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Rapid depletion of fossil fuel stock with a simultaneous rise in greenhouse gas emissions has led to an increase in the need for alternative energy. Cellulose based biofuels, especially bioethanol is a form of alternative energy that has the potential to replace petrol. The first step in cellulosic bioethanol production is the release of fermentable sugars via pre-treatment. Conventionally, physico-chemical and biological pre-treatment methods are energy intensive, environmentally unfavourable and expensive. This study, however reports on the use of a less energy consuming, cheap and environmental friendly alternative; photocatalysis, to produce fermentable sugars from cellulose. To achieve this, a range of photocatalysts were first screened based on their OH radical production rates using coumarin as a probe. TiO2 P25 was the photocatalyst that was found to have the highest OH radical production rate of 35.6 μM/hr, followed by Pt-C3N4 (0.88 μM/hr) and WO3 (0.28 μM/hr). LaCr-SrTiO3, Cr-SrTiO3 and yellow TiO2 did not produce any OH radicals due to their unsuitable electronic structure. P25 was further used for photocatalytic fermentable sugar production from cellulose. Photocatalytic cellulose I breakdown produced 0.04 % fermentable sugars whereas, with cellulose II feedstock the yield increased to 0.2 %. To further improve the yield, membrane bags were deployed which improved the sugar yields to 0.43 % and 0.71 % respectively from cellulose and cellulose II feedstocks. Photonic efficiencies followed the same trends as the sugar yields. Engineering design was further opted to enhance the sugar yields and hence a stacked frame photocatalytic reactor (SFPR) was designed. Various mixer configurations were designed and their mixing regime was determined using COMSOL Multiphysics 5.1 simulations. Amongst the mixers simulated, an 8-blade Rushton impeller was found to be the best configuration due its superior radial mixing profile and higher fluid velocity. The SFPR was then fabricated and operated with the impeller or a plus shaped magnetic bar as the mixer and the sugar yields were determined. Highest sugar yield and photonic efficiency was obtained from the cellulose II-impeller setup and was calculated to be 2.61 % and 9.45 % respectively. Respective lowest yields were obtained with cellulose I-stirrer bar setup and calculated to be 1.71 % and 5.64 %. Furthermore, the effect of H2O2 on fermentable sugar production was also tested. The cellulose II-stirrer bar configuration yielded 3.15 % fermentable sugars with the addition of 0.01 wt% H2O2 to the reaction mixture. The yield improved significantly to 14.1 % when the concentration of H2O2 was increased to 0.1 wt%.

Doctor of Philosophy / Department of Biological & Agricultural Engineering / Donghai Wang / Sweet sorghum accumulates high concentrations of fermentable sugars in the stem, produces significant amount of starch in the grain (panicle) and has shown to be a promising energy feedstock. Sweet sorghum has a short growing season so adding it to the sugar cane system would be good. The overall goal of this dissertation is to enhance the attractiveness of biofuel production from sweet sorghum to fully utilize fermentable sugars in the juice, starch in the panicle and structural carbohydrates in the stalk for high efficiency and low-cost ethanol production. Sweet sorghum juice was incorporated into the dry-grind process which increased ethanol yield by 28% increase of ethanol yield compared to the conventional ethanol method and decreased enzymatic hydrolysis time by 30 minutes. A very high gravity fermentation technique was applied using sweet sorghum juice and sorghum grain yielded 20.25% (v/v) of ethanol and 96% fermentation efficiency. Response surface methodology was applied in order to optimize diffusion conditions and to explore effects of diffusion time, diffusion temperature, and ratio of sweet sorghum biomass to grain on starch-to-sugar efficiency and total sugar recovery from sweet sorghum. Starch hydrolysis efficiency and sugar recovery efficiency of 96 and 98.5% were achieved, respectively, at an optimized diffusion condition of 115 minutes, 95 °C, and 22% grain loading. Extraction kinetics based on the optimized diffusion parameters were developed to describe the mass transfer of sugars in sweet sorghum biomass during the diffusion process. Ethanol obtained from fermented extracted sugars treated with granular starch hydrolyzing enzyme and those with traditional enzymes were comparable (14.5 – 14.6% v/v). Ethanol efficiencies also ranged from 88.92 –92.02%.

