Specific Substrate Uptake Rate Research Articles

Development of a defined medium for the heterotrophic cultivation of Metallosphaera sedula

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

KLa based scale-up cultivation of the extremophilic archaeon Sulfolobus acidocaldarius: from benchtop to pilot scale.

The two major scale-up criteria in continuously stirred bioreactors are 1) constant aerated power input per volume (Pg/Vl), and 2) the volumetric O2 mass transfer coefficient (kla). However, Pg/Vl is only influenced by the stirrer geometry, stirrer speed, aeration and working volume, while the kla is additionally affected by physiochemical properties of the medium (temperature, pH, salt content, etc.), sparging of gas and also by the bioreactor design. The extremophilic archaeon Sulfolobus acidocaldarius, thriving at 75°C and pH 3.0, has the potential for many biotechnological applications. However, previous studies imply that the family Sulfolobaceae might be affected by higher oxygen concentration in the headspace (>26%). Hence, adequate oxygen supply without being toxic has to be ensured throughout the different scales. In this study, the scale-up criteria Pg/Vl and kla were analyzed and compared in a 2L chemostat cultivation of S. acidocaldarius on a defined growth medium at 75°C and a pH value of 3.0, using two different types of spargers at the same aerated power input. The scale-up criterion kLa, ensuring a high specific growth rate as well as viability, was then used for scaleup to 20L and 200L. By maintaining a constant kla comparable dry cell weight, specific growth rate, specific substrate uptake rates and viability were observed between all investigated scales. This procedure harbors the potential for further scale-up to industrial size bioreactors.

Dynamic Feeding for Pichia pastoris.

The knowledge of certain strain-specific parameters of recombinant Pichia pastoris strains is required to be able to set up a feeding regime for fed-batch cultivations. These parameters are commonly determined either by time-consuming and labor-intensive continuous cultivations or by several, consecutive fed-batch cultivations. Here, we describe a fast method based on batch experiments with substrate pulses to extract certain strain characteristic parameters, which are required to set up a dynamic feeding strategy for P. pastoris strains based on the specific substrate uptake rate. We further describe in detail the course of actions, which have to be taken to obtain the desired dynamics during feeding.

Bioconversion study for xylitol and ethanol production by Debaryomyces hansenii: aeration, medium and substrate composition influence

Debaryomyces hansenii has been employed to study, initially, the influence of the oxygen availability on D-xylose to xylitol fermentation, as this parameter is considered as one of the most critical variables for this bio alcohol accumulation. Apart from the air supplied in the fermentation process through the stirring vortex (0.0 v/v/min), additional aeration rates (0.1–2.0 v/v/min) effects were discussed. Furthermore, a change in the fermentative medium composition as well as a comparative analysis of D. hansenii behavior with respect to fermentation of D-glucose and D-xylose mixtures solutions, with the aim of producing both xylitol and ethanol bioproducts, were performed. For these purposes, specific growth rates, biomass productivities, specific substrate-uptake rates, overall biomass yields, specific xylitol formation rates and overall xylitol yields values have been calculated, applying a differential method to the kinetic data. Aeration influence was clearly evinced since a faster D-xylose metabolism, for aeration values close to 1.0 v/v/min, was noted. This yeast exhibited a sequential substrate consumption, firstly D-glucose and then D-xylose. The maximum xylitol yield (0.32 kg kg– 1) was obtained for 0.5 v/v/min airflow, remarking a significant reduction of this parameter for both above and below the quoted air supply value.

A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h−1 on formate showed high specific substrate uptake rates (47 mmol g−1 h−1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7–16.4 mmol g−1 h−1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20–0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70–92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

Noninvasive online monitoring of Corynebacterium glutamicum fed-batch bioprocesses subject to spent sulfite liquor raw material uncertainty

In this study the use of a particle filter algorithm to monitor Corynebacterium glutamicum fed-batch bioprocesses with uncertain raw material input composition is shown. The designed monitoring system consists of a dynamic model describing biomass growth on spent sulfite liquor. Based on particle filtering, model simulations are aligned with continuously and noninvasively measured carbon evolution and oxygen uptake rates, giving an estimate of the most probable culture state. Applied on two validation experiments, culture states were accurately estimated during batch and fed-batch operations with root mean square errors below 1.1 g L−1 for biomass, 0.6 g L−1 for multiple substrate concentrations and 0.01 g g−1 h−1 for biomass specific substrate uptake rates. Additionally, upon fed-batch start uncertain feedstock concentrations were corrected by the estimator without the need of any additional measurements. This provides a solid basis towards a more robust operation of bioprocesses utilizing lignocellulosic side streams.

