Ethanol from lignocellulosic biomass is a promising alternative to rapidly depleting oil reserves. However, natural recalcitrance of lignocelluloses to biological and chemical treatments presents major engineering challenges in designing an ethanol conversion process. Current methods for pretreatment and hydrolysis of lignocelluloses generate a mixture of pentose (C5) and hexose (C6) sugars, and several microbial growth inhibitors such as acetic acid and phenolic compounds. Hence, an efficient ethanol production process requires a fermenting microorganism not only capable of converting mixed sugars to ethanol with high yield and productivity, but also having high tolerance to inhibitors. Although recombinant bacteria and yeast strains have been developed, ethanol yield and productivity from C5 sugars in the presence of inhibitors remain low and need to be further improved for a commercial ethanol production. The overarching objective of this work is to transform Zymomonas mobilis into an efficient whole-cell biocatalyst for ethanol production from lignocelluloses. Z. mobilis, a natural ethanologen, is ideal for this application but xylose (a C5 sugar) is not its 'natural' substrate. Back in 1995, researches at National Renewable Energy Laboratory (NREL) had managed to overcome this obstacle by metabolically engineering Z. mobilis to utilize xylose. However, even after more than a decade of research, xylose fermentation by Z. mobilis is still inefficient compared to that of glucose. For example, volumetric productivity of ethanol from xylose fermentation is 3- to 4- fold lower than that from glucose fermentation. Further reduction or complete inhibition of xylose fermentation occurs under adverse conditions. Also, high concentrations of xylose do not get metabolized completely. Thus, improvement in xylose fermentation by Z. mobilis is required. In this work, xylose fermentation in a metabolically engineered Z. mobilis was markedly improved by applying the technique of adaptive mutation. The adapted strain was able to grow on 10% (w/v) xylose and rapidly ferment xylose to ethanol within 2 days and retained high ethanol yield. Similarly, in mixed glucose-xylose fermentation, the strain produced a total of 9% (w/v) ethanol from two doses of 5% glucose and 5% xylose (or a total of 10% glucose and 10% xylose). Investigation was done to identify the molecular basis for efficient biocatalysis. An altered xylitol metabolism with reduced xylitol formation, increased xylitol tolerance and higher xylose isomerase activity were found to contribute towards improvement in xylose fermentation. Lower xylitol production in adapted strain was due to a single mutation in ZMO0976 gene, which drastically lowered the reductase activity of ZMO0976 protein. ZMO0976 was characterized as a novel aldo-keto reductase capable of reducing xylose, xylulose, benzaldehyde, furfural, 5-hydroxymethyl furfural, and acetaldehyde, but not glucose or fructose. It exhibited nearly 150-times higher affinity with benzaldehyde than xylose. Knockout of ZMO0976 was found to facilitate the establishment of xylose fermentation in Z. mobilis ZM4. Equipped with molecular level understanding of the biocatalytic process and insight into Z. mobilis central carbon metabolism, further genetic engineering of Z. mobilis was undertaken to improve the fermentation of sugars and lignocellulosic hydrolysates. These efforts culminated in construction of a strain capable of fermenting glucose-xylose mixture in presence of high concentration of acetic acid and another strain with a partially operational EMP pathway.

This study investigated the commissioning and maintenance of a Pennells two-tank bioreactor system with specific reference to its application in rural areas of KwaZulu- Natal, South Africa to treat sewage and generate biogas. The septic tank configuration was installed in a community which lacked electricity and domestic waste disposal. An artificial wetland was constructed at the outlet of the system to facilitate further treatment. Inefficient operation and maintenance of the system occurred due to various social/community-related problems which are typical of a field- and community-based project of this nature in a rural region of a Third World African country. These problems affected both maintenance and digester performance. The Pennells system was characterized by incomplete anaerobiosis which limited methanogenesis. Despite this, and attendant problems of low temperatures and elevated pH values, COD removal resulted. Laboratory-scale batch cultures, in conjunction with fluorescence and scanning electron microscopy, were used to identify a suitable anaerobic digester sludge for inoculation purposes. Perturbation experiments with locally used detergents and toxic compounds demonstrated the inimical effects of these agents. In contrast, low concentrations of penicillin and tetracycline promoted methanogenesis. Further analysis with light, fluorescence and scanning electron microscopy identified the acidogens as the predominant bacterial species, whilst fluorescence microscopy confirmed the absence of methanogens in the bioreactor. / Thesis (M.Sc)-University of Natal, Pietermaritzburg, 1997.

