Thesis (M.Sc. (Biochemistry, Microbiology & Biotechnology)) - Rhodes University, 2007.

abstract: Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations. / Dissertation/Thesis / Ph.D. Microbiology 2013

In an attempt to overcome the high maintenance and costs associated with traditional physico-chemical methods, much work is being done on the application of enzymes for the recovery of valuable metals from solutions and industrial effluents. One of the most widely studied enzymatic metal recovery systems uses hydrogenase enzymes, particularly from sulphate reducing bacteria (SRB). While it is known that hydrogenases from SRB mediate the reductive precipitation of metals, the mechanism of enzymatic reduction, however, is not yet fully understood. The main aim of the present study was to investigate the role of a hydrogenase enzyme in the removal of rhodium from both aqueous solution and industrial effluent. A quantitative analysis of the rate of removal of rhodium(III) by a resting SRB consortium under different initial rhodium and biomass concentrations, pH, temperature, presence and absence of SRB cells and electron donor, was studied. Rhodium speciation was found to be the main factor controlling the rate of removal of rhodium from solution. SRB cells were found to have a higher affinity for anionic rhodium species, as compared to both cationic and neutral species, which become abundant when speciation equilibrium was reached. Consequently, a pH-dependant rate of rhodium removal from solution was observed. The maximum SRB uptake capacity for rhodium was found to be 66 mg rhodium per g of resting SRB biomass. Electron microscopy studies revealed a time-dependant localization and distribution of rhodium precipitates, initially intracellularly and then extracellularly, suggesting the involvement of an enzymatic reductive precipitation process. A hydrogenase enzyme capable of reducing rhodium(III) from solution was isolated and purified by PEG, DEAE-Sephacel anion exchanger and Sephadex G200 gel exclusion. A distinct protein band with a molecular weight of 62kDa was obtained when the hydrogenase containing fractions were subjected to a 10% SDS-PAGE. Characterization studies indicated that the purified hydrogenase had an optimum pH and temperature of 8 and 40°C, respectively. A maximum of 88% of the initial rhodium in solution was removed when the purified hydrogenase was incubated under hydrogen. Due to the low pH of the industrial effluent (1.31), the enzymatic reduction of rhodium by the purified hydrogenase was greatly retarded. It was apparent that industrial effluent pretreatment was necessary before the application an enzymatic treatment method. In the present study, however, it has been established that SRB are good candidates for the enzymatic recovery of rhodium from both solution and effluent.

The enzymatic reduction of a high-valence form of metals to a low-valence reduced form has been proposed as a strategy to treat water contaminated with a range of metals and radionuclides. Metal reduction by sulphate reducing bacteria (SRB) is carried out either chemically (involving reduction by hydrogen sulphide) or enzymatically (involving redox enzymes such as the hydrogenases). While reduction of metal ions by hydrogen sulphide is well known, the enzymatic mechanism for metal reduction is poorly understood. The aims of this study were to investigate the role of SRB in facilitating platinum removal, and to investigate the role of a hydrogenase in platinum reduction in vitro. In order to avoid precipitation of platinum as platinum sulphide, a resting (non-growing) mixed SRB culture was used. The maximum initial concentration of platinum (IV), which SRB can effectively remove from solution was shown to be 50 mg.l⁻¹. Electron donor studies showed high platinum (IV) uptake in the presence of hydrogen, suggesting that platinum (IV) uptake from solution by SRB requires careful optimization with respect to the correct electron donor. Transmission electron microscopy (TEM) and energy dispersive X-ray (EDX) analysis indicated that platinum was being precipitated in the periplasm, a major area of hydrogenase activity in SRB. Purification of the hydrogenase by ammonium sulphate precipitation (65%), Toyopearl-Super Q 650S ion exchange and Sephacry 1 S-100 size exclusion chromatography revealed that the hydrogenase was monomeric with a molecular weight of 58 KDa, when analyzed by 12% SDS-PAGE. The purified hydrogenase showed optimal temperature and pH at 35°C and 7.5 respectively, and a poor thermal stability. In vitro investigation of platinum reduction by purified hydrogenase from mixed SRB culture showed that hydrogenase reduces platinum only in the presence of hydrogen. Major platinum (IV) reduction was observed when hydrogenase was incubated with cytochrome C₃ (physiological electron carrier in vivo) under hydrogen. The same observations were also noted with industrial effluent. Collectively these findings suggest that in vitro platinum reduction is mediated by hydrogenase with a concerted action of cytochrome C₃ required to shuttle the electron from hydrogenase.

