Synthesis and characterization of poly(3-hydroxyalkanoates) from Brassica carinata oil with high content of erucic acid and from very long chain fatty acids

Pseudomonas aeruginosa produced medium chain length poly(3-hydroxyalkanoates) (mcl-PHAs) when grown on substrates containing very long chain fatty acids (VLCFA, C>20). Looking for low cost carbon sources, we tested Brassica carinata oil (erucic acid content 35-48%) as an intact triglyceride containing VLCFA. Oleic (C18:1), erucic (C22:1), and nervonic (C24:1) acids were also employed for mcl-PHA production as model substrates. The polymers obtained were analyzed by GC of methanolyzed samples, GPC, 1H and 13C NMR, ESI MS of partially pyrolyzed samples, and DSC. The repeating units of such polymers were saturated and unsaturated, with a higher content of the latter in the case of the PHA obtained from B. carinata oil. Statistical analysis of the ion intensity in the ESI mass spectra showed that the PHAs from pure fatty acids are random copolymers, while the PHA from B. carinata oil is either a pure polymer or a mixture of polymers. Weight-average molecular weight varied from ca. 56,000 g/mol for the PHA from B. carinata oil and oleic acid, to about 120,000 g/mol for those from erucic and nervonic acids. The PHAs from erucic and nervonic acids were partially crystalline, with rubbery characteristics and a melting point (Tm) of 50°C, while the PHAs from oleic acid and from B. carinata oil afforded totally amorphous materials, with glass transition temperatures (Tg) of -52°C and -47°C, respectively.     

Biosynthesis and structural characterization of medium-chain-length poly(3-hydroxyalkanoates) produced by Pseudomonas aeruginosa from fatty acids

In this study, we investigated the ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize poly(3-hydroxyalkanoates) (PHAs) from saturated fatty acids with an even number of carbon atoms, from eight to 22, and from oleic acid. In a non-limiting medium, all carbon sources but docosanoic acid supported cell growth and PHA production, with eicosanoic acid giving the highest yield. In magnesium-limiting conditions, higher yields were obtained from sources with up to 16 carbon atoms. Composition was estimated by gas chromatography of methanolyzed samples and (13)C nuclear magnetic resonance. The 3-hydroxyalkanoate units extended from hexanoate to tetradecanoate or tetradecenoate, with octanoate and decanoate as the predominant components. Weight average molecular weights ranged from 78,000 to 316,000. Fast atom bombardment mass spectrometry of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.     

Production of polyesters consisting of medium chain length 3-hydroxyalkanoic acids by Pseudomonas mendocina 0806 from various carbon sources

Pseudomonas mendocina strain 0806 was isolated from oil-contaminated soil and found to produce polyesters consisting of medium chain length 3-hydroxyalkanoates (mclPHAs). The monomers of mclPHAs contained even numbers of carbon atoms, such as 3-hydroxyhexanoate (HHx or C₆), 3-hydroxyoctanoate (HO or C₈), and/or 3-hydroxydecanoate (HD or C₁₀) as major components when grown on many carbon sources unrelated to their monomeric structures, such as glucose, citric acid, and carbon sources related to their monomeric structures, such as myristic acid, octanoate, or oleic acid. On the other hand, PHA containing both even and odd numbers of hydroxyalkanoates (HA) monomers was synthesized when the strain was grown on tridecanoic acid. The molar ratio of carbon to nitrogen (C/N) had a significant effect on PHA composition: the strain produced PHAs containing 97–99% of HD monomer when grown in a glucose ammonium sulfate medium of C/N<20, and 20% HO, and 80% of the HD monomer when growth was conducted in media containing C/N>40. It was demonstrated that the HO/HD ratio in the polymers remained constant in media with a constant C/N ratio, regardless of the glucose concentration. Up to 3.6 g/L cell dry weight containing 45% of PHAs was produced when the strain was grown for 48 h in a medium containing 20 g/L glucose with a C/N ratio of 40.     

Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha

Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium octanoate as the carbon source in the presence of the fatty acid beta-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HA(MCL)) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, (1)H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HA(MCL) contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HA(MCL)-CoA thioesters were diverted from the fatty acid beta-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Microbial synthesis of poly(3-hydroxyalkanoates) by Pseudomonas aeruginosa from fatty acids: identification of higher monomer units and structural characterization

Pseudomonas aeruginosa ATCC 27853 accumulated poly(3-hydroxyalkanoates) (PHAs) after growth on saturated fatty acids with an odd number of carbon atoms. No nutrient limitation was required to induce PHA synthesis, although better yields were obtained when the medium was magnesium deprived. A comparative study was carried out between PHAs obtained from C-odd and those from C-even carbon sources. Repeating units identification was performed by gas chromatography (GC) and capillary liquid chromatography-electrospray mass spectrometry (LC-ESI MS) of methanolyzed samples. When C-odd n-alkanoic acids from nonanoic to pentadecanoic were used the lowest hydroxyalkanoate unit found was 3-hydroxyvalerate and the highest 3-hydroxypentadecanoate, whereas when C-even acids from octanoic to eicosanoic were used these were 3-hydroxycaproate and 3-hydroxyeicosanoate, respectively. Weight average molecular weights were in the range 187 000-596 000. DSC traces showed Tm and DeltaHm which varied from 43 to 58 degrees C and from 5.9 to 24.8 J/g, with the PHAs generated from C-odd carbon sources having lower values. ESI MS of partially pyrolyzed samples allowed the identification of oligomers up to heptamers, and statistical analysis of the ions intensity in the mass spectra showed that these PHAs are random copolyesters.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil

High-yield production of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha KCTC 2662 was investigated using soybean oil and γ-butyrolactone as carbon sources. In flask culture, it was shown that R. eutropha KCTC 2662 accumulated PHAs during the growth phase. The optimum carbon to nitrogen ratio (C/N ratio) giving the highest cell and PHA yield was 20 g-soybean oil/g-(NH(4))(2)SO(4). The 4-hydroxybutyrate (4HB) fraction in the copolymer was not strongly affected by the C/N ratio. In a 2.5-L fermentor, a homopolymer of poly(3-hydroxybutyrate) [P(3HB)] was produced from soybean oil as the sole carbon source by batch and fed-batch cultures of R. eutropha with dry cell weights of 15-32 g/L, PHA contents of 78-83 wt% and yields of 0.80-0.82 g-PHA/g-soybean oil used. By co-feeding soybean oil and γ-butyrolactone as carbon sources, a copolymer of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] could be produced with dry cell weights of 10-21 g/L, yields of 0.45-0.56 g-PHA/g-soybean oil used (0.39-0.50g-PHA/g-carbon sources used) and 4HB fractions of 6-10 mol%. Higher supplementation of γ-butyrolactone increased the 4HB fraction in the copolymer, but decreased cell and PHA yield.     

Industrial scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Large scale production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by Aeromonas hydrophila 4AK4 was examined in a 20,000 l fermentor. Cells were first grown using glucose as a carbon source, and polyhydroxyalkanoate (PHA) biosynthesis was triggered by the addition of lauric acid under conditions of limited nitrogen or phosphorus. When cells first grown in a medium containing 50 g glucose l(-1) were further cultivated after the addition of 50 g lauric acid l(-1) under phosphorus limitation, a final cell concentration, PHA concentration and PHA content of 50 g l(-1), 25 g l(-1), and 50 wt%, respectively, were obtained in 46 h, equivalent to PHA productivity of 0.54 g l(-1)t h(-1). The copolymer produced was found to be a random copolymer, and the 3HHx fraction was 11 mol%.     

Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans.

Pseudomonas resinovorans produced poly-beta-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose. In a chemostat culture, the PHA composition was beta-hydroxybutyrate (C4)-beta-hydroxyhexanoate (C6)-beta-hydroxyoctanoate (C8)-beta-hydroxydecanoate (C10) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62:23:7) on hexanoic acid. Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate.     

Chiral compounds from bacterial polyesters: sugars to plastics to fine chemicals.

A novel and efficient method for the production of enantiomerically pure (R)-(-)-hydroxycarboxylic acids by in vivo depolymerization of microbial polyester polyhydroxyalkanoates (PHAs) was developed. Using this method, several model compounds, (R)-(-)-3-hydroxyalkanoic acids, consisting of 4 to 12 carbon atoms, and (R)-(-)-3-hydroxy-5-phenylvaleric acid, could be prepared. In particular, (R)-(-)-3-hydroxybutyric acid could be efficiently prepared by this method. By providing the environmental condition in which cells possess high activity of intracellular PHA depolymerase and low activity of (R)-(-)-3-hydroxybutyric acid dehydrogenase, (R)-(-)-3-hydroxybutyric acid could be produced with a yield of 96% in only 30 min by in vivo depolymerization of polyhydroxybutyrate (PHB) accumulated in Alcaligenes latus.     

