The utilization of glycogen accumulating organisms for mixed culture production of polyhydroxyalkanoates

Production of polyhydroxyalkanoates (PHAs) by an open mixed culture enriched in glycogen accumulating organisms (GAOs) under alternating anaerobic–aerobic conditions with acetate as carbon source was investigated. The culture exhibited a stable enrichment performance over the 450‐day operating period with regards to phenotypic behavior and microbial community structure. Candidatus Competibacter phosphatis dominated the culture at between 54% and 70% of the bacterial biomass throughout the study, as determined by fluorescence in situ hybridization. In batch experiments under anaerobic conditions, PHA containing 3‐hydroxybutyrate (3HB) and 27 mol‐% 3‐hydroxyvalerate (3HV) was accumulated up to 49% of cell dry weight utilizing the glycogen pool stored in the SBR cycle. Under aerobic and ammonia limited conditions, PHA comprising only 3HB was accumulated to 60% of cell dry weight. Glycogen was consumed during aerobic PHA accumulation as well as under anaerobic conditions, but with different stoichiometry. Under aerobic conditions 0.31 C‐mol glycogen was consumed per consumed C‐mol acetate compared to 0.99 under anaerobic conditions. Both the PHA biomass content and the specific PHA production rate obtained were similar to what is typically obtained using the more commonly applied aerobic dynamic feeding strategy. Biotechnol. Bioeng. 2009; 104: 698–708 © 2009 Wiley Periodicals, Inc.     

Metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms under anaerobic conditions

This paper proposes a new metabolic model for acetate uptake by a mixed culture of phosphate- and glycogen-accumulating organisms (PAOs and GAOs) under anaerobic conditions. The model uses variable overall stoichiometry based on the assumption that PAOs may have the ability of using the glyoxylate pathway to produce the required reducing power for polyhydroxyalkonate (PHA) synthesis. The proposed model was tested and verified by experimental results. A sequencing batch reactor system was operated for enhanced biological phosphorus removal (EBPR) with acetate as the sole carbon source at different influent acetate/phosphate ratios. The resulting experimental data supported the validity of the proposed model, indicating the presence of GAOs for all tested HAc/P ratios, especially under P-limiting conditions. Strong agreement is observed between experimental values and model predictions for all model components, namely, PHB production, PHA composition, glycogen utilization, and P release.     

A metabolic model for acetate uptake under anaerobic conditions by glycogen accumulating organisms: Stoichiometry, kinetics, and the effect of pH

A metabolic model for the stoichiometry of acetate uptake under anaerobic conditions by an enriched culture of glycogen accumulating organisms (GAOs) was developed and tested by experimental studies. Glycogen served as the source of both reducing power and energy to drive the process of acetate uptake. The amount of glycogen consumed and poly-beta-hydroxyvalerate (PHV) accumulated in the cells increased with increasing pH, indicating that the energy requirements for acetate uptake increased with pH. The composition of the accumulated poly-beta-hydroxyalkanoates (PHAs) was adequately predicted using the assumption that acetyl-CoA and propionyl-CoA condense randomly to produce PHA. In addition, the rate of acetate uptake was strongly affected by the pH. The rate decreased with increasing pH and this dependence could be described with a saturation type of expression. A comparison of the rate of acetate uptake at low pH with the rates observed in enriched cultures of phosphorus accumulating organisms (PAOs) indicated that GAOs are able to compete effectively with PAOs in nutrient removal systems under certain conditions.     

Production of targeted poly(3-hydroxyalkanoates) copolymers by glycogen accumulating organisms using acetate as sole carbon source

