Submitted by Isabela Amendola (isabelaamendola@hotmail.com) on 2019-02-01T11:22:14Z No. of bitstreams: 1 Tese-Isabela-Amêndola.pdf: 2013510 bytes, checksum: 69df8d92f153155f71d6e8a4b37d5eb6 (MD5) / Approved for entry into archive by Silvana Alvarez null (silvana@ict.unesp.br) on 2019-02-01T17:50:14Z (GMT) No. of bitstreams: 1 amendola_i_dr_sjc.pdf: 2013510 bytes, checksum: 69df8d92f153155f71d6e8a4b37d5eb6 (MD5) / Made available in DSpace on 2019-02-01T17:50:14Z (GMT). No. of bitstreams: 1 amendola_i_dr_sjc.pdf: 2013510 bytes, checksum: 69df8d92f153155f71d6e8a4b37d5eb6 (MD5) Previous issue date: 2018-12-03 / A resistência microbiana aos antibióticos disponíveis é preocupação constante, devido à dificuldade no tratamento de infecções causadas por cepas resistentes, em decorrência do uso indiscriminado de antimicrobianos. Assim, a busca por terapias antimicrobianas alternativas tem sido crescente e necessária, sendo a fitoterapia umas das opções de escolha. O objetivo do presente estudo foi analisar a atividade antibacteriana de extratos glicólicos de Achyrocline satureioides (macela), Cynara scolymus (alcachofra), Hamamelis virginiana (hamamelis) e Persea americana (abacateiro), pelos períodos de 5 min e 24 h de exposição sobre bactérias anaeróbias Fusobacterium nucleatum subsp. nucleatum, Parvimonas micra, Porphyromonas endodontalis e Porphyromonas gingivalis, em culturas planctônica e biofilmes. As bactérias armazenadas a -80ºC foram ativadas em caldo Brucella enriquecido (hemina 1%, menadiona 1% e sangue de carneiro desfibrinado 5%) e incubadas em câmara de anaerobiose por 48 h a 37ºC por sete dias. A partir de culturas puras, o teste de microdiluição em caldo foi conduzido em microplacas por meio de suspensões bacterianas padronizadas em solução fisiológica estéril (NaCl 0,9%) e diluições dos extratos em caldo, sendo as placas incubadas por 48 h a 37ºC em anaerobiose. Alíquotas de cada poço foram semeadas em ágar Brucella enriquecido. Após incubação, a Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM) foram determinadas. As concentrações efetivas de cada extrato foram aplicadas sobre os biofilmes de cada espécie, formados em microplacas a partir de suspensões bacterianas puras padronizadas na escala 0,5 de McFarland. As microplacas foram incubadas por sete dias a 37ºC para formação dos biofilmes, sendo o meio trocado a cada 48 h. Os biofilmes foram tratados por 5 min e 24 h. Em seguida, foram lavados e desprendidos por homogeneizador ultrassônico. As suspensões diluídas foram adicionadas em ágar Brucella enriquecido. Após 48 h, as Unidades Formadoras de Colônia por mililitro (UFC/mL) foram determinadas. Os resultados foram analisados por ANOVA e teste de Tukey ou por Kruskal-Wallis e teste Dunns, ambos com nível de significância de 5% (p≤0,05). Sobre as culturas planctônicas, a CIM e CBM dos extratos foi determinada apenas para F. nucleatum. A CBM dos extratos de A. satureioides, C. scolymus e P. americana foi obtida sobre P. micra. Não foi obtida atividade bactericida para P. endodontalis e P. gingivalis. Sobre biofilmes, todas as espécies apresentaram reduções significativas quando expostas aos extratos em ambos os tempos. Pode-se concluir que os extratos testados apresentaram efeito bacteriostático sobre F. nucleatum. Atividade bactericida dos extratos foi observada sobre F. nucleatum, bem como sobre P. micra, exceto para H. virginiana. Os extratos avaliados também apresentaram efeito antibiofilme sobre F. nucleatum, P. micra, P. endodontalis e P. gingivalis por 5 min e 24 h de exposição. / Microbial resistance to antibiotics available is constant concern, due to the difficulty in treating infections caused by resistant strains as a result of the indiscriminate use of antimicrobials. Thus, the search for antimicrobial alternative therapies has been growing and necessary, being one option the herbal medicine. The objective of the present study was to analyze the antibacterial activity to Achyrocline satureioides glycolic extracts (macela), Cynara scolymus (artichoke), Hamamelis virginiana (Witch-Hazel) and Persea americana (avocado), for periods of 5 min and 24 h from exhibition on anaerobic bacteria Fusobacterium nucleatum subsp. nucleatum, Parvimonas micra, Porphyromonas gingivalis and Porphyromonas endodontalis in planktonic communities and biofilms. Bacteria stored at -80°C have been activated in Brucella broth enriched (hemin 1%, menadione 1% and defibrinated sheep blood 5%) and incubated in anaerobiose chamber for 48 h at 37° C for seven days. From pure cultures, the microdiluição test in broth was conducted in microplates through standardized bacterial suspensions in sterile saline solution (NaCl 0.9%) and dilution of the extracts