Produce Polyhydroxyalkanoates Production Research Articles

Potential of activated sludge-derived mixed microbial culture enriched on acetate to produce polyhydroxyalkanoates from various substrates

The use of waste activated sludge (WAS) as a biocatalyst to produce polyhydroxyalkanoates (PHA) from waste streams may help promote the beneficial use of WAS for low-carbon, sustainable wastewater treatment. However, it remains unclear which types of substrates can be used for efficient PHA production, and how the PHA production can be maximized. This study aimed to assess the substrate versatility of mixed microbial cultures (MMCs) constructed from WAS by enriching PHA-accumulating bacteria using an aerobic dynamic discharge (ADD) process fed with acetate. Twelve different substrates, including organic acids, saccharides, and alcohols, were selected as the test substrates. In single-batch assays, the highest PHA production (583–680 mg/L) was achieved using butyrate, acetate, and pyruvate. In fed-batch assays, > 30 wt% PHA content was achieved using acetate, butyrate, propionate, lactate, and ethanol, with the highest content (60.3 wt%) using acetate. These results indicate that acetate-fed MMC by the ADD process could efficiently produce PHA from volatile fatty acids, lactate, pyruvate, and ethanol. Polyhydroxybutyrate was preferentially produced from acetate, butyrate, pyruvate, lactate, and ethanol, whereas polyhydroxyvalerate was notably produced from propionate. The results suggest that PHA can be efficiently produced from a wide range of substrates using MMCs enriched on a single substrate.

Exploring polyhydroxyalkanoates biosynthesis using hydrocarbons as carbon source: a comprehensive review.

Environmental pollution caused by petrochemical hydrocarbons (HC) and plastic waste is a pressing global challenge. However, there is a promising solution in the form of bacteria that possess the ability to degrade HC, making them valuable tools for remediating contaminated environments and effluents. Moreover, some of these bacteria offer far-reaching potential beyond bioremediation, as they can also be utilized to produce polyhydroxyalkanoates (PHAs), a common type of bioplastics. The accumulation of PHAs in bacterial cells is facilitated in environments with high C/N or C/P ratio, which are often found in HC-contaminated environments and effluents. Consequently, some HC-degrading bacteria can be employed to simultaneously produce PHAs and conduct biodegradation processes. Although bacterial bioplastic production has been thoroughly studied, production costs are still too high compared to petroleum-derived plastics. This article aims to provide a comprehensive review of recent scientific advancements concerning the capacity of HC-degrading bacteria to produce PHAs. It will delve into the microbial strains involved and the types of bioplastics generated, as well as the primary pathways for HC biodegradation and PHAs production. In essence, we propose the potential utilization of HC-degrading bacteria as a versatile tool to tackle two major environmental challenges: HC pollution and the accumulation of plastic waste. Through a comprehensive analysis of strengths and weaknesses in this aspect, this review aims to pave the way for future research in this area, with the goal of facilitating and promoting investigation in a field where obtaining PHAs from HC remains a costly and challenging process.

Application of the solid-state fermentation process and its variations in PHA production: a review.

Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

Effect of cathode properties on the thermophilic electrosynthesis of PolyHydroxyAlkanoates by Kyrpidia spormannii

The recent discovery of the Knallgas bacterium Kyrpidia spormannii EA-1, able to produce PolyHydroxyAlkanoates (PHAs) on a cathode, is of great interest to produce bioplastics from electricity and CO2 waste streams. However, little is known on how to improve its electroautotrophic growth and performance in PHA production. We investigated the effect of cathode properties on biofilm formation and PHA synthesis, focusing on the choice of cathode material, the surface modification of a graphite cathode with different treatments or by electrodeposition of metal catalysts, and the distance between anode and cathode. The results show higher performance of iron-based electrodes, isopropanol and sonication treatment, and close distance between electrodes, with up to a 3-fold increase of PHA production, reaching a production of 117 mg·day−1·m−2, and a 10-fold increase in cell density of the biofilm (10.7 Log10 cells·cm−2).

Polyhydroxyalkanoate Production by Caenibius tardaugens from Steroidal Endocrine Disruptors.

The α-proteobacterium Caenibius tardaugens can use estrogens and androgens as the sole carbon source. These compounds are steroidal endocrine disruptors that are found contaminating soil and aquatic ecosystems. Here, we show that C. tardaugens, which has been considered as a valuable biocatalyst for aerobic steroidal hormone decontamination, is also able to produce polyhydroxyalkanoates (PHA), biodegradable and biocompatible polyesters of increasing biotechnological interest as a sustainable alternative to classical oil-derived polymers. Steroid catabolism yields a significant amount of propionyl-CoA that is metabolically directed towards PHA production through condensation into 3-ketovaleryl-CoA, rendering a PHA rich in 3-hydroxyvalerate. To the best of our knowledge, this is the first report where PHAs are produced from steroids as carbon sources.

