The research described in this thesis was aimed at understanding how particle size, porosity, and enzyme accessible surface area influence the rate of saccharification of microcrystalline cellulose. Microcrystalline cellulose (MCC) is a commonly used substrate for the study of cellulolytic enzymes. MCC preparations of different particle size are commercially available. In this study, MCC preparations having average particle sizes of 20, 50, and 90 μm were analyzed with respect to their enzyme accessible surface area, chemical and physical properties and rates of enzymatic saccharification. Saccharification studies were done using a commercially available cellulase preparation from Trichoderma reesei. Pore volume distributions were determined from solute exclusion experiments. Internal surface areas were calculated based on the application of the lamellae model to the pore volume distribution data. External surface areas were calculated based on the average particle size of each MCC preparation assuming that the particles could be represented as solid spheres. The different MCC preparations were found to have nearly equivalent enzyme accessible surface areas per unit weight. Greater than 99 % of the total enzyme accessible surface area for each MCC preparations was found to be within the porous structure of the particles. Enzymatic saccharification experiments demonstrated that the smaller particle size MCCs were more readily digested than those of larger particle size. The similarity of the three MCC preparations with respect to chemical and physical properties (other than particle size), pore volume distribution, and total enzyme accessible surface area suggests that a rate limiting factor in the enzymatic digestion of MCC is a resistance attributable to diffusion within the capillary network of these insoluble substrates. / Graduation date: 1996

Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl esterase from Aureobasidium pullulans were set as the primary objectives of this study. A further objective was to investigate a possible co-operative effect with other selected lignocellulolytic enzymes on substrates relevant to industry. In a comprehensive review, feruloyl esterases from various micro-organisms were compared both functionally and with regard to their primary structure, where applicable. Feruloyl esterases show intriguing differences in substrate specificity and sequence structure. Enzymes that are closely related regarding their amino acid sequence exhibit different substrate specificities. Sequence similarities can be found with a range of other enzyme families, including serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl hydrolases and xylanases. More data on the three dimensional structure of feruloyl esterases as well as an examination of all available feruloyl esterases with the same substrates is necessary before structure-function relationships can be established and before the feruloyl esterases can be organized into discrete families based on ancestral origins. The highest production levels of feruloyl esterase by A. pullulans are achieved when grown on birchwood xylan. Expression was not repressed when glucose or xylose was present in the medium. However, free ferulic acid supplemented to the medium affected fungal growth and therefore did not increase feruloyl esterase activity. It is also suggested that the synthesis of feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as naphthol AS-D chloroacetate as substrates. Feruloyl esterase from A. pullulans was purified to homogeneity using ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic interaction and ultimately gel filtration chromatography. With a molecular weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported to date. Kinetic data was produced using both synthetic and natural substrates. A. pullulans feruloyl esterase shows properties similar to other fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal sequence of A. pullulans feruloyl esterase was identified, but no similarities to known enzyme families were found. Peptide mass mapping did not reveal structural information. In an effort to evaluate the significance of feruloyl esterase from A. pullulans in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse were selectively treated using feruloyl esterase and hemicellulolytic enzymes. The enzymatic degradation reaction was monitored using microdialysis sampling, anion exchange chromatography, online desalting and mass spectrometry. It has been shown, that feruloyl esterase activity together with xylanase activity releases monosaccharides from both substrates. Sugars of higher degree of polymerisation were not released, giving evidence for the recalcitrance of the material. The fibre architecture of the substrates was apparently not accessible to the enzymes and therefore complete hydrolysis was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant is, bestaan. Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt van funksie en primêre struktuur omvattend met mekaar vergelyk, waar toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur van feruloïel esterase kan waargeneem word. Ensieme wat nou aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase, lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n analise van al die beskikbare feruloïel esterase met dieselfde substrate is nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat die feruloïel esterases in eie families op die grond van huloorsprong georganiseer kan word. Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3- naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is die ensiem die grootste van die feruloïel esterases tot dusver beskryf. Kinetiese data is met behulp van sintetiese en natuurlike substrate geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting gee nie. Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel esterase en lignosellulitiese ensieme behandel om die belang van feruloïel esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie, oplyn ontsouting en massaspektrometrie gemonitor. Wanneer die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele hidroliese verhinder.

