Producing poly-3-hydroxybutyrate with a high molecular mass from methane

Poly-3-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could provide a suitable substrate for the production of PHB. Methane is cheap and plentiful - not only as natural gas, but also as biogas. The methanotrophic strain Methylocystis sp. GB 25 DSMZ 7674 is able to accumulate PHB in a brief non-sterile process. The studies were carried out using a 7-l and a 70-l pressure bioreactor. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (ammonium, phosphorus or magnesium) in batch culture. The PHB content of biomass was as high as 51%; efficiency was highest during the first 5 h of the product formation process. The maximum PHB yield relative to the methane consumed was estimated to be 0.55 g g(-1). The PHB produced is of very high quality, having a high molecular mass of up to 2.5x10(6) Da.     

A metabolic model of the biological phosphorus removal process: I. Effect of the sludge retention time

The biological phosphorus removal process is a process which depends basically on three internal storage compounds. Poly-beta-hydroxybutyrate (PHB) produced during the anaerobic phase is used as substrate for biomass, polyphosphate, and glycogen formation. The reaction rates of the aerobic processes are primarily determined by the PHB content of the cells. This PHB content is highly dynamic due to the conversions during the anaerobic and aerobic phase of the cycle and the ratio between substrate addition and biomass present in the reactor. The amount of biomass present in the reactor is determined by the sludge retention time and growth rate. A metabolic model of the biological phosphorus removal process was developed and verified over a wide range of growth rates. The effect of different growth rates on the internal fractions of stored components was determined and described mathematically. One set of kinetic parameters was capable of describing the measured conversions of all components observed in the reactor as a function of the sludge retention time. (c) 1995 John Wiley & Sons, Inc.     

Poly-beta-hydroxybutyrate production by Pseudomonas sp. RZS 1 under aerobic and semi-aerobic condition

Pseudomonas sp. RZS1 was isolated from distillery effluent and identified based on phenotypic characters and 16s rRNA sequencing. It accumulated optimum amount (703.79 microg/mg of biomass) of poly-beta-hydroxybutyrate (PHB) under aerobic process of fermentation and 75 microg/mg of biomass under the anaerobic process of fermentation. Aerobic fermentation yielded 9.3-fold more PHB than semi-aerobic fermentation. Acetone alcohol method proved to be the best suitable recovery method as it gave 703.79 microg PHB per mg of biomass with a percentage recovery yield of 70.37. It started to accumulate PHB at the end of lag phase (from 6 h of incubation). Optimum amount of PHB (20 microg/ml) was reported during early stationary phase (30 h of incubation). Extracted PHB showed two peaks, minor one at 248 nm and major one at 365 nm. IR spectra revealed the presence of functional groups characteristics of PHB.     

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.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

Microbial production of polyhydroxybutyrate with tailor-made properties: an integrated modelling approach and experimental validation

The microbial production of polyhydroxybutyrate (PHB) is a complex process in which the final quantity and quality of the PHB depend on a large number of process operating variables. Consequently, the design and optimal dynamic operation of a microbial process for the efficient production of PHB with tailor-made molecular properties is an extremely interesting problem. The present study investigates how key process operating variables (i.e., nutritional and aeration conditions) affect the biomass production rate and the PHB accumulation in the cells and its associated molecular weight distribution. A combined metabolic/polymerization/macroscopic modelling approach, relating the process performance and product quality with the process variables, was developed and validated using an extensive series of experiments and measurements. The model predicts the dynamic evolution of the biomass growth, the polymer accumulation, the consumption of carbon and nitrogen sources and the average molecular weights of the PHB in a bioreactor, under batch and fed-batch operating conditions. The proposed integrated model was used for the model-based optimization of the production of PHB with tailor-made molecular properties in Azohydromonas lata bacteria. The process optimization led to a high intracellular PHB accumulation (up to 95% g of PHB per g of DCW) and the production of different grades (i.e., different molecular weight distributions) of PHB.     

Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

Utilization of oil extracted from spent coffee grounds for sustainable production of polyhydroxyalkanoates

Spent coffee grounds (SCG), an important waste product of the coffee industry, contain approximately 15 wt% of coffee oil. The aim of this work was to investigate the utilization of oil extracted from SCG as a substrate for the production of poly(3-hydroxybutyrate) (PHB) by Cupriavidus necator H16. When compared to other waste/inexpensive oils, the utilization of coffee oil resulted in the highest biomass as well as PHB yields. Since the correlation of PHB yields and the acid value of oil indicated a positive effect of the presence of free fatty acids in oil on PHB production (correlation coefficient R ²?=?0.9058), superior properties of coffee oil can be probably attributed to the high content of free fatty acids which can be simply utilized by the bacteria culture. Employing the fed-batch mode of cultivation, the PHB yields, the PHB content in biomass, the volumetric productivity, and the Y _P/S yield coefficient reached 49.4 g/l, 89.1 wt%, 1.33 g/(l h), and 0.82 g per g of oil, respectively. SCG are annually produced worldwide in extensive amounts and are disposed as solid waste. Hence, the utilization of coffee oil extracted from SCG is likely to improve significantly the economic aspects of PHB production. Moreover, since oil extraction decreased the calorific value of SCG by only about 9 % (from 19.61 to 17.86 MJ/kg), residual SCG after oil extraction can be used as fuel to at least partially cover heat and energy demands of fermentation, which should even improve the economic feasibility of the 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.     

The effect of dissolved oxygen on PHB accumulation in activated sludge cultures

Nitrogen removal from wastewater is often limited by the availability of reducing power to perform denitrification, especially when treating wastewaters with a low carbon:nitrogen ratio. In the increasingly popular sequencing batch reactor (SBR), bacteria have the opportunity to preserve reducing power from incoming chemical oxygen demand (COD) as poly-beta-hydroxybutyrate (PHB). The current study uses laboratory experiments and mathematical modeling in an attempt to generate a better understanding of the effect of oxygen on microbial conversion of COD into PHB. Results from a laboratory SBR with acetate as the organic carbon source showed that the aerobic acetate uptake process was oxygen-dependent, producing higher uptake rates at higher dissolved oxygen (DO) supply rates. However, at the lower DO supply rates (k(L)a 6 to 16 h(-1), 0 mg L(-1) DO), a higher proportion of the substrate was preserved as PHB than at higher DO supply rates (k(L)a 30, 51 h(-1), DO >0.9 mg L(-1)). Up to 77% of the reducing equivalents available from acetate were converted to PHB under oxygen limitation (Y(PHB/Ac) 0.68 Cmol/Cmol), as opposed to only 54% under oxygen-excess conditions (Y(PHB/Ac) 0.48 Cmol/Cmol), where a higher fraction of acetate was used for biomass growth. It was calculated that, by oxygen management during the feast phase, the amount of PHB preserved (1.4 Cmmol L(-1) PHB) accounted for an additional denitrification potential of up to 18 mg L(-1) nitrate-nitrogen. The trends of the effect of oxygen (and hence ATP availability) on PHB accumulation could be reproduced by the simulation model, which was based on biochemical stoichiometry and maximum rates obtained from experiments. Simulated data showed that, at low DO concentrations, the limited availability of adenosine triphosphate (ATP) prevented significant biomass growth and most ATP was used for acetate transport into the cell. In contrast, high DO supply rates provided surplus ATP and hence higher growth rates, resulting in decreased PHB yields. The results suggest that oxygen management is crucial to conserving reducing power during the feast phase of SBR operation, as excessive aeration rates decrease the PHB yield and allow higher biomass growth.     

Microbial Conversion of Methane into poly-β-hydroxybutyrate (PHB): Growth and intracellular product accumulation in a type II methanotroph

The biochemical pathways of methane to poly-β-hydroxybutyrate (PHB) in type II methanotrophs have been analyzed and used to propose stoichiometric equations for cell biomass and PHB. Conditions necessary for PHB accumulation in a batch culture of Methylocystis parvus OBBP were studied. In nitrogen limited cultures PHB started accumulating in the declining growth phase and maximum rate of PHB formation occurred during the late growth and early stationary phases. Inoculum age had an effect on maximum level of intracellular PHB, which increased with inoculum age up to ca. 70% of the cell dry weight using a 70 h inoculum. It has been shown that oxygen and methane requirements are high and pose a great stress on the mass transfer in the system; this limits the obtainment of high cell concentrations. When conditions for increased mass transfer of both CH₄ and O₂ were used, 5 g/l of cells could be produced. Finally an enrichment strategy was investigated so that cells would start accumulating PHB earlier during the growth phase. Alternatives to increase cell mass concentrations are discussed.     