Optimization of growth and electrosynthesis of PolyHydroxyAlkanoates by the thermophilic bacterium Kyrpidia spormannii

The recent discovery of the Knallgas bacterium Kyrpidia spormannii EA-1, able to produce PolyHydroxyAlkanoates (PHA) on a cathode, is of great interest to sustainably produce bioplastics from electricity and CO2. In this study, we investigated the effect of cathode properties on PHA synthesis, focusing on the choice of cathode material, the surface modification of a graphite cathode with different treatments or by electrodeposition of metal catalysts, and the distance between anode and cathode. The results show a particular high performance of iron-based electrodes with increased biofilm density and PHA production. Also, a simple treatment of the graphite cathode with isopropanol and sonication showed the best performance compared to more elaborate surface modifications treatments. Finally, the shorter the distance between anode and cathode was, the better the PHA production was obtained. These optimizations allow to increase by 5-fold the PHA production compared to initial conditions, reaching a production of 117 mg·day−1·m−2.

Enrichment of Methylocystis dominant mixed culture from rice field for PHB production

Presence of methanotrophs in diverse environmental habitats helps to reduce emissions of greenhouse gas like methane. Isolation and culture of undiscovered wealth of methanotrophic organisms can help in exploitation of these organisms in value added products. The present study focuses on the enrichment of methanotroph dominated mixed microbial community by use of three stage strategy of revival, proliferation, and segregation. During the enrichment process amplicon sequencing of 16 s rRNA V3-V4 region showed relative abundance of mixed culture comprising single methanotrophic species of Methylocystis genus (88.92%) along with only three other species. Methylocystis dominant mixed culture (MMI-11) was observed to produce polyhydroxyalkanoates (PHA). During studies to identify favourable culture conditions, nitrate was found to be preferred nitrogen source for growth and PHA production. Cell growth ability to produce PHA was also evaluated at 14 L fermentor by supplying gas using continuous bubbling and through pressurization in the headspace. The mixed methanotrophic culture was found to accumulate maximum of 22.20% polyhydroxybutyrate (PHB) under nitrate limited condition. The molecular weight of PHB was found to be 2.221 × 105 g mol−1 with polydispersity of 1.82.

Production of the Polyhydroxyalkanoate PHBV from Ricotta Cheese Exhausted Whey by Haloferax mediterranei Fermentation.

In the last decade, the dairy industry underwent a rapid expansion due to the increasing demand of milk-based products, resulting in high quantity of wastewater, i.e., whey and ricotta cheese exhausted whey (RCEW). Although containing high content of nutritional compounds, dairy by-products are still disposed as waste rather being reintroduced in a new production chain, hence leading to environmental and economic issues. This study proposes a new biotechnological approach based on the combination of membrane filtration and fermentation to produce poly-hydroxyalkanoates (PHA), biodegradable bioplastics candidate as an alternative to petroleum-derived plastics. The protocol, exploiting the metabolic capability Haloferax mediterranei to synthesize PHA from RCEW carbon sources, was set up under laboratory and pilot scale conditions. A multi-step fractionation was used to recover a RCEW fraction containing 12.6% (w/v) of lactose, then subjected to an enzymatic treatment aimed at releasing glucose and galactose. Fermentation conditions (culture medium for the microorganism propagation, inoculum size, time, and temperature of incubation) were selected according to the maximization of polymer synthesis, under in-flasks experiments. The PHA production was then tested using a bioreactor system, under stable and monitored pH, temperature, and stirring conditions. The amount of the polymer recovered corresponded to 1.18 g/L. The differential scanning calorimetry (DSC) analysis revealed the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as the polymer synthesized, with a relatively high presence of hydroxyvalerate (HV). Identity and purity of the polymer were confirmed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) and X-ray photoelectron (XPS) spectroscopy analyses. By combining the fractionation of RCEW, one of the most abundant by-products from the agri-food industry, and the use of the halophile Hfx mediterranei, the production of PHBV with high purity and low crystallinity has successfully been optimized. The process, tested up to pilot scale conditions, may be further implemented (e.g., through fed-batch systems) and used for large-scale production of bioplastics, reducing the economical and environmental issues related the RCEW disposal.

Polyhydroxyalkanoate synthesis by bacteria isolated from landfill and ETP with pomegranate peels as carbon source.

