Includes bibliographical references (leaves [259]-290) / xxviii, 290 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Investigates nitrogen assimilation and metabolism in rumen bacteria with the object of understanding the basic process and their controls. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Sciences, 1991

A variety of unicellular green algae have the ability to photo-produce molecular hydrogen (H2). Using sunlight to power the production of H2 from water is attractive due to the abundant supply of both resources and the potential for the technology to address global warming and energy supply concerns. Increasing levels of H2 production from those currently achievable with algal systems is a necessity for the technology to become economically feasible. Green unicellular algae are rare amongst organisms in that some have an ability to switch to an H2-producing metabolism when environmental conditions become anaerobic. The process of H2 production is greatly accentuated in the light due to the role of the photosynthetic apparatus directing electron flow to hydrogenase enzymes located in the chloroplast. Difficulties in maintaining continuous systems of H2 production largely result from the O2 sensitivity of hydrogenase enzymes. As O2 is generally produced through photosynthesis, the process of H2 production has always been short-lived. Recently, a process of inducing H2 production for several days was accomplished by depriving the growth medium of sulphur (Melis et al., 2000). Lacking sulphur, photosystem II activity diminishes to a point where any O2 evolved is consumed by respiration; this leads to the culture becoming anaerobic and to the onset of H2 production. The method of sulphur depletion has proven to be very useful for studies of H2 production due to enhanced rates over longer time periods being possible. This work was performed to search for new H2-producing Australian algal species and to shed light upon the molecular and biochemical interactions occurring when algal species move from aerobic photosynthetic growth to an anaerobic H2-producing status. An assay to test new species for an H2-producing ability was developed and implemented; leading to the isolation of new H2-producing species from Australian waters. The assay involved purging algal cultures in the dark with N2, sealing them in bioreactors and then exposing them to light. Metabolic profiling performed during this assay revealed cells to rapidly enter a fermentative metabolism upon the onset of anoxia. Acetate, formate and ethanol were key metabolites produced alongside H2 during this period. Metabolomics was used as a tool to understand the biochemical interactions occurring during 120 h of sulphur depleted H2 production. Extraction protocols were developed that allowed the detection and identification of over 100 metabolites using gas chromatography coupled to mass spectrometry, nuclear magnetic resonance spectroscopy and thin layer chromatography. Shifts in primary energy metabolism when cells switch from O2 production to H2 production were revealed. Indications are that both starch and triacylglyceride accumulate during the first 24 h of sulphur depletion prior to anoxia. Following the onset of anoxia, fermentative metabolism begins, H2 is produced and amino acids generally increase. A build-up of toxic fermentative end products and a lack of sulphur are believed to cause the termination of H2 production, rather than a lack of energy reserves. Key achievements of this work have been: • The establishment of an assay that can be used for future bio-prospecting work aimed at finding H2-producing algal species. • The isolation of new H2-producing green algal species from Australian waters. • The establishment of protocols for the extraction of metabolites from small volumes (1 ml) of Chlamydomonas reinhardtii cultures for analysis on a variety of analytical platforms. • The mapping of changes in metabolism of C. reinhardtii during the switch from an aerobic environment to an anaerobic H2-producing environment. • A range of recommendations for future research that may lead to higher H2 production.

Bibliography: leaves 122-138. Investigates the use of microbial inoculants to prevent the onset of acidosis in acutely grain fed animals; and, the most effective combination of virginiamycin and lactic acid utilising bacteria (selenomonas ruminantium subsp. lactilytica and Megasphaera elsdenii) in controlling lactic acid accumulations in vitro.

Includes bibliographies. This thesis describes the isolation of putative promoter sequences that can produce differential expression of a gene during anaerobic wine fermentations, the use of these sequences in the development of expression vectors and the application of this work to the production of genetically engineered wine yeasts for commercial purposes.

