Sedimento do reservatório hipereutrófico de Salto Grande, localizado na cidade de Americana, São Paulo, foi cultivado em condições anaeróbias em meio mineral adicionado de compostos orgânicos (ácidos voláteis e álcoois) com a finalidade de favorecer a metanogênese do sistema. Com a produção de 70% de metano o sedimento foi utilizado para o teste de degradação anaeróbica do TCE. Os testes foram realizados sob atmosfera de N2/CO2 (70:30%) em frascos reatores a 25ºC e agitação constante de 150 rpm. Os frascos reatores foram preparados com meio mineral, acrescido de fontes orgânicas (5 mM de ácidos acético, fórmico e butírico, mas 2,5 mM de ácido lático e 5 mM de etanol e metanol) e inoculado com 5 g de sólidos totais voláteis por litro. Foram preparados frascos com 12 e 6 mg de tricloroetileno por litro. Dois tipos de controles foram preparados, um sem tricoroetileno e outro sem inóculo. Análise da diversidade microbiana utilizando a metodologia do DGGE - Eletroforese em Gel com Gradiente Desnaturante - foram feitas com amostras dos frascos reatores no final do experimento. O DNA da comunidade foi extraído de acordo com o protocolo descrito por TSAI & OLSON (1991) e fragmentos do DNAr 16S foram amplificados com \"primers\" do Domínio Archaea e Bacteria. Os resultados dos testes de degradação do TCE demonstraram a remoção biótica de 68% e 66% nos reatores contendo 6 e 12 mg TCE/L, respectivamente, depois de 56 dias de incubação. No final do experimento morfologias similares aos gêneros Methanosarcina e Methanosaeta estavam presentes. A análise da diversidade microbiana não revelou uma significativa na comunidade após a adição do TCE, demonstrando que a microbiota enriquecida proveniente do reservatório de Salto Grande foi resistente à concentração do TCE estudada podendo ser responsável pelo processo de degradação sob metanogênese. / Sediments from the supereutrophic reservoir of Salto Grande, City of Americana, São Paulo State, Brazil, were cultivated under anaerobic conditions in a mineral medium added of organic compounds (volatile fatty acids and alcohols) in order to produce methane. Under 70% of methane production, sediment samples were used for tests of TCE anaerobic degradation. The tests were carried out under N2/CO2 (70:30%) atmosphere in reactor flasks, at 25°C, and constant shaking at 150 rpm. The reactor flasks were prepared with mineral medium, added with organic sources [5 mM of acetic, formic and butyric acids, plus 2.5 mM of lactic acid and 5 mM of ethanol and methanol each], and inoculated with 5 g of STV/L of the sediments. Amounts of 6 and 12 mg/L of TCE concentrations were evaluated. Two types of control reactors were prepared, without TCE and without sediments. Diversity analyses using the DGGE - Denaturing Gradient Gel Eletrophoresis - technique were done with samples from the reactor flasks at the end of the experiment. The community DNA was extracted as described by TSAl & OLSON (1991) and fragments of the 16SDNAr were magnified using the PCR methodology, with Bacteria and Archaea domain primers. The results showed degradation of 40% of TCE at concentrations of 6 mg/L and 12 mg/L after 13 days of incubation time, and complete organic acids removal with 40% of methane in the atmosphere. A second addition of 9 mM of the former organic acids indicated and 4.5 mM of lactic acid resulted in 90% of TCE removal, with 50% of methane, after 56 days of incubation time. Morphologies similar to the genera Methanosarcina, Methanosaeta and Methanospirillum were verified. The microbial diversity analysis did not reveal significant differences among Bacteria and Archaea domains under TCE additions. It was possible to assume that the enriched microbiota from the Salto Grande reservoir was resistant to the concentrations of TCE studied and can be responsible for the degradation processes under methanogenesis.