The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.

O objetivo deste trabalho foi enriquecer consórcio microbiano a partir de mistura de lodo granular de digestor anaeróbio de fluxo ascendente sob condições fototróficas anoxigênicas. Por meio de técnica de biologia molecular foi possível identificar 17 unidades taxonômicas operacionais (UTO) no consórcio microbiano, dentre as quais seqüências similares a Rhodobacter, gênero amplamente citado nos estudos de produção de gás hidrogênio por bactérias fototróficas. Exames microscópicos do consórcio fototrófico indicaram predomínio de bacilos Gram-negativos. Ensaios sob condições fototróficas foram realizados com dois meios de cultivo (RCVB e FANG) e os seguintes substratos orgânicos: ácido acético, butírico, cítrico, lático e málico, empregados como fonte de carbono, tanto para o crescimento celular, como para a produção do gás hidrogênio. A relação C/N inicial foi 30/4 e posteriormente 15/2, com o objetivo de favorecer o crescimento celular e a produção do \'H IND.2\'. A concentração dos substratos foi determinada de forma com que essa relação se mantivesse a mesma. O crescimento celular e consumo dos ácidos orgânicos foram similares para os dois meios de cultivo empregados. Entretanto, a produção do gás hidrogênio foi maior nos ensaios com o meio FANG. Dentre os substratos utilizados o consumo dos ácidos cítrico e málico foram os maiores (~100%), para concentrações iniciais de 3,3 g/L e 2,6 g/L, respectivamente. O menor consumo 25% foi observado em meio RCVB e ácido acético (2,5 g/L). O crescimento da biomassa variou de 0,06 g/L a 1,1 g/L, enquanto que a velocidade máxima específica de crescimento variou de 0,4 a 0,2 g SSV/L.d entre os substratos utilizados. A menor e maior concentração de hidrogênio foram de 8,5 e 22 mmol \'H IND.2\'/L, para os reatores alimentados com ácido lático e málico em meio FANG, respectivamente. Pôde-se concluir que o consórcio fototrófico enriquecido foi capaz de utilizar os ácidos orgânicos para produção do gás hidrogênio. / The aim of this work was enrich a mixture of granular sludge of an up flow anaerobic sludge blanket (UASB) under anoxygenic phototrophic conditions. The techniques of molecular biology identified 17 operational taxonomic units (UTO) in the microbial consortium among the sequences analised, which were similar to Rhodobacter, genus widely cited in studies of hydrogen gas production by phototrophic bacteria. Microscopic examinations of the phototrophic consortium showed predominance of Gram-negative bacilli. Tests were conducted under phototrophic conditions with two culture media (RCVB and FANG) and the following organic substrates: acetic, butyric, citric, lactic and malic acids that were used as carbon source for both cell growth and for the hydrogen gas production. The carbon nitrogen ratio (C/N) in the preliminaries tests was 30/4 and then it was changed to15/2 in order to improve the cell growth and hydrogen production. The concentration of substrates was determined for remain the same carbon/nitrogen ratio among the substrates. The cell growth and consumption of organic acids were similar for the two culture media used. However, the production of hydrogen gas was higher in trials with the medium FANG. Among the substrates used, the consumption of malic and citric acids were the highest (~100%) for initial concentrations of 3.3 g/L and 2.6 g/L, respectively. The shortest consumption (25%) was observed for the cells that grew on acetic acid, 2.5 g/L in RCVB culture medium. The growth of the biomass varied from 0.06 g/L to 1.1 g/L, whereas the maximum specific growth rate ranged from 0.4 to 0.2 g VSS/L.d between the substrates used. The lowest and highest concentrations of hydrogen were 8.5 and 22 mmol \'H IND.2\'/L for the reactor fed with lactic acid and malic acid in FANG\'s medium, respectively. It was concluded that the phototrophic consortium was able to use those organic acids for the production of hydrogen gas.