Thesis (MSc Food Sc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: The distillery industry produces large volumes of waste water with a high organic content throughout the year. These effluents must be treated in some manner before being discharged or recycled in the factory. Several treatment options are in use presently, but they all have disadvantages of some nature, such as long retention times, bad odours or the need for large areas of land. Considerable interest has been shown in the application of anaerobic digestion, especially the UASB design (upflow anaerobic sludge blanket), to treat this high strength waste water. Thus, the aim of this study was to investigate the efficiency of an upflow anaerobic sludge blanket (UASB) bioreactor using full-strength distillery effluent. The activity of the bacteria in the biogranules was also evaluated by developing an easy and reliable activity method to estimate the general biogas and methanogenic activity and to calibrate this method using different anaerobic granules from different sources. The influence of high strength distillery effluent on the anaerobic digestion process was investigated using a mesophilic lab-scale UASB bioreactor. During the experimental study, the organic loading rate (OLR) was gradually increased from 2.01 to 30.00 kgCOD.m-3.d-1, and simultaneously, the substrate pH was gradually lowered from 7.0 to 4.7. It was found that at an OLR of 30.00 kgCOD. m-3.d-1,the pH, alkalinity and biogas production stabilised to average values of 7.8, 6 000 mg.l-1 and 18.5 I.d-1 respectively. An average COD removal> 90% was found indicating excellent bioreactor stability. The low substrate pH holds considerable implications in terms of operational costs, as neutralisation of the biorector substrate is no longer necessary. The accumulation of fine solids present in the distillery substrate was found at the higher OLR's and resulted in the granular bed increasing with subsequent biomass washout and a lowering in efficiency parameters. However, a possible pre-treatment filtration of these fine solids would eliminate this problem. The success of the upflow anaerobic sludge bed (UASB) process is mainly due to the capability of retaining the active biomass in the reactor. Over the years, several methods have been developed to characterise and quantify sludge activity but each has advantages and disadvantages. There is thus an increasing need for a rapid method to evaluate the activity of the granular biomass. The activity method of Owen et al. (1979) as adapted by Lamb (1995), was thus evaluated in terms of efficiency and applicability in determining the activity of granular samples. The method was found to be inaccurate as well as time consuming and it was thus modified. Results obtained with the modified assay method were found to be more accurate and the impact of the different test substrates (glucose, lactate, acetate and formate) on activity, was more evident. The activity of seven different anaerobic granules, was subsequently evaluated. Biogas (Ss) and methanogenic (SM) activity was not measured in volume of gas produced per unit COD converted or volatile suspended solids (VSS), but as tempo of gas production (ml.h-1) in a standardised basic growth medium. The activity data obtained were also displayed as bar charts and "calibration scales". This illustrative depiction of activity data gave valuable information about population dynamics as well as possible substrate inhibition. The "calibration scales" can also be used to group the general biogas (Ss) and methanogenic activities (SM) of any new biogranule relative to active (O-type) and inactive (W-type) anaerobic granules, providing that the same method of activity testing is used. The "calibration scales" can thus be used to give a fast indication of how the activity value of one sample relates to the activity values of other granules, even when using different test substrates. / AFRIKAANSE OPSOMMING: Die stokery industrie produseer groot hoeveelhede afvalwater, wat hoë ladings van organiese materiaal gedurede die hele jaar bevat. Hierdie afvalwater moet op een of ander manier behandel word voordat dit gestort of vir hergebruik aangewend kan word. Daar is tans verskeie behandelingsmetodes wat gebruik kan word, maar elk het sy eie tekortkominge soos bv. lang retensie tye, onaangename reuke of die behoefte aan groot stukke oop grond. Groot belangstelling is getoon vir die gebruik van anaerobiese vertering, en meer spesifiek die "uflow anaerobic sludge blanket" UASB bioreaktor vir die behandeling van stokery uitvloeisels. Die doel van die studie was dus om die algehele effektiwiteit van 'n UASB bioreaktor, wat onverdunde stokery uitvloeisel behandel, te evalueer. Die methanogene- en algehele aktiwiteit van die bakterië in die biogranules was ook ge-evalueer deurdat 'n maklike en betroubare aktiwiteitsmetode omtwikkel is, waarna hierdie metode ook toegepas was op 'n reeks van verskillende tipe biogranules. Die invloed van volsterkte stokery uitvloeisel op die anaerobiese verteringsprosesse was ondersoek met die gebruik van 'n mesofiele laboratoriumskaal UASB bioreaktor. Gedurende die eksperimentele studie, was die organiese ladingstempo (OLT) verhoog van 2.01 na 30.00 kgCSB.m-3.d-1 (CSB = chemiese suurstof behoefte) met die gelyktydige verlaging in die pH van die bioreaktorsubstraat van 7.0 na 4.7. Dit was vasgestel dat met 'n OLT van 30.00 kgCSB.m-3.d-1, die pH, alkaliniteit en biogas geproduseer, gestabiliseer het na gemiddelde waardes van 7.8, 6000 mg.