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Unlike wastewater, pulp and paper mill effluents are generally severely deficient in bioavailable nitrogen and phosphorus. The influence of nitrogen and phosphorus limitations on steady-state or typical pulp and paper mill activated sludge floc properties and performance was studied using a bioreactor-fed synthetic raw mill effluent and seeded with mill activated sludge. Limitation of either nitrogen or phosphorus decreased growth, five-day biochemical oxygen demand, and suspended solids removal. Nitrogen limitation greatly enhanced activated sludge floc poly-beta3-hydroxybutyrate (PHB), but not carbohydrate or extracellular polymeric substances (EPS). In contrast, phosphorus limitation increased total floc carbohydrate and EPS, but not PHB. The flocs showed little ability to store either nitrogen or phosphorus. Nitrogen limitation, but not phosphorus limitation, produced much more negative net floc surface charge, increasing fines, while phosphorus limitation, but not nitrogen limitation, increased the floc bound water content and surface hydrophobicity and decreased fines.

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N 2 fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase ( nifH ) gene probing, and bacterial isolations. In situ N 2 fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH , of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH . In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N 2 -fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N 2 fixation (0.87 to 4.90 mg of N liter −1 day −1 ) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.

The white rot basidiomycete Trametes ( Coriolus ) versicolor can substantially increase the brightness and decrease the lignin content of washed, unbleached hardwood kraft pulp (HWKP). Monokaryotic strain 52J was used to study how HWKP and the lignin in HWKP affect the carbon metabolism and secretions of T. versicolor. Earlier work indicated that a biobleaching culture supernatant contained all components necessary for HWKP biobleaching and delignification, but the supernatant needed frequent contact with the fungus to maintain these activities. Thus, labile small fungal metabolites may be the vital biobleaching system components renewed or replaced by the fungus. Nearly all of the CO 2 evolved by HWKP-containing cultures came from the added glucose, indicating that HWKP is not an important source of carbon or energy during biobleaching. Carbon dioxide appeared somewhat earlier in the absence of HWKP, but the culture partial O 2 pressure was little affected by the presence of pulp. The presence of HWKP in a culture markedly increased the culture's production of a number of acidic metabolites, including 2-phenyllactate, oxalate, adipate, glyoxylate, fumarate, mandelate, and glycolate. Although the total concentration of these pulp-induced metabolites was only 4.3 mM, these compounds functioned as effective manganese-complexing agents for the manganese peroxidase-mediated oxidation of phenol red, propelling the reaction at 2.4 times the rate of 50 mM sodium malonate, the standard chelator-buffer. The presence of HWKP in a culture also markedly stimulated fungal secretion of the enzymes manganese peroxidase, cellulase, and cellobiose-quinone oxidoreductase, but not laccase (phenol oxidase) or lignin peroxidase.

The white rot basidiomycete Coriolus (Trametes) versicolor has been shown to decolorize the stable high-molecular-weight chromophores released by kraft mill bleacheries. This report looks at the fungal requirements for optimal growth and decolorization, and the mechanism of chromophore degradation. Simple carbohydrates were shown to be essential for effective decolorization, and a medium composed of inexpensive industrial by-products provided excellent growth and decolorization. The decolorization activity was entirely O2-dependent, but unaffected by scavengers of O-2, OH, and H2O2. Free extracellular H2O2 was not necessary for E1 effluent decolorization. Decolorization was also not measurably repressed or induced by a variety of factors reportedly regulating lignin peroxidase expression and secondary metabolism in both C. versicolor and the related fungus Phanerochaete chrysosporium. These included veratryl alcohol, H2O2, nitrogen and carbon limitation, culture age, and elevated trace metals. No extracellular peroxidases or hydrogen peroxide could be detected during decolorization, although substantial levels of laccase-type phenol oxidases were present. Thus, it appears that E1 effluent decolorization by C. versicolor proceeds via a mechanism not employing secreted H2O2 and peroxidase.

The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate d -3-hydroxybutyrate due to absence of the enzyme d -3-hydroxybutyrate dehydrogenase activity. Clones that conferred d -3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored d -3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was

The ability of 10 dikaryotic and 20 monokaryotic strains of Trametes ( Coriolus ) versicolor to bleach and delignify hardwood and softwood kraft pulps was assessed. A dikaryon (52P) and two of its mating-compatible monokaryons (52J and 52D) derived via protoplasting were compared. All three regularly bleached hardwood kraft pulp more than 20 brightness points (International Standards Organization) in 5 days and softwood kraft pulp the same amount in 12 days. Delignification (kappa number reduction) by the dikaryon and the monokaryons was similar, but the growth of the monokaryons was slower. Insoluble dark pigments were commonly found in the mycelium, medium, and pulp of the dikaryon only. Laccase and manganese peroxidase (MnP) but not lignin peroxidase activities were secreted during bleaching by all three strains. Their laccase and MnP isozyme patterns were compared on native gels. No segregation of isozyme bands between the monokaryons was found. Hardwood kraft pulp appeared to adsorb several laccase isozyme bands. One MnP isozyme (pI, 3.2) was secreted in the presence of pulp by all three strains, but a second (pI, 4.9) was produced only by 52P. A lower level of soluble MnP activity in one monokaryon (52D) was associated with reduced bleaching ability and a lower level of methanol production. Since monokaryon 52J bleached pulp better than its parent dikaryon 52P, especially per unit of biomass, this genetically simpler monokaryon will be the preferred subject for further genetic manipulation and improvement of fungal pulp biological bleaching.