Esta pesquisa teve como objetivo quantificar a emissão de metano (CH4) proveniente da vinhaça presente em diferentes sistemas de armazenamento e transporte e, adicionalmente, avaliar, por técnicas independentes de cultivo, a microbiota funcional relacionada à produção deste gás. Para atingir esta meta foram realizados três estudos complementares. O primeiro abordou a caracterização dos sistemas de armazenamento e transporte de vinhaça encontrados no Brasil baseado em um questionário aplicado às usinas produtoras de etanol. O segundo visou quantificar as emissões de CH4 em condições de campo provenientes da vinhaça nos canais e tanques e também em laboratório em um estudo de incubação. O terceiro estudo avaliou a microbiota funcional associada à emissão de CH4 através de técnicas independentes de cultivo, como PCR em tempo real (qPCR) e pirosequenciamento. As análises microbiológicas indicaram que as emissões de CH4 são produzidas, preferencialmente, através da decomposição anaeróbia do material orgânico dissolvido da vinhaça depositados no fundo dos sistemas. Estas emissões não são desprezíveis e devem ser consideradas nos cálculos de pegada de carbono do etanol. Nos canais sem revestimento a emissão média em dois anos safras consecutivos apresentou valor de 0,75 kg CO2 eq m-3 de vinhaça, aproximadamente 5 vezes superior às emissões na parte revestida. Nos tanques a emissão foi aproximadamente setenta vezes inferior quando comparada ao canal revestido. O experimento de incubação auxiliou no entendimento de que a vinhaça sozinha não produz quantidades significativas de CH4. Entende-se que os nichos microbianos metanogênicos provavelmente são formados no sedimento, enquanto que a vinhaça mantém as condições de anaerobiose do sedimento necessárias à metanogênese e fornece nutrientes para acelerar a reação. O gênero Methanobrevibacter se mostrou dominante na comunidade microbiana metanogênica, conforme demonstrado pelo pirosequenciamento do gene 16S rRNA. Houve correlação positiva entre a abundância do gene 16S rRNA de Arquéia e dos genes funcionais mcrA e mba com a emissão de CH4. As informações sobre produção e emissão de CH4 e das características da vinhaça constituem informações importantes para tomada de decisão sobre a mitigação e/ou aproveitamento do CH4 gerado para fins econômicos e ambientais. / This research aimed to quantify methane (CH4) emissions from the vinasse in different storage and transportation systems and, additionally, to evaluate the functional microbiota associated with the production of this gas by molecular biology approaches. Three complimentary studies were performed to reach this goal. The first one was related to the characterization of main vinasse storage and transportation systems adopted in Brazil based on a survey administered to the mills, in south-central region of Brazil, producing sugarcane etanol. The second aimed to quantify the CH4 emissions from vinasse in both, field - channels and thanks - and laboratory conditions. The third study evaluated the functional microbiota associated with the CH4 emission by molecular biology approaches like real time PCR ans pyrosequencing. Microbial analysis indicated that CH4 emissions are produced preferably by anaerobic decomposition of the organic material dissolved in the vinasse and deposited on the bottom of the systems. These emissions are not negligible and should be considered in ethanol\'s carbon footprint calculations. At the uncoated part of the channel, the average emission from two crop years was 0.75 kg CO2 eq m-3 of vinasse, about 5 times greater than the emissions at the coated part. Methane emissions from the tank were about seventy times lower than from the uncoated channel. The laboratory experiment supported the understanding that the vinasse alone produces no significant emission of CH4. The microbial methanogenic niches were probably formed in the sediment, while the vinasse keeps sediment anaerobic conditions necessary for methanogenesis and provides nutrients to speed up the reaction. The Methanobrevibacter genus showed dominant in methanogenic microbial community, as demonstrated by pyrosequencing of the 16S rRNA gene. There was a positive correlation between the abundance of 16S rRNA gene Archaea and the functional mcrA and mba genes with the emission of CH4. Information on production and emission of CH4 and vinasse characteristics are important for decision making on mitigation and/or use of gas generated for economic and environmental purposes.