-1 en 18.5l.d-1 , respektiewelik, sowel as 'n gemiddelde CSB verwydering van> 90%. Al hierdie waardes dui uitstekende bioreaktor stabiliteit aan. Die lae bioreaktorsubstraat pH kan van groot waarde wees vir die industrie, aangesien neutralisering van die uitvloeisel nie meer nodig is nie en kan sodoende die operasionele koste van die proses verlaag. Die konsentrering van fyn opgeloste soliedes in die bioreaktor by hoë OLT's, kan egter problematies raak, aangesien dit die granule-bed kan vergroot en veroorsaak dat van die biomassa uitspoel en kan verlore gaan. Die verlies van aktiewe biomassa kan die effektiwiteitsparameters negatief beinvloed, maar die plasing van 'n filterings stap voor die verterings stap, behoort hierdie probleem op te los. The sukses van die UASB-stelsel rus op die versekering dat die aktiewe biomassa in die reaktor behoue bly. Oor die jare was daar 'n verskeidenheid van aktiwiteitstoetsings-metodes ontwikkel, elk met sy eie nadele. Daar bestaan dus nog steeds 'n groot behoefte vir die daarstelling van 'n aktiwiteitstoetsings-metode wat vinnig en maklik is om uittevoer. Die aktiwiteitstoetsings-metode van Owen et al. (1979) wat deur Lamb (1995) aangepas is, was in terme van sy effektiwiteit en toepaslikheid ten opsigte van die gebruik daarvan vir aktiwiteitstoetsing vir biogranules, ge-evalueer. Dit is bevind dat die metode onakkuraat sowel as tydsrowend was en gevolglik dus aangepas. Die aangepaste metode het meer akkurate resultate gelewer en die impak van die verskillende toetssubstrate (glukose, laktaat, asetaat en formaat) op die granules het ook meer duidelik na vore gekom. Gevolglik was die aktiwiteit van sewe verskillende anaerobiese biogranules ondersoek. Die eenheid waarin atiwiteitsresultate aangegee is, was nie in volume gas geproduseer per eenheid CSB verwyder of per hoeveelheid gesuspendeerde vlugtige vetsure in die biomassa nie, maar as tempo van biogas (S8)- of metaan (SM)produksie (ml.h-1). Die data wat op hierdie wyse bekom was, is gebruik om staafdiagramme sowel as "kalibrasie skale" daar te stel. Hierdie illustrerende wyse om aktiwiteitsdata uit te beeld verskaf waardevolle informasie ten opsigte van die interaksies tussen die verskillende populasies in die granule en kan ook die aanwesigheid van moontlike substraat inhibisie aandui. Die "Kalibrasie skale" kan ook gebruik word om die algehele (SB) en methanogene (SM)aktiwiteite van einge nuwe biogranule vinnig te klassifiseer ten op sigte van 'n aktiewe (O-tipe) en 'n minder aktiewe (W-tipe) anaerobiese granules, mits dieselfde metode gebruik word om die aktiwiteits data te bekom.

Thesis (MSc)--Stellenbosch University, 2000. / ENGLISH ABSTRACT: The "classical" propionibacteria are used in a variety of natural dairy fermentations where they produce natural preservatives (propionic and acetic acids and bacteriocins) and large amounts of vitamin B12. The extracellular polysaccharide (ECP) producing ability of these bacteria also make them of special interest to the food and waste water management industries as the ECP has been illustrated to playa role in the initial granule formation in upflow anaerobic bioreactor systems. There is little known on the ECP production by propionibacteria and in this study different environmental conditions that influence ECP production were studied. Nineteen different Propionibacterium strains were examined in terms of ECP production and Propionibacterium strain 278 was identified as the best ECP producer. Further studies were only done on this strain because of its high ECP production and because it was originally isolated from an anaerobic digester. The influence of temperature, pH and sucrose concentration was determined through the measurement of ECP production and medium viscosity. It was found that more ECP was produced at temperatures lower than the optimum for growth with the optimum being between 22° and 25°C. Lower initial pH conditions of the growth medium (below pH 7.0) were found to inhibit ECP production and the influence when the initial pH values were between 7.0 and 8.5, was not significant. A higher carbon: nitrogen ratio, when 8% sucrose was added, was also found to enhance the ECP production. The upflow anaerobic sludge bed (UASB) bioreactor process depends on the upward movement of soluble matter through a blanket of active methanogenic granular sludge. The long start-up times as a result of the slow granulation process, as well as the need for a speedy replacement of granules once they have been washed out of the system, are limitations that restrict the general application of this excellent waste water treatment technology. Full exploitation of this biomass immobilisation technique can thus not be realised until the granule formation conditions are defined and optimised. The precise nature of the mechanisms involved in the formation of granules and the reason for their stability, is still not fully understood. It was hypothised by Britz et al. in 1999 that, through the implementation of environmental 'stress' conditions, a shift in the population dynamics of the anaerobic community can be obtained. This results in a concurrent increase in ECP formation that appears to