No description available.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: A laboratory-scale model was used to simulate biological sulfate removal. The focus of the research was microbial community response, such as the relative abundance of functional groups to changes in influent medium composition. Specific oligonucleotide probes were obtained that recognised sulfate reducing bacteria (SRB) within the biofilm community. Terminal restriction fragment length polymorphism (T-RFLP) and BIOLOG™ Ecoplate analyses were used to study the SRB community when provided with sodium lactate, sucrose or ethanol as carbon sources in complex Postgate C broth. These two analyses, as well as conventional methods, were applied to follow succession patterns in the laboratory scale reactors, and to determine the possible presence and relative abundance of microorganisms other than bacteria under sulfate reducing conditions. T-RFLP and BIOLOG™ Ecoplate analyses indicated a few dominant organisms in the community and a slight decline after a shift to another carbon source. Fluorescent hybridization showed higher numbers of SRB relative to the total microbial community than conventional culturing techniques. Furthermore, microscopic observations showed that not only SRB and other bacteria, but also yeast and filamentous fungi were integrated in a biofilm under sulfate reducing conditions. These microscopic observations were verified with fluorescent in situ hybridization (FISH) and yeast Live I Dead viability probes. / AFRIKAANSE OPSOMMING: Biologiese sulfaat-verwydering is met behulp van "n laboratoriumskaalmodel gesimuleer. Die doel van die navorsing was om die respons van "n mikrobiese gemeenskap met byvoorbeeld die relatiewe hoeveelheid van funksionele groepe op veranderinge in invloeiende medium samestelling te bestudeer. Spesifieke oligonukleotiedpeilers wat sulfaatreduserende bakterieë (SRB) in "n biofilmgemeenskap kan opspoor is gebruik. Die SRB gemeenskap is bestudeer met behulp van terminale-restriksiefragmentlengtepolimorfisme (TRFLP) en BIOLOGTM Ecoplate analise waar natriumlaktaat, sukrose of etanol as koolstofbronne toegevoeg is. Hierdie twee tipes analise en konvensionele metodes is aangewend om suksessiepatrone in die laboratoriumskaalreaktor te volg en die moontlike teenwoordigheid en relatiewe hoeveelheid van organismes, uitsluitende bakterieë, onder sulfaatreduserende kondisies te bepaal. Analise van T-RFLP en BIOLOGTM Ecoplate het aangedui dat In paar dominante organismes in die gemeenskap teenwoordig was, wat effens afgeneem het na verskuiwing na 'n ander koolstofbron. Fluoresserende hibridisasie het hoër getalle van SRB relatief tot die totale mikrobiese gemeenskap aangedui as konvensionele kultuur tegnieke. Mikroskopiese analises het verder getoon dat benewens SRB en ander bakterieë ook giste en filamentagtige swamme onder sulfaatreduserende kondisies in "n biofilm geïntegreer was. Hierdie mikroskopiese waarneminge is bevestig deur fluoresserende in situ hibridisasie (FISH) en gis Lewe / Dood lewensvatbaarheid peilers.

No description available.

The sulphate-reducing bacteria (SRB) are a diverse group of organisms which use sulphate as a terminal electron acceptor and produce the highly toxic gas, hydrogen sulphide. The deleterious effects of this include hydrocarbon reservoir souring, formation damage and microbial corrosion. The SRB are of major economic importance to the oil industry. However, knowledge of the microbial ecology of the deep subsurface remains limited. The aim of this project was to investigate whether organisms are indigenous to the hydrocarbon formation and/or are introduced during drilling operations. A range of molecular techniques such as 16S rDNA sequence analysis, probing with labelled oligonucleotides, and denaturing gradient gel electrophoresis (DGGE) were employed to investigate the microbial diversity in oil field samples. A wide range of bacterial 16S rDNA sequences were identified using these molecular methods. An analysis of drilling mud samples revealed a diverse range of bacterial 16S rDNA sequences confirming that bacteria, including SRB, can be introduced to the reservoir during drilling operations. A number of bacterial 16S rDNA sequences were recovered from a geological core sample taken from a depth of 9,770 feet. The microbial diversity was remarkable in such a high temperature, high pressure environment. This lends credence to the theory that certain bacteria may be indigenous to the subsurface environment. Scanning electron micrographs of core which had been incubated in growth medium indicated the presence of 'nannobacteria'. These tiny coccoids, with a diameter of only 0.1 μm are far smaller than the generally accepted minimum size for cellular life forms. The nannobacteria grew in regular colony shaped structures and were seen only in sections taken from inside the rock. This study indicates that hydrocarbon reservoirs provide an environment in which bacteria, if introduced during drill operations, may become established. However, the subsurface also contains complex indigenous microbial populations that demonstrate considerable species diversity and may include unrecognised life forms.