Effect of phosphate supply and aeration on poly-β-hydroxybutyrate production in Azotobacter chroococcum

The effects of phosphate supply and aeration on cell growth and PHB accumulation were investigated in Azotobacter chroococcum 23 with the aim of increasing PHB production. Phosphate limitation favoured PHB formation in Azotobacter chroococcum 23, but inhibited growth. Azotobacter chroococcum 23 cells demonstrated intensive uptake of orthophosphate during exponential growth. At the highest phosphate concentration (1·5 g/litre) and low aeration the amount of intracellular orthophosphate/g residual biomass was highest. Under conditions of fed-batch fermentation the possibility of controlling the PHB production process by the phosphate level in the cultivation medium was demonstrated. A 36 h fed-batch fermentation resulted in a biomass yield of 110 g/litre with a PHB cellular concentration of 75% dry weight, PHB content 82·5 g/litre, PHB yield Y_P/S = 0·24 g/g and process productivity 2·29 g/litre·h.     

Assay of Poly(3-Hydroxybutyrate) Depolymerase Activity and Product Determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.     

Assay of poly(3-hydroxybutyrate) depolymerase activity and product determination

Two methods for accurate poly(3-hydroxybutyrate) (PHB) depolymerase activity determination and quantitative and qualitative hydrolysis product determination are described. The first method is based on online determination of NaOH consumption rates necessary to neutralize 3-hydroxybutyric acid (3HB) and/or 3HB oligomers produced during the hydrolysis reaction and requires a pH-stat apparatus equipped with a software-controlled microliter pump for rapid and accurate titration. The method is universally suitable for hydrolysis of any type of polyhydroxyalkanoate or other molecules with hydrolyzable ester bonds, allows the determination of hydrolysis rates of as low as 1 nmol/min, and has a dynamic capacity of at least 6 orders of magnitude. By applying this method, specific hydrolysis rates of native PHB granules isolated from Ralstonia eutropha H16 were determined for the first time. The second method was developed for hydrolysis product identification and is based on the derivatization of 3HB oligomers into bromophenacyl derivates and separation by high-performance liquid chromatography. The method allows the separation and quantification of 3HB and 3HB oligomers up to the octamer. The two methods were applied to investigate the hydrolysis of different types of PHB by selected PHB depolymerases.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Application of self-cycling fermentation technique to the production of poly-beta-hydroxybutyrate

The production of poly-beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus DSM 545 in a cyclone bioreactor was compared using various culture methods: batch, fed-batch, and self-cycling fermentation (SCF) with and without extended periods of nutrient deprivation. SCF is a semi-continuous method that results in a nutrient limitation for every successive generation of cells and, therefore, may have advantages for products whose formation follow secondary metabolite kinetics. Use of the SCF technique without extended nutrient deprivation produced a PHB concentration of 1.2 g L(-1) as 40% of the biomass dry weight. With nitrogen deprivation for 4 or 6 h, the concentration of PHB decreased when compared to the standard SCF technique. However, nitrogen deprivation periods of 8 h resulted in an increase in PHB concentration to 2.7 g L(-1) or 59% of the biomass dry weight. The nutrient cycling may act to repress PHB accumulation during periods of nitrogen deprivation, unless a time threshold has been reached, after which PHB accumulation occurs as in normal batch culture. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 815-820, 1997.     

开放条件下高聚β-羟基丁酸酯含量的甲烷氧化混合菌群的富集

目的:在开放条件下以甲烷为底物富集高聚β-羟基丁酸酯(PHB)含量的甲烷氧化菌群,获得能够利用低成本碳源高产PHB的菌种。方法:采用丰盛-饥饿模式间歇供料,以甲烷为底物,好氧开放式培养甲烷氧化混合菌群,利用苏丹黑染色法动态检测丰盛和饥饿阶段胞内PHB含量的变化,以此为基础考察丰盛-饥饿期比例对富集高PHB含量的甲烷氧化菌群的影响。结果:丰盛-饥饿期比例为1∶3时,微生物PHB含量从17.7%增加到35.5%,且开放培养过程中菌群结构稳定。结论:通过丰盛-饥饿模式间歇供料开放式培养所得的高PHB含量的稳定的甲烷氧化菌群,具有工业生产PHB的应用价值。     

Formal kinetics of poly-β-hydroxybutyric acid (PHB) production in Alcaligenes eutrophus H 16 and Mycoplana rubra R 14 with respect to the dissolved oxygen tension in ammonium-limited batch cultures