The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

The biological degradation of lignocellulose into fermentable sugars for the production of liquid transportation fuels is feasible and sustainable, but equires a variety of enzymes working in synergy as lignocellulose is a complex and recalcitrant substrate. The cellulosome is a multi-enzyme complex (MEC) with a variety of cellulolytic and hemicellulolytic enzymes that appears to facilitate an enhanced synergy and efficiency, as compared to free enzymes, for the degradation of recalcitrant substrates such as lignocellulose and plant cell walls. Most of the studies on cellulosomes have focused on a few organisms; C. thermocellum, C. cellulovorans and C. cellulolyticum, and there is only limited knowledge vailable on similar complexes in other organisms. Some MECs have been identified in aerobic bacteria such as Bacillus circulans and Paenibacillus curdlanolyticus, but the nature of these MECs have not been fully elucidated. This study investigated the cellulolytic and emi-cellulolytic system of Bacillus licheniformis SVD1 with specific reference to the presence of a MEC, which has never been reported in the literature for B. licheniformis. A MEC of approximately 2,000 kDa in size, based on size exclusion chromatography using Sepharose 4B, was purified from a culture of B. licheniformis. When investigating the presence of enzyme activity in the total crude fraction as well as the MEC of a birchwood xylan culture, B. licheniformis was found to display a variety of enzyme activities on a range of substrates, although xylanases were by far the predominant enzyme activity present in both the crude and MEC fractions. Based on zymogram analysis there were three CMCases, seven xylanases, three mannanases and two pectinases in the crude fraction, while the MEC had two CMCases, seven xylanases, two mannanases and one pectinase. The pectinases in the crude could be identified as a pectin methyl esterase and a lyase, while the methyl esterase was absent in the MEC. Seventeen protein species could be detected in the MEC but only nine of these displayed activity on the substrates tested. The possible presence of a β-xylosidase in the crude fraction was deduced from thin layer chromatography (TLC) which demonstrated the production of xylose by the crude fraction. It was furthermore established that B. licheniformis SVD1 was able to regulate levels of enzyme expression based on the substrate the organism was cultured on. It was found that complexed xylanase activity had a pH optimum of between pH 6.0 and 7.0 and a temperature optimum of 55oC. Complexed xylanase activity was found to be slightly inhibited by CaCl2 and inhibited to a greater extent by EDTA. Complexed xylanase activity was further shown to be activated in the presence of xylose and xylobiose, both compounds which are products of enzymatic degradation. Ethanol was found to inhibit complexed xylanase activity. The kinetic parameters for complexed xylanase activity were measured and the Km value was calculated as 2.84 mg/ml while the maximal velocity (Vmax) was calculated as 0.146 U (μmol/min/ml). Binding studies, transmission electron microscopy (TEM) and a bioinformatic analysis was conducted to investigate whether the MEC in B. licheniformis SVD1 was a putative cellulosome. The MEC was found to be unable to bind to Avicel, but was able to bind to insoluble birchwood xylan, indicating the absence of a CBM3a domain common to cellulosomal scaffoldin proteins. TEM micrographs revealed the presence of cell surface structures on cells of B. licheniformis SVD1 cultured on cellobiose and birchwood xylan. However, it could not be established whether these cell surface structures could be ascribed to the presence of the MECs on the cell surface. Bioinformatic analysis was conducted on the available genome sequence of a different strain of B. licheniformis, namely DSM 13 and ATCC 14580. No sequence homology was found with cohesin and dockerin sequences from various cellulosomal species, indicating that these strains most likely do not encode for a cellulosome. This study described and characterised a MEC that was a functional enzyme complex and did not appear to be a mere aggregation of proteins. It displayed a variety of hemi-cellulolytic activities and the available evidence suggests that it is not a cellulosome, but should rather be termed a xylanosome. Further investigation should be carried out to determine the structural basis of this MEC.