Continuous Production of Long-Side-Chain Poly-beta-Hydroxyalkanoates by Pseudomonas oleovorans

Shake flask experiments showed that Pseudomonas oleovorans began to be growth inhibited at 4.65 g of sodium octanoate liter, with total inhibition at 6 g liter. In chemostat studies with 2 g of ammonium sulfate and 8 g of octanoate liter in the feed, the maximum specific growth rate was 0.51 h, and the maximum specific rate of poly-beta-hydroxyalkanoate (PHA) production was 0.074 g of PHA g of cellular protein h at a dilution rate (D) of 0.25 h. When the specific growth rate (mu) was <0.3 h, the PHA composition was relatively constant with a C(4)/C(6)/C(8)/C(10) ratio of 0.1:1.7:20.7:1.0. At mu > 0.3 h, a decrease in the percentage of C(8) with a concomitant increase in C(10) monomers as mu increased was probably due to the effects of higher concentrations of unmetabolized octanoate in the fermentor. At D = 0.24 h and an increasing carbon/nitrogen ratio, the percentage of PHA in the biomass was constant at 13% (wt/wt), indicating that nitrogen limitation did not affect PHA accumulation. Under carbon-limited conditions, the yield of biomass from substrate was 0.76 g of biomass g of octanoate consumed, the yield of PHA was 0.085 g of PHA g of octanoate used, and 7.9 g of octanoate was consumed for each gram of NH(4) supplied. The maintenance coefficient was 0.046 g of octanoate g of biomass h. Replacement of sodium octanoate with octanoic acid appeared to result in transport-limited growth due to the water insolubility of the acid.     

Effect of nitrogen limitation on long-side-chain poly-beta-hydroxyalkanoate synthesis by Pseudomonas resinovorans.

Pseudomonas resinovorans produced poly-beta-hydroxyalkanoates (PHAs) when grown on hydrocarbons but not on glucose. In a chemostat culture, the PHA composition was beta-hydroxybutyrate (C4)-beta-hydroxyhexanoate (C6)-beta-hydroxyoctanoate (C8)-beta-hydroxydecanoate (C10) (1:15:75:9) on octanoate and C4-C6-C8-C10 (8:62:23:7) on hexanoic acid. Contrary to the reported behavior of Pseudomonas oleovorans, the PHA accumulation rate increased under ammonium limitation on octanoate.     

Fourier transform infrared spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on sodium octanoate

Poly(hydroxyalkanoates) PHAs are synthesized by many bacteria as inclusion bodies and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. A method based on FT-IR was developed to carry out rapid qualitative and quantitative analysis of PHAs in Pseudomonas, when they were grown on sodium octanoate. Using absorbance of the ester band of PHAs, a rapid method was reported to distinguish PHB and PHO and to determine polymer content in intact bacteria. Relative areas in which the C=O area was normalized to the area of the peak representing the amid group (1656 cm(-1)) characteristic of bacteria were calibrated to the polymer content which was determined after solvent extraction. Polymer contents vary from 0% to 53% and depend on the nature of the bacteria. Among 27 strains of Pseudomonas belonging to the rRNA homology group I, a very low amount of bacteria were able to produce PHB. The majority of strains were able to produce a copolymer, PHO, in which the major constituent unit is 3-hydroxyoctanoate. The FT-IR results were further confirmed by gas chromatography analysis after methanolysis of polymer, but FT-IR method requires less preparation of sample than gas chromatography and it is very useful for screening a large variety of Pseudomonas.     

FabG mediates polyhydroxyalkanoate production from both related and nonrelated carbon sources in recombinant Escherichia coli LS5218

Polyhydroxyalkanoates (PHAs) composed of a mixture of short-chain-length-medium-chain-length (SCL-MCL) hydroxyacyl monomers are biologically produced polyesters that have properties ranging from thermoplastic to elastomeric, dependent on the molar ratio of SCL to MCL monomers incorporated into the copolymer. Because of the potential wide range of properties and applications for SCL-MCL PHA copolymers, it is important to develop and characterize novel metabolic pathways for SCL-MCL PHA production. The current study shows that coexpression of fabG genes from either E. coli or Pseudomonas sp. 61-3 with fabH(F87T) and PHA synthase genes enhances the production of SCL-MCL PHA copolymer from both related and nonrelated carbon sources in Escherichia coli LS5218, indicating the flexibility of FabG as a monomer-supplying enzyme for biological PHA production.     

