High Polyhydroxyalkanoate Content Research Articles

Comparison of batch, fed-batch and continuous operation modes for scalable polyhydroxyalkanoate (PHA) production and carbon sequestration from phenol

Polyhydroxyalkanoates (PHA) are biodegradable intracellular polymers, and a renewable alternative to conventional plastics. To reduce PHA's cost, this study combined an unconventional carbon source, i.e. a toxic aromatics phenol, with an acclimated consortium, to develop a scalable PHA production process. The resultant process performance was systematically compared across three operational modes: batch, fed-batch, and continuous. Phenol toxicity caused inhibition in the batch mode, reducing the PHA synthesis rate but resulting in a high PHA content in cells (PHA% > 50 %). Fed-batch feeding alleviated such inhibition, facilitating a high PHA titer and yield from the substrate. Interestingly, a higher feeding rate in the fed-batch reactor improved its performance, probably by providing a suitable food-to-microbe ratio for the biomass. Using a continuous mode also reduced phenol toxicity, but resulted in more synthesis of non-PHA cellular materials (NPCM). The difference might have been caused by different metabolic states and the presence of intermediates in the reactors. Comparing all 14 conditions under the three modes, feeding phenol more gradually tended to channel substrate away from PHA to NPCM synthesis. The volumetric productivity generally ranged from 3 to 12 mg L−1 min−1, while the biomass productivity was between 2 and 11 mg g initial CDW−1 min−1. A 3-stage, production-harvest and starvation-production process was then designed to simulate real industrialized manufacturing, where the operational modes had a long-term effect on biomass' productivity. Fed-batch mode performed the best, as it fixed 1/3 of phenol's organic carbon in PHA product, suggesting high potential for future application.

Polyhydroxyalkanoates production from lactic acid fermentation broth of agricultural waste without extra purification: The effect of concentrations

Polyhydroxyalkanoates (PHAs) production can potentially realize the high-value utilization of agricultural wastes and reduce the cost of commercial PHA production. This study aimed to explore the feasibility of converting lactic acid (LA) fermentation broth from agricultural wastes into PHAs. The LA fermentation broth from the co-fermentation of swine manure and apple waste and the simulated LA solution were used as feedstock. Effects of different LA concentrations of these feedstocks were explored in the PHA production. Results showed that PHA content and PHA yield of LA fermentation broth decreased with increase of concentrations of both feedstocks. The LA fermentation broth with LA concentration of 2420.8 mg/L resulted in the highest PHA content (55.4% dry cell weight) and PHA yield (0.48 g PHA-COD/g CODini). In addition, LA fermentation broth with LA concentration of 25084.5 mg/L had the highest concentration of PHA (1.04 g/L). The dominant bacteria for PHA production using both feedstocks were recognized as P. aminovorans. Using swine manure and apple waste to synthesize PHA could have the potential high benefits of economy and environment.

Microbial Valorization of Lignin to Bioplastic by Genome-Reduced Pseudomonas putida.

As the most abundant natural aromatic resource, lignin valorization will contribute to a feasible biobased economy. Recently, biological lignin valorization has been advocated since ligninolytic microbes possess proficient funneling pathways of lignin to valuable products. In the present study, the potential to convert an actual lignin stream into polyhydroxyalkanoates (PHAs) had been evaluated using ligninolytic genome-reduced Pseudomonas putida. The results showed that the genome-reduced P. putida can grow well on an actual lignin stream to successfully yield a high PHA content and titer. The designed fermentation strategy almost eliminated the substrate effects of lignin on PHA accumulation. Employing a fed-batch strategy produced the comparable PHA contents and titers of 0.35 g/g dried cells and 1.4 g/L, respectively. The molecular mechanism analysis unveiled that P. putida consumed more small and hydrophilic lignin molecules to stimulate cell growth and PHA accumulation. Overall, the genome-reduced P. putida exhibited a superior capacity of lignin bioconversion and promote PHA accumulation, providing a promising route for sustainable lignin valorization.

The First Insight into Polyhydroxyalkanoates Accumulation in Multi-Extremophilic Rubrobacter xylanophilus and Rubrobacter spartanus.

