Desulfurococcus fermentans is a hyperthermophilic archaeon growing optimally at 82??C. This microorganism is an obligate anaerobe with optimal growth pH of 6.0. It is capable of producing H2 as an end metabolic product using cellulose as growth substrate. The major goal of this study was to optimize the growth conditions for the production of H2 from various substrates such as cellulose, cellobiose, carboxymethyl cellulose (CMC), xylan, filter paper, avecil, starch and peptides. The highest cell density (2.83??108 cells/ml) was observed when yeast extract (0.2 g/L), starch (5 g/L) and xylan (4 g/L) were added to its growth media. The lowest generation time was shown to be 2.4 hours when yeast extract (0.2 g/L), starch (5 g/L) and cellobiose (4 g/L) were added to its growth medium. It was found that the growth of D. fermentans was obligately depended on the presence of yeast extract in the growth medium, and the H2 production was positively correlated to its growth. Cells of D. fermentans were cocci with diameters varying from 1 to 3 ??m. The largest cell size was observed using scanning electron microscopy when it grew in medium containing yeast extract (10 g/L) and starch (5 g/L). Maximum hydrogen production of 12% (v/v) was achieved when yeast extract (0.2 g/L), starch (5 g/L) and carboxymethyl cellulose (4 g/L) were added to the growth medium. Further studies are required to obtain the specific yield of H2 from various substrates through the quantification of both the consumption of substrates and the production of H2 by D. fermentans.

Faculty of Science School of Molecular and Cell Biology 9904041r lthompson@csir.co.za / The production of H2 was monitored using an automated, semi-continuously fed anaerobic fluidised bed bioreactor containing 2 facultatively anaerobic bacteria, Enterobacter cloacae (E. cloacae Ecl) and Citrobacter freundii (C. freundii Cf1). Shake flask tests using Endo formulation with modified C:N:P ratios, showed that a 334:28:5.6 ratio gave the highest attached counts of E. cloacae Ecl and C. freundii Cf1 in both single and binary species biofilms grown on granular activated carbon. Once the reactor had achieved steady state after 30 days using the modified C:N:P ratio, pH, redox potential, temperature, volatile fatty acids and the H2 production rate were monitored. The H2 production rate of 95 mmol H2 / (l x h) compared favourably with previous studies. Bacterial biofilms counts for both E. cloacae Ecl and C. freundii Cf1 remained high around 9.0 log cfu/g granular activated carbon, although biomass overgrowth could not be controlled in the reactor. The efficiency of converting sucrose into H2 was calculated at 20.5%. Therefore use of this technology to power a 5.0kW proton exchange fuel cell for a single rural household is currently not feasible due to the high organic load required. Pooling of wastewater generation capacity, improvement of bacterial strain selection and feed formulation, pH control, gas removal and purification are factors that need to be considered for future improvement of conversion efficiencies. Use of this technology would be most suited for industrial processes generating large volumes of wastewater high in carbohydrates. Alternatively, municipal wastewater treatment facilities could be converted into electricity generating facilities through the combination of this technology and proton exchange membrane fuel cells.

