Short Chain Length Polyhydroxyalkanoates Research Articles

Bioconversion of glycerol into polyhydroxyalkanoates through an atypical metabolism shift using Priestia megaterium during fermentation processes: A statistical analysis of carbon and nitrogen source concentrations

Polyhydroxyalkanoates (PHA) are bioplastics which are well known as intracellular energy storage compounds and are produced in a large number of prokaryotic species. These bio-based inclusions are biodegradable, biocompatible and environmental friendly. Industrial production of, short chain and medium chain length PHA, involves the use of microorganisms and their enzymes. Priestia megaterium previously known as Bacillus megaterium is a well-recognized bacterium for producing short chain length PHA. This study focuses to characterize this bacterium for the production of medium chain length PHA, and a novel blend of both types of monomers having enhanced properties and versatile applications. Statistical analyses and simulations were used to demonstrate that cell dry weight can be derived as a function of OD600 and PHA content. Optimization of growth conditions resulted in the maximum PHA production as: 0. 05 g. g-x. H−1, where the rate of PHA production was 0.28 g L−1. H−1 and PHA concentration was 4.94 g. L−1. This study also demonstrated FTIR to be a semi quantitative tool for PHA production. Moreover, conversion of scl-PHA to mcl-PHA with reference to time intermissions using GC-FID are shown.

Synthesis and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) multi-block copolymers comprising blocks of differing 3-hydroxyvalerate contents

Polyhydroxyalkanoates (PHAs) are attractive alternatives to commodity petroleum derived plastics, being bacterially derived, thermoprocessible and truly biodegradable under ambient conditions. However, the common short chain length PHAs - poly(3-hydroxybutyrate) (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) - are relatively stiff and brittle, and P3HB in particular has a narrow processing window. Block copolymer synthesis offers an opportunity to produce novel PHA materials comprised of different PHA phases, delivering a potential step-change improvement in PHA properties due to microphase separation. Therefore, this work demonstrates the synthesis and characterisation of novel high molecular weight PHBV-b-PHBV-b-PHBV block copolymers using isocyanate chemistry. High molecular weight, narrow compositional distribution, hydroxy-functionalised PHBV blocks of low (1 mol%) 3HV and high (67 mol%) 3HV contents were used as building blocks. The resulting block copolymers showed a very significant increase in elongation at break (to 76.0 %), as well as improved tensile toughness (to 15.0 MJ/m3) and tensile strength (to 18.5 MPa) compared to the starting materials or comparable blends. Based on the similarity in properties between the 1 mol% 3HV block copolymer products and their counterpart random copolymers, it is also shown that the introduction of urethane linkages had minimal effect on polymer properties. The morphologies of these novel PHBV materials were investigated, supporting the concept that the microphase separation of the different PHBV phases delivered these improved mechanical properties. This approach to PHA-b-PHA block copolymer synthesis offers an attractive generic platform for the production of novel biodegradable PHA materials for film and other applications.

Towards high-throughput screening (HTS) of polyhydroxyalkanoate (PHA) production via Fourier transform infrared (FTIR) spectroscopy of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99.

High-throughput screening (HTS) methods for characterization of microbial production of polyhydroxyalkanoates (PHA) are currently under investigated, despite the advent of such systems in related fields. In this study, phenotypic microarray by Biolog PM1 screening of Halomonas sp. R5-57 and Pseudomonas sp. MR4-99 identified 49 and 54 carbon substrates to be metabolized by these bacteria, respectively. Growth on 15 (Halomonas sp. R5-57) and 14 (Pseudomonas sp. MR4-99) carbon substrates was subsequently characterized in 96-well plates using medium with low nitrogen concentration. Bacterial cells were then harvested and analyzed for putative PHA production using two different Fourier transform infrared spectroscopy (FTIR) systems. The FTIR spectra obtained from both strains contained carbonyl-ester peaks indicative of PHA production. Strain specific differences in the carbonyl-ester peak wavenumber indicated that the PHA side chain configuration differed between the two strains. Confirmation of short chain length PHA (scl-PHA) accumulation in Halomonas sp. R5-57 and medium chain length PHA (mcl-PHA) in Pseudomonas sp. MR4-99 was done using Gas Chromatography-Flame Ionization Detector (GC-FID) analysis after upscaling to 50 mL cultures supplemented with glycerol and gluconate. The strain specific PHA side chain configurations were also found in FTIR spectra of the 50 mL cultures. This supports the hypothesis that PHA was also produced in the cells cultivated in 96-well plates, and that the HTS approach is suitable for analysis of PHA production in bacteria. However, the carbonyl-ester peaks detected by FTIR are only indicative of PHA production in the small-scale cultures, and appropriate calibration and prediction models based on combining FTIR and GC-FID data needs to be developed and optimized by performing more extensive screenings and multivariate analyses.