Actinobacteria belonging to the genus Rubrobacter are known for their multi-extremophilic growth conditions—they are highly radiation-resistant, halotolerant, thermotolerant or even thermophilic. This work demonstrates that the members of the genus are capable of accumulating polyhydroxyalkanoates (PHA) since PHA-related genes are widely distributed among Rubrobacter spp. whose complete genome sequences are available in public databases. Interestingly, all Rubrobacter strains possess both class I and class III synthases (PhaC). We have experimentally investigated the PHA accumulation in two thermophilic species, R. xylanophilus and R. spartanus. The PHA content in both strains reached up to 50% of the cell dry mass, both bacteria were able to accumulate PHA consisting of 3-hydroxybutyrate and 3-hydroxyvalerate monomeric units, none other monomers were incorporated into the polymer chain. The capability of PHA accumulation likely contributes to the multi-extremophilic characteristics since it is known that PHA substantially enhances the stress robustness of bacteria. Hence, PHA can be considered as extremolytes enabling adaptation to extreme conditions. Furthermore, due to the high PHA content in biomass, a wide range of utilizable substrates, Gram-stain positivity, and thermophilic features, the Rubrobacter species, in particular Rubrobacter xylanophilus, could be also interesting candidates for industrial production of PHA within the concept of Next-Generation Industrial Biotechnology.

Creating biotransformation of volatile fatty acids and octanoate as co-substrate to high yield medium-chain-length polyhydroxyalkanoate

Using mixed microbial consortium (MMC) to accumulate polyhydroxyalkanoate (PHA) is an effective strategy to solve high production cost and reduce the amount of excess sludge. In this study, a process for the production of short-chain-length and medium-chain-length PHA using volatile fatty acids (VFAs) from pretreated wood hydrolysate synergistic with octanoate as co-substrate was proposed. The effects of co-substrate ratios on PHA accumulation ability and physical properties were investigated. The incorporation of co-substrate accelerated the time of PHA and 3-hydroxyoctanoate reaching the maximum production (1834 and 280 mg COD/L). The highest PHA content was 53.0% (w/w), which was equivalent to that reported previously. The biopolymer films possessed high tensile strength, Young's modulus, and could be used in the field of water vapor barrier requirements. The accumulation strategy applied for converting fermentation products VFAs and octanoate co-substrate into high value and yield PHA could potentially demonstrate the valuable for low-cost large-scale production.

Bio-based conversion of volatile fatty acids from waste streams to polyhydroxyalkanoates using mixed microbial cultures

Production of polyhydroxyalkanoates is an important field in the biorefinery as bio-alternative to conventional plastics. However, its commercialization is still limited by high production cost. In this study, a process with the potential to reduce the production cost of polyhydroxyalkanoates was proposed. Mixed cultures accumulated polyhydroxyalkanoates using volatile fatty acid-rich effluents from waste streams, without pH and temperature control. In addition, the impact of two types of carbon sources was investigated by analyzing the microbial community as well as the polyhydroxyalkanoate accumulation capacity. Mixed cultures successfully adapted to different substrates, consuming the volatile fatty acids in their totality. The phyla Proteobacteria, Bacteroidetes and Firmicutes dominated the bacterial community. The highest polyhydroxyalkanoate content was 43.5% w/w, which is comparable to contents reported from mixed cultures using synthetic carbon sources. The biopolymer consisted of (R)-3-hydroxybutyrate 94.8 ± 1.7% w/w and (R)-3-hydroxyvaletare 5.2 ± 1.7% w/w.