enhance aggregate formation. In the second study it was found that, when 'stress' conditions were applied to already formed granules, the Gram-positive lactate-utilising acidogenic population gained an advantage and more propionic acid producing bacteria were present. The propionic and acetic acid concentrations were also found to increase, and concurrently, a decrease in the growth medium pH occurred. This confirms part of the granulation hypothesis that, when granules are 'stressed', the acidogenic population dynamics change and the lactate-utilising population responds to the gradual decrease in pH and the more acid-tolerant propionic acid producing bacteria gain a competitive advantage resulting in the increase in the propionic acid concentration. When propionibacteria were added to raw sludge during the granule production process, the granules were found to be more active than when nopropionibacteria had been added. This was probably due to the ECP formation by the propionibacteria that enhances the aggregation of the granules. Enhanced granulation was thus found in the batch systems with the fatty acids formed in correlation with the model for granulation. A good correlation was evident between the hypothesis and the experimental data and the hypothesis was partially verified in this study. / AFRIKAANSE OPSOMMING: Die "klassieke" propionibakterieë word in 'n verskeidenheid van natuurlike suiwel fermantasies gebruik waarin hulle verantwoordelik in vir die produksie van natuurlike voedsel preserveermiddels (propioonsuur, asynsuur en bakteriosiene) en groot hoeveelhede vitamiene B12. Die Ekstra Sellulêre Pollisakkaried (ESP) produserende eienskap van hierdie groep bakterieë maak hulle ook van belang in die voedsel en afvoerwater beheer industrieë, aangesien gevind is dat ESP 'n rol speel in die aanvanklike granule formasie in anaerobiese bioreaktor sisteme. Daar is nog baie min bekend oor die ESP produksie van propionibakterieë en in hierdie studie is verskeie omgewings faktore wat die ESP produksie beïnvloed, bestudeer. Negentien verskillende Propionibakterium stamme was bestudeer in terme van ESP produksie en Propionibakterium stam 278 was geïdentifiseer as die stam wat die meeste ESP produseer. Verdere studies was op hierdie stam gedoen na aanleiding van sy hoë ESP produksie en omdat dit oorspronklik uit 'n anaerobiese verteerder geisoleer is. Die invloed van termperatuur, pH en sukrose konsentrasie was bepaal deur die meting van die ESP produksie en die medium viskositeit. Dit was gevind dat meer ESP geproduseer was by temperature laer as die optimum vir groei, met die optimum temperatuur tussen 22° en 25°C. Dit is ook gevind dat laer aanvangs groei-medium pH (laer as pH 7.0), ESP produksie inhibeer. Die invloed van die aanvangs groei-medium pH tussen 7.0 en 8.5 was egter nie betekenisvol nie. Dit is ook gevind dat 'n hoër koolstof tot stikstof verhouding, verkry deur die byvoeging van 8% sukrose, die ESP produksie verhoog. Die "upflow anaerobic sludge blanket" (UASB) proses vind plaas as gevolg van die opwaarste beweging van opgeloste organiese materiaal deur 'n granule bed van aktiewe metanogeniese granulêre slyk. Die lang 'start-up' tye as gevolg van die stadige granulasie proses, en die nodigheid om 'n vinnige verplasing van granules te hê nadat dit uit die sisteem gewas is, is beperkings wat die algemene toepassing van hierdie fantastiese afvoerwater tegnologie, strem. Volle implementering van hierdie biomassa immobilisereings tegniek kan dus nie plaasvind voordat die granule formasie gedefinieer en geoptimiseer is nie. Die presiese eienskappe van die meganismes betrokke en die formasie van die granules en die rede vir hul stabiliteit word egter nog nie ten volle verstaan nie. Volgens 'n hipotese deur Britz et al. (1999), vind 'n verskuiwing in die populasie dinamika van die anaerobiese gemeenskap plaas tydens die implementasie van omgewings 'stress' toestande. Die resultaat is 'n verhoging in ESP produksie en 'n gevolglike verbetering in die granulasie proses. In die tweede studie was dit gevind dat, wanneer 'stress' toestande op die reeds gevormde granulasie toegepas word, die Gram-positiewe laktaat-benuttende asetogeniese populasie voordeel geniet en meer propioonsuur produserende bakterieë was teenwoordig. Die propioonsuur en asynsuur konsentrasies het ook verhoog en met 'n gevolglike daling in die groei-medium se pH. Dit bevestig 'n gedeelte van die hipotese dat, wanneer die granules onder 'stress' geplaas word, die asetogeniese populasie dinamika verander en die laktaat-benuttende populasie reageer tot die gedeeltelike afname in pH. Die meer suur-tolerante propioonsuur produserende bakterieë verkry 'n kompeterende voordeel en gevolglik is daar 'n verhoging in propioonsuur konsentrasie. Propionibakterieë was gevoeg by die onbehandelde slyk gedurende die granule produksie proses, en daar is gevind dat meer aktiewe granules gevorm word as andersins. Dit is moontlik as gevolg van die die ESP produksie van propionibakterieë wat die granulasie versnel het. Verbeterde granulasie was dus verkry in die sisteme waar propionibakterieë bygevoeg is. Vetsuur analises het gedui dat die gevormde vetsure ook in korrelasie was met die model van granulasie. Goeie korrelasie was dus verkry tussen die hipotese en die eksperimentele data en die hipotese is gedeeltelik bewys in hierdie studie.