The utilization of lignocellulosic residues to produce renewable energy is an interesting alternative to meet the increasing demand of fuels while at the same time reducing greenhouse gas emissions and climate change. Sweet sorghum bagasse is a lignocellulosic residue composed mainly of cellulose, hemicellulose, and lignin; and it is a promising substrate for ethanol production because its complex carbohydrates may be hydrolyzed and converted into simple sugars, and then fermented into ethanol. However, the utilization of lignocellulosic residues is difficult and inefficient. Lignocellulose is a very stable and compact complex structure, which is linked by β-1,4 and β-1,3 glycosidic bonds. Furthermore, the crystalline and amorphous features of cellulose fibers and the presence of hemicellulose and lignin make the conversion of lignocellulose into fermentable sugars currently impractical at commercial scale. The bioconversion of lignocellulose in nature is performed by microorganisms such as fungi and bacteria, which produce enzymes that are able to degrade lignocellulose. The present study evaluated the bioconversion of lignocellulosic residues of sweet sorghum into simple sugars using filamentous fungi directly in the hydrolysis of the substrate, without prior isolation of the enzymes. The fungus Neurospora crassa and some wild fungi (that grew naturally on sweet sorghum bagasse) were used in this investigation. The effect of the fungi on substrate degradation and the sugars released after hydrolysis were evaluated, and then compared with standard hydrolysis performed by commercial enzymes (isolated cellulases). In addition, different combinations of fungi and enzymes were used to determine the best approach. The main goal was to verify if the fungi were able to attack and break down the lignocellulose structure directly and at a reasonable rate, rather than by the current method utilizing isolated enzymes. The main finding of this study was that the fungi (N. crassa and wild fungi) were able to degrade sweet sorghum bagasse directly; however, in all of the cases, the hydrolysis process was not efficient because the hydrolysis rate was much lower than the enzymatic hydrolysis rate. Hydrolysis using a combination of fungus and commercial enzymes was a good approach, but still not efficient enough for practical use. The best results of combined hydrolysis were obtained when the substrate was under the fungus attack for three days and then, commercial enzymes with low enzymatic activity (7 FPU/g and 25 CBU/g) were added to the solution. These enzymes represent 10% of the current enzymatic activity recommended per gram of substrate. This process reached reasonable levels of sugars (close to 85% of sugars yield obtained by enzymatic hydrolysis); however, the conversion rate was still slower, making the process impractical and more expensive since it took twice the amount of time as commercial enzymes. Furthermore, the wild fungi able to degrade cellulose were isolated, screened, and identified. Two of them belong to the genus Aspergillus, one to the genus Acremonium, and one to the genus Rhizopus. Small concentration of spores-0.5mL- (see Table 4, CHAPTER III- for specific number of spores per mL) did not show any sugar released during hydrolysis of sweet sorghum bagasse. However, when the concentration of spores was increased (to 5mL and 10mL of solution), citric acid production was detected. This finding indicates that those wild fungi were able to degrade lignocellulose, even though no simple sugars were measured, citric acid production is an indicator of fungi growing and utilization of lignocellulose as nutrient. It is assumed that the fungi consume the sugars at the same time they are released, thus they are not detected. The maximum concentration of citric acid (~14.50 mg/mL) was achieved between days 8-11 of hydrolysis. On the other hand, before using lignocellulose, the substrate needed to be pretreated in order to facilitate its decomposition and subsequent hydrolysis. Sweet sorghum bagasse was washed three times to remove any soluble sugars remaining after the juice was extracted from the stalks. Then, another finding of this study was that the first wash solution could be used for ethanol production since the amount of sugars present in it was close to 13°Brix. The ethanol yield after 48 hours of fermentation was in average 6.82mg/mL, which is close to the theoretical ethanol yield. The other two washes were too dilute for commercial ethanol production. In terms of pretreatments, the best one to break down sweet sorghum bagasse was 2% (w/v) NaOH. This pretreatment shows the highest amounts of glucose and xylose released after hydrolysis. Unwashed and untreated bagasse (raw bagasse) did not show any sugar released. In terms of ethanol, 74.50% of the theoretical yield was reached by enzymatic hydrolysis, while 1.10% was reached by hydrolysis using the fungus N. crassa. Finally, it is important to remark that further investigation is needed to improve the direct conversion of lignocellulose into fermentable sugars by fungal enzymes. This approach is a promising technology that needs to be developed and improved to make it efficient and feasible at commercial scale.

