Polyhydroxybutyrate Content Research Articles

Polyhydroxybutyrate production accompanied by the effective reduction of chemical oxygen demand (COD) and biological oxygen demand (BOD) from industrial effluent

Industrial effluents are major pollution-causing agents for our environment. Our study focuses on utilizing effluents from different industries for efficient production of Polyhydroxybutyrate (PHB). Presence of PHB was identified by Sudan Black staining method. The PHB production parameters for Pseudomonas aeruginosa MTCC 4673 were studied critically, and it was found that glucose with 8.5 mg/L (0.0550 g PHB/g substrate) PHB concentration yielded the highest among the carbon sources used. Peptone with 8.9 mg/L (0.0524 g PHB/g substrate) of PHB concentration, an incubation period of 48 h and at a pH of 7 yielded the optimum results. These studies were compared with those of Alcaligens latus MTCC 2311. Dairy effluents (DE) and tannery effluents (TE) were considered for the best possible substrate, for the production of PHB in an optimized media. The results indicated that the dairy effluents gave a higher yield of PHB. Amongst various dilution levels studied from 10–100% (v/v), 50% (v/v) concentration of the dairy effluent showed maximum PHB productivity of 0.0582 g PHB/g substrate. A comparison of the chemical oxygen demand (COD) and biological oxygen demand (BOD) from the results, showed a significant removal percentage of 78.97% BOD and 53.482% COD, which highlighted the importance of utilizing effluents for PHB production, in order to reduce the risk of toxic effluent discharge. FT-IR analysis was carried out to confirm the presence of PHB.

Polyhydroxybutyrate production by direct use of waste activated sludge in phosphorus-limited fed-batch culture

Polyhydroxybutyrate (PHB) production directly by waste activated sludge (WAS) was investigated in aerobic fed-batch conditions using acetic acid as substrate. PHB production was induced by phosphorus limitation. WAS of different origin were tested with various degrees of phosphorus limitation and PHB contents of up to 70% (gCOD PHB/gCOD particulate) were obtained. This strategy showed the importance of maintaining cell growth for PHB production in order to increase PHB concentration and that the degree of phosphorus limitation has a direct impact on the quantity of PHB produced. Pyrosequencing of 16S rRNA transcripts showed changes in the active bacteria of the WAS microbial community as well as the acclimation of populations depending on sludge origin. The monitoring of the process appeared as the key factor for optimal PHB production by WAS. Different strategies are discussed and compared in terms of carbon yield and PHB content with the feast and famine selection process.

Chemical inhibition of acetyl coenzyme A carboxylase as a strategy to increase polyhydroxybutyrate yields in transgenic sugarcane

Polyhydroxybutyrate (PHB) is a naturally occurring bacterial polymer that can be used as a biodegradable replacement for some petrochemical-derived plastics. Polyhydroxybutyrate is produced commercially by fermentation, but to reduce production costs, efforts are underway to produce it in engineered plants, including sugarcane. However, PHB levels in this high-biomass crop are not yet commercially viable. Chemical ripening with herbicides is a strategy used to enhance sucrose production in sugarcane and was investigated here as a tool to increase PHB production. Class A herbicides inhibit ACCase activity and thus reduce fatty acid biosynthesis, with which PHB production competes directly for substrate. Treatment of PHB-producing transgenic sugarcane plants with 100 μM of the class A herbicide fluazifop resulted in a fourfold increase in PHB content in the leaves, which peaked ten days post-treatment. The minimum effective concentration of herbicide required to maximize PHB production was 30 μM for fluazifop and 70 μM for butroxydim when applied to saturation. Application of a range of class A herbicides from the DIM and FOP groups consistently resulted in increased PHB yields, particularly in immature leaf tissue. Butroxydim or fluazifop treatment of mature transgenic sugarcane grown under glasshouse conditions increased the total leaf biomass yield of PHB by 50%-60%. Application of an ACCase inhibitor in the form of a class A herbicide to mature sugarcane plants prior to harvest is a promising strategy for improving overall PHB yield. Further testing is required on field-grown transgenic sugarcane to more precisely determine the effectiveness of this strategy.

