Biosynthesis of Poly(3-Hydroxyalkanoic Acid) Copolymer from CO(inf2) in Pseudomonas acidophila through Introduction of the DNA Fragment Responsible for Chemolithoautotrophic Growth of Alcaligenes hydrogenophilus

Pseudomonas acidophila is a bacterial strain producing a poly(3-hydroxyalkanoic acid) (PHA) copolymer from low-molecular-weight organic compounds such as formate and acetate. The genes responsible for PHA production were cloned in cosmid pIK7 containing a 14.8-kb HindIII fragment of P. acidophila DNA. With the aim of developing a means of producing a PHA copolymer from CO(inf2), cosmid pIK7 was introduced into a polymer-negative mutant of the chemolithoautotrophic bacterium Alcaligenes eutrophus PHB(sup-)4. However, the recombinant strain produced a homopolymer of 3-hydroxybutyric acid (polyhydroxybutyric acid) from CO(inf2). Since it was thought that the composition of the accumulated polymer might depend not on the PHA biosynthetic genes but on the metabolism of the host strain, a recombinant plasmid, pFUS, containing the genes for chemolithoautotrophic growth of the hydrogen-oxidizing bacterium A. hydrogenophilus was introduced into P. acidophila by conjugation. The recombinant plasmid pFUS was stably maintained in P. acidophila in the absence of chemolithoautotrophic or antibiotic selection. This pFUS-harboring strain possessed the ability to grow under a gas mixture of H(inf2), O(inf2), and CO(inf2) in a mineral salts medium, and PHA copolymer accumulation was confirmed by nuclear magnetic resonance spectral analysis. A gas chromatogram obtained by gas chromatography-mass spectrometry showed the composition of the polymer to be 52.8% 3-hydroxybutyrate, 41.1% 3-hydroxyoctanoate, and 6.1% 3-hydroxydecanoate. This is the first report of the production of a PHA copolymer from CO(inf2) as sole carbon source.     

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products

AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.     

Autotrophic synthesis of polyalkanoates by Alcaligenes eutrophus in presence of carbon monoxide]

The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophus was found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide. The gas mixtures that contained from 5 to 20 vol% CO did not inhibit the key enzymes of PHA synthesis--beta-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase. In the presence of CO, cells accumulated up to 70-75 wt% PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate. Chromatographic-mass spectrometric analysis showed that the PHA synthesized by A. eutrophus is a copolymer containing more than 99 mol% beta-hydroxybutyrate and trace amounts of beta-hydroxyvalerate. The PHA synthesized under the conditions described did not differ from that synthesized by A. eutrophus cells from electrolytic hydrogen.     

The Autotrophic Synthesis of Polyhydroxyalkanoate by Alcaligenes eutrophusin the Presence of Carbon Monoxide

The CO-resistant strain B5786 of the hydrogen-oxidizing bacterium Alcaligenes eutrophuswas found to be able to synthesize polyhydroxyalkanoates (PHAs) under the conditions of growth limitation by nitrogen deficiency (the factor that promotes PHA synthesis) and growth inhibition by carbon monoxide. The gas mixtures that contained from 5 to 20 vol % CO did not inhibit the key enzymes of PHA synthesis–-ketothiolase, acetoacetyl-CoA reductase, hydroxybutyrate dehydrogenase, and PHA synthase. In the presence of CO, cells accumulated up to 70–75 wt % PHA (with respect to the dry biomass) without any noticeable increase in the consumption of the gas substrate. Chromatographic–mass spectrometric analysis showed that the PHA synthesized by A. eutrophusis a copolymer containing more than 99 mol % -hydroxybutyrate and trace amounts of -hydroxyvalerate. The PHA synthesized under the conditions described did not differ from that synthesized by A. eutrophuscells from electrolytic hydrogen.     

Gene dosage effects on polyhydroxyalkanoates synthesis from n-alcohols in Paracoccus denitrificans

