Medium Chain Length Polyhydroxyalkanoates Research Articles

Increased sedimentation of a Pseudomonas–Saccharomyces microbial consortium producing medium chain length polyhydroxyalkanoates

Abstract Concerns about feasibility, separability, settleability, efficiency once hampered studies on polyhydroxyalkanoates (PHAs) production, which mainly focused on single strain microorganism or activated sludge rather than artificial microbial consortia. Here, a medium chain length PHAs (mcl-PHAs) producing Pseudomonas–Saccharomyces consortium with xylose as the main substrate was studied. Mcl-PHAs accumulation increased from 12.69 mg·L−1 to 152.3 mg·L−1 without any optimization method. The presence of Saccharomyces cerevisiae, though in a relatively low concentration, improved the sedimentation of cell mass of the mixed culture by 60%. Reasons for better sedimentation of the consortium were complex: first, the length of Pseudomonas putida increased two to three times in the consortium; second, the positive surface charge of P. putida was neutralized by S. cerevisiae; third, the adhesion proteins on the surface of S. cerevisiae interacted with the P. putida.

Biosynthesis and characterization of a novel, biocompatible medium chain length polyhydroxyalkanoate by Pseudomonas mendocina CH50 using coconut oil as the carbon source.

This study validated the utilization of triacylglycerides (TAGs) by Pseudomonas mendocina CH50, a wild type strain, resulting in the production of novel mcl-PHAs with unique physical properties. A PHA yield of 58% dcw was obtained using 20 g/L of coconut oil. Chemical and structural characterisation confirmed that the mcl-PHA produced was a terpolymer comprising of three different repeating monomer units, 3-hydroxyoctanoate, 3-hydroxydecanoate and 3-hydroxydodecanoate or P(3HO-3HD-3HDD). Bearing in mind the potential of P(3HO-3HD-3HDD) in biomedical research, especially in neural tissue engineering, in vitro biocompatibility studies were carried out using NG108-15 (neuronal) cells. Cell viability data confirmed that P(3HO-3HD-3HDD) supported the attachment and proliferation of NG108-15 and was therefore confirmed to be biocompatible in nature and suitable for neural regeneration.

Application of cider by-products for medium chain length polyhydroxyalkanoate production by Pseudomonas putida KT2440

In this study the technical feasibility of using cider by-products as a substrate for the production of medium chain length polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas putida KT2440 is demonstrated. The untreated apple pomace was found to be composed of two main residual sugars, glucose and fructose, making it a promising cheap growth substrate. A PHA concentration of 1.1 g L−1 and a PHA accumulation of 25.5% were obtained in flask experiments using apple pomace as the sole carbon source without any pretreatment, employing a fed batch strategy. The biopolymer was extracted from the biomass and a detailed characterization was carried out. The solution-casted film was found to be a mcl-PHA copolymer composed by different saturated and unsaturated ester monomers with elastomeric characteristics making it suitable for applications in biomedicine or the processing of value added co-products such as environmentally friendly adhesives or biodegradable films for agricultural purposes.

Manipulating the structure of medium-chain-length polyhydroxyalkanoate (MCL-PHA) to enhance thermal properties and crystallization kinetics

We investigated the effects of the structure of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) on their thermal properties and crystallization kinetics. The predominantly homopolymeric poly(3-hydroxydecanoate), P(3HD)-98, and the poly(3-hydroxydodecanoate), P(3HDD), exhibited sharp crystallization peaks upon cooling, with the latter exhibiting faster crystallization rates. A chemical modification strategy involving reaction with dicumyl peroxide and triallyl trimesate coagent was implemented to introduce branching and enhance the crystallization kinetics of P(3HD-98). Increases in the exothermic crystallization temperature by 8 °C and in the overall crystallinity of the P(3HD)-98 were observed upon chemical modification. The Avrami crystallization kinetic parameters obtained by fitting the isothermal crystallization data revealed a significant increase in the crystallization rate of the modified P(3HD)-98.

