Isolation of poly-3-hydroxybutyrate metabolism genes from complex microbial communities by phenotypic complementation of bacterial mutants.

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 <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful.

Effects of nitrogen and phosphorus limitation on the activated sludge biomass in a kraft mill biotreatment system.

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.