O objetivo do presente estudo foi identificar entre 10 óleos essenciais, os dois com maior capacidade mitigadora das emissões de metano proveniente da fermentação in vitro da microbiota ruminal de bovinos da raça Nelore (Etapa I), e o subsequente impacto da adoção destes dois óleos essenciais como estratégia nutricional para mitigação das emissões de metano in vivo por bovinos Nelore (Etapa II). Na primeira etapa, pela técnica de produção de gases in vitro foram avaliados a produção de gases acumulada, a degradabilidade, o fator de partição, a concentração de nitrogênio amoniacal, a produção de ácidos graxos de cadeia curta e a produção de metano com o uso dos óleos essenciais extraídos das seguintes plantas: anis estrelado (Illicium verum), citronela (Cymbopogon winterianus), cravo botão (Eugenia caryophyllus), eucalipto staigeriana (Eucalyptus staigeriana), eucalipto globulus (Eucalyptus globulus), gengibre (Zingiber officinale), ho wood (Cinnamomum camphora), melaleuca (Melaleuca alternifolia), orégano (Origanum vulgare) e tomilho branco (Thymus vulgaris). Todos os óleos essenciais foram testados em quatro doses; 0, 50, 250 e 500 mg/l de meio de incubação. O delineamento experimental foi em blocos casualizados, com quatro blocos, 10 tratamentos, quatro doses e duas réplicas dentro de cada bloco. Os procedimentos estatísticos foram obtidos com auxílio do programa SAS 9.3. Os resultados foram submetidos a análise de variância e regressão, com graus de liberdade desdobrados em efeitos linear e quadrático. A produção de gases acumulada sofreu efeito linear negativo (P<0,05) com o aumento dos níveis de inclusão dos óleos essenciais de citronela, ho wood, orégano e tomilho. A degradabilidade do substrato, a produção total de ácidos graxos de cadeia curta e a concentração de nitrogênio amoniacal não foram afetadas (P>0,05) por nenhum dos tratamentos. As menores produções líquidas de metano estiveram associadas às menores produções acumuladas de gases, nas quais os óleos essenciais de orégano e de tomilho tiveram maior poder mitigador de metano em doses acima de 250 mg/l. Esses mesmos óleos essenciais (tomilho e orégano) mostraram ser os mais promissores para o uso em dietas de bovinos por apresentarem as maiores eficiências na mitigação da produção de metano sem afetar a degradação do substrato, sugerindo provável efeito inibitório sobre a população de arqueias metanogênicas. Na etapa II, os óleos essenciais de orégano e de tomilho foram avaliados através de um delineamento experimental em quadrados latinos 3x3 replicados com três períodos de 21 dias cada, no qual seis bovinos da raça Nelore foram alimentados com dieta total composta de 70% silagem de milho e 30% concentrado a base de milho e farelo de soja. Os óleos essenciais foram inclusos na dieta na quantidade de 3 ml por kg de concentrado. Foram mensurados o consumo, a digestibilidade aparente total (da MS, da FDN e da FDA), os parâmetros ruminais (pH, concentração de nitrogênio amoniacal e de ácidos graxos de cadeia curta) e a emissão de metano (através da técnica do gás traçador SF6). As análises estatísticas foram realizadas com auxílio do programa SAS 9.3, utilizando o procedimento MIXED. O efeito de tratamento foi separado pelo teste de Tukey a 5% de probabilidade. A inclusão dos óleos essenciais de orégano ou de tomilho na dosagem avaliada in vivo, não foi suficiente para promover mudanças significativas (P>0,05) em nenhum dos parâmetros avaliados. / The objective of this study was to identify two out of 10 essential oils with the greatest capacity to mitigate methane emissions through an in vitro fermentation assay using ruminal microorganisms of Nellore cattle (Step I), and the subsequent impact of the use of these two essential oils as a nutritional strategy to mitigate methane emissions in vivo trial with Nellore cattle (Step II). In the step 1, it was evaluated through in vitro gas production technique the accumulated gas production, degradability, partitioning factor, ammoniacal nitrogen concentration, short chain fatty acid production and methane production, with the use of essential oils extracted from the following plants: anise (Illicium verum), citronella (Cymbopogon winterianus), clove (Eugenia caryophyllus), eucalyptus staigeriana (Eucalyptus staigeriana), eucalyptus globulus (Eucalytus globulus), ginger (Zingiber officinale), melaleuca (Melaleuca alternifolia), oregano (Origanum vulgare) and