Dissertations / Theses on the topic 'Purple non-sulfur photosynthetic bacteria'

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.<br>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.

Mercury (Hg) is a global pollutant and potent neurotoxin that bioaccumulates in aquatic and terrestrial food webs as monomethylmercury (MeHg). Anaerobic microbes are largely responsible for MeHg production, which depends on the bioavailability of inorganic Hg substrates to methylators. Hg redox cycling pathways such as Hg reduction play a key role in determining Hg’s availability in the environment. Although abiotic photochemical Hg reduction typically dominates in oxic surface environments, Hg reduction pathways mediated by photosynthetic and anaerobic microbes are thought to play an important role in anoxic habitats where light is limited and MeHg production occurs. Currently, the physiological mechanisms driving phototrophic and anaerobic Hg reduction remain poorly understood. The main objective of my thesis is to provide mechanistic details on novel anaerobic and phototrophic Hg reduction pathways. I used a combination of physiological, biochemical and trace Hg analytical techniques to study Hg reduction pathways in a variety of anaerobic and photosynthetic bacteria. I demonstrated that Hg redox cycling was directly coupled to anoxygenic photosynthesis in aquatic purple non-sulphur bacteria that reduced HgII when cells incurred a redox imbalance. I discovered that terrestrial fermentative bacteria reduced Hg through pathways that relied on the generation of reduced redox cofactors. I also showed that sulphur assimilation controlled Hg reduction in an anoxygenic phototroph isolated from a rice paddy. In addition, I developed methods to explore cryptic anaerobic Hg redox cycling pathways using Hg stable isotope fractionation. At its core, my thesis underscores the intimate relationship between cell redox state and microbial Hg reduction and suggests a wide diversity of microbes can participate in anaerobic Hg redox cycling.

The movement towards a more sustainable energy economy may require not only the generation of cleaner fuel sources, but the conversion of waste streams into value-added products. Phototrophic purple non-sulfur bacteria are capable of metabolizing VFAs (volatile fatty acids)and generate hydrogen as a byproduct of nitrogen fixation using energy absorbed from light. VFAs are easily produced from dark anaerobic fermentation of food, agricultural, and municipal wastes, which could then be fed into photobioreactors of purple bacteria for hydrogen production. The process of photofermentation by purple bacteria for hydrogen production remains attractive due to the capability of reaching high substrate conversions under mild operating conditions, but increasing the efficiency of converting light energy into hydrogen remains challenging. Purple bacteria cannot utilize the entire solar spectrum, and the dominant region of absorption lies in the near-infrared region above 800 nm. In this work, the model purple non-sulfur bacteria Rhodopseudomonas palustris was used to study different strategies to increase light utilization and hydrogen production. Near-infrared LED arrays were selected to match the target bacteriochlorophyll absorption range, and were tested to be used as a sole illumination source for photofermentation. Additionally, plasmonic nanoparticles with resonant frequencies matching bacterial absorbance were added in solution to increase light utilization through scattering and near field electric enhancement effects at intensities around 100 W/m2 . Both of these approaches proved to increase cellular growth rate and hydrogen production, which opens the door to utilizing more advanced photonic structures for use in bacterial phototrophic processes.

Biological hydrogen production is a renewable, carbon-neutral and clean route for hydrogen production. Purple non-sulfur (PNS) bacteria have the ability to produce biohydrogen via photofermentation process. The type of the bacterial strain used in photofermentation is known to have an important effect on hydrogen yield. In this study, the effect of different co-cultures of PNS bacteria on photofermentation process was investigated in search of improving the hydrogen yield. For this purpose, growth, hydrogen production and substrate utilization of single and co-cultures of different PNS bacteria (R. capsulatus (DSM 1710), R. capsulatus hup- v (YO3), R. palustris (DSM 127) and R. sphaeroides O.U.001 (DSM 5864)) were compared on artificial H2 production medium in 150 mL photobioreactors under continuous illumination and anaerobic conditions. In general, higher hydrogen yields were obtained via co-cultivation of two different PNS bacteria when compared with single cultures. Further increase in hydrogen yield was observed with co-cultivation of three different PNS bacteria. Co-cultures of two different PNS bacteria have resulted in up to 1.4 and 2.1 fold increase in hydrogen yield and hydrogen productivity. Whereas co-cultures of three different PNS bacteria have resulted in up to 1.6 and 2.0 fold increase in hydrogen yield and hydrogen productivity compared to single cultures. These results indicate that, defined co-cultures of PNS bacteria produce hydrogen at a higher yield and productivity, due most probably to some synergistic relationship. Further studies regarding the physiological and molecular changes need to be carried out for deeper understanding of the mechanism of hydrogen production in co-cultures.

Some purple bacteria species, such as Rhodopseudomonas palustris 2.1.6, produce light harvesting antenna (LH2) with unusual absorption spectra when they are grown under low-light intensities. This ability is often related to the presence of multiple genes encoding the LH2 apoproteins. This thesis describes isolation of pure stable LH2s from Rhodopseudomonas palustris 2.1.6 grown at different light intensities, determination of the polypeptide composition of high- (HL) and low-light (LL) LH2 complexes and characterisation their spectroscopic properties using various optical spectroscopies. The question of whether rings with a heterogeneous apoprotein composition exist has been addressed by single-molecule spectroscopy. For the first time, direct evidence that individual LL LH2 complexes have a heterogeneous αβ-apoprotein composition has been found. Such mixed rings feature Bchl a molecules with both B820-like and B850-like site-energies. This finding was supported by a femtosecond study on the energy transfer reactions and exciton relaxations within both HL and LL LH2 complexes. This thesis also describes attempts to crystallise the HL and LL LH2s. Even though three-dimensional crystals of both HL and LL LH2 complexes only diffracted to low resolution, it was possible to use molecular replacement to obtain structures that suggest both these types of LH2s are nonamers.