Putative promoters of polyhydroxyalkanoate (PHA)-synthetic genes of Paracoccus denitrificans were identified. Gene dosage effects for PHA synthesis were investigated in recombinants of P. denitrificans with increased expression levels of each PHA synthetic enzyme. In the cultivation of shake flasks using ethanol or n-pentanol as carbon source, a self-cloning recombinant of the phaC-encoding PHA synthase showed the highest contents [(g PHA). (g total biomass)-1] and the highest rates of PHA accumulation [(g PHA). (g residual biomass)-1. h-1] among these recombinants. The PHA content and PHA accumulation rate (g PHA/g residual biomass. h-1) of the self-cloning recombinant was 2 and 2.7 times higher, respectively, than that of the wild strain. This result strongly suggests that the step of PHA synthase is limited in in vivo PHA synthesis from n-pentanol via 3-ketovaleryl-CoA through beta-oxidation, and from ethanol via acetyl-CoA. Studies on fed-batch cultures keeping the alcohol concentration constant (0.02%) in a 5-L bioreactor showed that the ability of PHA biosynthesis was improved by the gene dosage of PHA synthase, although the growth rate of cells during the growth-associated PHA synthesis phase was retarded. The molecular weight of the polymer isolated from the strain, dosed by the PHA synthase gene, was lower than that of the polymer from the wild strain, indicating that the amount of PHA synthase in vivo affects the molecular weight of the polymer.     

Physiological and biochemical characteristics and capacity for polyhydroxyalkanoates synthesis in a glucose-utilizing strain of hydrogen-oxidizing bacteria, Ralstonia eutropha B8562

The physiological, biochemical, genetic, and cultural characteristics of the glucose-utilizing mutant strain Ralstonia eutropha B8562 were investigated in comparison with the parent strain R. eutropha B5786. The morphological, cultural, and biochemical characteristics of strain R. eutropha B8562 were similar to those of strain R. eutropha B5786. Genetic analysis revealed differences between the 16S rRNA gene sequences of these strains. The growth characteristics of the mutant using glucose as the sole carbon and energy source were comparable with those of the parent strain grown on fructose. Strain B8562 was characterized by high yields of polyhydroxyalkanoate (PHA) from different carbon sources (CO2, fructose, and glucose). In batch culture with glucose under nitrogen limitation, PHA accumulation reached 90% of dry weight. In PHA, beta-hydroxybutyrate was predominant (over 99 mol %); beta-hydroxyvalerate (0.25-0.72 mol %) and beta-hydroxyhexanoate (0.008-1.5 mol %) were present as minor components. The strain has prospects as a PHA producer on glucose-containing media.     

Study of Ralstonia eutropha culture producing polyhydroxyalkanoates on products of coal processing

Kinetic indices of growth, polyhydroxyalkanoate (PHA) accumulation, and gas exchange have been studied in a culture of the carbon monoxide-resistant hydrogen strain Ralstonia eutropha B-5786 grown on a gaseous substrate (GS) obtained by lignite gasification. The GS was shown to be suitable for PHA production. To increase the degree of GS consumption, various modes of gas supply to the culture were tested. Based on the results, an algorithm was developed for calculating and controlling gas-exchange parameters in the PHA-accumulating culture of Ralstonia eutropha, grown on a new GS allowing high polymer yields (up to 75%) and degrees of the substrate utilization (up to 90%).     

Study of a Ralstonia eutropha Culture Producing Polyhydroxyalkanoates on Products of Coal Processing

Kinetic indices of growth, polyhydroxyalkanoate (PHA) accumulation, and gas exchange were studied in a culture of the carbon monoxide-resistant hydrogen strain Ralstonia eutropha B-5786 grown on a gaseous substrate (GS) obtained by lignite gasification. The GS was shown to be suitable for PHA production. To increase the degree of GS consumption, various modes of gas supply to the culture were tested. Based on the results, an algorithm was developed for calculating and controlling gas-exchange parameters in the PHA-accumulating culture of Ralstonia eutropha, grown on a new GS allowing high polymer yields (up to 75%) and degrees of substrate utilization (up to 90%).     

Polyhydroxybutyrate production in halophilic marine bacteria Vibrio proteolyticus isolated from the Korean peninsula

Polyhydroxybutyrates (PHB) are biodegradable polymers that are produced by various microbes, including Ralstonia, Pseudomonas, and Bacillus species. In this study, a Vibrio proteolyticus strain, which produces a high level of polyhydroxyalkanoate (PHA), was isolated from the Korean marine environment. To determine optimal growth and production conditions, environments with different salinity, carbon sources, and nitrogen sources were evaluated. We found that the use of a medium containing 2% (w/v) fructose, 0.3% (w/v) yeast extract, and 5% (w/v) sodium chloride (NaCl) in M9 minimal medium resulted in high PHA content (54.7%) and biomass (4.94 g/L) over 48 h. Addition of propionate resulted in the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(HB-co-HV)) copolymer as propionate acts as a precursor for the HV unit. In these conditions, the bacteria produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) containing a 15.8% 3HV fraction with 0.3% propionate added as the substrate. To examine the possibility of using unsterilized media with high NaCl content for PHB production, V. proteolyticus was cultured in sterilized and unsterilized conditions. Our results indicated a higher growth, leading to a dominant population in unsterilized conditions and higher PHB production. This study showed the conditions for halophilic PHA producers to be later implemented at a larger scale.