white thyme (Thymus vulgaris). All essential oils were tested in four doses; 0, 50, 250 and 500 mg / l of incubation solution. The experimental design was in randomized blocks, with four blocks, 10 treatments, four doses and two replicates within each block. The statistical procedures were done with the help of the SAS 9.3 program. The results were analyzed by analysis of variance and regression, with degrees of freedom unfolded in linear and quadratic. Accumulated gas production had a linear negative effect (P <0.05) with increasing the inclusion level of the essential oils of citronella, ho wood, oregano and thyme. Substrate degradability, total short chain fatty acid production and ammoniacal nitrogen concentration were not affected (P> 0.05) by treatments. The lower liquid methane yields were associated with lower accumulated gas production, in which oregano and thyme essential oils were the more potent at doses greater than 250 mg / l. These essential oils (thyme and oregano) were the most promising for use in cattle diets because they were the most effective for methane mitigation without affecting substrate degradation, suggesting a probable inhibitory effect on the methanogenic arqueas. In step II, the essential oils of oregano and thyme were evaluated through an experimental design in replicate 3x3 Latin squares with three periods of 21 days each, in which six Nellore cattle were distributed in the control, oregano oil and thyme oil treatments, were fed a total diet composed of 70% corn silage and 30% of concentrate based on ground corn and soybean meal. The essential oils were included in the diet 3 ml per kg of concentrate. Intake, total apparent digestibility (DM, NDF and ADF), ruminal parameters (pH, concentration of ammoniacal nitrogen and short chain fatty acids) and methane emission (through SF6 tracer gas) were measured. The inclusion of the essential oils of oregano or thyme in the dosage evaluated was not sufficient to promote significant changes (P> 0.05) in any of the outcomes evaluated.

O sedimento marinho da Península Antártica representa uma área sensível a mudanças ambientais. No entanto, pouco se conhece sobre as comunidades microbianas que habitam esse ecossistema, incluindo a sua diversidade, distribuição e variações temporais. O objetivo foi determinar a estrutura das comunidades microbianas nos sedimentos marinhos da Baía do Almirantado, Ilha Rei George, Península Antártica. Sedimentos da Baía apresentam uma predominância dos Filos Proteobacteria, Firmicutes e Actinobacteria. Análise temporal revelou que comunidades microbianas em sedimentos próximos à estação Ferraz são mais estáveis quando comparadas aos sedimentos em áreas de menor atividade antrópica. No gradiente de profundidade foi observado que a estrutura de comunidade não mudou, indicando tolerância a variações de pressão hidrostática. Organismos heterotróficos dos gêneros Psychrobacter, Psychromonas e Loktanella foram os mais abundantes, sugerindo uma alta concentração de matéria orgânica disponível. O enriquecimento de culturas metanogênicas produziu até 1,70 mmol de CH4 após 120 dias de incubação. Este estudo sugere que as condições dos sedimentos favorecem organismos psicrofílicos de metabolismo heterotrófico. / Marine sediment of the Antarctic Peninsula is a susceptible area to environmental changes. However, little is known about the microbial communities inhabiting this ecosystem, including its diversity, distribution and variations over time. The aim of this study was to determine the structure of microbial communities present in marine sediments of Admiralty Bay, King George Island, on the Antarctic Peninsula. Sediments from Admiralty Bay shown a predominance of the Proteobacteria, Firmicutes, and Actinobacteria phyla. Temporal analysis revealed that microbial communities in sediment, near Ferraz station, are more stable compared to that in the sediments in areas of lower human activity. No variation on the community structure was observed in depth gradient, indicating tolerance to hydrostatic pressure variations. Heterotrophic organisms of the genera Psychrobacter, Psychromonas and Loktanella were the most abundant, suggesting a high concentration of organic matter in the sediment. Enrichment of methanogenic cultures enrichment yielded 1.70 mmol of CH4. This study suggests that conditions in sediments favoring metabolism of heterotrophic and psychrophilic organisms.

