Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: The microbial degradation of the lignin carbohydrate complex of plant material is only partially understood. Lignin carbohydrate complex was extracted from wheat straw and subsequently analysed. An adjustment to the standard protocol was required to increase the amount of lignin carbohydrate complex extracted from wheat straw. Characterization of the lignin carbohydrate complex after trifluoacetic acid hydrolysis was done by capillary electrophoresis. HPLC proved ineffective, as baseline separation could not be achieved. Characterization of the lignin carbohydrate complex revealed that a large portion (68 %) consisted of carbohydrate and lignin (20 %). Capillary electrophoresis of the trifluoroacetic acid hydrolysates of the lignin carbohydrate complex revealed that the carbohydrates consisted of mannose, xylose, arabinose, galactose, glucose and rhamnose. The major monosaccharide present in the lignin carbohydrate complex was mannose which made up 34 % of the total carbohydrate composition. Ferulic and p-coumaric acid were present in the lignin carbohydrate complex, but in concentrations less than 1 % of the lignin carbohydrate complex. The lignin carbohydrate complex of wheat straw probably had a heterogenous structure consisting of a variety of molecules, as molecular weight determination could not be accurately determined. An estimated molecular weight of 5.9 kOa was determined. Ten fungal strains (Aspergillus niger, Aureobasidium pul/u/ans, Bjerkandera adusta, Corio/us versicolor, Lenzitus betu/ina, Phanerochaete chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 and Trichoderma reeseï; were evaluated for growth on the lignin carbohydrate complex. All strains except B. adusta showed growth after 5 days with A. niger, A. pul/u/ans, C. versicolor, P. chrysosoporium and T. reesei showing the best growth on the lignin carbohydrate complex. The culture fluid revealed a number of proteins secreted by these organisms. The protein determination was confirmed by SOS-PAGE which revealed an array of proteins ranging from 8 kOa to 180 kOA. Prominent bands between 26 kOa and 80 kOa could be observed in the culture fluid of A. niger, A. pul/ulans and T. reesei, but not in C. versicolor. Activity studies on the culture fluid of these four strains revealed activity for xylanase, xylosidase, arabinofuranosidase, ferulic acid esterase and laccase, with vast differences between the activities of the various fungi. The enzymes of these fungal strains were also evaluated for their ability to degrade xylan and sugar cane bagasse using capillary electrophoresis. It appeared that all the organisms produced enzymes to degrade birchwood xylan. However, the electropherograms revealed that the degradation patterns of birchwood xylan differed among these organisms over the same time interval, as xylotetraose, xylotriose, xylobiose and xylose were released in various concentrations. The electropherograms obtained from the enzyme hydrolysates of sugar cane bagasse, pointed to the substrate being inaccessible. Electropherograms of the culture fluid of A. niger, A. pul/ulans, C. versicolor and T. reesei, when incubated on the lignin carbohydrate complex indicated similar peaks to those obtained and identified in the trifluoroacetic acid hydrolysates. However, the electropherograms of the culture fluid of these organisms revealed additional smaller peaks which could not be identified. The electropherograms of the culture fluid of the various organisms also indicated a complete release of some sugars, using the trifluoacetic acid hydrolysate of the lignin carbohydrate complex as a control for the amount of sugars present. HPLC analyses revealed that after 72 h, no apparent degradation of the lignin carbohydrate complex took place as peak height and areas appeared to be similar. These peaks could however not be identified due to a lack of standards as well as baseline separation which could not be achieved. / AFRIKAANSE OPSOMMING: Tans word die mikrobiese afbraak van die lignienkoolhidraatkompleks van plant materiaal slegs gedeeltelik verstaan. Lignienkoolhidraatkompleks