Polyhydroxybutyrate Content Research Articles

Production of polyhydroxybutyrate by activated sludge performing enhanced biological phosphorus removal

In this study, polyhydroxybutyrate (PHB) – a biodegradable plastics material – was produced by activated sludge performing enhanced biological phosphorus removal (EBPR) in batch experiments under anaerobic, aerobic and anaerobic/aerobic conditions. Under anaerobic conditions, the maximum PHB content of the dry biomass was 28.8% by weight, while under aerobic or anaerobic/aerobic conditions, the maximum PHB content was about 50%. The PHB production rate with respect to the volatile suspended solids (VSS) was: (i) 70 mg/(g VSS) h under aerobic conditions that followed anaerobic conditions, (ii) 156 mg/(g VSS) h under anaerobic condition, and (iii) 200 mg/(g VSS) h under aerobic conditions with energy also supplied from polyphosphate. A side stream, with initially anaerobic conditions for PHB accumulation and phosphorus release, and then aerobic conditions for PHB accumulation, was proposed. In this side stream, biomass with a high PHB content and a high PHB production rate could be both achieved.

Zobellella denitrificans strain MW1, a newly isolated bacterium suitable for poly(3-hydroxybutyrate) production from glycerol

To search for new bacteria for efficient production of polyhydroxyalkanoates (PHAs) from glycerol. Samples were taken from different environments in Germany and Egypt, and bacteria capable of growing in mineral salts medium with glycerol as sole carbon source were enriched. From a wastewater sediment sample in Egypt, a Gram-negative bacterium (strain MW1) was isolated that exhibited good growth and that accumulated considerable amounts of polyhydroxybutyrate (PHB) from glycerol and also from other carbon sources. The 16S rRNA gene sequence of this isolate exhibited 98.5% and 96.2% similarity to Zobellella denitrificans strain ZD1 and to Zobellella taiwanensis strain ZT1 respectively. The isolate was therefore affiliated as strain MW1 of Z. denitrificans. Strain MW1 grows optimally on glycerol at 41 degrees C and pH 7.3 and accumulated PHB up to 80.4% (w/w) of cell dry weight. PHB accumulation was growth-associated. Although it was not an absolute requirement, 20 g l(-1) sodium chloride enhanced both growth (5 g cell dry weight per litre) and PHB content (87%, w/w). Zobellella denitrificans strain MW1 is also capable to accumulate the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer if sodium propionate was used as cosubstrate in addition to glycerol. A new PHB-accumulating strain was isolated and identified. This strain is able to utilize glycerol for growth and PHB accumulation to high content especially in the presence of NaCl that will enable the utilization of waste glycerol from biodiesel industry. This study is the first report on accumulation of PHA in a member of the new genus Zobellella. Furthermore, utilization of glycerol as the sole carbon source for fast growth and PHB biosynthesis, growth in the presence of NaCl and high PHB contents of the cells will make this newly isolated bacterium a potent candidate for industrial production of PHB from crude glycerol occurring as byproduct during biodiesel production.

Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium

This research aimed at increasing the cell density and production of the homopolymer polyhydroxybutyrate (PHB) by Bacillus megaterium BA-019, using renewable and inexpensive bioresources as a substrate. A higher cell density and a greater PHB production level were obtained by using sugarcane molasses and urea as carbon and nitrogen sources, respectively. The limitation of nitrogen at a C/N molar ratio of 25 resulted in enhanced cell growth and PHB production in batch cultures. Fed-batch cultivation with the feeding nutrient composed of MSM with sugarcane molasses, urea and trace elements, and controlled by a pH-stat feeding control, lead to a significantly enhanced cell concentration and PHB production. The optimal feeding medium in this system required a higher total sugar concentration (400 g/l) and a C/N molar ratio of 10 mol/mol. Under these conditions the highest attained cell mass (72.6 g/l DW) and PHB content (42% of cell dry wt.) were achieved in a short cultivation time (24 h), leading to improved PHB productivity (1.27 g/l/h). However, dissolved oxygen was limiting and thus the system is likely to be suboptimal and capable of even further improvements to the PHB production rate.