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy.

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

Optimal process design space to ensure maximum viability and productivity in Penicillium chrysogenum pellets during fed-batch cultivations through morphological and physiological control

BackgroundBiomass growth of Pencillium chrysogenum is characterised by a distinct pellet morphology consisting of compact hyphal agglomerates. Fungal pellets are advantageous in industrial process control due to rheological advantages but lead to biomass degradation due to diffusional limitations of oxygen and substrate in the pellet’s core. Several fermentation parameters are known to affect key pellet characteristics regarding morphology, viability and productivity. Pellet morphology and size are affected by agitation. Biomass viability and productivity are tightly interlinked with substrate uptake and dissolved oxygen concentration.ResultsThe goal of this study was to study the impact of the fermentation parameters power input, dissolved oxygen content and specific substrate uptake rate on morphology, biomass viability and productivity. A design of experiments (DoE) approach was conducted and corresponding responses were analysed using novel morphological descriptors analysed by a previously established flow cytometry method. Results clearly display inverse correlations between power input and pellet size, specific morphological parameters related to pellet density can be increased in direct proportion to power input. Biomass viability and productivity are negatively affected by high specific substrate uptake rates.ConclusionsBased upon multiple linear regression, it was possible to obtain an optimal design space for enhanced viability and productivity at beneficial morphological conditions. We could maintain a high number of pellets with favourable morphology at a power input of 1500 W/m3. A sound compromise between viability and high productivity is possible at a specific glucose uptake rate of 0.043 g/g/h at dissolved oxygen levels of 40% minimum.

Continuous operation, a realistic alternative to fed-batch fermentation for the production of recombinant lipase B from Candida antarctica under the constitutive promoter PGK in Pichia pastoris

In this work, continuous and fed-batch operational modes for producing the recombinant protein lipase B from Candida antarctica under the new constitutive promoter PGK in the cell factory Pichia pastoris was assessed. For this purpose, the influence of the specific growth rate (μ) was examined from 0.05 to 0.16 h−1 on various key bioprocess parameters.The overall biomass-to-substrate yield YX/S and specific substrate uptake rate (qs) were found to be independent of the particular operational mode. However, the μ-dependence of specific production rate (qp) differed with the cultivation strategy used. Thus, qp was linearly related in the continuous mode but suggested saturation in fed-batch cultures.The highest volumetric and specific productivity were obtained at the highest μlevels in the continuous mode, which provided volumetric and specific productivities (QP and Qe, respectively) roughly 1.5 and 3 times greater than the fed-batch mode. The continuous mode also proved more effective in the long rung; thus, CalB production after 6 weeks was estimated to be about 5.8 times greater than with the fed-batch mode.Continuous operation with the novel constitutive promoter PGK is thus a realistic alternative to the standard fed-batch cultivation at a high cell density typically used in industrial bioprocesses for heterologous protein production in Pichia pastoris.

Custom made inclusion bodies: impact of classical process parameters and physiological parameters on inclusion body quality attributes

BackgroundThe bacterium E. coli is a major host for recombinant protein production of non-glycosylated products. Depending on the expression strategy, the recombinant protein can be located intracellularly. In many cases the formation of inclusion bodies (IBs), protein aggregates inside of the cytoplasm of the cell, is favored in order to achieve high productivities and to cope with toxic products. However, subsequent downstream processing, including homogenization of the cells, centrifugation or solubilization of the IBs, is prone to variable process performance or can be characterized by low extraction yields as published elsewhere. It is hypothesized that variations in IB quality attributes (QA) are responsible for those effects and that such attributes can be controlled by upstream process conditions. This contribution is aimed at analyzing how standard process parameters, such as pH and temperature (T) as well as different controlled levels of physiological parameters, such as specific substrate uptake rates, can vary IB quality attributes.ResultsClassical process parameters like pH and T influence the expression of analyzed IB. The effect on the three QAs titer, size and purity could be successfully revealed. The developed data driven model showed that low temperatures and low pH are favorable for the expression of the two tested industrially relevant proteins. Based on this knowledge, physiological control using specific substrate feeding rate (of glucose) qs,Glu is altered and the impact is tested for one protein.ConclusionsTime dependent monitoring of IB QA—titer, purity, IB bead size—showed a dependence on classical process parameters pH and temperature. These findings are confirmed using a second industrially relevant strain. Optimized process conditions for pH and temperature were used to determine dependence on the physiological parameters, the specific substrate uptake rate (qs,Glu). Higher qs,Glu were shown to have a strong influence on the analyzed IB QAs and drastically increase the titer and purity in early time stages. We therefore present a novel approach to modulate—time dependently—quality attributes in upstream processing to enable robust downstream processing.