Many bacteria produce polyhydroxyalkanoates (PHAs) when exposed to stressful conditions. It is a known fact that PHAs have the potential to replace petrochemical-based plastics as they are biodegradable, biocompatible, and thermoprocessible materials. The study focusses on producing PHA from microbes isolated from polluted environments and pomegranate peels were utilized as a unique carbon source. This was done to ensure reduction in the cost of the substrate that has not yet been reported as a substrate for PHA production. A comparative study was also done with Cupriavidus necator, the reference strain. Out of many bacterial strains, isolated, eight of these were found to have ability to produce PHA. Pomegranate peel was substituted as carbon source in the medium and all bacterial isolates along with reference strain were used to test their ability to produce PHA from waste. Cupriavidus necator, the reference strain, yielded 71% PHA. Bacillus halotolerans DSM8802 yielded 83% at 1:1:: C:N ratio at 72h.

Effect of High Pressure on Paracoccus denitrificans Growth and Polyhydroxyalkanoates Production from Glycerol.

The performance of fermentation under non-conventional conditions, such as high pressure (HP), is a strategy currently tested for different fermentation processes. In the present work, the purpose was to apply HP (10-50MPa) to fermentation by Paracoccus denitrificans, a microorganism able to produce polyhydroxyalkanoates (PHA) from glycerol. In general, cell growth and glycerol consumption were both reduced by HP application, more extensively at higher pressure levels, such as 35 or 50MPa. PHA production and composition was highly dependent on the pressure applied. HP was found to decrease polymer titers, but increase the PHA content in cell dry mass (%), indicating higher ability to accumulate these polymers in the cells. In addition, some levels of HP affected PHA monomeric composition, with the polymer produced at 10 and 35MPa showing considerable differences relative to the ones obtained at atmospheric pressure. Therefore, it is possible to foresee that the changes in polymer composition may also affect its physical and mechanical properties. Overall, the results of this study demonstrated that HP technology (at specific levels) can be applied to P. denitrificans fermentations without compromising the ability to produce PHA, with potentially interesting effects on polymer composition.

Influence of removal of microbial inhibitors on PHA production from spent coffee grounds employing Halomonas halophila

The valorization of food waste is the sustainable way how to handle resources wisely. Spent coffee grounds (SCGs) are waste products of the instant coffee production and coffee brewing. The potential of Halomonas halophila to produce polyhydroxyalkanoates (PHA) from fermentable sugars derived from SCGs has been studied. This organism was able to process SCG hydrolysates as a carbon source for its growth. Diluted acid hydrolysis (4.0 vol.% sulfuric acid, 120 min, 100 °C) has been employed, and three different SCG hydrolysates from non-modified SCGs, defatted SCGs and defatted SCGs with the eliminated phenolics have been prepared. However, in shake flask fermentation cultures, the growth of H. halophila was entirely inhibited on all the hydrolysates. Therefore, the hydrolysates have been detoxified using sorbent based on styrene-divinylbenzene based resins. When H. halophila was grown on detoxified hydrolysates, poly(3-hydroxybutyrate) (P3HB) was accumulated during 72 h. The biopolymer was characterized by gas chromatography, and size exclusion chromatography coupled with multiangle light scattering and differential refractometry. PHB titers reached 0.95 g/L with PHB content in bacteria cell dry mass 27% (wt/wt), the molecular weight of the produced polymer was about 440–825 kDa. This study demonstrates that at least the detoxification of SCG hydrolysates with sorbent was necessary to promote the fermentation of H. halophila. Moreover, the extraction of coffee oil and phenolics from SCG as a detoxification pretreatment step contributes to the increase of economic and environmental values of spent coffee grounds in the case of the cascading utilization and resource recovery.

Cyanobacterial PHA Production-Review of Recent Advances and a Summary of Three Years' Working Experience Running a Pilot Plant.

Cyanobacteria, as photoautotrophic organisms, provide the opportunity to convert CO2 to biomass with light as the sole energy source. Like many other prokaryotes, especially under nutrient deprivation, most cyanobacteria are able to produce polyhydroxyalkanoates (PHAs) as intracellular energy and carbon storage compounds. In contrast to heterotrophic PHA producers, photoautotrophic cyanobacteria do not consume sugars and, therefore, do not depend on agricultural crops, which makes them a green alternative production system. This review summarizes the recent advances in cyanobacterial PHA production. Furthermore, this study reports the working experience with different strains and cultivating conditions in a 200 L pilot plant. The tubular photobioreactor was built at the coal power plant in Dürnrohr, Austria in 2013 for direct utilization of flue gases. The main challenges were the selection of robust production strains, process optimization, and automation, as well as the CO2 availability.

