Thesis (M.S. in chemical engineering)--Washington State University, August 2005. / Includes bibliographical references.

Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.

A unique, previously undescribed Gram-negative bacterium was isolated from several soils in Texas and extensively characterized in this study. The cells measured 1-2 by 4-6 μm. The distinguishing characteristic of the bacterium is the extraordinary capsular material which surrounds the cells. The new isolates are aerobic, mesophilic, non motile and have the ability to utilize a variety of organic compounds as the sole source of carbon and energy. The organism grows optimally at 30° C and the optimal pH lies between 7.0-8.0. The isolates produce catalase but oxidase is not produced. They do not produce indole or hydrogen sulfide. The organism can hydrolyze gelatin and Tween 80 but not starch, esculin and casein. The major cellular fatty acid is anteiso 15:0. The guanine and cytosine content is 58-62 mole%. The organism's taxonomic position was further established by specific gene probes, 16S rRNA homology, DNA homology and "ribotyping." These data showed that it was most closely related to members of the genus Paenibacillus, although somewhat divergent from other species classified in this genus. After careful evaluation of the results obtained during this study, it is proposed that this unique bacterium be named Paenibacillus velasolus sp. nov.

A cellulose-decomposing bacterium isolated from bovine feces was purified, identified as a member of the genus, Cellvibrio, and the new species name gilvus was proposed. Cellulose decomposition was demonstrated and cellobiose was the only hydrolytic sugar product. Excellent growth was obtained on mineral salts medium containing cellobiose, a vitamin mixture, and organic nitrogen (casein hydrolyzate) which was required for growth. Volatile and non-volatile acids, volatile neutral compounds, and carbon dioxide constituted the fermentation products. A preference for cellobiose was shown by a 30 to 46% greater growth rate than resulted on glucose. An investigation was conducted to explain this disaccharide preference. Intact cells oxidized glucose and cellobiose immediately and the rate of glucose oxidation was 10% less than obtained from cellobiose. Thus, hypotheses that adaptive hexokinase, hexokinase deficiency or impermeability to glucose could explain the preference were dispelled. Oxygen assimilation ratios of equivalent amounts of glucose, cellobiose and a mixture of these were 100:110:140. Alternate metabolic pathways were indicated. Resting cells grown on cellobiose esterified inorganic phosphate in the presence of glucose or cellobiose showed that these sugars were metabolized to phosphate esters. Relative rates of utilization of both sugars revealed that 130% more cellobiose was used at pH 6.5 and 590% more at pH 7.0. On a molar basis 30% more acid was formed from glucose than from cellobiose. A phosphorylase was indicated by the stimulation of cellobiose respiration by inorganic phosphate. Disaccharide preference was associated with the intracellular enzymes because soluble enzymes utilized 7.25 uM of cellobiose but only 2.8 uM of glucose. The adenosine triphosphate requirement for glucose utilization indicated hexokinase activity. Inorganic phosphate increased cellobiose utilization two-fold and was accompanied by esterification. Soluble enzymes from glucose-grown cells produced a constitutive cellobiose enzyme. Phosphate depressed glucose utilization while adenosine triphosphate depressed cellobiose utilization. Fructose-6-phosphate was the only ester detected in cells grown on either sugar. Cellobiose-grown cells contained 6.5 mg total P/2 g cells whereas glucose-grown cells contained only 2.1 mg. Glucose was converted to glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-diphosphate by cell-free enzymes in the presence of adenosine triphosphate. Fructose-1,6-diphosphate was metabolized to pyruvic acid. This was evidence for an Embden-Meyerhof pathway. A cellobiose phosphorylase was demonstrated as a reversible reaction producing glucose and alpha-D-glucose-1-phosphate. Conversion of glucose-1-phosphate to glucose-6-phosphate was not obtained but fructose-6-phosphate was formed. Ground cells oxidized fructose-1,6-diphosphate and fructose-6-phosphate in the presence of diphosphopyridine nucleotide, coenzyme-A and methylene blue. These preparations in which oxidizing systems were saturated with fructose-1,6-diphosphate and cofactors oxidized glucose and cellobiose at greatly increased rates above that obtained from the ester. Gluconic acid was detected in the final reaction mixtures. An explanation of the disaccharide preference resides in several factors. The very active direct phosphorylation of cellobiose yielding a hexose phosphate at less expense energy-wise than the adenosine triphosphate-requiring phosphorylation of glucose showed cellobiose to be a more efficient energy-yielding substrate. The weak hexokinase would limit the formation of hexose phosphates which in turn could impede the energy obtained as high energy phosphate compounds necessary for growth and reproduction. A portion of available glucose was probably wasted in the direct oxidation to gluconic acid. Finally, a proposed scheme for the metabolic pathways of glucose and cellobiose in Cellvibrio gilvus was presented. / Ph. D.