A proprietary technology owned by Texas A

La bio-transformation naturelle des médicaments peut produire des métabolites toxiques; l’identification de ces métabolites est essentielle dans la stratégie de choix de molécules thérapeutiques. En appliquant les technologies de fermentation en bioréacteur des cellules hétérologues (souches d’E. coli recombinantes exprimant une iso-enzyme de cytochrome P450 humain avec la réductase humain), la bioconversion du substrat (principe actif) en ses métabolites de dégradation, a été réalisée à grande échelle (g-g). Notre choix s’est porté sur le complexe hCYP3A4/HR fonctionnel produit par un hôte E. coli. Les cellules intactes ou les membranes cellulaires peuvent être exploitées comme biocatalyseur dans un système bioréacteur. Cependant, la faible solubilité des principes actifs dans des milieux de bioconversion aqueuse limitent le rendement. Un bioréacteur biphasique a été étudié. En solution, plusieurs combinaisons eau/solvants organiques conciliant la viabilité des cellules, la solubilité des principes actifs et produits de réaction et la catalyse des complexes enzymatiques ont conduit à l’établissement d’un mélange approprié. Cependant, ces combinaisons présentent toujours une inhibition importante du pouvoir catalytique des complexes enzymatiques. Pour minimiser un effet dénaturant possible des solvants sur le système enzymatique, ce dernier a été maintenu dans un environnement aqueux en immobilisant les cellules et/ou les membranes cellulaires dans une matrice hydrophile. L’alginate de calcium apparaît être une matrice d’immobilisation idéale pour les membranes assurant la fonctionnalité du complexe CYP/HR et permettant en outre un stockage à long terme des préparations. Par contre, l’immobilisation des cellules dans diverses matrices, si elle permet une viabilité et une conservation à long terme des souches recombinantes, ne permet aucune expression de l’activité enzymatique présente dans les cellules. La combinaison d’une localisation du complexe hCYP/HR fonctionnel dans la membrane interne et d’une perméabilité réduite des cellules d’E. coli (immobilisées) en est une explication possible mais non-démontrée. Entre-temps, cette technologie de bioréacteur homogène biphasique ou par immobilisation des membranes cellulaires a été utilisée plusieurs reprises pour produire des métabolites humains à partir de divers principes actifs. Ces métabolites ont été purifiés avec succès, démontrant que cette approche technologique est compétitive comparée aux procédures conventionnelles. Néanmoins, de nouvelles pistes de recherche seraient extrêmement intéressantes. La localisation des complexes enzymatiques recombinants en surface des cellules permettrait de concilier les propriétés hydrophobes des principes actifs et l’environnement hydrophile nécessaire aux enzymes. D’autre part une investigation de complexes enzymatiques résistant aux solvants pourrait remplacer avantageusement l’immobilisation.

Studies Directed to the Optimization of Fermentation of Rhodococcus sp. DAP 96253 and Rhodococcus rhodochrous DAP 96622 by GENE KIRK DRAGO Under the Direction of George E. Pierce ABSTRACT Bench- and pilot plant scale fed-batch fermentations were performed in stirred-tank bioreactors (STBR) with Rhodococcus sp. DAP 96253 and R. rhodochrous DAP 96622 in an attempt to elucidate parameters that may affect the optimization of a fermentation process for high biomass production and high inducible expression of cobalt-high-molecular-mass nitrile hydratase (Co-H-NHase. The effects of these factors on amidase (AMDase) activity were also investigated. Biomass and NHase production were inhibited by a total addition of acetonitrile and acrylonitrile (AC / AN) at 500 ppm during a 48 h run. Biomass and enzyme activity were uncoupled when the inoculum mass was increased from 4 g (wet weight) to ¡Ý 19 g. Other factors that allowed for the uncoupling of biomass production from enzyme activity were the reduction of the AC / AN feed rate from a step-addition at 2500 ¦Ìl / min to a continuous addition at 80 ¨C 120 ¦Ìl / min, and the delay to 18 h post-inoculation the time of initial inducer addition. The inhibition of both biomass production and NHase activity was relieved when both the total concentration of AC / AN was reduced to ¡Ü 350 ppm and the AC / AN feedrate was reduced. The factors with the greatest influence were shown to be the inducer, the inducer concentration, inoculum mass and source as well as the major carbohydrate and nitrogen source. In addition, this lab is the first to report high AN-specific NHase induction by asparagine (1300 ppm) in a fed-batch fermentation system. Prior to this program, 250 mg of cells (wet weight) per liter could be provided in 4 ¨C 10 days with an activity of 1 U NHase per mg of cells (dry weight). Current production is > 50 g / L in 48 h with an NHase activity > 150 U / mg of dry cell weight. INDEX WORDS: Amidase, Asparagine, Biodetoxification, Fermentation, Nitrile, Nitrile Hydratase, Rhodococcus

