PHB Content Research Articles

Production of PHB from Crude Glycerol

AbstractCrude glycerol – a by‐product of the large scale production of diesel oil from rape – is examined for its possible use as a cheap feedstock for the biotechnological synthesis of poly(3‐hydroxybutyrate) (PHB). The glycerol samples of various manufacturers differ in their contamination with salts (NaCl or K2SO4), methanol or fatty acids. At high cell density fermentation these pollutants could possibly accumulate to inhibiting concentrations. The bacteria used were Paracoccus denitrificans and Cupriavidus necator JMP 134, which accumulate PHB from pure glycerol to a content of 70 % of cell dry mass. When using crude glycerol containing 5.5 % NaCl, a reduced PHB content of 48 % was observed at a bacterial dry mass of 50 g/L. Furthermore the PHB yield coefficient was reduced, obviously due to osmoregulation. The effect of glycerol contaminated with K2SO4 was less pronounced. The molecular weight of PHB produced with P. denitrificans or C. necator from crude glycerol varies between 620000 and 750000 g/mol which allows the processing by common techniques of the polymer industry.

Utilization of agricultural residues for poly(3-hydroxybutyrate) production by Halomonas boliviensis LC1

Utilization of cheap and readily available agricultural residues as cheap carbon sources for poly(3-hydroxybutyrate) (PHB) production by Halomonas boliviensis. Wheat bran was hydrolysed by a crude enzyme preparation from Aspergillus oryzae NM1 to provide a mixture of reducing sugars composed mainly of glucose, mannose, xylose and arabinose. Growth of H. boliviensis using a mixture of glucose (0.75% w/v) and xylose (0.25% w/v) in the medium led to a PHB content and concentration of 45 wt% and 1 g l(-1), respectively, after 30 h. A similar PHB concentration was attained when H. boliviensis was grown on wheat bran hydrolysate but with a lower PHB content, 34 wt%. In a batch cultivation mode in a fermentor, using 1.8% (w/v) reducing sugars, the maximum PHB accumulation by H. boliviensis was attained in 20 h, but was reduced to about 30 wt%. By adding butyric acid (0.8% v/v), sodium acetate (0.8% w/v) and decreasing the reducing sugars concentration to 1 x 0% w/v in the medium, PHB accumulation and concentration were increased to 50 wt% and 4 g l(-1), respectively, after 20 h. Butyric acid and sodium acetate for PHB production could also be provided by anaerobic digestion of solid potato waste. Cheap and readily available agricultural residues can be used as substrates to produce PHB. The production of PHB by H. boliviensis using wheat bran hydrolysate as source of carbon is expected to reduce the production cost and motivates further studies. Large-scale commercial utilization of PHB is mainly hampered by its high production cost. Carbon source for PHB production accounts up to 50% of the total production costs. Thus, the use of waste agricultural residues can substantially reduce the substrate cost (and in turn even provide value to the waste), and can downsize the production costs. This improves the market competitiveness. Studies on PHB production by moderate halophiles were recently initiated with H. boliviensis and findings show that it has potential for commercial exploitation. PHB production by H. boliviensis using wheat bran and potato waste is hence interesting.

High poly(β-hydroxybutyrate) production by Pseudomonas fluorescens A2a5 from inexpensive substrates

A strain of poly(β-hydroxybutyrate) (PHB)-accumulating bacterium was isolated from the soil in Alaska of USA, identified as Pseudomonas fluorescens, and designated as strain A2a5. The organism grew at temperatures below 30 °C, and accumulated a large amount of granules in its cells when it was cultured in the sugarcane liquor medium. The purified sample from cells was determined as PHB by gas chromatographic and nuclear magnetic resonance analysis of polyesters. The cell density of the culture in shaking bottles reached OD 600 = 155 with PHB concentration of 31 g l −1. In 5 l bioreactor, a maximum cell dry weight (CDW) of 32 g l −1 with a PHB concentration of 22 g l −1 was obtained, and the PHB content was up to 70% and productivity was 0.23 g l −1 h −1.