O Brasil é o segundo maior produtor de etanol do mundo. Na safra 2015/16, foram produzidos 30,4 bilhões de litros. Na produção de etanol realizada no Brasil é gerado grande volume de vinhaça, cerca de 10 a 13 litros desse resíduo por litro de etanol produzido. Isso significa uma produção anual de cerca de 300 bilhões de litros desse material residual. Em função disso e do risco de contaminação ambiental por esse material, há necessidade de reduzir o volume desse resíduo. Com isso minimizar o custo com a gestão desse passivo ambiental. Isso pode ser feito mediante a concentração da vinhaça. Além disso, o mercado de energia elétrica tem se apresentado com uma boa oportunidade para o setor sucroenergético. Por esses motivos, o objetivo deste projeto foi a produção de metano a partir do uso de vinhaça concentrada. Para tal, a vinhaça concentrada foi obtida em uma usina no Estado de São Paulo que utiliza concentrador de vinhaça de múltiplos efeitos. Utilizaram-se dois reatores (R1 e R2) do tipo UASB (reator anaeróbio de fluxo ascendente e manta de lodo) com capacidade para 50 L e volume reacional de 34,5 L cada. Os reatores foram operados à 38 ºC, com tempo de detenção hidráulica de 24 horas por 103 dias. O reator R1 recebeu cargas orgânicas volumétricas (COV) crescentes durante 9 fases (3,94; 3,5; 3,89; 4,66; 4,94; 5,33; 5,66; 6,08; 6,38 e 8,96 g DQO (L d)-1), com DQO afluente variando de 23.927 mg L-1 a 44.060 mgL-1. O reator R2 operou como tratamento controle, com COV variando entre 3,83 e 4,32 g DQO (L d)-1. A avaliação do processo de biodigestão anaeróbia foi realizada com base na produção de biogás, eficiência de remoção de DQO, pH, alcalinidade, concentração de ácidos voláteis totais (AVT), concentração de sólidos no lodo e atividade metanogênicas específica do lodo (AME). R1 mostrou-se mais eficiente, com pH e alcalinidade dentro da faixa ideal e efluente com menor concentração de AVT. O desempenho do processo mostrouse estável, atingindo eficiência máxima de remoção de DQO de 90% em R1. A maior produção volumétrica de biogás ocorreu concomitantemente com a maior produção específica de biogás, quando a DQO afluente foi de 42.003 mg L-1, a produção volumétrica de biogás foi de 135,24 L dia-1, e a produção de biogás em relação a DQO removida foi de 0,362 LbiogásgDQOremov-1. No fim do experimento, a AME de R1 foi de 1,19 gDQOCH4 gSTV.d-1, 40% superior a R2, que teve 0,85 gDQOCH4 gSTV.d-1. A alta eficiência de remoção de matéria orgânica e produção de biogás no reator alimentado com alta concentração de DQO foi obtida através da adaptação gradual do consórcio microbiano. / Brazil is the second largest producer of ethanol in the world. In the 2015/16 crop, 30.4 billion liters were produced. In Brazil\'s ethanol production, a large volume of vinasse is generated, about 10 to 13 liters of this residue per liter of ethanol produced. This means an annual output of about 300 billion liters of this waste material. Because of this and the risk of environmental contamination by this material, there is a need to reduce the volume of this waste. This will minimize the cost of managing this environmental liability. This can be done by concentrating the vinasse. In addition, the electricity market has presented itself with a good opportunity for the sugar-energy sector. For these reasons, the objective of this study was to produce methane from concentrated vinasse. For this purpose, concentrated vinasse was obtained from a mill that uses a multi-purpose vinasse concentrator in São Paulo State. Two reactors (R1 and R2) of UASB type (up-flow anaerobic sludge blanket) with capacity for 50 L and reactional volume of 34.5 L each one. The reactors were operated at 38 °C, with hydraulic retention time of 24 hours for 103 days. The reactor R1 received increasing organic load rate (OLR) during 9 phases (3.94, 3.5, 3.89, 4.66, 4.94, 5.33, 5.66, 6.08, 6.38 And 8.96 g COD (L d)-1), with affluent COD ranging from 23,927 mg L-1 to 44,060 mg L-1. The