Enabling electrospinning of medium-chain length polyhydroxyalkanoates (PHAs) by blending with short-chain length PHAs

Polyhydroxyalkanoates (PHAs) have recently attracted significant attention in medical applications. Electrospinning of short chain-length (scl-)PHAs has been extensively investigated, while medium chain length (mcl-)PHAs are not suitable for electrospinning since they are elastomeric at room temperature. We improved the electrospinability of an mcl-PHA poly (3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHOHHx) by blending with a scl-PHA poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV25, with 25 mol% HV). Morphology of electrospun PHBV25/PHOHHx blends at various ratios was investigated. Effects of various processing parameters on nanofiber morphology were investigated, such as solution concentration, feeding rate and applied voltage. Crystallinity, chemical structure, and mechanical properties of the electrospun mats were also studied.

The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis.

Polyhydroxyalkanoates (PHAs) are a diverse class of bio-polymers synthesized by bacteria, usually during imbalanced growth conditions. Optimizing PHA productivity is highly dependent on the bioreactor oxygen transfer rate (OTR), which is an important consideration for process performance and economics, particularly with increasing scale. Relatively few in-depth studies are available regarding the effect of OTR and dissolved oxygen content (DOC) on PHA formation, synthesis rates, composition, and characteristics. This review examines past research studies on the effect of low DOC environments on production of short-chain length (scl-) PHAs, synthesized by both pure and mixed cultures, in order to identify opportunities and gaps concerning the effect of DOC on production of medium-chain length (mcl-) PHAs, an area that has not been studied in detail. The literature indicates that production of scl-PHA (a reductive process) acts as an electron sink allowing cells to maintain balanced redox state at low DOC. Conversely, production of mcl-PHA via fatty acid de novo synthesis (also a reductive process) does not occur to any significant extent in low DOC environments, while mcl-PHA synthesis from fatty acids (an oxidative process) can be promoted in low DOC environments. The monomer composition, molecular mass, as well as physical and thermal properties of the polymer can change in response to OTR, but further research in this area is required for both scl- and mcl-PHAs. Process design and management of bioreactor OTR in PHA production might therefore be directed by the final application of the polymer rather than cost considerations.

In Vivo Tracking and 1H/19F Magnetic Resonance Imaging of Biodegradable Polyhydroxyalkanoate/Polycaprolactone Blend Scaffolds Seeded with Labeled Cardiac Stem Cells.

Medium-chain length polyhydroxyalkanoates (MCL-PHAs) have demonstrated exceptional properties for cardiac tissue engineering (CTE) applications. Despite prior work on MCL-PHA/polycaprolactone (PCL) blends, optimal scaffold production and use as an alternative delivery route for controlled release of seeded cardiac progenitor cells (CPCs) in CTE applications in vivo has been lacking. We present herein applicability of MCL-PHA/PCL (95/5 wt %) blends fabricated as thin films with an improved performance compared to the neat MCL-PHA. Polymer characterization confirmed the chemical structure and composition of the synthesized scaffolds, while thermal, wettability, and mechanical properties were also investigated and compared in neat and porous counterparts. In vitro cytocompatibility studies were performed using perfluorocrown-ether-nanoparticle-labeled murine CPCs and studied using confocal microscopy and 19F magnetic resonance spectroscopy and magnetic resonance imaging (MRI). Seeded scaffolds were implanted and studied in the postmortem murine heart in situ and in two additional C57BL/6 mice in vivo (using single-layered and double-layered scaffolds) and imaged immediately after and at 7 days postimplantation. Superior MCL-PHA/PCL scaffold performance has been demonstrated compared to MCL-PHA through experimental comparisons of (a) morphological data using scanning electron microscopy and (b) contact angle measurements attesting to improved CPC adhesion, (c) in vitro confocal microscopy showing increased SC proliferative capacity, and (d) mechanical testing that elicited good overall responses. In vitro MRI results justify the increased seeding density, increased in vitro MRI signal, and improved MRI visibility in vivo, in the double-layered compared to the single-layered scaffolds. Histological evaluations [bright-field, cytoplasmic (Atto647) and nuclear (4′,6-diamidino-2-phenylindole) stains] performed in conjunction with confocal microscopy imaging attest to CPC binding within the scaffold, subsequent release and migration to the neighboring myocardium, and increased retention in the murine myocardium in the case of the double-layered scaffold. Thus, MCL-PHA/PCL blends possess tremendous potential for controlled delivery of CPCs and for maximizing possible regeneration in myocardial infarction.