As necessidades de energia global são, na sua maioria, dependentes de combustíveis fósseis. Hidrogênio é uma energia limpa alternativa a esses combustíveis fósseis. Bactérias fototróficas produzem hidrogênio a partir de compostos orgânicos por meio de processo anaeróbio dependente de luz. Assim, este trabalho visou avaliar o efeito das concentrações iniciais de ácido acético e biomassa, e a influência da intensidade luminosa, na produção de hidrogênio por bactéria fototrófica púrpura não-sulfurosa. Foram utilizados reatores em batelada de 2000 mL, com volume útil de 1000 mL e headspace de 1000 mL preenchido com hélio. Nos reatores foi adicionado ácido acético e glutamato de sódio (0,8 mmol/L) como fontes de carbono e nitrogênio, respectivamente, e cultura de bactéria fototrófica púrpura não-sulfurosa previamente purificada. O aumento da concentração inicial de ácido acético de 10 mmol/L para 17 mmol/L não promoveu mudanças significativas tanto no crescimento celular, quanto, na produção de hidrogênio (8,3 mL \'H IND.2\'/g massa seca.h e 8,8 mL \'H IND.2\'/g massa seca.h, respectivamente), para intensidade luminosa de 9000 - 10.000 lux. Nessa mesma intensidade luminosa, o aumento da concentração de biomassa inicial de 0,02 g/L para 0,04 g/L favoreceu o aumento da produção de hidrogênio de 8,8 mL \'H IND.2\'/g massa seca.h para 10,6 mL \'H IND.2\'/g massa seca.h, respectivamente. A produção de hidrogênio diminuiu acentuadamente de 10,6 mL \'H IND.2\'/g massa seca.h para 1,0 mL \'H IND.2\'/g massa seca.h com a diminuição da intensidade luminosa de 9000 - 10.000 lux para 4000 - 5000 lux. Na ausência de luz não ocorreu crescimento e produção de hidrogênio. A cultura manteve-se predominantemente avermelhada e as análise microscópicas mostraram a predominância de bacilos curvos, gram-negativos, aglomerados em formações de roseta; características típicas de alguns gêneros de bactérias fototróficas púrpuras não-sulfurosas. Todos os ensaios foram realizados à temperatura de 30 \'+ OU -\' 1 grau Celsius. A análise da estrutura da comunidade microbiana foi realizada por reação de polimerização em cadeia (PCR) de fragmentos de gene RNAr 16S, seguida de eletroforese em gel de gradiente desnaturante (DGGE), e revelou que não houve variações relevantes na estrutura das populações microbianas em função das diferentes condições de cultivo.<br>The global energy requirements are mostly dependent on fossil fuels. Hydrogen is a clean energy alternative to these fuels. Phototrophic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. Therefore, this study aimed at to evaluate the effect of the initial concentrations of acetic acid and biomass, and the influence of the light intensity on hydrogen production by purple non-sulfur phototrophic bacteria. The experiments were performed in batch operation, in reactors of 2000 mL, with culture volume of 1000 mL and headspace of 1000 mL, filled with helium. Acetic acid and sodium glutamate (0.8 mmol/L) were used as sources of carbon and nitrogen, respectively, and culture of purple non-sulfur phototrophic bacteria previously purifided. The increase of the initial acetic acid concentration from 10 mmol/L to 17 mmol/L did not promote significant changes in the cell growth and in the hydrogen production (8.3 mL \'H IND.2\'/g dry weight.h and 8.8 mL \'H IND.2\'/g dry weight.h, respectively), under a light intensity of 9000 - 10,000 lux. In this same light intensity, the increase of the initial biomass concentration from 0.02 g/L to 0.04 g/L resulted in an increase in the hydrogen production from 8.8 mL \'H IND.2\'/g dry weight.h to 10.6 mL \'H IND.2\'/g dry weight.h, respectively. The hydrogen production suddenly decreased from 10.6 mL \'H IND.2\'/g dry weigh.h to 1.0 mL \'H IND.2\'/g dry weight.h with the reduction of the light intensity from 9000 - 10,000 lux to 4000 - 5000 lux. Hydrogen production was not observed in absence of light. The culture remained predominantly purple and the microscopic analysis showed the predominance of rod-shaped cells, gram-negative, accumulated in formation of rosettes; typical characteristics of some types of purple non-sulfur phototrophic bacteria. The analysis of the structure of the microbial community was carried out by reaction of polymerization in chain (PCR) of the RNAr 16S, followed of denaturing gradient gel electrophoresis (DGGE), and reveled that the structure of the microbial populations did not change significantly in function of the different conditions of culture.

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.<br>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.