Correlating the biochemical methane potential of bio-P sludge with its polyhydroxyalkanoate content

Abstract The new paradigm in wastewater treatment aims at diverting as much organic matter as possible to anaerobic digestion to boost energy recovery (i.e. biogas production) and to decrease operational costs (i.e. aeration requirements and sludge generation). To achieve this, short sludge retention time (SRT) systems could be implemented in the first stage of the wastewater treatment plants (WWTPs) that include biological phosphorus removal. This enables efficient removal of both carbon and phosphorus. This paper focuses on the biochemical methane potential (BMP) that could be reached when using bio-P sludge with different polyhydroxyalkanoate (PHA) content obtained under different SRTs and under anaerobic and aerobic environments. The PHA content of the biomass increased from 3.15 to 9.80 mmol C/g VSS when the SRT was decreased from 15 to 5 days and, on average, anaerobic sludge contained 45% more PHA than the aerobic sludge. BMP was correlated with the PHA content, following an approximate linear trend: BMP (mL CH4/g VSS) = 240 + 15.3 * PHA (mmol C/g VSS), independently of SRT and the anaerobic or aerobic extraction. The maximum BMP obtained was 401 mL CH4/g VSS with the biomass with the highest PHA content (9.80 mmol C/g VSS). When operating at low SRT and purging from the anaerobic phase, 28% of the influent COD content could be recovered as methane. This represented up to 2.5 times more energy than in the more conventional scenario tested: operating at high SRT with conventional aerobic purge.

Reconsideration on the effect of nitrogen on mixed culture polyhydroxyalkanoate production toward high organic loading enrichment history

In most of the operating strategies for mixed microbial cultures polyhydroxyalkanoate (PHA) production, moderate organic loads and low nitrogen concentrations are used, however, the real waste streams contain variable concentrations of carbon and nitrogen. To evaluate the effect of enrichment history on PHA producer and production the various carbon and nitrogen levels were utilized during the accumulation phase. Different operating strategies were applied in three sequencing batch reactors (SBRs) subjected to aerobic dynamic feeding. The maximum PHA production of the enriched cultures under nutrient excess, limitation and starvation (Cmol/Nmol ratio of 8, 40 and 1, respectively) was evaluated in batch assays. A higher PHA content and biomass growth were achieved in the nutrients presence in comparison to the nutrient starvation condition. The cultures from the SBR treated under short sludge retention time, high organic loading rate, short cycle length (SBR#3) and nutrient excess reached the maximum PHA content (54.9%) and biomass increase (38.9%). Under nutrient limitation, the negative biomass growth was observed under nutrient starvation because of the sampling loss. The succession of microbial communities in SBRs and batch assays was analyzed using terminal restriction fragment length polymorphism. The SBR#3 had the best overall PHA production performance considering its high PHA content and productivity in all nutrient content, it indicates that nitrogen has a substantial impact on PHA yield especially when high organic loading rate enrichment history is involved.

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 (4.94g/L) over 48h. 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.

Enhanced polyhydroxyalkanoate production by mixed microbial culture with extended cultivation strategy

Low biomass output is a crucial reason for low polyhydroxyalkanoate (PHA) production in mixed microbial cultures (MMCs) PHA process. In this research, an extended cultivation strategy was proposed to rapidly expand the biomass yield of PHA accumulating MMCs and conserve the PHA accumulating ability simultaneously. High PHA content of the cultivated MMCs of 71.4% and 66.7% (higher than 62.1% of the seed biomass) in batch assays and biomass magnification of 43 and 52 were obtained after 10days of extended cultivation with and without sludge discharge, respectively. By embedding the extended cultivation process into the production process, a highly competitive PHA production performance in terms of overall PHA storage yield (0.49g CODPHA/gCODVFA) and volumetric productivity (1.21gPHA/L/d with final cell density of 17.22g/L) was achieved. The proposed PHA production process based on the extended cultivation can be a promising choice in industrial scale practice.

Exploring the Potential of 1-Pentanol and Oleic Acid for Optimizing the Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Copolymer by Cupriavidus sp. USMAA1020

The potential of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV) copolymer production has remained unexplored for Cupriavidus sp. USMAA1020. This study is devoted to a full range exploration on the biosynthesis of polyhydroxyalkanoate (PHA) containing 3HV monomer, from the screening of carbon sources to the optimization of fermentation parameters. Fatty acid (oleic acid) and alcohol (1-pentanol) was found to be the best combination which resulted in high cell dry weight (CDW) (9.4 g/L), as well as high PHA accumulation of 76 wt%. Alcohols were found to be less toxic compared to other 3HV precursors; meanwhile, oleic acid contributed to sufficient amount of acetyl-CoAs as the building block for PHA thus contributing to high PHA content. Improvement was strategized by focusing on 1-pentanol concentration, incubation period and temperature which are the major factors affecting the copolymer production. An optimization study has resulted in high CDW (13.3 g/L) and PHA concentration (10.23 g/L), which increased significantly to 45 and 43%, respectively, compared to pre-optimized culture. This study warrants the usage of oleic acid and 1-pentanol as one of the viable strategies for industrial scale production of P(3HB-co-3HV) copolymer.