Ph.D., Faculty of Science, University of the Wiwatersrand, 2011 / Biological production of hydrogen gas has received increasing interest from the international community during the last decade. Most studies on biological fermentative hydrogen production from carbohydrates using mixed cultures have been conducted in conventional continuous stirred tank reactors (CSTR) under mesophilic conditions. Investigations on hydrogen production in reactor systems with attached or self-immobilized microbial growth have also appeared recently in the literature. These investigations on attached or self-immobilised bacteria involve hydrogen production in the mesophilic and thermophilic temperature range. The present study investigated the design and operational features of anaerobic fluidized granular bed bioreactor (AFGB) system which would facilitate the simultaneous achievement of high productivities (HPs) and high hydrogen yields (HYs).Where high HPs is greater than 120 mmol H2 /(L.h) and HYs greater than 4 mol H2/mol glucose. Theoretical maximum yield for an exponentially growing non-granulated bacterial monoculture will always be less than the thermodynamic maximum of 4 mol H2 /mol glucose: C6H12O6 +4H2O → 2CH3COO- + 4H2 + 4H+ + 2HCO3. The design features included reducing the total non-working or dead volume of bioreactor system. The operational improvements included application of thermophilic temperatures and high rates of de-gassed effluent recycling through the fluidized granular bed. An example of an optimal ratio of effluent recycle rate (R) to bioreactor working volume (V) was (3.0 L/min)/(3.2 L/min) = 0.94 minutes. Under conditions where temperatures were maximised and V/R were minimized the HPs increased to 21.58 L H2 /h. Also under these conditions the HYs increased above 3.0 mol H2/mol glucose. Specific hydrogen productivity for the fluidized granular bed increased from 0.25 L H2 / (g BM.h) or 8.83 mmol H2 / (g BM.h) at 45 oC to 0.525 L H2 / (g BM.h) or 18.03 mmol H2 / ( g BM.h) at 70 oC. A 3.64 fold increase in hydrogen yield occurred with an increase in temperature from 45 oC to 70 oC. XX When expressed in terms of glucose, this represents an increase from 1.34 mol H2 /mol glucose to 4.65 mol H2 /mol glucose. Finally, an evaluation of the net energy production by the AFGB system revealed a positive energy balance, making thermophilic biohydrogen production energetically viable from a commercial perspective.

The recent surge in biodiesel production has resulted in a huge surplus of crude glycerol, a by-product of the process to the level of 10% by weight. This is turn has caused the price of glycerol to fall dramatically, and there are now few economically viable channels for using this resource: waste glycerol is usually combusted. Therefore, much interest has arisen in the possibility of making use of glycerol with biotechnology, as this would not only be a more efficient use of resources but also make biodiesel itself more commercially viable. The purple bacterium Rhodopseudomonas palustris is able to metabolize glycerol through photofermentation and thereby produce hydrogen, a commercially useful commodity. R. palustris is of particular interest for this purpose as, in contrast to many other species which have been investigated with a view to fermenting glycerol, it is highly tolerant of crude glycerol. The feedstock requires little purification or dilution to be made suitable for cultivation of R. palustris. Furthermore, the hydrogen gas produced by R. palustris when grown on glycerol is of high purity, and the organism's great metabolic diversity suggests it may be a useful strain for remediation of other waste materials. However, much groundwork is needed to establish R. palustris as a viable chassis organism for biotechnological purposes. This work sets out to establish optimal conditions for cultivating R. palustris in the laboratory, including the design of a suitable batch photobioreactor system. It also determines optimal conditions for electroporation of R. palustris for the purpose of knocking out endogenous genes or introducing heterologous genes. Furthermore, the introduction of heterologous genes is attempted in order to demonstrate the possibility of producing other high-value compounds with R. palustris, and several deletion strains with potential benefits for hydrogen production are created. Finally, several existing deletion strains are investigated to establish their suitability as chassis strains for further genetic manipulation.