碩士<br>元智大學<br>生物科技與工程研究所<br>99<br>Purple non-sulfur bacteria (PNSB) possess diverse metabolic pathway to survive in different environments, including photoautotrophic, photoheterotrophic, chemoautotrophic as well as chemoheterotrophic metabolisms. PNSB have been widely used in the fields of environmental protection, agricultural production, aquaculture and medical application, etc. The unique physiological characteristics of PNSB have also attracted the attention of researchers to investigate the possible application of these microorganisms. In this study, we have isolated and identified several strains of photosynthetic bacteria from sediments or soil from different environment in northern Taiwan, including pond water, rice field and channel. Enrichment technology was employed for isolation of PNSB with anaerobic incubation under light condition. The isolates were identified by 16S rRNA gene sequence analysis and several strains of Rhodobacter sp. were chosen for further evaluation for their applications such as production of coenzyme Q10 and bacterial pigments.

碩士<br>國立中興大學<br>環境工程學系所<br>105<br>Conversions of organic substrates to biohydrogen through separated dark-fermentation or photo- fermentation are both being recognized as promising clean energy production techniques. With or without illumination, both fermentations could produce hydrogen gas under anaerobic condition. Therefore, it is possible to enforce dark- and photo-fermentations to occur in the same reactor and it might have advantages over separated fermentation by reducing overall reactor volume since hydrogen-producing PNSB (Photosynthetic purple non-sulfur bacteria) could directly utilize the VFAs formed by dark fermentation. With this motivation, experiments of separated fermentation mode were established. CH5 is a strain of dark-fermentation bacteria (DFB), Clostridium pasteurianum, was found to have optimally cumulative amount of hydrogen when fed with 15 g L-1 glucose and it also produced significant amount of butyrate and acetic. Selecting two PNSB were C6 and G11, Rhodopseudomonas palustris, utilize not only butyrate but also acetate for bio-hydrogen. In the same time, when butyrate was used as sole carbon source, the two isolated PNSB strains could successfully produce 208.8 and 183.1 mL H2 L-1 culture (1 g/L butyric acid), its hydrogen yield were 0.82 and 0.98 mol H2 mol-1 butyrate, respectively. The growth status of hydrogen-producing microorganism in the time of combination was still not clearly explored. In this study, the stable growth period and the logarithmic growth phase of the strain were selected respectively. The anaerobic bioreactor were incubated at 30°C, at 120 rpm on an incubating shaker under 6-7 klux of light illumination using LED lamps. The inoculate at DFB/PNSB ratio were 1:1 (v/v) in co-culture medium (15 g/L glucose). There are maximum hydrogen yield 2.54 and 1.74 mol H2 mol-1 glucose were obtained by log phase of CH5+C6 and CH5+G11, respectively. Stationary phase of CH5+C6 yield maximum accumulative hydrogen production of 1485 mL H2 L-1 culture. C6 is better than G11 go further co-culture with CH5 by shelter of bioreactor experiment. Log phase test has the higher hydrogen yield, after culture 60 hours gas performance was approaching flat. Stationary phase test has the highest accumulative hydrogen production, it could continue to produce gas for 100 hours. For acquisition more accumulative hydrogen production, stationary phase of CH5+C6 got on subsequent study. In order to investigate the effect of light on co-culture fermentation, the shelter of bioreactor by differ cover area in this study. Through differ bioreactor cover area look forward to creating different environment for bacteria co-fermentation. 0% shelter of CH5 has 26% less accumulative hydrogen production than 100% shelter of CH5, it need more study and test to provide whether light supply can change the metabolism of CH5. 100% shelter of C6 has 55% less accumulative hydrogen production and use up more 4.56 g L-1 glucose than 0% shelter of C6. The 69% of gas composition was carbon dioxide, but only 16% was hydrogen produced by 100% shelter of C6. It speculated the 100% shelter of C6 that without light offering energy to promote electronic chain on membrane of PNSB, there are no enough ATP for cell growth not to mention for bio-hydrogen produced. 50% shelter of CH5+C6 promoted 67% accumulative hydrogen production than 0% shelter of CH5+C6, it also upgraded 588% accumulative hydrogen production than 100% shelter of CH5+C6. It could remove 60% glucose in 50% shelter of CH5+C6 and 100% shelter of CH5+C6. Main gas composition in 0% shelter of CH5+C6 and 50% shelter of CH5+C6 were hydrogen, in 100% shelter of CH5+C6 was carbondioxide. It is success for shelter of bioreactor to promote bio-hydrogen of co-culture fermentation. The 50% shelter of CH5+C6 consumed 26% sCOD, the accumulative hydrogen production of 1581 mL H2 L-1 culture, and the hydrogen yield was 1.309 mol H2 mol-1 glucose.

碩士<br>嘉南藥理大學<br>環境工程與科學系<br>107<br>Swine wastewater contains a large amount of organic matter and a high concentration of ammonia, which is toxic to the aquatic organisms, is one of the main water pollution in Taiwan. Direct discharge will pollute river and lake quality. In this study, the efficiency of swine wastewater treatment by photosynthetic purple non-sulfur bacteria (PNS, obtained from Food Industry Development Institute) was investigated. The changes of chemical properties and nutrient salt durin the treatment of swine wastewater by supplement of different proportions of PNS culture. The changes of pH, EC value, TOC, NO3-, SO42-, PO43-, NH4+ and OD864nm of wastewater were determined during the test. Furthermore, effects of additional nutrient (carbon and nitrgen) on the efficiency of swine wastewater by PNS were also discussed. Comparasion of efficiency of swine wastewater treatment by addition of carbon sources (including glucose, sucrose and fructooligosaccharides) or nitrogen source (including potassium nitrate, ammonium chloride, yeast extract) could provide a reference for the biological treatment of swine wastewater. Inoculation of PNS culture could slightly increase the pH value of swine wastewater(p<0.05); the high proportion (20%) of PNS culture could also remove 28.48% of TOC until the 9th day of culture. In the early stage of wastewater treatment, the content of ammonia-N and sulfate in wastewater were significantly reduced due to the growth of microorganisms(p<0.05). Addition of carbon source and nitrogen source could not increase the TOC removal rate of wastewater; and the addition of carbon source also had a certain effect on the removal of nitrate in wastewater. Furthermore, addition of carbon source and nitrogen source could increase the sulfate removal rate of wastewater