Feasibility study on production of biodegradable polymer and wastewater treatment using Aeromonas strains for materials recycling

With consideration of wastewater treatment and materials recycling for cradle-to-cradle (C2C) sustainable development, this treatability study analyzed the capability of poly 3-hydroxybutyrate (PHB) production in wastewater-laden media using indigenous dye-decolorizing Aeromonas hydrophila NIU01, KB23, Aeromonas salmonicida 741. Compared to paper-container, frozen food, wine manufacturing wastewater, wastewater generated from printing and dyeing industry was found to be more appropriate to efficiently produce PHB for materials recycling. Due to lack of sufficient essential inorganic nutrients provided for cell propagation, dye-decolorized wastewater with augmented MR media in different ratios was used to explore toxicity potency of mixed media and to present PHB-producing capability of cells. In particular, when MR media were completely replaced by decolorized culture broth, significant stimulating effect on PHA-production was shown (ca. 52.5% PHB content). This study clearly revealed the promising feasibility of simultaneous wastewater treatment and biopolymer production for cradle-to-cradle sustainable development.

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3h−1) and maximal specific non-growth PHB production rate (0.16gg−1h−1) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016gL−1), glycerol (0.05gL−1), non-growth related PHB synthesis (0.011gL−1) and nitrogen/PHB related inhibition constant (0.405gL−1), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23h) and better PHB yield (0.347gg−1). Initial concentration of biomass 16.8gL−1 and glycerol concentration in broth between 3 and 5gL−1 were decisive factors for increasing of productivity.

Polyhydroxybutyrate production from oil palm empty fruit bunch using Bacillus megaterium R11

Oil palm empty fruit bunch (OPEFB), contains abundant cellulose and hemicelluloses and can be used as a renewable resource for fuel and chemical production. This study, as the first attempt, aims to convert OPEFB derived sugars to polyhydroxybutyrate (PHB). OPEFB collected from a Malaysia palm oil refinery plant was chemically pretreated and enzymatically hydrolyzed by an in-house prepared cellulase cocktail. The PHB producer, Bacillus megaterium R11, was isolated in Singapore and could accumulate PHB up to 51.3% of its cell dry weight (CDW) from both glucose and xylose. Tryptone was identified as its best nitrogen source. PHB content and production reached 58.5% and 9.32 g/L, respectively, for an overall OPEFB sugar concentration of 45 g/L. These respectively reached 51.6% and 12.48 g/L for OPEFB hydrolysate containing 60 g/L sugar with a productivity of 0.260 g/L/h.

Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillus megaterium BA-019

This study demonstrated the improved polyhydroxybutyrate (PHB) production via high cell density cultivation of Bacillus megaterium BA-019 with balanced initial total sugar concentration and carbon to nitrogen (C/N) weight ratio. In the 10L stirred fermentor operated at 30°C, pH 7.0, 600rpm, and 1.0vvm air, with the initial total sugar concentration of 60g/L and urea at the C/N weight ratio of 10:1, 32.48g/L cell biomass with the corresponding PHB weight content of 26.94% and volumetric productivity of 0.73g/Lh were obtained from batch cultivation. Continuing cultivation by intermittent feeding of the sugarcane molasses along with urea at the C/N weight ratio of 12.5:1 gave much improved biomass and PHB production (90.71g/Lbiomass with 45.84% PHB content and 1.73g/Lh PHB productivity). Similar biomass and PHB yields were obtained in the 90L stirred fermentor when using the impeller tip speed as the scale-up criterion.

Bioconversion of restaurant waste into Polyhydroxybutyrate (PHB) by recombinant E. coli through anaerobic digestion

The effect of temperature (30°C, 37°C, uncontrolled) and initial pH adjustment at pH 7 in the anaerobic digestion process was investigated to enhance the production of organic acids from restaurant waste. The highest organic acid level obtained was 39.6 g/L on the fifth day of fermentation conducted at 30°C and initial pH 7. The acids produced corresponded to 39.4% of the yield based on the initial concentration of substrate. The main organic acids produced were lactic and acetic acids. Using organic acids from fermented restaurant waste, recombinant Escherichia coli pnDTM2 gave PHB concentration, PHB content and PHB productivity of 9.2 g/L, 44% w/w and 0.54 g/L/h, respectively, in a pH stat fed-batch culture.

