Polyhydroxyalkanoate Formation Research Articles

Biosynthesis of polyhydroxyalkanoates (PHAs) with continuous feeding of mixed organic acids as carbon sources by Ralstonia eutropha

Feeding strategies of feeding mixed organic acids as carbon sources for the biosynthesis of polyhydroxyalkanoates (PHAs) by Ralstonia eutropha were investigated. A feeding strategy was proposed according to the singular optimization control theory after comparing several feeding strategies of mixed acids for PHAs formation which referred to that the culture was absent of carbon sources firstly, and mixed acids was fed throughout the procedure at varying rates, additionally, to keep the cell more active, nitrogen source (sulfate ammonia) was also fed from about the 12th hour of the fermentation. In contrast with the batch culture in which the maximum dry cell weight and PHAs concentration were only 3.89 and 1.45 g/l, the corresponding values of the continuous feeding fed-batch culture were increased to 14.35 and 6.89 g/l, respectively.

Kinetics modeling of inhibition and utilization of mixed volatile fatty acids in the formation of polyhydroxyalkanoates by Ralstonia eutropha

Acetic, propionic and butyric acids are the major fermentation acids produced on acidogenesis of organic wastes such as food scraps. They can be utilized by Ralstonia eutropha as sole carbon sources for cell growth and polyhydroxyalkanoate (PHA) synthesis. The acids, however, are inhibitory and toxic to the bacterium depending on the medium pH and the total acid concentration. The inhibition and utilization kinetics of the total acids and individual acids in mixed acid media were investigated in flask batch cultures, and simulated by modified Michaelis–Menten models based on variable cell activity. Cell activity, a function of the total acid concentration, is defined as the active fraction of the measurable residual biomass (RBM) under specific culture conditions, which might be as low as 20% at high acid concentrations. The overall production rate of PHA is contributed from the utilization rates of individual acids as well as the interaction of two acids such as acetate and propionate, depending on the predominant acids in the medium. R. eutropha preferred propionic acid for cell mass synthesis and butyric acid for polymer synthesis, the latter giving the highest polymer yield (0.39 g/g) among the three acids. In mixtures of predominant acetic and butyric acids, a high PHA formation rate (60 mg/g RBM per h) was achieved, compared with the relatively low PHA formation rate (35 mg/g RBM per h) in the mixtures of predominant acetic and propionic acids. Acetic acid in acid mixtures reduced the metabolism of propionic acid, which resulted in a high rate ratio (up to 0.8) of hydroxyvalerate (HV) formation to the overall PHA formation.

A dynamic study and modeling of the formation of polyhydroxyalkanoates combined with treatment of high strength wastewater.

Production of biodegradable thermoplastics, polyhydroxyalkanoates (PHAs), from organic wastes may provide multiple benefits to environmental conservation efforts. In this study, microfiltration-coupled reactors were set up to study the dynamic behavior of a typical PHA-producing bacteria, Ralstonia eutropha, fed with a real acidic solution from starch acidogenesis in a continuous flow system. The majorfermentation acids (butyric and acetic acids) were utilized by the PHA producers at a high conversion rate (>95%) when the cells were suspended in a small volume of mineral solution (pH 7), but at a low conversion rate (<10%) when the cells were suspended in an acidic solution (pH 4). The acids were consumed mainly for PHA synthesis and maintenance energy, which resulted in slow growth of PHA-producing cells and a washout of the cells in the continuous flow system. PHAs, however, were continuously synthesized and accumulated during the washout. A simple dynamic model is proposed for estimation of specific growth rates and PHA formation rates during the washout at two hydraulic retention times (HRT) or dilution rates. The net specific growth rate of PHA-producing cells was near zero at a hydraulic retention time (HRT) of around 30 h, but it increased to 0.01 h(-1) when the HRT was reduced to 18 h. The model also reveals that PHA was synthesized faster based on the active biomass (ABM) during the short HRT (10.3 mg PHA/g ABM.h) than during the long HRT (3.4 mg PHA/g ABM.h).