Polyhydroxyalkanoate (PHA) Synthesis by Glycerol-based Mixed Culture and Its Relation with Oxygen Uptake Rate (OUR)

Polyhydroxyalkanoate (PHA) is one of the most promising biopolymers with the advantages of biodegradable biocompatible and renewable. A glycerol-fed PHA-producing mixed culture was enriched in a feast-famine regime. The substrate versatility and PHA production capacity of the mixed culture were studied using acetate,propionate,butyric, glucose and lactate as substrate, respectively. The results showed a high PHA content with lactate and acetate as the sole substrate, respectively. When acetate and propionate were used as mixed substrates to produce PHA, the PHA contents increased with the proportion of acetate and the PHA content was the highest as the ratio acetate/propionate was 3:1.The results based on the modeling of simultaneously storage and growth and the linear fitting under three different acetate/propionate ratios showed that there was a linear relation between PHA synthesis and OUR. Thus, it is feasible to estimate PHA content based on in-situ real-time monitoring data of OUR.

PHA production from the organic fraction of municipal solid waste (OFMSW): Overcoming the inhibitory matrix

Leachate from the source separated organic fraction of municipal solid waste (OFMSW) was evaluated as a substrate for polyhydroxyalkanoates (PHA) production. Initially, the enrichment step was conducted directly on leachate in a feast-famine regime. Maximization of the cellular PHA content of the enriched biomass yielded to low PHA content (29 wt%), suggesting that the selection for PHA-producers was unsuccessful. When the substrate for the enrichment was switched to a synthetic volatile fatty acid (VFA) mixture -resembling the VFA carbon composition of the leachate-the PHA-producers gained the competitive advantage and dominated. Subsequent accumulation with leachate in nutrient excess conditions resulted in a maximum PHA content of 78 wt%. Based on the experimental results, enriching a PHA-producing community in a “clean” VFA stream, and then accumulating PHA from a stream that does not allow for enrichment but does enable a high cellular PHA content, such as OFMSW leachate, makes the overall process much more economically attractive. The estimated overall process yield can be increased four-fold, in comparison to direct use of the complex matrix for both enrichment and accumulation.

Efficient polyhydroxyalkanoate (PHA) accumulation by a new continuous feeding mode in three-stage mixed microbial culture (MMC) PHA production process

A new continuous feeding mode was developed to optimize the polyhydroxyalkanoates (PHAs) accumulation step in the three-stage PHAs production process by mixed microbial cultures (MMCs) using mixed volatile fatty acids (VFAs) as substrate. The continuous feeding assays under low biomass loading rate (BLR) (in the range of 3.5–5.5Cmol VFA/Cmol X/d) can be directly fed with feedstock with pH from 5.0 to 10.0 without any pH regulation. The maximum intracellular PHA content of 70.4% and system PHA yield (YPHA/Ssys) of 0.81Cmol PHA/Cmol VFA were achieved under the continuous feeding mode at BLR of 3.86Cmol VFA/Cmol X/d when directly fed with substrate at pH 5. These values were higher than those under the pulse wise feeding mode with the same substrate and active biomass concentration. The results indicated that a self-balanced pH state was formed in the continuous feeding system. Further studies on the reaction pH and kinetics of the MMCs in the continuous feeding reactor were conducted to understand the pH variation mechanism and accumulation process. The optimal BLR to get high PHA content and YPHA/Ssys was 3.5–5.5Cmol VFA/Cmol X/d.

