Distributed state simulation of endogenous processes in biological wastewater treatment

Distributed state-type simulations (based on modeling of individual bacteria as they move through a reactor system) predicted a greater sensitivity of enhanced biological phosphorus removal (EBPR) performance to endogenous degradation than did conventional, "lumped"-type simulations (based on average biomass compositions). Recent research has indicated that the variable hydraulic residence times experienced by individual microbial storage product accumulating bacteria in systems with completely mixed reactors tend to produce populations with diverse microbial storage product contents (distributed states). Endogenous degradation in EBPR systems is of particular interest because the polyphosphate accumulating organisms (PAOs) responsible for EBPR rely on the accumulation of three different storage products that may be endogenously degraded. Simulations indicated that as endogenous degradation rates of microbial storage products were increased, EBPR performance decreased more rapidly according to the distributed approach than according to the lumped approach. State profile analysis demonstrated that as these rates increased, the population fraction with depleted storage products also increased, and this tended to increase the error in calculated biokinetic rates by the lumped approach. Simulations based on recently reported endogenous rate coefficients also suggested large differences between distributed and lumped predictions of EBPR performance. These results demonstrated that endogenous decay processes may play a more important role in EBPR than predicted by the lumped approach. This suggests a need for further research to determine endogenous process rates, and for incorporation of this information to distributed-type simulators, as this should lead to improved accuracy of EBPR simulations.     

Process hydraulics, distributed bacterial states, and biological phosphorus removal from wastewater

Hydraulic characteristics of biological wastewater treatment systems were shown to affect bacterial state distributions and system performance through mathematical simulations. The term "state" is used here to mean the microbial storage product and biomass content of a bacterium. The traditional approach to simulating biological treatment processes assumes "lumped" (average) states, rather than accounting for variable states across bacterial populations. Distributed states were previously suggested as critical to enhanced biological phosphorus removal (EBPR), but the factors that cause distributed states were not evaluated. A primary driver for distributed state development is variable hydraulic experiences of bacteria as they cycle through completely mixed reactors, and so process characteristics that affect hydraulics were hypothesized to affect state distributions. Two design characteristics affecting system hydraulics were evaluated using a new distributed state simulation program (DisSimulator 1.0): total hydraulic residence time (HRT) and numbers of reactors in series. Distributed predictions consistently predicted worse EBPR performance than did the lumped approach. Increasing HRTs (with constant solids retention times) tended to increase state distributions, to increase the differences between lumped and distributed simulation predictions, and to decrease predicted EBPR performance. As the numbers of reactors in series increased, distributed predictions tended to converge with lumped simulation predictions. Distributed simulations tended to predict a greater benefit to using reactors in series than did lumped simulations. This work provides guidance for new strategies to improve EBPR by minimizing state distributions. The targeted hydraulic characteristics may be more important to EBPR than previously recognized due to their effects on distributed states.     

A review and update of the microbiology of enhanced biological phosphorus removal in wastewater treatment plants

Enhanced biological phosphorus removal (EBPR) from wastewater can be more-or-less practically achieved but the microbiological and biochemical components are not completely understood. EBPR involves cycling microbial biomass and influent wastewater through anaerobic and aerobic zones to achieve a selection of microorganisms with high capacity to accumulate polyphosphate intracellularly in the aerobic period. Biochemical or metabolic modelling of the process has been used to explain the types of carbon and phosphorus transformations in sludge biomass. There are essentially two broad-groupings of microorganisms involved in EBPR. They are polyphosphate accumulating organisms (PAOs) and their supposed carbon-competitors called glycogen accumulating organisms (GAOs). The morphological appearance of microorganisms in EBPR sludges has attracted attention. For example, GAOs as tetrad-arranged cocci and clusters of coccobacillus-shaped PAOs have been much commented upon and the use of simple cellular staining methods has contributed to EBPR knowledge. Acinetobacter and other bacteria were regularly isolated in pure culture from EBPR sludges and were initially thought to be PAOs. However, when contemporary molecular microbial ecology methods in concert with detailed process performance data and simple intracellular polymer staining methods were used, a betaproteobacteria called ‘Candidatus Accumulibacter phosphatis’ was confirmed as a PAO and organisms from a novel gammaproteobacteria lineage were GAOs. To preclude making the mistakes of previous researchers, it is recommended that the sludge ‘biography’ be well understood – i.e. details of phenotype (process performance and biochemistry) and microbial community structure should be linked. This revised version was published online in August 2006 with corrections to the Cover Date.     