Production of Polyhydroxyalkanoate from Paenibacillus durus BV-1 Isolated from Oil Mill Soil

Polyhydroxyalkanoates (PHAs) are polyesters naturally synthesized by numerous bacteria as intracellular energy storage materials during unbalanced growth. These polyesters find many applications in medicine, veterinary practice, tissue engineering, food packaging, etc. In the present study, spent wash and oil mill soil samples were screened for the isolation of bacteria producing polyhydroxyalkanoates. Different bacterial isolates were screened by PCR using primers designed for Bacillus megaterium. Isolate-4 reacted positively giving amplicons similar in size to that of Bacillus megaterium. Based on 16S rRNA gene sequences, isolate-4 was identified as Paenibacillus durus. Nitrogen limited mineral salt medium with fructose as chief carbon source was used for production of Polyhydroxybutyrate (PHB) in shake flask studies. The effect of various carbon and nitrogen sources on PHB production was evaluated, with respect to yield and PHB content. The study revealed that fructose and peptone gave better PHB accumulation and growth rates for the isolate and the reference strain, and hence, were the best among the carbon and nitrogen sources used. Further studies are needed to optimize the production conditions.

Pentose-rich hydrolysate from acid pretreated rice straw as a carbon source for the production of poly-3-hydroxybutyrate

The aim of this work was to evaluate three different bacterial strains for their ability to accumulate poly-3-hydroxybutyrate (PHB) using pentose sugar rich hydrolysate generated from acid pretreated rice straw as the sole carbon source. Out of these, Bacillus firmus NII 0830 showed maximum PHB production. Acid pretreated black liquor contained sugars and sugar degradation products such as formic acid, acetic acid, furfural and hydroxymethyl furfural. The bacterium grew in the hydrolysate medium without any detoxification and it could accumulate 1.9g/l biomass with 1.697g/l PHB and the PHB content in the cell was 89%. This was the highest value ever reported from Bacillus species. The optimum conditions for the fermentation media were an inoculum concentration of 6.5%, 90h of incubation and 0.75% of xylose concentration. The characterization of extracted polymer was carried out by FTIR, 1H and 13C NMR which showed characteristics similar to that of the standard PHB from Sigma.

Bulking sludge for PHA production: Energy saving and comparative storage capacity with well-settled sludge

Two acetate-fed sequencing batch reactors (SBR) were operated under an aerobic dynamic feeding (ADF) model (SBR #2) and with anaerobic phase before aerobic phase (SBR #1) to select mixed cultures with a high polyhydroxyalkanoates (PHA) storage response. Although kinetic selection based on storage response should bring about a predominance of floc-formers, a bulking sludge with storage response comparable to well-settled sludge was steadily established. An anaerobic phase was introduced before the aerobic phase in the ADF model to improve the sludge settleability (SBR #1), however, due to the consequent increased feast/famine ratio, the performance of SBR #1, in terms of both the maximum PHB (polyhydroxybutyrate) cell content and ΔPHB, was lower than that of SBR #2. SBR #2 gradually reached a steady state while SBR #1 failed suddenly after 50 days of operation. The maximum specific substrate uptake rate and storage rate for the selected bulking sludge were 0.4 Cmol Ac/(Cmol X·hr) and 0.18 Cmol Ac/(Cmol PHB·hr), respectively, resulting a yield of 0.45 Cmol PHB/(Cmol Ac) in SBR #2 in the culture enrichment phase. A maximum PHB content of 53% of total suspended solids and PHB storage rate of 1.36 Cmol Ac/(Cmol PHB·hr) was achieved at 10.2 hr in batch accumulation tests under nitrogen starvation. The results indicated that it was feasible to utilize filamentous bacteria to accumulate PHA with a rate comparable to well-settled sludge. Furthermore, the lower dissolved oxygen demand of filamentous bacteria would save energy required for aeration in the culture enrichment stage.