Modified Anaerobic Digestion Model No. 1 for modeling methane production from food waste in batch and semi-continuous anaerobic digestions

A modified Anaerobic Digestion Model No. 1 (ADM1) with optimized kinetic parameters was presented to model methane production in the anaerobic digestion of food waste. Experimental data from batch and semi-continuous fermentations were used to calibrate and verify the model. Modified ADM1 simulation was carried out using AQUASIM 2.0 software. Sensitivity analysis was used to identify and evaluate the most sensitive kinetic parameters during biogas production. The decay constant of microorganisms, the disintegration constant, the hydrolysis constant of carbohydrates, the Monod maximum specific substrate uptake rate, and the half-saturation constants affected biogas production significantly. The optimized values of these parameters were 0.001, 0.16, 3, 1 and 0.23, respectively. Optimization results were validated using batch and semi-continuous experiments. The modified ADM1 well-predicted methane production, with R2 values for the validation experiments all above 90%. These results can be used as basic data to simulate methane production in full-scale reactors.

One-Stage vs. Two-Stage Bioprocesses: Comparative Computational and Experimental Studies and Consequences for Strain Design

Biotechnological processes for the production of (high-volume) chemicals are often based on one-stage or two-stage processes (OSPs and TSPs, respectively). Many classical bioprocesses are OSPs in which growth and product synthesis are coupled, however, OSPs often exhibit low volumetric productivities due to inherent trade-offs between biomass and product yields. Here, TSPs, where growth and production are separated, may help to improve the productivity of bioprocesses and with rapidly growing genetic toolboxes for dynamic metabolic control TSPs are becoming more attractive. However, there are only few theoretical and experimental studies systematically comparing OSPs and TSPs in terms of their process metrics (productivity, yield etc.).We have constructed E. coli strains for heterologous production of itaconate for both OSP and TSP. We used computer-aided design to build a mutant strain (ita23) for growth-coupled synthesis of itaconate. In an OSP, this strain reached the highest product yield and titer that have ever been reported for heterologous itaconate production. For increasing the volumetric productivity via a TSP, we constructed a new strain (ita36A) allowing a temperature-dependent switch from fast growth to production. Indeed, a TSP with ita36A increased the peak productivity of ita23 by 91% and the titer by 46%. Despite of this successful implementation of a TSP, we observed that the productivity, after reaching a peak value, decreased quickly in the production phase due to low substrate uptake and product synthesis rates. We therefore used mathematical models to systematically compare productivities of TSPs and OSPs. Our simulations show that the overall volumetric productivity of a TSP can in fact be lower than of a corresponding OSP due to the sharp decrease of the specific substrate uptake rate in (non-growth) TSP production phases. This raises a (often neglected) big challenge for designing competitive TSPs. We propose approaches to improve substrate utilization rates in the production phase by which TSPs really outperform OSPs. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSP vs. TSP.

Impact of Glycerol as Carbon Source onto Specific Sugar and Inducer Uptake Rates and Inclusion Body Productivity in E. coli BL21(DE3).

The Gram-negative bacterium E. coli is the host of choice for a multitude of used recombinant proteins. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures using isopropyl β-d-1 thiogalactopyranoside as inducer are often referred to cause stress reactions, leading to a phenomenon known as “metabolic” or “product burden”. These high expressions of recombinant proteins mainly result in decreased growth rates and cell lysis at elevated induction times. Therefore, approaches tend to use “soft” or “tunable” induction with lactose and reduce the stress level of the production host. The usage of glucose as energy source in combination with lactose as induction reagent causes catabolite repression effects on lactose uptake kinetics and as a consequence reduced product titer. Glycerol—as an alternative carbon source—is already known to have positive impact on product formation when coupled with glucose and lactose in auto-induction systems, and has been referred to show no signs of repression when cultivated with lactose concomitantly. In recent research activities, the impact of different products on the lactose uptake using glucose as carbon source was highlighted, and a mechanistic model for glucose-lactose induction systems showed correlations between specific substrate uptake rate for glucose or glycerol (qs,C) and the maximum specific lactose uptake rate (qs,lac,max). In this study, we investigated the mechanistic of glycerol uptake when using the inducer lactose. We were able to show that a product-producing strain has significantly higher inducer uptake rates when being compared to a non-producer strain. Additionally, it was shown that glycerol has beneficial effects on viability of cells and on productivity of the recombinant protein compared to glucose.