Medium chain length polyhydroxyalkanoates as potential matrix materials for peripheral nerve regeneration.

Polyhydroxyalkanoates are natural, biodegradable, thermoplastic and sustainable polymers with a huge potential in fabrication of bioresorbable implantable devices for tissue engineering. We describe a comparative evaluation of three medium chain length polyhydroxyalkanoates (mcl-PHAs), namely poly(3-hydroxyoctanoate), poly(3-hydroxyoctanoate-co-3-hydoxydecanoate) and poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate), one short chain length polyhydroxyalkanoate, poly(3-hydroxybutyrate), P(3HB) and synthetic aliphatic polyesters (polycaprolactone and polylactide) with a specific focus on nerve regeneration, due to mechanical properties of mcl-PHAs closely matching nerve tissues. In vitro biological studies with NG108-15 neuronal cell and primary Schwann cells did not show a cytotoxic effect of the materials on both cell types. All mcl-PHAs supported cell adhesion and viability. Among the three mcl-PHAs, P(3HO-co-3HD) exhibited superior properties with regards to numbers of cells adhered and viable cells for both cell types, number of neurite extensions from NG108-15 cells, average length of neurite extensions and Schwann cells. Although, similar characteristics were observed for flat P(3HB) surfaces, high rigidity of this biomaterial, and FDA-approved polymers such as PLLA, limits their applications in peripheral nerve regeneration. Therefore, we have designed, synthesized and evaluated these materials for nerve tissue engineering and regenerative medicine, the interaction of mcl-PHAs with neuronal and Schwann cells, identifying mcl-PHAs as excellent materials to enhance nerve regeneration and potentially their clinical application in peripheral nerve repair.

Genomic insights into the metabolic potential of a novel lignin-degrading and polyhydroxyalkanoates producing bacterium Pseudomonas sp. Hu109A

Lignin is the most abundant heterogeneous aromatic polymer present on planet Earth and is recalcitrant to degradation due to its complex structure, therefore, imposing a challenge to biorefinery procedures. Identifying new microbial strains with the potential to valorize lignin into useful compounds is indispensable to achieving green sustainable consumption. In this study, a novel Pseudomonas strain designated as Hu109A was isolated from the termite gut and the genome was sequenced and analyzed further. The genome contains a circular chromosome with the size of 5,131,917 bp having a GC content of 62.6% and 4698 genes. Genome annotation reveals that the strain possesses lignin-oxidizing enzymes such as DyP-type peroxidases, laccase, dioxygenase, and aromatic degradation gene clusters. The genome also contains O-methyltransferases which function in accelerating the lignin degradation by methylating the free hydroxyl phenolic compounds which in high concentration can inhibit the lignin peroxidase. Furthermore, the genome exhibits two gene clusters encoding the enzymes related to polyhydroxyalkanoates (PHA) synthesis. Pseudomonas strains are generally assumed to produce medium chain length PHAs (mcl-PHAs) only, however, strain Hu109A contains both Class II PHA synthase genes involved in mcl-PHAs and Class III PHA synthase gene involved in short-chain length PHAs (scl-PHAs). Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that using 1 g/L lignin as the sole carbon source, the maximum production of PHA observed was 103.68 mg/L, which increased to 186 mg/L with an increase in lignin concentration to 3 g/L. However, PHA production while using glucose as the sole carbon source was significantly lower than the lignin source, and maximum production was 125.6 mg/L with 3 g/L glucose. The strain Hu109A can tolerate a broad range of solvents including methanol, isopropanol, dimethylformamide, and ethanol, revealing its potential for industrial applications.