Thermophilic bacterium Caldimonas taiwanensis produces poly(3-hydroxybutyrate- co-3-hydroxyvalerate) from starch and valerate as carbon sources

Caldimonas taiwanensis accumulated polyhydroxybutyrate (PHB) at 55 °C from gluconate, fructose, maltose, and glycerol under nitrogen-limited condition. The PHB content peaked at 14 h after inoculation from gluconate. C. taiwanensis did not grow or accumulate PHA from fatty acids as the sole carbon source; however, it incorporated 3-hydroxyvalerate (3-HV) into PHB polymer from gluconate and valerate as a mixed carbon source. By adjusting the valerate concentration, the molar fraction of 3-HV could be modulated from 10 mol% to 95 mol%. Fatty acid valerate substantially inhibited cell growth and PHA accumulation with the addition of as little as 5 mM to the medium. Supplementing the medium with yeast extract overcame the inhibition, which enhanced not only the yield of biomass but also PHA productivity. The in vivo substrate specificity of PHA synthase ranged from C 4 to C 6. In addition, C. taiwanensis also incorporated a wide range of 3-HV into PHA from soluble starch and valerate as a mixed carbon source. Food-grade starches made from cassava, corn, potato, sweet potato and wheat respectively mixed with valerate were studied for poly(3-hydroxybutyrate- co-3-hydroxyvalerate) [P(3HB- co-3HV)] production. In this study, C. taiwanensis exhibited high promise for reducing the production cost of P(3HB- co-3HV).

Determination of optimum operating conditions for production of polyhydroxybutyrate by activated sludge submitted to dynamic feeding regime

Polyhydroxybutyrate (PHB) was produced by activated sludge in a two-stage process. In the first stage, the sludge with high storage potential was enriched in a sequencing batch reactor (SBR) operated by a dynamic feeding regime. The biosynthesis of PHB was sequentially accomplished in a batch reactor in the second stage. This article focused on the optimization of PHB content by evaluating the effects of three critical factors: Dissolved oxygen, pH and food-to-microorganism (F/M) ratio in the batch reactor. The results showed that the rate of substrate uptake, as well as the yield and content of PHB increased with the increase of dissolved oxygen concentration. The control of pH was not necessary under the weak alkaline condition for improvement of PHB content, except for the case in which the pH value was out of rang of 7.0 to 10.0. The enhanced F/M ratio favored PHB accumulation in the range of 1.0 to 4.5 C-mmol/C-mmol and the highest PHB content, 64%, was achieved at 4.5 C-mmol/C-mmol. When F/M ratio was enhanced to 6.0 C-mmol/C-mmol, the phenomenon of utilizing simultaneously intracellular PHB and external substrate by sludge was observed, which resulted in a sharp decrease of PHB content. The microscopic observation clearly confirmed that dynamic feeding regime was effective for selecting and enriching the sludge with a high storage potential.

On-line monitoring of PHB production by mixed microbial cultures using respirometry, titrimetry and chemometric modelling

This work describes a method for on-line monitoring of biomass production, acetate consumption and intracellular polyhydroxybutyrate (PHB) storage by mixed microbial cultures (MMC). The method is based on reliable and easily available on-line measurements, namely pH, dissolved oxygen, dissolved carbon dioxide, on-line respirometry and on-line titrimetric analysis. Biomass production refers to active biomass growth and also to the synthesis of extracellular polymeric substances (EPS). The composition and kinetics of EPS synthesis has high variability depending on the culture enrichment protocol. Since the metabolism for EPS production is rather difficult to define, it was not possible to develop a reliable estimation model based on metabolic principles only. Instead, projection of latent structures (PLS) linear regression constrained by steady state carbon balance was employed. PHB concentration and biomass production rate were directly estimated by the PLS model, whereas acetate concentration was indirectly estimated through the carbon balance. The method was validated experimentally with data of four experiments carried out in a SBR. Accurate on-line estimations were obtained with regression coefficients ( r 2) of 0.986 and 0.980 for biomass concentration, 0.976 and 0.999 for PHB and 0.992 and 0.999 for acetate concentration in calibration and validation, respectively. These results confirm the ability of the proposed methodology for on-line monitoring of the state variables in PHB production process by MMC.

Effect of growth conditions on the molecular weight of poly-3-hydroxybutyrate produced by Azotobacter chroococcum 7B

It has been shown that poly-3-hydroxybutyrate (PHB) of predetermined molecular weight can be obtained by varying the growth conditions of the producer strain, Azotobacter chroococcum 7B: pH, temperature, aeration, presence of sodium acetate as an additional carbon source, or growth on crude complex carbon sources (molasses, vinasse, or starch). High-molecular-weight polymer can be obtained at pH 7.0, optimal for the culture (1485 kDa), temperature 30-37 degrees C (1600-1450 kDa, respectively), and low aeration (2215 kDa). The following factors decrease PHB MW: pH deviation to the acidic (pH 6.0, 476 kDa) or alkaline (pH 8.0, 354 kDa) range or lower temperature (20 degrees C, 897 kDa). Introduction of additional carbon source (sodium acetate) at concentrations in the medium varying from 0 to 5 g/l provides an original method of production of PHB with predetermined MW in a wide range, from 270 to 1515 kDa, with high PHB content in the cell.