La recherche de sources d’énergie fiables ayant un faible coût environnemental est en plein essor. L’hydrogène, étant un transporteur d’énergie propre et simple, pourrait servir comme moyen de transport de l’énergie de l’avenir. Une solution idéale pour les besoins énergétiques implique une production renouvelable de l’hydrogène. Parmi les possibilités pour un tel processus, la production biologique de l’hydrogène, aussi appelée biohydrogène, est une excellente alternative. L’hydrogène est le produit de plusieurs voies métaboliques bactériennes mais le rendement de la conversion de substrat en hydrogène est généralement faible, empêchant ainsi le développement d’un processus pratique de production d’hydrogène. Par exemple, lorsque l’hydrogène est produit par la nitrogénase sous des conditions de photofermentation, chaque molécule d’hydrogène constituée requiert 4 ATP, ce qui rend le processus inefficace. Les bactéries photosynthétiques non sulfureuses ont la capacité de croître sous différentes conditions. Selon des études génomiques, Rhodospirillum rubrum et Rhodopseudomonas palustris possèdent une hydrogénase FeFe qui leur permettrait de produire de l’hydrogène par fermentation anaérobie de manière très efficace. Il existe cependant très peu d’information sur la régulation de la synthèse de cette hydrogénase ainsi que sur les voies de fermentation dont elle fait partie. Une surexpression de cette enzyme permettrait potentiellement d’améliorer le rendement de production d’hydrogène. Cette étude vise à en apprendre davantage sur cette enzyme en tentant la surexpression de cette dernière dans les conditions favorisant la production d’hydrogène. L’utilisation de résidus organiques comme substrat pour la production d’hydrogène sera aussi étudiée.<br>The search for alternative energy sources with low environmental impact is in great expansion. Hydrogen, an elegant and simple energy transporter, could serve as means of transporting energy in the future. An ideal solution to the increasing energy needs would imply a renewable production of hydrogen. Out of all the existing possibilities for such a process, the biological production of hydrogen, also called biohydrogen, is an excellent alternative. Hydrogen is the end result or co-product of many pathways in bacterial metabolism. However, such pathways usually show low yields of substrate to hydrogen conversion, which prevents the development of efficient production processes. For example, when hydrogen is produced via nitrogenase under photofermentation conditions, each hydrogen molecule produced requires 4 molecules of ATP, rendering the process very energetically inefficient. Purple non-sulfur bacteria are highly adaptive organisms that can grow under various conditions. According to recent genomic analyses, Rhodospirillum rubrum and Rhodopseudomonas palustris possess, within their genome, an FeFe hydrogenase that would allow them to produce hydrogen via dark fermentation quite efficiently. Unfortunately, very little information is known on the regulation of the synthesis of this enzyme or the various pathways that require it. An overexpression of this hydrogenase could potentially increase the yields of substrate to hydrogen conversion. This study aims to increase our knowledge about this FeFe hydrogenase by overexpressing it in conditions that facilitate the production of hydrogen. The use of organic waste as substrate for hydrogen production will also be studied.

碩士<br>國立中興大學<br>環境工程學系<br>93<br>Biological phosphorus removal from wastewater had been accomplished in activated sludge reactors by proper process design. The potential PAOs in EBPR system were identified as the Rhodocyclus-related microorganisms which belongs to the Rhodosiprillums in recently researches, and the role they play in systems are not yet fully understood. The other researches had observed the phototrophic purple nonsulfur bacteria are 2-fold capability of polyphosphate accumulating than PAOs in both anaerobic and aerobic condition, and the model of biological phosphorus removal were much different with the model described by scientists. Augmentation of phototrophic purple nonsulfur bacteria, Rhodopseudomonas palustris, was applized in this research had a better capability of polyphosphate accumulating in both anaerobic and aerobic under a laboratory scale EBPR system and in an activated sludge system. The results of bioagumentaiton demostrated that the P-removal efficiency was between 15% to 30% as the reactor was operated under a cycle of 7 days, and the microorganism would exsit in the system after bioagumentation. When the efficiency of P removal in anaerobic step was enhanced, the released P contents was decreased 15% compared to the EBPR process without augmentation, however P uptake contents was not decrease but increased in aerobic process. It suggests that Rhodopseudomonas palustris could accumulate polyphosphate under anaerobic period in EBPR process, but the P-removal efficiency were unstable due to the competition with the originally existe PAOs in the system during the initial period of bioaugmentation. After added photo-source the augmentated strain were competited other microorganisms, and the enhanced P removal efficiency was apparently decreased. In the non-phosphorus removal process, results of batch experiments showed the P-removal efficiency was between 10% to 20% after augmentation, but Rhodopseudomonas palustris would be washed out due to the lower ability of competition in the continuous influent system.