was vanaf koringstrooi geïsoleer en gevolglik geanaliseer. Daar moes van die standaard prosedure vir die ekstraksie van lignienkoolhidraatkompleks afgewyk word ten einde beter lignienkoolhidraatkompleks opbrengs te lewer. Karakterisering van die lignienkoolhidraatkompleks na trifluoroasynsuurvertering was deur kapillêre elektroforese bepaal. Dit wou voorkom asof kapillêre elektroforese "n beter opsie vir die analise van die verteerde monster van lignienkoolhidraatkompleks is, vergeleke met hoëdruk vloeistof chromatografie. Daar was gevind dat die lignienkoolhidraatkompleks uit 68 % koolhidraat en 20 % lignien bestaan. Kapillêre elektroforese het die teenwoordigheid van die volgende suikers bevestig naamlik, mannose, xilose, arabinose, glukose, galaktose en ramnose. Mannose was die dominerende suiker in die lignienkoolhidraatkompleks wat 34 % van die totale koolhidraat opbrengs uitgemaak het. Ferulien- en p-kumaarsuur kon ook identifiseer word, maar die twee sure het minder as 1 % van die totale inhoud van die lignienkoolhidraatkompleks uitgemaak. Vanuit resultate bekom wil dit voorkom dat die lignienkoolhidraatkompleks "n heterogene molekuul is omdat die molekulêre gewig daarvan nie akkuraat bepaal kon word nie. 'n Geskatte molekulêre grootte van ongeveer 5.9 kDa was bepaal met verwysing na die hoogste piek wat in die chromatogram waargeneem was. Tien fungus kulture was in die studie gebruik om hul vermoë te toets om op die lignienkoolhidraatkompleks te groei, naamlik Aspergillus niger, Aureobasidium pullulans, Bjerkandera adusfa, Goriolus versicolor, Lenziius betuline. Phanerochaefe chrysosporium, Pycnoporus coccineus, Pycnoporus sanguineus 294, Pycnoporus sanguineus K5-2-3 en Trichoderma reesei. B. eauste het nie groei na 5 dae getoon nie, en dit wou voorkom asof A. niger, A. pul/ulans, G. versicolor, P. chrysosoporium en T. reesei die beste kon groei op die substraat na 5 dae. Die kultuurvloeistof van die vier kulture het getoon dat proteïene deur hierdie organisms uitgeskei was. Hierdie proteinbepaling was ook bevestig deur SOS-PAGE, wat bande tussen 8 kDa en 180 kDa gelewer het. Prominente bande tussen 26 kDa en 80 kDa kon waargeneem word in die kultuurvloeistof van A. niger, A. pul/ulans, en T. reesei, maar nie in die kultuurvloeistof van C. versicolor nie. Aktiwiteitstudies op die kultuur vloeistowwe het getoon dat daar aktiwiteit was vir die volgende ensieme, naamlik xilanase, xilosidase, arabinofuranosidase en feruliensuur esterase. Hierdie aktiwiteit het aansienlik verskil tussen die verskillende organismes. Die ensieme van die vier organismes was ook gebruik om hul vermoë te toets om xilaan en suikerriet af te breek. Daar was gevind dat xilaanafbraak verskillend was vir die organisms oor dieselfde tydperk. Xilotetraose, xilotriose, xilobiose en xilose was in verskillende konsentrasies gevind vir die verskillende organismes. Die elektroferogramme van die kultuurvloeistof op suikerriet van die verskillende organismes het getoon dat die substraat nie toeganklik vir die ensieme was nie. Die elektroferogramme van die kultuurvloeistof op lignienkoolhidraatkompleks van die verskillende organismes het dieselfde pieke getoon soos geïdentifiseer in die elektroferogramme van die trifluoroasynsuur vertering. Die elektroferogramme met die ensiem vertering het egter addisionele pieke getoon wat nie sigbaar op die elektroferogramme van die trifluoroasynsuur vertering was nie. Hierdie pieke het min of meer dieselfde tyd ge-elueer as die monosakkariede. Kapillêre elektroforese het ook getoon dat die ensiematiese afbraak van die lignienkoolhidraatkompleks gelei het tot algehele vrystelling van sommige suikers, wanneer die trifluoroasynsuur vertering as maatstaaf dien vir die hoeveelheid suikers teenwoordig in die lignienkoolhidraatkompleks. Hoëdruk vloeistof chromatografie het getoon dat geen sigbare afbraak na 72 h van inkubasie met die ensieme op die lignienkoolhidraatkompleks plaasgevind het nie aangesien die piek hoogtes konstant gebly het. Hierdie pieke kon egter nie geïdentifiseer word nie as gevolg van lae resolusie van die pieke asook standaarde wat nie beskikbaar was nie.