Synthesis of novel biodegradable elastomers based on poly[3-hydroxy butyrate] and poly[3-hydroxy octanoate] via transamidation reaction

Poly(3-hydroxyalkanoate)s (PHAs) are a class of polymers receiving attention because of their potential as renewable, biodegradable and high-technology properties. Unlike most short chain length (scl) PHAs such as poly(3-hydroxybutyrate) (PHB), medium chain length (mcl) PHAs such as poly(3-hydroxyoctanoate) (PHO) exhibit low crystallinity and are elastomeric in character. PHB-b–PEG-b–PHO block copolymers can combine both properties in block copolymer matrix. In this study, we report the synthesis of the block copolymers combining the PHB and PHO blocks. Transamidation reactions of PHB with polyethylene glycol with primary amine yield equimolar amounts and PHB with amine ends. PHO reacts with the modified PHB containing the amine end to give PHB-b–PEG-b–PHO block copolymers. Structural analysis of the products was performed by using 1H–, 13C, heteronuclear single quantum coherence NMR techniques. Thermal and mechanical properties of the block polymers were also evaluated.

Carbon flux to growth or polyhydroxyalkanoate synthesis under microaerophilic conditions is affected by fatty acid chain-length in Pseudomonas putida LS46.

Economical production of medium-chain length polyhydroxyalkanoates (mcl-PHA) is dependent on efficient cultivation processes. This work describes growth and mcl-PHA synthesis characteristics of Pseudomonas putida LS46 when grown on medium-chain length fatty acids (octanoic acid) and lower-cost long-chain fatty acids (LCFAs, derived from hydrolyzed canola oil) in microaerophilic environments. Growth on octanoic acid ceased when the oxygen uptake rate was limited by the oxygen transfer rate, and mcl-PHA accumulated to 61.9% of the cell dry mass. From LCFAs, production of non-PHA cell mass continued at a rate of 0.36gL-1h-1 under oxygen-limited conditions, while mcl-PHA accumulated simultaneously to 31% of the cell dry mass. The titer of non-PHA cell mass from LCFAs at 14h post-inoculation was double that obtained from octanoic acid in bioreactors operated with identical feeding and aeration conditions. While the productivity for octanoic acid was higher by 14h, prolonged cultivation on LCFAs achieved similar productivity but with twice the PHA titer. Simultaneous co-feeding of each substrate demonstrated the continued cell growth under microaerophilic conditions characteristic of LCFAs, and the resulting polymer was dominant in C8 monomers. Furthermore, co-feeding resulted in improved PHA titer and volumetric productivity compared to either substrate individually. These results suggest that LCFAs improve growth of P. putida in oxygen-limited environments and could reduce production costs since more non-PHA cell mass, the cellular factories required to produce mcl-PHA and the most oxygen-intensive cellular process, can be produced for a given oxygen transfer rate.

Screening of polyhydroxyalkanoate-producing bacteria and PhaC-encoding genes in two hypersaline microbial mats from Guerrero Negro, Baja California Sur, Mexico.