A significant trend in the modern day industrial biotechnology is the utilization and application of renewable resources, and ecofriendly approach to industrial processes and waste management. As a consequence thereof, the biotechnology of holocellulases: cellulase and xylanase and, enzymatic hydrolysis of renewable and abundant lignocellulosic biomass to energy and value added products are rapidly increasing; hence, cost effective enzyme system is imperative. In that context, exploration of microbiota for strains and enzymes with novel industrial properties is vital for efficient and commercially viable enzyme biotechnology. Consequent on the complex characteristics of high salinity, variable pressure, temperature and nutritional conditions, bacterial strains from the marine environment are equipped with enzyme machinery of industrial importance for adaptation and survival. In this study, bacterial strains were isolated form Nahoon beach and optimized for holocellulase production. Three isolates selected for lignocellulolytic potential were identified by 16S ribosomal deoxyribonucleic acid (rDNA) sequence analysis. Isolate FS1k had 98 percent similarity with Streptomyces albidoflavus strain AIH12, was designated as Streptomyces albidoflavus strain SAMRC-UFH5 and deposited in the GenBank with accession number KU171373. Similarly, isolates CS14b and CS22d with respective percentage similarity of 98 and 99 (percent) with Bacillus cereus strains and Streptomyces sp. strain WMMB251 were named Bacillus cereus strain SAMRC-UFH9 and Streptomyces sp. strain SAMRC-UFH6; and were deposited in the GenBank with accession number KX524510 and KU171374 respectively. Optimal pH, temperature and agitation speed for cellulase production by S. albidoflavus strain SAMRC-UFH5, and B. cereus strain SAMRC-UFH9 were 6 and 7; 40 and 30 (°C); and 100 and 150 (rpm) respectively; while xylanase production was optimal at pH, temperature and agitation speed of 8 and 7; 40 and 30 (°C); and 150 and 50 (rpm) respectively. Maximum cellulase activity of 0.26 and 0.061(U/mL) by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 were attained at 60 h respectively, while maximal xylanase activity of 18.54 and 16.6 (U/mL) was produced by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 at 48 h and 60 h respectively. Furthermore, xylanase production by S. albidoflavus strain SAMRC-UFH5 and B. cereus strain SAMRC-UFH9 was maximally induced by wheat straw and xylan respectively, while cellulase production was best induced by mannose and carboxymethyl cellulose respectively. On the other hand, cellulase and xylanase production by Streptomyces sp. strain SAMRC-UFH6 was optimal at pH, temperature and agitation speed of 7 and 8, 40 °C and 100 rpm, respectively. Highest production of cellulase and xylanase was attained at 84 and 60 h with respective activity of 0.065 and 6.34 (U/mL). In addition, cellulase and xylanase production by the strain was best induced by beechwood xylan. Moreover, xylanase produced by Streptomyces sp. strain SAMRC-UFH6 at optimal conditions was characterized by optimal pH and temperature of 8 and 80-90 °C respectively; retaining over 70 percent activity at pH 5-10 after 1 h and 60 percent of initial activity at 90 °C after 90 min of incubation. In all, optimization improved cellulase and xylanase production yields, being 40 and 95.5, 10.89 and 72.17, and 10 and 115- fold increase by S. albidoflavus strain SAMRC-UFH5, B. cereus strain SAMRC-UFH9 and Streptomyces sp. SAMRC-UFH6 respectively. The results of this study suggest that the marine bacterial strains are resource for holocellulase with industrial applications.

Lignocellulosic biomass is a potential source of fermentable sugars such as glucose. Enzymatic hydrolysis of cellulose is a viable method of solubilizing the glucose from biomass, but the cellulose fraction of native lignocellulosic material is shielded from enzymatic attack by the lignin-hemicellulose matrix surrounding it. Pretreating lignocellulosic biomass with dilute sulfuric acid at high temperatures solubilizes hemicellulose, rendering the cellulose fraction more susceptible to enzymatic hydrolysis. Evaluation of dilute-acid, high-temperature pretreatments depends on polysaccharide analysis of the two fractions resulting from a pretreatment, prehydrolyzed solids(PHS) and prehydrolyzate liquid(PH). The polysaccharide analysis is based on a method described by the National Renewable Energy Laboratory and involves a two-stage sulfuric acid hydrolysis followed by HPLC quantification using ion-moderated partition chromatography and refractive index detection. The subject of this thesis is identifying and quantifying the sources of error associated with the polysaccharide analysis and the error associated with the evaluation of the effects of pretreatment on the polysaccharide fractions of switchgrass and poplar. This was addressed by conducting replicate polysaccharide analyses on single samples of native biomass, PHS, and PH. The variability associated with these measurements was compared to the variability associated with replicate analyses of identically pretreated biomass. It was found that the use of sugar standards to correct for sugar destroyed during the analysis adds error and most likely overestimates the amount of sugar from biomass actually destroyed. It is evident that assuming a volume after neutralization of the hydrolyzed biomass sample is more reproducible than measuring the volume. When using a batch-type reactor and the temperature and acid parameters used in this study,140°C-180°C/ 0.6-1.2 % sulfuric acid (w/w), it is evident that the major source of error in evaluating pretreatment conditions is the pretreatment itself, not the analysis. / Graduation date: 1995