published_or_final_version / abstract / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Civil Engineering / Doctoral / Doctor of Philosophy

Thesis (MTech (Environmental Management))--Cape Peninsula University of Technology, 2015. / This study was conducted to investigate the occurrence of PAH degrading microorganisms in two river systems in the Western Cape, South Africa, and their ability to degrade two PAH compounds (acenaphthene and fluorene). A total of 19 bacterial isolates were obtained from the Diep- and Plankenburg Rivers. These microorganisms were first identified phenotypically on various selective and general media (such as nutrient agar, Eosine Methylene Blue and Mannitol Salts Agar), followed by staining and biochemical testing, followed by molecular identification using 16S rRNA and PCR. The isolates were then tested for acenaphthene and fluorene degradation first at flask scale and then in a Stirred Tank Bioreactor at varying temperatures (25ºC, 30ºC, 35ºC, 37ºC, 38ºC, 40ºC and 45ºC). All experiments were run without the addition of supplements, bulking agents, biosurfactants or any other form of biostimulants. Four of the 19 isolated microorganisms were identified as acenaphthene and fluorene degrading isolates. Three of the four microorganisms identified as PAH degrading isolates were Gram negative isolates. Results showed that Raoultella ornithinolytica, Serratia marcescens, Bacillus megaterium and Aeromonas hydrophila efficiently degraded fluorene (99.90%, 97.90%, 98.40% and 99.50%) and acenaphthene (98.60%, 95.70%, 90.20% and 99.90%) at 37ºC, 37ºC, 30ºC and 35ºC, respectively. The degradation of fluorene was found to be more efficient and rapid compared to that of acenaphthene and degradation at Stirred Tank Bioreactor scale was more efficient for all treatments. Throughout the biodegradation experiments, there was an exponential increase in microbial plate counts ranging from 5 x 104 to 9 x 108 CFU/ml. The increase in plate count was observed to correlate with the efficient degradation temperature profiles and percentages. The PAH degrading microorganisms isolated during this study significantly reduced the concentrations of acenaphthene and fluorene and can be used on a larger, commercial scale to bioremediate PAH contaminated river systems. Other factors that influence the optimal expression of biodegradative potential of microorganisms other than temperature and substrate (nutrient) availability, such as pH, moisture and salinity will be investigated in future studies, as well as the factors contributing to the higher fluorene degradation compared to acenaphthene. Furthermore, the structure and toxicity of the by-products and intermediates produced during microbial metabolism of acenaphthene and fluorene should be investigated in further studies.

Ng, Tsz Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 86-98). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vii / List of Figures --- p.xiii / List of Plates --- p.XV / List of Tables --- p.xxi / Abbreviations --- p.xxii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- "Pollution, toxicity and environmental impact of azo dye" --- p.1 / Chapter 1.2 --- Common treatment methods for dyeing effluent --- p.2 / Chapter 1.2.1 --- Physicochemical methods --- p.2 / Chapter 1.2.1.1 --- Coagulation/ flocculation --- p.2 / Chapter 1.2.1.2 --- Adsorption --- p.3 / Chapter 1.2.1.3 --- Membrane filtration --- p.4 / Chapter 1.2.1.4 --- Fenton reaction --- p.4 / Chapter 1.2.1.5 --- Ozonation --- p.5 / Chapter 1.2.1.6 --- Photocatalytic oxidation --- p.6 / Chapter 1.2.2 --- Biological treatments --- p.7 / Chapter 1.2.2.1 --- Degradation of azo dyes by bacteria --- p.8 / Chapter 1.2.2.1.1 --- Anaerobic conditions --- p.8 / Chapter 1.2.2.1.2 --- Aerobic conditions --- p.9 / Chapter 1.2.2.1.3 --- Combined anaerobic and aerobic conditions --- p.10 / Chapter 1.2.2.2 --- Decolourization of azo dyes by fungi --- p.11 / Chapter 1.2.2.3 --- Mechanisms of azo dye reduction by microorganisms --- p.12 / Chapter 1.3 --- "Chromium species, toxicity and their impacts on environment" --- p.14 / Chapter 1.4 --- Common treatment methods for chromium --- p.16 / Chapter 1.4.1 --- Chemical and physical methods --- p.16 / Chapter 1.4.2 --- Biological methods --- p.17 / Chapter 1.4.2.1 --- Chromium reduction by aerobic bacteria --- p.17 / Chapter 1.4.2.2 --- Chromium reduction by anaerobic bacteria --- p.18 / Chapter 1.5 --- Studies concerning azo dye