Production of polyhydroxybutyrate (PHB) by Alcaligenes latus using sugarbeet juice

The practicality of using sugarbeet juice as medium to grow Alcaligenes latus (ATCC 29714) for production of polyhydroxybutyrate (PHB), a biodegradable plastic, was explored in this study. Dilute sugarbeet juice, sugarbeet juice with partial and complete addition of nutrients other than sugar were used as culture media. Media with partial nutrient addition was shown to be optimal for PHB production, with final dry cell weight (DCW) 10.30±1.01g/L, PHB concentration 4.01±0.95g/L, PHB content 38.66±7.28%, Yp/x (gPHB produced per g dry cell weight) 0.39±0.07 and a maximum PHB productivity of 0.22±0.01g/Lh. The melting temperature of PHB extracted from sugarbeet juice-grown cells supplemented with partial nutrients was measured to be 151.46°C with crystallinity of 43.12% and the corresponding crystallinity temperature of 45.42°C. Thermal degradation of extracted PHB occurred from 255.14 to 283.69°C with the degradation peak at 273.86°C.

Upstream process optimization of polyhydroxybutyrate (PHB) by Alcaligenes latus using two-stage batch and fed-batch fermentation strategies

This research focused on optimizing the upstream process time for production of polyhydroxybutyrate (PHB) from sucrose by two-stage batch and fed-batch fermentation with Alcaligenes latus ATCC 29714. The study included selection of strain, two-stage batch fermentations with different time points for switching to nitrogen limited media (14, 16 or 18 h) and fed-batch fermentations with varied time points (similar to two stage) for introducing nitrogen limited media. The optimal strain to produce PHB using sucrose as carbon source was A. latus ATCC 29714 with maximum-specific growth rate of 0.38 ± 0.01 h(-1) and doubling time of 1.80 ± 0.05 h. Inducing nitrogen limitation at 16 h and ending second stage at 26 h gave optimal performance for PHB production, resulting in a PHB content of 46.7 ± 12.2 % (g PHB per g dry cell weight) at the end of fermentation. This was significantly higher (P ≤ 0.05) (approximately 7 %) than the corresponding fed batch run in which nitrogen limitation was initiated at 16 h.

PHB production by Methylocystis hirsuta from natural gas in a bubble column and a vertical loop bioreactor

Biosynthesis of poly-3-hydroxybutyrate (PHB) by Methylocystis hirsuta from natural gas in two different media was studied for the first time. After selection of the suitable medium, the effect of two key parameters such as methane to air ratio and nitrogen content on the PHB production was determined in a bubble column bioreactor using a full factorial design. It was found that both of these factors had a significant effect on the PHB accumulation (42.5% w/w of cell dry weight). PHB production by M. hirsuta, unlike other methanotrophic bacteria, was found to be a growth associated metabolite. Subsequently, the PHB production was carried out in a forced-liquid vertical tubular loop bioreactor (VTLB) at optimum condition determined in the bubble column bioreactor. The PHB content of biomass was 51.6% w/w of cell dry weight (CDW) in the VTLB. These results indicated that the loop bioreactors specially are suitable candidates for PHB production from natural gas.

Polyhydroxybutyrate production in Bacillus mycoides DFC1 using response surface optimization for physico-chemical process parameters

The production of polyhydroxybutyrate (PHB) by Bacillus sp. is most often growth associated and is influenced by various physico-chemical parameters. Imbalanced nutrient conditions were often found to result in sporulation and low PHB production in Bacillus sp. In the present investigation, Bacillus mycoides DFC1 strain isolated from garden soil was studied for PHB production in glucose–peptone broth. The effect of glucose/peptone ratio on biomass yield, PHB production and sporulation was investigated. Central composite rotatable design was used to study the interactive effects of three variables: glucose, peptone and pH on cell growth and PHB production. The optimized medium conditions with the constraint ‘to maximize’ cell growth and PHB content were glucose 17.34 g/l, peptone 7.03 g/l at pH 7.3. A maximum dry cell weight of 4.35 g/l and PHB yield of 3.32 g/l amounting to 76.32 % (w/w) of dry cell weight with negligible sporulation at the end of 72 h resulted in a significant increase (1.83–3.32 g/l or 1.82-fold) in the production of PHB in comparison to the medium used in preliminary studies.