Dans la nature, les microorganismes s’organisent en communauté où la concertation de leur métabolisme leur permet de coloniser des endroits peu propices. La biodégradation de la matière organique nécessite un couplage métabolique entre les différents microorganismes impliqués et constitue un modèle de choix pour l'étude des interactions où leurs relations restent mal définies et nécessitent d’être mieux caractérisées. Le décryptage du mécanisme mis en jeu permettrait d'optimiser la production de composés bioénergétiques comme l'hydrogène. Nous avons étudié un consortium composé de Desulfovibris vulgaris Hildenborough, une bactérie sulfato-réductrice et de Clostridium acetobutylicum, une bactérie fermentaire. Ces deux bactéries sont retrouvées dans des consortia naturels impliqués dans la dégradation de la biomasse. Des approches de microbiologie, de métabolisme et de microscopie ont permis de démontrer l’existence d’une interaction physique et d'un échange de molécules cytoplasmiques entre les deux bactéries. Ceci s'associe à une réorientation du flux de carbone dans la bactérie C. acetobutylicum qui se traduit par une production d’hydrogène accrue. Ce comportement est lié aux conditions de stress nutritionnel pour la bactérie D. vulgaris. De plus, des molécules signal de type AI-2 jouent un rôle important dans la mise place de l'interaction physique. Un inhibiteur de cette interaction, produit par D. vulgaris dans certaines conditions, a été découvert. Ce travail a permis d'acquérir de nouvelles connaissances sur les relations métaboliques et les interactions physiques entre les bactéries impliquées dans la biodégradation de la biomasse dans un consortium. / In nature microorganisms live in communities, in which the complementarity of their metabolism allows them to colonize less favourable ecological niches. Biodegradation of organic matter requires tight metabolic coupling between the different microorganisms involved, and constitutes an ideal model for studying the interactions between them, which are still not well established and require further characterization. Furthermore, deciphering the metabolic couplings established between the partners would allow optimization of this process for production of compounds of biotechnological interest, such as hydrogen. During the course of this work we have studied an artificial consortium constituted by Desulfovibris vulgaris Hildenborough sulphate-reducing bacterium, and Clostridium acetobutylicum a fermentative bacterium; both of them are found in natural consortia involved in biomass degradation. Microbiological, metabolic and microscopic approaches allowed us to show the existence of a physical interaction, with exchange of cytoplasmic molecules, between the two bacteria. This is associated with reorientation of the carbon flux in Clostridium acetobutylicum, resulting in increased hydrogen production. This behaviour is linked with the nutritional stress of D. vulgaris. Moreover, AI-2 type signal molecules produced in these conditions are crucial for the physical interaction between the two bacterial partners. An inhibitor produced by D. vulgaris in certain conditions has been discovered. This work has allowed us to acquire new knowledge about metabolic relations and physical interactions between bacteria involved in biomass degradation in a consortium

No description available.

Production of hydrogen from biomass through the use of dark and photofermentative bacteria will be applicable in the future and a promising route. The aim of this study is to develop and to scale-up solar panel photobioreactors for the biological hydrogen production by photosynthetic purple non sulfur (PNS) bacteria on artificial substrates and on real dark fermentation effluent of molasses. The parameters studied are light intensity, temperature, feed stock, feed rate, pH, cell density, light and dark cycle and carbon to nitrogen ratio on hydrogen production. Continuous hydrogen production has been achieved on artificial medium and dark fermentor effluent of molasses containing acetate and lactate by Rhodobacter capsulatus wild type and (hup-) mutant strains in panel photobioreactors in indoor and outdoor conditions by fed batch operation. Laboratory (from 4 to 8 liters) and large scale (20 L) panel photobioreactors by using various designs and construction materials were developed. In this photobioreactors continuous hydrogen production was achieved by feeding. Na2CO3 can be used as buffer to keep the pH stable during long term operation on molasses dark fermentor effluent. The adjustment of the feedstock by dilution and buffer addition were found to be essential for the long term stability of pH, biomass and H2 production for both in indoor and outdoor applications.

By the integration of dark and photo fermentative hydrogen production processes, higher yields of hydrogen can be obtained from biomass. In the first step, biomass is utilized for hydrogen production by dark fermentation and in the second step, the effluent of dark fermentation is further utilized for hydrogen production by photofermentation using photosynthetic purple non-sulfur bacteria. The purpose of this study was to develop a solar pilot scale tubular photobioreactor (PBR) for continuous photo fermentative hydrogen production from the effluent of dark fermentation. This study demonstrated the implementation of the solar pilot tubular PBR for this new technology for the first time and successful continuous operations were performed in different seasons. Two different strains of Rhodobacter capsulatus were used for the operations. It was showed that even in winter, pure hydrogen could be produced in the pilot PBR with an average productivity of 0.3 mol H2/m3.h, when circulation of the PBR was continuous. Productivity obtained by the mutant strain was 0.2 mol H2/m3.h with periodical circulation. The integration between dark and photo fermentation was proven at pilot scales by using real dark fermenter effluents of molasses and thick juice. DFE of thick juice yielded a maximum productivity of 0.27 mol H2/m3.h whereas the maximum productivity obtained from DFE of molasses was 0.12 mol H2/m3.h. The most important factor affecting productivity is found to be the total received light energy and a yield factor (mmol H2/g dry cell weight) was correlated with total received light energy. Acetic acid consumption rates were found to be first order for daytime and zero order for nights. Furthermore acetic acid utilization for different metabolic pathways were estimated and by-product, poly- &beta / - hydroxybutyrate, specific rates of product formation were determined.

