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Hydrogen is an environmentally friendly and highly efficient energy source. Fermentative hydrogen production is an exciting R&D area that offers a means to produce hydrogen from a variety of renewable resources or even wastewaters. However, the development of fermentative hydrogen production processes has been hampered due to their low yield and relatively high costs. The aim of this thesis was to improve fundamental knowledge of hydrogen-producing bacteria, provide genetic information associated with the hydrogen evolution, and to optimise operating conditions to enhance hydrogen yield. Isolation and identification of hydrogen producing bacteria from activated sludge were conducted using 16S rRNA gene-directed PCR-denaturing gradient gel electrophoresis (DGGE), clone library and heterotrophic plate isolation. The results showed that Clostridium sp. were dominant and active hydrogen producers. For the first time, three hydrogen producers, which harboured the [FeFe] hydrogenase gene, were characterised by 16S rDNA sequencing, and further physiologically identified as Clostridium sp. (W1), Clostridium butyricum (W4) and Clostridium butyricum (W5). The structure of the putative [FeFe] hydrogenase gene cluster of C. butyricum W5 was also described. The changes in [FeFe] hydrogenase mRNA expression of C. butyricum W5 during fermentation were monitored. Statistical analysis showed that both the [FeFe] hydrogenase mRNA expression level and cell growth have positive relationships with hydrogen production. The newly isolated C. butyricum W5 demonstrated highly promising hydrogen fermentation performance and was therefore used as the working strain. Optimization of operating conditions in terms of carbon and nitrogen sources, pH, temperature and inoculum size was carried out in a laboratory scale batch system. Use of molasses and NH₄NO₃ resulted in a high hydrogen production yield. Under the optimized fermentation conditions, 100g/L molasses, 1.2g/L NH₄NO₃, and 9×10⁴ cell/ml initial cell number at 39°C and pH 6.5, a maximum hydrogen yield of 1.85 mol H₂/ mol hexose was achieved. This corresponded to a hydrogen production rate of 17.38 mmol/h/L. Acetic, lactic and butyric acids were found to be the main by-products of the fermentation. The interrelations between the hydrogen yield and other yields of metabolites were statistically analysed corresponding to the variation in operating conditions. The dry cell weight was found to have a power relationship with hydrogen production. The results from this study have provided a better understanding of metabolic processes and gene expression involved in fermentative hydrogen production, and an improved bioengineering process for hydrogen production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339837 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

Hydrogen is an environmentally friendly and highly efficient energy source. Fermentative hydrogen production is an exciting R&D area that offers a means to produce hydrogen from a variety of renewable resources or even wastewaters. However, the development of fermentative hydrogen production processes has been hampered due to their low yield and relatively high costs. The aim of this thesis was to improve fundamental knowledge of hydrogen-producing bacteria, provide genetic information associated with the hydrogen evolution, and to optimise operating conditions to enhance hydrogen yield. Isolation and identification of hydrogen producing bacteria from activated sludge were conducted using 16S rRNA gene-directed PCR-denaturing gradient gel electrophoresis (DGGE), clone library and heterotrophic plate isolation. The results showed that Clostridium sp. were dominant and active hydrogen producers. For the first time, three hydrogen producers, which harboured the [FeFe] hydrogenase gene, were characterised by 16S rDNA sequencing, and further physiologically identified as Clostridium sp. (W1), Clostridium butyricum (W4) and Clostridium butyricum (W5). The structure of the putative [FeFe] hydrogenase gene cluster of C. butyricum W5 was also described. The changes in [FeFe] hydrogenase mRNA expression of C. butyricum W5 during fermentation were monitored. Statistical analysis showed that both the [FeFe] hydrogenase mRNA expression level and cell growth have positive relationships with hydrogen production. The newly isolated C. butyricum W5 demonstrated highly promising hydrogen fermentation performance and was therefore used as the working strain. Optimization of operating conditions in terms of carbon and nitrogen sources, pH, temperature and inoculum size was carried out in a laboratory scale batch system. Use of molasses and NH₄NO₃ resulted in a high hydrogen production yield. Under the optimized fermentation conditions, 100g/L molasses, 1.2g/L NH₄NO₃, and 9×10⁴ cell/ml initial cell number at 39°C and pH 6.5, a maximum hydrogen yield of 1.85 mol H₂/ mol hexose was achieved. This corresponded to a hydrogen production rate of 17.38 mmol/h/L. Acetic, lactic and butyric acids were found to be the main by-products of the fermentation. The interrelations between the hydrogen yield and other yields of metabolites were statistically analysed corresponding to the variation in operating conditions. The dry cell weight was found to have a power relationship with hydrogen production. The results from this study have provided a better understanding of metabolic processes and gene expression involved in fermentative hydrogen production, and an improved bioengineering process for hydrogen production. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339837 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

4 pp. / Vaccinating for clostridial diseases is an important part of a ranch health program. These infections can have significant economic impacts on the ranch due to animal losses. There are several diseases caused by different organisms from the genus Clostridia, and most of these are preventable with a sound vaccination program. Many of these infections can progress very rapidly; animals that were healthy yesterday are simply found dead with no observed signs of sickness. In most cases treatment is difficult or impossible, therefore we rely on vaccination to prevent infection. The most common organisms included in a 7-way or 8-way clostridial vaccine are discussed below. By understanding how these diseases occur, how quickly they can progress, and which animals are at risk you will have a chance to improve your herd health and prevent the potential economic losses that come with a clostridial disease outbreak.