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.     

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.
     

聚磷菌和聚糖菌的竞争影响因素研究进展

目前, 强化生物除磷工艺(EBPR)以其经济有效而得到广泛的应用, 该工艺关键在于聚磷菌的富集。然而已经发现, 有一类细菌—聚糖菌(GAOs)能够和聚磷菌(PAOs)竞争, 导致除磷效果恶化。关于PAOs-GAOs的竞争, 研究已经很多, 但是其结论有趋同也有矛盾, 有必要对此进行分析讨论。根据近年来国内外的相关报道, 阐述了聚磷菌与聚糖菌的竞争影响因素, 其中碳磷比、碳源种类、温度、pH值是关键因素, 而污泥龄、溶解氧以及水力停留时间等因素对于PAOs和GAOs的竞争也起一定的作用。此外, 在EBPR系统中, 缺氧条件下, 存在反硝化聚磷菌(DPB)和反硝化聚糖菌(DGAO)也会对聚磷菌富集和系统除磷产生影响。最后对EBPR系统未来的发展方向进行了展望。     

Effect of aspartate and glutamate on the fate of enhanced biological phosphorus removal process and microbial community structure

This study investigated the fate of enhanced biological phosphorus removal (EBPR) and changes in microbial speciation in a sequencing batch reactor (SBR) fed with aspartate and glutamate. It involved SBR operation for 288 days, batch tests for observation of metabolic functions together with microscopic and phylogenetic analyses. Polyphosphate accumulating organisms (PAOs) were observed in abundance with complete removal of phosphorus. Fluorescence in situ hybridization (FISH) combined with 4′,6-dia-midino-2-phenylindole (DAPI) staining confirmed the accumulation of polyphosphate by Rhodocyclus-related and Actinobacterial PAOs. Aspartate seemed to favor the competitive growth of Rhodocyclus-related PAOs since EBPR population used the common biochemical pathways followed by Rhodocyclus-related PAOs in the aspartate fed batch tests. In the glutamate fed batch reactors, however, Actinobacterial PAOs appeared to be competitively selected which explains the lower levels of PHA generation. Even though operational conditions did not change, effective EBPR could not be maintained during the latter part of the study.     

Microbial selection on enhanced biological phosphorus removal systems fed exclusively with glucose

The microbial selection on an enhanced biological phosphorus removal (EBPR) system was investigated in a laboratory-scale sequencing batch reactor fed exclusively with glucose as the carbon source. Fluorescence In Situ Hybridization analysis was performed to target two polyphosphate accumulating organisms (PAOs) (i.e., Candidatus Accumulibacter phosphatis and Microlunatus phosphovorus) and two glycogen accumulating organisms (GAOs) (i.e., Candidatus Competibacter phosphatis and Micropruina glycogenica). The results show that glucose might not select for Candidatus Accumulibacter phosphatis. However, Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica might be selected. The highest percent relative abundance (% RA) of Candidatus Accumulibacter phosphatis was about 42%; this occurred at the beginning of the experimental period when phosphorus removal was efficient. However, the % RA of these bacteria decreased, reaching below 4% at the end of the run. The maximum % RA of Microlunatus phosphovorus, Candidatus Competibacter phosphatis, and Micropruina glycogenica was about 21, 37, 17%, respectively. It appears that a higher glucose concentration might be detrimental for Microlunatus phosphovorus and Micropruina glycogenica. Results also indicate a dominance of GAOs over PAOs when EBPR systems are fed with glucose. It is possible that the GAOs outcompete the PAOs at low pH values; it has been reported that at low pH, GAOs use glycogen as the energy source to uptake glucose. As a result, P-removal deteriorated. Therefore, glucose is not a strong candidate as a carbon source to supplement EBPR systems that do not contain sufficient volatile fatty acids.     