Thesis (MSc Food Sc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The wine industry significantly contributes to South Africa's water demand and subsequent pollution of the limited resource. Wastewater is produced throughout the year with an increase in volume and organic load during the vintage season. Anaerobic digestion (AD), specifically the upflow anaerobic sludge bed (UASB) technology has been shown to be feasible in the treatment of cellar wastewater. However, the legal standard for chemical oxygen demand (COD) for disposal in a natural water resource (75 rnq.L") is often not met. The aim of the study was to conduct a laboratory-scale investigation into the feasibility of combining pre- and post-ozonation processes with AD in order to achieve a final COD closer to the legal disposal limit. While acclimatising an UASB bioreactor containing mixed anaerobic granules to a cellar wastewater with a pH set at 8.0, stable-state conditions were not reached. Sucrose additions to the substrate, increased substrate loads, heattreatment of the substrate and an addition of isolated cellar effluent bacteria to facilitate degradation prior to AD, were all unsuccessful in maintaining stable-state in terms of COD removal efficiency. Once the substrate pH was re-set to 7.5, the reactor stabilised. The lowest efficient operational pH was found to be 5.73 resulting in a COD removal of 88% at a substrate COD < 5 000 rnq.L". At a substrate pH of 6.0, the lowest efficient operational hydraulic retention time (HRT) and corresponding organic loading rate (OLR) were 19.7 hand 9.75 kg COD.m-3d-1 , respectively, with the COD removal being maintained around 84%. The reactor effluent still had a final COD of 1280 rnq.L", which was well above the legal South African limit. Dominant bacteria were isolated from raw cellar wastewater and identified as Acinetobacter haemolyticus, Burkholderia cepacia and Cryseomonas luteola. In order to investigate the possibility that ozonation improved biodegradability, the growth of the isolates at 35°C was monitored over 24 h in sterile ozonated and non-ozonated substrates from the vintage and non-vintage seasons. All the isolates increased by at least 1.5 log cycles in the control substrates from both seasons. Ozonation of the wastewater batches for 10 min at a rate of 73 rnq.L" led to slightly increased growth of the inoculants in the substrate batch from the vintage season. For the substrates from the non-vintage season, ozonation had an inhibitory effect on the bacterial growth. A 5 min ozonation treatment at a concentration of 73 rnq.L" was found to be optimal for both a pre- and post-treatment to UASB-treatment of cellar wastewater. Both UASB treatment and ozonation were effective in reducing the COD by 85% and 20%, respectively. The COD reduction was improved to 88% when UASB treatment was combined with post-ozonation. The total reduction in total suspended solids (TSS) for the combined process was 97%, compared to 80% for UASB and 73% for an ozone treatment alone. The reduction for volatile suspended solids (VSS) was 98% compared to 81% for UASB and 73% for the ozone treatment alone. The total reduction when using a pre-ozonation UASB treatment combination was an average of 86% for COD. The TSS and VSS were both reduced by 95%. Biogas production increased from 1.4 L.d-1 to 3.8 L.d-1 when an ozonated wastewater was used as substrate. When the UASB treatment was combined with both a pre- and post-ozonation treatment process, the COD was reduced by 89% while TSS and VSS were both reduced by 99%. This study showed that pre- and post-ozonation treatment processes could successfully be utilised to improve UASB treatment of cellar wastewater. Although the legal limits for discarding into a natural resource were not met, significant progress was made in reducing COD levels. Cellar wastewaters do however, vary according to season and the wastewater composition could affect the efficiency of a pre-ozonation process. / AFRIKAANSE OPSOMMING: Die wynindustrie maak "n beduidende bydrae tot die eise wat aan Suid-Afrika se waterbronne gestel word en gevolglik die besoedeling van die beperkte hulpbron. Afloopwater, wat in volume en organiese lading gedurende die parstyd toeneem, word reg deur die jaar opgelewer. Anaërobiese vertering (AV), spesifiek die "Upflow anaerobic sludge blanket" (UASB) tegnologie, is alreeds suksesvol gebruik om kelderafloop te behandel. Die wetlike vereiste vir chemiese suurstof behoefte (CSB) vir storting in "n natuurlike hulpbron (75 rnq.L"), word egter dikwels nie bereik nie. Die doel van die studie was om in "n laboratorium-skaal ondersoek AV te kombineer met voor- en na-osoneringsprossesse, om sodoende te poog om "n CSB nader aan die wetlike standaard te verkry. Terwyl"n UASB bioreaktor wat gemengde anaerobiese granules bevat het, geakklimatiseer is tot kelderafloop met "n pH gestel tot 8.0, kon stabiele toestande nie bereik word nie. Die byvoeging van sukrose tot die substraat, verhoogde substraatladings, hitte-behandeling van die substraat en die byvoeging van geïsoleerde kelderafloop bakterië om substraatafbraak voor AV aan te help, was onsuksesvol om stabiliteit in terme van CSB-verwydering, te handhaaf. "n Verstelling van die substraat pH na 7.5, het gelei tot reaktorstabiliteit. By die laagste doeltreffende bedryfs-pH van 5.73 en substraat CSB < 5 000 rnq.L", was die CSB-verwydering 88%. By "n substraat pH van 6.0 was die laagste doeltreffende bedryfs-hidroliese retensie tyd en -organiese ladingstempo 19.7 h en 9.75 kg CSB.m-3d-1, onderskeidelik, terwyl die CSB verwydering rondom 84% gehandhaaf is. Die CSB van die reaktoruitvloesel van 1 280 rnq.L", was steeds ver bo die wetlike vereiste. Dominante bakterië is uit kelderafloop geïsoleer en as Acinetobacter haemolyticus, Burkholderia cepacia en Cryseomonas luteola, geïdentifiseer. Die moontlikheid dat osonering bioafbreekbaarheid bevorder, is ondersoek deur die groei van die isolate by 35°C oor 24 h in steriele geësoneerde en ongeësoneerde substrate te monitor. Die substrate is berei vanaf kelderafloop wat in die parsseisoen sowel as die nie-parsseisoen versamel is. AI die isolate het met ten minste 1.5 log siklusse in die kontrole substrate van beide seisoene, vermeerder. Vir die kelderafloop wat in die parsseisoen versamel is, het osonering vir 10 min teen 73 rnq.L" gelei tot effens verbeterde groei van die innokulante. Osonering het 'n onderdrukkende effek op die groei van bakterië in die afloopwater versamel in die nie-parsseisoen, gehad. Osonering vir 5 min teen 'n konsentrasie van 73 rnq.L" is as optimum vir beide voor- en na-osoneringsbehandeling tot UASB-behandeling van die kelderafloop, gevind. UASB-behandeling en osonering het die CSB met 85 en 20% onderskeidelik, verminder. Die vermindering kon tot 88% verhoog word wanneer UASB-behandeling met na-osonering gekombineer is. Die vermindering in totale gesuspendeerde vastestowwe (TGV) vir die gekombineerde proses was 97%, in vergelyking met 80% vir UASB- en 73% vir osoonbehandeling alleen. Die vermindering in vlugtige gesuspendeerde vastestowwe (VGV) was 98% in vergelyking met 81% vir UASB- en 73% vir osoonbehandeling alleen. Die totale CSB verwydering vir 'n voor-osonerings UASB kombinasie was gemiddeld 86%. Die TGV en VGV is beide met 95% verminder. Biogasproduksie het ook vermeerder vanaf 1.4 L.d-1 tot 3.8 L.d-1 toe geosoneerde afloopwater as substraat gebruik is. Die kombinasie van UASB-behandeling met voor-osonering, sowel as na-osonering het gelei tot 'n CSB-verwydering van 89% terwyl TGV en VGV beide met 99% verminder is. Hierdie studie het getoon dat voor- en na-osonering suksesvol gebruik kan word om UASB-behandeling van kelderafloop te verbeter. Hoewel wetlike vereistes vir storting in 'n natuurlike hulpbron nie bereik is nie, is beduidende vordering gemaak in die verlaging van CSB-vlakke. Die verskil in die samestelling van kelderafloop gedurende die onderskeie seisoene, kan egter die doeltreffendheid van die voor-osoneringsproses beïnvloed.

