水生态系统中宇宙发展现状

水生态系统中宇宙指人工构建的河流、湖泊、湿地、海洋等水生态系统近自然模拟装置,可以控制一定的物理、化学要素和生境条件,开展生物群落响应研究。中宇宙实验可以弥补传统实验室小型装置模拟要素过于简单,而野外现实系统复杂不可控制和难以开展对比实验的局限。本文列举了当前国际上多个科研机构长期运行的水生态系统中宇宙,将其归纳为流水型中宇宙、围隔装置、陆基模拟池、移动式水槽4类,各类中宇宙一般包括物理-生物模拟系统、自动调控系统、监测分析系统、中央控制系统。近年来,我国水生态系统的研究逐渐从单一环境要素向生态系统整体转变,对中宇宙模拟实验的需求愈加迫切。建议我国水生态系统中宇宙建设应提高结构复杂性,并与野外现场监测相结合,共同支撑更大尺度系统的整体模拟。     

Biocalorimetry- supported analysis of fermentation processes

The close relation between metabolic activity and heat release means that calorimetry can be successfully applied for on-line monitoring of biological processes. Since the use of available calorimeters in biotechnology is difficult because of technical limitations, a new sensitive heat-flux calorimeter working as a laboratory fermenter was developed and tested for different aerobic and anaerobic fermentations with Saccharomyces cerevisiae and Zymommonas mobilis. The aim of the experiments was to demonstrate the abilities of the method for biotechnological purposes. Fermentations as well as the corresponding heat, substrate and product analyses were reproducible. During experiments the heat signal was used as a sensitive and fast indicator for the response of the organisms to changing conditions. One topic was the monitoring of diauxic growth phenomena during batch fermentations, which may affect process productivity. S. cerevisiae was used as the test organism and a protease-excreting Bacillus licheniformis strain as an industrial production system. Other experiments focused on heat measurements in continuous culture under substrate-limiting conditions in order to analyse bacterial nutrient requirements. Again, Z. mobilis was used as the test organism. Ammonium, phosphate, magnesium, biotin and panthothenate, as important substrate compounds, were varied. The results indicate that these nutrients are required in lower amounts for growth than formerly suggested. Thus, a combination of heat measurements and other methods may rapidly improve our knowledge of nutrient requirements even for a well-known microorganism like Z. mobilis. *** DIRECT SUPPORT *** AG903062 00004     

Development of a microbial time/temperature indicator prototype for monitoring the microbiological quality of chilled foods

A time/temperature indicator (TTI) system based on the growth and metabolic activity of a Lactobacillus sakei strain was developed for monitoring food quality throughout the chilled-food chain. In the designed system, an irreversible color change of a chemical chromatic indicator (from red to yellow) progressively occurs due to the pH decline that results from microbial growth and metabolism in a selected medium. The relation of the TTI response (color change) to the growth and metabolic activity (glucose consumption, lactic acid production, pH decrease) of L. sakei was studied. In addition, the temperature dependence of the TTI kinetics was investigated isothermally in the range of 0 to 16 degrees C and modeled with a system of differential equations. At all temperatures tested, the pH and color changes of the TTI system followed closely the growth of L. sakei, with the endpoint (the time at which a distinct visual color change to the final yellow was observed) of the TTI coinciding with a population level of 10(7) to 10(8) CFU/ml. The endpoint decreased from 27 days at 0 degrees C to 2.5 days at 16 degrees C, yielding an activation energy of 97.7 kJ/mol, which was very close to the activation energy of the L. sakei growth rate in the TTI substrate (103.2 kJ/mol). Furthermore, experiments conducted on the effect of the inoculum level showed a negative linear relationship between the level of L. sakei inoculated in the system medium and the endpoint of the TTI. For example, the endpoint at 8 degrees C ranged from 6 to 2 days for inoculum levels of 10(1) and 10(6) CFU/ml, respectively. This relationship allows the easy adjustment of the TTI endpoint at a certain temperature according to the shelf life of the food product of concern by using an appropriate inoculum level of L. sakei. The microbial TTI prototype developed in the present study could be used as an effective tool for monitoring shelf life during the distribution and storage of food products that are spoiled primarily by lactic acid bacteria or other bacteria exhibiting similar kinetic responses and spoilage potentials. Apart from the low cost, the main advantage of the proposed TTI is that its response closely matches the loss of the quality of a food product by simulating the microbial spoilage process in particular environments.     