and Cr(VI) co-treatment --- p.19 / Chapter 1.6 --- Response surface methodology --- p.21 / Chapter 1.6.1 --- Response surface methodology against one-factor-at-a-time design --- p.22 / Chapter 1.6.2 --- Phases of response surface methodology --- p.25 / Chapter 1.6.3 --- 2 - level factorial design --- p.26 / Chapter 1.6.4 --- Path of steepest ascent --- p.27 / Chapter 1.6.5 --- Central composite design --- p.28 / Chapter 2. --- Objectives --- p.30 / Chapter 3. --- Materials and Methods --- p.31 / Chapter 3.1 --- Isolation of bacterial strains --- p.31 / Chapter 3.1.2 --- Azo dye decolourization --- p.33 / Chapter 3.1.3 --- Chromate reduction --- p.34 / Chapter 3.2 --- Identification of selected bacterial strains --- p.35 / Chapter 3.2.1 --- Gram stain --- p.35 / Chapter 3.2.2 --- Sherlock® Microbial Identification System --- p.35 / Chapter 3.2.3 --- 16S ribosomal RNA sequencing --- p.37 / Chapter 3.3 --- Optimization of dye decolourization and chromate reduction efficiency with response surface methodology --- p.38 / Chapter 3.3.1 --- Minimal-run resolution V design --- p.38 / Chapter 3.3.2 --- Path of steepest ascent --- p.40 / Chapter 3.3.3 --- Central composite design --- p.41 / Chapter 3.3.4 --- Statistical analysis --- p.43 / Chapter 3.3.5 --- Experimental validation of the optimized conditions --- p.43 / Chapter 3.4 --- Determination of the performance of the selected bacterium in different conditions --- p.43 / Chapter 3.5 --- Determination of azoreductase and chromate reductase activities --- p.44 / Chapter 3.5.1 --- Preparation of cell free extract --- p.44 / Chapter 3.5.2 --- Azoreductase and chromate reductase assay --- p.45 / Chapter 3.6 --- Determination and characterization of degradation intermediates --- p.45 / Chapter 3.6.1 --- Isolation and concentration of the purple colour degradation intermediate --- p.45 / Chapter 3.6.2 --- Mass spectrometry analysis --- p.47 / Chapter 3.6.3 --- Atomic absorption spectrometry analysis --- p.48 / Chapter 4. --- Results --- p.49 / Chapter 4.1 --- Azo dye decolourizing and chromate reducing ability of the isolated bacterial strain --- p.49 / Chapter 4.2 --- Identification of selected bacterium --- p.50 / Chapter 4.3 --- Optimization of dye decolourization and chromate reduction efficiency with response surface methodology --- p.50 / Chapter 4.3.1 --- Minimal-run resolution V design --- p.50 / Chapter 4.3.2 --- Path of the steepest ascend --- p.54 / Chapter 4.3.3 --- Central composite design --- p.55 / Chapter 4.3.4 --- Validation of the predicted model --- p.62 / Chapter 4.4 --- Performance of the selected bacterium in different conditions --- p.62 / Chapter 4.4.1 --- Chromate and dichromate --- p.62 / Chapter 4.4.2 --- Initial pH --- p.63 / Chapter 4.4.3 --- Low and high salt concentration --- p.63 / Chapter 4.4.4 --- Initial K2CrO4 concentration --- p.63 / Chapter 4.4.5 --- Initial Acid Orange 7 concentration --- p.63 / Chapter 4.4.6 --- Nutrients limitation --- p.64 / Chapter 4.5 --- Chromate reductase and azoreductase activities --- p.67 / Chapter 4.6 --- Determination of degradation intermediates --- p.67 / Chapter 4.6.1 --- Mass spectrum of the degradation intermediate --- p.68 / Chapter 4.6.2 --- Chromium content of the degradation intermediate --- p.70 / Chapter 5. --- Discussion --- p.71 / Chapter 5.1 --- Characteristic of Brevibacterium linens --- p.71 / Chapter 5.2 --- Optimization of dye decolourization and chromate reduction with response surface methodology --- p.72 / Chapter 5.3 --- Performance of Brevibacterium linens under different culture conditions --- p.75 / Chapter 5.4 --- Postulation of mechanisms --- p.76 / Chapter 5.4.1 --- Possible reasons of unexpected results of the effect of initial Acid Orange 7 and K2CrO4 concentration --- p.76 / Chapter 5.4.2 --- Properties of the purple colour degradation intermediate --- p.78 / Chapter 5.4.3 --- Mechanisms likely responsible for the chromate reduction --- p.80 / Chapter 5.4.4 --- Explanation of the unexpected results --- p.80 / Chapter 6. --- Conclusions --- p.83 / Chapter 7. --- References --- p.86 / Chapter 8. --- Appendices --- p.99 / Chapter 8.1 --- Definition and calculation of different terms in 2-level factorial design --- p.99 / Chapter 8.2 --- Definition and calculation of different terms in ANOVA table --- p.100 / Chapter 8.3 --- Aliases of terms and resolution --- p.103 / Chapter 8.4 --- Moving of factors in path of steepest ascent --- p.105 / Chapter 8.5 --- Estimation of the parameters in linear regression models --- p.106 / Chapter 8.6 --- Definition and calculation of different terms in test of fitness --- p.109