     

Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations

Improved production costs will accelerate commercialization of polyhydroxyalkanoate (PHA) polymer and PHA-based products. Plant oils are considered favorable feedstocks, due to their high carbon content and relatively low price compared to sugars and other refined carbon feedstocks. Different PHA production strategies were compared using a recombinant strain of Ralstonia eutropha that produces high amounts of P(HB-co-HHx) when grown on plant oils. This R. eutropha strain was grown to high cell densities using batch, extended batch, and fed batch fermentation strategies, in which PHA accumulation was triggered by nitrogen limitation. While extended batch culture produced more biomass and PHA than batch culture, fed batch cultivation was shown to produce the highest levels of biomass and PHA. The highest titer achieved was over 139 g/L cell dry weight (CDW) of biomass with 74% of CDW as PHA containing 19 mol% HHx. Our data suggest that the fermentation process is scalable with a space time yield (STY) better than 1 g PHA/L/h. The achieved biomass concentration and PHA yield are among the highest reported for the fermentation of recombinant R. eutropha strains producing P(HB-co-HHx).     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.     

利用微生物混合培养技术生产聚羟基烷酸(PHA)研究

研究了圆褐固氮菌(Azotobacter chroococcum)突变株G3与巨大芽孢杆菌(Bacillus megaterium)G6发酵生产聚羟基烷酸(PHA)的人工可配伍性,确定了它们混合培养的适宜条件,先将G3菌株发酵培养24～28 h后,再以15%(v/v)接种量接入G6菌株并同时补加05%(g/g)蛋白胨(FP)和0.5%(g/g)NH4NO3,继续混合培养42～46h,细胞干重达32 g/L,PHA含量为80%,再结合补料技术最终生物量可达53 g/L,PHA产生量达42.4 g/L。糖对PHA的转化率为0.32。人工混合培养成功地解决了固氮菌发酵生产PHA过程中,发酵液粘度过高,传质较差,补糖总量上不去等技术问题。     

Polyhydroxyalkanoate degradation is associated with nucleotide accumulation and enhances stress resistance and survival of Pseudomonas oleovorans in natural water microcosms

Pseudomonas oleovorans GPo1 and its polyhydroxyalkanoic acid (PHA) depolymerization-minus mutant, GPo500 phaZ, residing in natural water microcosms, were utilized to asses the effect of PHA availability on survival and resistance to stress agents. The wild-type strain showed increased survival compared to the PHA depolymerase-minus strain. The appearance of a round cellular shape, characteristic of bacteria growing under starvation conditions, was delayed in the wild type in comparison to the mutant strain. Percent survival at the end of ethanol and heat challenges was always higher in GPo1 than in GPo500. Based on these results and on early experiments (H. Hippe, Arch. Mikrobiol. 56:248-277, 1967) that suggested an association of PHA utilization with respiration and oxidative phosphorylation, we investigated the association between PHA degradation and nucleotide accumulation. ATP and guanosine tetraphosphate (ppGpp) production was analyzed under culture conditions leading to PHA depolymerization. A rise in the ATP and ppGpp levels appeared concomitant with PHA degradation, while this phenomenon was not observed in the mutant strain unable to degrade the polymer. Complementation of the phaZ mutation restored the wild-type phenotype.     

Poly(ethylene glycol)-mediated molar mass control of short-chain- and medium-chain-length poly(hydroxyalkanoates) from Pseudomonas oleovorans