Dissimilatory sulphate reduction, brought about by the action of sulphate reducing bacteria (SRB) was used in the treatment of acid mine drainage (AMD) in a fluidised bed bioreactor. Biologically produced hydrogen sulphide and bicarbonate ions, by SRB, facilitated the precipitation of heavy metals and the generation of alkalinity in the synthetic acid mine water, respectively. The SRB that had been selected were able to utilize acetate as the sole carbon source and were capable of growing in the bioreactors at low pHs, facilitating an increase in the influent pH from 2.75-7.0 to 5.4-7.8, after a 24-hour hydraulic retention time (HRT). The precipitation efficiencies for Fe, Mn, Zn, Cu, Cr and Al after a HRT of 24 h as metal sulphides ranged between 84- 99% for influent pH values of between 4 and 7, and above 54% for influent pH values between 2.75 and 4. Microbial metabolic activity decreased with decreasing influent pH. This was inferred from the decreasing differences in chemical oxygen demand (COD) depletion rate over a 24 h HRT, as influent acidity levels approached pH 2.75. Molecular studies, using PCR-DGGE analysis on the microbial consortium in the bioreactor, revealed the presence of at least 8 different bacterial species in the consortium. Attempts at sequencing these bands yielded inconclusive results, with the bands showing sequence homology to a large number of previously uncultured and undescribed bacteria. Scanning electron microscopy confirmed the presence of bacteria of different morphology, as well as the presence of biofilms, which account for the heavy metal and low pH tolerances that the bacteria sustained.

This research was completed to assess passive treatment methods for mitigation of acid mine drainage (AMD) at a former mine site in British Columbia. The objectives were to determine if suitable passive treatment methods were available, and if concentrations of Cd, Zn, and other key contaminants in groundwater could be reduced to below regulatory standards during bench-scale testing. Biological treatment with sulphate reducing bacteria (SRB) was selected, and bench-scale treatment testing was conducted using columns amended with low cost organic sources. Removal of more than 99% Cd, 93% Co, 96 % Cu, 86% Ni and 98% Zn was observed, resulting in metals concentrations in treated effluent consistently lower than applicable groundwater standards. Sustainability attributes of treatment with SRB and the potential to recover valuable metals are discussed, and recommendations for further testing and implementation are provided.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Many industrial activities discharge sulphate- and metal-containing wastewaters, including the manufacture of pulp and paper, mining and mineral processing, and petrochemical industries. Acid mine drainage (AMD) is an example of such sulphate- and metal-containing waste streams. Formation of AMD is generally the result of uncontrolled oxidation of the sulphide minerals present in the terrain in which the drainage flows with concomitant leaching of the metals. Acid mine drainage (AMD) and other sulphate- and metal-containing waste streams are amenable to active biological treatment. Anaerobic reduction of sulphate, reaction of produced sulphide with metal ions present in the waste stream, and biooxidation of excess sulphide are three main sub-processes involved in the active biotreatment of AMD. Anaerobic reduction of sulphate can be achieved in continuous stirred tank bioreactors with freely suspended cells or in immobilized cell bioreactors. The application of freely suspended cells in a continuous system dictates a high residence time to prevent cell wash-out, unless a biomass recycle stream is used. In an immobilized cell system biomass residence time becomes uncoupled from the hydraulic residence time, thus operation of bioreactor at shorter residence times becomes possible. In the present work, kinetics of anaerobic sulphate reduction was studied in continuous immobilized cell packed-bed bioreactors. Effects of carrier matrix, concentration of sulphate in the feed and sulphate volumetric loading rate on the performance of the bioreactor were investigated. The bioreactor performance, in terms of sulphate reduction rate, was dependent on the nature of the carrier matrix, specifically the total surface area which was provided by the matrix for the establishment of biofilm. Among the three tested carrier matrices, sand displayed the superior performance and the maximum volumetric reduction rate of 1.7 g/L-h was achieved at the shortest residence time of 0.5 h. This volumetric reduction rate was 40 and 8 folds faster than the volumetric reduction rates obtained with glass beads (0.04 g/L-h; residence time: 28.6 h) and foam BSP (0.2 g/L-h; residence time: 5.3 h), respectively. Further kinetic studies with sand as a carrier matrix indicated that the extent of volumetric reduction rate was dependent on the feed sulphate concentration and volumetric loading rate. At a constant feed sulphate concentration, increases in volumetric loading rate caused the volumetric reduction rate to pass through a maximum, while increases in feed sulphate concentrations from 1.0 g/L to 5.0 g/L led to lower volumetric reduction rates. The maximum volumetric reduction rates achieved in the bioreactors fed with initial sulphate concentration of 1.0, 2.5 and 5.0 g/L were 1.71, 0.82 and 0.68 g/L-h, respectively. The coupling of lactate utilization to sulphate reduction was observed in all experimental runs and the rates calculated based on the experimental data were in close agreement with calculated theoretical rates, using the stoichiometry of the reactions involved. The maximum volumetric reduction rates achieved in the immobilized cell bioreactors were significantly faster than those reported for freely suspended cells employed in the stirred tank bioreactors.