碩士<br>東吳大學<br>微生物學系<br>95<br>Purple non-sulfur bacteria are a group of extraordinary metabolic diverse bacteria. They can grow with photoautotroph, photoheterotroph, chemoheterotroph or chemoautotroph. Under various conditions, they can enjoy exceptional flexibility within each of these modes of metabolism. Due to the special physical characteristics properties, they had attracted scientist’s attention in resent years. These bacterias are widely distributed in nature such as lakes, water ponds, seas, or farm, etc. We isolated 39 strains bacteria from different aqueous phase of northern Taiwan. We discussed the diversity of their morphology and genetic diversity of their 16S-23S ribosomal intergenic spacer region(ISR) . First, dependent on cell shapes, we classed these strains into seven types: coccus, oval, ring, horse's hoof, and three size rods. Colors of their cell suspension are brown, pink, purple and red-brown, etc. Their cultural characteristics almost are circular forms , convex elevation and entire margins. Then, according to above characteristics, we classed 39 strains in to twenty-six clusters. Taking 16S rRNA sequence of these 26 representatives to blast with DataBank , the similarity all are larger than 99%，which reveals they were related to Rhodobacter sphaeroides, Rhodovulum sulfidophilum and Rhodopseudomonas sp. and revealed there were highly diversity of their morphology . In other hand, the16S-23S ISR of 39 strains were amplified by PCR and digested with PvuII, AluI, HaeIII, resulting in 22 RFLP profiles. This results showed that 39 purple nonsulfur bacterias could be classed into 22 groups. 16S-23S ISR-RFLP revealed that there was a genotype diversity within Rba. sphaeroides and Rhodovulum sp. strain MB263,which there were different 7 and 2 RFLP profiles respectively. This result showed that there was genotype diversity of 16S-23S ISR with both species ,or may that there was not enough database of 16S rRNA sequence in GenBank to blast accurately to the species. Hence, analysis of 16S-23S ISR-RFLP was more accurate than analysis of 16S rRNA sequences for identification of purple non-sulfur bacteria. By this method, 16S-23S ISR-RFLP, we can set up a characterization system of purple non-sulfur bacteria.

Phototrophic Fe(II)-oxidizing bacteria use electrons from ferrous iron [Fe(II)] and energy from light to drive reductive CO2 fixation. This metabolism is thought to be ancient in origin, and plays an important role in environmental iron cycling. It has been implicated in the deposition of Banded Iron Formations, a class of ancient sedimentary iron deposits. Consistent with this hypothesis, we discovered that hydrogen gas, a thermodynamically favorable electron donor to Fe(II), in an Archean atmosphere would not have inhibited phototrophic Fe(II) oxidation. To understand this physiology and the connection to BIF formation at the molecular level, the mechanisms of phototrophic Fe(II) oxidation were examined in two purple non-sulfur bacteria, Rhodopseudomonas palustris TIE-1 and Rhodobacter sp. SW2. Important advances were made in elucidating genes critical to phototrophic Fe(II) oxidation. In R. palustris TIE-1, the first genetically tractable phototrophic Fe(II) oxidizer isolated, transposon mutagenesis identified a putative integral membrane protein and a potential cobalamin (vitamin B12) biosynthesis protein involved in Fe(II) oxidation. Increased expression of a putative decaheme c-type cytochrome, encoded by pioA, was observed when cells were grown under Fe(II)-oxidizing conditions. Two genes located immediately downstream of pioA in the same operon, pioB and pioC, encode a putative outer membrane beta-barrel protein and a putative high potential iron-sulfur protein, respectively. Deletion studies demonstrated that all three genes are involved in phototrophic Fe(II) oxidation. In Rhodobacter sp. SW2, a three-gene operon, foxEYZ, was found to be involved in phototrophic Fe(II) oxidation through heterologous expression in a close relative, Rhodobacter capsulatus SB1003. The first gene, foxE, encodes a novel c-type cytochrome located in the periplasm. Expression of foxE alone confers light-dependent Fe(II) oxidation activity to SB1003, but maximal activity is achieved when foxE is co-expressed with foxY and foxZ. FoxY appears to contain the redox cofactor pyrroloquinoline quinone and FoxZ a cytoplasmic membrane transporter. Recombinant PioC was overexpressed and partially purified from Escherichia coli. This research presents a detailed study of the physiology and genetics of phototrophic Fe(II) oxidation in two purple non-sulfur bacteria, and provides our first insight into the molecular mechanisms of this metabolism.

碩士<br>國立中興大學<br>環境工程學系所<br>94<br>Biological phosphorus removal from wastewater had been accomplishing in activated sludge reactors by proper process design. One of the potential PAOs in EBPR system was believed to be the Rhodocyclus-related microorganism which was identified phylogenetically close to the Rhodosiprillums. However, the role they play in systems have not been fully understood yet. Previous studies from our research demonstrated that the purple nonsulfur bacteria isolated from activated sludge could remove phosphorus from wastewater by accumulate it as intracellular polyphosphate. Polyphosphate-accumulating ability of the phototrophic purple nonsulfur bacteria was found to be twice stronger than other PAOs in both anaerobic and aerobic condition, and this model of biological phosphorus removal were different with the PAOs model described by scientists. Furthermore, the results also showed that most of the isolated pure cultures could accumulate polyphosphate under both anaerobic-only and aerobic-only condition. This observation was not only never been reported on the literatures but also quite different from the well-known anaerobic/aerobic cycling phosphorus accumulation model. It was demonstrated that bioaugmentation of purple nonsulfur bacteria with high polyphosphate accumulating ability into lab-scale activated sludge system could enhance the overall efficiency of phosphorus removal. All of these results prompted an interesting research subject on the phosphorus accumulating mechanism the purple nonsulfur bacteria. In this proposal, polyphosphate accumulating phenomenon performed by the purple nonsulfur bacteria and the pathway of this polyphosphate metabolism were studied. Results of this study showed that Rhodopseudomonas palustris GN11 could accumulate polyphosphate when anaerobicly incubated, and it started accumulating polyphosphate when entering the stationary phase. Results of this study will be helpful to understand and promote to establish a polyphosphate accumulating mechanism by purple nonsulfur bacteria.