One of the main limitations in bacterial polyhydroxyalkanoate (PHA) production with mixed cultures is the fact that primarily polyhydroxybutyrate (PHB) homopolymers are generated from acetate as the main carbon source, which is brittle and quite fragile. The incorporation of different 3-hydroxyalkanoate (HA) components into the polymers requires the addition of additional carbon sources, leading to extra costs and complexity. In this study, the production of poly(3-hydroxybutyrate (3HB)-co-3-hydroxyvalerate (3HV)-co-3-hydroxy-2-methylvalerate (3HMV)), with 7-35C-mol% of 3HV fractions from acetate as the only carbon source was achieved with the use of glycogen accumulating organisms (GAOs). An enriched GAO culture was obtained in a lab-scale reactor operated under alternating anaerobic and aerobic conditions with acetate fed at the beginning of the anaerobic period. The production of PHAs utilizing the enriched GAO culture was investigated under both aerobic and anaerobic conditions. A polymer content of 14-41% of dry cell weight was obtained. The PHA product accumulated by GAOs under anaerobic conditions contained a relatively constant proportion of non-3HB monomers (30+/-5C-mol%), irrespective of the amount of acetate assimilated. In contrast, under aerobic conditions, GAOs only produced 3HB monomers from acetate causing a gradually decreasing 3HV fraction during this aerobic feeding period. The PHAs were characterized by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC). The data demonstrated that the copolymers possessed similar characteristics to those of commercially available poly(3HB-co-3HV) (PHBV) products. The PHAs produced under solely anaerobic conditions possessed lower melting points and crystallinity, higher molecular weights, and narrower molecular-weight distributions, compared to the aerobically produced polymers. This paper hence demonstrates the significant potential of GAOs to produce high quality polymers from a simple and cheap carbon source, contributing considerably to the growing research body on bacterial PHA production by mixed cultures.     

Model-based analysis of anaerobic acetate uptake by a mixed culture of polyphosphate-accumulating and glycogen-accumulating organisms

An increasing number of studies shows that the glycogen-accumulating organisms (GAOs) can survive and may indeed proliferate under the alternating anaerobic/aerobic conditions found in EBPR systems, thus forming a strong competitor of the polyphosphate-accumulating organisms (PAOs). Understanding their behaviors in a mixed PAO and GAO culture under various operational conditions is essential for developing operating strategies that disadvantage the growth of this group of unwanted organisms. A model-based data analysis method is developed in this paper for the study of the anaerobic PAO and GAO activities in a mixed PAO and GAO culture. The method primarily makes use of the hydrogen ion production rate and the carbon dioxide transfer rate resulting from the acetate uptake processes by PAOs and GAOs, measured with a recently developed titration and off-gas analysis (TOGA) sensor. The method is demonstrated using the data from a laboratory-scale sequencing batch reactor (SBR) operated under alternating anaerobic and aerobic conditions. The data analysis using the proposed method strongly indicates a coexistence of PAOs and GAOs in the system, which was independently confirmed by fluorescent in situ hybridization (FISH) measurement. The model-based analysis also allowed the identification of the respective acetate uptake rates by PAOs and GAOs, along with a number of kinetic and stoichiometric parameters involved in the PAO and GAO models. The excellent fit between the model predictions and the experimental data not involved in parameter identification shows that the parameter values found are reliable and accurate. It also demonstrates that the current anaerobic PAO and GAO models are able to accurately characterize the PAO/GAO mixed culture obtained in this study. This is of major importance as no pure culture of either PAOs or GAOs has been reported to date, and hence the current PAO and GAO models were developed for the interpretation of experimental results of mixed cultures. The proposed method is readily applicable for detailed investigations of the competition between PAOs and GAOs in enriched cultures. However, the fermentation of organic substrates carried out by ordinary heterotrophs needs to be accounted for when the method is applied to the study of PAO and GAO competition in full-scale sludges.     

A new interpretation of ASM2d for modeling of SBR performance for enhanced biological phosphorus removal under different P/HAc ratios

This study evaluated the prediction capability of Activated Sludge Model No. 2d (ASM2d), for the enhanced biological phosphorus removal (EBPR) performance of a sequencing batch reactor (SBR) receiving variable influent phosphate load. For this purpose, a laboratory-scale SBR was operated with a synthetic feed containing acetate as the sole carbon source. The experiments were conducted in four different Runs to ensure a range of different phosphate/acetate ratios in the influent. Model evaluations were carried out using concentration profiles measured throughout a representative cycle at steady state. An iterative calibration methodology was developed based on sensitivity analysis and applied to four different sets of experimental data on relevant model parameters reflecting SBR performance. ASM2d was able to predict the steady state behavior of the SBR system receiving variable influent phosphate loads only with the recalibrated parameter set. The regular changing pattern of the coefficients could be interpreted with the ability of the SBR system to sustain glycogen accumulating microorganisms, GAOs, which can store substrate under anaerobic conditions without polyphosphate energy, but deriving energy from the degradation of glycogen. Thus they are capable of prevailing at lower P/Ac ratios. The results indicate the need to include glycogen and GAOs as model components for processes involving both phosphate accumulating organisms, (PAOs) and GAOs, in order to obtain a better prediction of X(PHA) and oxygen uptake rate (OUR) profiles in the system.     

Analysis of the microbial community structure and function of a laboratory scale enhanced biological phosphorus removal reactor

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.     