Thesis submitted in fulfilment of the requirements for the degree Master of Technologae: Chemical Engineering in the Faculty of Engineering at the CAPE PENINSULA UNIVERSITY OF TECHNOLOGY 2014 / Most softdrink industries in developing countries are moving towards wastewater reuse or recycling. Water and wastewater reutilization, costs of treatment and disposal guidelines, remain the most critical factors for the development of sustainable water use for softdrink industries. Wastewater reuse or recycle has potential in the softdrink industry, depending on the wastewater characteristics’ concentration and volume. During this study, an integrated laboratory scale anaerobic/aerobic sidestream membrane bioreactor (MBR) system was used for treating softdrink industry wastewater (SDIW). The aim was to evaluate the system’s performance, and identify potential opportunities to recycle the water, and therefore reduce freshwater intake and minimise wastewater production. The objectives were to: evaluate: 1) treatment efficiencies for the individual stages; 2) biogas production in the anaerobic stage; and 3) the overall performance of the integrated system under different operating conditions. The SDIW used in this study was classified as medium to high strength wastewater with a total chemical oxygen demand (CODt) ranging between 2 242 and 11 717 mg/L and a biological oxygen demand (BOD) of up to 1 150 mg/L. The major pollutants in the SDIW were caustic soda; dissolved sugars, namely fructose (1 071 mg/L) and sucrose (6 900 mg/L); with the pH ranging between 6.1 and 12. The SDIW was characterized by total suspended solids (TSS) of 66 mg/L, as well as fats, oils and greases (FOG) of 40 mg/L. The maximum turbidity and colour was 65.3 NTU and 42 mg Pt/L, respectively. All the physiochemical properties and inorganic parameters were within the within the City of Cape Town’s (CCT’s) industrial wastewater quality discharge standards by-law (South Africa, 2006). Excluding the total dissolved solids (TDS) and electrical conductivity (EC) with maximum values were 1 050 mg/L and 1 483 μS/cm, respectively. Anaerobic pre-treatment of this SDIW was studied using a laboratory-scale expanded granular sludge bed (EGSB) reactor maintained at mesophilic temperature of between 35 to 37˚C. An organic loading rate (OLR), upflow velocity (Vup) and hydraulic retention time (HRT) of 10.9 kg COD/m3d, 0.85 m/h and ~11.8 h, respectively, resulting in COD treatment efficiencies of up to 93% CODt. An increase in nitrate (NO3-) in the EGSB product stream was an indication of an anaerobic ammonium (NH4+) oxidation (ANAMMOX) process. Anaerobic digestion (AD) of SDIW in the EGSB resulted in biogas production with methane (CH4), carbon dioxide (CO2), nitrogen (N2), and oxygen (O2), concentrations of up to 70%, 11%, 14.8%, and 4.1%, respectively. At the OLR and Vup of 10.9 kg COD/m3d and 0.85 m/h, respectively, the EGSB produced 16.7 L/d of biogas. The EGSB anaerobic pre-treatment resulted in stable treatment efficiencies for the removal of organic constituents, as well as biogas production without adding an external carbon source. The MBR post-treatment satisfactorily operated at a feed flowrate of up to 33.7 L/d, OLR of 2.3 and 3.1 kg COD/m3d for the anoxic and aerobic zones, respectively, and an HRT of approximately 0.41 h for both zones. The average CODt removal achieved was 86%. The dissolved oxygen (DO) concentration of 2.1 mg/L in the anoxic zone combined with an aeration rate and DO concentration of 11.8 L/min and 5.7 mg/L, in the aerobic zone resulted in NH4+; NO3-; and orthophosphate (PO43-), removal rates up to 90%; 55% and 39%, respectively. However, the MBR post-treatment did not decrease the orthophosphate concentration to within the SANS 241:2011 drinking water standards. The integrated EGSB-MBR treatment for SDIW was able to achieve an overall CODt removal efficiency of up to 94%. Although the MBR performance was successful the EC, TDS, PO43-, and colour concentrations in the ultrafiltration (UF) permeate did not meet the CCT’s industrial wastewater standards by-law (2006) as well as the SANS’ drinking water standards 241:2011 and required further treatment for reuse.