碩士<br>東吳大學<br>微生物學系<br>100<br>Purple non-sulfur bacteria are a group of extraordinary metabolic diverse bacteria, therefore may widely exist in the environment. The study the diversity of freshwater environment of the purple non-sulfur bacteria have been many reported, however, the diversity of the hemophilic purple non-sulfur bacteria are less studied. So this study target separation of pure strains of purple non-sulfur bacteria in different environments of Taiwan western coast, and the use of molecular biological methods for classification, differences strains caused by different environmental , isolated producing hydrogen and Polyhydroxybutyrate (PHB) capacity assessment. Experimental screening out 34 purple non-sulfur bacteria, of which three for halotolerant of purple non-sulfur bacteria, others were all the halophilic of purple non-sulfur bacteria. These bacteria using 16S rDNA sequences sequencing, they were all Rhodovulum sulfidphilum. Strain differences in colony size and bacteria liquid on the color rendering. Use 16S-23S ITS RFLP, 16S rDNA sequence similarity and relationship between phylogenetic tree, 16S-23S ITS (ribosomal intergenic spacer;ITS) sequence similarity and genetic relationship between trees and multi locus sequence typing (the Multiocus sequence type) trying to categorize strains do,but it can do no further grouping. Hydrogen parts, first detected using PCR method screening while nifD and nifH gene of strains, and gas production test, found under the strains of malic acid as carbon sources capable of producing gas. For gas analysis, recognizing the hydrogen gas generated, then selection two strains one was hydrogen yield the highest R. sulfidphilum SSa7, another was gas yield the higher then others R. sulfidphilum SSa3. Maximize hydrogen production efficiency of 6.31ml/h/L and 2.72/ml/h/L, respectively. PHB production parts, strain detected phaC gene using PCR method and then quantitative PHB. Isolated PHB yield were 6~15 (mg/g fresh wet) with a known PHB production ability of Alcaligenes latus 61.60 (mg/g fresh wet) comparative ability is poor. Wastewater section, since waste water usually family livelihood is a fresh water environment. Using halotolerant strains R. sulfidphilum SSa7 to test whether reduce the total organic carbon and to produce hydrogen and producing PHB. It could reduce total organic carbon and produced PHB without hydrogen in wastewater. The future can expect R. Sulfidphilum whole genome sequencing, we are convenient to classification this strain. PHB production and hydrogen-producing part trying to find the most suitable conditions to increase the yield. Strains are using in food or a leather factory, to test if it is capable of producing hydrogen and producing PHB.

碩士<br>國立中興大學<br>環境工程學系所<br>95<br>Phototrophic purple non-sulfur bacteria had been applied to treat waste water with high concentration of organic matters and degrade aromatic hydrocarbons, such as benzoate or 3-chlorobenzoate, in waste water or soil. Besides contaminants removal, purple non-sulfur bacteria was also used in the study of biological H2 production. Other researches had observed that purple non-sulfur bacteria would accumulate polyphosphate (poly-P) inside the cells, and the capability of poly-P accumulating was 2 to 3 times higher than PAOs. Therefore, purple non-sulfur bacteria was a valuable microbial community for the environmental engineering field. Not only species and physiological characteristics but also microbial population is important for studying environmental microbiology. In the past researches, quantification of microorganisms by traditional methods always led to underestimate. A new molecular biotechnique, Real-Time PCR, was expected to overcome this disadvantage, which with faster, reliable, sensitive and convenient advantage on cell quantification, and has recently been applied to quantify environmental microorganisms. Nevertheless, quantification of purple non-sulfur bacteria by molecular biotechnologies has not been reported to date. For this reason, the primarily goal of this study is to develop a Real-Time PCR assay to quantify the purple non-sulfur bacteria which is based on enumerating the copy number of pufM gene. At first, known quantities of pufM gene inserted plasmid DNA was used as standard DNA, a series of 10-fold diluted standard DNA analyzed by Real-Time PCR for obtaining cycle threshold (CT), and then a standard curve was generated from these data. Due to the effect of sample DNA preparation on quantitative accuracy, six different commercial DNA extraction kits were used to compare the extracted DNA quality based on measuring nucleic acid assay (A260/A280). One of the commercial kit was finally chosen for further experimental using. After Real-Time PCR assay was established, purple non-sulfur bacteria within both pure culture and environmental samples were quantified by using Real-Time PCR and other quantitative methods. From the quantification results of pure culture Rhodopseudomonas palustris GN11, there is no obvious difference between Real-Time PCR, DAPI staining and plate count methods. Real-Time PCR assay overcame the disadvantages which would lead cells losing on DAPI staining and plate count, and obtained more accurate amount. Quantification results of environmental samples indicated that date from Real-Time PCR were 101 to 104-folds higher than that from plate count method. This experiment confirmed that microorganisms in environmental samples would compete to each other while using traditional culture; moreover, a portion of purple non-sulfur bacteria probably couldn’t grow on sodium acetate based medium. Using plate count method actually resulted in data underestimate; on the other hand, results from Real-Time PCR should more reliable. Quantification of purple non-sulfur bacteria by using plate count method, cost at least 5 to 7 days, but less than 1 hour using Real-Time PCR method. Besides more reliable, quantification using Real-Time PCR method was faster. For this reason, Real-Time PCR method was really a proper method for the quantification of phototrophic purple non-sulfur bacteria.