Hypersaline microbial mats develop through seasonal and diel fluctuations, as well as under several physicochemical variables. Hence, resident microorganisms commonly employ strategies such as the synthesis of polyhydroxyalkanoates (PHAs) in order to resist changing and stressful conditions. However, the knowledge of bacterial PHA production in hypersaline microbial mats has been limited to date, particularly in regard to medium-chain length PHAs (mcl-PHAs), which have biotechnological applications due to their plastic properties. The aim of this study was to obtain evidence for PHA production in two hypersaline microbial mats of Guerrero Negro, Mexico by searching for PHA granules and PHA synthase genes in isolated bacterial strains and environmental samples. Six PHA-producing strains were identified by 16S rRNA gene sequencing; three of them corresponded to a Halomonas sp. In addition, Paracoccus sp., Planomicrobium sp. and Staphylococcus sp. were also identified as PHA producers. Presumptive PHA granules and PHA synthases genes were detected in both sampling sites. Moreover, phylogenetic analysis showed that most of the phylotypes were distantly related to putative PhaC synthases class I sequences belonging to members of the classes Alphaproteobacteria and Gammaproteobacteria distributed within eight families, with higher abundances corresponding mainly to Rhodobacteraceae and Rhodospirillaceae. This analysis also showed that PhaC synthases class II sequences were closely related to those of Pseudomonas putida, suggesting the presence of this group, which is probably involved in the production of mcl-PHA in the mats. According to our state of knowledge, this study reports for the first time the occurrence of phaC and phaC1 sequences in hypersaline microbial mats, suggesting that these ecosystems may be a novel source for the isolation of short- and medium-chain length PHA producers.

Binary polyhydroxyalkanoate systems for soft tissue engineering

Progress in tissue engineering is dependent on the availability of suitable biomaterials. In an effort to overcome the brittleness of poly(3-hydroxybutyrate), P(3HB), a natural biodegradable polyester, and widen its biomedical applications, plasticising of P(3HB) with oligomeric substances of related structure has been studied. A biosynthesised medium-chain-length polyhydroxyalkanoate (mcl-PHA) copolymer, the plasticiser precursor, was obtained using vegetable waste frying oil as a sole carbon source. The mcl-PHA was transformed into an oligomeric derivative by acid hydrolysis. The plasticising effect of the oligomeric mcl-PHA on P(3HB) was studied via characterisation of thermal and mechanical properties of the blends in the course of ageing at ambient conditions. Addition of oligomeric mcl-PHA to P(3HB) resulted in softer and more flexible materials based entirely on PHAs. It was shown that the oligomeric mcl-PHA transformed highly crystalline P(3HB) into materials with a dominant amorphous phase when the content of oligomeric mcl-PHA exceeded 10 wt%. In vitro biocompatibility studies of the new binary PHA materials showed high viability and proliferation of C2C12 myoblast cells. Thus, the proposed approach for P(3HB) plasticisation has the potential for the generation of more pliable biomaterials based on P(3HB) which can find application in unique soft tissue engineering applications where a balance between stiffness, tensile strength and ductility is required. Statement of SignificancePolyhydroxyalkanoates, a broad family of natural biodegradable and biocompatible polymers, have emerged as highly promising biomaterials both for bulk and biomedical applications. Here we describe an approach to tune the mechanical properties of stiff and brittle poly(3-hydroxybutyrate) and thereby to expand its potential biomedical applications. Plasticisation, a common practice in the plastic industry to modify polymer mechanical properties, has been used very cautiously for biomedical applications due to plasticiser toxicity and migration. We have developed a plasticiser for poly(3-hydroxybutyrate) based on a structurally related but softer and pliable medium chain length polyhydroxyalkanoate. Additives of oligomeric derivatives of this polymer improved ductility of poly(3-hydroxybutyrate), greatly widening the future applicability of this well-established biomaterial. In parallel, the binary polyhydroxyalkanoate materials also exhibited improved cell attachment and proliferation, a highly desirable outcome.