Cai, Qinhong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 142-166). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of contents --- p.viii / List of figures --- p.xv / List of plates --- p.xix / List of tables --- p.xxi / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Pollution generated from dyeing industry --- p.1 / Chapter 1.2 --- Occurrence and pollution of chromium azo dyes --- p.2 / Chapter 1.3 --- Common treatment methods for dyeing effluents --- p.7 / Chapter 1.3.1 --- Physicochemical methods --- p.7 / Chapter 1.3.2 --- Chemical methods --- p.9 / Chapter 1.3.2.1 --- Ozonation --- p.10 / Chapter 1.3.2.2 --- Fenton reaction --- p.11 / Chapter 1.3.2.3 --- Sodium hypochlorite (NaOCl) --- p.12 / Chapter 1.3.2.4 --- Photocatalytic oxidation (PCO) --- p.13 / Chapter 1.3.3 --- Physical methods --- p.14 / Chapter 1.3.3.1 --- Adsorption --- p.14 / Chapter 1.3.3.2 --- Membrane filtration --- p.15 / Chapter 1.3.4 --- Biological treatments --- p.16 / Chapter 1.3.4.1 --- Decolorization of azo dyes by bacteria --- p.16 / Chapter 1.3.4.1.1 --- Under anaerobic conditions --- p.18 / Chapter 1.3.4.1.2 --- Under anoxic conditions --- p.19 / Chapter 1.3.4.1.3 --- Under aerobic conditions --- p.21 / Chapter 1.3.4.2 --- Mechanisms of azo dye reduction by bacteria --- p.23 / Chapter 1.3.4.3 --- Decolorization of azo dyes by fungi and algae --- p.27 / Chapter 1.4 --- Chromium species and their impacts on environment --- p.27 / Chapter 1.4.1 --- Chromium toxicology and speciation --- p.28 / Chapter 1.4.2 --- Common treatment methods for chromium --- p.31 / Chapter 1.5 --- Studies concerning treatment of chromium azo dyes --- p.32 / Chapter 1.6 --- Response surface methodology (RSM) --- p.33 / Chapter 1.6.1 --- RSM vs. one factor-at-a-time (OFAT) design --- p.36 / Chapter 1.6.2 --- Phases of RSM --- p.39 / Chapter 1.6.3 --- Two level factorial design --- p.40 / Chapter 1.6.4 --- Path of steepest ascent (PSA) --- p.43 / Chapter 1.6.5 --- Central composite design (CCD) --- p.44 / Chapter 1.6.6 --- Estimation of the parameters in linear regression models --- p.45 / Chapter 1.6.7 --- Test of fitness --- p.47 / Chapter 2. --- Objectives and significance of the project --- p.49 / Chapter 3. --- Materials and methods --- p.50 / Chapter 3.1 --- Chemicals --- p.50 / Chapter 3.1.1 --- Chemicals for preparation of bacterial culture media --- p.50 / Chapter 3.1.2 --- Chemicals for identification of bacteria --- p.50 / Chapter 3.1.3 --- Chemicals for chromium speciation --- p.51 / Chapter 3.1.4 --- Chemicals for immobilization of bacterial cells --- p.52 / Chapter 3.2 --- Sludge samples --- p.53 / Chapter 3.3 --- Characterization of Acid Yellow 99 --- p.54 / Chapter 3.4 --- Monitor of azo dye decolorization --- p.55 / Chapter 3.5 --- "Isolation of bacterial strains, which can degrade Acid Yellow 99" --- p.55 / Chapter 3.6 --- Identification of selected bacterial strains --- p.58 / Chapter 3.6.1 --- Gram stain --- p.58 / Chapter 3.6.2 --- Sherlock® microbial identification system --- p.58 / Chapter 3.6.3 --- Biolog® microstation system --- p.59 / Chapter 3.6.4 --- Selection of the most effective bacterial strains --- p.59 / Chapter 3.6.5 --- 16S ribosomal RNA sequencing --- p.60 / Chapter 3.7 --- Chromium speciation with interferences of chromium organic complexes --- p.60 / Chapter 3.7.1 --- Instrumentation --- p.60 / Chapter 3.7.2 --- Column preparation --- p.61 / Chapter 3.7.3 --- Determination of percentage retained and recovery --- p.62 / Chapter 3.7.4 --- "Speciation of Cr(VI), ionic Cr(III) and chromium azo dye" --- p.63 / Chapter 3.7.4 --- Preparation of Cr(III)-organic complexes --- p.65 / Chapter 3.7.5 --- Preparation of a microbial degraded chromium azo dye sample --- p.65 / Chapter 3.8 --- Chromium distribution in a treated solution --- p.66 / Chapter 3.9 --- Distribution of AY99 in a treated solution --- p.68 / Chapter 3.10 --- Optimization of decolorization process with response surface methodology (RSM) --- p.70 / Chapter 3.10.1 --- Correlation of cell mass and cell density of selected bacteria --- p.70 / Chapter 3.10.2 --- Preliminary investigation of the optimum conditions --- p.70 / Chapter 3.10.3 --- Minimal run resolution V (MR5) design --- p.71 / Chapter 3.10.4 --- Path of steepest ascent (PSA) --- p.74 / Chapter 3.10.5 --- Central composite design (CCD) and RSM --- p.75 / Chapter 3.10.6 --- Statistical analysis --- p.76 / Chapter 3.10.7 --- Experimental validation of the optimized conditions --- p.77 / Chapter 3.11 --- Immobilization of bacterial cells --- p.77 / Chapter 3.11.1 --- Immobilization by polyvinyl alcohol (PVA) gels --- p.77 / Chapter 3.11.2 --- Immobilization by polyacrylamide gels --- p.78 / Chapter 3.11.3 --- Performance of immobilized cells and free cells --- p.79 / Chapter 3.11.5 --- Storage stabilities of immobilized cells and free cells --- p.80 / Chapter 3.12 --- Performance of a laboratory scale bioreactor --- p.80 / Chapter 3.12.1 --- Chromium distribution in the bioreactor --- p.82 / Chapter 3.12.2 --- Distribution of AY99 in the bioreactor --- p.82 / Chapter 3.12.3 --- Fourier transform infrared spectroscopy (FT-IR) analysis of suspended particles in the treated solution --- p.84 / Chapter 4. --- Results --- p.85 / Chapter 4.1 --- Characterization of AY99 --- p.85 / Chapter 4.2 --- Identification of isolated bacterial strains --- p.86 / Chapter 4.3 --- Selection of the most effective bacterial strains --- p.89 / Chapter 4.4 --- Chromium speciation with interferences of chromium organic complexes --- p.91 / Chapter 4.4.1 --- Effect of pH --- p.91 / Chapter 4.4.2 --- Speciation of Cr(VI)，ionic Cr(III) and chromium azo dye --- p.92 / Chapter 4.4.3 --- Effect of other Cr(III)-organic complexes --- p.93 / Chapter 4.4.4 --- Limit of detection --- p.94 / Chapter 4.4.5 --- Capacity of Amberlite XAD-4 resin --- p.94 / Chapter 4.4.6 --- Determination of Cr(VI) in a microbial degraded chromium azo dye solution --- p.95 / Chapter 4.5 --- Chromium distribution in a free cells treated solution --- p.95 / Chapter 4.6 --- Distribution of AY99 in free cells treated solution --- p.96 / Chapter 4.7 --- Optimization of decolorization process with RSM --- p.98 / Chapter 4.7.1 --- Correlation of cell mass and cell density of selected bacteria --- p.98 / Chapter 4.7.2 --- MR5 design --- p.100 / Chapter 4.7.3 --- Path of steepest ascent (PSA) --- p.102 / Chapter 4.7.4 --- Central composite design (CCD) and RSM --- p.103 / Chapter 4.8 --- Immobilization of bacterial cells --- p.106 / Chapter 4.8.1 --- Performance of immobilized cells and free cells --- p.106 / Chapter 4.8.2 --- Storage stabilities of immobilized cells and free cells --- p.108 / Chapter 4.9 --- Performance of the laboratory scale bioreactor --- p.108 / Chapter 4.9.1 --- Treatment efficiencies of the bioreactor --- p.108 / Chapter 4.9.2 --- Performance stability of the bioreactor in 5 consecutive runs --- p.111 / Chapter 4.9.3 --- Chromium distribution in the bioreactor --- p.114 / Chapter 4.9.4 --- Distribution of AY99 in the bioreactor --- p.115 / Chapter 4.9.5 --- FT-IR analysis of suspended particles in the treated solution --- p.115 / Chapter 5. --- Discussion --- p.117 / Chapter 5.1 --- Chromium speciation with interferences of chromium organic complexes --- p.117 / Chapter 5.2 --- Chromium distribution --- p.117 / Chapter 5.3 --- Distribution of AY99 --- p.122 / Chapter 5.4 --- Optimization of decolorization process with RSM --- p.124 / Chapter 5.4.1 --- MR5 design --- p.124 / Chapter 5.4.2 --- Path of steepest ascent (PSA) --- p.125 / Chapter 5.4.3 --- Central composite design (CCD) and RSM --- p.126 / Chapter 5.5 --- Immobilization of bacterial cells --- p.126 / Chapter 5.5.1 --- Performance of immobilized cells and free cells --- p.126 / Chapter 5.5.2 --- Storage stability of immobilized cells and free cells --- p.128 / Chapter 5.6 --- Performance of the laboratory scale bioreactor --- p.130 / Chapter 5.6.1 --- Treatment efficiencies of the bioreactor --- p.130 / Chapter 5.6.2 --- Performance stability of the bioreactor in 5 consecutive runs --- p.131 / Chapter 5.6.3 --- FT-IR analysis of suspended particles in the treated solution --- p.132 / Chapter 5.6.4 --- Post treatments of bioreactor treated effluents / Chapter 6. --- Conclusions --- p.136 / Chapter 7. --- References --- p.142