Polyhydroxyalkanoates from Pseudomonas sp. using synthetic and olive mill wastewater under limiting conditions

The present study aimed at investigating the ability of bacteria isolated from an enriched mixed culture to produce polyhydroxyalkanoates (PHAs) and examining the effect of nitrogen and dual nitrogen–oxygen limitation on PHAs production, by using both synthetic and olive mill wastewater (OMW). PHAs production was performed through batch experiments using both the enriched culture and the isolated strains (belonging to the genus of Pseudomonas) aiming to compare PHAs accumulation capacity, yields and rates. The use of enriched culture and synthetic wastewater under nitrogen limitation resulted in the highest PHA accumulation, i.e. 64.4%gPHAs/g of cell dry mass (CDM). However, when OMW was used, PHAs accumulation significantly decreased, i.e. 8.8%gPHAs/g CDM. The same trend was followed by the isolated strains, nevertheless, their ability to synthesize PHAs was lower. Although, dual nitrogen–oxygen limitation generally slowed down PHAs biosynthesis, in certain strains PHAs production was positively affected.

Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses

BackgroundRalstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16.ResultsTotal RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition.ConclusionsThe results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.

Impact of carbon source and variable nitrogen conditions on bacterial biosynthesis of polyhydroxyalkanoates: Evidence of an atypical metabolism in Bacillus megaterium DSM 509

Twenty bacterial strains were examined on their ability to produce polyhydroxyalkanoates (PHA) from different carbon sources under rich and depleted nitrogen conditions. Preliminary experiments with glucose as sole carbon source allowed to select PHA producing bacteria using FTIR spectroscopy. They were further tested with eight additional carbon substrates including organic, fatty acids or sugars. PHA content and monomer composition of four chosen strains (Pseudomonas putida mt-2, Bacillus megaterium DSM 90 and DSM 509, Corynebacterium glutamicum DSM 20137) were assessed by gas chromatography techniques for two cultural conditions: during growth phase on a mineral medium (MM) and after transfer of cells on a fresh MM without nitrogen (MM-N). For several carbon substrates, substantial amounts of PHA (up to 53% of the cell dry weight: CDW) were already obtained in MM for C.glutamicum DSM 20137 and the two B.megaterium strains; after transfer in MM-N, PHA contents remained constant except for B.megaterium DSM 509 where PHA production increased whatever the carbon source. P.putida mt-2 synthesized PHA under deprived nitrogen conditions. Highest PHA accumulation reached 48 and 77% of CDW with octanoic acid as substrate in B.megaterium DSM 90 and P.putida mt-2, respectively. Surprisingly, an atypical metabolic shift was observed for B.megaterium DSM 509 cultivated with nearly all unrelated carbon sources: whereas short chain length PHA (scl-PHA) were synthesized in MM, medium chain length PHA (mcl-PHA) were produced after transfer of cells into MM-N supplemented with the same substrate.

Polyhydroxyalkanoates production from effluent of hydrogen fermentation process by Cupriavidus sp. KKU38

This study investigated the use of the residual sugar and volatile fatty acids (VFAs) in the effluent of the hydrogen production process to produce polyhydroxyalkanoates (PHAs) by Cupriavidus sp. KKU38 in batch fermentation. VFAs in the effluent were lactic, butyric, acetic and propionic acids with a total VFA concentration of 1725 mg/L. The C/N ratio of effluent was 100:2.5, which is defined as the excess carbon and limited nitrogen condition suitable for PHA production. The experiments were conducted in 250-mL Erlenmeyer flasks with a 100 mL working volume. The inoculum size was 30% (v/v) with the initial number of cells 106 cells/mL. Residual sugars and acetic acid in the effluent were the major substrates used to produce PHAs while lactic and butyric acids in the effluent were used for biomass synthesis. The maximum PHA concentration and PHA content obtained were 0.85 g/L and 71.42% (w/w) of dry biomass weight, respectively. After fermentation, carbon oxygen demand (COD) in the effluent was reduced by up to 82.73%.