Surfactant-mediated hydrothermal pretreatment of Ryegrass followed by enzymatic saccharification for polyhydroxyalkanoate production

Lignocellulosic biomass from non-food crops is an attractive renewable source of sugars. These sugars can be used as feedstock for the microbial production of different industrially relevant compounds. Delignification of lignocellulosic biomass is a major challenge in sugar extraction. In the present study, ryegrass biomass (non-food), which is high in carbohydrates, was pre-treated with the anionic surfactant sodium dodecyl sulphate (SDS) for effective delignification. Enzymatic hydrolysis of the pre-treated biomass led to the extraction of high sugar levels compared with the raw biomass. Furthermore, these sugars were used for the production of poly-hydroxyalkanoates (PHA), which is an alternative to synthetic polymers. Ryegrass treated with 1% SDS solution at 180°C showed the maximum delignification. This pre-treated biomass was characterised by TGA, FTIR and SEM and showed successful lignin removal. Enzymatic hydrolysis of the resultant pre-treated biomass showed 87% conversion into sugars and was used as a carbon source for the production of medium chain length (mcl) PHAs using two Pseudomonas strains. Both strains accumulated approximately 30% of their cell dry weight in mcl-PHAs. Hence, this study shows for the first time that surfactant pretreatment is effective for lignin solubilisation; this process increased the biomass surface area and improved enzyme actions, resulting in higher sugar extraction.

Analysis of Medium-Chain-Length Polyhydroxyalkanoate-Producing Bacteria in Activated Sludge Samples Enriched by Aerobic Periodic Feeding.

Analysis of mixed microbial populations responsible for the production of medium-chain-length polyhydroxyalkanoates (MCL-PHAs) under periodic substrate feeding in a sequencing batch reactor (SBR) was conducted. Regardless of activated sludge samples and the different MCL alkanoic acids used as the sole external carbon substrate, denaturing gradient gel electrophoresis analysis indicated that Pseudomonas aeruginosa was the dominant bacterium enriched during the SBR process. Several P. aeruginosa strains were isolated from the enriched activated sludge samples. The isolates were subdivided into two groups, one that produced only MCL-PHAs and another that produced both MCL- and short-chain-length PHAs. The SBR periodic feeding experiments with five representative MCL-PHA-producing Pseudomonas species revealed that P. aeruginosa has an advantage over other species that enables it to become dominant in the bacterial community.

Medium Chain-Length Polyhydroxyalkanoate Copolymer Modified by Bacterial Cellulose for Medical Devices.

Medium chain-length polyhydroxyalkanoates (mPHAs) are flexible elastomeric biopolymers with valuable properties for biomedical applications like artificial arteries and other medical implants. However, an environmentally friendly and high productivity process together with the tuning of the mechanical and biological properties of mPHAs are mandatory for this purpose. Here, for the first time, a melt processing technique was applied for the preparation of bionanocomposites starting from poly(3-hydroxyoctanoate) (PHO) and bacterial cellulose nanofibers (BC). The incorporation of only 3 wt % BC in PHO improved its thermal stability with 25 °C and reinforced it, increasing the Young's modulus with 76% and the tensile strength with 44%. The percolation threshold calculated with the aspect ratio of the fibers after melt processing was very low and close to 3 wt %. We showed that this bionanocomposite is able to preserve the ductile behavior during storage, no important aging being noted between 3 h and one month after compression-molding. Moreover, this study is the first to investigate the melt processability of PHO nanocomposite for tube extrusion. In addition, biocompatibility study showed no proinflammatory immune response and better cell adhesion for PHO/BC nanocomposite with 3 wt % BC and demonstrated the high feasibility of this bionanocomposite for in vivo application of tissue-engineered blood vessels.

Poly(3-hydroxyoctanoate), a promising new material for cardiac tissue engineering.