R2 reactor was operated as a control treatment, with OLR ranging from 3.83 to 4.32 g COD (L d)-1. The evaluation of the anaerobic biodigestion process was carried out based on biogas production, COD removal efficiency, pH, alkalinity, total volatile acid concentration (TVA), sludge solids concentration and sludge specific methanogenic activity (SMA). R1 showed to be more efficient, with pH and alkalinity within the ideal range and effluent with lower concentration of TVA. The process performance was stable, achieving maximum COD removal efficiency of 90% in R1. The higher volume of biogas production occurred concurrently with the higher specific biogas production, when the COD was 42.003 mg L-1, the volumetric biogas production was 135.24 L day-1, and the biogas production in relation to removed COD was 0.362 Lbiogas gCODremoved -1. At the end of the experiment, the SMA of R1 was 1.19 gCODCH4 gTVS.day-1, 40% higher than R2, which had 0.85 gCODCH4 gTVS.day-1. The high efficiency of organic matter removal and biogas production in the reactor fed with high COD concentration was obtained through the gradual adaptation of the microbial consortium.

Sedimento do reservatório hipereutrófico de Salto Grande, localizado na cidade de Americana, São Paulo, foi cultivado em condições anaeróbias em meio mineral adicionado de compostos orgânicos (ácidos voláteis e álcoois) com a finalidade de favorecer a metanogênese do sistema. Com a produção de 70% de metano o sedimento foi utilizado para o teste de degradação anaeróbica do TCE. Os testes foram realizados sob atmosfera de N2/CO2 (70:30%) em frascos reatores a 25ºC e agitação constante de 150 rpm. Os frascos reatores foram preparados com meio mineral, acrescido de fontes orgânicas (5 mM de ácidos acético, fórmico e butírico, mas 2,5 mM de ácido lático e 5 mM de etanol e metanol) e inoculado com 5 g de sólidos totais voláteis por litro. Foram preparados frascos com 12 e 6 mg de tricloroetileno por litro. Dois tipos de controles foram preparados, um sem tricoroetileno e outro sem inóculo. Análise da diversidade microbiana utilizando a metodologia do DGGE - Eletroforese em Gel com Gradiente Desnaturante - foram feitas com amostras dos frascos reatores no final do experimento. O DNA da comunidade foi extraído de acordo com o protocolo descrito por TSAI & OLSON (1991) e fragmentos do DNAr 16S foram amplificados com \"primers\" do Domínio Archaea e Bacteria. Os resultados dos testes de degradação do TCE demonstraram a remoção biótica de 68% e 66% nos reatores contendo 6 e 12 mg TCE/L, respectivamente, depois de 56 dias de incubação. No final do experimento morfologias similares aos gêneros Methanosarcina e Methanosaeta estavam presentes. A análise da diversidade microbiana não revelou uma significativa na comunidade após a adição do TCE, demonstrando que a microbiota enriquecida proveniente do reservatório de Salto Grande foi resistente à concentração do TCE estudada podendo ser responsável pelo processo de degradação sob metanogênese. / Sediments from the supereutrophic reservoir of Salto Grande, City of Americana, São Paulo State, Brazil, were cultivated under anaerobic conditions in a mineral medium added of organic compounds (volatile fatty acids and alcohols) in order to produce methane. Under 70% of methane production, sediment samples were used for tests of TCE anaerobic degradation. The tests were carried out under N2/CO2 (70:30%) atmosphere in reactor flasks, at 25°C, and constant shaking at 150 rpm. The reactor flasks were prepared with mineral medium, added with organic sources [5 mM of acetic, formic and butyric acids, plus 2.5 mM of lactic acid and 5 mM of ethanol and methanol each], and inoculated with 5 g of STV/L of the sediments. Amounts of 6 and 12 mg/L of TCE concentrations were evaluated. Two types of control reactors were prepared, without TCE and without sediments. Diversity analyses using the DGGE - Denaturing Gradient Gel Eletrophoresis - technique were done with