An investigation of the biodegradation of complex hydrocarbon wastes, with emphasis on chlorinated aromatic compounds, in an anaerobic/aerobic bioprocess environment was made. A reactor configuration was developed consisting of linked anaerobic and aerobic reactors which served as the model for a proposed bioremediation strategy targeting subterranean soil/sediment/aquifer chlorinated phenol-contaminated environments. Here oxygen is frequently limited and sulphate is readily available, as occurs especially in marine sediment and intertidal habitats. In the anaerobic system the successful transformation and mobilization of the model contaminant, 2,4,6-trichlorophenol, was shown to rely on reductive dechlorination by a sulphate-reducing dependent dechlororespiring co-culture. This was followed in the aerobic system by degradation of the pollutant and its metabolites, 2,4-dichlorophenol, 4-chlorophenol and phenol, by immobilized white-rot fungi.The strategy was initially investigated separately in laboratory bench- and intermediate scale reactors whereafter reactors were linked to simulate the integrated biodegradation strategy. The application of the fungal reactor to treat an actual waste stream by degrading complex mixtures of hydrocarbons in a waste oil recycling effluent was also investigated. The mineralization of phenol and 2,4,6-TCP by immobilized fungal cultures was studied in pinewood chip and foam glass bead-packed trickling reactors. The reactors were operated in sequencing batch format. Removal efficiency increased over time and elevated influent phenol and TCP (800 and 85 mg.L⁻¹) concentrations were degraded by > 98 % in 24 – 30 h batch cycles. Comparable performance between the packing materials was shown. Uptake by the packing was negligible and stripping of compounds induced by aeration had a minimal effect on biodegradation efficiency. Reactor performances are discussed in relation to sequencing batch operation and nutrient requirements necessary to sustain fungal activity in inert vs. organic material packed systems. It was shown that a co-culture consisting of sulphate-reducing and dechlororespiring bacteria established in fed-batch and soil flasks, as well as pine chip-packed fluidized bed reactors. Results showed reductive dechlorination of 2,4,6-TCP to be in strict dependence on the activity of the sulphate-reducing population, sulphate and lactate concentrations. Transformation to 2,4-DCP, 4-CP and phenol was enhanced in sulphate deficient conditions. Dechlororespiring activity was found to be dependent on the fermentative activity of sulphate-reducing bacteria, and the culture was also shown to mobilize and dechlorinate TCP in soils contaminated with the pollutant. Linking the systems achieved degradation of the compound by > 99 % through fungal mineralization of metabolites produced in the dechlororespiring stage of the system. pH correction to the anaerobic reactor was found to be necessary since acidic effluent from the fungal reactor inhibited sulphate reduction and dechlorination. The fungal reactor system was evaluated at intermediate-scale using a complex waste oil recycling effluent. Substantial COD reduction (> 96 % in 48 h batch cycles) and removal of specific effluent hydrocarbon components was shown in diluted, undiluted (COD > 37 g.L⁻¹) and 2,4,6-TCP-spiked effluents. Industrial application of the fungal reactor was evaluated in a 14 m³ pilot plant operated on-site at a waste oil processing plant.

M.S.

A proteína Cpn60 faz parte de um grupo de proteínas altamente conservadas que estão envolvidas em funções celulares essenciais. camundongos BALB\\c foram imunizados com 5 ou 10 µg da proteína recombinante (Cpn60r) sozinha ou adicionada à vacina DTP sem hidróxido de alumínio (NADTP). A vacina DTP do Instituto Butantan (DTP) foi usada como controle. Foi avaliada a produção de citocinas por células esplênicas após reestímulo in vitro com a Cpn60r. Os animais foram desafiados após o protocolo de imunização. A Cpn60r sozinha ou misturada à vacina NADTP foi capaz de induzir níveis de anticorpos contra pertussis mais altos do que os induzidos pela DTP. Os níveis de IgG1 e IgG2a foram similares para todos os grupos. Pôde-se observar a produção de de IL-6 e IFN-&#947 nos grupos imunizados com Cpn60r. Os grupos imunizados com Cpn60r+NADTP apresentaram um índice de proteção entre 60 e 80% contra o desafio pela bactéria virulenta, semelhante ao grupo imunizado com DTP. A proteína Cpn60r é bastante promissora não somente como imunógeno, mas também como adjuvante. / The Cpn60 protein is a member of a group of higly conserved proteins linked to essencial cell functions. The Cpn60 was cloned, expressed and its immune response has been evaluated. BALB\\c mice were immunized with 5 or 10 µg of the recombinant protein (Cpn60r) alone or mixed with DPT vaccine without aluminum hidroxyde (NADPT). The DPT vaccine from Instituto Butantan was used as control. We evaluated the cytokines production by spleen cells after they have been reestimulated in vitro with Cpn60r. The animals were challenged after the immunization protocol. The Cpn60r alone or mixed with NADPT vaccine was able to induce higher antibodies levels than DPT. IgG1 and IgG2a levels were similar in all groups. We could detect levels of IL-6 and IFN-&#947 on groups immunized with Cpn60r. The groups immunized with Cpn60r+NADTP showed a 60 and 80% protection rate against the challenge with the live bacteria, similar to the group immunized with DPT. These results show the immune response of the recombinant protein that could be included in immunization protocols for pertussis.