With the State of Alagoas as the third largest citrus producer in the Northeast region of Brazil, specifically cultivating Laranja Lima, being of fundamental importance the study of the hydrolyzate of Laranja Lima albedo for the planning of bioethanol production. The characterization of the Lima Orange albedo from the Cooplal juice industry of the municipality of Santana do Mundaú was carried out at the Bioflex 01 Industrial Laboratory of GRANBIO. For the hydrolysis process of the biomass were used the acids: hydrochloric, nitric, phosphoric and sulfuric with concentrations of 0.5 and 1.0% and periods of 30, 60, 90 and 120 minutes. The experimental design was completely randomized in the 4 x 2 x 4 factorial scheme, with three replications. Comparisons of the acid type averages within the concentrations and within the evaluation periods were done through the Tukey test at 5% probability. The hydrolysis with sulfuric acid obtained the best results for all variables studied. It is concluded that the hydrolysis process makes fermentable sugars of cellulose and hemicellulose available for the fermentation process. / Tendo o Estado de Alagoas como o terceiro maior produtor de citrus da região Nordeste do Brasil, cultivando especificamente Laranja Lima, sendo de fundamental importância o estudo do hidrolisado do albedo de Laranja Lima para o planejamento da produção de bioetanol. A caracterização do albedo da Laranja Lima da indústria de suco da Cooplal do município de Santana do Mundaú, foi realizado no Laboratório Industrial Bioflex 01 da GRANBIO. Para o processo de hidrólise da biomassa foram utilizados os ácidos: clorídrico, nítrico, fosfórico e sulfúrico com concentrações de 0,5 e 1,0% e períodos de 30, 60, 90 e 120 minutos. O delineamento experimental utilizado foi o inteiramente casualizado no esquema fatorial de 4 x 2 x 4, com três repetições. As comparações das médias de tipos de ácidos dentro das concentrações e dentro dos períodos de avaliação foram feitas através pelo teste de Tukey a 5% de probabilidade. A hidrólise com o ácido sulfúrico obteve os melhores resultados para todas variáveis estudados. Conclui-se que o processo de hidrólise torna os açúcares fermentescíveis da celulose e hemicelulose disponíveis para o processo fermentativo.

This research was conducted in order to enable primary producers to maximize the use of cheap roughage sources while still maintaining body weight during dry winter months when the crude protein (CP) content of roughage sources are at a minimum. The data obtained from this study will give an economic advantage when formulating supplements to be used during this time of the year. The aim of this study was to determine the optimum level of non-protein-nitrogen (NPN) and fermentable metabolizable energy (FME) to increase microbial protein synthesis, optimize rumen fermentation and increase digestibility of dry matter (DM) and neutral detergent fibre (NDF) in sheep fed on poor quality forages. A metabolic trial was conducted where intake of DM, organic matter (OM), NDF and CP was recorded; rumen volatile fatty acid (VFA) production was recorded as well as rumen pH over the different treatments. Microbial protein synthesis was determined by analysing purine derivatives in the urine. An in situ trial was also done to determine changes in ruminal digestibility of DM and NDF on different treatments. Five treatments were used. Treatment 1 consisted of NPN and FME balanced according to the NRC (2007) requirements for a 50kg whether, and served as a control. Treatment. Treatment 2 consisted 15% less NPN than control but the same amount of FME than control while treatment 3 consisted 15% more NPN than the control but the same amount of FME as the control treatment. Treatment 4 consisted of 15% less FME, but the same amount of NPN, than the control treatment, while treatment 5 consisted of 15% more FME, but the same amount of NPN than the control treatment. A 5 x 5 Latin square design was used in this study. Five Merino wethers were allowed to adapt to supplements which were infused directly into the rumen at 9:00 and 15:30 every day. After adaptation animals were placed in individual metabolic crates for three and given three day to adapt to crate environment. After the initial three days the sampling period commenced. Results obtained indicated that treatment had no effect on DM, OM, NDF and water intake but intake of CP was significantly increased for treatment 3 when compared to treatment 2. When intake of DM, OM, NDF and CP, related to metabolic bodyweight (W0.75) was calculated, treatment 5 resulted in lower