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Clostridia are a class of gram-positive, anaerobic bacteria best known for their powerful toxins. These bacteria cause many diseases that are difficult to treat and often deadly, including colitis, botulism, tetanus and gas gangrene. These diseases are caused by the secretion of specific toxins, though current treatments do little to nullify these toxins and better therapeutics are urgently needed. The development of such treatments is hindered by our poor understanding of clostridial toxin secretion, which is itself hindered by the innate characteristics of these bacteria that make them difficult to study. Of the pathogenic clostridia, Clostridium perfringens is relatively easy to culture and straddles the line between pathogen and commensal, making it an attractive model organism for studying clostridial toxin secretion. C. perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals that can also cause disease. C. perfringens produces more toxins than any other bacterium, and these toxins generally function as a means to lyse host cells so the bacteria may scavenge their intracellular nutrients. The primary focus of the research in this dissertation is the secretion of the toxin TpeL by a small membrane protein, TpeE. Preceding the study of TpeL secretion were two other projects, which are discussed in Chapters 2 and 3. Chapter 2 describes an experimental plan to characterize the genes involved in muscle cell adherence as a very basic model to mimic skeletal muscle attachment in gas gangrene. Like many other bacteria, C. perfringens can produce T4P, extracellular filaments that are synthesized, extended and retracted from the cell by the concerted effort of many proteins. Results from initial, proof-of-concept adherence assays are presented and demonstrate that statistical significance was lost when data were compiled. Despite efforts to troubleshoot this, robust test output was not achieved and the project was discontinued November 2016. Chapter 3 describes the experimental plan and initial findings of a project where a link between T4P and virulence was investigated. Such a link had been demonstrated in the T4P model organism Pseudomonas aeruginosa, where PilT, the T4P retraction ATPase, was shown to sense surface attachment and initiate virulence. In C. perfringens, PilT demonstrates a number of characteristics that lead us to think it may also function as a sensor, coordinating host cell attachment and colonization by alternatively associating with PilM and FtsA. We developed an experimental plan to determine if PilT binds both PilM and FtsA by co-immunoprecipitation with live-cell fluorescence imaging. However, we were unable to demonstrate the functionality of a PilT-fluorescent protein fusion with an anti-pilin ELISA assay, nor were we able to detect PilT or FtsA overexpression by immunoblotting, and the project was discontinued in November 2017. In retrospect, these experiments likely failed because of an inactive promoter region in the overexpression plasmid. Though clostridial diseases require secreted toxins, their secretion mechanisms are largely uncharacterized, and Chapter 4 describes the investigation of a potentially conserved toxin secretion mechanism. TpeL is a recently discovered C. perfringens toxin that is associated with chicken necrotic enteritis, a disease that costs the poultry industry billions of dollars each year. TpeL belongs to a subset of clostridial toxins characterized by their large size and conserved structure, the large clostridial toxins. The gene for tpeL and nearly all other large clostridial toxins lies next to a gene encoding a small membrane protein. Since bacterial genes with a shared function are often found in close proximity, it is suspected that these small proteins share some function with these toxins, and another research group has shown the two large clostridial toxins in C. difficile need this small membrane protein for their secretion. We isolated the small membrane protein and toxin genes tpeE and tpeL from native regulatory elements and overexpressed them heterologously in a different strain of C. perfringens. By immunoblotting, we found rapid TpeL secretion requires TpeE, and secretion was abolished when C-terminal sections of either protein were mutated. By immunoblotting and growth curve analyses, we found that TpeE is maintained at low concentrations and is not lethal in C. perfringens, but was expressed to high levels and was lethal in Escherichia coli. Our results, in conjunction with those from other research groups strongly suggest a conserved secretion mechanism dependent on small, membrane proteins. Our findings further the understanding of toxin secretion, a key step toward novel and effective clostridial disease strategies. Chapter 5 describes the outcome of an experimental approach where tpeE and