir pressure and temperature. The microbial ecology of the samples was examined using molecular, DNA-based techniques. The results from these studies were also compared across the sites to determine any existing trends. Results reveal that increasing CO2 leads to decreased DNA concentrations regardless of the site, suggesting that microbial processes will be significantly hindered or absent nearest the CO2 injection/leakage plume where CO2 concentrations are highest. At CO2 exposures expected downgradient from the CO2 plume, selected microorganisms emerged as dominant in the CO2 exposed conditions. Results suggest that the altered microbial community was site specific and highly dependent on pH. The site-dependent results suggests no ability to predict the emerging dominant species for other CO2exposed environments. This body of work improves the understanding of how a subsurface microbial community may respond to conditions expected from geologic carbon storage and CO2 leakage. This is the first step for understanding how a CO2 altered microbial community may impact injectivity, permanence of stored CO2, and subsurface water quality. .

It may be possible to tailor crop management to encourage diverse soil microbial communities and beneficial microorganisms, and produce high quality food products. Studies were carried out in 2005-2007 to evaluate the impact of spring wheat (Triticum aestivum L.) cultivar choice and crop polycultures on soil microbial communities in organic and conventional systems, and subsequent wheat quality. Five wheat cultivars were grown organically and conventionally to evaluate grain breadmaking quality and micronutrient content and their impact on the soil microbial community. Organic grain yields were roughly half of conventional yields, but quality levels were all acceptable for Canadian Western Hard Red Spring wheat. Measured soil (0-15 cm) microbial profiles (by phospholipid fatty acid analysis) differed between the two management systems, and amongst cultivars in the conventional system. The most recent cultivar in the study, AC Superb, exhibited the highest levels of fungi suggesting that breeding efforts in conventionally managed environments may have resulted in cultivating mycorrhizal dependence in that environment. In general, many of the studied grain micronutrients were greater in the organically grown wheat system, possibly due in part to decreased grain yield and smaller grain size. Maximizing grain micronutrient content through wheat cultivar choice was dependent on management system. The presence of fungi biomarkers appears to have improved uptake of Mn and Cu. Monocultures and polycultures of common annual crops were grown organically and conventionally in 2006-2007. Intercrops exhibited an ability to overyield in an organic system, largely through weed suppression, but intercrops also overyielded in a conventional system where weeds were controlled through herbicides. As intercrop complexity decreased, the instances of improved weed suppression declined. Management systems and wheat cultivars can alter the composition of the soil microbial community. Annual crop polycultures did not alter soil microbial communities in this study, but showed evidence of agronomic benefits in both organic and conventional systems. / Plant Science

The structure and function of microbial communities are critical to the maintenance and sustainability of terrestrial ecosystem processes. Consequently, there is substantial interest in assessing how microbial communities respond to various land management practices, and if alterations to the characteristics of microbial communities has the potential to disrupt ecosystem processes. This thesis was conducted to identify the long term effects of fertilisation and different levels of post-harvest organic matter removal on the characteristics of the FH litter and soil microbial communities in six, second rotation Pinus radiata plantation forests located around New Zealand. The six sites, established between 1986 and 1994, were sampled in 2002 and 2003. Various physical and chemical properties of the sites were measured, and litterfall production was determined. The microbial biomass in the FH litter layer and soil was determined by chloroform fumigation-extraction, and Biolog plates were used to assess the relative differences in microbial community diversity, based on patterns of substrate utilisation. Fertilisation substantially altered the physical and chemical properties of the forest floor, including FH litter moisture content, mass, carbon content, nitrogen content and carbon: nitrogen ratio and soil pH, nitrogen content and carbon: nitrogen ratio. The same range of FH litter and soil properties were also significantly changed by different levels of organic matter removal. The biomass and diversity of the FH litter and soil microbial communities were significantly altered by fertilisation and organic matter removal, and the differences in the microbial community characteristics were significantly correlated to the effects of the fertilisation and organic matter removal treatments on the physical and chemical environment in the majority of cases. The physical and chemical properties of the sites were significantly correlated to estimates of wood production, and it was also found that the characteristics of the microbial community were strongly related to productivity at several sites. The results demonstrated that fertilisation and organic matter removal regimes have had long term effects on the microbial communities at the sites. The persistence of the effects of the organic matter removal treatments were particularly noteworthy, as these treatments were applied at site establishment, and despite no subsequent reinforcement over the life of the trials, were still substantially influencing the physical, chemical and microbiological properties of the FH litter and soil up to 17 years later. The results of this thesis also emphasised the value of long-term experiments in assessing the effects of disturbance on the physical, chemical and microbiological characteristics of forest ecosystems. Further research into the specific nature of the relationship between site productivity and microbial community characteristics was suggested as an important focus for future studies.