Mutations Derived from the Thermophilic Polyhydroxyalkanoate Synthase PhaC Enhance the Thermostability and Activity of PhaC from Cupriavidus necator H16

The thermophile Cupriavidus sp. strain S-6 accumulated polyhydroxybutyrate (PHB) from glucose at 50°C. A 9.0-kbp EcoRI fragment cloned from the genomic DNA of Cupriavidus sp. S-6 enabled Escherichia coli XL1-Blue to synthesize PHB at 45°C. Nucleotide sequence analysis showed a pha locus in the clone. The thermophilic polyhydroxyalkanoate (PHA) synthase (PhaC(Csp)) shared 81% identity with mesophilic PhaC of Cupriavidus necator H16. The diversity between these two strains was found dominantly on their N and C termini, while the middle regions were highly homologous (92% identity). We constructed four chimeras of mesophilic and thermophilic phaC genes to explore the mutations related to its thermostability. Among the chimeras, only PhaC(H16β), which was PhaC(H16) bearing 30 point mutations derived from the middle region of PhaC(Csp), accumulated a high content of PHB (65% [dry weight]) at 45°C. The chimera phaC(H16)(β) and two parental PHA synthase genes were overexpressed in E. coli BLR(DE3) cells and purified. At 30°C, the specific activity of the chimera PhaC(H16β) (172 ± 17.8 U/mg) was 3.45-fold higher than that of the parental enzyme PhaC(H16) (50 ± 5.2 U/mg). At 45°C, the half-life of the chimera PhaC(H16β) (11.2 h) was 127-fold longer than that of PhaC(H16) (5.3 min). Furthermore, the chimera PhaC(H16β) accumulated 1.55-fold (59% [dry weight]) more PHA content than the parental enzyme PhaC(H16) (38% [dry weight]) at 37°C. This study reveals a limited number of point mutations which enhance not only thermostability but also PhaC(H16) activity. The highly thermostable and active PHA synthase will provide advantages for its promising applications to in vitro PHA synthesis and recombinant E. coli PHA fermentation.

Correlation between bio-hydrogen production and polyhydroxybutyrate (PHB) synthesis by Rhodopseudomonas palustris WP3-5

The aim of this study was to determine the competition between H2 production and polyhydroxybutyrate (PHB) accumulation of Rhodopseudomonas palustris WP3-5 when grown on six different substrates. From the results, strain WP3-5 can utilize acetate, propionate, malate, and lactate to produce H2 but can only synthesize PHB on acetate and propionate. The substrate conversion efficiency (SCE) on acetate and propionate increased significantly after the maximum PHB content was achieved, illustrating a competition for reducing power when PHB synthesis occurred. However, when strain WP3-5 was cultivated at suboptimal pH values on acetate, the synthesized PHB prevented strain WP3-5 from the stress of the inappropriate pH and retained H2 producing efficiency as at optimal pH value. Consequently, although PHB synthesis does compete with H2 production in R. palustris WP3-5, it is still conducive to H2 production when strain WP3-5 is in a stressful condition.

On the control of molecular weight distribution of polyhydroxybutyrate in Azohydromonas lata cultures