The need to reduce the cost of adsorption technology has led scientists to explore the use of many low cost adsorbents especially those from renewable resources. Lignocellulose and montmorillonite clay have been identified as potentially low cost and efficient adsorbent materials for the removal of toxic heavy metals and organic substances from contaminated water. Montmorillonite clay has good adsorption properties and the potential for ion exchange. Lignocellulose possesses many hydroxyl, carbonyl and phenyl groups and therefore, both montmorillonite and lignocellulose are good candidates for the development of effective and low cost adsorbents in water treatment and purification. The aim of this study was to prepare composite materials based on lignocellulose and montmorillonite clay and subsequently evaluate their efficacy as adsorbents for heavy metal species and organic pollutants in aqueous solution. It was also important to assess the adsorption properties of the modified individual (uncombined) lignocellulose and montmorillonite. Lignocellulose and sodium-exchanged montmorillonite (NaMMT) clay were each separately modified with methyl methacrylate (MMA), methacrylic acid (MAA) and methacryloxypropyl trimethoxysilane (MPS) and used as adsorbents for the removal of heavy metals and dyes from aqueous solution. The lignocellulose and NaMMT were modified with MMA, MAA and MPS through free radical graft polymerisation and/or condensation reactions. NaMMT was also modified through Al-pillaring to give AlpMMT. The materials were characterised by fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and small angle X-ray scattering (SAXS) and characterisation results showed that the modification of the montmorillonite with MAA, MMA and MPS was successful. The modified lignocellulose and montmorillonite materials were evaluated for the adsorption of heavy metal ions (Cd2+ and Pb2+) from aqueous solution by the batch method. The adsorption isotherms and kinetics of both Cd2+ and Pb2+ onto the NaMMT clay, AlpMMT and lignocellulose materials are presented. The Langmuir isotherm was found to be the best fit for the adsorption of both heavy metals onto all the adsorbents. AlpMMT showed very poor uptake for heavy metals (both Cd2+ and Pb2+). PMMAgMMT, PMAAgMMT, PMAAgLig and PMPSgLig showed improved adsorption for both heavy metals. The mechanism of heavy metal adsorption onto the adsorbents was best represented by the pseudo second-order kinetic model. PMPSgLig, NaMMT and AlpMMT showed relatively high adsorption capacities for methyl orange, while the adsorption of neutral red was comparable for almost all the adsorbents. Neither the Langmuir model nor the Freundlich model was found to v adequately describe the adsorption process of dyes onto all the adsorbents. The pseudo second-order model was found to be the best fit to describe the adsorption mechanism of both dyes onto all the adsorbents. The modification of lignocellulose and montmorillonite with suitable organic groups can potentially produce highly effective and efficient adsorbents for the removal of both heavy metals and dyes from contaminated water. Novel adsorbent composite materials based on lignocellulose and montmorillonite clay (NaMMT) were also prepared and evaluated for the removal of pollutants (dyes and heavy metals) from aqueous solution. The lignocellulose-montmorillonite composites were prepared by in situ intercalative polymerisation, using methyl methacrylate, methacrylic acid and methacryloxypropyl trimethoxysilane (MPS) as coupling agents. The composite materials were characterised by FTIR, TGA, TEM and SAXS. SAXS diffractograms showed intercalated nanocomposites of PMMAgLig-NaMMT and PMAAgLig-NaMMT, whereas PMPSgLig-NaMMT showed a phase-separated composite and the same results were confirmed by TEM. The lignocellulose-montmorillonite composites were assessed for their adsorption properties for heavy metal ions (Cd2+ and Pb2+) and dyes (methyl orange and neutral red) from aqueous solution. Among these composite materials, only PMAAgLig-NaMMT showed a marked increase in the uptake of both Cd2+ and Pb2+ relative to lignocellulose and montmorillonite when used independently. The adsorption data were fitted to the Langmuir and Freundlich isotherms, as well as to the pseudo first-order and pseudo second-order kinetic models. The data were best described by the Langmuir isotherm and the pseudo second-order kinetic model. On the adsorption of dyes, only PMPSgLig-NaMMT showed enhanced adsorption of methyl orange (MetO) compared with lignocellulose and montmorillonite separately. The enhanced adsorption was attributed to the synergistic adsorption due to the presence of MPS, lignocellulose and NaMMT. Competitive adsorption studies were carried out from binary mixtures of MetO and Cd2+ or Pb2+ in aqueous solution. The adsorption process of MetO onto the composite material was found to follow the Freundlich adsorption model, while the mechanism of adsorption followed both the pseudo first-order and pseudo second-order models. This particular composite can be used for the simultaneous adsorption of both heavy metals and organic dyes from contaminated water. The adsorption of neutral red to the composite materials was comparable and the pseudo second-order kinetic model best described the adsorption mechanism.