Formation of aerobic granules and their PHB production at various substrate and ammonium concentrations

Aerobic granular sludge rich in polyhydroxybutyrate (PHB) was cultivated in a sequencing batch reactor (SBR) by seeding anaerobic granular sludge. The PHB content in aerobic granules was investigated and the experimental results reveal that both influent chemical oxygen demand (COD) and ammonium concentrations had a significant effect on the morphological characteristics and the PHB production of the aerobic granular sludge. At a COD and ammonium concentration of 750 mg/L and 8.5 mg/L, respectively, the PHB content of the granules reached 44%, but their poor settling ability, as evidenced by a high sludge volume index, was observed. This was attributed to the outgrowth of filamentous bacteria on the granule surface. However, an increase in the ammonium concentration resulted in an elevated sludge concentration and a decrease in the PHB content in the granules. In this case, the aerobic granular sludge with a regular and compact structure was formed. The results suggest that, through controlling the COD and ammonium concentrations in the influent, the PHB-rich aerobic granular sludge with good settling ability could be cultivated.

Macromolecular composition and anaerobic degradation of the sludge produced in a sequencing batch reactor

The effect of sequencing batch reactor (SBR) operating conditions on sludge macromolecular composition and the effect of sludge macromolecular composition on the anaerobic degradation of the sludge produced in SBR was investigated in this work. A SBR, fed with synthetic wastewater, was operated at different air flow rates. The resulting sludge was analyzed in terms of protein, carbohydrate, phospholipid and polyhydroxybutyrate concentrations. Methane production during anaerobic digestion of the sludge was also measured. Ammonium, nitrite, nitrate, dissolved oxygen and chemical oxygen demand (COD) track studies in the SBR were carried out in order to relate SBR performance and sludge macromolecular composition. The lowest air flow rate at which the SBR was operated was 2 l min -1 , in which case the dissolved oxygen concentration was lower than 0.5 mg l-1 in the SBR and partial denitrification occurred during the feeding phase. An increased air flow rate caused a decrease in protein concentration, as well as an increase in carbohydrate concentration. Polyhydroxybutyrate (PHB) concentration in the sludge was independent of air flow rate. At different air flows, the methane production rates were similar, but the total volume of methane was greater during anaerobic digestion of the sludge produced at low air flow rates. These results indicate a strategy by which changes in sludge composition can optimize the operation of anaerobic sludge digesters.

Nutrient optimization for production of polyhydroxybutyrate from halotolerant photosynthetic bacteria cultivated under aerobic-dark condition

Three halotolerant bacterial strains; Rhodobacter sphaeroides ES16 (the wild type) and the two mutant strains of R. sphaeroides ES16, namely N20 and U7, were cultivated in glutamate-malate (GM) medium and screened for production of polyhydroxybutyrate (PHB). The mutant strains N20 and U7 were found to accumulate PHB (53.9 and 42.0% of DCW, respectively) 3.6 and 2.8 times higher than the wild type strain (19.5% of DCW), respectively. R. sphaeroides N20 were selected for studies on the effects of nutrient and environmental conditions on PHB accumulation. The optimal condition was 4 g/l acetate, 0.02 g/l (NH 4 ) 2 SO 4 , C/N ratio of 6:1, 1.0 g/l K 2 HPO 4 , 1.0 g/l KH 2 PO 4 and 3% NaCl with initial pH at 7.0. Under this optimal condition, the maximum PHB accumulation increased from 53.9% to 88% of DCW and 9.11 ± 0.08 g/l biomass, 8.02 ± 0.10 g/l PHB concentration were achieved after 60 hrs cultivation at 37oC. These results are the highest values ever obtained from photosynthetic bacteria reported so far.