Durante a exploração de petróleo offshore (fora da costa), a injeção de água do mar no processo de recuperação secundária de petróleo, ocasiona a produção de sulfeto de hidrogênio (H2S) pela presença das bactérias redutoras de sulfato (BRS), que reduzem o sulfato presente na água em sulfeto. A produção intensiva de H2S tem sido um dos maiores problemas das indústrias petrolíferas, pois constitui-se uma das principais causas de corrosão em linhas de produção (tubulações), equipamentos e tanques metálicos. Os principais micro-organismos presentes em amostras salinas provenientes de tanques de armazenamento de água e óleo da indústria do petróleo são as bactérias anaeróbias heterotróficas totais (BANHT) e as bactérias redutoras de sulfato (BRS). Atualmente, a quantificação desses grupos microbianos é realizada através da técnica do Número Mais Provável (NMP) que estima o resultado em aproximadamente 28 dias. Neste trabalho foi utilizada a metodologia de produção semi-contínua de sulfetos biogênicos por 15 dias, numa tentativa de correlacionar com os resultados de quantificação de BANHT e BRS através da técnica convencional do NMP. Nesse caso, avaliou-se as condições mais adequadas para a produção biogênica de sulfetos em tanques, alterando-se parâmetros tais como salinidade, temperatura e composição do meio de cultura. Verificou-se que os aumentos da salinidade e da temperatura do meio implicaram na diminuição da atividade biogênica semi-contínua de geração de sulfetos. E conforme dilui-se o meio de cultura, o crescimento de bactérias foi reduzido, assim como a geração de sulfetos. A quantificação de BRS e BANHT foi avaliada pela técnica do NMP de acordo com o método do FDA em 2011 e de Harrigan em 1998. Este último subestima a população microbiana, desconsiderando os limites e erros provenientes da técnica / During off-shore oil recovery, seawater injection for secondary oil recovery, produces hydrogen sulphide (H2S), due to the presence of sulphate reducing bacteria (SRB), that reduces sulphate from seawater to sulphide. The massive production of H2S is presently one of the main problems in the petroleum industries, constituting one of the main causes of corrosion in production lines (dutes), equipments and tanks. The main microbial species found in this type of saline samples comes from water and oil storage tanks in the petroleum industry, being general anaerobic heterotrophic bacteria (GAHB) and sulphate reducing bacteria (SRB). Presently, the quantification of those microbial groups is made through a technique known as Most Probable Number (MPN). This quantification is obtained in around 28 days. In the present work a methodology based on the semi-continuous production of sulphide during 15 days was conducted, in an attempt to correlate these results with the quantification of GAHB and SRB cells through conventional MPN technique. In this case, the most suitable conditions for biogenic sulphide production in tanks, was studied through the evaluation of environmental parameters such as salinity, temperature and culture medium composition. It could be observed that an increase in salinity and temperature of the medium produced a marked decrease in the semi-continuous biogenic production of sulphide. In relation to the culture medium, its dilution promoted a decreased in cell growth, consequently in the production of sulphides. The quantification of SRB and GAHB was evaluated through the MPN technique, according to procedures suggested by FDA in 2011 and Harrigan in 1998. It was observed that this last procedure underestimated the microbial population, due to the lack of information about limits and standard deviations