Cellulose is the most abundant and renewable biopolymer on earth. Generally, it is well known for its structure properties or its natural and industrial derivatives due to its biodegradability or easily controlled transformation. By contrast, non-biodegradable cellulose is relatively unusual and not intensively investigated. The discovery of well preserved Miocene fossil woods during a recent excavation from the Entre-Sambre-et- Meuse (ESEM) karsts (southern Belgium) gives a rare opportunity to investigate resistant cellulose. The wood specimens were examined using physicochemical and biochemical techniques in order to correlate the exceptional preservation of these fossilized remains after 15 million years to the non-biodegradability of their lignocellulose content. Structural and chemical changes were assessed by comparing the structural features of the fossil samples with those of their modern counterpart, Metasequoia. Solid state 13C nuclear magnetic resonance (NMR) and microscopic analysis showed good preservation of the cellulose structure in the fossil wood from the ESEM peat deposit. Moreover, there also appears to be a complete loss of hemicelluloses in the fossil wood structure maybe due to their branched structure and their lower molecular weight. According to several authors, the peatification, the initial biochemical stage of coalification, is characterized by a complete loss of hemicelluloses and a significant reduction in cellulose, which is completely degraded after a short span of geological time. However, the cellulose content, similar to that found in the living species Metasequoia, suggests that the ESEM fossil wood is at a very early stage of peatification. Compared to the lignin in modern Metasequoia, the lignin structure has undergone slight chemical alteration such as demethoxylation, cleavage of some b-O-4¢ linkages and alkylation of the resulting catechol-like structures. Furthermore, since uncondensed structures were degraded in the fossil woods, the condensed structures already present in the original lignin, became more dominant. Despite the substantial cellulose fraction available in the fossil tissue, degradation tests showed that the fossil cellulose could not be degraded by cellulases and microorganisms usually involved in the biodegradation of organic matter. This lack of bioavailability could be due to the structural features of the fossil biopolymers (cellulose, extractives and lignin) or the burial environment. In our work, we have analyzed the influence of the cristallinity, the size of crystallites, the type of crystal lattice and the ratio of two allomorphs I a/I b as well as the morphology of the cellulose microfibrilles on the cellulose digestibility. These cellulose structural features seemed to have no effect on cellulose biodegradability in the Miocene woods samples. On the other hand, the wood extractives (tannins, resin acids, terpenes,) may contribute to some resistance properties of the fossil cellulose but their action is not sufficient to explain the inhibition of the cellulose degradation. On the basis of our observations, we suggest that the presence of a modified lignin structure greatly influences cellulose biodegradability. Indeed, by altering the lignin structure with various delignification pretreatments and studying the effect of the resulting changes on enzymes efficiency, we showed that the disintegration of the condensed structures, linked to the drastic reduction of the uncondensed structures, could have significant impact on the major improvement of the cellulose bioavailability. In conclusion, the nature and/ or the proportion of intermolecular substructures could be the key of the cellulose protection. La cellulose est le biopolymère le plus abondant et le plus renouvelable sur terre. Généralement, il est reconnu pour ses propriétés structurales ou ses dérivés naturels et industriels, eux-mêmes caractérisés par leur biodégradabilité ou leur mise en uvre facilement contrôlée. Par contraste, une cellulose non-biodégradable est relativement inhabituelle et peu étudiée. La découverte de bois fossiles du Miocène extrêmement bien préservés au cours dexploitations récentes de karsts de lEntre-Sambre-et-Meuse (ESEM, Belgique) offre une rare opportunité détudier une cellulose résistante. Les spécimens de bois ont été examinés à laide de techniques physicochimiques et biochimiques en vue de corréler la préservation exceptionnelle de ces macrofossiles après 15 millions dannées à la non-biodégradabilité de leur contenu cellulosique. Les modifications structurales et chimiques ont été attribuées en comparant les caractéristiques des échantillons fossiles avec ceux de leur équivalent moderne, le Metasequoia. Les analyses microscopiques et par résonance magnétique nucléaire C13 de létat solide ont révélé une préservation importante de la structure cellulosique dans les bois fossiles des dépôts argilo-tourbeux de lESEM. De plus, la concentration