Optimization of polyhydroxybutyrate production from a wild type and two mutant strains of Rhodobacter sphaeroides using statistical method

Interaction studies using central composite design (CCD) gave the optimum concentrations of acetate at 4 g l −1 and (NH 4) 2SO 4 at 0.01 g l −1 with an optimum temperature of 35 °C. Rhodobacter sphaeroides N20 gave the highest PHB (7.8 g l −1) and biomass (DCW) (8.2 g l −1) values compared to the wild type strain and the mutant strain U7. The CCD results predicted that the optimum medium for the mutant strain N20 consisted of 3.90 g l −1 acetate, 0.01 g l −1 (NH 4) 2SO 4 at 33.5 °C ( R 2 = 0.985). Validation of this model by culturing the mutant strain in this optimum medium exhibited similar values of PHB (7.76 g l −1), biomass (8.32 g l −1) and the PHB content in the cell 93.2% of DCW. Similar amounts of PHB were also obtained in batch fermentations using a 5-l bioreactor. The effect of pH and aeration rate was also studied and the optimum values were found to be pH 7.0 with an aeration rate of 1.0 vvm. Under these optimal conditions, strain N20 produced the highest amount of PHB production (8.76 g l −1), PHB content (95.4% of DCW) as well as the product yield ( Y p/ x ) (0.72). These results are the highest values ever obtained from photosynthetic bacteria reported so far.

The synthesis of poly(3‐hydroxybutyrate)‐g‐poly(methylmethacrylate) brush type graft copolymers by atom transfer radical polymerization method

AbstractBrush type of poly (3‐hydroxy butyrate), PHB, copolymer synthesis has been reported. Natural PHB was chlorinated by passing chlorine gas through PHB solution in CHCl3/CCl4 mixture (75/25 v/v) to prepare chlorinated PHB, PHB‐Cl, with the chlorine contents varying between 2.18 and 39.8 wt %. Toluene solution of PHB‐Cl was used in the atom transfer radical polymerization (ATRP) of methyl methacrylate, MMA, in the presence of cuprous bromide (CuBr)/2,2′‐bipyridine complex as catalyst, at 90°C. This “grafting from” technique led to obtain poly (3‐hydroxybutyrate)‐g‐poly(methylmethacrylate) (PHB‐g‐PMMA) brush type graft copolymers (cylindrical brush). The polymer brushes were fractionated by fractional precipitation methods and the γ values calculated from the ratio of the volume of nonsolvent to volume of solvent of brushes were ranged between 2.8 and 9.5 depending on the molecular weight, grafting density, and side chain length of the brushes, while the γ values of PHB, PHB‐Cl, and homo‐PMMA were 2.7–3.8, 0.3–2.4, and 3.0–3.9, respectively. The fractionated brushes were characterized by gel permeation chromatography, 1H‐NMR spectrometry, thermogravimetric analysis (TGA), and differential scanning calorimetry techniques. PHB‐g‐PMMA brush type graft copolymers showed narrower molecular weight distribution (mostly in range between 1.3 and 2.2) than the PHB‐Cl macroinitiator (1.6–3.5). PHB contents in the brushes were calculated from their TGA thermograms and found to be in range between 22 and 42 mol %. The morphologies of PHB‐g‐PMMA brushes were also studied by scanning electron microscopy. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

Optimizing conditions for poly(β-hydroxybutyrate) production by Halomonas boliviensis LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(beta-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l(-1) CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l(-1) and 14 g l(-1), respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l(-1) in 17 h but reduced the PHB content in the cells to 52 wt.%.

