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The recent development of culture-independent methods has revolutionized the study of complex microbial communities such as those present in activated sludge treatment systems. DNA probes that hybridize to genes coding for key enzymes that catalyze microbial processes have been widely used. Can such probes be used to quantify target genes and thus quantify the potential of a microbial community to carry out a reaction of interest? / Optimal conditions for DNA extraction, probe validation, hybridization, and activity measurements were determined for the pulp and paper treatment system environment under study. Using gene probes for key denitrification genes (nirS, nirK), the correlation between denitrifiers and denitrification activity in an enrichment culture and activated sludge samples was tested. The same correlation between nitrogen fixation and nitrogen-fixing bacteria in primary clarifiers was assessed using a probe for the gene encoding a component of the nitrogenase enzyme (nifH). This work was successful in establishing the correlation between gene numbers and their corresponding enzymatic activity and thus supports the quantitative hybridization approach for the monitoring of microbial communities. (Abstract shortened by UMI.)

Many low molecular weight chlorinated organic compounds produced by the pulp and paper industry during kraft bleaching of the wood pulp are toxic. Mass balance studies suggest that mineralization of chlorinated organics is occurring in pulp and paper mill wastewater treatment systems. To understand the nature of dechlorination activity, molecular tools such as oligonucleotide primers and corresponding DNA probes were developed to monitor the presence of microorganisms possessing key genes (dehalogenases) responsible for the degradation of chloroaliphatic organics in kraft pulp mill effluent treatment systems. Oligonucleotide primers designed from the coding sequence of known dehalogenases and a methane monooxygenase gene, which is known to catalyze dehalogenation reactions, were used for polymerase chain reaction (PCR) analysis, using genomic DNA extracted from dehalogenating bacterial isolates and total community DNA extracted from water and sediments of lagoon treatment systems. PCR amplification with dhlB oligonucleotide primers, designed from the haloacid dehalogenase of Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similar results were obtained with mmoX primers designed from the methane monooxygenase gene of Methylococcus capsulatus. DNA sequence analysis of several PCR fragments showed significant similarity to known dehalogenase genes. The molecular tools developed in this study revealed the presence of different types of microorganisms with dechlorination potential in the microbial community of pulp mill effluents.

Knowledge of price responsiveness of energy is important for designing effective price-based controls to curb the GHG emissions in Canada. The translog and logit models are developed in this study to analyze the demand for four types of energy inputs: coal, electricity, natural gas and refined petroleum products in Ontario's pulp and paper industry. The results suggest that the industry is inelastic to price change of energy consumed. Tests indicate that the translog model behaves slightly better than the logit model. The translog model was then applied to study the feasibility of imposing a carbon tax on energy inputs on Ontario's pulp and paper industry, which indicated that this sector does not seem to response to changes in energy inputs prices. Therefore, a carbon tax does not seem to be a good policy option for decreasing greenhouse gas emissions in this sector.

The influence of nitrogen (N) and phosphorus (P) limitation on pulp and paper mill activated sludge (AS) floc properties was studied using a bioreactor fed with synthetic Kraft mill effluent. The bioreactor and synthetic effluent were designed and shown to perform like the real mill system providing the AS, establishing the in vivo relevance of the results. Limitation of either N or P produced inadequate effluent biotreatment, shown by poor BOD5 and suspended solids removal, and by decreased biomass health, performance, and floc settling. Greatly enhanced poly-beta-hydroxybutyrate (PHB) (but not carbohydrate or extracellular polymeric substances [EPS]) synthesis was the common response of the floc microbial community to N limitation over many days. In contrast, P-limitation increased total carbohydrate and EPS, but not PHB. / N limitation, but not P limitation, caused the net floc surface charge to be much more negative, while P-limitation, but not N-limitation, increased the floc bound water content and surface hydrophobicity. Thus, in real pulp and paper mill AS systems, careful manipulation of N or P additions may be useful to optimize the key process of charged polymer-assisted AS dewatering.