Efficiently unsterile polyhydroxyalkanoate production from lignocellulose by using alkali-halophilic Halomonas alkalicola M2

The alkali-halophilic Halomonas alkalicola M2 was isolated and developed for an open unsterile polyhydroxyalkanoate (PHA) fermentation from lignocellulose at pH 10.0 and NaCl 70 g/L. The alkaline pretreatment liquid (APL) was converted into PHA by the strain, which was significantly affected by the cultural conditions, including pH, NaCl concentration, nitrogen source, and APL concentration. The extracted PHA was composed of three monomers and similar in physicochemical properties to conventional short chain-length PHA. A record yield of 2.1 and 5.9 g of PHA was accumulated from 100 g dry bamboo powder (BP) by using APL and APL combined with hydrolysate during a 48-h open unsterile fermentation process, respectively. In summary, the alkali-halophilic H. alkalicola M2 achieved the open unsterile fermentation for lignocellulose efficient bioconversion into PHA under high alkalinity and salinity conditions and would be an ideal producer in the field.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its β-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

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.

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.

Modification and Potential Application of Short-Chain-Length Polyhydroxyalkanoate (SCL-PHA).

As the only kind of naturally-occurring biopolyester synthesized by various microorganisms, polyhydroxyalkanoate (PHA) shows a great market potential in packaging, fiber, biomedical, and other fields due to its biodegradablity, biocompatibility, and renewability. However, the inherent defects of scl-PHA with low 3HV or 4HB content, such as high stereoregularity, slow crystallization rate, and particularly the phenomena of formation of large-size spherulites and secondary crystallization, restrict the processing and stability of scl-PHA, as well as the application of its products. Many efforts have focused on the modification of scl-PHA to improve the mechanical properties and the applicability of obtained scl-PHA products. The modification of structure and property together with the potential applications of scl-PHA are covered in this review to give a comprehensive knowledge on the modification and processing of scl-PHA, including the effects of physical blending, chemical structure design, and processing conditions on the crystallization behaviors, thermal stability, and mechanical properties of scl-PHA.

Performance of Short Chain Length Polyhydroxyalkanoate Production by Comamonas sp EB172 in Large Scale Bioreactors Based on Constant Impeller Tip Speed

Aims: In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) or PHBV, a short chain length polyhydroxyalkanoates (PHAs) production by a local isolate Comamonas sp. EB172 using 500 g/L mixed organic acids were investigated in 10 L and 150 L stirred tank bioreactors based on constant impeller tip speed (minimum 0.56 m/s and maximum 2.22 m/s). Original Research Article Mumtaz et al.; BBJ, 13(2): 1-11, 2016; Article no.BBJ.24684 2 Results: The ratio of the three acids i.e., acetic, butyric, and propionic, were 2:1:1, mimicking clarified mixed acids obtained from anaerobically treated palm oil mill effluent. In pH controlled fedbatch cultivation, in 10 L bioreactor, the strain could accumulate 84% PHBV with biomass concentration of about 4.08 g/L under dual nutrient limitation strategy i.e., dissolved oxygen and nitrogen limited conditions. By maintaining similar tip speed in 150 L bioreactor, a final biomass concentration of 5.35 g/L with a PHBV content of 72% and volumetric productivity of 0.083 g/L/h were obtained. The calculated yield was 0.259 g PHBV/g mixed acids. Conclusion: Both the yield and PHA content in 150 L bioreactor were comparable to that obtained in 10 L scale where PHBV content ranged from 7090% (w/w) of the cell with yield of 0.270.4 g PHBV/g mixed acid, respectively. This is the first report of utilizing mixed organic acids for PHBV biosynthesis in large scale.