en cellulose est proche de celle présente dans lespèce moderne, Metasequoia. Par contre, une perte complète des structures hémicellulosiques est constatée. Celle-ci pourrait être attribuée à leurs structures branchées et leurs faibles poids moléculaires. Ces observations suggèrent que les bois fossiles de lESEM se situent à un stade très précoce de la turbification. En effet, selon différents auteurs, la turbification, le stade biochimique initial de la coalification, est caractérisée par une perte complète des hémicelluloses et une réduction significative de la cellulose, qui est complètement dégradée après un très court intervalle de temps géologique. Par comparaison avec la lignine de lespèce moderne, la structure ligneuse fossile a subi de légères altérations chimiques telles que des déméthoxylations, le clivage de certains liens intermoléculaires (b-O-4¢ en majorité) et lalkylation des structures catéchols résultantes. En outre, les structures condensées déjà présentes dans la lignine dorigine, sont devenues plus dominantes par rapport aux structures non-condensées qui ont subi de fortes dégradations dans les bois fossiles. Malgré la fraction cellulosique substantiellement accessible dans les tissus fossiles végétaux, les tests de dégradation ont montré que la cellulose fossile ne pouvait être que faiblement dégradée par des cellulases et les microorganismes impliqués habituellement dans les processus de biodégradation de la matière organique. Ce manque de réactivité pourrait être lié aux caractéristiques structurales des biopolymères fossiles (cellulose, composés extractibles et lignine) ou à la matrice denfouissement. Limplication de celle-ci dans le processus de préservation de la cellulose a été écarté car les conditions environnementales présentes actuellement dans les karsts auraient du favoriser la biodégradabilité de ces bois. Au cours de nos recherches, nous avons analysé linfluence de la cristallinité, la taille des cristallites, le type de réseau cristallin et le rapport entre les allomorphes I a/I b ainsi que la morphologie des microfibrilles de cellulose sur la digestibilité de la cellulose. Ces paramètres structuraux associés à la cellulose ne semblent pas avoir deffet sur la biodégradabilité de la cellulose dans les échantillons de bois du Miocène. Dautre part, les composés extractibles (tannins, acides résiniques, terpènes,) peuvent contribuer à la capacité de résistance à la décomposition de la cellulose fossile mais leur action nest pas suffisante pour expliquer linhibition importante de la dégradation de la cellulose. Sur base de nos observations, nous suggérons que la présence dune structure ligneuse modifiée influence fortement la biodégradabilité de la cellulose. En effet, en altérant la structure ligneuse avec différents prétraitements de délignification et en étudiant limpact des transformations structurales sur lefficacité de la dégradation enzymatique, nous avons montré que la désagrégation des structures condensées, liée à la diminution drastique des structures non-condensées, pourrait avoir un impact significatif sur lamélioration majeure de la bioaccessibilité du substrat cellulosique. En conclusion, la nature et/ou la proportion des sous-unités intermoléculaires présentes dans la structure ligneuse pourrait être la clé de la protection exercée par la lignine.

Graduation date: 1999

Microcosm studies were conducted beginning with three xylene isomers: ortho-xylene, meta-xylene and para-xylene; and continued with the four mononitroxylene (MNX) isomers, culminating with testing ten dinitroxylene (DNX) isomers. Soil samples were obtained from a historically contaminated site with high levels of dinitrotoluene (DNT), trinitrotoluene (TNT) and dinitroxylene (DNX) and used as the inoculum for microcosm tests. The microcosm method of different isomers was based on the previous work on biodegradation of nitrotoluene. As it was demonstrated previously that 2,4-DNT degrading bacteria were present at the site, it was hypothesized that these may be capable of transforming or cometabolizing some of DNX isomers. Thus, DNX cometabolism studies were conducted in the presence of 2,4-DNT degrading bacteria. The presence of xylene and 2,4-DNT degrading was confirmed in this thesis. Meanwhile, several MNX and DNX isomers showed degradability in microcosm studies. Cometabolism studies showed that four DNX isomers could be cometabolized by 2,4-DNT enrichment.

No description available.