Summary Under chemolithoautotrophic growth conditions with the organism Alcaligenes eutrophus H16 the exponential growth phase is characterized by two different growth rates, each associated with different specific rates of ammonium consumption. On the basis of the analytical determination of Poly--hydroxybutyric acid (PHB), it can be conclusively shown that PHB is synthesized even during the exponential growth phase at a specific rate proportional to the specific growth rates of total biomass. After complete consumption of ammonium, the increase of biomass is exclusively due to PHB synthesis, whereas protein and rest biomass (cell dry weight minus PHB) remain constant. After an extended period of fermentation, the PHB content reaches a saturation value. The transient phase between the growth and the storage phase is very short in comparison to the duration of the whole fermentation. In the case of Alcaligenes eutrophus, strain H 16, high concentrations of dissolved oxygen strongly influence growth as well as PHB synthesis.Abbrevations ^cO₂,L concentration of oxygen in the liquid phase (dissolved oxygen tension: d.o.t) - ^cH₂,L concentration of hydrogen in the liquid phase - ^cCO₂,L concentration of carbon dioxide in the liquid phase - S limiting substrate, concentration of - X total biomass, concentration of; total cell dry weight - P product; PHB, concentration of - R rest biomass: X-P, concentration of - ^rX dX/dt growth rate - ^rP dP/dt rate of PHB synthesis - ^rR dR/dt rate of rest biomass production - ^r0 ^dcO₂,L/dt rate of oxygen consumption - X dX/dt·1/X=r_X·1/X specific growth rate - P dP/dt·1/P=r_P·1/P specific rate of product formation - R dR/dt·1/R=r_R·1/R specific rate of rest biomass formation - r₀/R specific respiration rate     

Production of poly(3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli.

Recombinant Escherichia coli XL1-Blue harboring a high-copy-number plasmid containing the Alcaligenes eutrophus polyhydroxyalkanoate synthesis genes could efficiently synthesize poly(3-hydroxybutyrate) (PHB) in a complex medium containing yeast extract and tryptone but not in a defined medium. One of the reasons for the reduced PHB production in a defined medium was thought to be severe filamentation of cells in this medium. By overexpressing an essential cell division protein, FtsZ, in recombinant E. coli producing PHB, filamentation could be suppressed and PHB could be efficiently produced in a defined medium. A high PHB concentration of 149 g/liter, with high productivity of 3.4 g of PHB/liter/h, could be obtained by the pH-stat fed-batch culture of the filamentation-suppressed recombinant E. coli in a defined medium. It was also found that insufficient oxygen supply at a dissolved oxygen concentration (DOC) of 1 to 3% of air saturation during active PHB synthesis phase did not negatively affect PHB production. By growing cells to the concentration of 110 g/liter and then controlling the DOC in the range of 1 to 3% of air saturation, a PHB concentration of 157 g/liter and PHB productivity of 3.2 g of PHB/liter/h were obtained. For the scale-up studies, fed-batch culture was carried out in a 50-liter stirred tank fermentor, in which the DOC decreased to zero when cell concentration reached 50 g/liter. However, a relatively high PHB concentration of 101 g/liter and PHB productivity of 2.8 g of PHB/liter/h could still be obtained, which demonstrated the possibility of industrial production of PHB in a defined medium by employing the filamentation-suppressed recombinant E. coli.     

Study on the flocs poly-β-hydroxybutyrate production and process optimization in the bio-flocs technology system

The bio-flocs technology (BFT) was applied in the sequencing batch reactor (SBR) to treat aquaculture wastewater for flocs poly-β-hydroxybutyrate (PHB) accumulation with alternant anaerobic and aerobic conditions. The statistical modeling approach was used to evaluate system performance and to optimize the flocs PHB yield at batch mode. The results show that all variables have significant impact on the response objective, as well as the interactions of the C/N ratio with the flocs biomass concentration (VSS) and anaerobic time, respectively. By process optimization, approximately 150-200 PHB/VSS (mg·g) of flocs PHB yield was achieved in the range of 4-7 g/l of flocs biomass concentration, 15-18 of the C/N ratio and 50-85 min of anaerobic time in the BFT systems. The results demonstrated that a suitable flocs PHB yield can be obtained via optimizing the ex-situ operating strategy, which have potential prebiotic value and practical implication for the sustainable aquaculture.