Characterisation of polyhydroxyalkanoate copolymers with controllable four-monomer composition

Polyhydroxyalkanoate (PHA) copolymers comprising the four monomers 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylvalerate (3HMV) and 3-hydroxy-2-methylbutyrate (3HMB) were generated using the recently discovered Defluviicoccus vanus-related glycogen accumulating organisms (DvGAOs) under anaerobic conditions without applying any nutrient limitations. The composition could be manipulated in a defined range by modifying the ratio of propionate and acetate provided in the feed stream. The PHAs produced were characterised as random copolymers (from propionate alone) or a mixture of random copolymers (from mixture of propionate and acetate) through microstructure analysis using 13C NMR spectroscopy. The sequence distribution of all eight comonomer pairs in the carbonyl region of 3HB and 3HV was identified and assigned with confidence utilising two-dimensional heteronuclear multiple bond coherence (HMBC) spectroscopy. Weight average molecular weights were in the range 390-560 kg/mol. Differential scanning calorimetry (DSC) traces showed that the melting temperature (Tm) varied between 70 and 161 degrees C and glass transition temperature (Tg) ranged from -8 to 0 degrees C. The incorporation of considerable amounts of 3HMV and 3HMB monomer units introduced additional "defects" into the PHBV copolymer structure and hence greatly lowered the crystallinity. The data indicate the potential of these four-monomer PHAs to be employed for practical applications, considering their favourable properties and the cost-effective production process using a mixed culture and simple carbon sources.     

The biodegradation of poly-3-hydroxyalkanoates,PHAs, with long alkyl substitutents byPseudomonas maculicola

Utilizing a quantitative clear zone technique, the activity of an extracellular depolymerase system fromPseudomonas maculicola was investigated. Polymer degradation was influenced by the amount and availability of secondary carbon sources, with a simultaneous utilization of both sources. The initial carbon source in the liquid preculture also affected the eventual colony growth and polymer degradation. The enzyme solution was determined to readily degrade poly-3-hydroxyalkanoates (PHAs) with relatively long alkyl substituents at the 3 position: poly-3-hydroxyoctanoate (PHO), poly-3-hydroxynonanoate (PHN), and their copolymers (P[HO-co-HN]) and poly-3-hydroxyundecanoate (PHU). However, the system was unable to degrade either PHAs with shorter alkyl groups, including poly-3-hydroxybutyrate (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-co-HV]) or PHAs with unusual substituents such as poly(3-hydroxy-5-phenylvaleric acid) (PHPV). It is proposed that degradation of these more bulky side chain polymers was prevented by the inability of the bacteria to assimilate their monomeric components, which inhibited the successful utilization of secondary carbon sources and thus inhibited colony growth.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Production of polyhydroxyalkanoates from agricultural waste and surplus materials

To be competitive with common plastics, the production costs of polyhydroxyalkanoates (PHAs) have to be minimized. Biotechnological polymer production occurs in aerobic processes; therefore, only about 50% of the main carbon sources and even a lower percentage of the precursors used for production of co-polyesters end up in the products wanted. A second cost factor in normally phosphate-limited production processes for PHAs is the costs for complex nitrogen sources. Both cheap carbon sources and cheap nitrogen sources are available from agricultural waste and surplus materials and make a substantial contribution for minimizing PHA production costs. In this study, fermentations for PHA production were carried out in laboratory-scale bioreactors on hydrolyzed whey permeate and glycerol liquid phase from the biodiesel production using a highly osmophilic organism. Without any precursor, the organism produced a poly[3(hydroxybutyrate-co-hydroxyvalerate)] copolyester on both carbon sources. During the accumulation phases, a constant 3-hydroxyvalerate content of 8-10% was obtained at a total PHA concentration of 5.5 g/L (on hydrolyzed whey permeate) and 16.2 g/L (glycerol liquid phase). In an additional fermentation, an expensive nitrogen source was substituted by meat and bone meal beside the glycerol liquid phase as a carbon source, resulting in a final PHA concentration of 5.9 g/L.