Biosynthesis of poly-3-hydroxybutyrate with a high molecular weight by methanotroph from methane and methanol

Poly-3-hydroxybutyrate (PHB) can be produced by various species of bacteria. Among the possible carbon sources, both methane and methanol could be a suitable substrate for the production of PHB. Methane is cheap and plentiful not only as natural gas, but also as biogas. Methanol can also maintain methanotrophic activity in some conditions. The methanotrophic strain Methylosinus trichosporium IMV3011 can accumulate PHB with methane and methanol in a brief nonsterile process. Liquid methanol (0.1%) was added to improve the oxidization of methane. The studies were carried out using shake flasks. Cultivation was performed in two stages: a continuous growth phase and a PHB accumulation phase under the conditions short of essential nutrients (ammonium, nitrate, phosphorus, copper, iron (III), magnesium or ethylenediamine tetraacetate (EDTA)) in batch culture. It was found that the most suitable growth time for the cell is 144 h. Then an optimized culture condition for second stage was determined, in which the PHB concentration could be much increased to 0.6 g/L. In order to increase PHB content, citric acid was added as an inhibitor of tricarboxylic acid cycle (TCA). It was found that citric acid is favorable for the PHB accumulation, and the PHB yield was increased to 40% (w/w) from the initial yield of 12% (w/w) after nutrient deficiency cultivation. The PHB produced is of very high quality with molecular weight up to 1.5×10 6Da.

Biodegradable double‐layer films based on biological resources: Polyhydroxybutyrate and cellulose

AbstractA novel biocomposites double‐layer films using polyhydroxybutyrate (PHB) and cellulose paper were produced. The biocomposites were prepared by the solvent‐casting method (with chloroform). Films of the blends were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), scanning electron micrograph (SEM), Fourier transform infrared spectroscopy (FTIR), X‐ray (XR) and surface roughness measurements. Moisture absorption, water absorption and water vapor permeation of films has been investigated. The PHB impregnated the fibers of cellulose paper and it was on the valley of paper structure. The double‐layer films showed diminutions in the moisture absorption, in the water abortion, in water vapor permeation and in surface roughness. According to contact angle, surface free energies and the barrier properties of the films can be considered more hydrophobic. The experimentally observed tensile properties (modulus and tensile strength) of double‐layer films with different content of PHB were determined. Measurements show the tensile strength and modulus of cellulose paper increases with the percentage of PHB. SEM photomicrographs of the fractured films surfaces were also analyzed. It was possible to obtain a biodegradable material with little amount of PHB to improve the barrier and the mechanical properties of cellulose paper, taking advantage of the good properties of both materials. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l −1 with a PHB concentration of 6.1 g l −1 in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h −1 over the value obtained in batch cultivation (PHB production rate – 0.4 g h −1).

Rapid microwave assisted esterification method for the analysis of poly-3-hydroxybutyrate in Alcaligenes latus by gas chromatography

In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3-hydroxybutyrate (Me-3HB) in acidified methanol (H 2SO 4, 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assisted method was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was comparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventional method, affording a significant saving of time and energy.

Study of controlled tetracycline release from porous calcium phosphate/polyhydroxybutyrate composites

AbstractPorous calcium phosphate ceramics were prepared by sintering of mixtures of nanocrystalline apatitic calcium phosphate and fibrous natural cotton cellulose after pressing at temperatures of 1150 °C and 1250 °C. Micro-and macropores were present in microstructures of ceramic samples. The microstructures of porous ceramics were similar to those observed in bone tissues and fiber-like randomly oriented texture was observed in ceramics. Polyhydroxybutyrate (PHB) biopolymer layers are distributed homogeneously in the samples after evaporation of the diluent (chloroform) from the PHB vacuum impregnated porous samples. The tetracycline (TTC) release rate decreases with the content of polyhydroxybutyrate in the ceramic samples, which corresponds to the rise in amount of biopolymer displaced in the pores of ceramics. The concentration of TTC in the phosphate buffer saline solution varies almost linearly with time after the first seven hours from the start of the release of the calcium phosphate ceramic samples with 2.4 mass % of polyhydroxybutyrate. The initial burst effect was significantly depressed by the preparation method used.

Cultivation of polyhydroxybutyrate-rich aerobic granular sludge in a sequencing batch reactor

In this study a two-step strategy was adopted to cultivate Polyhydroxybutyrate (PHB)-rich aerobic granular sludge in a sequencing batch reactor (SBR) fed with a synthetic wastewater. In the first step both oxygen and ammonia were initially limited, in order to enhance the PHB-storage ability of sludge. In the second step granular sludge was cultivated to get a high PHB volumetric productivity. The PHB content of sludge increased to 43.1±2.0% in the first step. During the sludge granulation, the PHB content was constant at 40±4.6%. With the granulation, the settling ability of the PHB-rich sludge continuously improved, as evidenced by a decreased sludge volume index. The matured PHB-rich granular sludge presented a buff color and regular morphology with elliptical and flat shape.