Made available in DSpace on 2015-03-26T13:51:47Z (GMT). No. of bitstreams: 1 texto completo.pdf: 700292 bytes, checksum: f54866bb9d53611e8feef9c8d9a5b216 (MD5) Previous issue date: 2009-07-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The sulphate reducing bacteria (SRB) are considered the principal group of microorganisms involved in the formation of biofilms and biocorrosion of pipelines. The prevention and the control of the growth of that bacterial group have been accomplished mainly by the use of biocide or bacteriostatic agents, as well as for the use of metabolic inhibitors. In this work the effect of mixtures was evaluated containing the biosurfactant rhamnolipid, the inhibitor metabolic molybdate of sodium and the biocide tetrakis (hydroxymethyl) phosphonium sulfate (THPS) about the metabolic activity of Desulfotomaculum nigrificans as well as about your adhesion to coupons of stainless steel AISI 304. The effect of the mixtures about the metabolic activity was evaluated by the estimate of viable cells and for the determination of residual sulphate in the medium of culture. The evaluation of the adhesion to coupons of stainless steel was accomplished by the direct count to the epifluorescence microscope. The experimental planning was accomplished according to Central Design Compost Rotational (DCCR) and the Methodology of Surface of Answer (MSR) was used to analyze the experimental design. There was not significant difference among the effects caused by the three compounds in the cellular viability in 6 and 18 hours after the addition of the compounds to the culture of D. nigrificans. All the appraised compounds were capable to inhibit the growth of D. nigrificans in anaerobic medium Baars to 55 ºC. The data of concentration of residual sulphate confirmed the effect of inhibition of the compounds also tested about the metabolic activity of that bacterium. Among the appraised compounds, the biosurfactant rhamnolipid was what presented larger effect of inhibitation in the adhesion of D. nigrificans to coupons of stainless steel, when cultivated in medium Baars to 55 ºC. It was the only compounds with effect significant statistics appeared in the times of 24, 48 and 96 hours of treatment evaluated. The Methodology of Surface of Answer revealed that the largest inhibition of the adhesion happens when the biosurfactant is added in concentrations from 1,0 to 1,6 times the value of your concentration critical micelar. The results suggest the potential of application of the biosurfactant rhamnolipid in the control of the adhesion and, eventually, of the biocorrosion caused by D. nigrificans. / As bactérias redutoras de sulfato (BRS) são consideradas o principal grupo de microrganismos envolvidos na formação de biofilmes e biocorrosão de oleodutos. A prevenção e o controle do crescimento desse grupo bacteriano têm sido realizados principalmente pela utilização de agentes biocidas ou bacteriostáticos, bem como pelo uso de inibidores metabólicos. Neste trabalho foi avaliado o efeito de misturas contendo o biossurfactante ramnolipídeo, o inibidor metabólico molibdato de sódio e o biocida sulfato de tetrakis (hidroximetil)fosfônio (THPS) sobre a atividade metabólica de Desulfotomaculum nigrificans, bem como sobre a sua adesão a cupons de aço inoxidável AISI 304. O efeito das misturas sobre a atividade metabólica foi avaliado pela estimativa de células viáveis e pela determinação de sulfato residual no meio de cultura. A avaliação da adesão a cupons de aço inoxidável foi realizada pela contagem direta em microscópio de epifluorescência. O planejamento experimental foi realizado segundo o Delineamento Composto Central Rotacional (DCCR) e a Metodologia de Superfície de Resposta (MSR) foi utilizada para se analisar o delineamento experimental. Não houve diferença significativa entre os efeitos causados pelos três compostos na viabilidade celular em 6 e 18 horas após a adição dos compostos à cultura de D. nigrificans. Todos os compostos avaliados foram capazes de inibir o crescimento de D. nigrificans em meio Baars anaeróbio a 55 ºC. Os dados de concentração de sulfato residual confirmaram o efeito inibitório dos compostos testados também sobre a atividade metabólica dessa bactéria. Dentre os compostos avaliados, o biossurfactante ramnolipídeo foi o que apresentou maior efeito inibitório na adesão de D. nigrificans a cupons de aço inoxidável, quando cultivada em meio Baars a 55 ºC. Ele foi o único composto com efeito estatisticamente significativo nos tempos de 24, 48 e 96 horas de tratamento avaliados. A Metodologia de Superfície de Resposta revelou que a maior inibição da adesão ocorre quando o biossurfactante é adicionado em concentrações de 1,0 a 1,6 vezes o valor da sua concentração micelar crítica. Os resultados sugerem o potencial de aplicação do biossurfactante ramnolipídeo no controle da adesão e, eventualmente, da biocorrosão causada por D. nigrificans.