Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch r... more Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with 31 P and 13 C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, ...
A process is provided for increasing the production of PHA in a mixed culture biomass. In a first... more A process is provided for increasing the production of PHA in a mixed culture biomass. In a first stage of the process, organic material associated with a substrate is converted to volatile fatty acids. In the case of a wastewater treatment process, if the wastewater includes sufficient volatile fatty acids (VFAs) to support the process, then it is unnecessary to convert organic material to VFAs. In a second stage of the process, an anaerobic-aerobic selection process is utilized to select glycogen accumulating organisms that cause these organisms to proliferate and dominate the open mixed culture biomass. By providing relatively high organic loading in the form of VFAs in the anaerobic treatment phase of the selection process, glycogen accumulating organisms having a relatively high level of stored glycogen are produced. In a third stage, the PHA accumulation process is practiced where the glycogen rich organisms are fed VFAs under anaerobic or aerobic conditions or combinations th...
A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Deflu... more A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Defluviicoccus vanus-related organisms presenting a Glycogen Accumulating Organisms (GAO) phenotype. Initial batch tests were performed using anaerobic/aerobic conditions to assess the capacity of different carbon sources utilization frequently abundant in wastewater: acetate, propionate, butyrate, valerate and glucose. Acetate and propionate were totally consumed in the anaerobic phase as well as butyrate and valerate, though these last ones with a very low consumption rate. All substrates were converted to polyhydroxyalkanoates (PHA). Glucose had a very slight anaerobic consumption but failed to disclose a typical GAO phenotype. In aerobic conditions, again all carbon sources were readily consumed except for glucose, with acetate and propionate having the higher consumption rates. Therefore, glucose seems not be used by this type of organisms. Acetate and propionate consumption rates indicated that these GAOs could reveal good competition advantages in EBPR systems where these carbon sources are available, especially propionate. Volatile Fatty Acid (VFA) uptake in aerobic phase and consequential PHA production indicate these organisms as possible candidates for PHA production.
In this work we study the optimization of a polyhydroxyalkanoates (PHA) production process by mix... more In this work we study the optimization of a polyhydroxyalkanoates (PHA) production process by mixed cultures based on a detailed hybrid metabolic model. The metabolic network under consideration was first decomposed into its fundamental pathways using the elementary flux modes (EFM) technique. Then, a dynamical hybrid semi-parametric model was formulated, which allowed to identify the EFM kinetics from experimental data of 7 batch runs. The EFM fluxes were interpreted in terms of metabolic consistency. The final model allowed to characterize the metabolism dynamics, namely of how the relative weight of pathways evolves in time in a typical batch or fed-batch run. The present technique is a step forward for the integration of systems biology and bioprocess control.
Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegr... more Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.
In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in acti... more In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in activated sludge during both the anaerobic and the aerobic stages. By supplying a 13C label on the methyl group of acetate at the beginning of the anaerobic ...
This study investigated the link between the process performance of two denitrifying phosphorus (... more This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.
The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater... more The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve.
Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus ... more Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.
Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed cultur... more Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed culture enriched in glycogen accumulating organisms (GAOs) with fermented sugar cane molasses as substrate was studied. The produced polymers contained five types of monomers, namely 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV) and the medium chain length monomer 3-hydroxyhexanoate (3HHx). With fermented molasses as substrate, PHA was produced under concurrent consumption of stored glycogen with yields of 0.47-0.66 C-mol PHA per C-mol of total carbon substrate and with rates up to 0.65 C-mol/C-molX h. In order to investigate the role of glycogen during aerobic PHA accumulation in GAOs, synthetic single volatile fatty acids (VFAs) were used as substrates and it was found that the fate of glycogen was dependent on the type of VFA being consumed. Aerobic PHA accumulation occurred under concurrent glycogen consumption with acetate as substrate and under minor concurrent glycogen production with propionate as substrate. With butyrate and valerate as substrates, PHA accumulation occurred with the glycogen pool unaffected. The composition of the PHA was dependent on the VFA composition of the fermented molasses and was 56-70 mol-% 3HB, 13-43 mol-% 3HV, 1-23 mol-% 3HHx and 0-2 mol-% 3H2MB and 3H2MV. The high polymer yields and production rates suggest that enrichment of GAOs can be a fruitful strategy for mixed culture production of PHA from waste substrates.
The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well ad... more The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.
