Characterization of temperature-sensitive and lipopolysaccharide overproducing transposon mutants of Pseudomonas putida CA-3 affected in PHA accumulation.

A library of 20 000 transposon (Tn5) mutants of the gram-negative bacterium Pseudomonas putida CA-3 was generated and screened for adverse affects in polyhydroxyalkanoates (PHA) accumulation. Two mutants of interest were characterized phenotypically. CA-3-126, a mutant disrupted in a stress-related protein Clp protease subunit ClpA, demonstrated greater decreases in PHA accumulation compared with the wild type at reduced and elevated temperatures under PHA-accumulating growth conditions. CA-3-M, which is affected in the aminotransferase class I enzyme, accumulated reduced levels of PHA relative to the wild type and had lower growth yields on all carbon sources tested. Mutant CA-3-M produced up to 10-fold higher levels of lipopolysaccharide relative to the wild type and exhibited 1.2-fold lower aminotransferase activity with phenylalanine as a substrate compared with the wild-type strain. The composition of the lipopolysaccharide produced by the mutant differed from that produced by the wild-type strain. Growth and PHA accumulation by CA-3-M was the same as the wild type when the nitrogen concentration in the medium was increased to 265 mg N L(-1).

Improvement of the conversion of polystyrene to polyhydroxyalkanoate through the manipulation of the microbial aspect of the process: a nitrogen feeding strategy for bacterial cells in a stirred tank reactor.

Pseudomonas putida CA-3 has been shown to accumulate the biodegradable plastic polyhydroxyalkanoate (PHA) when fed styrene or polystyrene pyrolysis oil as the sole carbon and energy source under nitrogen limiting growth conditions (67 mg nitrogen per litre at time 0). Batch fermentation of P. putida CA-3 grown on styrene or polystyrene pyrolysis oil in a stirred tank reactor yields PHA at 30% of the cell dry weight (CDW). The feeding of nitrogen at a rate of 1mg N/l/h resulted in a 1.1-fold increase in the percentage of CDW accumulated as PHA. An increase in the rate of nitrogen feeding up to 1.5mg N/l/h resulted in further increases in the percentage of the cell dry weight composed of PHA. However, feeding rates of 1.75 and 2mg N/l/h resulted in dramatic decreases in the percentage of cell dry weight composed of PHA. Interestingly nitrogen was not detectable in the growth medium after 16 h, in any of the growth conditions tested. A higher cell density was observed in cells supplied with nitrogen and thus further increases in the overall production of PHA were observed through nitrogen feeding. The highest yield of PHA was 0.28 g PHA per g styrene supplied with a nitrogen feeding rate of 1.5mg/l/h.

A two step chemo-biotechnological conversion of polystyrene to a biodegradable thermoplastic.

A novel approach to the recycling of polystyrene is reported here; polystyrene is converted to a biodegradable plastic, namely polyhydroxyalkanoate (PHA). This unique combinatorial approach involves the pyrolysis of polystyrene to styrene oil, followed by the bacterial conversion of the styrene oil to PHA by Pseudomonas putida CA-3 (NCIMB 41162). The pyrolysis (520 degrees C) of polystyrene in a fluidized bed reactor (Quartz sand (0.3-0.5 mm)) resulted in the generation of an oil composed of styrene (82.8% w/w) and low levels of other aromatic compounds. This styrene oil, when supplied as the sole source of carbon and energy allowed for the growth of P. putida CA-3 and PHA accumulation in shake flask experiments. Styrene oil (1 g) was converted to 62.5 mg of PHA and 250 mg of bacterial biomass in shake flasks. A 1.6-fold improvement in the yield of PHA from styrene oil was achieved by growing P. putida CA-3 in a 7.5 liter stirred tank reactor. The medium chain length PHA accumulated was comprised of monomers 6, 8, and 10 carbons in length in a molar ratio of 0.046:0.436:1.126, respectively. A single pyrolysis run and four fermentation runs resulted in the conversion of 64 g of polystyrene to 6.4 g of PHA.

Genetic characterization of accumulation of polyhydroxyalkanoate from styrene in Pseudomonas putida CA-3.

Pseudomonas putida CA-3 is capable of accumulating medium-chain-length polyhydroxyalkanoates (MCL-PHAs) when growing on the toxic pollutant styrene as the sole source of carbon and energy. In this study, we report on the molecular characterization of the metabolic pathways involved in this novel bioconversion. With a mini-Tn5 random mutagenesis approach, acetyl-coenzyme A (CoA) was identified as the end product of styrene metabolism in P. putida CA-3. Amplified flanking-region PCR was used to clone functionally expressed phenylacetyl-CoA catabolon genes upstream from the sty operon in P. putida CA-3, previously reported to generate acetyl-CoA moieties from the styrene catabolic intermediate, phenylacetyl-CoA. However, the essential involvement of a (non-phenylacetyl-CoA) catabolon-encoded 3-hydroxyacyl-CoA dehydrogenase is also reported. The link between de novo fatty acid synthesis and PHA monomer accumulation was investigated, and a functionally expressed 3-hydroxyacyl-acyl carrier protein-CoA transacylase (phaG) gene in P. putida CA-3 was identified. The deduced PhaG amino acid sequence shared >99% identity with a transacylase from P. putida KT2440, involved in 3-hydroxyacyl-CoA MCL-PHA monomer sequestration from de novo fatty acid synthesis under inorganic nutrient-limited conditions. Similarly, with P. putida CA-3, maximal phaG expression was observed only under nitrogen limitation, with concomitant PHA accumulation. Thus, beta-oxidation and fatty acid de novo synthesis appear to converge in the generation of MCL-PHA monomers from styrene in P. putida CA-3. Cloning and functional characterization of the pha locus, responsible for PHA polymerization/depolymerization is also reported and the significance and future prospects of this novel bioconversion are discussed.