碩士<br>國立中興大學<br>生命科學系所<br>96<br>Environmental contaminants, such as anthropogenic chlorinated compounds tri-chloroethylene (TCE) and tetrachloroethylene (PCE), can be reductively dehalogenated to less chlorinated ethenes or innocuous compounds by micro-organisms through halorespiration under anaerobic condition. The halorespiration is composed of electron donor, electron carrier, and electron acceptor. Most of these microorganisms can use hydrogen as electron donor to proceed reductive dehalogenation. The decomposition of chlorinated compound as the major targets for bioremediation with hydrogen-producing microorganisms by using co-culture. Here we construct the genes enconding cytochrome P450cam gene (camC, camA, and camB) expressed in a photosynthetic hydrogen-producing microorganisms Rhodo-pseudomonas palustris CGA009. Under anoxic condition, pentachloroethane (PCA) that bind at the camphor-binding site could be reduced by the camC of cytochrome P450cam gene. We use pentachloroethane as a model substrate for characterizing genetically engineered bacteria containing theses enzyme systems. The Rhodopseudomonas palustris CGA009 express gene including pMG105, pMG-camC, and pMG-camCAB were analyzed by using RT-PCR to monitor RNA expression. In order to analysis the biodegradation of pentachloroethane, the engineered strain carried the pMG-camCAB plasmid exhibited the predominant rate of the PCA degradation and production of hydrogen rather than pMG105 and pMG105-camC strains. Two strains of pMG105 and pMG-camCAB absence of the ability to grow on 3-Chlorobenzoate and 3-Chlorobenzoate plus Benzoate as carbon source. Probably no chlorinated benzoate or benzoate were degraded by Rhodopseudomonas palustris CGA009 two strains.