SummaryGlycogen‐accumulating organisms (GAOs) are found in enhanced biological phosphorus removal... more SummaryGlycogen‐accumulating organisms (GAOs) are found in enhanced biological phosphorus removal systems where they compete with polyphosphate‐accumulating organisms for external carbon substrates. 13C nuclear magnetic resonance (13C‐NMR) was used to elucidate the metabolic pathways operating in an enriched GAO culture dominated by two known GAOs (81.2%). The experiments consisted of adding 13C‐acetate (labelled on position 1 or 2) to the culture under anaerobic conditions, and operating the culture through a cycle consisting of an anaerobic, an aerobic and a further anaerobic phase. The carbon transformations over the cycle were monitored using in vivo13C‐NMR. The two‐carbon moieties in hydroxybutyrate and hydroxyvalerate were derived from acetate, while the propionyl precursor of hydroxyvalerate was primarily derived from glycogen, with only a small fraction originating from acetate. Comparison of the labelling patterns in hydroxyvalerate at the end of the first and the second an...
ABSTRACT A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phos... more ABSTRACT A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.
Background This paper presents a metabolic model describing the production of polyhydroxyalkanoat... more Background This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA) copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. Results The model was validated with experimental data collected in a sequencing batch reactor (SBR) operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments), and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichmen...
A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7... more A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under g~s (H2S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated .acetic acid concentrations of approximately 54 mg/L.
Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch r... more Phosphorus and carbon metabolism in Microlunatus phosphovorus was investigated by using a batch reactor to study the kinetics of uptake and release of extracellular compounds, in combination with 31 P and 13 C nuclear magnetic resonance (NMR) to characterize intracellular pools and to trace the fate of carbon substrates through the anaerobic and aerobic cycles. The organism was subjected to repetitive anaerobic and aerobic cycles to induce phosphorus release and uptake in a sequencial batch reactor; an ultrafiltration membrane module was required since cell suspensions did not sediment. M. phosphovorus fermented glucose to acetate via an Embden-Meyerhof pathway but was unable to grow under anaerobic conditions. A remarkable time shift was observed between the uptake of glucose and excretion of acetate, resulting in an intracellular accumulation of acetate. The acetate produced was oxidized in the subsequent aerobic stage. Very high phosphorus release and uptake rates were measured, ...
A process is provided for increasing the production of PHA in a mixed culture biomass. In a first... more A process is provided for increasing the production of PHA in a mixed culture biomass. In a first stage of the process, organic material associated with a substrate is converted to volatile fatty acids. In the case of a wastewater treatment process, if the wastewater includes sufficient volatile fatty acids (VFAs) to support the process, then it is unnecessary to convert organic material to VFAs. In a second stage of the process, an anaerobic-aerobic selection process is utilized to select glycogen accumulating organisms that cause these organisms to proliferate and dominate the open mixed culture biomass. By providing relatively high organic loading in the form of VFAs in the anaerobic treatment phase of the selection process, glycogen accumulating organisms having a relatively high level of stored glycogen are produced. In a third stage, the PHA accumulation process is practiced where the glycogen rich organisms are fed VFAs under anaerobic or aerobic conditions or combinations th...
A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Deflu... more A reactor was successfully enriched (90% as shown by Fluorescence in situ Hybridization) in Defluviicoccus vanus-related organisms presenting a Glycogen Accumulating Organisms (GAO) phenotype. Initial batch tests were performed using anaerobic/aerobic conditions to assess the capacity of different carbon sources utilization frequently abundant in wastewater: acetate, propionate, butyrate, valerate and glucose. Acetate and propionate were totally consumed in the anaerobic phase as well as butyrate and valerate, though these last ones with a very low consumption rate. All substrates were converted to polyhydroxyalkanoates (PHA). Glucose had a very slight anaerobic consumption but failed to disclose a typical GAO phenotype. In aerobic conditions, again all carbon sources were readily consumed except for glucose, with acetate and propionate having the higher consumption rates. Therefore, glucose seems not be used by this type of organisms. Acetate and propionate consumption rates indicated that these GAOs could reveal good competition advantages in EBPR systems where these carbon sources are available, especially propionate. Volatile Fatty Acid (VFA) uptake in aerobic phase and consequential PHA production indicate these organisms as possible candidates for PHA production.
In this work we study the optimization of a polyhydroxyalkanoates (PHA) production process by mix... more In this work we study the optimization of a polyhydroxyalkanoates (PHA) production process by mixed cultures based on a detailed hybrid metabolic model. The metabolic network under consideration was first decomposed into its fundamental pathways using the elementary flux modes (EFM) technique. Then, a dynamical hybrid semi-parametric model was formulated, which allowed to identify the EFM kinetics from experimental data of 7 batch runs. The EFM fluxes were interpreted in terms of metabolic consistency. The final model allowed to characterize the metabolism dynamics, namely of how the relative weight of pathways evolves in time in a typical batch or fed-batch run. The present technique is a step forward for the integration of systems biology and bioprocess control.
Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegr... more Activated sludge was submitted to aerobic dynamic substrate feeding for the production of biodegradable plastics. Two sequencing batch reactors were operated with acetate or propionate as sole carbon substrates. With acetate a homopolymer of polyhydroxybutyrate (PHB) was obtained and with propionate a copolymer of hydroxybutyrate and hydroxyvalerate P(HB/HV) was produced. Three main morphotypes were identified in both sludges: two belong to the Alphaproteobacteria class and the third to the Betaproteobacteria class. Bacilli belonging to Betaproteobacteria were shown by FISH analysis, applied in combination with Nile Blue post-staining, to be the main responsible for PHAs storage. The latter were affiliated to Azoarcus genus within Betaproteobacteria.
In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in acti... more In vivo13C-NMR, 31P-NMR techniques were applied to study phosphorus and carbon metabolism in activated sludge during both the anaerobic and the aerobic stages. By supplying a 13C label on the methyl group of acetate at the beginning of the anaerobic ...
This study investigated the link between the process performance of two denitrifying phosphorus (... more This study investigated the link between the process performance of two denitrifying phosphorus (P) removal systems and their microbial community structure. Two sequencing batch reactors (SBRs) were operated with either acetate or propionate as the sole carbon source, and were gradually acclimatised from anaerobic-aerobic to anaerobic-anoxic conditions. It was found that the propionate SBR was able to sustain denitrifying P removal after acclimatisation, while the enhanced biological phosphorus removal (EBPR) activity in the acetate reactor collapsed after the aerobic phase was eliminated. The results suggested that the anoxic glycogen production rate in the acetate SBR was insufficient to support the anaerobic glycogen demand for acetate uptake. The chemical transformations in each SBR suggested that different types of polyphosphate-accumulating organisms (PAOs) were present in each system, possessing different affinities for nitrate. Microbial characterisation with fluorescence in situ hybridisation (FISH) revealed that Accumulibacter was the dominant organism in each reactor, although different cell morphotypes were observed. A coccus morphotype was predominant in the acetate SBR while the propionate SBR was enriched in a rod morphotype. It is hypothesised that the coccus morphotype corresponds to an Accumulibacter strain that is unable to use nitrate as electron acceptor but is able to use oxygen, and possibly nitrite. The rod morphotype is proposed to be a PAO able to use nitrate, nitrite and oxygen. This hypothesis is in agreement with literature studies focussed on the identity of denitrifying PAOs (DPAOs), as well as a recent metagenomic study on Accumulibacter.
The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater... more The enhanced biological phosphorus removal (EBPR) process has been implemented in many wastewater treatment plants worldwide. While the EBPR process is indeed capable of efficient phosphorus (P) removal performance, disturbances and prolonged periods of insufficient P removal have been observed at full-scale plants on numerous occasions under conditions that are seemingly favourable for EBPR. Recent studies in this field have utilised a wide range of approaches to address this problem, from studying the microorganisms that are primarily responsible for or detrimental to this process, to determining their biochemical pathways and developing mathematical models that facilitate better prediction of process performance. The overall goal of each of these studies is to obtain a more detailed insight into how the EBPR process works, where the best way of achieving this objective is through linking together the information obtained using these different approaches. This review paper critically assesses the recent advances that have been achieved in this field, particularly relating to the areas of EBPR microbiology, biochemistry, process operation and process modelling. Potential areas for future research are also proposed. Although previous research in this field has undoubtedly improved our level of understanding, it is clear that much remains to be learned about the process, as many unanswered questions still remain. One of the challenges appears to be the integration of the existing and growing scientific knowledge base with the observations and applications in practice, which this paper hopes to partially achieve.
Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus ... more Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.
Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed cultur... more Batch production of polyhydroxyalkanoates (PHAs) under aerobic conditions by an open mixed culture enriched in glycogen accumulating organisms (GAOs) with fermented sugar cane molasses as substrate was studied. The produced polymers contained five types of monomers, namely 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxy-2-methylbutyrate (3H2MB), 3-hydroxy-2-methylvalerate (3H2MV) and the medium chain length monomer 3-hydroxyhexanoate (3HHx). With fermented molasses as substrate, PHA was produced under concurrent consumption of stored glycogen with yields of 0.47-0.66 C-mol PHA per C-mol of total carbon substrate and with rates up to 0.65 C-mol/C-molX h. In order to investigate the role of glycogen during aerobic PHA accumulation in GAOs, synthetic single volatile fatty acids (VFAs) were used as substrates and it was found that the fate of glycogen was dependent on the type of VFA being consumed. Aerobic PHA accumulation occurred under concurrent glycogen consumption with acetate as substrate and under minor concurrent glycogen production with propionate as substrate. With butyrate and valerate as substrates, PHA accumulation occurred with the glycogen pool unaffected. The composition of the PHA was dependent on the VFA composition of the fermented molasses and was 56-70 mol-% 3HB, 13-43 mol-% 3HV, 1-23 mol-% 3HHx and 0-2 mol-% 3H2MB and 3H2MV. The high polymer yields and production rates suggest that enrichment of GAOs can be a fruitful strategy for mixed culture production of PHA from waste substrates.