Three strains of Pseudomonas oleovorans, a well known poly(hydroxyalkanoate) (PHA) producer, were tested for the ability to control PHA molar mass and end group structure by addition of poly(ethylene glycol) (PEG) to the fermentation medium. Each strain of P. oleovorans - NRRL B-14682 (B-14682), NRRL B-14683 (B-14683), and NRRL B-778 (B-778) - synthesized a different type of PHA from oleic acid when cultured under identical growth conditions. Strain B-14682 produced poly(3-hydroxybutyrate) (PHB), while B-14683 synthesized a medium-chain-length PHA ( mcl-PHA) with a repeat unit composition ranging from C4 to C14 and some mono-unsaturation in the C14 alkyl side chains. Strain B-778 synthesized a mixture of PHB (95 mol%) and mcl-PHA (5 mol%). The addition of 0.5% (v/v) PEG (M(n) =200 g/mol, PEG-200) to the fermentation broth of strains B-14682 and B-778 resulted in chain termination through esterification at the carboxyl terminus of the PHB with PEG chain segments, thus reducing the molar mass by 54% and 23%, respectively. The molar mass of the mcl-PHA produced by strains B-14683 and B-778 also showed a 34% and 47% reduction in the presence of PEG-200, respectively, but no evidence of esterification was present. PEG-400 (M(n) =400 g/mol) had a reduced effect on PHA molar mass. In fact, the molar masses of the mcl-PHA derived from strain B-14683 and both the PHB and mcl-PHA from B-778 were unchanged by PEG-400. In contrast, the PHB produced by B-14682 showed a 35% reduction in molar mass in the presence of PEG-400.     

Production of interleukin-1-alpha and interleukin-2 by mononuclear cells in healthy adults in relation to different experimental conditions and to the presence of growth hormone

Several reports support the view that the growth hormone (GH) possesses a number of important immunomodulatory properties. This study was undertaken to determine in vitro the role of the GH on interleukin (IL) production. Cultures of blood peripheral lymphocytes obtained from human normal adults were performed in RPMI medium in the presence or absence of phytohemagglutinin (PHA), heated normal serum (NHS) 1% and GH 12.5-500 microgram/l. After incubation from 15 h to 4 days at 37 degrees C in a humidified atmosphere containing 5% CO2, cells were discarded and the supernatants were tested for their contents of IL-1 alpha and IL-2 measured using the Amersham radioimmunoassay system. The results of these in vitro experiments show that: (1) the bulk cultures from peripheral lymphocytes are suitable to study the IL-1 and IL-2 production; (2) in optimal conditions for IL production (incubation during 48 h in the presence of PHA and NHS) no effect of GH was observed on IL production; (3) in the absence of PHA GH acts at physiological doses (less than 100 ng/ml) by increasing the IL production. This effect of GH was optimized with a short time of incubation (16 h) and in the simplest conditions of cultures, that is to say in the absence of serum and of PHA: thus in the presence of GH 100 ng/ml the IL-1 production increases from 0.53 to 3.86 fmol (tubes) and IL-2 increases from 0.18 to 3.22 fmol (tubes). These differences are significant (p less than 0.001). We conclude that GH acts in vitro on mononuclear cells to induce IL production.     

Recovery of poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) from Ralstonia eutropha cultures with non‐halogenated solvents

Reduced downstream costs, together with high purity recovery of polyhydroxyalkanoate (PHA), will accelerate the commercialization of high quality PHA‐based products. In this work, a process was designed for effective recovery of the copolymer poly(hydroxybutyrate‐co‐hydroxyhexanoate) (P(HB‐co‐HHx)) containing high levels of HHx (>15 mol%) from Ralstonia eutropha biomass using non‐halogenated solvents. Several non‐halogenated solvents (methyl isobutyl ketone, methyl ethyl ketone, and butyl acetate and ethyl acetate) were found to effectively dissolve the polymer. Isoamyl alcohol was found to be not suitable for extraction of polymer. All PHA extractions were performed from both dry and wet cells at volumes ranging from 2 mL to 3 L using a PHA to solvent ratio of 2% (w/v). Ethyl acetate showed both high recovery levels and high product purities (up to 99%) when using dry cells as starting material. Recovery from wet cells, however, eliminates a biomass drying step during the downstream process, potentially saving time and cost. When wet cells were used, methyl isobutyl ketone (MIBK) was shown to be the most favorable solvent for PHA recovery. Purities of up to 99% and total recovery yields of up to 84% from wet cells were reached. During polymer recovery with either MIBK or butyl acetate, fractionation of the extracted PHA occurred, based on the HHx content of the polymer. PHA with higher HHx content (17–30 mol%) remained completely in solution, while polymer with a lower HHx content (11–16 mol%) formed a gel‐like phase. All PHA in solution could be precipitated by addition of threefold volumes of n‐hexane or n‐heptane to unfiltered PHA solutions. Effective recycling of the solvents in this system is predicted due to the large differences in the boiling points between solvent and precipitant. Our findings show that two non‐halogenated solvents are good candidates to replace halogenated solvents like chloroform for recovery of high quality PHA. Biotechnol. Bioeng. 2013; 110: 461–470. © 2012 Wiley Periodicals, Inc.