samples from the reactor flasks at the end of the experiment. The community DNA was extracted as described by TSAl & OLSON (1991) and fragments of the 16SDNAr were magnified using the PCR methodology, with Bacteria and Archaea domain primers. The results showed degradation of 40% of TCE at concentrations of 6 mg/L and 12 mg/L after 13 days of incubation time, and complete organic acids removal with 40% of methane in the atmosphere. A second addition of 9 mM of the former organic acids indicated and 4.5 mM of lactic acid resulted in 90% of TCE removal, with 50% of methane, after 56 days of incubation time. Morphologies similar to the genera Methanosarcina, Methanosaeta and Methanospirillum were verified. The microbial diversity analysis did not reveal significant differences among Bacteria and Archaea domains under TCE additions. It was possible to assume that the enriched microbiota from the Salto Grande reservoir was resistant to the concentrations of TCE studied and can be responsible for the degradation processes under methanogenesis.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

The most common strategy for handling paper mill solid waste is typically disposal in landfills. However, several drawbacks are associated with this type of solid waste management, such as increasing costs due to oil price rise, governmental restrictions on land use, and environmental concerns such as leaching of disposed contaminants into groundwater, as well as methane generation of and release to the atmosphere, thus contributing to global warming. An alternative to reduce solids prior to disposal and to recover methane as a renewable fuel is anaerobic digestion, but it is not yet clear whether such an approach is feasible in paper mills. In this study, the anaerobic digestion of paper mill waste streams was evaluated for a paper plant located in Belen, Costa Rica, to investigate up to what extent certain waste streams can be anaerobically digested, to what extent energy can be produced in the form of methane for implementation in a wastewater treatment plant and to evaluate the conditions that will favor methane generation from select waste streams. Batch assays were performed to evaluate the biodegradability of single and combined waste samples under ideal, laboratory conditions. Samples were obtained from the manufacturing plant as well as the wastewater treatment plant at the paper mill under study. The ultimate biodegradability ranged 25 to 85% in terms of volatile solids destruction, corresponding to the waste activated sludge (WAS) and Flotation Cell rejects, respectively. The COD destruction of single samples ranged from 45 to 63%, corresponding to WAS and wastewater treatment plant (WWTP) dissolved air flotation (DAF) skimmings, respectively. Methane generation ranged from 80 to 190 ml at 35oC/g COD added for all single samples (excluding underflows). In combination Feed 1 was reduced by 46 and 52% and Feed 2 by 27 and 38%, respectively. Two combinations of two single samples each (Feed 1 and 2), formulated according to plant operational data, and their solids and COD destruction as well as methane generation in semicontinuous flow anaerobic digesters were evaluated at different solids retention times (30, 20, 15, and 7 days). Nutrients (N, and P) availability as well as alkalinity in the plant waste streams were evaluated and minimum supplements were used to support an efficient anaerobic digestion process. The reactors reached stable operation at all retention times evaluated. Methanogenesis was the predominant, terminal metabolic process under anaerobic, mesophilic conditions, but the overall process rate was determined by the hydrolysis of the particulate substrate. Reactors fed with Feed 1 achieved the highest level of destruction, which amounted to 85% of phosphorus that is typically present in paper mill wastes. Alkalinity addition to the feed (3.5 mg NaHCO3/L) was necessary to maintain the reactors pH above 6.9.

