Polyhydroxyalkanoates Yield Research Articles

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.

Effects of phosphorus limitation and temperature on PHA production in activated sludge

The study was designed to investigate the effects of temperature and phosphorus limitation on polyhydroxyalkanoate (PHA) production and storage by activated sludge biomass. The two-stage operation approach, i.e. a growth phase followed by a nutrient limitation phase, was applied to induce PHA accumulation. The pre-selected temperatures of 10, 20 and 30 degrees C were investigated under phosphorus limitation conditions using three four-litre fully aerobic SBR systems operated at an SRT of 10 days with cycle time and HRT of 6 and 10 hours. PHA production was greater in the 10 degrees C system than in the 20 degrees C and 30 degrees C systems but there was little difference between the two higher temperatures. The maximum PHA fractions of the sludge were 52, 45 and 47%TSS for the three temperatures from low to high, and the maximum PHA concentrations in the mixed liquors were 1,491, 1,294 and 1,260 mg/l, respectively. However, it was observed that very low values of PHA yield per unit COD consumed were obtained, i.e., 0.05, 0.03 and 0.04 mgPHA/mgCODu, for the 10, 20 and 30 degrees C reactors, respectively. This was because all three systems required several days to reach maximum PHA accumulation in their mixed liquor biomasses. It is probable the bacteria still had some stored poly-P in their cells upon initiation of the phosphorus limited influent, and PHA accumulation was delayed until the stored phosphorus was depleted. Also, PHA productivity was reduced by the large amounts of biomass lost from the systems because of sludge bulking.

Polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha cells: a computer simulation.

Computer simulation of polyhydroxyalkanoate (PHA) granule formation in vivo could help to design strategies to optimize the fermentation process and achieve higher yields of PHA. It could also suggest biotechnological approaches to control the granule size and molecular weight of the polymer. A computer program simulating the formation of PHA granules inside a Ralstonia eutropha cell was developed, based on published experimental data. The results are applicable to R. eutropha cells or other microorganisms and transgenic plants, where polyhydroxybutyrate production is made possible by heterologous expression systems. The simulation starts at the outset of the PHA accumulation phase when the cells are small and contain no PHA granules. In the presence of abundant glucose, the cell responds to phosphorus limitation by producing 3-hydroxybutyryl-CoA which undergoes polymerization on the few PHA synthase molecules present in the cytoplasm. The amphiphilic PHA synthase-PHA complex attracts additional PHA synthase molecules and granules begin to grow from these initiation sites. Phosphorus limitation and the appearance of PHA in the cytoplasm also stimulate production of phasin molecules that attach themselves to the growing granules. As the granules grow bigger, they begin to touch each other and move to optimize their packing. The phasin coat prevents the granules from coalescing. The size of the cell increases and its prolate ellipsoid shape becomes closer to spherical. The accumulation process stops either when the supply of glucose is exhausted or when the granules become tightly packed within the cell, so that access to their surface is limited. All important variables, such as cell dimensions, granule size, counts of granule-associated molecules, PHA yield, degree of polymerization of the PHA molecules, etc., are recorded in real time during the simulation. Examples of virtual experiments with the cell and their results are shown.

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K 2HPO 4 (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K 2HPO 4/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K 2HPO 4 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K 2HPO 4 1.0 g/l and inoculum 150.58 ml/l.

Metabolic analysis on fatty acid utilization by Pseudomonas oleovorans: mcl-poly(3-hydroxyalkanoates) synthesis versus β-oxidation

Medium chain length (mcl) polyhydroxyalkanoates (PHAs) are a type of natural elastomer with potential applications in tissue engineering and drug delivery. Pseudomonas oleovorans was cultivated in a 2 l fermenter for investigation of mcl-PHAs synthesis and β-oxidation of two mcl fatty acids, nonanoic acid (NA) and octanoic acid (OA), and the regulation role of acetic acid as a co-substrate. With a maximum PHA content around 34 wt.% of cell mass, the polyesters produced were co-polymers and the composition was dependent on both mcl-fatty acids and acetic acid. Acetic acid was the major extracellular metabolic intermediate by P. oleovorans on pure NA (or OA), released at high NA consumption rate (up to 1.9 mmol g −1 h −1), but taken up at low NA concentration levels. The specific NA consumption rate was a sum of specific PHA synthesis rate ( r PHA) and specific formation rate of valeryl-CoA ( r V-CoA) due to β-oxidation. The ratio of r PHA to r V-CoA was a good indicator of PHA synthesis efficiency on NA because valeryl-CoA was not incorporated into the polymer instead exclusively for energy supply and cell mass synthesis. This ratio could be improved by adding acetic acid as a co-substrate to the mcl fatty acids, and the yield of PHA on NA was increased from 0.24 to 0.33 g g −1. The polymer composition was also changed with more 3-hydroxynonanoate and less 3-hydroxyheptanonate in the copolymers because of the depressed β-oxidation in the presence of external acetic acid. Similar results were also obtained when P. oleovorans was fed with OA, an even-carbon-number fatty acid, with or without acetic acid as a co-substrate.