Substrate versatility of polyhydroxyalkanoate producing glycerol grown bacterial enrichment culture

Waste-based polyhydroxyalkanoate (PHA) production by bacterial enrichments generally follows a three step strategy in which first the wastewater is converted into a volatile fatty acid rich stream that is subsequently used as substrate in a selector and biopolymer production units. In this work, a bacterial community with high biopolymer production capacity was enriched using glycerol, a non-fermented substrate. The substrate versatility and PHA production capacity of this community was studied using glucose, lactate, acetate and xylitol as substrate. Except for xylitol, very high PHA producing capacities were obtained. The PHA accumulation was comparable or even higher than with glycerol as substrate. This is the first study that established a high PHA content (≈70 wt%) with glucose as substrate in a microbial enrichment culture. The results presented in this study support the development of replacing pure culture based PHA production by bacterial enrichment cultures. A process where mixtures of substrates can be easily handled and the acidification step can potentially be avoided is described.

Polyhydroxyalkanoates accumulation by Methylobacterium organophilum CZ-2 during methane degradation using citrate or propionate as cosubstrates

Methylobacterium organophilum CZ-2 synthesized polyhydroxyalkanoates (PHAs) under nitrogen limitation with CH4 as carbon source and when either citrate or propionate was added as cosubstrates. The highest PHAs content (yPHA) in closed flasks was obtained in the CH4-citrate and CH4-propionate experiments attaining values of 0.82 and 0.68, respectively. M. organophilum CZ-2 cultivated in bioreactors with citrate and continuous CH4 addition yielded a final PHAs concentration of 143gm−3 containing hydroxybutyrate (HB), hydroxyvalerate (HV) and hydroxyoctanoate (HO), in a 55:35:10 ratio, with, yPHA of 0.88 and a CH4 elimination capacity (EC) of 20gm−3h−1. With propionate, the yPHA was 0.3 and the EC around 8gm−3h−1. From 1H and 13C NMR experiments it was found that the polymer produced with CH4-citrate contained six different monomers: 3HB, 3HV, 4HV, 4-hydroxyheptanoate (4HH), 3HO and 4HO, showing the great versatility of this PHAs producing bacterium.

Utilization of vinasse for production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei

Vinasse, a highly polluting waste of the ethanol industry was utilized for the production of polyhydroxyalkanoate (PHA) by the extremely halophilic archaeon, Haloferax mediterranei in shake-flasks. Following pre-treatment through adsorption on activated carbon, 25%-50% (v/v) pre-treated vinasse was utilized leading to 70% maximum accumulation of PHA. Maximum PHA concentration of 19.7 g/l, product yield coefficient (based on total carbohydrates) of 0.87 and 0.21 g/l h volumetric productivity were achieved. Concomitant lowering of BOD5 of pre-treated vinasse by at least 78% and COD by at least 80% was attained at the end of this process. The PHA was recovered by osmotic lysis of the cells and purification by sodium hypochlorite and organic solvents. Through UV–vis spectroscopy, gas chromatography, differential scanning calorimetry and nuclear magnetic resonance spectroscopy, the PHA was identified as poly-3-(hydroxybutyrate-co-hydroxyvalerate). The 3-hydroxyvalerate content was 12.36 mol % (utilizing 25% pre-treated vinasse) and 14.09 mol % (utilizing 50% pre-treated vinasse). High salt concentration in the medium allowed this process without sterile conditions and thus reduction in costs of sterilization can be envisaged. Activated charcoal pre-treatment of vinasse is economical than competing processes such as ultrafiltration of whey, extrusion and enzymatic treatment of rice and corn starch. Without impacting sugar prices, this process can easily be integrated into a distillery that has fermentation equipment and trained personnel. High PHA content, productivity, zero-cost carbon source, low-cost isolation of a high-purity product and potential integration into ethanol manufacturing unit with concomitant wastewater treatment should merit further development of this process to higher scales.