Production of polyhydroxyalkanoates byBurkholderia cepaciaATCC 17759 using a detoxified sugar maple hemicellulosic hydrolysate

Sugar maple hemicellulosic hydrolysate containing 71.9g/l of xylose was used as an inexpensive feedstock to produce polyhydroxyalkanoates (PHAs) by Burkholderia cepacia ATCC 17759. Several inhibitory compounds present in wood hydrolysate were analyzed for effects on cell growth and PHA production with strong inhibition observed at concentrations of 1g/l furfural, 2g/l vanillin, 7g/l levulinic acid, and 1M acetic acid. Gradual catabolism of lower concentrations of these inhibitors was observed in this study. To increase the fermentability of wood hydrolysate, several detoxification methods were tested. Overliming combined with low-temperature sterilization resulted in the highest removal of total inhibitory phenolics (65%). A fed-batch fermentation exhibited maximum PHA production after 96h (8.72g PHA/L broth and 51.4% of dry cell weight). Compositional analysis by NMR and physical-chemical characterization showed that PHA produced from wood hydrolysate was composed of polyhydroxybutyrate (PHB) with a molecular mass (M (N)) of 450.8kDa, a melting temperature (T (m)) of 174.4°C, a glass transition temperature (T (g)) of 7.31°C, and a decomposition temperature (T (decomp)) of 268.6°C.

Engineering polyhydroxyalkanoate content and monomer composition in the oleaginous yeast Yarrowia lipolytica by modifying the ß-oxidation multifunctional protein

Recombinant strains of the oleaginous yeast Yarrowia lipolytica expressing the PHA synthase gene (PhaC) from Pseudomonas aeruginosa in the peroxisome were found able to produce polyhydroxyalkanoates (PHA). PHA production yield, but not the monomer composition, was dependent on POX genotype (POX genes encoding acyl-CoA oxidases) (Haddouche et al. FEMS Yeast Res 10:917-927, 2010). In this study of variants of the Y. lipolytica β-oxidation multifunctional enzyme, with deletions or inactivations of the R-3-hydroxyacyl-CoA dehydrogenase domain, we were able to produce hetero-polymers (functional MFE enzyme) or homo-polymers (with no 3-hydroxyacyl-CoA dehydrogenase activity) of PHA consisting principally of 3-hydroxyacid monomers (>80%) of the same length as the external fatty acid used for growth. The redirection of fatty acid flux towards β-oxidation, by deletion of the neutral lipid synthesis pathway (mutant strain Q4 devoid of the acyltransferases encoded by the LRO1, DGA1, DGA2 and ARE1 genes), in combination with variant expressing only the enoyl-CoA hydratase 2 domain, led to a significant increase in PHA levels, to 7.3% of cell dry weight. Finally, the presence of shorter monomers (up to 20% of the monomers) in a mutant strain lacking the peroxisomal 3-hydroxyacyl-CoA dehydrogenase domain provided evidence for the occurrence of partial mitochondrial β-oxidation in Y. lipolytica.

Intracellular biopolymer productions using mixed microbial cultures from fermented POME

This study aimed to produce polyhydroxyalkanoates (PHAs) from organic wastes by mixed bacterial cultures using anaerobic-aerobic fermentation systems. Palm oil mill effluent (POME) was used as an organic source, which was cultivated in a two-step-process of acidogenesis and acid polymerization. POME was operated in a continuous flow anaerobic reactor to access volatile fatty acids (VFAs) for PHAs production. During fermentation, VFA concentration was produced in the range of 5 to 8 g/L and the COD concentration reduced up to 80% from 65 g/L. The VFA from anaerobic fermentation was then utilised for PHA production using a mixed culture in availability of aerobic bioreactor. Production of PHAs was recorded high when using a high volume of substrates because of the higher VFA concentration. Even though the maximum PHA content was observed at only 40% of the cell dried weight (CDW), their production and performance are significant in mixed microbial culture.

A Proposal for Zero Emission from Palm Oil Industry Incorporating the Production of Polyhydroxyalkanoates from Palm Oil Mill Effluent.

In our previous reports, we have described the production of organic acids from palm oil mill effluent (POME) which were used as fermentation substrates to produce polyhydroxyalkanoates (PHA). Here we propose a zero emission from palm oil industry incorporating the production of PHA from POME. Our results showed that by evaporation, the organic acids could be concentrated to about 100 g·l–1 for use as substrates for the fed-batch PHA fermentation. Upon condensation of the steam, the water had a COD of 80 ppm, low enough for it to be either recycled or discharged. It was confirmed that the energy for the evaporation can be provided by combusting solid wastes such as shells and empty oil palm bunches. The concentrated organic acids were successfully converted to PHA by Ralstonia eutropha strain ATCC 17699 under a non-sterile fermentation system when the initial cell density was kept high at 4 g·l–1. After 150 hours, 20 g·l–1 cells were obtained with more than 50% PHA content. A repeated fed-batch system was also performed to obtain a high cell inoculum and to mimic the operation of a large PHA production fermentor at C/N ratios of 15 and 30 respectively, with only acetic and propionic acids as carbon sources. It was suggested that the energy for this proposed process could be sufficiently supplied by combustion of the solid wastes from the palm oil mill.