New plants

Response Surface Methodology (RSM) was used to investigate how the crude glycerin concentration and the carbon to nitrogen (C:N) ratio in the culture medium affect four indicators of polyhydroxyalkanoates (PHAs) accumulation by mixed microbial cultures (MMC): the observed coefficient of active-biomass yield (Yobs,BA), the observed coefficient of PHA yield (Yobs,PHA), the PHA content in biomass (XPHA) and the volumetric productivity (PrV). The C:N ratio had the largest effect on Yobs,BA and Yobs,PHA. When the C:N ratio was increased, Yobs,BA decreased and Yobs,PHA increased, regardless of the concentration of crude glycerin in the culture medium. The C:N ratio also had the largest effect on the PHA content, whereas volumetric productivity was strongly affected by both the C:N ratio and the crude glycerin concentration. The optimal conditions for PHA accumulation were a crude glycerin concentration of 8954 mg COD/L with a C:N ratio of 15.9 mg C/mg N-NH4, which gave a Yobs,BA of 0.29 mg CODBA/mg COD, a Yobs,PHA of 0.28 mg CODPHA/mg COD, a XPHA of 55.6% VSS and a PrV of 757.3 mg CODPHA/L⋅d (550.0 mg PHA/L⋅d). The accumulated PHAs consisted mainly of 3-hydroxybutyrate. By using RSM, it was possible to predict crude glycerin concentrations and C:N ratios not tested here that will allow desirable values of PHA content in biomass or PHA productivity, which can be useful for designing PHA production with MMC.

The Ralstonia eutropha PhaR protein couples synthesis of the PhaP phasin to the presence of polyhydroxybutyrate in cells and promotes polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are polyoxoesters that are produced by many bacteria and that accumulate as intracellular granules. Phasins (PhaP) are proteins that accumulate during PHA synthesis, bind PHA granules, and promote further PHA synthesis. Interestingly, PhaP accumulation seems to be strictly dependent on PHA synthesis, which is catalyzed by the PhaC PHA synthase. Here we have tested the effect of the Ralstonia eutropha PhaR protein on the regulation of PhaP accumulation. R. eutropha strains with phaR, phaC, and/or phaP deletions were constructed, and PhaP accumulation was measured by immunoblotting. The wild-type strain accumulated PhaP in a manner dependent on PHA production, and the phaC deletion strain accumulated no PhaP, as expected. In contrast, both the phaR and the phaR phaC deletion strains accumulated PhaP to higher levels than did the wild type. This result implies that PhaR is a negative regulator of PhaP accumulation and that PhaR specifically prevents PhaP from accumulating in cells that are not producing PHA. Transfer of the R. eutropha phaR, phaP, and PHA biosynthesis (phaCAB) genes into a heterologous system, Escherichia coli, was sufficient to reconstitute the PhaR/PhaP regulatory system, implying that PhaR both regulates PhaP accumulation and responds to PHA directly. Deletion of phaR caused a decrease in PHA yields, and a phaR phaP deletion strain exhibited a more severe PHA defect than a phaP deletion strain, implying that PhaR promotes PHA production and does this at least partially through a PhaP-independent pathway. Models for regulatory roles of PhaR in regulating PhaP and promoting PHA production are presented.

Factors affecting the economics of polyhydroxyalkanoate production by bacterial fermentation

Polyhydroxyalkanoates (PHA) have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. In this paper, several factors affecting the production cost of PHA, such as PHA productivity, content and yield, the cost of the carbon substrate, and the recovery method were reviewed. A sensitivity analysis was also carried out with respect to these factors and with a view to scale-up. Several production processes were designed on the basis of the reported fermentation and recovery results, and were economically evaluated. PHA productivity only affects equipment-related costs, but PHA content has multiple effects on the process economics. Development of an economical and efficient recovery method is also important to the overall economics of PHA production.

Effect of oligosaccharides on glucose consumption by Rhodobacter sphaeroides in polyhydroxyalkanoate production from enzymatically treated crude sago starch

A method of producing a bacterial polyester, polyhydroxyalkanoate (PHA), from enzymatically treated crude sago starch using the photosynthetic bacterium Rhodobacter sphaeroides (IFO 12203) is described. When α-amylase and glucoamylase were used in sequence to saccharify sago starch, the isomaltose thus produced was found to enhance glucose consumption by the cells, resulting in increased volumetric PHA productivity. On the other hand, treatment with a mixture of glucoamylase and pullulanase during the saccharification of sago starch resulted in the production of glucose but no isomaltose. Using this solution, glucose consumption by R. sphaeroides was lower than that using the solution treated with α-amylase+glucoamylase. These results suggested that isomaltose (with 1–6 bonding) contributed to the enhancement of the glucose consumption rate and PHA yield. This was experimentally confirmed by cultivating R. sphaeroides in a medium containing glucose with either isomaltose or malto-oligosaccharides without 1–6 bonding.

Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1

The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C12:0) and myristic (C14:0) acids, while oleic acid (C18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C6 to C14, with C8 as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers.