Cardiac tissue engineering (CTE) is currently a prime focus of research because of an enormous clinical need. In the present work, a novel functional material, poly(3-hydroxyoctanoate), P(3HO), a medium chain-length polyhydroxyalkanoate (PHA), produced using bacterial fermentation, was studied as a new potential material for CTE. Engineered constructs with improved mechanical properties, crucial for supporting the organ during new tissue regeneration, and enhanced surface topography, to allow efficient cell adhesion and proliferation, were fabricated. Results showed that the mechanical properties of the final patches were close to that of cardiac muscle. Biocompatibility of neat P(3HO) patches, assessed using neonatal ventricular rat myocytes (NVRM), showed that the polymer was as good as collagen in terms of cell viability, proliferation and adhesion. Enhanced cell adhesion and proliferation properties were observed when porous and fibrous structures were incorporated into the patches. In addition, no deleterious effect was observed on adult cardiomyocyte contraction when cardiomyocytes were seeded on the P(3HO) patches. Hence, P(3HO)-based multifunctional cardiac patches are promising constructs for efficient CTE. This work will have a positive impact on the development of P(3HO) and other PHAs as a novel new family of biodegradable functional materials with huge potential in a range of different biomedical applications, particularly CTE, leading to further interest and exploitation of these materials. Copyright © 2016 John Wiley & Sons, Ltd.

Heterologous expression of phaC2 gene and poly-3-hydroxyalkanoate production by recombinant Cupriavidus necator strains using canola oil as carbon source

Many heterologous transformation studies have been carried out using the Cupriavidus necator PHB−4 strain to investigate the expression characteristics of various polyhydroxyalkanoate (PHA) synthase enzymes. In this study, we generated a recombinant C. necator PHB−4 strain by transforming a plasmid (pMRC03) harbouring the synthetic phaC2 gene of Pseudomonas putida CA-3. Under conditions favourable for expression of the phaC2 P.putCA-3 gene, canola oil was used as carbon source for the synthesis of PHAs. The expressed synthase polymerised monomers of 3-hydroxybutyrate (3-HB), 3-hydroxyvalerate (3-HV) and 3-hydroxyhexanoate (3-HHx) in the recombinant C. necator PHB−4 (pMRC03) strain. We then co-expressed the phaC2P.putCA-3 gene with the native phaC1C.ne gene in wild type Cupriavidus necator H16 (C. necator H16 (pMRC03)). This co-expression produced a PHA blend of 3-HB, 3-HV, 3-HHx and 3-hydroxyoctanoate (3-HO) monomers in the presence of canola oil. Gas chromatography analysis revealed the presence of 94mol% 3-HB, 1mol% 3-HV, 4mol% 3-HHx and 1mol% 3-HO in a tetra-polymer. Thus, we confirmed that a synthetic phaC2 gene encoding the synthase enzyme is functionally active with substrates ranging from short to medium chain length PHAs.

Polyhydroxybutyrate and polyhydroxydodecanoate produced by Burkholderia contaminans IPT553.

In this paper, we introduce a new Burkholderia contaminans capable of producing a newly characterized polymer. CG-MS and magnetic nuclear resonance 1 H and 13 C were used to determine the constitution of polymers produced in glucose, glucose with casein, sucrose and sucrose with casein. Three pairs of primers were used to find the polyhydroxyalkanoates (PHA) synthase class and sequence. The synthesized polymers were composed by short-chain length PHA (scl-PHA), especially polyhydroxybutyrate (PHB), and medium chain length PHA (mcl-PHA), especially polyhydroxydodecanoate (PHDd), and their concentration, constitution and molecular weight depend on carbon source used. The bacterium showed only class I synthase which could not explain the mcl-PHA production. Burkholderia contaminans has a class I PHA synthase and produces PHB combined to PHDd when cultivated in sucrose or glucose, and PHDd concentration is affected when casein is used. PHA are natural polymers produced by a wide range of bacteria. The presence of PHDd monomers confers to the polymer elastomeric properties. Previously, PHDd was only obtained when bacteria were cultivated in related carbon source. In this work, B. contaminansIPT553 produced PHB with PHDd using simple and low-cost carbon sources that can make possible the cheaper production of a more flexible biopolymer with crystallinity and elasticity different from the more common PHAs.