Eucalyptus, pine and wattle are the predominant exotic wood species used in the production of dissolving pulp in South Africa. On entering the mill, wood is chipped and stored in outdoor piles where it becomes vulnerable to microbial degradation and spontaneous combustion. Major losses of stored chips are due to high temperatures and combustion caused by heat energy released by microbial fermentation. Changes in the chemistry of the wood chips caused by the metabolic activity of indigenous microflora combined with the inherent chemical characteristics of each wood species could have a potential impact on final pulp quality and yield. Therefore the objective of this study was to analyse the microbial (bacteria and fungi) communities present in commercial wood chip piles and correlate this with changes in the chemistry of the wood in summer and winter. The molecular fingerprinting technique of Denaturing Gradient Gel Electrophoresis (DGGE) was optimized for the detection of microbial diversity in commercial wood chips. Wood chips were collected from an industrial wood yard and milled to different specifications. A total of four primer sets with GC-clamps were tested in nested PCR for DGGE analysis. 16S and 18S rRNA genes were amplified using 338f-GC/518r; 933F-GC/1387R (bacteria) and NS26/518R-GC; EF4F/518R-GC (fungi), respectively. Several gel gradients were examined to determine optimal separation of bacterial (40/60%, 35/50%, 30/60%) and fungal (35/50%, 20/45%, 25/50%) PCR-DGGE products. Comparison of the DGGE profiles revealed greater diversity in the milled wood chips amplified using primer sets; 338F-GC/518R (16S) and NS26/518R-GC (18S) with gradients of 30/60% (16S) and 25/50% (18S). Once optimized, this standardized protocol was tested against five samples to assess its applicability to woodyard samples. 16S and 18S DGGE profiles were generated and amplicons excised from gels, re-amplified, sequenced and the microorganism from which the DNA originated was determined. In the second phase a cross-sectional study of wood chip piles from a commercial dissolving pulp mill was conducted with sample collected in summer and winter using the optimized PCR-DGGE technique. Microbial strains were identified after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Chemical characteristics of the wood chips were evaluated by conducting extractive analyses using HPLC. Due to unpredictable combinations of different wood species in commercial wood chip piles, the third phase involved the investigation of individual Eucalyptus species. The microflora indigenous to the two Eucalyptus species (E. dunnii and E. nitens) and a combination of the two were subjected to winter and summer simulations for one month during which samples were tested for wood chemistry properties, microflora and the final samples were used to generate dissolving pulp. Using the PCR-DGGE method eighteen bacterial and twelve fungal species were identified from the five samples collected from the commercial wood chip pile, compared to the ten bacterial and nine fungal isolates which were identified using the culturing technique and standard 16S and 18S rRNA gene sequence analysis. Predominant genera in the optimization phase of this study were Klebsiella spp. (×3), Bacillus spp. (×2), Pantoea spp. (×2), Pseudomonas spp. (×2) and Paecilomyces spp. (×2). Application of the optimized DGGE technique to samples collected from the commercial pulping mill in summer and winter revealed variable profiles indicating a range of bacterial and fungal strains that varied in intensity in the areas and seasons sampled. Seventy nine (45 in summer and 34 in winter) and 29 (20 in summer and 9 in winter) distinct amplicons representing bacteria and fungi, respectively, were visualized. Predominant genera in summer were Pantoea rodasii, Inquilinus limosus, Streptococcus sp., Klebsiella spp., Diversispora sp., Boletaceae sp., Scutellospora sp., and Ophiostoma bicolour. In winter the prevailing genera were Leuconostoc palmae, Streptococcus sp., Bacillus spp., Diversispora sp., Boletaceae sp., and Bullera sp. Lower cellulose levels in summer correlated significantly with high microbial loads and the predominance of Bacillus spp., suggesting that in warm humid environments storage should not exceed 1-2 weeks. No correlations were determined between the decreased hot water levels in winter and microbial activity, however they were correlated to increased exposure of those samples to environmental factors. Chemistry data on the wood chips imparts the quality of the wood which only permitted projection of final pulp quality. This inadequacy was addressed in the third phase which included identification of microbial strains, originating from the individual Eucalyptus species, after sequencing of 16S and 18S rRNA amplicons separated by DGGE. Fungal and bacterial species were also isolated, cultured, identified and screened for lignocellulolytic enzyme activity. Ninety two and 88% of the fungi isolated were capable of producing cellulase and xylanase, respectively. Significant correlations exist between the microflora, seasons (greater diversity and loading in summer) and the chemical and physical properties of wood chips (lower cellulose and viscosity in summer) as well as Eucalyptus species (significantly higher cellulose and viscosity for the combination and E. nitens). Indigenous microflora of each wood species may be one of the contributing factors to poor/good pulp quality, as significant correlations were made between enzyme production of microorganisms and wood chemistry which ultimately has an impact on the final pulp quality and yields. This investigation provides proof of concept that combining wood species with different deterioration rates results in an overall improvement in pulp quality and thus paves the way for a practical and applicable approach to managing quality of chips. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

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