Ecophysiology of a group of uncultured Gammaproteobacterial glycogen-accumulating organisms in full-scale enhanced biological phosphorus removal wastewater treatment plants

The presence of glycogen-accumulating organisms (GAOs) in enhanced biological phosphorus removal (EBPR) plants can seriously deteriorate the biological P-removal by out-competing the polyphosphate-accumulating organisms (PAOs). In this study, uncultured putative GAOs (the GB group, belonging to the Gammaproteobacteria) were investigated in detail in 12 full-scale EBPR plants. Fluorescence in situ hybridization (FISH) revealed that the biovolume of the GB bacteria constituted 2-6% of total bacterial biovolume. At least six different subgroups of the GB bacteria were found, and the number of dominant subgroups present in each plant varied between one and five. Ecophysiological investigations using microautoradiography in combination with FISH showed that, under aerobic or anaerobic conditions, all subgroups of the GB bacteria could take up acetate, pyruvate, propionate and some amino acids, while some subgroups in addition could take up formate and thymidine. Glucose, ethanol, butyrate and several other organic substrates were not taken up. Glycolysis was essential for the anaerobic uptake of organic substrates. Polyhydroxyalkanoates (PHA) but not polyphosphate (polyP) granules were detected in all GB bacterial cells. Polyhydroxyalkanoate formation after anaerobic uptake of acetate was confirmed by measuring the increase in fluorescence intensity of PHA granules inside GB bacterial cells after Nile blue staining. One GB subgroup was possibly able to denitrify, and several others were able to reduce nitrate to nitrite. PAOs were also enumerated by FISH in the same treatment plants. Rhodocyclus-related PAOs and Actinobacteria-related PAOs constituted up to 7% and 29% of total bacterial biovolume respectively. Rhodocyclus-related PAOs always coexisted with the GB bacteria and showed many physiological similarities. Factors of importance for the competition between the three groups of important bacteria in EBPR plants are discussed.     

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.     

增强型生物除磷过程中聚磷酸盐积累微生物的研究进展

从磷污染控制、污水脱磷和磷资源角度论述了生物除磷的作用,并着重论述了增强型生物除磷过程中聚磷酸盐微生物(PAO)的研究历史、代谢特征及研究方法．聚磷酸盐广泛存在于自然界,但只有少数PAO微生物被分离、培养、鉴定出来．培养基能否分离出PAO和PAO能否在实验室条件下表现出polyP积累特征,均至关重要．糖原积累微生物(GAO)与PAO对碳源存在竞争关系,影响EBPR的效率．原位荧光分子杂交、激光共聚焦扫描电镜、微量放射自显影术、活体核磁共振光谱等现代科学技术的发展。使我们能够观察原位微生物群落组成、空间结构和功能变化．对PAO的深入研究,可改进污水脱磷的效率,提高对磷在环境中迁移转化的认识     

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.     

Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

生物除磷系统中聚糖菌代谢机理的研究进展

强化生物除磷（EBPR）被认为是一种最经济、可持续的污水除磷工艺。近年来大量研究报道,系统中聚糖菌的大量繁殖会使除磷工艺性能变差或完全失败。介绍了聚糖菌的代谢机理和影响聚糖菌与聚磷菌之间竞争的因素（如进水基质、P／C、pH值、温度和泥龄等）,便于更好地理解聚糖菌的特性,从而实现提高生物除磷系统运行的性能与稳定性。     

The microbiology of biological phosphorus removal in activated sludge systems

Activated sludge systems are designed and operated globally to remove phosphorus microbiologically, a process called enhanced biological phosphorus removal (EBPR). Yet little is still known about the ecology of EBPR processes, the microbes involved, their functions there and the possible reasons why they often perform unreliably. The application of rRNA-based methods to analyze EBPR community structure has changed dramatically our understanding of the microbial populations responsible for EBPR, but many substantial gaps in our knowledge of the population dynamics of EBPR and its underlying mechanisms remain. This review critically examines what we once thought we knew about the microbial ecology of EBPR, what we think we now know, and what still needs to be elucidated before these processes can be operated and controlled more reliably than is currently possible. It looks at the history of EBPR, the currently available biochemical models, the structure of the microbial communities found in EBPR systems, possible identities of the bacteria responsible, and the evidence why these systems might operate suboptimally. The review stresses the need to extend what have been predominantly laboratory-based studies to full-scale operating plants. It aims to encourage microbiologists and process engineers to collaborate more closely and to bring an interdisciplinary approach to bear on this complex ecosystem.     