Le microbiote digestif, composé de 1012 à 1014 bactéries par gramme de selle, est dominé par les bactéries anaérobies. Ces dernières, qui dépassent largement les aérobies, ont été découvertes en 1865 par Louis Pasteur dans ses travaux sur la fermentation. Les bactéries anaérobies représentent un intérêt médical particulier par leur implication dans les maladies infectieuses et métaboliques. Les anaérobies sont caractérisés par leur difficulté à être cultivés, car nécessitant une absence ou des concentrations faibles d’oxygène. Ainsi les connaissances sur ces microorganismes étaient limitées. Cependant, avec l’avènement des outils de biologie moléculaire notamment le séquençage à haut débit et le concept culturomics associé à l’identification par spectrométrie de mass MALDI-TOF, la connaissance des microorganismes anaérobies a été accentuée. Dans ce travail, nous nous sommes attelés dans un premier temps à la mise au point d’un milieu de culture efficace pour la culture des bactéries anaérobies et les tests d’activités antimicrobiennes. Nous avons montré dans un deuxième temps que culturomics, associé au MALDI-TOF, était un outil puissant dans l’identification des bactéries anaérobies en microbiologie clinique, mais également dans l’exploration de la diversité microbienne du tube digestif. Cette technique nous a permis d’isoler 19 nouvelles espèces de bactéries anaérobies dont 9 ont été décrites dans la troisième partie de ce travail. / The human gut microbiota is known to contain around 1012 to 1014 bacteria per gram of feces, with the majority being anaerobic. The latters were first discovered in 1865 by Louis Pasteur while working on fermentation. Anaerobic bacteria are known to play an important role in health and diseases and thus have taken a lot of attention in the medical field, especially in infectiousdiseases and metabolism. These bacteria are known for its sophisticated culture system because its growth requires little to no oxygen. Nevertheless, few is known about these type of microorganisms but with the advancement of molecular biology and sequencing techniques, its study became wider. Culturomics, is a recently developed culture-based approach that relies on optimizing culture conditions for bacterial growth and its identification by MALDI-TOF MS and 16S rRNA sequencing. The present work aims to create and optimized culture condition for anaerobic bacteria along with testing its anaerobic activities. Also, we aim to demonstrate the efficiency of Culturomics and MALDI-TOF in culturing, identifying and describing anaerobic bacteria in clinical microbiology and in the human gut. This approach allowed us to isolate 19 new anaerobic species out of which 9 has been described in this work.Keywords: Human gut microbiota, culture of anaerobic bacteria, culturomics, MALDI-TOF.