The effect of simple and complex on the composition and diversity of microbial communities and on end-product (biogas and VFAs) synthesis was investigated using an anaerobic batch respirometer at 37 °C and pH 7.2. These experiments, simple substrates were chemically pure and contain a single carbon source (glucose or α-cellulose), while complex substrates were chemically “impure” substrates containing a mixture of two or three carbon sources (biodiesel-derived glycerol or wheat straw) with a substrate/inoculum ratio 6g chemical oxygen demand (COD)/ g volatile solids (VS) seed and 100g of pre-treated dairy manure digestate (DMD), respectively. Concentrations of hydrogen, carbon dioxide, acetate, butyrate, propionate, and ethanol synthesized by different communities selected by growth on the different substrates were measured and confirmed the growth of the microbial communities. 16S rDNA illumina sequencing revealed that DMD without substrates was more diverse than the microbiota cultured by fermentation reactions containing D-glucose, glycerol α-cellulose or wheat straw. The data confirmed that substrates play a crucial role in determining the diversity of species in microbial communities. Dominant operational taxonomic units (OTUs) belonging to families Clostridiaceae, Ruminococcaceae, and Enterobacteriaceae, and the genera Clostridium, Ruminococcus, Sporolactobacillus, and Syntrophomonas were potentially responsible for changes in end-product synthesis patterns in communities cultured with simple and complex substrates. / October 2015