in broth, being incubated plates for 48 h at 37° C in anaerobiosis. Aliquots of each well were sown in Brucella agar enriched. After incubation, the Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) were determined. Effective concentrations of each extract were applied on the biofilms of each species, formed in microplates from pure bacterial suspensions in 0.5 McFarland scale standard. The microplates were incubated for 7 days at 37°C for the formation of biofilms, being the culture medium replaced every 48 h. Biofilms were treated for 5 min and 24 h have been washed and given off by ultrasonic homogenizer. Dilute suspensions were added in Brucella agar enriched. After 48 h, the Colony Forming Units per milliliter (CFU/ml) were determined. The results were analyzed by ANOVA and Tukey test, or Kruskal-Wallis test and Dunns, both with a significance level of 5% (p ≤ 0.05). On the planktonic cultures, CIM and CBM of extracts was determined only to F. nucleatum. The CBM of the extracts of A. satureioides, C. scolymus and P. americana was obtained on P. micra. Bactericidal activity was not obtained for P. endodontalis and P. gingivalis. About biofilms, all species exhibited significant reductions when exposed to the extracts in both times. It can be concluded that the extracts tested showed bacteriostatic effect on F. nucleatum. Bactericidal activity of extracts was observed on F. nucleatum and P. micra, except for H. virginiana. The extracts evaluated also presented antibiofilme effect on F. nucleatum, P. micra, P. endodontalis and P. gingivalis for 5 min and 24 h.

The biogenic reduction of Mn(IV) oxides is one of the most favorable anaerobic electron transfer processes in aquatic systems and likely plays an important role in the redox cycle of both carbon and nitrogen in anaerobic environments; yet, the different pathways involved in the microbial transformation of Mn(IV) oxides remain unclear. The coupling between the reduction of Mn(IV) to Mn(II) and the oxidation of organic carbon to CO₂ is largely catalyzed by microorganisms in various environments such as redox stratified water columns and sediments. The recent discovery that soluble Mn(III) exists in natural systems and is formed during biological oxidation of Mn(II) implies the possibility that Mn(III) is formed as an intermediate during the microbial reduction of Mn(IV). In this dissertation, mutagenesis studies and kinetic analysis were combined to study the mechanism of microbial reduction of Mn(IV) by Shewanella oneidensis MR-1, one of the most studied metal-respiring prokaryotes. We show for the first time that the microbial reduction of Mn(IV) proceeds step-wise via two successive one-electron transfer reactions with soluble Mn(III) as intermediate produced in solution. The point mutant strain Mn3, generated via random chemical mutagenesis, presents a unique phenotype that reduces solid Mn(IV) to Mn(III) but not to Mn(II), suggesting that these two reduction steps proceed via different electron transport pathways. Mutagenesis studies on various in-frame deletion mutant strains demonstrate that the reduction of both solid Mn(IV) and soluble Mn(III) occurs at the outer membrane of the cell and Mn(IV) respiration involves only one of the two potential terminal reductases (c-type cytochrome MtrC and OmcA) involved in Fe(III) respiration. Interestingly, only the second electron transfer step is coupled to the respiration of organic carbon, which opposes the long-standing paradigm that microbial reduction of Mn(IV) proceeds via the single transfer of two electrons coupled to the mineralization of carbon substrates. The coupling between anaerobic nitrification and Mn reduction has been demonstrated to be thermodynamically favorable. However, the existence of this process in natural system is still in debate. In this dissertation, characterization of coastal marine sediments was combined with laboratory incubations of the same sediments to investigate the effect of Mn oxides on the redox cycle of nitrogen. Our slurry incubations demonstrate that anaerobic nitrification is catalyzed by Mn oxides. In addition, mass balance calculations on NH₄⁺ link the consumption of NH₄⁺ to anaerobic ammonium oxidation in the presence of Mn oxides and confirm the occurrence of Mn(IV)-catalyzed anaerobic nitrification. The activity of anaerobic nitrification is greatly affected by the initial ratio of Mn(IV) to NH₄⁺, the reactivity of Mn oxides, and the reducing potential of the system. Overall, Mn(IV)-catalyzed anaerobic nitrification may be an important source of nitrite/nitrate in anaerobic marine sediments and provide an alternative pathway for subsequent nitrogen losses in the marine nitrogen cycle.