The objective of this study was to determine the effects of method of barley grain processing (dry-rolled vs. pelleted barley) and source of oilseed (ground canola vs. ground flaxseed), arranged as a 2 x 2 factorial, on feed intake, ruminal fermentation, nutrient flow to the duodenum, and milk production and composition in dairy cows. Eight Holstein cows (655 ± 69 kg; 83 ± 16 DIM) were used in a replicated 4 x 4 Latin square with 28-d periods. Cows in one square were fitted with ruminal and duodenal cannulae. Cows fed dry-rolled barley consumed 1.8 to 3.5 kg/d more (P = 0.02) DM than those fed pelleted barley; however, source of supplemental dietary fat had no effect on DM intake. Ruminal pH was lower (P = 0.045) in cows fed pelleted barley compared to those fed dry-rolled barley. Ruminal concentration of acetate was greater (P = 0.001), whereas ruminal concentration of propionate tended to be lower (P = 0.11), in cows fed dry-rolled barley compared to those fed pelleted barley; consequently, the acetate:propionate ratio was higher (P = 0.01) in cows fed dry-rolled barley compared to those fed pelleted barley. Ruminal concentration of total VFA was unaffected (P > 0.05) by diet. Source of dietary fat had no effect on ruminal digestion of OM, NDF, ADF or starch; however, ruminal starch digestion was slightly higher in cows fed pelleted barley compared to those fed dry-rolled barley (90.8 vs. 89.5%). Total dietary fatty acid intake was higher (P < 0.05) in cows consuming dry-rolled barley compared to those fed pelleted barley. Duodenal flow of C18:0 was lower, whereas that of C18:2n6c was higher (P < 0.05) in cows fed pelleted barley compared to those fed dry-rolled barley. Feeding flaxseed increased duodenal flows of C18:3n3, cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid compared to feeding canola. Milk yield was unaffected (P > 0.05) by diet; however, milk fat content was higher (P = 0.004) in cows fed dry-rolled barley compared to those fed pelleted barley. Milk fat content of C18:3 was higher (P = 0.005) in cows fed canola compared to those fed flax. Milk fat content of C18:3 and cis-9, trans-11 C18:2 were higher in cows fed pelleted barley compared to those fed dry-rolled barley with flax as the source of oilseed, but not with canola (interaction, P < 0.01). Milk fat content of saturated fatty acids decreased (P < 0.001) and that of polyunsaturated fatty acids increased (P = 0.003) in cows fed pelleted barley compared to those fed dry-rolled barley. In summary, milk fatty acid profiles were altered by method of grain processing and source of oilseed.