Methane is a greenhouse gas and a major contributor to climate change. Methanogenic Archaea produce more than 1 billion tons of this gas each year through methanogenesis, the anaerobic reduction of CO₂ to methane. Coenzyme B (CoB) is one of eight coenzymes required for methanogenesis and it is unique to methanogens. Therefore, this coenzyme is a potential target for inhibiting methanogenesis. To further elucidate the CoB biosynthetic pathway, genes from Methanocaldococcus jannaschii were cloned and expressed in an effort to identify the CoB homoaconitase. From this study, the MJ0499-MJ1277 pair of proteins was identified as the methanogen isopropylmalate isomerase involved in leucine and isoleucine biosynthesis. The MJ1003-MJ1271 pair of proteins was characterized as the homoaconitase required for CoB biosynthesis. This enzyme exhibited broad substrate specificity, catalyzing the isomerization of cis-unsaturated tri-carboxylates with [gamma]-chains of 1-5 methylenes in length. Previously characterized homoaconitases only catalyzed half of the predicted reactions in the isomerization of homocitrate. The MJ1003-MJ1271 proteins function as the first homoaconitase described to catalyze the full isomerization of homocitrate to homoisocitrate. Also, the CoB homoaconitase was identified as specific for (R)-homocitrate and cis-unsaturated intermediates, contrary to a previous study that suggested the substrate specificity of this enzyme included (S)-homocitrate and trans-homoaconitate. The M. jannaschii isopropylmalate isomerase and homoaconitase share more than 50% sequence identity and catalyze analogous reactions. Site directed mutagenesis of the MJ1271 protein was used to identify residues involved in substrate specificity. Arg26 of MJ1271 was critical for the specificity of the CoB homoaconitase. Mutation of this residue to the analogous residue in the M. jannaschii isopropylmalate isomerase, Val28, altered the substrate specificity of the homoaconitase to include the substrates of isopropylmalate isomerase. These homologs of aconitase require a [4Fe-4S] cluster for coordinating their respective substrates at the enzyme active site. However, methanogens lack most of the proteins required for iron-sulfur cluster assembly. Therefore, genes homologous to the Salmonella enterica ApbC iron-sulfur scaffold protein were characterized from methanogens. The MMP0704, MJ0283, and SSO0460 proteins from Methanococcus maripaludis, M. jannaschii, and Solfolobus solfataricus, respectively, were identified as scaffold proteins involved in methanogen iron-sulfur cluster biosynthesis. / text

Natural gas is an important transitional energy source to replace more carbon intensive coal combustion in the face of climate change and increasing global energy demands. A significant proportion of natural gas reserves (~20%) were recently generated by microorganisms that degrade organic-rich formations (i.e. coal, shale, oil) in-situ to produce methane. Recent studies have shown that these microbial communities may be potentially stimulated to generate more methane to extend the lifetime (~10 years) of existing biogenic gas wells. This dissertation investigates how microbial coalbed methane (CBM) systems are impacted by geochemical conditions, microbial community composition, and groundwater recharge. The first study is a review and synthesis of existing basic research and commercial activities on enhancement of microbial CBM generation, and identification of key knowledge gaps that need to be addressed to advance stimulation efforts. The second study couples water and gas geochemistry with characterization of microbial communities in coalbeds in the Powder River Basin (PRB), Wyoming to investigate the influence of microbiology on water and gas geochemistry. Geochemistry results indicated that nutrients are likely source in situ from coal, and that all sulfate must be removed from the system before methanogenesis will commence. Increased archaeal (i.e. methanogens) diversity was observed with decreasing sulfate concentration, while sulfate reducing bacterial communities were different in wells with high sulfate concentrations (sulfate reducing conditions) when compared to wells with low sulfate concentrations (methanogenic conditions). The third study uses noble gases to constrain the residence time of groundwater associated with CBM in the PRB. Measured diffusional release rates of 4He from PRB coals were ~800 times greater than typical rates observed in sandstone or carbonate aquifers, and measured 4He values far exceeded expected values from in-situ decay of U and Th. Groundwater 4He residence times ranged from <1 to ~800 years using the measured diffusion rates versus ~130 to 190,000 years using a standard model. Coal waters with the longest residence time had the highest alkalinity concentrations, suggesting greater extents of microbial methanogenesis, although there was no relationship between groundwater "age" and methane concentrations or isotopic indicators of methanogenesis. Constraining the relationship between microbial activity (e.g. mechanisms of coal biodegradation and methane generation), environmental geochemical conditions, and groundwater flow is important to better understand subsurface hydrobiogeochemical processes and to ensure the success of future projects related to stimulation of microbial CBM.