The PLFA profiling method was adopted and used to determine changes in microbial community structure and abundance along natural and human-induced environmental gradients. The presented studies were based on field sampling campaigns combined with targeted laboratory experiments. According to the aims of particular studies, microbial PLFA fingerprinting was combined with the auxiliary below- and aboveground ecosystem characteristics to identify the drivers of microbial responses to environmental changes or with 13C-labelling and metagenomics to obtain more complex information about running processes and involved microorganisms.

Various aspects of the ecophysiology of rootless carnivorous Utricularia plants were assessed, with focus on nutrient acquisition and mutualistic interactions within trapping organs. The study includes extracellular enzyme measurements, the evaluation of commensal microbial community structure and function, stable isotope labelling to determine plant carbon allocation, and ion chromatography analyses of trap fluid composition

abstract: This thesis research focuses on phylogenetic and functional studies of microbial communities in deep-sea water, an untapped reservoir of high metabolic and genetic diversity of microorganisms. The presence of photosynthetic cyanobacteria and diatoms is an interesting and unexpected discovery during a 16S ribosomal rRNA-based community structure analyses for microbial communities in the deep-sea water of the Pacific Ocean. Both RT-PCR and qRT-PCR approaches were employed to detect expression of the genes involved in photosynthesis of photoautotrophic organisms. Positive results were obtained and further proved the functional activity of these detected photosynthetic microbes in the deep-sea. Metagenomic and metatranscriptomic data was obtained, integrated, and analyzed from deep-sea microbial communities, including both prokaryotes and eukaryotes, from four different deep-sea sites ranging from the mesopelagic to the pelagic ocean. The RNA/DNA ratio was employed as an index to show the strength of metabolic activity of deep-sea microbes. These taxonomic and functional analyses of deep-sea microbial communities revealed a `defensive' life style of microbial communities living in the deep-sea water. Pseudoalteromonas sp.WG07 was subjected to transcriptomic analysis by application of RNA-Seq technology through the transcriptomic annotation using the genomes of closely related surface-water strain Pseudoalteromonas haloplanktis TAC125 and sediment strain Pseudoalteromonas sp. SM9913. The transcriptome survey and related functional analysis of WG07 revealed unique features different from TAC125 and SM9913 and provided clues as to how it adapted to its environmental niche. Also, a comparative transcriptomic analysis of WG07 revealed transcriptome changes between its exponential and stationary growing phases. / Dissertation/Thesis / Ph.D. Civil and Environmental Engineering 2013