Acid mine drainage is a well known environmental pollutant, not only in South Africa, but throughout the world, and the use of microbial processes in the treatment of these wastes has been the subject of investigation over past decades. Lignocellulose packed-bed reactors have been used in passive treatment systems, and, although effective initially, they show early decline in performance while the packing material remains largely un-utilized. Little is known about this phenomenon which remains a severe constraint in the development of efficient passive mine water treatment systems. It has been proposed that the degradation pathways of the complex lignocellulose substrate may be limited in some way in these systems during the manifestation of this effect. This study has addressed the problem using a molecular microbial ecology methodology in an attempt to relate trophic functions of the microbial population to the physico-chemical data of the system. A field-scale lignocellulose packed-bed reactor located at Vryheid Coronation Colliery (Northern Kwa-Zulu Natal province, South Africa) was monitored for six years and the results showed the classic profile of performance decline related to a slowdown in sulphate reduction and alkalinity production. The reactor was decommissioned , comprehensive samples were collected along the depth profile and the microbial populations investigated by means of 16S rRNA gene methodology. The population was found to include cellulolytic Clostridia spp., CytophagaIFlavobacterlBacteroidetes, Sphingomonadaceae and as yet uncultured microorganisms related to microbiota identified in the rumen and termite gut. These are all known to be involved as primary fermenters of cellulose. Oesulphosporosinus was present as sulphate reducer. A comparison of substrata sampling and population distribution suggested that spatial and temporal gradients within the system may become established over the course of its operation. Based on these findings, a laboratory-scale reactor was constructed to simulate the performance of the packed-bed reactor under controlled experimental conditions. The laboratory-scale reactor was operated for 273 days and showed comparable performance to that in the field in both biomolecular and physicochemical data. Clearly defined trophic niches were observed. These results suggested that a sequence of events does occur in lignocellulose degradation over time. Based on the spatial and temporal column studies, a descriptive model was proposed to account for these events. It was found that fermentative organisms predominate in the inlet zone of the system using easily extractable compounds from the wood, thus providing feedstock for sulphate reduction occurring in the succeeding compartments. Production of sulphide and alkalinity appears to be involved in the enhancement of lignin degradation and this, in turn, appears to enhance access to the cellulose fraction. However, once the readily extractables are exhausted, the decline in sulphide and alkalinity production leads inexorably to a decline in the overall performance of the system as a sulphate reducing unit operation. These observations led to the proposal that with the addition of a limited amount of a readily available carbon source, such as molasses, in the initial zone of the the reactor, the ongoing generation of sulphide would be sustained and this in turn would sustain the microbial attack on the lignocellulose complex. This proposal was tested in scale-up studies and positive results indicate that the descriptive model may, to some extent, provide an account of events occurring in these systems. The work on sustaining lignocellulose degradation through the maintenance of sulphate reduction in the initial stages of the reactor flow path has led to the development of the Degrading Packed-bed Reactor concept and that, has subsequently been successfully evaluated in the field.

No description available.

South Africa is renowned for its mining industry. The period over which the polluted waters from the existing and abandoned mines will require treatment has driven research into the development of passive treatment systems. These waters are characterised by a low pH, high concentrations of heavy metals, high levels of sulphate salts and low concentrations of organic material. The biological treatment of these waters has been a subject of increasing focus as an alternative to physicochemical treatment. The utilisation of lignocellulose as a carbon source has been restricted by the amount of reducing equivalents available within the lignocellulose matrix. After a few months of near 100% sulphate reduction, it was found that although there was a large fraction of lignin and cellulose remaining, sulphate reduction was reduced to less than 20%. The present study demonstrated that lignocellulose can be utilised as a carbon source for sulphate reduction. It was established that lignocellulose degradation was enhanced under biosulphidogenic conditions and that lignin could be degraded by a sulphate reducing microbial consortium. It was established using lignin model compounds synthesized in our laboratory, that the bonds within the lignin polymer can be cleaved within the sulphidic environment. The presence of cellulolytic enzymes, using CMCase as a marker enzyme, was detected within the sulphate reducing microbial consortium. Based on the results obtained a descriptive model was formulated for the degradation of lignocellulose under biosulphidogenic conditions. It was determined that the initial reduction in sulphate observed using lignocellulose as a carbon source was due to the easily extractable components. The degradation of which resulted in the production of sulphide, which aided in the degradation of lignin, allowing greater access to cellulose. Once the easily extractable material is exhausted, the cycle is halted, unless the sulphide production can be maintained. This is the focus of an ongoing project, testing the hypothesis that an easy to assimilate carbon source added after exhaustion of the easily extractable material, can maintain the sulphide production.