Pentaerythritol tetranitrate (PETN), a nitrate ester, is widely used as a powerful explosive and is classified as a munitions constituent of great concern by DoD in U.S.A. It is an environmental concern and poses a threat to ecosystem and human health. Our objective was to examine potential remediation strategies for both PETN-contaminated water and soil. Flow-through iron columns were used to determine the potential for using granular iron to degrade PETN in aqueous phase. PETN transformation in both a 100% iron column and a 30% iron and 70% silica sand column followed pseudo-first-order kinetics, with average half-lives of 0.26 and 1.58 minutes, respectively. Based on the identified intermediates and products, the reaction pathway was proposed to be a sequential denitration process, in which PETN was stepwisely reduced to pentaerythritol with the formation of pentaerythritol trinitrate (PETriN) and pentaerythritol dinitrate (PEDN). Although pentaerythrito mononitrate was not detected, an approximately 100% nitrogen mass recovery indicated that all nitro groups were removed from PETN. Nitrite was released in each denitration step and subsequently reduced to NH4+ by iron. Nitrate was not detected during the experiment, suggesting that hydrolysis was not involved in PETN degradation. Furthermore, batch experiments showed that PETN dissolution was likely a rate-limiting factor for PETN degradation, especially in the case with high amount of iron. Using 50% methanol as a representative co-solvent, PETN solubility was greatly enhanced and thus the removal efficiency was improved. The results demonstrate the use of granular iron to remediate PETN-contaminated water. The biodegradability of aqueous PETN was examined with a mixed microbial culture from a site contaminated with PETN. The mixed culture was enriched and selected using a mineral medium containing acetate and yeast extract as carbon and nutrient sources in the presence of nitrate or sulfate. The final enrichment cultures were used as inocula for studying PETN biodegradation under nitrate-reducing and sulfate-reducing conditions. In addition, PETN degradation was tested using the original microbial culture under the mixed electron acceptor conditions of nitrate and sulfate. The results showed that under all conditions tested, PETN was sequentially reduced, apparently following the same pathway as the abiotic reduction by granular iron. Pentaerythritol mononitrate, a suspected intermediate in the abiotic degradation by iron, was identified in this experiment. The presence of nitrate seemed not to affect the kinetics of PETN degradation, with both PETN and nitrate degrading simultaneously. However, the rate of nitrate reduction was much faster than PETN degradation. With respect to sulfate, its presence did not have an adverse effect on PETN degradation, indicated by the very similar degradation rates of PETN in the presence and absence of sulfate. Under all conditions, PETN appeared to act as a terminal electron acceptor for energy generation during biodegradation. A utilization sequence by bacteria in the order of nitrate, PETN, PETriN, PEDN and sulfate was clearly observed. The study in this phase demonstrated that under anaerobic conditions, with carbon sources provided, PETN can be effectively biodegraded by indigenous bacteria in contaminated soil, most likely by denitrifying bacteria. Based on the successful demonstration of abiotic and biotic degradation of PETN in the aqueous phase, both methods were further tested for remediating PETN-contaminated soil in both laboratory and pilot scale. In the laboratory, a systematic soil microcosm experiment was conducted using soil from a contaminated site and additions of either granular iron or organic materials, with deoxygenated Millipore water. Because of the high concentration in the contaminated soil, solid-phase of PETN was initially present in the microcosms. Two types of DARAMEND products, D6390Fe20 (containing 20% iron + 80% botanical materials) and ADM-298500 (100% botanical materials), were used as sources of carbon and other nutrients. During the 84-day incubation period, more than 98% was removed in all DARAMEND treatments, following pseudo-first-order kinetics with half-lives ranging between 8 and 18 days. The results clearly demonstrated that PETN can be effectively degraded under anaerobic conditions with the addition of carbon and possibly nutrients. As in the aqueous tests, the sequence of microbial utilization was nitrate followed by PETN and sulfate. In contrast to the tests with aqueous PETN, iron was not effective in removing PETN in the contaminated soil, due to iron passiviation caused by the presence of high levels of nitrate in the soil. In addition, a slight enhancement was observed in a combined system of iron and biodegradation over biodegradation only. However, the extent of enhancement is not believed to be significant relative to the extra cost for iron addition. A pilot scale test was conducted at a PETN-contaminated site at Louviers, CO, a waste pond which had received waste water from PETN manufacture for over 20 years. The test involved 10 treatments, one control without amendment, one amended with iron (10%), eight with different types and amounts of organic carbon (1%, 2% and 4% of D6390Fe20; 2% and 4% of ADM-298500 and 1%, 2% and 4% of brewers grain). Each treatment was performed in a plastic tub (45 cm wide × 90 cm long × 25 