The aim of this study was to optimize methane production by investigating hydrothermal pretreatment of sugarcane bagasse impregnated with acid (H2SO4) and alkaline H2O2 using substrate (g kg-1) -inoculum (g kg-1) ratio of 1:2. Batch reactors were realized under mesophilic conditions (37 °C). A central composite design (CCD) involving three factors; temperature (°C), time (min), and chemical compound concentration (H2O2 (%v/v) and H2SO4 (%w/v)) was utilized to optimize hydrothermal pretreatment. Thirty-two hydrothermal pretreatments were conducted according to CCD. H2O2 assisted hydrothermal pretreatment resulted in higher solid recovery (93.13%), higher percent glucan increase (139.52%), and lower lignin recovery (76.48%) in pretreated solid fraction in comparison to H2SO4 impregnated hydrothermal pretreatment. In the latter case, lower solid yield (12.27%), glucan recovery (187.01%) and higher lignin recovery (358.85%) was recorded. Higher COD solubilization (25.20 g L-1), lower total phenolic (content 658.13 ppm), higher sulfate (7240 mg L-1), furfural (925.77-2216.47 mg L-1) and 5-hydroxymehtylfurfural (70.95-970.08 mg L-1) were observed in liquid hydrolysate of H2SO4 assisted hydrothermal pretreatment. While lower COD solubilization (17.75 mg L-1), higher total phenolic content (3005.63 ppm), lower concentration of furfural (0-56.91 mg L-1) and 5-hydroxymethylfurfural (2.56-56.60 mg L-1) was recorded with H2O2 assisted hydrothermal pretreatment. Concerning methane production for H2O2 assisted conditions, 5.59 Nmmol g-1 TVS (2% H2O2) to 13.49 Nmmol g-1 TVS (6% H2O2) was recorded. However, pretreatment with 7.36% H2O2 resulted in 14.43 Nmmol g-1 TVS, which was 118.16% higher comparing to untreated sugarcane bagasse (6.60 Nmmol g-1 TVS). Methanogenic inhibition was recorded for most of the H2SO4.pretreated reactor (1 – 3 %w/v H2SO4). Minimum CH4 production observed was 0.58 Nmmol g-1 TVS in pretreatment O-HSO (2% w/v H2SO4). Acetic acid was the predominant volatile fatty acid observed in digestion process of H2O2 treated batch reactor however was not recorded in H2SO4 treated batch reactors. Microbial community analysis indicated the prevalence of unclassified AUTHM297, Clostridium, and Treponema related genera in H2O2 treated reactors. Genera related aromatic compound degradation were identified and abundant in H2SO4 treated reactors. Methanolinea, Methanobacterium, and Methanosaeta were abundant methanogens in both pretreatments. Hydrogen peroxide assisted hydrothermal pretreatment was verified as a better choice for methane production comparing to sulfuric acid assisted hydrothermal pretreatment primarily on account of higher lignin solubilization, higher glucan recovery, and lower furanic compounds production. / O objetivo deste estudo foi otimizar a produção de metano investigando as condições do pré-tratamento hidrotérmico assistido do bagaço da cana de açúcar sob impregnação de ácido (H2SO4) e álcali (H2O2) utilizando-se a razão substrato (g kg-1) inóculo (g kg-1) de 1:2. Os reatores em batelada foram mantidos em condições mesofílicas (37 ºC). Para otimizar as condições de pré-tratamento hidrotérmico, o design de composto central rotacional (DCCR) foi realizado utilizando três fatores: temperatura (ºC), tempo (min) e concentração do composto químico (H2O2 (%v/v) e H2SO4 (% p/v)). Trinta e dois pré-tratamentos hidrotérmicos foram realizados de acordo com a concepção do DCCR. O pré-tratamento hidrotérmico assistido do bagaço, com H2O2 resultou em maior recuperação de sólidos (93,13%), elevado percentual de glicana (139,52%) e menor recuperação de lignina (76,48%) da fração sólida pré-tratada, se comparada aquele com H2SO4. Nesse último caso, observou-se menor rendimento de sólidos (12,27%) e glucanas (187,01%) e maior recuperação de lignina (358,85%). No líquido hidrolisado do pré-tratamento hidrotérmico assistido do bagaço com H2SO4 foi observada elevada solubilização de DQO (25,20 g L-1), menor teor de fenóis totais (658,13 mg L-1), elevado sulfato (7240 mg L-1), furfural (925,77 - 2216,47 mg L-1 e 5-hidroximetilfurfural (70,95 - 970,08 mg L-1). Enquanto, foi registrado menor solubilização de DQO (17,27 g L-1), maior teor de fenóis totais (3005,63 ppm), e menor concentração de furfural (0 - 56.91 mg L-1), 5-hidroximetilfurfural (2,56 - 50,60 mg L--1 com H2O2. Em relação ao a produção de metano nas condições com H2O2, observou-se 5.59 Nmmol g-1 STV (2%v/v H2O2) a 13.49 Nmmol g-1 STV (6%v/v H2O2). No tratamento com 7.36% de H2O2 observou-se 14,43 Nmmol g-1 STV que foi 118.16% maior se comparado com o bagaço não-tratado (6,60 Nmmol g-1 STV). Inibição metanogênica foi observada na maioria dos reatores pré-tratados com H2SO4 (1 – 3% p/v), e a produção mínima observada foi de 0.58 Nmmol g-1 TVS no pré-tratamento com 2% p/v de H2SO4. Ácido acético foi o principal ácido orgânico volátil observado somente no reatores por tratamento de H2O2. Por meio da A análise da comunidade microbiana, para o domínio Bacteria, foi observado prevalência dos gêneros AUTHM297, Clostridium e Treponema nos reatores cujo substrato foi pré-tratado com H2O2. Gêneros relacionados à degradação de compostos aromáticos foram identificados e estiveram em maior abundância nos reatores cujo substrato foi pré-tratado com H2SO4. Methanolinea, Methanobacterium, e Methanosaeta foram os microrganismos do domínio Archaea mais abundantes e identificados em ambos os pré-tratamentos. O pré-tratamento hidrotérmico assistido com H2O2 foi a melhor opção em relação ao H2SO4, devido a maior solubilização de lignina, maior recuperação de glucano e baixa produção de compostos furânicos.

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