Development of novel scl-mcl polyhydroxyalkanoates blends for soft tissue engineering

Event Abstract Back to Event Development of novel scl-mcl polyhydroxyalkanoates blends for soft tissue engineering Pooja P. Basnett1, Barbara B. Lukasiewicz1, Rinat R. Nigmatullin1 and Ipsita Roy1 1 University Of Westminster, Faculty of science and technology, United Kingdom Introduction: Polyhydroxyalkanoates (PHAs) are group of natural biopolymers produced by various microorganisms as a reserve of carbon and energy under nutrient limiting conditions and excess of carbon source[1]. PHAs are biodegradable, non-toxic and biocompatible[2]. Furthermore PHAs as biomaterials are characterized by a wide diversity of monomeric structures. In fact depending on the number of carbon atoms present in the monomer, PHAs are classified into (1) short chain length (SCL) PHA which contains C3 to C5 carbon atoms and (2) medium chain length (MCL) PHA that contains C6 to C14 carbon atoms. This wider variance in their chemical composition enables PHA family to exhibit a broad spectrum of material properties. For instance, SCL-PHAs are brittle and semi-crystalline, whereas MCL-PHAs are highly elastomeric[3]. All these positive attributes make PHAs a potential candidate for a variety of applications, particularly in the area of medicine such as wound management, drug delivery, medical devices, regeneration of both hard and soft tissues etc. engineering[4]-[6]. In this study we have presented results obtained from blending two different types of Polyhydroxyalkanoates: poly-3-hydroxybutyrate P(3HB) an SCL PHA with hydrolysed P(3HHx-3HO-3HD-3HDD) an MCL PHA to develop matrix amenable for soft tissue engineering application. Experimental Methods: MCL-PHAs copolymer was produced using Pseudomonas sp. with waste frying oil as the sole carbon source. Similarly, P(3HB) was produced using Bacillus sp. and glucose as the carbon source. Bacterial cultures were grown on mineral salt medium under nitrogen limitation with excess of carbon. Polymer was extracted from biomass by solid phase extraction. Pure polymers were analysed by Fourier Transformed Infrared Spectroscopy (FTIR) and Gas Chromatography – Mass Spectrometry (GC-MS). Acidic hydrolysis of the mcl-PHA was performed for 20 hours. Growth profile of bacteria and polymer content has been monitored. Three different blends of P(3HB) and hydrolysed P(3HHx-3HO-3HD-3HDD) were prepared by solvent cast method (95/5, 90/10 and 80/20). Studies were then carried out to look into aspects such as thermal, mechanical, microstructural and protein adsorption of the developed blend systems. Biocompatibility assessment was performed by carrying out in vitro interaction of the films with the mouse myoblasts cell line (C2C12). Fluorescence staining by Calcein AM and Ethidium homodimer-1 were performed for confocal microscope. Results and Discussion: Polymers were produced and chemically identified by FTIR, GC-MS and NMR as P(3HB) and P(3HHx-3HO-3HD-3HDD). Additionally P(3HHx-3HO-3HD-3HDD) copolymer was hydrolysed to reduce molecular weight and thereby increase the compatibility between the two polymers within the blend matrix With the increase in the concentration of hydrolysed mcl-PHA copolymer, the glass transition temperature dropped and melting temperature decreased. The surface roughness of blend films was higher when compared to the neat P(3HB). The highest surface roughness (Rq=1.22±0.017μm) was obtained for the blend with 20 wt% addition of P(3HHx-3HO-3HD-3HDD). With the increased concentration of the mcl-PHAs, protein adsorption was higher for the blend composition 80/20 when compared with their blends and neat P(3HB). In vitro biocompatibility studies confirmed higher cell proliferation on the all blends in comparison with TCP at day 1, 3 and 7. SEM images and confocal micrographs confirm high biocompatibility of the cells for the blend films in all the compositions (Figure 1a-c). Figure 1a-c. SEM images of the C2C12 cells seeded on the blend films P(3HB)/P(3HHx-3HO-3HD-3HDD): 95/5, 90/10 and 80/20 at day 7. Conclusion: Both SCL and MCL PHAs were successfully produced and chemically characterised as P(3HB) and P(3HHx-3HO-3HD-3HDD). Three different blend compositions were fabricated and further studied. The most favourable blend for cell proliferation and adhesion was with the maximum amount of mcl-PHAs, 20%wt. In fact blend material demonstrated greater adhesion of the C2C12 cells on its surface when compared to the TCP control. Taken together, these results suggest the potential of the developed SCL-MCL blend system as promising new matrix for soft tissue engineering application. The authors would like to thank technical staff from University College London and ReBiostent EU FP7 project funding. ReBioStent is supported by grant agreement No 604251 from the European Union’s Seventh Programme for research, technological development and demonstration (FP7).