Molecular dynamics of amorphous/crystalline polymer blends studied by broadband dielectric spectroscopy

The molecular dynamics of poly(vinyl acetate), PVAc, and poly(hydroxy butyrate), PHB, as an amorphous/crystalline polymer blend has been investigated using broadband dielectric spectroscopy over wide ranges of frequency (10 −2 to 10 5 Hz), temperature, and blend composition. Two dielectric relaxation processes were detected for pure PHB at high and low frequency ranges at a given constant temperature above the T g. These two relaxation peaks are related to the α and α′ of the amorphous and rigid amorphous regions in the sample, respectively. The α′-relaxation process was found to be temperature and composition dependent and related to the constrained amorphous region located between adjacent lamellae inside the lamellar stacks. In addition, the α′-relaxation process behaves as a typical glass relaxation process, i.e., originated from the micro-Brownian cooperative reorientation of highly constraints polymeric segments. The α-relaxation process is related to the amorphous regions located between the lamellar crystals stacks. In the PHB/PVAc blends, only one α-relaxation process has been observed for all measured blends located in the temperature ranges between the T g’s of the pure components. This last finding suggested that the relaxation processes of the two components are coupled together due to the small difference in the T g’s (Δ T g = 35 °C) and the favorable thermodynamics interaction between the two polymer components and consequently less dynamic heterogeneity in the blends. The T g’s of the blends measured by DSC were followed a linear behavior with composition indicating that the two components are miscible over the entire range of composition. The α′-relaxation process was also observed in the blends of rich PHB content up to 30 wt% PHB. The molecular dynamics of α and α′-relaxation processes were found to be greatly influenced by blending, i.e., the dielectric strength, the peak broadness, and the dielectric loss peak maximum were found to be composition dependent. The dielectric measurements also confirmed the slowing down of the crystallization process of PHB in the blends.

Biotechnological conversion of agro-industrial wastewaters into biodegradable plastic, poly β-hydroxybutyrate

Waste activated sludge generated from a combined dairy and food processing industry wastewater treatment plant was evaluated for its potential to produce biodegradable plastic, poly β-hydroxybutyric acid (PHB). Deproteinized jowar grain-based distillery spentwash yielded 42.3% PHB production (w/w), followed by filtered rice grain-based distillery spentwash (40% PHB) when used as substrates. Addition of di-ammonium hydrogen phosphate (DAHP) resulted in an increase in PHB production to 67% when raw rice grain-based spentwash was used. Same wastewater, after removal of suspended solids by filtration and with DAHP supplementation resulted in lower PHB production (57.9%). However, supplementing other wastes with DAHP led to a substantial decrease in PHB content in comparison to what was observed in the absence of DAHP.

New Biodegradable Thermogelling Copolymers Having Very Low Gelation Concentrations

New biodegradable multiblock amphiphilic and thermosensitive poly(ether ester urethane)s consisting of poly[(R)-3-hydroxybutyrate] (PHB), poly(ethylene glycol) (PEG), and poly(propylene glycol) (PPG) blocks were synthesized, and their aqueous solutions were found to undergo a reversible sol-gel transition upon temperature change at very low copolymer concentrations. The multiblock poly(ether ester urethane)s were synthesized from diols of PHB, PEG, and PPG using 1,6-hexamethylene diisocyanate as a coupling reagent. The chemical structures and molecular characteristics of the copolymers were studied by GPC, 1H NMR, 13C NMR, and FTIR. The thermal stability of the poly(PEG/PPG/PHB urethane)s was studied by thermogravimetry analysis (TGA), and the PHB contents were calculated based on the thermal degradation profile. The results were in good agreement with those obtained from the 1H NMR measurements. The poly(PEG/PPG/ PHB urethane)s presented better thermal stability than the PHB precursors. The water soluble poly(ether ester urethane)s had very low critical micellization concentration (CMC). Aqueous solutions of the new poly(ether ester urethane)s underwent a sol-gel-sol transition as the temperature increased from 4 to 80 degrees C, and showed a very low critical gelation concentration (CGC) ranging from 2 to 5 wt %. As a result of its multiblock architecture, a novel associated micelle packing model can be proposed for the sol-gel transition for the copolymer gels of this system. The new material is thought to be a promising candidate for injectable drug systems that can be formulated at low temperatures and forms a gel depot in situ upon subcutaneous injection.