cm deep), containing approximately 18 cm thick layer of soil and 6-8 cm of standing water. Over 74 days, very little consistent reduction of PETN was found in the iron treatment, which was also due to iron passivation in the presence of nitrate in the soil. In contrast, significant removal of PETN (11,200 to 33,400 mg/kg) was observed in the treatments amended with organic materials, and the extent of removal increased with increasing amounts of organic materials. The pilot test was consistent with the results of the laboratory experiments for iron and biodegradation with carbon addition. For biological treatment, the stoichiometric estimation suggests that the complete remediation in many of the treatments will be ultimately limited by carbon sources. Results of this study showed the great potentials of using granular iron to degrade PETN in solution and using indigenous bacteria present in contaminated soils to biodegrade PETN in both the solution and soil phase. Both iron and biodegradation with carbon addition represent viable approaches for remediation of PETN-contaminated water and soil, though iron may not be appropriate in the presence of high concentration of nitrate.

Pentaerythritol tetranitrate (PETN), a nitrate ester, is widely used as a powerful explosive and is classified as a munitions constituent of great concern by DoD in U.S.A. It is an environmental concern and poses a threat to ecosystem and human health. Our objective was to examine potential remediation strategies for both PETN-contaminated water and soil. Flow-through iron columns were used to determine the potential for using granular iron to degrade PETN in aqueous phase. PETN transformation in both a 100% iron column and a 30% iron and 70% silica sand column followed pseudo-first-order kinetics, with average half-lives of 0.26 and 1.58 minutes, respectively. Based on the identified intermediates and products, the reaction pathway was proposed to be a sequential denitration process, in which PETN was stepwisely reduced to pentaerythritol with the formation of pentaerythritol trinitrate (PETriN) and pentaerythritol dinitrate (PEDN). Although pentaerythrito mononitrate was not detected, an approximately 100% nitrogen mass recovery indicated that all nitro groups were removed from PETN. Nitrite was released in each denitration step and subsequently reduced to NH4+ by iron. Nitrate was not detected during the experiment, suggesting that hydrolysis was not involved in PETN degradation. Furthermore, batch experiments showed that PETN dissolution was likely a rate-limiting factor for PETN degradation, especially in the case with high amount of iron. Using 50% methanol as a representative co-solvent, PETN solubility was greatly enhanced and thus the removal efficiency was improved. The results demonstrate the use of granular iron to remediate PETN-contaminated water. The biodegradability of aqueous PETN was examined with a mixed microbial culture from a site contaminated with PETN. The mixed culture was enriched and selected using a mineral medium containing acetate and yeast extract as carbon and nutrient sources in the presence of nitrate or sulfate. The final enrichment cultures were used as inocula for studying PETN biodegradation under nitrate-reducing and sulfate-reducing conditions. In addition, PETN degradation was tested using the original microbial culture under the mixed electron acceptor conditions of nitrate and sulfate. The results showed that under all conditions tested, PETN was sequentially reduced, apparently following the same pathway as the abiotic reduction by granular iron. Pentaerythritol mononitrate, a suspected intermediate in the abiotic degradation by iron, was identified in this experiment. The presence of nitrate seemed not to affect the kinetics of PETN degradation, with both PETN and nitrate degrading simultaneously. However, the rate of nitrate reduction was much faster than PETN degradation. With respect to sulfate, its presence did not have an adverse effect on PETN degradation, indicated by the very similar degradation rates of PETN in the presence and absence of sulfate. Under all conditions, PETN appeared to act as a terminal electron acceptor for energy generation during biodegradation. A utilization sequence by bacteria in the order of nitrate, PETN, PETriN, PEDN and sulfate was clearly observed. The study in this phase demonstrated that under anaerobic conditions, with carbon sources provided, PETN can be effectively biodegraded by indigenous bacteria in contaminated soil, most likely by denitrifying bacteria. Based on the successful demonstration of abiotic and biotic degradation of PETN in the aqueous phase, both methods were further tested for remediating PETN-contaminated soil in both laboratory and pilot scale. In the laboratory, a systematic soil microcosm experiment was conducted using soil from a contaminated site and additions of either granular iron or organic materials, with deoxygenated Millipore water. Because of the high concentration in the contaminated soil, solid-phase of PETN was initially present in the microcosms. Two types of DARAMEND products, D6390Fe20 (containing 20% iron + 80% botanical materials) and ADM-298500 (100% botanical materials), were used as sources of carbon and other nutrients. During the 84-day incubation period, more than 98% was removed in all DARAMEND treatments, following pseudo-first-order kinetics with half-lives