O objetivo deste trabalho foi enriquecer consórcio microbiano a partir de mistura de lodo granular de digestor anaeróbio de fluxo ascendente sob condições fototróficas anoxigênicas. Por meio de técnica de biologia molecular foi possível identificar 17 unidades taxonômicas operacionais (UTO) no consórcio microbiano, dentre as quais seqüências similares a Rhodobacter, gênero amplamente citado nos estudos de produção de gás hidrogênio por bactérias fototróficas. Exames microscópicos do consórcio fototrófico indicaram predomínio de bacilos Gram-negativos. Ensaios sob condições fototróficas foram realizados com dois meios de cultivo (RCVB e FANG) e os seguintes substratos orgânicos: ácido acético, butírico, cítrico, lático e málico, empregados como fonte de carbono, tanto para o crescimento celular, como para a produção do gás hidrogênio. A relação C/N inicial foi 30/4 e posteriormente 15/2, com o objetivo de favorecer o crescimento celular e a produção do \'H IND.2\'. A concentração dos substratos foi determinada de forma com que essa relação se mantivesse a mesma. O crescimento celular e consumo dos ácidos orgânicos foram similares para os dois meios de cultivo empregados. Entretanto, a produção do gás hidrogênio foi maior nos ensaios com o meio FANG. Dentre os substratos utilizados o consumo dos ácidos cítrico e málico foram os maiores (~100%), para concentrações iniciais de 3,3 g/L e 2,6 g/L, respectivamente. O menor consumo 25% foi observado em meio RCVB e ácido acético (2,5 g/L). O crescimento da biomassa variou de 0,06 g/L a 1,1 g/L, enquanto que a velocidade máxima específica de crescimento variou de 0,4 a 0,2 g SSV/L.d entre os substratos utilizados. A menor e maior concentração de hidrogênio foram de 8,5 e 22 mmol \'H IND.2\'/L, para os reatores alimentados com ácido lático e málico em meio FANG, respectivamente. Pôde-se concluir que o consórcio fototrófico enriquecido foi capaz de utilizar os ácidos orgânicos para produção do gás hidrogênio. / The aim of this work was enrich a mixture of granular sludge of an up flow anaerobic sludge blanket (UASB) under anoxygenic phototrophic conditions. The techniques of molecular biology identified 17 operational taxonomic units (UTO) in the microbial consortium among the sequences analised, which were similar to Rhodobacter, genus widely cited in studies of hydrogen gas production by phototrophic bacteria. Microscopic examinations of the phototrophic consortium showed predominance of Gram-negative bacilli. Tests were conducted under phototrophic conditions with two culture media (RCVB and FANG) and the following organic substrates: acetic, butyric, citric, lactic and malic acids that were used as carbon source for both cell growth and for the hydrogen gas production. The carbon nitrogen ratio (C/N) in the preliminaries tests was 30/4 and then it was changed to15/2 in order to improve the cell growth and hydrogen production. The concentration of substrates was determined for remain the same carbon/nitrogen ratio among the substrates. The cell growth and consumption of organic acids were similar for the two culture media used. However, the production of hydrogen gas was higher in trials with the medium FANG. Among the substrates used, the consumption of malic and citric acids were the highest (~100%) for initial concentrations of 3.3 g/L and 2.6 g/L, respectively. The shortest consumption (25%) was observed for the cells that grew on acetic acid, 2.5 g/L in RCVB culture medium. The growth of the biomass varied from 0.06 g/L to 1.1 g/L, whereas the maximum specific growth rate ranged from 0.4 to 0.2 g VSS/L.d between the substrates used. The lowest and highest concentrations of hydrogen were 8.5 and 22 mmol \'H IND.2\'/L for the reactor fed with lactic acid and malic acid in FANG\'s medium, respectively. It was concluded that the phototrophic consortium was able to use those organic acids for the production of hydrogen gas.