Two Polyhydroxyalkanoate Synthases from Distinct Classes from the Aromatic Degrader Cupriavidus pinatubonensis JMP134 Exhibit the Same Substrate Preference.

Cupriavidus pinatubonensis JMP134 utilizes a variety of aromatic substrates as sole carbon sources, including meta-nitrophenol (MNP). Two polyhydroxyalkanoate (PHA) synthase genes, phaC1 and phaC2, were annotated and categorized as class I and class II PHA synthase genes, respectively. In this study, both His-tagged purified PhaC1 and PhaC2 were shown to exhibit typical class I PHA synthase substrate specificity to make short-chain-length (SCL) PHA from 3-hydroxybutyryl-CoA and failed to make medium-chain-length (MCL) PHA from 3-hydroxyoctanoyl-CoA. The phaC1 or phaC2 deletion strain could also produce SCL PHA when grown in fructose or octanoate, but the double mutant of phaC1 and phaC2 lost this ability. The PhaC2 also exhibited substrate preference towards SCL substrates when expressed in Pseudomonas aeruginosa PAO1 phaC mutant strain. On the other hand, the transcriptional level of phaC1 was 70-fold higher than that of phaC2 in MNP-grown cells, but 240-fold lower in octanoate-grown cells. Further study demonstrated that only phaC1 was involved in PHA synthesis in MNP-grown cells. These findings suggested that phaC1 and phaC2 genes were differentially regulated under different growth conditions in this strain. Within the phaC2-containing gene cluster, a single copy of PHA synthase gene was present clustering with genes encoding enzymes in the biosynthesis of PHA precursors. This is markedly different from the genetic organization of all other previously reported class II PHA synthase gene clusters and this cluster likely comes from a distinct evolutionary path.

High polyhydroxybutyrate production in Pseudomonas extremaustralis is associated with differential expression of horizontally acquired and core genome polyhydroxyalkanoate synthase genes.

Pseudomonas extremaustralis produces mainly polyhydroxybutyrate (PHB), a short chain length polyhydroxyalkanoate (sclPHA) infrequently found in Pseudomonas species. Previous studies with this strain demonstrated that PHB genes are located in a genomic island. In this work, the analysis of the genome of P. extremaustralis revealed the presence of another PHB cluster phbFPX, with high similarity to genes belonging to Burkholderiales, and also a cluster, phaC1ZC2D, coding for medium chain length PHA production (mclPHA). All mclPHA genes showed high similarity to genes from Pseudomonas species and interestingly, this cluster also showed a natural insertion of seven ORFs not related to mclPHA metabolism. Besides PHB, P. extremaustralis is able to produce mclPHA although in minor amounts. Complementation analysis demonstrated that both mclPHA synthases, PhaC1 and PhaC2, were functional. RT-qPCR analysis showed different levels of expression for the PHB synthase, phbC, and the mclPHA synthases. The expression level of phbC, was significantly higher than the obtained for phaC1 and phaC2, in late exponential phase cultures. The analysis of the proteins bound to the PHA granules showed the presence of PhbC and PhaC1, whilst PhaC2 could not be detected. In addition, two phasin like proteins (PhbP and PhaI) associated with the production of scl and mcl PHAs, respectively, were detected. The results of this work show the high efficiency of a foreign gene (phbC) in comparison with the mclPHA core genome genes (phaC1 and phaC2) indicating that the ability of P. extremaustralis to produce high amounts of PHB could be explained by the different expression levels of the genes encoding the scl and mcl PHA synthases.