The microbial production of polyhydroxybutyrate (PHB) has been reported by several studies dealing with the identification and optimisation of key-process variables and fermentation strategies for the maximisation of polymer yield and productivity. However, little work has been done on the control of the molecular weight distribution (MWD) of PHB, as a product quality variable that significantly affects its end-use properties and processing characteristics. In this study, batch and fed-batch cultures of Azohydromonas lata, as a PHB host bacterium, are conducted in flasks and in a lab-scale bioreactor, where a number of culture nutritional and operational variables (i.e., medium composition, dissolved oxygen concentration, harvesting time, feeding policy and post-treatment conditions) are examined in a comprehensive way for their effect on the time evolution of the molecular properties of the produced PHB. Under specific nutritional conditions, batch or fed-batch cultivation mode and pulse or continuous nutrients addition policies, a wide range of number and weight average molecular weights of PHB was attained. Moreover, the harvested culture post-treatment conditions were assessed and found to greatly affect the MWD of PHB. Finally, the optimal nutritional and operational (i.e., feeding policy) conditions were established for the maximisation of the intracellular PHB content and productivity. In general, the PHB is efficiently produced in A. lata bacteria with relatively narrow and unimodal MWD, however, under extreme conditions favouring the enzymic or mechanical degradation of PHB, a broad and bimodal MWD can be effected

Comparing poly-3-hydroxybutyrate accumulation in Azospirillum brasilense strains Sp7 and Sp245: The effects of copper(II)

Intracellular accumulation of poly-3-hydroxybutyrate (PHB) was comparatively studied in the plant-growth-promoting bacterium (PGPB) Azospirillum brasilense (epiphytic strain Sp7 and endophytic strain Sp245) grown microaerobically for 2 days under nitrogen deficiency in the standard malate salt medium in the absence (control) or presence of copper (0.1mM Cu2+). For quantitative determination of PHB content of cells, diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy of whole-cell biomass samples was used. After 2 days in control cells, PHB accumulation in strain Sp7 reached ca. 24% of dry cell mass (d.c.m.). In strain Sp245, the PHB content in the control was over 1.3-fold higher (32% d.c.m.) than in strain Sp7. In the presence of copper(II), PHB accumulation was notably enhanced in strain Sp7 (over 1.6-fold, up to ca. 39% d.c.m.), whereas in strain Sp245 it changed insignificantly (from 32% up to ca. 35% d.c.m.). The levels of copper(II) uptake were comparable in both strains. These findings are in line with our earlier observations that even in rich NH4+-supplemented medium, some heavy metals, including copper(II), induce PHB biosynthesis in A. brasilense strain Sp7, but not in Sp245. The dissimilarities in the levels of PHB accumulation and in the effects induced by copper(II) in the two strains are attributed to their different adaptive potentials owing to different ecological niches they occupy in the rhizosphere.

Utilization of biodiesel waste as a feedstock for the production of polyhydroxybutyrate by Cupriavidus necator

This study aimed to investigate the potential of using wastewater and crude glycerol from biodiesel refinery to produce polyhydroxyalkanoates (PHAs) through fermentation of Cupriavidus necator TISTR 1095. The result indicates that crude glycerol yielded high cell growth (35 to 37 g/L) and poly-3-hydroxybutyrate (PHB; 17.85 to 19.98 g/L). However, no cell growth obtained from biodiesel-wastewater due to high Na + presented. Among medium and experimental factors influencing PHB accumulation, crude glycerol, (NH 4 ) 2 SO 4 and trace element concentration revealed significant effects (P<0.1). Their optimal values were 60 g/L crude glycerol, 1.32 g/L (NH 4 ) 2 SO 4 and 2.0 g/L trace element. Under these optimal conditions, the strain TISTR 1095 produced the highest biomass (46.25±2.10 g/L) and PHB concentration of 24.98±1.87 g/L with PHB content of 54.01% of DCW. Effect of experimental conditions including aeration rate and agitation speed as well as sterile condition on PHB accumulation was also studied. The optimal aeration rate (2 vvm) and agitation speed (150 rpm) under septic condition during cultivation gave slightly increase of biomass and PHB. The maximum biomass (46.96±0.28 g/L) and PHB concentration of 25.32±0.20 g/L (53.92% of DCW) was achieved in 20-L fermentor. Moreover, the purified PHB from C. necator TISTR 1095 was partially characterized; their properties were similar to commercial PHB. Key words : Biodiesel, Cupriavidus necator , glycerol, optimization, polyhydroxybutyrate, poly-3-hydroxybutyrate (PHB).