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ABSTRACTThe obligate predatorBdellovibrio bacteriovorusHD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 ofB. bacteriovorusHD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBdsuggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBdhas been extracellularly produced using different hypersecretor Tol-pal mutants ofEscherichia coliandPseudomonas putidaas recombinant hosts. The recombinant PhaZBdhas been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBdis an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow.

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a b s t r a c tSeveral studies have dealt with the microwave-induced pyrolysis of organic residues as an effective technology for maximizing the production of syngas at the expense of the production of oily tars. This liquid fraction is composed of a complex mixture of organic compounds making it difficult to upgrade for use as a biofuel and containing hazardous compounds such as polycyclic aromatic hydrocarbons (PAH). In this study the effect of the type of residue (municipal solid wastes, sewage sludges, plastic wastes and agroresidues) and the heating mechanism on the product distribution within the oil fraction has been investigated by means of GC-MS technique. Two different approaches have been performed: direct GC-MS analysis and methanolysed-oils GC-MS analysis, the later determining the non-volatile compounds. In general, quite different distributions are attained when microwave pyrolysis is conducted, providing lighter compounds. In some cases, such as the plastic-derived oil, microwave pyrolysis lead to a potential source of chemicals such as benzene, toluene and xylenes.

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