Engineering Escherichia coli for Improved Production of Short-Chain-Length-co-Medium-Chain-Length Poly[(R)-3-hydroxyalkanoate] (SCL-co-MCL PHA) Copolymers from Renewable Nonfatty Acid Feedstocks

Polyhydroxyalkanoates (PHAs) are biorenewable and biodegradable polyesters that have garnered attention as alternatives to more common petroleum-based polymers. One of the current limitations for the widespread use of PHAs is the inability to produce PHA polymers with desired material properties. Previous studies have shown that PHA copolymers consisting primarily of one short-chain-length (SCL) repeating unit and a small concentration of medium-chain-length (MCL) repeating units have physical properties resembling the petroleum-based plastic polyethylene. In addition, these SCL-co-MCL PHA copolymers have been investigated for biomedical applications such as tissue engineering. However, bacterial production of these SCL-co-MCL PHA copolymers is often at a much lower yield compared to SCL PHA biosynthesis produced from simple sugars such as glucose. Here, we report the highest yield to date of SCL-co-MCL PHA copolymers produced from glucose. Two separate biosynthetic pathways for SCL and MCL PHAs were introduced into Escherichia coli LS5218, and copolymer production experiments were carried out in batch fermentations. The PHA copolymers produced consisted of repeating units with 4, 6, 8, 10, and 12 carbons at mol % concentrations similar to that of other SCL-co-MCL PHA copolymers reported to have desirable physical properties. The PHA repeating unit compositions, structures, and linkages between individual repeating unit types were analyzed by GC and NMR. The thermal properties of purified PHA copolymers were also examined. The engineered strain developed in this study (E. coli LS5218-STQKABGK) provides a platform to further increase PHA copolymer yields from unrelated carbon sources in a non-native PHA producing bacterial strain.

Biopolymers production from mixed cultures and pyrolysis by-products

Polyhydroxyalkanoates (PHAs) production from low value substrates and/or byproducts represents an economical and environmental promising alternative to established industrial manufacture methods. Bio-oil resulting from the fast-pyrolysis of chicken beds was used as substrate to select a mixed microbial culture (MMC) able to produce PHA under feast/famine conditions. In this study a maximum PHA content of 9.2% (g/g cell dry weight) was achieved in a sequencing batch reactor (SBR) operated for culture selection. The PHA obtained with bio-oil as a carbon source was a copolymer composed by 70% of hydroxybutyrate (HB) and 30% of hydroxyvalerate (HV) monomers. Similar results have been reported by other studies that use real complex substrates for culture selection indicating that bio-oil can be a promising feedstock to produce PHAs using MMC. To the best of our knowledge this is the first study that demonstrated the use of bio-oil resulting from fast pyrolysis as a possibly feedstock to produce short chain length polyhydroxyalkanoates.

Native granule associated short chain length polyhydroxyalkanoate synthase from a marine derived Bacillus sp. NQ-11/A2

A rapidly growing marine derived Bacillus sp. strain NQ-11/A2, identified as Bacillus megaterium, accumulated 61% polyhydroxyalkanoate by weight. Diverse carbon sources served as substrates for the accumulation of short chain length polyhydroxyalkanoate. Three to nine granules either single or attached as buds could be isolated intact from each cell. Maximum activity of polyhydroxyalkanoate synthase was associated with the granules. Granule-bound polyhydroxyalkanoate synthase had a K(m) of 7.1 x 10(-5) M for DL-beta-hydroxybutyryl-CoA. Temperature and pH optima for maximum activity were 30 degrees C and 7.0, respectively. Sodium ions were required for granule-bound polyhydroxyalkanoate synthase activity and inhibited by potassium. Granule-bound polyhydroxyalkanoate synthase was apparently covalently bound to the polyhydroxyalkanoate-core of the granules and affected by the chaotropic reagent urea. Detergents inhibited the granule-bound polyhydroxyalkanoate synthase drastically whilst glycerol and bovine serum albumin stabilized the synthase.