Modulation of talA gene in pentose phosphate pathway for overproduction of poly-β-hydroxybutyrate in transformant Escherichia coli harboring phbCAB operon

The talA gene encoding transaldolase, the key enzyme in the nonoxidative pentose phosphate pathway, was amplified in a transformant Escherichia coli harboring the phbCAB operon to shift the metabolic flux of the hexose mono-phosphate shunt to the odd-ball biosynthesis pathway for poly-beta-hydroxybutyrate overproduction. The PHB content in the transformant E. coli coharboring the phbCAB operon and talA gene increased from 28.2% to 52.3%, and the retarded cell growth was overcome. This increase seems to be mainly due to the concomitant supplies of the intermediates NADPH and acetyl-CoA, which are from the activated pentose phosphate pathway through the modulation of the talA gene and from the Embden-Meyerhof pathway.

Compositional study and cytotoxicity of biodegradable poly(ester urethane)s consisting of poly[( R)-3-hydroxybutyrate] and poly(ethylene glycol)

Biodegradable PHB–PEG multi-block polyurethane copolymers comprising PHB blocks ( M n: 1100, 1740 and 3240) and PEG blocks ( M n: 1960, 3250, 4150 or 7950) were synthesized followed by characterization by GPC, 1H NMR, and FT-IR. The PHB contents ranged from 9 to 62% by weight. The copolymers displayed improved thermal stabilities compared with their respective precursors. The morphological structures of the copolymers were studied by FT-IR, DSC and XRD. FT-IR revealed the existence of amorphous and crystalline phases of PHB. Both DSC and XRD analyses showed that separate crystalline phases are formed by PEG and PHB blocks in the copolymers. Upon annealing, the melting transition temperature ( T m), melting enthalpy (Δ H m) and the fractional crystallinity ( X c) of the PEG block increased when the length of PEG incorporated into the copolymer increased. These values were higher when the PHB block length is shorter as the shorter PHB chain does not disrupt the crystallization of PEG as much as the longer PHB chain. A similar disruptive effect on the crystallization of PHB segments was observed by varying PEG chain lengths but the effect is less pronounced compared with the PEG segments. A comparison of the swelling properties of the poly(ester urethane)s showed that the length and crystalline properties of the PHB block significantly affects the water uptake properties of the copolymers. The crystalline properties and the water uptake capacities of the copolymers could be fine-tuned by consideration of the length of the PHB and PEG block incorporated. The results of the cytotoxicity tests demonstrated that the poly(PHB/PEG) urethanes were non-cytotoxic and could potentially be used for biomedical purposes.

Effect of Overexpression of a Soluble Pyridine Nucleotide Transhydrogenase (UdhA) on the Production of Poly(3-hydroxybutyrate) in Escherichia coli

A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phb operon from Alcaligenes eutrophus H16 and the native udhA from E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.

Optimization of nutrient feed concentration and addition time for production of poly(β-hydroxybutyrate)

PHB is an intracellular storage compound produced by many organisms under conditions of nutrient limitation (N, P, etc.) when carbon source is present in excess. Model-based fed-batch cultivation is one of the techniques to induce desired nutrient limitations intelligently for over production of such partially growth-associated polymeric products. In the present study, batch kinetics of PHB synthesis by Wautersia eutropha was established in a bioreactor. Batch kinetics featured a biomass concentration of 19.68 g/l with a PHB content of 10.89 g/l in 60 h. A mathematical model was then developed, which adequately described the batch kinetics. The model equations were extrapolated to fed-batch cultivation by incorporating the dilution terms due to fresh nutrient (carbon and nitrogen) feeding. Model-based fed-batch fermentation was conducted by feeding nitrogen (7 g/l) from 20–30 h at the rate of 70 ml/h and fructose (300 g/l) from 24–34 h at the rate of 80 ml/h constantly which yielded 32 g/l biomass containing 14 g/l PHB in 50 h. The productivity was 1.5-times greater than that obtained in batch increasing from 0.18 to 0.28 g/l-h. The model proved to be highly instrumental in the design of suitable fed-batch cultivation.

Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA mutants in microaerobiosis.