“Candidatus Dechloromonas phosphoritropha” and “Ca. D. phosphorivorans”, novel polyphosphate accumulating organisms abundant in wastewater treatment systems

Members of the genus Dechloromonas are often abundant in enhanced biological phosphorus removal (EBPR) systems and are recognized putative polyphosphate accumulating organisms (PAOs), but their role in phosphate removal is still unclear. Here, we used 16S rRNA gene sequencing and fluorescence in situ hybridization (FISH) to investigate the abundance and distribution of Dechloromonas spp. in Danish and global wastewater treatment plants. The two most abundant species worldwide revealed in situ dynamics of important intracellular storage polymers, measured by FISH-Raman in activated sludge from four full-scale EBPR plants and from a lab-scale reactor fed with different substrates. Moreover, seven distinct Dechloromonas species were determined from a set of ten high-quality metagenome-assembled genomes (MAGs) from Danish EBPR plants, each encoding the potential for polyphosphate (poly-P), glycogen, and polyhydroxyalkanoates (PHA) accumulation. The two species exhibited an in situ phenotype in complete accordance with the metabolic information retrieved by the MAGs, with dynamic levels of poly-P, glycogen, and PHA during feast-famine anaerobic–aerobic cycling, legitimately placing these microorganisms among the important PAOs. They are potentially involved in denitrification showing niche partitioning within the genus and with other important PAOs. As no isolates are available for the two species, we propose the names Candidatus Dechloromonas phosphoritropha and Candidatus Dechloromonas phosphorivorans.Subject terms: Water microbiology, Microbial ecology     

Kinetic and stoichiometric characterization for efficient enhanced biological phosphorus removal (EBPR) process at high temperatures

A recently reported stable and efficient EBPR system at high temperatures around 30 °C has led to characterization of kinetic and stoichiometric parameters of the Activated Sludge Model no. 2d (ASM2d). Firstly, suitable model parameters were selected by identifiability analysis. Next, the model was calibrated and validated. ASM2d was found to represent the processes well at 28 and 32 °C except in polyhyroxyalkanoate (PHA) accumulation of the latter. The values of the kinetic parameters for PHA storage (q _PHA), polyphosphate storage (q _PP) and growth (μ _PAO) of polyphosphate-accumulating organisms (PAOs) at 28 and 32 °C were found to be much higher than those reported by previous studies. Besides, the value of the stoichiometric parameter for the requirement of polyphosphate for PHA storage (Y _PO4) was found to decrease as temperature rose from 28 to 32 °C. Values of two other stoichiometric parameters, i.e. the growth yield of heterotrophic organisms (Y _H) and PAOs (Y _PAO), were high at both temperatures. These calibrated parameters imply that the extremely active PAOs of the study were able to store PHA, store polyphosphate and even utilize PHA for cell growth. Besides, the parameters do not follow the Arrhenius correlation due to the previously reported unique microbial clade at 28 and 32 °C, which actively performs EBPR at high temperatures.     

Post-anoxic denitrification driven by PHA and glycogen within enhanced biological phosphorus removal

The objective of this research was to interrogate and develop a better understanding for a process to achieve post-anoxic denitrification without exogenous carbon augmentation within enhanced biological phosphorus removal (EBPR). Sequencing batch reactors fed real wastewater and seeded with mixed microbial consortia were operated under variable anaerobic-aerobic-anoxic and organic carbon loading conditions. The process consistently achieved phosphorus and nitrogen removal, while the observed specific denitrification rates were markedly higher than expected for post-anoxic systems operated without exogenous organic carbon addition. Investigations revealed that post-anoxic denitrification was predominantly driven by glycogen, an intracellular carbon storage polymer associated with EBPR; moreover, glycogen reserves can be significantly depleted post-anoxically without compromising EBPR. Success of the proposed process is predicated on providing sufficient organic acids in the influent wastewater, such that residual nitrate carried over from the post-anoxic period is reduced and polyhydroxyalkanoate (PHA) synthesis occurs.     

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms

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.