Production and characterization of bacterial polyhydroxyalkanoate copolymers and evaluation of their blends by fourier transform infrared spectroscopy and scanning electron microscopy

Rhizobium meliloti produced a copolymer of short chain length polyhydroxyalkanoate (scl-PHA) on sucrose and rice bran oil as carbon substrates. Recombinant Escherichia coli (JC7623ABC1J4), bearing PHA synthesis genes, was used to synthesize short chain length-co-medium chain length PHA (scl-co-mcl-PHA) on glucose and decanoic acid. Fourier transform infrared spectroscopy (FTIR) spectra of the PHAs indicated strong characteristic bands at 1282, 1723, and 2934 cm(-1) for scl-PHA and at 2933 and 2976 cm(-1) for scl-co-mcl-PHA polymer. Differentiation of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate-P(HB-co-HV) copolymer was obseverd using FTIR, with absorption bands at 1723 and 1281 for PHB, and at 1738, 1134, 1215 cm(-1) for HV-copolymer. The copolymers were analyzed by GC and (1)H NMR spectroscopy. Films of polymer blends of PHA produced by R. meliloti and recombinant E. coli were prepared using glycerol, polyethylene glycol, polyvinyl acetate, individually (1:1 ratio), to modify the mechanical properties of the films and these films were evaluated by FTIR and scanning electron microscopy.

Investigating the structure–property relationship of bacterial PHA block copolymers

Mechanical testing of solvent cast films consisting of short-chain-length (SCL) polyhydroxyalkanoate (PHA) films suggested that films consisting of block copolymers retained more elasticity over time with respect to films of similar random copolymers of comparable composition. Two experimental techniques, wide angle X-ray scattering (WAXS) and uniaxial extension, were used to quantitatively investigate the structure–property relationship of bacterially synthesized PHA block copolymers of poly(3-hydroxybutyrate) (PHB) homopolymer and poly(3-hydroxybutyrate- co-3-hydroxyvalerate) random copolymer (PHBV) segments. Uniaxial testing experiments yielded the Young's modulus, ultimate tensile strength, and the elongation until fracture of the films. Percent crystallinity was determined by deconvolution of amorphous and crystalline scattering peaks obtained from WAXS. Two PHBV films containing either 8% 3-hydroxyvalerate monomer (3HV) or 29% 3HV exhibited a quick transition to brittle behavior, decreasing to less than 20% percent elongation at fracture within a few days after annealing. Conversely, the block copolymer samples remained higher than 100% elongation at fracture a full 3 months after annealing. Because block copolymers covalently link polymers that would otherwise form thermodynamically separate phases, the rates and degrees of crystallization of the block copolymers are less than the random copolymer samples. These differences translate into materials that extend the property space of biologically synthesized SCL PHA.

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442: CHARACTERIZATION OF A PARADIGMATIC ENZYME

Polyhydroxyalkanoates (PHAs) can be catabolized by many microorganisms using intra- or extracellular PHA depolymerases. Most of our current knowledge of these intracellular enzyme-coding genes comes from the analysis of short chain length PHA depolymerases, whereas medium chain length PHA (mcl-PHA) intracellular depolymerization systems still remained to be characterized. The phaZ gene of some Pseudomonas putida strains has been identified only by mutagenesis and complementation techniques as putative intracellular mcl-PHA depolymerase. However, none of their corresponding encoded PhaZ enzymes have been characterized in depth. In this study the PhaZ depolymerase from P. putida KT2442 has been purified and biochemically characterized after its overexpression in Escherichia coli. To facilitate these studies we have developed a new and very sensitive radioactive method for detecting PHA hydrolysis in vitro. We have demonstrated that PhaZ is an intracellular depolymerase that is located in PHA granules and that hydrolyzes specifically mcl-PHAs containing aliphatic and aromatic monomers. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. We have modeled the three-dimensional structure of PhaZ complexed with a 3-hydroxyoctanoate dimer. Using this model, we found that the enzyme appears to be built up from a corealpha/beta hydrolase-type domain capped with a lid structure with an active site containing a catalytic triad buried near the connection between domains. All these data constitute the first biochemical characterization of PhaZ and allow us to propose this enzyme as the paradigmatic representative of intracellular endo/exo-mcl-PHA depolymerases.