ranging between 8 and 18 days. The results clearly demonstrated that PETN can be effectively degraded under anaerobic conditions with the addition of carbon and possibly nutrients. As in the aqueous tests, the sequence of microbial utilization was nitrate followed by PETN and sulfate. In contrast to the tests with aqueous PETN, iron was not effective in removing PETN in the contaminated soil, due to iron passiviation caused by the presence of high levels of nitrate in the soil. In addition, a slight enhancement was observed in a combined system of iron and biodegradation over biodegradation only. However, the extent of enhancement is not believed to be significant relative to the extra cost for iron addition. A pilot scale test was conducted at a PETN-contaminated site at Louviers, CO, a waste pond which had received waste water from PETN manufacture for over 20 years. The test involved 10 treatments, one control without amendment, one amended with iron (10%), eight with different types and amounts of organic carbon (1%, 2% and 4% of D6390Fe20; 2% and 4% of ADM-298500 and 1%, 2% and 4% of brewers grain). Each treatment was performed in a plastic tub (45 cm wide × 90 cm long × 25 cm deep), containing approximately 18 cm thick layer of soil and 6-8 cm of standing water. Over 74 days, very little consistent reduction of PETN was found in the iron treatment, which was also due to iron passivation in the presence of nitrate in the soil. In contrast, significant removal of PETN (11,200 to 33,400 mg/kg) was observed in the treatments amended with organic materials, and the extent of removal increased with increasing amounts of organic materials. The pilot test was consistent with the results of the laboratory experiments for iron and biodegradation with carbon addition. For biological treatment, the stoichiometric estimation suggests that the complete remediation in many of the treatments will be ultimately limited by carbon sources. Results of this study showed the great potentials of using granular iron to degrade PETN in solution and using indigenous bacteria present in contaminated soils to biodegrade PETN in both the solution and soil phase. Both iron and biodegradation with carbon addition represent viable approaches for remediation of PETN-contaminated water and soil, though iron may not be appropriate in the presence of high concentration of nitrate.

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

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

No description available.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban Institute Of Technology, 2005 xxiii, 172 p. : ill. ; 30 cm / The provision of safe and sanitary water is a constitutional right and above all, a necessity of life. As a result of the rapid urbanisation and the past policies of apartheid, a large population of South Africa dwell in informal settlements, where there is very little hope of development, as the government does not possess the resources that are necessary for a full-scale sanitation programme. Therefore, on-site treatments have been considered to provide sanitation in these dense peri-urban areas. The anaerobic baffled reactor (ABR) is one such sanitation system. This reactor utilises the phenomenon of anaerobic digestion to degrade substrates. One of the major disadvantages of any anaerobic treatment processes is the extreme sensitivity of the bacterial communities, thus inducing slow recovery rates following toxic shocks. Therefore, an understanding of these microbial consortia is essential to effectively control, operate and optimise the anaerobic reactor. Fluorescence in situ hybridization, 4’,6-diamidino-2-phenylindole (DAPI) staining and DNA sequencing techniques were applied to determine the microbial consortium, as well as their reactions to daily operating conditions. With an understanding of these populations and their responses to perturbations within the system, it is possible to construct an anaerobic system that is successful in its treatment of domestic wastewater. In situ hybridizations were conducted for three operating periods, each characterised by specific flow rates. Results showed Eubacterial population dominance over the Archaeal population throughout both of the operating periods investigated. However, these cells cumulatively consisted of 50% of the total biomass fraction, as determined by DAPI staining. Group-probes utilised revealed a high concentration of fermentative acidogenic bacteria, which lead to a decrease in the pH values. It was noted that the ABR did not separate the acidogenic and methanogenic phases, as expected. Therefore, the decrease in pH further inhibited the proliferation of Archaeal acetoclastic methanogens, which were not present in the second operating period. DNA sequencing results revealed the occurrence of the hydrogenotrophic Methanobacterium and Methanococcus genera and confirmed the presence of Methanosarcina. Sequencing of the bacterial DNA confirmed the presence of the low G+ C Gram Positives (Streptococcus), the high G+C Gram Positives (Propionibacterium) and the sulfate reducing bacteria (Desulfovibrio vulgaris). However, justifications were highly subjective due to a lack of supportive analytical data, such as acetate, volatile fatty acids and methane concentrations. Despite this, findings served to add valuable information, providing details on the specific microbial groups associated with ABR treatment processes.