Improving carbon and energy distribution by coupling growth and medium chain length polyhydroxyalkanoate production from fatty acids by Pseudomonas putida KT2440

The production of medium chain length polyhydroxyalkanoates by Pseudomonas putida KT2440 from fatty acids leads to the loss of a large proportion of carbon. We studied the possibility of a shift of potentially available energy and carbon towards monitored residual growth during the production phase. A Fed-Batch culture achieving 125.6 g/L of total biomass containing 54.4% (g/g) of medium chain length polyhydroxyalkanoates was carried out leading to an overall experimental carbon yield of 0.7 Cmole/Cmole. The analysis of modeling fluxes deduced from experimental data indicated how carbon and reduced cofactors (NADH and FADH2) were managed to conclude that part of the carbon and reduced cofactors made available by polymer production were used in anabolic pathways. The strategy which consisted in coupled growth and medium chain length polyhydroxyalkanoate production enhanced the global yields compared to growth followed by a production phase. The understanding of carbon and energy fluxes distribution allowed deducing optimized culture strategy to perform the highest reported in the literature.

Microaerophilic environments improve the productivity of medium chain length polyhydroxyalkanoate biosynthesis from fatty acids in Pseudomonas putida LS46

Medium chain length polyhydroxyalkanoate (mcl-PHA, a commercially desirable biopolymer) synthesis from fatty acids is an oxidative process. Growth and mcl-PHA synthesis were studied in Pseudomonas putida LS46 when subjected to a spectrum of oxygen transfer rates, which were intended to simulate dissolved oxygen (DO) gradients likely to be encountered during process scale-up. It was found that maintaining DO at 1–5% (of air saturation at 30 °C) initiated significant mcl-PHA synthesis and concurrent growth at <0.5 μmax was also observed. Further reductions in DO resulted in nearly negligible growth, but improved biopolymer synthesis. When the volumetric oxygen mass transfer coefficient (kLa) was reduced to 38 h−1, DO was below detectable limits and both the biopolymer content (57.3 ± 3.8% CDM) and yield (0.62 ± 0.06 g mcl-PHA g octanoic acid−1) reached the maximum observed values. Under these conditions, the maximum rate of polymer synthesis was also observed (227.9 ± 9.8 mg PHA g residual cell mass−1 h−1), and this value was higher than that which could be achieved using nitrogen limitation. Further reductions in oxygen transfer rate did not improve productivity, and PHA synthesis completely ceased when aeration was replaced with N2 gassing. These results suggest that manipulation of the bioreactor oxygen transfer rate during the PHA accumulation phase could be a strategy used to improve overall productivity during a fed batch or continuous feed process.

Medium chain length polyhydroxyalkanoates biosynthesis in Pseudomonas putida mt-2 is enhanced by co-metabolism of glycerol/octanoate or fatty acids mixtures

The synthesis of medium chain length polyhydroxyalkanoates (mcl-PHAs) by Pseudomonas putida mt-2 was investigated under nitrogen-rich then deficient conditions with glycerol/octanoate or long-chain fatty acids (LCFAs) as carbon sources. When mixed, glycerol and octanoate were co-assimilated regardless of nitrogen availability but provided that glycerol uptake has been already triggered under non-limiting nutrient conditions. This concomitant consumption allowed to enhance mcl-PHAs accumulation (up to 57% of cell dry weight (CDW)) under both non-limiting and nitrogen deficient conditions. Octanoate then mostly drove anabolism of the polyester with 3-hydroxyoctanoate (3HO) synthesized as the main monomer (83%). If the preferred PHA precursor octanoate was supplied, glycerol was mainly involved in cell growth and/or maintenance but very little in PHA production even under nitrogen starvation. P. putida cells accumulated higher amounts of mcl-PHAs when grown on mixtures of LCFAs compared to LCFAs supplied as single substrate (25% and 9% of CDW, respectively). However, only a weak enrichment of the polyester was observed after transfer of cells in a fresh nitrogen-free medium containing the same combination of LCFAs. Some typical units within the polyester were related to the LCFAs ratio supplied in the medium indicating that tailor-made monomers could be synthesized.