Improvement of the production of poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate‐co‐4‐hydroxybutyrate) terpolyester by manipulating the culture condition

AbstractBACKGROUND: The aim of this work is to enhance the production of terpolyester poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate‐co‐4‐hydroxybutyrate) (P(3HB‐co‐3HV‐co‐4HB)) produced by a locally isolated bacterium, Cupriavidus sp. USMAA2‐4. The monomer composition was varied by supplementing different carbon precursors and by manipulating the culture condition through one‐stage cultivation. The effect of C/N ratio and different concentrations of carbon source and precursors were investigated in order to produce higher content of this terpolyester. Although research on this biodegradable polyester is abundant, studies on terpolyester P(3HB‐co‐3HV‐co‐4HB) are still limited.RESULTS: Supplementation of oleic acid in accumulation medium increased the bacterial growth and polyhydroxyalkanoate (PHA) accumulation. It was also shown that medium consisting of assorted carbon precursors at C/N 20 gave relatively high dry cell weight and P(3HB‐co‐3HV‐co‐4HB) content. Various compositions of terpolyester were obtained when the concentration of oleic acid and 4HB precursors were manipulated. The combination of oleic acid with γ‐butyrolactone and 1‐pentanol was found to be the best combination to produce high PHA content (81 wt%). The composition of monomer in P(3HB‐co‐3HV‐co‐4HB) was produced in the range 8–13 mol% for 3HV and 9–24 mol% for 4HB, respectively.CONCLUSIONS: The production of P(3HB‐co‐3HV‐co‐4HB) in shake‐flasks successfully produced 81 wt% of PHA content. This manipulated culture condition can be used at larger scale to provide modeling for the production of terpolyester in a bioreactor. Copyright © 2012 Society of Chemical Industry

Biosynthesis and Characterization of Poly(3-hydroxybutyrate-co-3- hydroxyhexanoate) from Palm Oil Products in a Wautersia eutropha Mutant

Palm kernel oil, palm olein, crude palm oil and palm acid oil were used for the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) [P(3HB-co-3HHx)] by a mutant strain of Wautersia eutropha (formerly Ralstonia eutropha) harboring the Aeromonas caviae polyhydroxyalkanoate (PHA) synthase gene. Palm kernel oil was an excellent carbon source for the production of cell biomass and P(3HB-co-3HHx). About 87% (w/w) of the cell dry weight as P(3HB-co-3HHx) was obtained using 5 g palm kernel oil/l. Gravimetric and microscopic analyses further confirmed the high PHA content in the recombinant cells. The molar fraction of 3HHx remained constant at 5 mol % regardless of the type and concentration of palm oil products used. The small amount of 3HHx units was confirmed by 13C NMR analysis. The number average molecular weight (M(n)) of the PHA copolymer produced from the various palm oil products ranged from 27 0000 to 46 0000 Da. The polydispersity was in the range of 2.6-3.9.

Bacterial synthesis of polyhydroxyalkanoates containing aromatic and aliphatic monomers by Pseudomonas putida CA-3

Pseudomonas putida CA-3 has the ability to accumulate to high levels unique polyhydroxyalkanoate (PHA) heteropolymers composed of aromatic and aliphatic monomers. The majority of monomers are aromatic making up 98% of the polymer. ( R)-3-hydroxyphenylvalerate and ( R)-3-hydroxyphenylhexanoate are the most abundant monomers found in polymers accumulated from phenylalkanoic acids with an uneven and even number of carbons on the acyl side chain respectively. PHAs accumulated from phenylvaleric and phenylhexanoic acid were partially crystalline while all other PHAs were amorphous. Significant differences in the yield and PHA content of the cells occurred when different phenylalkanoic acids were supplied as growth substrates. Increasing the initial concentration of the growth substrate increased both the PHA content of the cells and the overall yield (g PHA/g carbon supplied) of PHA accumulated by P. putida CA-3 cells. The highest PHA content (% cell dry wt.) from an aromatic carbon source was 59% when 15 mM phenylvaleric acid was supplied as the sole source of carbon and energy. This corresponded to a maximum PHA yield of 0.42 g PHA/g carbon supplied. In and attempt to increase the level of PHA accumulated from related growth substrates acrylic acid was added to the growth medium. However, the addition of various concentrations of acrylic acid to the growth medium had either no effect or decreased the PHA content of the cell accumulated from phenylalkanoic acids by P. putida CA-3.