Due to its availability, low-price, and higher degree of reduction than lignocellulosic sugars, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. However, this high degree of reduction of carbon atoms in glycerol also results in significant challenges in regard to its utilization under fermentative conditions. Therefore, in order to unlock the full potential of microorganisms for the fermentative conversion of glycerol into fuels and chemicals, a detailed understanding of the anaerobic fermentation of glycerol is required. The work presented here highlights a comprehensive experimental investigation into fermentative glycerol metabolism in Escherichia coli, which has elucidated several key pathways and mechanisms. The activity of both the fermentative and respiratory glycerol dissimilation pathways was found to be important for maximum glycerol utilization, a consequence of the metabolic cycle and downstream effects created by the essential involvement of PEP-dependent dihydroxyacetone kinase (DHAK) in the fermentative glycerol dissimilation pathway. The decoupling of this cycle is of central importance during fermentative glycerol metabolism, and while multiple decoupling mechanisms were identified, their relative inefficiencies dictated not only their level of involvement, but also implicated the activity of other pathways/enzymes, including fumarate reductase and pyruvate kinase. The central role of the PEP-dependent DHAK, an enzyme whose transcription was found to be regulated by the cyclic adenosine monophosphate (cAMP) receptor protein (CRP)-cAMP complex, was also tied to the importance of multiple fructose 1,6-bisphosphotases (FBPases) encoded by fbp, glpX, and yggF. The activity of these FBPases, and as a result the levels of fructose 1,6-bisphosphate, a key regulatory compound, appear to also play a role in the involvement of several other enzymes during fermentative glycerol metabolism including PEP carboxykinase. Using this improved understanding of fermentative glycerol metabolism as a platform, E. coli has been engineered to produce high yields and titers of ethanol (19.8 g/L, 0.46 g/g), co-produced along with hydrogen, and 1,2-propanediol (5.6 g/L, 0.21 g/g) from glycerol, demonstrating its potential as a carbon source for the production of fuels and reduced chemicals.

The objective of this study was to determine the effects of method of barley grain processing (dry-rolled vs. pelleted barley) and source of oilseed (ground canola vs. ground flaxseed), arranged as a 2 x 2 factorial, on feed intake, ruminal fermentation, nutrient flow to the duodenum, and milk production and composition in dairy cows. Eight Holstein cows (655 ± 69 kg; 83 ± 16 DIM) were used in a replicated 4 x 4 Latin square with 28-d periods. Cows in one square were fitted with ruminal and duodenal cannulae. Cows fed dry-rolled barley consumed 1.8 to 3.5 kg/d more (P = 0.02) DM than those fed pelleted barley; however, source of supplemental dietary fat had no effect on DM intake. Ruminal pH was lower (P = 0.045) in cows fed pelleted barley compared to those fed dry-rolled barley. Ruminal concentration of acetate was greater (P = 0.001), whereas ruminal concentration of propionate tended to be lower (P = 0.11), in cows fed dry-rolled barley compared to those fed pelleted barley; consequently, the acetate:propionate ratio was higher (P = 0.01) in cows fed dry-rolled barley compared to those fed pelleted barley. Ruminal concentration of total VFA was unaffected (P > 0.05) by diet. Source of dietary fat had no effect on ruminal digestion of OM, NDF, ADF or starch; however, ruminal starch digestion was slightly higher in cows fed pelleted barley compared to those fed dry-rolled barley (90.8 vs. 89.5%). Total dietary fatty acid intake was higher (P < 0.05) in cows consuming dry-rolled barley compared to those fed pelleted barley. Duodenal flow of C18:0 was lower, whereas that of C18:2n6c was higher (P < 0.05) in cows fed pelleted barley compared to those fed dry-rolled barley. Feeding flaxseed increased duodenal flows of C18:3n3, cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid compared to feeding canola. Milk yield was unaffected (P > 0.05) by diet; however, milk fat content was higher (P = 0.004) in cows fed dry-rolled barley compared to those fed pelleted barley. Milk fat content of C18:3 was higher (P = 0.005) in cows fed canola compared to those fed flax. Milk fat content of C18:3 and cis-9, trans-11 C18:2 were higher in cows fed pelleted barley compared to those fed dry-rolled barley with flax as the source of oilseed, but not with canola (interaction, P < 0.01). Milk fat content of saturated fatty acids decreased (P < 0.001) and that of polyunsaturated fatty acids increased (P = 0.003) in cows fed pelleted barley compared to those fed dry-rolled barley. In summary, milk fatty acid profiles were altered by method of grain processing and source of oilseed.