intake of both water and NDF as compared to treatment 4. Differences between levels of FME and NPN in this study was insufficient to have an influence on DMD, OMD or NDFD however, CP degradability was increased for treatment 3 and treatment 5. Ruminal pH was unaffected by treatment. Increased levels of NH3-N for treatment 3 when compared to treatment 1 and 2, was observed. Both treatments 2 and 5 resulted significant decreases in rumen NH3-N. Treatments had no effect on the proportions of VFA produced or on the Acetate to Propionate produced ratio. Treatment 3 caused an improvement in CP an N balance when compared to treatment 1 and 2. Treatment 3, when compared to treatment 1 and 2, lead to an increase in N balance/kgW0.75. Treatment 5 caused a higher microbial protein synthesis in contrast to treatment 4. Results from the in situ trial showed a decreased a-value (solubility) for the NDF fraction of treatment 3 when compared to treatment 2. The rate of degradability (c) of both DM and NDF was increased for treatment 2 compared with treatment 3. The b, ED and PD values showed no response to treatment. / Dissertation (MSc Agric)--University of Pretoria, 2013. / gm2014 / Animal and Wildlife Sciences / unrestricted

In this paper, replacing complete or partly of growth mediums with secondarily wastewater was studied. Lipid content of Neochloris oleoabundans grown in a 0.3 X SE medium and autoclaved secondarily treated wastewater mixture was 22.27 % (w/w). The maximum biomass concentration of N. oleoabundans grown in wastewater with no additional nutrients was 0.636 g/L with 33% (w/w) glucose. Two culture lines, MA, and NA were isolated within our laboratory and could grow in secondarily treated wastewater with no additional nutrients. The maximum biomass concentration of MA in batch culture was 0.860 g/L and the sum of glucose and xylose was 40% (w/w). The maximum biomass concentration of NA was 1.562 g/l and the sum of glucose and xylose was 33.8% (w/w). The maximum specific growth rates of NA and MA were determined to be 0.0566 and 0.0337 per hour.

La santé digestive du lapin est difficile à maîtriser en élevage, notamment en période de sevrage où des troubles digestifs sont fréquents et peuvent entraîner des pertes importantes. Bien que l’utilisation d’antibiotiques diminue, afin d’éviter l’apparition de problèmes de résistance, des solutions alternatives doivent être trouvées. Nos principaux objectifs étaient : i) d’étudier l’influence de l’incorporation de fibres rapidement fermtentescibles (FRF) dans un aliment distribué précocement dès 15 jours d’âge, en comparaison d’un aliment avec antibiotiques (tiamuline et apramycine) utilisés pour lutter contre l’entéropathie épizootique du lapin ; ii) de déployer des méthodologies en génomique ciblant l’espèce lapin (amélioration et réannotation d’un microarray) et son microbiote digestif (pipeline d’analyse du gène de l’ARNr 16S), pour du phénotypage moléculaire. L’originalité de notre travail réside ainsi dans l’acquisition de données par trois approches complémentaires : phénotypique, transcriptomique, et métagénomique. Au niveau phénotypique, la stimulation de l’activité fermentaire par les FRF est vérifiée avec une hausse de la concentration caecale en AGV (+20%) et une plus forte acidité du biotope caecal. De plus, les FRF tendent à réduire la mortalité en post-sevrage et améliorent l’efficacité alimentaire. Au niveau transcriptomique, nous avons tout d’abord apporté de nouvelles connaissances sur l’immunité du lapin adulte avec la stimulation in vitro de cellules mononuclées du sang périphérique par du LPS et de la PMA-ionomycine. Cette étude confirme l’importance du lapin comme animal modèle pour la recherche biomédicale. Chez le jeune lapin, la nouvelle version du microarray nous a permis de montrer que les fonctions biologiques sont plus rapidement mises en place avec les FRF qu’avec un aliment standard, et que les antibiotiques nivellent l’expression génique. De plus, les FRF contribueraient à limiter l’inflammation intestinale. Au niveau métagénomique et en ciblant l’ARNr 16S, nous montrons qu’une augmentation de l’activité microbienne caecale n’est pas associée à une modification majeure de la composition du microbiote, à l’exception de la stabilisation de l’abondance des Campylobacteraceae, qui limiterait l’inflammation digestive. Nos travaux, encore préliminaires, n’ont pas permis d’identifier des corrélations significatives entre l’expression génique dans le sang et/ou l’iléon, et les profils taxonomiques (OTUs) majoritaires du microbiote digestif. L’ensemble de nos résultats