When Do Two-Stage Processes Outperform One-Stage Processes?

Apart from product yield and titer, volumetric productivity is a key performance indicator for many biotechnological processes. Due to the inherent trade-off between the production of biomass as catalyst and of the actual target product, yield and volumetric productivity cannot be optimized simultaneously. Therefore, in combination with genetic techniques for dynamic regulation of metabolic fluxes, two-stage fermentations (TSFs) with separated growth and production phase have recently gained much interest because of their potential to improve the productivity of bioprocesses while still allowing high product yields. However, despite some successful case studies, so far it has not been discussed and analyzed systematically whether or under which conditions a TSF guarantees superior productivity compared to one-stage fermentation (OSF). In this study, we use mathematical models to demonstrate that the volumetric productivity of a TSF is not automatically better than of a corresponding OSF. Our analysis reveals that the sharp decrease of the specific substrate uptake rate usually observed in (non-growth) production phases severely impacts the volumetric productivity and thus raises a big challenge for designing competitive TSF processes. We discuss possible approaches such as enforced ATP wasting to improve substrate utilization rates in the production phase by which TSF processes can become superior to OSF. We also analyze additional factors influencing the relative performance of OSF and TSF and show that OSF processes can be more appropriate if a high product yield is an economic constraint. In conclusion, a careful assessment of the trade-offs between substrate uptake rates, yields, and productivity is necessary when deciding for OSF vs. TSF processes.

Impact of phosphate limitation on PHA production in a feast-famine process

Double-limitation systems have shown to induce polyhydroxyalkanoates (PHA) production in chemostat studies limited in e.g. carbon and phosphate. In this work the impact of double substrate limitation on the enrichment of a PHA producing community was studied in a sequencing batch process. Enrichments at different C/P concentration ratios in the influent were established and the effect on the PHA production capacity and the enrichment community structure was investigated. Experimental results demonstrated that when a double substrate limitation is imposed at a C/P ratio in the influent in a range of 150 (C-mol/mol), the P-content of the biomass and the specific substrate uptake rates decreased. Nonetheless, the PHA storage capacity remained high (with a maximum of 84 wt%). At a C/P ratio of 300, competition in the microbial community is based on phosphate uptake, and the PHA production capacity is lost. Biomass specific substrate uptake rates are a linear function of the cellular P-content, offering advantages for scaling-up the PHA production process due to lower oxygen requirements.

Bioprocess development workflow: Transferable physiological knowledge instead of technological correlations.

Microbial bioprocesses need to be designed to be transferable from lab scale to production scale as well as between setups. Although substantial effort is invested to control technological parameters, usually the only true constant parameter is the actual producer of the product: the cell. Hence, instead of solely controlling technological process parameters, the focus should be increasingly laid on physiological parameters. This contribution aims at illustrating a workflow of data life cycle management with special focus on physiology. Information processing condenses the data into physiological variables, while information mining condenses the variables further into physiological descriptors. This basis facilitates data analysis for a physiological explanation for observed phenomena in productivity. Targeting transferability, we demonstrate this workflow using an industrially relevant Escherichia coli process for recombinant protein production and substantiate the following three points: (1) The postinduction phase is independent in terms of productivity and physiology from the preinduction variables specific growth rate and biomass at induction. (2) The specific substrate uptake rate during induction phase was found to significantly impact the maximum specific product titer. (3) The time point of maximum specific titer can be predicted by an easy accessible physiological variable: while the maximum specific titers were reached at different time points (19.8±7.6 h), those maxima were reached all within a very narrow window of cumulatively consumed substrate dSn (3.1±0.3 g/g). Concluding, this contribution provides a workflow on how to gain a physiological view on the process and illustrates potential benefits. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:261-270, 2017.