La fermentation sombre et les piles à combustible microbiennes (MFC) sont deux technologies émergentes respectivement pour la conversion biologique de l'énergie chimique des composés organiques en hydrogène (H2) et en électricité. En raison des avantages cinétiques et thermodynamiques, la température élevée peut être la clé pour augmenter à la fois la production d'H2 de fermentation sombre et la production d'électricité dans les MFC. Par conséquent, cette thèse se concentre sur la manière dont la température influence la production biologique de H2 et d'électricité à partir d'eaux usées contenant du carbone organique. Deux inocula traités thermiquement (à boues activées fraîches et digérées) ont été comparés pour la production de H2 à partir de xylose à 37, 55 et 70 °C. A la fois à 37 et 55 °C, on obtient un meilleur rendement en H2 par les boues activées fraîches comparé aux boues digérées tandis qu'un très faible rendement en H2 est obtenu par les deux inocula à 70 °C. Ensuite, quatre prétraitements d'inoculum différents (chocs acides, alcalins, thermiques et de congélation) ont été évalués pour créer une efficace communauté productrice de H2 mésophile (37 °C) ou thermophile (55 °C). Les chocs acides et alcalins ont sélectionné des micro-organismes producteurs de H2, appartenant aux Clostridiaceae, au détriment des bactéries produisant du lactate, ce qui a donné respectivement le rendement en H2 le plus élevé à 37 et 55 °C. Bien que le choc thermique ait abouti à un faible rendement en H2 dans un seul lot, il a été montré que la production de H2 par les boues activées fraîches traitées thermiquement augmentait dans l'expérience avec quatre cycles consécutifs. Des boues activées fraîches et traitées thermiquement ont été sélectionnées comme inoculum pour la production continue de H2 à partir d'une eau usée synthétique contenant du xylose dans un réacteur à lit fluidisé (FBR) mésophile (37 °C) et thermophile (55-70 °C, augmenté par étapes). Un rendement en H2 plus élevé a été obtenu dans le FBR thermophile que dans le FBR mésophile. En outre, la production de H2 à 70 °C, qui a échoué dans l'étude précédente, a été couronnée de succès dans le FBR, avec un rendement stable de 1.2 mol H2 mol-1 xylose. La température de fonctionnement de 70 °C s'est également révélée optimale pour la production de H2 à partir d'eaux usées thermomécaniques (TMP) dans un incubateur à gradient de température, car la culture en batch à 70 ° C. Une approche de l'ARN a été utilisée pour étudier la structure et le rôle des communautés microbiennes attachées à l'anode, attachées à la membrane et planctoniques dans un MFC mésophile (37 °C) et thermophile (55 °C) alimenté au xylose. Une communauté anodine dominée par Geobacteraceae a soutenu la production d'électricité à 37 °C, alors que l'établissement de micro-organismes méthanogènes et H2 oxydants a entraîné une faible production d'électricité à 55 °C. Cependant, le développement d'une communauté exoélectrogène thermophile peut être favorisé en appliquant une stratégie de démarrage qui comprend l'imposition d'un potentiel négatif à l'anode et l'inhibition chimique des méthanogènes. Une communauté exoélectrogénique mésophile a également été montré pour produire de l'électricité à partir d'eaux usées de TMP dans un MFC à flux ascendant exploité à 37 °C. En conclusion, une production de H2 plus élevé et plus stable peut être obtenu dans une fermentation sombre thermophile plutôt que mésophile. La fermentation sombre à 70 °C est particulièrement appropriée pour le traitement des eaux usées de TMP car elle est libérée à haute température (50-80 °C) et pourrait être traitée sur site. Les eaux usées de TMP peuvent également être utilisées comme substrat pour la production d'électricité dans les MFC mésophiles. La production d'électricité dans les MFC thermophiles est faisable, mais l'enrichissement des micro-organismes exoélectrogènes thermophiles peut nécessiter une longue période de démarrage / Dark fermentation and microbial fuel cells (MFCs) are two emerging technologies for biological conversion of the chemical energy of organic compounds into hydrogen (H2) and electricity, respectively. Due to kinetic and thermodynamic advantages, high temperature can be the key for increasing both dark fermentative H2 production and electricity production in MFCs. Therefore, this thesis focuses on delineating how temperature influences biological production of H2 and electricity from organic carbon-containing wastewaters. Two heat-treated inocula (fresh and digested activated sludge) were compared, for H2 production from xylose at 37, 55 and 70 °C. At both 37 and 55 °C, a higher H2 yield was achieved by the fresh than digested activated sludge, whereas a very low H2 yield was obtained by both inocula at 70 °C. Then, four different inoculum pretreatments (acidic, alkaline, heat and freezing shocks) were evaluated for creating an efficient mesophilic (37 °C) or thermophilic (55 °C) H2 producing community. Acidic and alkaline shocks selected known H2 producing microorganisms belonging to Clostridiaceae at the expenses of lactate producing bacteria, resulting in the highest H2 yield at 37 and 55 °C, respectively. Although a heat shock resulted in a low H2 yield in a single batch, H2 production by the heat-treated fresh activated sludge was shown to increase in the experiment with four consecutive batch cycles.Heat-treated fresh activated sludge was selected as inoculum for continuous H2 production from a xylose-containing synthetic wastewater in a mesophilic (37 °C) and a thermophilic (55-70 °C, increased stepwise) fluidized bed reactor (FBR). A higher H2 yield was obtained in the thermophilic than in the mesophilic FBR. Furthermore, H2 production at 70 °C, which failed in the earlier batch study, was successful in the FBR, with a stable yield of 1.2 mol H2 mol-1 xyloseadded. Operation temperature of 70 °C was also found optimal for H2 production from thermomechanical pulping (TMP) wastewater in a temperature gradient incubator assay.A RNA approach was used to study the structure and role of the anode-attached, membrane-attached and planktonic microbial communities in a mesophilic (37 °C) and a thermophilic (55 °C) two-chamber, xylose-fed MFC. An anode attached community dominated by Geobacteraceae sustained electricity production at 37 °C, whereas the establishment of methanogenic and H2 oxidizing microorganisms resulted in a low electricity production at 55 °C. However, the development of a thermophilic exoelectrogenic community can be promoted by applying a start-up strategy which includes imposing a negative potential to the anode and chemical inhibition of methanogens. A mesophilic exoelectrogenic community was also shown to produce electricity from TMP wastewater in an upflow MFC operated at 37 °C. In conclusion, a higher and more stable H2 yield can be achieved in thermophilic rather than mesophilic dark fermentation. Dark fermentation at 70 °C is particularly suitable for treatment of TMP wastewater as it is released at high temperature (50-80 °C) and could be treated on site. TMP wastewater can be also used as substrate for electricity production in mesophilic MFCs. Electricity production in thermophilic MFCs is feasible, but enrichment of thermophilic exoelectrogenic microorganisms may require a long start-up period