Metabolic influence of lead on polyhydroxyalkanoates (PHA) production and phosphate uptake in activated sludge fed with glucose or acetic acid as carbon source

Sludge in a sequential batch reactor (SBR) system was used to investigate the effect of lead toxicity on metabolisms of polyphosphate accumulating organisms (PAOs) and glycogen accumulating organisms (GAOs) communities fed with acetic acid or glucose as their sole carbon source, respectively. Results showed that the effect of lead on substrate utilization of both PAOs and GAOs was insignificant. However, lead substantially inhibited both of phosphate release and uptake of PAOs. In high concentration of acetic acid trials, an abnormal aerobic phosphate release was observed instead of phosphate uptake and the release rate increased with increasing lead concentration. Results also showed that PAOs could normally synthesize polyhydroxybutyrate (PHB) in the anaerobic phase even though lead concentration was 40 mg L⁻¹. However, they could not aerobically utilize PHB normally in the presence of lead. On the other hand, GAOs could not normally metabolize polyhydroxyvalerate (PHV) in both the anaerobic and aerobic phases.     

Experimental evaluation of decrease in the activities of polyphosphate/glycogen‐accumulating organisms due to cell death and activity decay in activated sludge

Decrease in bacterial activity (biomass decay) in activated sludge can result from cell death (reduction in the amount of active bacteria) and activity decay (reduction in the specific activity of active bacteria). The goal of this study was to experimentally differentiate between cell death and activity decay as the cause of decrease in bacterial activity. By means of measuring maximal anaerobic phosphate release rates, verifying membrane integrity by live/dead staining and verifying presence of 16S rRNA with fluorescence in situ hybridization (FISH), the decay rates and death rates of polyphosphate‐accumulating organisms (PAOs) in a biological nutrient removal (BNR) system and a laboratory phosphate removing sequencing batch reactor (SBR) system were determined, respectively, under famine conditions. In addition, the decay rate and death rate of glycogen‐accumulating organisms (GAOs) in a SBR system with an enrichment culture of GAOs were also measured under famine conditions. Hereto the maximal anaerobic volatile fatty acid uptake rates, live/dead staining, and FISH were used. The experiments revealed that in the BNR and enriched PAO‐SBR systems, activity decay contributed 58% and 80% to the decreased activities of PAOs, and that cell death was responsible for 42% and 20% of decreases in their respective activities. In the enriched GAOs system, activity decay constituted a proportion of 74% of the decreased activity of GAOs, and cell death only accounted for 26% of the decrease of their activity. Biotechnol. Bioeng. 2010; 106: 399–407. © 2010 Wiley Periodicals, Inc.     

Metabolic pathway for propionate utilization by phosphorus-accumulating organisms in activated sludge: 13C labeling and in vivo nuclear magnetic resonance

In vivo 13C and 31P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-13C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV5, 59%; HV4, 5.0%; HV3, 1.1%; HV2, 3.5%; and HV1, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV4 is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.     

Microbial population response to changes of the operating conditions in a dynamic nutrient-removal sequencing batch reactor

A nutrient-removal sequencing batch reactor operated with short anaerobic/aerobic cycles was subjected to different operating conditions, namely, cycle length, feeding pattern and feed composition. The changes in microbial population, as well as the contribution of microbial groups to the total nutrient removal, were estimated using the kinetic parameters obtained in this study. Denitrifying polyphosphate-accumulating organisms (DPAOs) were detected in the system, representing a fraction of 23% of phosphorus-accumulating organisms (PAOs). The results suggest that DPAOs and non-DPAOs are different microorganisms. The presence of nitrate in the feed stimulated DPAOs to predominate over non-DPAOs. Feeding the reactor with a mixture of organic substrates also stimulated DPAOs. Glycogen-accumulating organisms (GAOs) were likely to be present in the system and their development over PAOs was apparently favoured by increasing the aeration time and feeding during the aerobic phase. In contrast, the presence of propanoate in the feed apparently favoured PAOs over GAOs.     