We assessed the effects of different arcA mutations on poly(3-hydroxybutyrate) (PHB) synthesis in recombinant Escherichia coli strains carrying the pha synthesis genes from Azotobacter sp. strain FA8. The arcA mutations used were an internal deletion and the arcA2 allele, a leaky mutation for some of the characteristics of the Arc phenotype which confers high respiratory capacity. PHB synthesis was not detected in the wild-type strain in shaken flask cultures under low-oxygen conditions, while ArcA mutants gave rise to polymer accumulation of up to 24% of their cell dry weight. When grown under microaerobic conditions in a bioreactor, the arcA deletion mutant reached a PHB content of 27% +/- 2%. Under the same conditions, higher biomass and PHB concentrations were observed for the strain bearing the arcA2 allele, resulting in a PHB content of 35% +/- 3%. This strain grew in a simple medium at a specific growth rate of 0.69 +/- 0.07 h(-1), whereas the deletion mutant needed several nutritional additives and showed a specific growth rate of 0.56 +/- 0.06 h(-1). The results presented here suggest that arcA mutations could play a role in heterologous PHB synthesis in microaerobiosis.

Synthesis of polyblends from polypropylene and poly(R,S)‐β‐hydroxybutyrate, and their characterization

AbstractBlends of modified polypropylene (PP) with poly(R,S)‐β‐hydroxybutyrate (PHB) were prepared by casting polymer solutions, followed by compression molding into thin films. The modified polypropylene was obtained by oxidation with hydrogen peroxide. Oxidation of polypropylene produced new functional groups such as carbonyl and hydroxyl groups on the polymer chain, and a decrease in molecular weight and crystallinity of the polymers. Maximum crystallinity and mechanical properties of the polyblends were found with a PP/PHB ratio of 90/10 (w/w), and then decreased with increasing PHB content in the polyblends. Biodegradability of the polyblends was lower than that of bacterial and synthetic PHBs. Furthermore, an increase of PHB proportion in the polyblends resulted in highly non‐compatible polyblends. Hence only PHB and small parts of the polyblends were decomposed by microorganisms. Copyright © 2006 Society of Chemical Industry

Preparation and properties of biodegradable thermoplastic starch/poly(hydroxy butyrate) blends

AbstractThe vital differences using three types of thermoplastic starches (TPS), including potato starch, corn starch, and soluble potato starch, with two different gelatinization degrees to blend with poly(hydroxy butyrate) (PHB) are thoroughly discussed in this study. For blends containing a certain amount of PHB, thermal stability remains in a certain degree. In all cases of this study, mechanical properties of TPS blended with PHB confer higher performance than those of pristine TPS. In particular, a significant increase on tensile strength and tear strength is observed for TPS (potato starch) blended with PHB at low gelatinization degree. A suitable degree of gelatinization of starch is critical to achieve optimum performance. The investigation on the morphological observation partly features the supporting evidence of the above findings. The assessment of biodegradability indicates that the values of water absorption and weight loss increase with increasing treatment period and glycerol content, but decrease with increasing amount of PHB content. Among three types of starches investigated, the TPS (soluble starch)/PHB blend gives the highest level of water absorption and weight loss. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 2371–2379, 2006

Hydrophilicity Impact upon Physical Properties of the Environmentally Friendly Poly(3‐hydroxybutyrate) Blends: Modification Via Blending

AbstractWe have integrated scientific research of polymer blends on the base of poly‐3‐hydroxybutyrate (PHB and its copolymers) with bench testing in blend preparation by both solvent casting and melt extrusion. As a second component, we have used traditional synthetic macromolecules with various hydrophilicity degree and hence with different morphologies and physical behavior. Besides, variation of polymer hydrophilicity permits to control both the service characteristic and the rate of (bio)degradation operating in the presence of water. Therefore, a substantial part of our work is devoted to water transport in the parent PHB and its blends. Combining the morphology knowledge (SEM, WAXS, FTIR tecynique), transport characteristics (permeability cells and McBain spring microbalance), and mechanical testing, we propose that blending of the parent biodegradable polymer with synthetic macromolecules is a perspective tool to design novel materials with improved characteristics. Both the water transport coefficients and the mechanical characteristics are essentially sensitive to structure and morphology of the blends. Hydrophilicity variation in the order LDPE < SPEU < PVA at blending with PHB shows that the morphology transformation in immicsible or partly miscible blends shifted along the PHB concentration scale as LDPE (at ∼16 wt% PHB) < PVA (∼30 wt% PHB) < SPEU (∼50 wt% PHB) Our instrumental monitoring the structural hierarchy of parent polymers and their blends as well as , simultaneously, the study of transport processes, their modeling, and computer simulating open up the way to understanding the precepts of polymer operation in corrosive and bioactive media.