A leptospirose é uma zoonose causada por bactérias do gênero Leptospira. A patogênese da doença em humanos é observada principalmente no pulmão, fígado e rins. Neste trabalho, foi avaliado o papel da resposta imune inata na proteção contra a leptospirose usando camundongos como modelo experimental. Os animais foram infectados com L. interrogans e o desenvolvimento da doença foi acompanhado, observando-se a morte de animais C3H/HeJ, enquanto C3H/HePas apresentou icterícia e BALB/c não apresentou sintomas. O perfil de mRNA foi medido por qPCR nas amostras de rim, fígado e pulmão e as concentrações de proteinas TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 foram analisadas por ELISA em extratos dos tecidos e no soro. Os resultados demonstraram que L. interrogans estimula a expressão prematura de TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 na linhagem BALB/c resistente à infecção. A análise histológica indica que estes mediadores podem estar relacionados com o influxo de diferentes células do sistema imune desempenhando importantes funções na proteção contra leptospirose. / Leptospirosis is a worldwide zoonosis caused by Leptospira. The pathogenesis in humans is mainly observed in lungs, livers and kidneys. In this work the role of innate immune response in protection against leptospirosis is being studied using different mice models. The animals were infected intraperitoneally with virulent cells of L. interrogans serovar Copenhageni and the development of the disease was followed, being observed mortality of C3H/HeJ mice, whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. Samples of liver, kidney, lungs and sera were analyzed following the profiles of mRNA and protein of the cytokines TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and CXCL1/IL-8. We showed that Leptospira infection stimulates early expression of cytokine TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and IL-8 in the resistant mice strain BALB/c. Histological analysis indicates that the expression of those molecules can be related to the influx of distinct immune cells, which play a role in the naturally acquired protective immunity.

A proteína Cpn60 faz parte de um grupo de proteínas altamente conservadas que estão envolvidas em funções celulares essenciais. camundongos BALB\\c foram imunizados com 5 ou 10 µg da proteína recombinante (Cpn60r) sozinha ou adicionada à vacina DTP sem hidróxido de alumínio (NADTP). A vacina DTP do Instituto Butantan (DTP) foi usada como controle. Foi avaliada a produção de citocinas por células esplênicas após reestímulo in vitro com a Cpn60r. Os animais foram desafiados após o protocolo de imunização. A Cpn60r sozinha ou misturada à vacina NADTP foi capaz de induzir níveis de anticorpos contra pertussis mais altos do que os induzidos pela DTP. Os níveis de IgG1 e IgG2a foram similares para todos os grupos. Pôde-se observar a produção de de IL-6 e IFN-&#947 nos grupos imunizados com Cpn60r. Os grupos imunizados com Cpn60r+NADTP apresentaram um índice de proteção entre 60 e 80% contra o desafio pela bactéria virulenta, semelhante ao grupo imunizado com DTP. A proteína Cpn60r é bastante promissora não somente como imunógeno, mas também como adjuvante. / The Cpn60 protein is a member of a group of higly conserved proteins linked to essencial cell functions. The Cpn60 was cloned, expressed and its immune response has been evaluated. BALB\\c mice were immunized with 5 or 10 µg of the recombinant protein (Cpn60r) alone or mixed with DPT vaccine without aluminum hidroxyde (NADPT). The DPT vaccine from Instituto Butantan was used as control. We evaluated the cytokines production by spleen cells after they have been reestimulated in vitro with Cpn60r. The animals were challenged after the immunization protocol. The Cpn60r alone or mixed with NADPT vaccine was able to induce higher antibodies levels than DPT. IgG1 and IgG2a levels were similar in all groups. We could detect levels of IL-6 and IFN-&#947 on groups immunized with Cpn60r. The groups immunized with Cpn60r+NADTP showed a 60 and 80% protection rate against the challenge with the live bacteria, similar to the group immunized with DPT. These results show the immune response of the recombinant protein that could be included in immunization protocols for pertussis.