Production of PHA from starchy wastewater via organic acids

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60–80%), propionic (10–30%) and butyric (5–40%) acids. The total volatile fatty acids reached 4000 mg l −1 at a chemical oxygen demand (COD) loading rate of 25–35 g l −1 day −1. A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l −1 day −1 gave a high propionic acid content (29% wt) and a high rate around 26 g l −1 day −1 led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80–100 mg NH 3 per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8–9 g l −1) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from γ-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a γ-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 291–299, 2000.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation fromγ-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.

Anaerobic phosphate release from activated sludge with enhanced biological phosphorus removal. A possible mechanism of intracellular pH control

The biochemical mechanisms of the wastewater treatment process known as enhanced biological phosphorus removal (EBPR) are presently described in a metabolic model. We investigated details of the EBPR model to determine the nature of the anaerobic phosphate release and how this may be metabolically associated with polyhydroxyalkanoate (PHA) formation. Iodoacetate, an inhibitor of glycolysis, was found to inhibit the anaerobic formation of PHA and phosphate release, supporting the pathways proposed in the EBPR metabolic model. In the metabolic model, it is proposed that polyphosphate degradation provides energy for the microorganisms in anaerobic regions of these treatment systems. Other investigations have shown that anaerobic phosphate release depends on the extracellular pH. We observed that when the intracellular pH of EBPR sludge was raised, substantial anaerobic phosphate release was caused without volatile fatty acid (VFA) uptake. Acidification of the sludge inhibited anaerobic phosphate release even in the presence of VFA. From these observations, we postulate that an additional possible role of anaerobic polyphosphate degradation in EBPR is for intracellular pH control. Intracellular pH control may be a metabolic feature of EBPR, not previously considered, that could have some use in the control and optimisation of EBPR. Copyright 1999 John Wiley & Sons, Inc.

PHA production by activated sludge

The production of polyhydroxyalkanoate by anaerobic–aerobic activated sludge was reviewed concentrating on the biochemical mechanisms and on the trials to increase polyhydroxyalkanoate (PHA) content in activated sludge. The anaerobic–aerobic activated sludge system selects microorganisms with the capabilities to couple glycolysis, polyphosphate degradation, and PHA accumulation for anaerobic substrate uptake. Some of the PHA-related metabolisms observed there have not been seen in pure cultures so far. Such metabolisms are the formation of PHA containing 3-hydroxy-2-methylvalerate, and ‘3-hydroxyvalerate fermentation’ in which glucose or glycogen is converted to 3-hydroxyvalerate-rich PHA while yielding energy. The PHA content of activated sludge can be increased up to 62% by applying a microaerophilic–aerobic activated sludge process. PHA production by activated sludge is worth investigation.

Effect of carbon source on the formation of polyhydroxyalkanoates (PHA) by a phosphate-accumulating mixed culture

In the present work, attention was devoted to understand how different carbon substrates and their concentration can influence the production of PHA by polyphosphate-accumulating bacteria. Acetate, propionate, and butyrate were tested independently. The composition of the polymers formed was found to vary with the substrate used. Acetate leads to the production of a copolymer of hydroxybutyrate (HB) and hydroxyvalerate (HV) with the HB units being dominant. With propionate, HV units are mainly produced and only a small amount of HB is synthesized. When butyrate is used, the amount of polymer formed is much lower with the HB units being produced to a higher extent. The yield of polymer produced per carbon consumed ( Y P/S) was found to diminish from acetate (0.97) to propionate (0.61) to butyrate (0.21). Using a mixture of acetate, propionate, and butyrate and increasing the carbon concentration, although maintaining the relative concentration of each substrate, propionate is primarily consumed and consequently, PHA synthesized was enriched in HV units. The polymers obtained in all experiments were copolymers with the average molecular weight of the most representative fraction higher when hydroxybutyrate units were present in considerable amounts. All the polymers synthesized were found to be quite homogeneous and their average molecular weight is of the same order of magnitude as the ones commercially available.