suggère d’une part que les FRF peuvent être proposées comme une nouvelle stratégie nutritionnelle péri-sevrage, avec une amélioration de l’efficacité alimentaire et une protection accrue de la santé digestive, et d’autre part que des approches génomiques sont prometteuses pour qualifier finement les phénotypes d’intérêt chez le lapin. / Digestive health of rabbits is difficult to manage in breeding systems, especially around weaning where digestive problems are very common and can result in significant losses. The use of antibiotics decreases in order to avoid the emergence of problems with antibiotic resistance, but alternatives must be found. The objectives of this work were: i) to study effects of the incorporation of rapidly fermentable fiber (FRF) in diets fed to rabbits from 15 days of age, and compared to a feed with antibiotics (tiamulin and apramycin), used to fight against epizootic rabbit enteropathy; ii) to deploy genomics methodologies targeting the rabbit species (improvement and re-annotation of a microarray) and the digestive microbiota (pipeline analysis of 16S rRNA gene), for molecular phenotyping. An innovative approach was used in this work through the combination of three complementary approaches: phenotypic, transcriptomic, and metagenomic. At the phenotypic level, the stimulation of the fermentation activity by FRF is verified with higher cecal VFA concentrations (+ 20%) and higher cecal acidity. Moreover, the FRF tend to reduce mortality post-weaning and improve feed efficiency. At the transcriptomic level, we obtained new data on the immunity of the adult rabbit with in vitro stimulation of peripheral blood mononuclear cells with LPS and PMA-ionomycin. This study confirms the importance of the rabbit as an animal model for biomedical research. In young rabbits, the new version of the microarray allowed us to show that biological functions are more quickly implemented with FRF than a standard diet, and that antibiotics level out the gene expression. In addition, FRF help to limit intestinal inflammation. At the metagenomic level, the targeting of the 16S rRNA, we showed that an increase in cecal microbiota activity is not associated with major changes of microbiota composition, with the exception of the stabilization of the relative abundance of Campylobacteraceae, which limits gastrointestinal inflammation. Our preliminary results did not identify significant correlations between gene expression in blood and/or ileum, and principal taxonomic profiles of the digestive microbiota. Our results suggest that FRF can be proposed as a new nutrition strategy peri-weaning, with improved feed efficiency and increased protection of digestive health, and secondly that genomic approaches are promising to describe more precisely phenotypes of interest in rabbits.

Epigenetic regulation of gene expression occurs at a number of levels including covalent DNA and histone modifications, nucleosome positioning and ATP-dependent chromatin remodelling as well as higher order chromatin folding and 3D genome organisation. ATP-dependent chromatin remodelling complexes modulate nucleosome structure, positioning and chromatin de-compaction and are involved in gene activation and repression. SWI/SNF ATP-dependent chromatin remodelling complexes contain either BRG1 or BRM as the core ATPase together with other common and variable subunits. BRG1 is required for terminal epidermal differentiation in mice and humans and for hair follicle stem cell activation during mouse hair follicle regeneration and cutaneous wound healing. However, the role of SWI/SNF complexes in human hair growth and wound healing remain unknown. Here it is demonstrated that genes encoding SWI/SNF complex subunits are expressed in human hair follicles. It also highlights that siRNA mediated suppression of SWI/SNF complexes in hair follicle culture has no effect on hair growth, or anagen-catagen transition in the short term, but a significant increase in proliferation of the outer root sheath keratinocytes was seen. The data also documents the expression of several SWI/SNF subunits in human epidermis and that siRNA mediated SMARCA4 gene suppression in primary human keratinocyte monolayers defined the requirements of BRG1 for wound closure through control of cell migration, but not proliferation. In summary, this data revealed a diverse SWI/SNF complex subunit composition in human epidermis and hair follicle, and an essential role of the core complex ATPase BRG1 in keratinocyte migration during wound closure and re-epithelisation.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.