Due to a combination of selection criteria, sludges from upflow anaerobic digesters treating industrial waste waters consist primarily of well-settling, dense agglomerates called granules. Quantification of the component mixed microbial populations of these granules has been severely restricted by the inability of researchers to disrupt them without concomitantly destroying numerous cells. In situ quantification using light and electron microscopy is complicated by the high cell numbers and bacterial diversity; the small cell size; and the destructive nature of electron microscopy preparative techniques preventing the viewing of more than a small percentage of the population at a time. For these reasons, in this investigation, standardization of qualitative electron microscopic techniques was performed prior to their application to granules. Isolation and electron and light microscopic techniques were applied to granules from a fullscale clarigester treating effluent from a maize-processing factory. In addition, a method using montaged transmission electron micrographs (TEMs) taken along a granule radius, and image analysis, was developed for bacterial quantification within granules. This method, together with antibody probe quantification, was applied to granules from an upflow anaerobic sludge blanket (UASB) digester treating a brewery effluent. The clarigester granules contained a metabolically and morphologically diverse population of which many members were not isolated or identified. By contrast, the UASB digester granules consisted primarily of morphotypes resembling Methanothrix, Methanobacterium and Desulfobulbus, in order of predominance. However, only about one-third of the population reacted with antibody probes specific to strains of bacterial species expected to occur within these granules. According to the antibody probe library used, the Methanobacterium-like cells observed in TEMs were probably Methanobrevibacter arboriphilus. From this study it is apparent that different anaerobic digester designs, operational parameters, and the chemical composition of the waste water purified, are factors which influence the formation and maintenance of granules differing with respect to their microbial populations. Until the difficulties associated with quantification are overcome, the processes governing granule formation and/or population selection will remain obscure. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1995.

Interacting microbial associations capable of utilizing thiosulphate as an energy source were enriched/isolated from activated sludge, landfill site [mal covering soil and soil from an acid mine water drainage site. The isolates were designated Lf-I, Ws-2 and Am-3, respectively. Although hydrogen sulphide was the target molecule for gas biofiltration, thiosulphate, which is a key oxidized intermediate, was used in this study due to the difficulty of working with a toxic gas. Together with thiosulphate oxidation, the microbial associations were assessed for their abilities to oxidize dissolved sulphide to elemental sulphur. Physiological analyses (temperature, pH and substrate concentration optimization) were made with closed and open cultures while morphological characterization and species compositional changes were monitored by light and scanning electron microscopy (SEM). To investigate further functional and structural responses to physiological changes, denaturing-gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S DNA gene fragments and Biolog GN microtitre plates were used. The associations were found to be active metabolically between 0 and 35°C, 15 and 50°C, and 15 and 45°C, with optimum temperatures of 25, 40 and 35°C for Lf-l, Ws-2 and Am-3, respectively. The optimum pH range for microbial association Lf-l was between 3 and 4. The maximum specific growth rates of associations Lf-l , Ws-2 and Am-3 were 0.08, 0.06 and 0.03 h~l , respectively. Components of all three Gram negative rod-dominated associations were motile and displayed anaerobiosis. During open culture cultivation the species complement of Lf-l , as determined by morphological analysis, changed. The same association oxidized sulphide (40 ppm) to sulphur although Ws-2 and Am-3 did not have this capacity. Biolog GN plates detected pH-effected species compositional changes in Lf-l and these were confirmed by DGGE. The same technique showed that enrichment had occurred in the Biolog GN wells. Species composition changes also resulted in response to different pH values (2 to 9), temperatures (5 to 40°C) and dilution rates (0.003 to 0.09 h-1 ), but activity changes were not always accompanied by population profile changes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.