tpeL were expressed from two different expression system plasmids. A number of off-target effects materialized with this approach which confounded our experimental results. The predominantly confounding effect was off-target protein secretion, found by immunoblotting to be associated with one of the expression systems. Despite efforts to minimize these effects, it became clear results from this approach would be uninterpretable and the two-plasmid approach for TpeE and TpeL expression was abandoned. A cut-and-paste strategy using the historical, single inducible expression system was implemented in its place. The exact mechanism for TpeL secretion by the small membrane protein TpeE is unclear. Chapter 6 outlines some hypotheses towards this mechanism and a nascent plan to uncover it. An efficient starting point is to determine if the two proteins are in close enough proximity to one another to interact in vivo. We developed a strategy to determine this by crosslinking and immunoblotting, using the size differential between the proteins to our advantage. Though the results of this study were confounded by an inability of TpeL to solubilize in buffer, the groundwork is laid for future endeavors. / Doctor of Philosophy / Clostridium perfringens is a bacterium found naturally in soils and in the gastrointestinal tracts of humans and animals worldwide. C. perfringens is an important organism to study due to its roles as a decomposer in our ecosystem and its ability to cause a number of diseases. These diseases cause considerable harm to livestock and poultry industries, as well as to human and animal life. Though these diseases vary wildly, they share this in common: they are defined by specific toxins and what defines a harmful lineage of C. perfringens is its ability to produce and secrete these toxins. In fact, this is the common denominator to all clostridial diseases, including the notorious diseases C. difficile-associated colitis, tetanus, botulism, and gas gangrene. Of primary concern in diseases caused by C. perfringens and other clostridia is that effective, novel therapies are grossly lacking. The effects of these powerful toxins can outpace antibiotic therapy and this often leads to extended periods of suffering, even in favorable cases. Of all the pathogenic clostridia, C. perfringens is easy to test in the laboratory and may even be used in place of more dangerous and difficult to work with bacteria. This is useful in developing better treatments and for studying treatment applications for the toxins themselves! Indeed, bacterial toxins have beneficial applications, Botox being a good example, as well as in cancer treatments. Like many other bacteria, C. perfringens can produce strong, rope-like appendages called pili that are made and extended and retracted from the cell, similar to a lasso, by the concerted effort of many different proteins. These pili confer a number of advantages for bacteria, one being a means for attachment to host cells, an important first step in establishing an infection. With the overarching goal toward sowing future therapeutic developments, Chapter 2 describes an experimental plan to identify and understand the genes for the proteins that allow C. perfringens to attach to muscle cells. Preliminary results are presented for a proof-of-concept method, which was ultimately discontinued November 2016 because reliable, robust results were not obtained. In addition to host cell attachment, pili have been shown to function a sensor for cell. For example, in another bacterium, pili "sense" a suitable surface for attachment and interpret this signal so the bacterium can attach and "set up shop" by releasing toxins. Based on considerable evidence, we thought C. perfringens might also "sense" a surface with its pili and interpret this signal for attachment and cell growth by means of interactions between three specific proteins. We designed a series of experiments to test this hypothesis, but due to the failure of important, initial studies, this project was discontinued in November 2017. Even though all clostridial diseases are caused by toxins, which must be secreted outside the bacterium to do harm, how these toxins are secreted is poorly understood. In Chapter 4, we investigate a toxin secretion method where a certain type of toxin is thought to be secreted through a temporary hole formed by many copies of a small, partner. First, we forced C. perfringens bacteria to artificially produce both the small protein and the toxin and found that the toxin needs this small protein to be secreted. We then deleted parts of both the toxin and the small protein and determined which parts of each are essential for this secretion method by linking an absence of secretion in bacteria whose proteins are missing essential parts. Further, we determined that production of this small, partner protein was kept