For the purpose of sulphide removal in aquaculture ponds, three strains (name: TH21, QN71, QN51) were isolated and selected with the highest sulphide removal activity from Thanh Hoa and Quang Ninh coastal zones. These strains have identified and tested in a number of aquaculture ponds in different areas with good water quality results. With the objective of purple non sulfur bacteria biomass production containing 3 selected strains for wide application and suitable price for farmers, in this study, we study on optimum conditions of mixed purple non sulfur bacteria biomass production at pilot scale. The results showed that the sources of substrates were soybean meal (1g/l) and acetate (0.5g/l). These substrates are low cost, easy to find, convenient in large culture. The mixture of photosynthetic bacteria can be cultured in glass tanks, under micro aerobic and natural lighting conditions that produce highly concentrated photosynthetic bacteria and lowest rest media. / Nhằm mục tiêu xử lý sulphide trong môi trường nuôi trồng thủy sản, chúng tôi đã phân lập và lựa chọn được ba chủng vi khuẩn tía quang hợp có khả năng loại bỏ sulphide cao nhất ký hiệu TH21, QN71, QN52 từ các vùng ven biển Thanh Hóa và Quảng Ninh. Các chủng này đã được định loại và thử nghiệm tại một số ao nuôi thủy sản ở các vùng khác nhau thu được kết quả tốt về chất lượng nước. Để tạo chế phẩm vi khuẩn tía quang hợp từ 3 chủng lựa chọn được ứng dụng rộng rãi và có giá thành phù hợp cho nông hộ, trong nghiên cứu này, chúng tôi nghiên cứu tối ưu hóa các điều kiện sản xuất sinh khối hỗn hợp 3 chủng vi khuẩn tía quang hợp ở quy mô pilot. Kết quả cho thấy đã tìm kiếm được nguồn cơ chất là bột đậu tương (1g/l) và acetate (0.5g/l) là những chất có giá thành thấp, dễ tìm kiếm, thuận tiện trong nhân nuôi ở quy mô lớn. Hỗn hợp vi khuẩn tía quang hợp có thể nuôi trong các bể kính, ở điều kiện vi hiếu khí, có ánh sáng chiếu tự nhiên có thể sản xuất được chế phẩm vi khuẩn tía quang hợp có mật độ cao, cơ chất còn lại sau sản xuất là ít nhất.

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The process of photosynthesis begins with a capture of sunlight and its quick conversion into the chemical energy. Both these primary processes take place in a specially designed photosynthetic apparatus that is present in cells of all photosynthetic organisms. In green sulfur bacteria the apparatus consists of a massive light-harvesting antenna (chlorosome), intermediate antennas (baseplate complex and FMO proteins) and of the reaction center, where the conversion into the chemical energy occurs. The energy flow through the apparatus can be monitored by time-resolved spectroscopy techniques. Coherent two-dimensional electronic spectroscopy (2DES) in one of such techniques, which combines high temporal and spectral resolution, and therefore it is especially well suited for studying multichromophoric systems such as photosynthetic apparatus. This thesis describes the principles of the 2DES technique and outlines the basic facts about the photosynthetic apparatus of green sulfur bacterium Chlorobaculum tepidum. Finally, it summarizes the investigation of the photosynthetic machinery using 2DES. Results presented in this thesis provide new insights into the exciton diffusion and vibrational coherences within chlorosomes, excitonic structure of the baseplate and the overall energy flow through the entire...