A periodontite é uma resposta inflamatória desencadeada por um complexo biofilme constituído por diversos microrganisnos, particularmente por bactérias anaeróbias, tais como Porphyromonas spp. e Fusobacterium spp. Essas bactérias produzem vários fatores de virulência capazes de atuar no início e progressão da doença. Cem amostras subgengivais foram analisadas, sendo 50 de cães com periodontite e 50 de cães sadios, obtendo-se 144 isolados bacterianos identificados como pertencentes aos gêneros Porphyromonas e Fusobacterium. A produção de hemolisinas e hemaglutininas, susceptibilidade aos soros humano, canino e equino, e susceptibilidade a dez antibióticos foram avaliadas, assim como a presença dos genes prtC (colagenase), fimA (fímbrias), tetQ e tetM (resistência à tetraciclina). Igualmente, a capacidade quimiotática de neutrófilos e a produção de IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 e IL-17, foram avaliadas. <font face=\"Symbol\">&#946-hemólise foi produzida por P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis e P. circumdentaria. Dos 144 isolados, 21 P. gulae e 2 F. nucleatum aglutinaram eritrócitos humanos. A maioria dos isolados foi resistente à ação dos soros humano, equino ou canino. Todos os 144 isolados foram sensíveis à amoxicilina, clindamicina, tetraciclina, amoxicilina/clavulanato, cefoxitina e penicilina G. Três P. gulae, 2 P.macacae e 2 P. canifelinum abrigaram o gene tetQ e apenas um F. nucleatum e um P. catoniae foram positivos para a presença do gene tetM. As espécies P. gulae, P. cangingivalis e P. circumdentaria abrigaram o gene prtC. A maioria dos isolados abrigou o gene fimA tipo I e nenhum deles abrigou o gene fimA tipo V. Somente nas P. gulae foi observada a presença de cápsula pela microscopia eletrônica de transmissão. Todos os isolados estimularam quimiotaxia dos neutrófilos. Interleucinas IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 e IL-11 foram detectadas após estímulo com as espécies de Porphyromonas. As espécies de Fusobacterium não estimularam a produção de IL-11 e P. gulae induziu elevada concentração dessa citocina. Nenhum dos isolados estimulou a liberação de IL-17. / Periodontitis is an inflammatory response caused by a complex microbial biofilm, including anaerobic bacteria such as Porphyromonas spp. and Fusobacterium spp. Those species present several virulence factors which act on early step and during the disease evolution. One hundred of subgingival samples were analyzed, obtained from 50 dogs with and 50 without periodontitis. One hundred and forty four bacterial species were isolated belonging to both Porphyromonas and Fusobacterium genus. Hemolysin and hemagglutinin production, susceptibility to human, equine and canine sera, and antimicrobial susceptibility to 10 antibiotics were evaluated. In addition, the presence of gene prtC (collagenase), fimA (fimbriae), tetQ and tetM (tetracycline resistance), as well as neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945, IL-11 and IL-17 production were also determined. <font face=\"Symbol\">&#946-hemolysis was produced by P. gulae, P. crevioricanis, P. cangingivalis, P. gingivicanis and P. circumdentaria. Of the 144 isolates, 21 P. gulae and 2 F. nucleatum were able to agglutinate human erythrocytes. Most of the isolates were resistant to the action of human, equine or canine serum. All isolates were susceptible to amoxicillin, clindamycin, tetracycline, amoxicillin/clavulanate, cefoxitin and penicillin G. Three P. gulae, 2 P.macacae and 2 P. canifelinum harbored tetQ, and only one F. nucleatum and one P. catoniae were tetM positive. P. gulae, P. cangingivalis and P. circumdentaria harbored prtC. Most of the isolates harbored fimA I gene and none of them harbored fimA V. Only P. gulae showed capsule through transmission electron microscopy. All isolates induced neutrophils chemotaxis and IL-1<font face=\"Symbol\">&#946, IL-8, TNF-<font face=\"Symbol\">&#945 and IL-11 were produced when neutrophils were stimulated by Porphyromonas spp. Fusobacterium spp. did not stimulate IL-11 production and P. gulae induced high concentration of cytokine. None of the isolates stimulated IL-17.

A sequência de eventos bacterianos que ocorre durante a colonização do trato gastrointestinal pode afetar o futuro da saúde do hospedeiro, particularmente no que diz respeito à regulação do sistema imunológico. Um entendimento claro do processo de colonização do intestino humano neonatal nos países em desenvolvimento está faltando, porque os poucos estudos disponíveis foram, em sua maioria, realizados utilizando técnicas de cultura. O objetivo deste estudo foi detectar e quantificar as bactérias dos gêneros Bifidobacterium, Lactobacillus, Eubacterium e Lactococcus, importantes componentes anaeróbios da microbiota intestinal usando PCR em tempo real. O grupo de estudo foi composto por 10 crianças, acompanhadas durante o primeiro ano de vida, vivendo em baixas condições sócio-econômicas em São Paulo, Brasil. Amostras de fezes foram avaliadas em períodos de 24 horas, 7 dias, 30 dias, 3 meses, 6 meses e 1 ano. Durante o primeiro ano de vida, há um aumento da quantidade de Bifidobacterium spp., quando comparada com as outras bactérias anaeróbias estudadas, com médias variando de 8,27x1010 a 2,51x1012 número de cópias de DNA/g de fezes. Lactobacillus spp. também foi encontrado em todos os pontos de tempo estudado, com médias variando de 4,03x108 a 1,46x1010 número de cópias de DNA/g de fezes. Lactococcus spp. foi o gênero bacteriano encontrado em quantidades menores. As contagens máximas desses gêneros foram encontradas entre o terceiro e sexto mês de vida. Embora o gênero Eubacterium seja descrito como um dos principais membros da microbiota intestinal, este foi encontrado em amostras de apenas duas crianças. A inclusão de dieta sólida e a mudança do tipo de amamentação influenciam a composição da microbiota. No entanto, não se pode estabelecer um padrão para a presença destes micro-organismos ao longo dos meses, mostrando que a microbiota é única e está sujeita a interferências ambientais. / The sequence of bacterial events that occurs during the colonization of the gastrointestinal tract may affect the future health of the host, particularly with respect to the regulation of the naive immune system. A clear understanding of the colonization process of the human neonatal gut in developing countries is lacking because the few available studies were mostly performed using culture techniques. The aim of this study was to detect and quantify the bacterial genera Bifidobacterium, Eubacterium, Lactobacillus and Lactococcus, important anaerobic components of the intestinal microbiota using real-time PCR. The study group comprised 10 children followed during the first year of life, living in low socio-economic conditions in São Paulo, Brazil. Fecal samples were evaluated at times of 24 hours, 7 days, 30 days, 3 months, 6 months and 1 year. During the first year of life, there is an increased amount of Bifidobacterium spp. compared to others studied anaerobic bacteria, with averages ranging from 8,27x1010 to 2,51x1012 DNA copy number/g of feces. Lacotbacillus was also found in all studied time point, with averages ranging from 4,03x108 to 1,46x1010 DNA copy number/g of feces. Bacterial genus Lactococcus is found in smaller quantities. The maximum counts of these genera were found between the third to sixth month of life. Although the genus Eubacterium is described as one of the leading members of the intestinal microbiota, this was found in samples of only 2 children. The inclusion of solid diet and change of type of feeding influences the composition of the microbiota, however, could not set a standard for the presence of these micro-organisms over the months, showing that the microbiota is unique and is subject to environmental interference.