to low levels and was harmless in C. perfringens, but was lethal in a different, unrelated bacterium, Escherichia coli, implying that C. perfringens bacteria have an ability control this hypothetical hole in themselves that E. coli does not. Our results, in conjunction with those from previous groups, suggest a pattern for secretion of this type using these small proteins. This information is a key first step towards developing better therapies for clostridial diseases, since without toxin secretion, clostridial diseases cannot occur. Chapter 5 describes the surprising outcome of an experimental approach where the toxin and small partner protein are produced in the bacterium by two different mechanisms. We found a number of off-target effects associated with this approach, one of which was the strange facilitation of off-target protein secretion. These off-target effects confused our experimental results and since it was likely that future experiments would also be uninterpretable, we abandoned this approach and used a simpler one instead. The mechanisms for toxin (TpeL) secretion by its small, partner protein (TpeE) are unclear. A key, initial step towards understanding this mechanism is to determine if the two proteins are in close enough proximity to one another in the bacterium. We developed a strategy to determine if the proteins are close enough together in the cell that takes advantage of the considerable size difference between the two proteins. Presented in this chapter are several initial experiments that can enable this experiment in the future.

Interactions of microorganisms, both prokaryotic and eukaryotic, with the metal antimony were studied. Of particular interest was the process of biomethylation. Volatilisation of trimethylantimony from inorganic antimony substrate by mixed inoculum (of environmental source) enrichment cultures was demonstrated to occur. Trimethylantimony was the sole volatile antimony species detected in incubations designed to promote the growth of clostridia, no stibine or other volatile methylated species were detected. Two Clostridium sp. were isolated from environmental enrichment incubations and three characterised Clostridium sp. were demonstrated to possess a biomethylating capability. Up to 21 μg. 1-1 involatile methylantimony species were detected in the culture medium of monoseptic incubations of the characterised Clostridium sp. The relative quantities of involatile mono-, di- and trimethylantimony species produced during the course of the cultivation period is consistent with trimethylantimony oxide being a final product of antimony biomethylation, with monoand dimethylantimony species appearing transiently in the cultures as intermediates of an antimony biomethylation pathway. The fungi Cryptococcus humicolus, Candida boidinii, Candida tropicalis, Geotrichum candidum and Saccharomyces cerevisiae were all demonstrated to possess a similar antimony biomethylating capability. Volatile and involatile methylantimony species were detected, with involatile species being the predominant form. Both stibine and trimethylantimony were detected in culture headspace gases of fungal incubations. Levels of trimethylantimony were higher in incubations supplied with antimony III substrate, whilst stibine was the predominant volatile antimony species in incubations supplied with V valency substrate. S. cerevisiae demonstrated the highest stibine generating capability with up to 0.3% substrate being transformed. Regardless of substrate, overall antimony biomethylation efficiency (to both volatile and involatile species) was low, indicating that this biotransformation does not form the primary mode of resistance to the metal. Less than 0.1% of antimony III substrate was biomethylated by C. humicolus, the most productive species in terms of formation of methylantimony compounds. The intracellular accumulation of methylated antimony species further belies the theory that antimony biomethylation constitutes a resistance mechanism. Study of C. humicolus revealed the biomethylation process to be enzymatic and inducible by arsenic but not by antimony. This may indicate that the enzymes of the arsenic biomethylation pathway are the likely biocatalysts for the biomethylation of antimony. The low efficiency of antimony biomethylation indicates that this is most likely a fortuitous process. A number of Gram-positive cocci isolated from soil and sediment were demonstrated to bioreduce antimonate to an unknown inorganic antimony III compound concurrently with lactate oxidation and biomass formation (as measured by protein). Up to 48% of the supplied antimonate was bioreduced. The demonstration of dissimilatory antimonate respiration adds this metal to the increasing list of known "unusual" electron acceptors such as uranium, arsenic, selenium, iron and manganese. These studies reveal some of the microbial interactions of microorganisms with the metal antimony, demonstrating the potential that microorganisms have to contribute to the biogeochemical cycling of antimony through biotransformation processes