Generic biomass estimation methods targeting physiologic process control in induced bacterial cultures

Advanced bioprocess development strategies focus on the control of physiological entities, which rely on accurate real‐time determination of the biomass concentration. Various methods have been proposed in literature but up to this date a comprehensive and differentiated comparison of biomass estimation approaches for early stage bioprocess development is missing. In this study, we compared hard sensor, soft‐sensor, and data‐driven approaches for real‐time biomass estimation in respect to accuracy, transferability, and costs. The outlined methods were tested with two different microbial strains and recombinant products using Escherichia coli. To investigate the applicability of the outlined methods, method performance was assessed in correspondence to metabolic activity. Based on statistical descriptors the methods were compared and discussed. The results indicate no significant impact of strain or biomass estimation approach on the measurement quality. The average relative error of 11–13% can be greatly reduced by over 85% combining the outlined methods by the means of weighted average. This approach proved to be highly robust even during highly dynamic process conditions of oscillating specific substrate uptake rates. Concluding, the combination of low cost first principle soft‐sensor approaches in combination with a hybrid soft‐sensor yields the best information‐to‐effort ratio.

Fed-Batch Production of Bacterial Ghosts Using Dielectric Spectroscopy for Dynamic Process Control.

The Bacterial Ghost (BG) platform technology evolved from a microbiological expression system incorporating the ϕX174 lysis gene E. E-lysis generates empty but structurally intact cell envelopes (BGs) from Gram-negative bacteria which have been suggested as candidate vaccines, immunotherapeutic agents or drug delivery vehicles. E-lysis is a highly dynamic and complex biological process that puts exceptional demands towards process understanding and control. The development of a both economic and robust fed-batch production process for BGs required a toolset capable of dealing with rapidly changing concentrations of viable biomass during the E-lysis phase. This challenge was addressed using a transfer function combining dielectric spectroscopy and soft-sensor based biomass estimation for monitoring the rapid decline of viable biomass during the E-lysis phase. The transfer function was implemented to a feed-controller, which followed the permittivity signal closely and was capable of maintaining a constant specific substrate uptake rate during lysis phase. With the described toolset, we were able to increase the yield of BG production processes by a factor of 8–10 when compared to currently used batch procedures reaching lysis efficiencies >98%. This provides elevated potentials for commercial application of the Bacterial Ghost platform technology.

Application of ADM1 for modeling of biogas production from anaerobic digestion of Hydrilla verticillata

The present study focused on the application of anaerobic digestion model no. 1 (ADM1) to simulate biogas production from Hydrilla verticillata. Model simulation was carried out by implementing ADM1 in AQUASIM 2.0 software. Sensitivity analysis was used to select the most sensitive parameters for estimation using the absolute–relative sensitivity function. Among all the kinetic parameters, disintegration constant (kdis), hydrolysis constant of protein (khyd_pr), Monod maximum specific substrate uptake rate (km_aa, km_ac, km_h2) and half-saturation constants (Ks_aa, Ks_ac) affect biogas production significantly, which were optimized by fitting of the model equations to the data obtained from batch experiments. The ADM1 model after parameter estimation was able to well predict the experimental results of daily biogas production and biogas composition. The simulation results of evolution of organic acids, bacteria concentrations and inhibition effects also helped to get insight into the reaction mechanisms.

Strategy to reduce the acclimation period for enrichment of PHA accumulating cultures

A selective sludge discharge (SSD) process is proposed and evaluated in this study to accumulate the polyhydroxyalkanoate (PHA) accumulating bacteria in mixed microbial cultures (MMCs). Sequencing batch reactor (SBR) PHA production in the SSD mode was compared to the conventional aerobic dynamic feeding (ADF) mode. Aerobic granular sludge rich in PHA was cultivated in a SBR by seeding the MMCs. The granular sludge was formed on 14 and 8 d of the operation in SBR #1 and SBR #2, respectively, however, the PHA production performance of SBR #1 fluctuated during the 30 d. The MMCs in SBR #2 showed the best PHA accumulating ability in terms of the yield of PHA, specific substrate uptake rate and PHA storage rate, a maximum PHA content and average PHA storage rate of 58.2% and 0.33 Cmol PHA/(Cmol X/h), respectively, were achieved on the day 30 of enrichment operation in the batch assays.