Submitted in complete fulfillment for the degree of Doctor of Philosophy (Biotechnology), Durban University of Technology, Durban, South Africa, 2015. / Anaerobic digestion, a proven and highly efficient biological process for treating industrial wastewater and biogas generation is an underutilized technology in South Africa. Some of the industries that have on-site anaerobic reactors tend to face problems in operating these reactors due to poor understanding of the process and implementation of the technology. This has resulted in high pollutant loads in their final effluents and low energy recovery. In this study, an on-site full–scale upflow anaerobic sludge blanket (UASB) reactor treating brewery wastewater was extensively monitored in order to evaluate the efficiency in terms of effluent quality, biogas production and microbial structure. Furthermore, developed and adopted kinetic models were used to optimize the performance of the full–scale UASB reactor using a combined Pareto differential evolution (CPMDE) algorithm. A preliminary analysis of the raw wastewater has shown that the wastewater produced from the brewery industry was high in organic matter with a total chemical oxygen demand (COD) between 1096.41 to 8926.08 mg/L. The average removal efficiency of COD from the UASB reactor after treatment was 79% with a methane (CH4) production of 60-69% at temperature ranges of 28-32˚C and hydraulic retention time (HRT) of 12 h within the optimal pH range for anaerobic bacteria (6.6 and 7.3) under various organic loading rates. However, the results also showed an increase in total suspended solids (TSS), nitrogen (N2), ammonia (NH3) and orthophosphate concentrations when comparing the influent to the effluent, which indicated the necessity for further optimization of the reactor condition in order to reduce these effluent parameters to acceptable standards and to increase CH4 production. In order to optimize the process, a thorough understanding of microbial interaction was essential. A combination of different molecular techniques viz., fluorescence in–situ hybridization (FISH), polymerase chain reaction (PCR) and quantitative real-time PCR (QPCR) were employed to understand the microbial community structure of the granular sludge samples using species specific primers and probes. The results revealed that the dominance of diverse groups of eubacteria belonging to phyla Proteobacteria, Firmicutes and Chloroflexi and an uncultured candidate division WS6 with four different orders of methanogenic Archaea viz., Methanomicrobiales, Methanococcales, Methanobacteriales and Methanosarcinales belonging to hydrogenotrophic and aceticlastic methanogens were within the reactor samples. Quantification of the 16S rDNA copies of eubacteria and methanogenic Archaea using species-specific primers further confirmed the spatial distribution of these microorganisms within the different compartments of the reactor where, the upper compartments were dominated by eubacteria and the lower compartments by methanogenic Archaea. The concentration of Archaea per nanogram of DNA was much higher (96.28%) than eubacteria (3.78%) in lower compartments, while, the eubacteria concentration increased to 98.34% in upper compartments with a decrease in Archaea quantity (1.66%). A modified kinetic methane generation model (MMGM) was developed on the basis of mass balance principles with respect to substrate (COD) degradation and the endogenous decay rate to predict CH4 production efficiency of the reactor. Furthermore, a Stover–Kincannon kinetic model was adopted with the aim of predicting the final effluent quality in terms of COD concentration and model coefficients were determined using the data collected from the full–scale reactor. Thereafter, a model-based multi-objective optimization was carried out using the CPMDE algorithm with three–objective functions namely; maximization of volumetric CH4 production rate; minimization of effluent substrate concentration and minimization of biomass washout, in order to increase the overall efficiency of the UASB reactor. Important decision variables and constraints related to the process were set for the optimization. A set of non-dominated solutions with high CH4 production rates of between 2.78 and 5.06 L CH4/g COD/day at low biomass washout concentrations were obtained at almost constant solution for the effluent COD concentration. A high COD removal efficiency (85-87%) at ~30-31˚C and 8-9 h HRT was obtained for the multi-objective optimization problem formulated. This study could significantly contribute towards optimization of a full–scale UASB reactor treating brewery wastewater for better effluent quality and biogas production. Knowledge on the activity and performance of microbial community present in the granular sludge taken from the full–scale UASB reactor would contribute significantly to future optimization strategies of the reactor. In addition, optimization using an evolutionary algorithm under different operational conditions would help to save both time and resources wasted in operating anaerobic bioreactors.

"February 2003." Addendum and corrigenda inserted at back Includes bibliographical references (leaves 249-272) Aims to identify and characterise potential virulence-associated factors from the locus of enterocyte effacement-negative shiga-toxigenic Escherichia coli O113:H21 strain 98NK2 which was responsible for an outbreak of haemolytic uremic syndrome. Particular attention was focused on putative virulence genes encoded on the megaplasmid of this strain.