Production of polyhydroxybutyrate in sugarcane

We report here the production of the bacterial polyester, polyhydroxybutyrate (PHB), in the crop species sugarcane (Saccharum spp. hybrids). The PHB biosynthesis enzymes of Ralstonia eutropha [beta-ketothiolase (PHAA), acetoacetyl-reductase (PHAB) and PHB synthase (PHAC)] were expressed in the cytosol or targeted to mitochondria or plastids. PHB accumulated in cytosolic lines at trace amounts, but was not detected in mitochondrial lines. In plastidic lines, PHB accumulated in leaves to a maximum of 1.88% of dry weight without obvious deleterious effects. Epifluorescence and electron microscopy of leaf sections from these lines revealed that PHB granules were visible in plastids of most cell types, except mesophyll cells. The concentration of PHB in culm internodes of plastidic lines was substantially lower than in leaves. Western blot analysis of these lines indicated that expression of the PHB biosynthesis proteins was not limiting in culm internodes. Epifluorescence microscopy of culm internode sections from plastidic lines showed that PHB granules were visible in most cell types, except photosynthetic cortical cells in the rind, and that the lower PHB concentration in culm internodes was probably a result of dilution of PHB-containing cells by the large number of cells with little or no PHB. We discuss strategies for producing PHB in mitochondria and mesophyll cell plastids, and for increasing PHB yields in culms.

Influence of electron acceptor, carbon, nitrogen, and phosphorus on polyhydroxyalkanoate (PHA) production by Brachymonas sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH4Cl), and phosphorus (KH2PO4) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g−1 biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g−1 biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g−1 biomass h−1, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.

Identification and Characterization of the Bacillus thuringiensi s phaZ Gene, Encoding New Intracellular Poly-3-Hydroxybutyrate Depolymerase

A gene that codes for a novel intracellular poly-3-hydroxybutyrate (PHB) depolymerase has now been identified in the genome of Bacillus thuringiensis subsp. israelensis ATCC 35646. This gene, previously annotated as a hypothetical 3-oxoadipate enol-lactonase (PcaD) gene and now designated phaZ, encodes a protein that shows no significant similarity with any known PHB depolymerase. Purified His-tagged PhaZ could efficiently degrade trypsin-activated native PHB granules as well as artificial amorphous PHB granules and release 3-hydroxybutyrate monomer as a hydrolytic product, but it could not hydrolyze denatured semicrystalline PHB. In contrast, purified His-tagged PcaD of Pseudomonas putida was unable to degrade trypsin-activated native PHB granules and artificial amorphous PHB granules. The B. thuringiensis PhaZ was inactive against p-nitrophenylpalmitate, tributyrin, and triolein. Sonication supernatants of the wild-type B. thuringiensis cells exhibited a PHB-hydrolyzing activity in vitro, whereas those prepared from a phaZ mutant lost this activity. The phaZ mutant showed a higher PHB content than the wild type at late stationary phase of growth in a nutrient-rich medium, indicating that this PhaZ can function as a PHB depolymerase in vivo. PhaZ contains a lipase box-like sequence (G-W-S(102)-M-G) but lacks a signal peptide. A purified His-tagged S102A variant had lost the PHB-hydrolyzing activity. Taken together, these results indicate that B. thuringiensis harbors a new type of intracellular PHB depolymerase.

Polyhydroxybutyrate production from a novel feedstock derived from a wheat-based biorefinery

A wheat-based biorefining strategy was evaluated for polyhydroxybutyrate (PHB) production by Cupriavidus necator (formerly classified as Ralstonia eutropha but currently designated Wautersia eutropha). Wheat was bioconverted into two feedstock streams, wheat hydrolysate (WH) and fungal extract (FE) that were rich in glucose and nitrogen, respectively. WH and FE were mixed in appropriate proportions to provide media with varying glucose (5–26 g l −1) and free amino nitrogen (FAN) (0.1–1.2 g l −1) concentrations for batch shake flask fermentations. Increasing FAN concentration resulted in higher microbial growth and less PHB accumulation. The consumption of various carbon sources (carbohydrates, amino acids, peptides) resulted in high growth yields (up to 1.07 g cells (g glucose) −1) as related to glucose. Specific growth rates up to 0.16 h −1 were observed. Three WH with similar glucose (200–220 g l −1) and varying FAN (0.3–1.48 g l −1) concentrations were evaluated in fed-batch shake flask fermentations for C. necator growth and PHB accumulation. The medium with the highest nitrogen concentration (WH3) gave the highest microbial biomass concentration (29.9 g l −1), growth yield (0.28 g residual microbial biomass (g glucose) −1) and PHB yield (0.43 g (g glucose) −1). WH2 gave the highest PHB concentration (51.1 g l −1) and content (0.7 g g −1).