Staining and quantification of poly‐3‐hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry

Poly [(R)-3-hydroxybutyric acid] (PHB) is a prokaryote storage material for carbon and energy that accumulates in cells under unbalanced growth conditions. Because this class of biopolymers has plastic-like properties, it has attracted considerable interest for biomedical applications and as a biodegradable commodity plastic. Current flow cytometric techniques to quantify intracellular PHB are based on Nile red. Here, an improved cytometric technique for cellular PHB quantification utilizing BODIPY 493/503 staining was developed. This technique was then automated using an automated flow cytometry system. Using flow cytometry, the fluorescence of Saccharomyces cerevisiae and Cupriavidus necator with varying PHB content after staining with BODIPY 493/503 and Nile red was compared, and automated staining techniques were developed for both cultures. BODIPY 493/503 staining had less background staining, higher sensitivity and specificity to PHB, and higher saturation values than did Nile red staining. The developed automated staining procedure was capable of analyzing the PHB content of a bioreactor sample every 25 min and measured the average PHB content with accuracy comparable to offline GC analysis. BODIPY 493/503 produced an overall better staining for PHB than did Nile red. When combined with the automated system, this technique provides a new method for the online monitoring and control of bioreactors.

Crystallization behavior and biodegradation of poly(3-hydroxybutyrate) and poly(ethylene glycol) multiblock copolymers

Block copolymerization by using isocyanates is an effective method for incorporating PHB and PEG because it can prepare copolymers with good properties, such as toughness, strength, and so on. In this study, we adopted soil suspension system to estimate the biodegradability of a series of PHB/PEG multiblock copolymers with different compositions and block lengths. In the degradation process, the changes in weight loss, molecular weight, and tensile strength were periodically measured to determine the biodegradability, and the surface morphology was also observed by SEM. In contrast to pure PHB, the weight loss of the copolymer was relatively lower. On the other hand, the tensile strength and molecular weight experienced apparent decrease, and for BHG1000-3-1, they reached 46.7% and 77.7% of the initial value, respectively. SEM observation showed that the surface was covered with numerous erosion pits. All these indicate that the degradation indeed took place and long-chain molecules have been hydrolyzed into shorter ones. The crystallization behavior was also investigated by DSC and WAXD. The results showed that both the segments, PEG and PHB, can form crystalline phases at lower PHB contents ranging from 29% to 44%, and when PHB component was more than 60%, only PHB phase can crystallize.

Long-term aeration management for improved N-removal via SND in a sequencing batch reactor

Management of the aeration length in a sequencing batch reactor (SBR) can improve N-removal by minimising the amount of organic substrate that is oxidised aerobically. This study investigates the long-term effect of aeration control on N-removal via simultaneous nitrification and denitrification (SND) by a mixed culture in a 2 L acetate-fed SBR, using PHB as the electron donor for denitrification. The reactor was operated continuously with automated termination of the aerobic phase after ammonium depletion, using the specific oxygen uptake rate (SOUR) as the control parameter. This resulted in an increase of the organic loading rate (OLR) from 0.33 to 0.59 g BOD g −1 d −1. Over the first 12 cycles of operation, the PHB content of the biomass increased three-fold and resulted in a progressively increasing SOUR, which allowed an increased amount of nitrogen removal via SND from 34% to 52%. After one month of continuous operation with controlled aeration, the settling efficiency of the biomass had significantly improved (SVI 70 mL g −1 X). Long-term oxygen management resulted in biomass with a higher capacity for N-removal via SND and improved settling characteristics. Our results may help to explain long-term historical effects of N-removal capabilities in WWTPs and assist design engineers in choosing an appropriate aeration length and OLR.