Genomic DNA fragments encoding the silent type B neurotoxin gene from Clostridium botulinum NCTC 2916 have been cloned and the complete nucleotide sequence determined. The translated sequence revealed that the gene encoded a neurotoxin which was closely related to type B neurotoxin genes from Group I Clostridium botulinum. However among the nucleotide sequence differences, aG to T transition has interrupted the coding sequence with the formation of a stop codon. In addition the deletion of an adenine residue has resulted in a frame-shift mutation. Analysis of the DNA sequence contiguous with the silent type B neurotoxin gene revealed the presence of a gene encoding a Nontoxic-Nonhaemagglutinin protein which appears to share a bicistronic mRNA transcript with the type B neurotoxin gene. In the reverse orientation, the partial sequence of a gene encoding a haemagglutinin protein was found, typical of type A and B botulinal neurotoxin complexes. Separating the genes encoding the 'components of the neurotoxin complex was a gene of 178 amino acids which possessed features commonly associated with transcriptional factors. To facilitate the in vivo study of botulinal neurotoxin complex regulation, a gene transfer system using clostridial components has been developed. The minimal replicon of the cryptic plasmid pCB 102 from Clostridium butyricum NCIB 7423 was located to 1.6 kb DNA fragment by deletion analysis, enabling the identification of hitherto undiscovered putative ORFs and secondary structures, consistent with a replicative function. The replicon has been incorporated in to a number of Escherichia coli vectors resulting in a versatile series of shuttle vectors which have demonstrated high structural and segregational stabilities in a heterologous host Clostridium beyerinckii NCI NIB 8052. Gene transfer of a Group I Clostridium botulinum type A strain was demonstrated with a representative pCB 102-derived shuttle vector, pMTL540E. In addition, a 5.9 kb plasmid indigenous to C. hotulimun NCTC 2916 was cloned and the complete nucleotide sequence determined. Eight putative ORFs have been identified, including a putative replication protein and recombinase.

Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).

Consolidated bioprocessing, a method that involves cellulase production, substrate hydrolysis, and fermentation all in one step, requires lower energy input and aims at achieving reduced biofuel production costs than traditional processes. It is an economically appealing strategy for the efficient production of biofuels such as ethanol or H2. At present, the yields of fermentative hydrogen and ethanol production are less than the theoretical maximum and vary between anaerobic Clostridia due to the presence of highly branched metabolic pathways. With the recent advancements in ‘Omic technologies, the selected cellulolytic species, in this case, C. termitidis, was extensively studied to identify the key enzymes that are involved in hydrogen and ethanol synthesis pathways in both the genome and proteome under different culture conditions. Metabolic characterization involving growth and end-product synthesis patterns were performed on 2 g L-1 cellobiose and α-cellulose under batch conditions to determine its metabolic potential for hydrogen and/or ethanol production. Initial characterization has shown the ability of C. termitidis to produce hydrogen, ethanol, and various other end-products on the two susbtrates. Continous N2 sparging in the pH-controlled bioreactors with cellobiose and α-cellulose showed a consistent increase in the H2 synthesis and lowered ethanol production compared to batch studies, with the H2 yields of 1.03 and 1.34 mol product per mol hexose equivalent added, respectively. Shotgun 2-D proteome analyses were performed to compare cellulose versus cellobiose grown cultures across exponential and stationary phases of growth. Most of the glycolytic proteins were detected in the proteome with some exceptions and no significant change was observed across both growth conditions. Hydrogen synthesis was regulatd via PFOR and ferredoxin-dependent hydrogenase, where as ethanol synthesis was regulated primarily via bifunctional AdhE activity. Proteomic analyses of C. termitidis cultured on hexose sugars in the absence of xylose suggested possible sequential utilization of xylose and cellobiose for the first time. Putative proteins consistent with xylose fermentation were observed at high levels. The hypothesis that C. termitidis can sequentially utilize xylose and cellobiose was further validated using batch fermentations tests on pure (xylose, cellobiose, xylan) and mixed substrates (xylose + cellobiose).