Enzymatic total synthesis of gibberellin A₄ from acetate

Natural terpenoids have elaborate structures and various bioactivities, making difficult their synthesis and labeling with isotopes. We report here the enzymatic total synthesis of plant hormone gibberellins (GAs) with recombinant biosynthetic enzymes from stable isotope-labeled acetate. Mevalonate (MVA) is a key intermediate for the terpenoid biosynthetic pathway. 13C-MVA was synthesized from 13C-acetate via acetyl-CoA, using four enzymes or fermentation with a MVA-secreted yeast. The diterpene hydrocarbon, ent-kaurene, was synthesized from 13C-acetate and 13C-MVA with ten and six recombinant enzymes in one test tube, respectively. Four recombinant enzymes, P450 monooxygenases and soluble dioxygenases involved in the GA? biosynthesis from ent-kaurene via GA?? were prepared in yeast and Escherichia coli. All intermediates and the final product GA? were uniformly labeled with 13C without dilution by natural abundance when [U-13C?] acetate was used. The 13C-NMR and MS data for [U-13C??] ent-kaurene confirmed 13C-13C coupling, and no dilution with the 12C atom was observed.     

Fully coupled activated sludge model (FCASM): Model development

A sub-microscopic mechanism model named Fully Coupled Activated Sludge Model (FCASM) about biological nutrient removal in the wastewater treatment process was developed in the present study. The functional organisms existing simultaneously in the activated sludge system were separated into eight groups, including aerobic heterotrophic organisms, nitrite reducing organisms, nitrate reducing organisms, ammonium oxidizing autotrophs, nitrite oxidizing autotrophs, non-denitrifying phosphorus-accumulating organisms (PAOs), denitrifying phosphorus-accumulating bacteria (DPB), and glycogen-accumulating organisms (GAOs). In FCASM, the interaction relationships of the eight functional microorganisms were taken fully into account. FCASM could model biological nitrogen removal via nitrite by splitting nitrification process and denitrification process into two-step reactions, and the autotrophs and denitrifying organisms were divided into two groups, respectively. What’s important, FCASM included the anaerobic maintenance processes of sequential utilization of polyphosphate followed by glycogen for PAOs and DPB and glycolysis of the intracellular stored glycogen for GAOs.     

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells

The advantage of using ¹³C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that ¹³C-[2] acetate or ¹³C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using ¹³C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/¹³C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.     

Decolorization of azo-reactive dye by polyphosphate- and glycogen-accumulating organisms in an anaerobic-aerobic sequencing batch reactor

An anaerobic-aerobic sequencing batch reactor with a sludge age of 8 days and anaerobic + aerobic + settling times of 18 + 5 + 1 h, was used to decolorize an azo-reactive dye wastewater. The nutrient broth (NB) and sodium acetate (SA) solution at 500 + 0, 350 + 150, 250 + 250 and 0 + 500 mg/l as COD was fed to the system to promote the polyphosphate-accumulating organisms (PAOs), while only glucose (500 mg/l COD) was used as a glycogen-accumulating organisms (GAOs) promoting substrate. The decolorization capability of the process was about 73-77 and 59-64% in terms of ADMI for the systems which the PAOs and GAOs proliferated, respectively. The color reduction was mainly achieved within the first 2 h of the anaerobic stage.     

Metabolic Characteristics of a Glycogen-Accumulating Organism in Defluviicoccus Cluster II Revealed by Comparative Genomics

Glycogen-accumulating organisms (GAOs) may compete with phosphate-accumulating organisms (PAOs) for short-chain fatty acids (VFAs) in anaerobic polyhydroxyalkanoates (PHA) synthesis, but no consequently aerobic polyphosphate accumulation in enhanced biological phosphorus removal (EBPR) process, thus deteriorating the EBPR process. They are detected frequently in the deteriorated EBPR process, but their metabolisms are still far from our comprehensions for there is seldom pure culture. In this study, a nearly complete draft genome of a GAOs in Defluviicoccus cluster II, GAO-HK, is recruited from the metagenome of activated sludge in a full-scale industrial anoxic/aerobic wastewater plant. Comparative genomics reveal similar metabolisms of PHA and glycogen in GAOs of GAO-HK, Defluviicoccus tetraformis TFO71 (TFO71) and Competibacter phosphatis clade IIA (CPIIA), and PAOs of Accumulibacter clade IIA UW-1 (UW-1) and Tetrasphaera elongata Lp2 (Lp2). Although there are similar gene cassettes related with polyphosphate metabolism in these GAOs and PAOs, especially for Defluviicoccus-relative bacteria and UW-1, ppk1 in GAOs are diverse from those in the identified PAOs, implying the difference of polyphosphate metabolism in GAOs and PAOs. Additionally, genes related to the dissimilatory denitrification are absent in TFO71 and GAO-HK, implying that additional nitrate or nitrite may favor PAOs over Defluviicoccus-relative GAOs. Therefore, PAOs suffering from competition of Defluviicoccus-relative GAOs might be rescued with the additional nitrate/nitrite, which is important to improve the stability of EBPR processes.     