Effect of organic acid profiles during anaerobic treatment of palm oil mill effluent on the production of polyhydroxyalkanoates by Rhodobacter sphaeroides

A two-stage process for the production of polyhydroxy-alkanoates (PHA) from palm oil mill effuent (POME) is proposed. In the first stage, anaerobic treatment of POME by palm oil sludge was carried out to obtain organic acids, particularly acetic and propionic acids. The organic acids were then converted to PHA by a phototrophic bacterium, Rhodobacter sphaeroides (IFO 12203), in the second stage. During anaerobic treatment of POME by sludge, formic acid was formed when the pH was maintained at less than 4. By controlling the pH at different values, different organic acid profiles were obtained. This study was carried out to determine the effect of formic acid on the production of PHA from acetic and propionic acids by R. sphaeroides. The results clearly indicated that the presence of formic acid substantially decreased PHA yield (g·g −1-organic acid consumed) and PHA content in cells (g·g −1-cell weight). In the absence of formic acid, a PHA yield of 0.50 g·g −1 and a PHA content in cells of 67% were obtained. With increasing formic acid concentration, the PHA yield and content dropped to as low as 0.21 and 18%, respectively. Thus it is suggested that if POME is used for PHA production, a pH should be maintained at 7 in the anaerobic sludge treatment step, so that no formic acid is produced which would affect the maximum yield of PHA.

The role of β-oxidation of short-chain alkanoates in polyhydroxyalkanoate copolymer synthesis inAzotobacter vinelandiiUWD

Valerate and other short-chain, uneven-length fatty acids promoted the formation of the polyhydroxyalkanoate (PHA) copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Azotobacter vinelandii UWD growing in glucose medium. The uptake of valerate was inducible, being repressed by acetate but not by glucose. A likely route that would direct valerate into PHA synthesis involved the β-oxidation pathway. The short-chain fatty acids butyrate, valerate, trans.-2-pentenoate, crotonate, hexanoate, heptanoate, and octanoate induced the coordinate production of the β-oxidation enzymes enoyl-CoA hydratase (EGH) and L-(+)-3-hydroxybutyryl-CoA dehydrogenase (HAD).trans-3-Pentenoate was the best inducer of these activities, which suggested that the isomerase of the β-oxidation complex also was present. However, 3-hydroxyacyl-CoA epimerase activity of the β-oxidation complex was not detected. 3-Ketoacyl-CoA thiolase activity was constitutive in A. vinelandii and appeared to associate only loosely with the 73 000 Da ECH–HAD complex. Thus, 3-ketoacyl-CoA, the end product of HAD activity, could be directed into PHA synthesis through acetoacetyl-CoA reductase generating the 3-hydroxyvalerate subunit of the polymer. When valerate was the sole carbon source, the incorporation of valerate into the polymer was normal, but most of the valerate was directed into metabolism and very little PHA was formed. When glucose also was present, the β-oxidation of short-chain alkanoates inhibited the specific activity of acetoacetyl-CoA reductase and 3-ketothiolase and the PHA yield. A model for PHA synthesis was developed that suggests that the use of fatty acids to promote PHA copolymer formation in A. vinelandii will inevitably result in decreased PHA yield.Key words: β-oxidation, poly(β-hydroxyalkanoate) synthesis, short chain fatty acids, regulation.

Production of polyhydroxyalkanoates byAzotobacter vinelandiiUWD in beet molasses culture

Beet molasses (BM) has proven to be an excellent feedstock for polyhydroxyalkanoate (PHA) production by Azotobacter vinelandii UWD. The substrate-cost for PHA production from BM in fed-batch culture was one-third of that using glucose. Copolymers containing β-hydroxyvalerate are readily formed in BM medium when valerate is used as a precursor. The origin of the hydroxyvalerate monomer was most likely a β-ketoacyl-CoA intermediate in the β-oxidation of the odd-length n-alkanoates. BM also contained unidentified factors that stimulated PHA production to a greater extent than cell growth. Analysis of BM fractions has suggested that amino-N compounds may be required for PHA-yield-promotion. Thus the addition of a small amount of commercial peptone to mineral salts medium containing pure or other impure sugar sources has led to significantly increased PHA yields.

Production of polyhydroxyalkanoates by Azotobacter vinelandii UWD in beet molasses culture

Beet molasses (BM) has proven to be an excellent feedstock for polyhydroxyalkanoate (PHA) production by Azotobacter vinelandii UWD. The substrate-cost for PHA production from BM in fed-batch culture was one-third of that using glucose. Copolymers containing β-hydroxyvalerate are readily formed in BM medium when valerate is used as a precursor. The origin of the hydroxyvalerate monomer was most likely a β-ketoacyl-CoA intermediate in the β-oxidation of the odd-length n-alkanoates. BM also contained unidentified factors that stimulated PHA production to a greater extent than cell growth. Analysis of BM fractions has suggested that amino-N compounds may be required for PHA-yield-promotion. Thus the addition of a small amount of commercial peptone to mineral salts medium containing pure or other impure sugar sources has led to significantly increased PHA yields.