A l’heure actuelle, les biocarburants renouvelables et qui ne nuit pas à l'environnement sont à l'étude intensive en raison de l'augmentation des problèmes de santé et de la diminution des combustibles fossiles. H2 est l'un des candidats les plus prometteurs en raison de ses caractéristiques uniques, telles que la densité d'énergie élevée et la génération faible ou inexistante de polluants. Une façon attrayante pour produire la H2 est par les bactéries photosynthétiques qui peuvent capter l'énergie lumineuse pour actionner la production H2 avec leur système de nitrogénase. L'objectif principal de cette étude était d'améliorer le rendement de H2 des bactéries photosynthétiques pourpres non sulfureuses utilisant une combinaison de génie métabolique et le plan des expériences. Une hypothèse est que le rendement en H2 pourrait être améliorée par la redirection de flux de cycle du Calvin-Benson-Bassham envers du système de nitrogénase qui catalyse la réduction des protons en H2. Ainsi, un PRK, phosphoribulose kinase, mutant « knock-out » de Rhodobacter capsulatus JP91 a été créé. L’analyse de la croissance sur des différentes sources de carbone a montré que ce mutant ne peut croître qu’avec l’acétate, sans toutefois produire d' H2. Un mutant spontané, YL1, a été récupéré qui a retenu l'cbbP (codant pour PRK) mutation d'origine, mais qui avait acquis la capacité de se développer sur le glucose et produire H2. Une étude de la production H2 sous différents niveaux d'éclairage a montré que le rendement d’YL1 était de 20-40% supérieure à la souche type sauvage JP91. Cependant, il n'y avait pas d'amélioration notable du taux de production de H2. Une étude cinétique a montré que la croissance et la production d'hydrogène sont fortement liées avec des électrons à partir du glucose principalement dirigés vers la production de H2 et la formation de la biomasse. Sous des intensités lumineuses faibles à intermédiaires, la production d'acides organiques est importante, ce qui suggère une nouvelle amélioration additionnel du rendement H2 pourrait être possible grâce à l'optimisation des processus. Dans une série d'expériences associées, un autre mutant spontané, YL2, qui a un phénotype similaire à YL1, a été testé pour la croissance dans un milieu contenant de l'ammonium. Les résultats ont montré que YL2 ne peut croître que avec de l'acétate comme source de carbone, encore une fois, sans produire de H2. Une incubation prolongée dans les milieux qui ne supportent pas la croissance de YL2 a permis l'isolement de deux mutants spontanés secondaires intéressants, YL3 et YL4. L'analyse par empreint du pied Western a montré que les deux souches ont, dans une gamme de concentrations d'ammonium, l'expression constitutive de la nitrogénase. Les génomes d’YL2, YL3 et YL4 ont été séquencés afin de trouver les mutations responsables de ce phénomène. Fait intéressant, les mutations de nifA1 et nifA2 ont été trouvés dans les deux YL3 et YL4. Il est probable qu'un changement conformationnel de NifA modifie l'interaction protéine-protéine entre NifA et PII protéines (telles que GlnB ou GlnK), lui permettant d'échapper à la régulation par l'ammonium, et donc d'être capable d'activer la transcription de la nitrogénase en présence d'ammonium. On ignore comment le nitrogénase synthétisé est capable de maintenir son activité parce qu’en théorie, il devrait également être soumis à une régulation post-traductionnelle par ammonium. Une autre preuve pourrait être obtenue par l'étude du transcriptome d’YL3 et YL4. Une première étude sur la production d’ H2 par YL3 et YL4 ont montré qu'ils sont capables d’une beaucoup plus grande production d'hydrogène que JP91 en milieu d'ammonium, qui ouvre la porte pour les études futures avec ces souches en utilisant des déchets contenant de l'ammonium en tant que substrats. Enfin, le reformage biologique de l'éthanol à H2 avec la bactérie photosynthétique, Rhodopseudomonas palustris CGA009 a été examiné. La production d'éthanol avec fermentation utilisant des ressources renouvelables microbiennes a été traitée comme une technique mature. Cependant, la plupart des études du reformage de l'éthanol à H2 se sont concentrés sur le reformage chimique à la vapeur, ce qui nécessite généralement une haute charge énergetique et résultats dans les émissions de gaz toxiques. Ainsi le reformage biologique de l'éthanol à H2 avec des bactéries photosynthétiques, qui peuvent capturer la lumière pour répondre aux besoins énergétiques de cette réaction, semble d’être plus prometteuse. Une étude précédente a démontré la production d'hydrogène à partir d'éthanol, toutefois, le rendement ou la durée de cette réaction n'a pas été examiné. Une analyse RSM (méthode de surface de réponse) a été réalisée dans laquelle les concentrations de trois facteurs principaux, l'intensité lumineuse, de l'éthanol et du glutamate ont été variés. Nos résultats ont montré que près de 2 moles de H2 peuvent être obtenus à partir d'une mole d'éthanol, 33% de ce qui est théoriquement possible.<br>Currently, renewable and environmentally friendly biofuels are under intensive study due to increasing health concerns and diminishing fossil fuels. H2 is one of the most promising candidates due to its unique characteristics, such as a high energy density and low to non-existent generation of pollutants. One attractive way to produce H2 is through photosynthetic bacteria which can capture light energy to drive H2 production with their nitrogenase system. The major aim of this study was to improve H2 yield of the purple non-sulfur photosynthetic bacteria using a combination of metabolic engineering and design of experiments. One hypothesis was that H2 yield could be improved by redirection of Calvin-Benson-Bassham cycle flux to the nitrogenase system which catalyzes the reduction of protons to H2. Thus, a PRK, phosphoribulose kinase, knock out mutant of Rhodobacter capsulatus JP91 was created. Analysis of growth with different carbon sources showed that this mutant could only grow in acetate medium without, however, producing any H2. A spontaneous mutant, YL1, was recovered which retained the original cbbP (encoding PRK) mutation, but which had gained the ability to grow on glucose and produce H2. A study of H2 production under different illumination levels showed that the yield of YL1 was 20-40% greater than the wild type JP91 strain. However, there was no appreciable improvement of the H2 production rate. A kinetic study showed that growth and hydrogen production are strongly linked with electrons from glucose being mostly directed to H2 production and biomass formation. Under low to intermediate light intensities, the production of organic acids was significant, suggesting further improvement of H2 yield is possible by process optimization. In a related series of experiments, another spontaneous mutant, YL2, which has a similar phenotype to YL1, was tested for growth in ammonium-containing media. The results showed that YL2 could only grow with acetate as carbon source, again, without producing any H2. Prolonged incubation in media not supporting growth of YL2 enabled the isolation of two interesting secondary spontaneous mutants, YL3 and YL4. Western blot analysis showed that both strains had constitutive nitrogenase expression under a range of ammonium concentrations. The genomes of YL2, YL3 and YL4 were sequenced in order to find the mutations responsible for this phenomenon. Interestingly, mutations of nifA1 and nifA2 were found in both YL3 and YL4. It is likely that a conformational change of NifA alters the protein-protein interaction between NifA and PII proteins (such as GlnB or GlnK), enabling it to escape regulation by ammonium and thus to be capable of activating nitrogenase transcription in the presence of ammonium. It is not clear how the synthesized nitrogenase is able to maintain its activity since in theory it should also be subject to posttranslational regulation by ammonium. Further evidence could be obtained by studying the transcriptome of YL3 and YL4. An initial study of H2 production by YL3 and YL4 showed that they are capable of much greater hydrogen production than JP91 in ammonium medium, which opens the door for future studies with these strains using ammonium-containing wastes as substrates. Finally, the biological reformation of ethanol to H2 with the photosynthetic bacterium, Rhodopseudomonas palustris CGA009 was examined. Ethanol production with microbial fermentation using renewable resources has been treated as a mature technique. However, most studies of the reformation of ethanol to H2 have focused on chemical steam reforming, which usually requires a high energy input and results in toxic gas emission. Thus biological reformation of ethanol to H2 with photosynthetic bacteria, which can capture light to meet the energy requirement of this reaction, seems to be more promising. A previous study had demonstrated hydrogen production from ethanol, however, the yield or the duration of this reaction were not examined. A RSM (response surface methodology) analysis was carried out in which three key factors, light intensity, ethanol and glutamate concentrations were varied. Our results showed that nearly 2 moles of H2 could be obtained from one mole of ethanol, 33% of what is theoretically possible.