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The relationship between the abundance of three functional genes and their corresponding biochemical reaction rates was investigated in several activated sludge and mill effluent microbial communities. Gene probes were prepared for two key denitrification genes (nirS and nirK) and for one nitrogen-fixation gene (nifH) and were validated using a variety of strains of known nir and nif genotype. ATP-based measures of viable cell numbers were used to provide total population sizes. In certain microbial communities (activated sludge enrichment cultures and multiple samples taken from the same mill primary clarifier), a strong correlation was observed between gene abundance and biochemical activity rates. However, when comparing several different nonenriched activated sludge bioreactors and separate primary clarifier microbial communities, the ratio of specific gene abundance to biochemical activity rates varied widely. These results suggest that in cases where a microbial community is not fully induced for a given biochemical activity or when very different communities are compared, quantitative gene probing can give a better measure of a community's potential to carry out the encoded function than can the relevant biochemical assay. However, the gene quantitation method employed here probably underestimated the true number of probed genes present in the microbial communities due to nirS and nifH genes in the communities having reduced DNA sequence similarity with the probes used.Key words: denitrification, nitrogen fixation, quantitative hybridization, activated sludge, primary clarifier.

Coliform bacteria have long been used to indicate fecal contamination of water and thus a health hazard. In this study, the in-mill water and external effluent treatment systems of seven typical Canadian pulp and paper mills were all shown to support the growth of numerous coliforms, especially Klebsiella Spp., Escherichia coli, Enterobacter spp., and Citrobacter spp. In all mills and most sampled locations, klebsiellas were the predominant coliforms. Although all but one of the mills had no sewage input and most disinfected their feed (input) water, all contained the most typical fecal indicator bacterium, E. coli. Many of the mill coliforms were classified as fecal coliforms by standard ''MPN'' and metabolic tests, but this was shown to be due to their thermotolerance, not their origin. Mill coliforms were shown not to be just simple transients from feedwater or furnish (wood), but to be continuously growing, especially in some of the primary clarifiers. Isolated mill coliforms grew very well on a sterilized raw combined mill effluent. The fecal streptococci (enterococci), alternative indicators of fecal health hazards, were common in all mills in the absence of sewage. Ten strains of E. coli isolated from four mills were all shown to be non-toxigenic strains of harmless serotypes. No salmonellas were found. Therefore, the use of total coliform, fecal coliform, enterococci, or E. coli counts as indicators of fecal contamination, and thus of health hazard in pulp and paper mill effluents or biosolids (sludges) known to be free of fecal input is invalid. # 2001 Elsevier Science Ltd. All rights reserved

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.

The transition metal manganese is considered to be a minor micronutrient in both pro- and eukaryotes, usually being required from the environment at subnanomolar levels. Until recently, Mn was only known to function in cells as a cofactor for a few enzymatic reactions. A notable exception has been reported in many lactic acid bacterial species which require micromolar medium Mn levels for growth and contain up to 35 mM Mn. These high Mn concentrations are accompanied by the near or complete absence of intracellular iron and superoxide dismutase (SOD). Lacking hemes, Lactobacillus plantarum and related species contain a unique Mn-cofactored catalase as well as millimolar Mn(II) in a nonenzymic complex performing the function of the micromolar superoxide dismutase found in most other aerotolerant cells. The high Mn(II) levels are accumulated via an efficient active transport system and are stored intracellularly in a high molecular weight complex. Study of Lactobacillus plantarum has provided an interesting example of the substitution of Mn for Fe in several of the biological roles of Fe, an alternative mechanism of aerotolerance, and a better understanding of the unique biochemistry of the lactic acid bacteria.

The hypoferremic response in C57 black mice during EL4 lymphoma cell growth was followed to determine if a lowered transferrin iron saturation inhibited iron acquisition and growth by these tumor cells. Although the lymphoma induced a strong hypoferremic response, it occurred late in the disease when the tumor burden was high and did not appear to effectively limit tumor cell growth, as all mice subsequently died. The induction of a hypoferremic response early in the disease with subcutaneous injections of turpentine also did not inhibit tumor cell growth. Parallel in vitro cell growth experiments revealed iron to be readily available to the lymphoma cells, regardless of whether the transferrin pool was 100, 50, or 10% saturated with iron. The dynamics of 59Fe uptake by the lymphoma cells from 50% saturated ferritransferrin were followed and saturation of iron uptake was found to occur at 150 nM iron and transferrin, far below physiological levels. Lowering transferrin saturation from 50 to 10% did not result in a proportional decrease in 59Fe uptake. Thus the ability of the EL4 tumor cells to obtain iron appeared to exceed the ability of a vigorous murine hypoferremic response to sequester it, suggesting that the hypoferremic response does not effectively limit EL4 ascites tumor cell growth.