The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well ad... more The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.
SummaryGlycogen‐accumulating organisms (GAOs) are found in enhanced biological phosphorus removal... more SummaryGlycogen‐accumulating organisms (GAOs) are found in enhanced biological phosphorus removal systems where they compete with polyphosphate‐accumulating organisms for external carbon substrates. 13C nuclear magnetic resonance (13C‐NMR) was used to elucidate the metabolic pathways operating in an enriched GAO culture dominated by two known GAOs (81.2%). The experiments consisted of adding 13C‐acetate (labelled on position 1 or 2) to the culture under anaerobic conditions, and operating the culture through a cycle consisting of an anaerobic, an aerobic and a further anaerobic phase. The carbon transformations over the cycle were monitored using in vivo13C‐NMR. The two‐carbon moieties in hydroxybutyrate and hydroxyvalerate were derived from acetate, while the propionyl precursor of hydroxyvalerate was primarily derived from glycogen, with only a small fraction originating from acetate. Comparison of the labelling patterns in hydroxyvalerate at the end of the first and the second an...
ABSTRACT A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phos... more ABSTRACT A laboratory scale sequencing batch reactor (SBR) operating for enhanced biological phosphorus removal (EBPR) and fed with a mixture of volatile fatty acids (VFAs) showed stable and efficient EBPR capacity over a four-year-period. Phosphorus (P), poly-beta-hydroxyalkanoate (PHA) and glycogen cycling consistent with classical anaerobic/aerobic EBPR were demonstrated with the order of anaerobic VFA uptake being propionate, acetate then butyrate. The SBR was operated without pH control and 63.67 +/- 13.86 mg P l-1 was released anaerobically. The P% of the sludge fluctuated between 6% and 10% over the operating period (average of 8.04 +/- 1.31%). Four main morphological types of floc-forming bacteria were observed in the sludge during one year of in-tensive microscopic observation. Two of them were mainly responsible for anaerobic/aerobic P and PHA transformations. Fluorescence in situ hybridization (FISH) and post-FISH chemical staining for intracellular polyphosphate and PHA were used to determine that 'Candidatus Accumulibacter phosphatis' was the most abundant polyphosphate accumulating organism (PAO), forming large clusters of coccobacilli (1.0-1.5 micro m) and comprising 53% of the sludge bacteria. Also by these methods, large coccobacillus-shaped gammaproteobacteria (2.5-3.5 micro m) from a recently described novel cluster were glycogen-accumulating organisms (GAOs) comprising 13% of the bacteria. Tetrad-forming organisms (TFOs) consistent with the 'G bacterium' morphotype were alphaproteobacteria, but not Amaricoccus spp., and comprised 25% of all bacteria. According to chemical staining, TFOs were occasionally able to store PHA anaerobically and utilize it aerobically.
Background This paper presents a metabolic model describing the production of polyhydroxyalkanoat... more Background This paper presents a metabolic model describing the production of polyhydroxyalkanoate (PHA) copolymers in mixed microbial cultures, using mixtures of acetic and propionic acid as carbon source material. Material and energetic balances were established on the basis of previously elucidated metabolic pathways. Equations were derived for the theoretical yields for cell growth and PHA production on mixtures of acetic and propionic acid as functions of the oxidative phosphorylation efficiency, P/O ratio. The oxidative phosphorylation efficiency was estimated from rate measurements, which in turn allowed the estimation of the theoretical yield coefficients. Results The model was validated with experimental data collected in a sequencing batch reactor (SBR) operated under varying feeding conditions: feeding of acetic and propionic acid separately (control experiments), and the feeding of acetic and propionic acid simultaneously. Two different feast and famine culture enrichmen...
A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7... more A culture of SRB growing in lactate was incubated at different pH values in the range of 5.8 to 7.0. Highest growth rates were observed at pH 6.6. Under g~s (H2S) stripping conditions the specific growth rate decreased with the undissociated acetic acid produced. An inhibition of SRB growth of 50% was observed for undissociated .acetic acid concentrations of approximately 54 mg/L.
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Papers by Paulo Lemos