Competition between polyphosphate and glycogen accumulating organisms in enhanced biological phosphorus removal systems with acetate and propionate as carbon sources

Enhanced biological phosphorus removal (EBPR) is a widely used process for achieving phosphorus removal from wastewater. A potential reason for EBPR failure is the undesirable growth of glycogen accumulating organisms (GAOs), which can compete for carbon sources with the bacterial group responsible for phosphorus removal from wastewater: the polyphosphate accumulating organisms (PAOs). This study investigates the impact of carbon source on EBPR performance and the competition between PAOs and GAOs. Two sequencing batch reactors (SBRs) were operated during a 4-6 month period and fed with a media containing acetate or propionate, respectively, as the sole carbon source. It was found that the acetate fed SBR rarely achieved a high level of phosphorus removal, and that a large portion of the microbial community was comprised of "Candidatus Competibacter phosphatis", a known GAO. The propionate fed SBR, however, achieved stable phosphorus removal throughout the study, apart from one brief disturbance. The bacterial community of the propionate fed SBR was dominated by "Candidatus Accumulibacter phosphatis", a known PAO, and did not contain Competibacter. In a separate experiment, another SBR was seeded with a mixture of PAOs and a group of alphaproteobacterial GAOs, both enriched with propionate as the sole carbon source. Stable EBPR was achieved and the PAO population increased while the GAOs appeared to be out-competed. The results of this paper suggest that propionate may provide PAOs with a selective advantage over GAOs in the PAO-GAO competition, particularly through the minimisation of Competibacter. Propionate may be a more suitable substrate than acetate for enhancing phosphorus removal in EBPR systems.     

Metabolic Pathway for Propionate Utilization by Phosphorus-Accumulating Organisms in Activated Sludge: 13C Labeling and In Vivo Nuclear Magnetic Resonance

In vivo ¹³C and ³¹P nuclear magnetic resonance techniques were used to study propionate metabolism by activated sludge in enhanced biological phosphorus removal systems. The fate of label supplied in [3-¹³C]propionate was monitored in living cells subjected to anaerobic/aerobic cycles. During the anaerobic phase, propionate was converted to polyhydroxyalkanoates (PHA) with the following monomer composition: hydroxyvalerate, 74.2%; hydroxymethylvalerate, 16.9%; hydroxymethylbutyrate, 8.6%; and hydroxybutyrate, 0.3%. The isotopic enrichment in the different carbon atoms of hydroxyvalerate (HV) produced during the first anaerobic stage was determined: HV₅, 59%; HV₄, 5.0%; HV₃, 1.1%; HV₂, 3.5%; and HV₁, 2.8%. A large proportion of the supplied label ended up on carbon C-5 of HV, directly derived from the pool of propionyl-coenzyme A (CoA), which is primarily labeled on C-3; useful information on the nature of operating metabolic pathways was provided by the extent of labeling on C-1, C-2, and C-4. The labeling pattern on C-1 and C-2 was explained by the conversion of propionyl-CoA to acetyl-CoA via succinyl-CoA and the left branch of the tricarboxylic acid cycle, which involves scrambling of label between the inner carbons of succinate. This constitutes solid evidence for the operation of succinate dehydrogenase under anaerobic conditions. The labeling in HV₄ is explained by backflux from succinate to propionyl-CoA. The involvement of glycogen in the metabolism of propionate was also demonstrated; moreover, it was shown that the acetyl moiety to the synthesis of PHA was derived preferentially from glycogen. According to the proposed metabolic scheme, the decarboxylation of pyruvate is coupled to the production of hydrogen, and the missing reducing equivalents should be derived from a source other than glycogen metabolism.     

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.     

强化生物除磷系统主要微生物及其代谢机理研究进展

强化生物除磷(enhanced biological phosphorus removal,EBPR)工艺在废水除磷处理中应用广泛.主要功能微生物及其代谢机理的研究是有效调控EBPR工艺稳定运行与效能提升的基础.本文选取EBPR系统中最主要的两类微生物(聚磷菌和聚糖菌),从底物吸收机制、糖酵解途径、TCA途径的贡献以及聚磷菌和聚糖菌的代谢相似性等方面对这些微生物的代谢机理进行综述,评价了分子生物学技术在研究EBPR系统微生物学及其代谢机理方面的应用现状,在此基础上对EBPR系统今后的研究方向进行了展望.