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For a sustainable economy, biodegradable biopolymers polyhydroxyalkanoates (PHA) are desirable substitutes to petroleum-based plastics that contaminate our environment. Medium-chain-length (MCL) PHA bioplastics are particularly interesting due to their thermoplastic properties. To hamper the high cost associated to PHA production, the use of bacterial mixed cultures cultivated in open systems and using cheap resources is a promising strategy. Here, we studied the operating conditions favouring direct MCL accumulation by activated sludge, using oleic acid as a model substrate and phosphorus limitation in fed-batch bioreactors. Our results confirm the presence of PHA-accumulating organisms (PHAAO) in activated sludge able to accumulate MCL from oleic acid. A positive correlation between phosphorus (P) limitation and PHA accumulation was demonstrated, allowing up to 26% PHA/total biomass accumulation, and highlighted its negative impact on the MCL/PHA fraction in the polymer. Diversity analysis through 16S rRNA amplicon sequencing revealed a differential selection of PHAAO according to the P-limitation level. A differential behaviour for the orders Pseudomonadales and Burkholderiales at increasing P-limitation levels was revealed, with a higher abundance of the latter at high levels of P-limitation. The PHA accumulation observed in activated sludge open new perspectives for MCL-PHA production system based on P-limitation strategy applied to mixed microbial communities. Key points: • Direct accumulation of MCL-PHA in activated sludge was demonstrated. • MCL-PHA content is negatively correlated with P-limitation. • Burkholderiales members discriminate the highest P-limitation levels. [ABSTRACT FROM AUTHOR]

Polyhydroxyalkanoates (PHAs) can be produced by microorganisms from renewable resources and are regarded as promising bioplastics to replace petroleum-based plastics. A medium-chain-length PHAs (mcl-PHA)-producing strain Pseudomonas mendocina NK-01 was isolated previously by our lab and its whole-genome sequence is currently available. Morphology engineering of manipulating cell morphology-related genes has been applied for enhanced accumulation of the intracellular biopolymer short-chain-length PHAs (scl-PHA). However, it has not yet been reported to improve the yield of mcl-PHA by morphology engineering so far. In this work, several well-characterized cell morphology-related genes, including the cell fission ring (Z-ring) location genes minCD, peptidoglycan degradation gene nlpD, actin-like cytoskeleton protein gene mreB, Z-ring formation gene ftsZ, and FtsZ inhibitor gene sulA, were intensively investigated for their impacts on the cell morphology and mcl-PHA accumulation by gene knockout and overexpression in P. mendocina NKU, a upp knockout mutant of P. mendocina NK-01. For a minCD knockout mutant P. mendocina NKU-∆minCD, the average cell length was obviously increased and the mcl-PHA production was improved. However, the nlpD knockout mutant had a shorter cell length and lower mcl-PHA yield compared with P. mendocina NKU. Overexpression of mreB in P. mendocina NKU resulted in spherical cells. When ftsZ was overexpressed in P. mendocina NKU, the cell division was accelerated and the mcl-PHA titer was improved. Furthermore, mreB, ftsZ, or sulA was overexpressed in P. mendocina NKU-∆minCD. Consequently, the mcl-PHA titers were all increased compared with P. mendocina NKU-∆minCD carrying the empty vector. The multiple fission pattern was finally achieved in ftsZ-overexpressing NKU-∆minCD. In this work, improved production of mcl-PHA in P. mendocina NK-01 has been achieved by morphology engineering. This work provides an alternative strategy to enhance mcl-PHA accumulation in mcl-PHA-producing strains. [ABSTRACT FROM AUTHOR]

The industrial production of low value-added biological products poses significant challenges due to cost pressures. In recent years, it has been argued that synthetic biology approaches will lead to breakthroughs that eliminate price bottlenecks for the production of a wide range of biological products including bioplastics and biofuels. One significant bottleneck lies in the necessity to break the tough cell walls of microbes in order to release intracellular products. We here report the implementation of the first synthetic biology standard part based on the lambda phage SRRz genes and a synthetic ribosome binding site (RBS) that works in Escherichia coli and Halomonas campaniensis, which enables the producer strains to induce lysis after the addition of small amounts (1-5 %) of solvents or to spontaneously lyse during the stresses of downstream processing, and thus has the potential to eliminate the mechanical cell disruption step as both an efficiency bottleneck and a significant capex barrier when implementing downstream bioprocesses. [ABSTRACT FROM AUTHOR]

A process for the conversion of post consumer (agricultural) polyethylene (PE) waste to the biodegradable polymer medium chain length polyhydroxyalkanoate (mcl-PHA) is reported here. The thermal treatment of PE in the absence of air (pyrolysis) generated a complex mixture of low molecular weight paraffins with carbon chain lengths from C8 to C32 (PE pyrolysis wax). Several bacterial strains were able to grow and produce PHA from this PE pyrolysis wax. The addition of biosurfactant (rhamnolipids) allowed for greater bacterial growth and PHA accumulation of the tested strains. Some strains were only capable of growth and PHA accumulation in the presence of the biosurfactant. Pseudomonas aeruginosa PAO-1 accumulated the highest level of PHA with almost 25 % of the cell dry weight as PHA when supplied with the PE pyrolysis wax in the presence of rhamnolipids. The change of nitrogen source from ammonium chloride to ammonium nitrate resulted in faster bacterial growth and the earlier onset of PHA accumulation. To our knowledge, this is the first report where PE is used as a starting material for production of a biodegradable polymer. [ABSTRACT FROM AUTHOR]

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases. [ABSTRACT FROM AUTHOR]