Control of polyhydroxyalkanoate synthesis in Azotobacter vinelandii strain UWD

To better understand the reasons for the hyper-accumulation of polyhydroxyalkanoate (PHA) in mutant Azotobacter vinelandii UWD, the kinetic properties of 3-ketothiolase, acetoacetyl-CoA reductase, and β-hydroxybutyrate dehydrogenase were examined. The regulation of the condensation of acetyl-CoA mediated by 3-ketothiolase was normal, in that it was negatively regulated by free CoA, but inhibition was overcome by higher concentrations of acetyl-CoA. Acetoacetyl-CoA from this reaction was reduced to 3-hydroxybutyryl-CoA by an NADPH-specific acetoacetyl-CoA reductase. This enzyme also reduced 3-ketovaleryl-CoA derived from the β-oxidation of C5, C7 or C9 n-alkanoates, but at only 16% of the rate found with the C4-substrate. The acetoacetyl-CoA reductase was determined to be an allosteric enzyme that bound NADPH and acetoacetyl-CoA at multiple binding sites in a general hybrid Ping-Pong random mechanism. The enzyme was negatively regulated by acetoacetyl-CoA, but this was overcome at high concentrations of NADPH. The activity of pyridine nucleotide transhydrogenase was determined to be important for the conversion of NADH in these mutant cells to NADPH and for decreasing the availability of NADP+, which was a negative regulator of the acetoacetyl-CoA reductase. The combination of high acetoacetyl-CoA, the UWD mutation, transhydrogenase activity, and high NADPH appeared to be the conditions promoting PHA formation by strain UWD during active growth on glucose. Degradation of PHA in strain UWD did not appear to be regulated at the level of β-hydroxybutyrate dehydrogenase. This enzyme was unaffected by NADH, was inhibited only 13% by pyruvate and its activity was enhanced by NADPH. The thiolysis of acetoacetyl-CoA also was unusual, in that 3-ketothiolase was not inhibited by acetoacetyl-CoA, but free CoA was a competitive inhibitor in a bireactant Ping-Pong mechanism. This inhibition was overcome by higher concentrations of the normal first substrate, acetoacetyl-CoA. Thus a single thiolase was used for the condensation of acetyl-CoA and the thiolysis of acetoacetyl-CoA, derived from PHA depolymerization or from the β-oxidation of n-alkanoates.

Uptake of Organic Substrates and Accumulation of Polyhydroxyalkanoates Linked with Glycolysis of Intracellular Carbohydrates under Anaerobic Conditions in the Biological Excess Phosphate Removal Processes

Anaerobic uptake mechanisms of acetate, propionate and lactate by the sludge developed in an anaerobic aerobic biological phosphate removal process was investigated. Anaerobic batch experiments were conducted with these substrates, and mass balances of materials involved in substrate uptake metabolism were calculated. These substrates were accumulated in the sludge as polyhydroxyalkanoates composed of 3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxy-2-methylbutyrate and 3-hydroxy-2-methylvalerate. The reducing power necessary for the formation of polyhydroxyalkanoates from acetate and/or propionate was shown to be supplied from glycolysis of intracellular carbohydrates. Lactate was converted to 3-hydroxyvalerate rich polyhydroxyalkanoate via acetyl-CoA and propionyl-CoA by using propionate fermentation pathway. Redox balance was found to be a factor regulating the anaerobic uptake of substrates and their further metabolism. Glycogen seemed to serve not only as a source of reducing power necessary for the redox balance regulation but also as an important energy source, because glycolysis can provide both reducing power and energy. It was indicated that some bacteria might be able to get energy necessary for substrate uptake under strict anaerobic conditions not by the polyphosphate hydrolysis but by the consumption of intracellular carbohydrates.

Metabolism of Organic Substances in Anaerobic Phase of Biological Phosphate Uptake Process

Biochemical mechanisms of anaerobic uptake of organic substrate by anaerobic-aerobic activated sludge were investigated experimentally. As the organic polymers stored in the cell, 3H2MB and 3H2MV were newly identified besides PHB and PHV. Conceptual pathways for the formation of PHA (polyhydroxyalkanoates) were postulated. It was suggested that the conversion of glycogen into PHA should be important for the mechanisms of biological excess phosphate removal.