Advanced electrochemical sensors for cell cancer monitoring

The possibility of using minimally invasive analytical instruments to monitor cancerous cells and their interactions with analytes provide great advances in cancer research and toxicology. The real success in the development of a reliable sensor for cell monitoring depends on the ability to design powerful instrumentation that will facilitate efficient signal transduction from the biological process that occurs in the cellular environment. The resulting sensor should not affect cell viability and must function as well as adapt the system to the specific conditions imposed by the cell culture. Due to their performance, electrochemical biosensors could be used as an effective instrument in cell cancer research for studying biochemical processes, cancer development and progression as well as toxicity monitoring. Current research in this direction is conducted through high-throughput, compact, portable, and easy to use sensors that enable measurement of cells' activity in their optimum environment. This paper discusses the potential of a high-throughput electrochemical multisensor system, so-called the DOX system for monitoring cancerous cells and their interaction with chemical toxins. We describe the methodology, experiments, and the operation principle of this device, and we focus on the challenges encountered in optimizing and adapting the system to the specific cell-culture conditions. The DOX system is also compared with conventional cell-culture techniques.     

A small metabolic flux model to identify transient metabolic regulations in Saccharomyces cerevisiae

The understanding of dynamic metabolic regulations is important for physiological studies and strain characterization tasks. The present study combined transient experiments with online metabolic flux analysis (MFA) in order to quantify metabolic regulations, namely carbon catabolite repression of respiration and transient acetic-acid production, in Saccharomyces cerevisiae during aerobic growth on glucose. The aim was to investigate which additional information can be gained from using a small metabolic flux model to study transient growth provoked by shift-up and shift-down experiments, compared to online monitoring alone. The MFA model allowed us to propose new correlations between pathways of the central metabolism. A linear correlation between glycolytic flux and respiratory capacity holds for shift-down and shift-up experiments. This confirmed that respiratory functions were subjected to carbon catabolite repression and suggested that respiratory capacity is controlled by the glycolytic flux rather than the glucose influx. Furthermore, the model showed that control of repression of respiration by the glycolytic flux was a dynamic phenomenon. Co-factor balancing within the MFA model showed that transient acetic-acid production indicated a transient limitation in another part of the central metabolism but not in oxidative phosphorylation. However, at super-critical growth rates and when coupling of anabolism and catabolism is resumed, the limitation shifts to oxidative phosphorylation, with the consequence that ethanol is formed. The online application of small metabolic flux models to transient experiments enhanced the physiological insight into transient growth and opens up the use of transient experiments as an efficient tool to understand dynamic metabolic regulations.     

Strategies for quantitation of phosphoproteomic data

Recent developments in phosphoproteomic sample-preparation techniques and sensitive mass spectrometry instrumentation have led to large-scale identifications of phosphoproteins and phosphorylation sites from highly complex samples. This has facilitated the implementation of different quantitation strategies in order to study the biological role of protein phosphorylation during disease progression, differentiation or during external stimulation of a cellular system. In this article, a brief summary of the most popular strategies for phosphoproteomic studies is given; however, the main focus will be on different quantitation strategies. Methods for metabolic labeling, chemical modification and label-free quantitation and their applicability or inapplicability in phosphoproteomic studies are discussed.     

Stability of mutations in a Sphingomonas strain.

Sphingomonas sp. strain RW1 is able to mineralise dibenzofuran and dibenzo-p-dioxin. Three mutants were constructed that could not use dibenzofuran or dibenzo-p-dioxin as a carbon source but were able to grow with the succeeding metabolites of the pathway. Two different mutagenic agents were applied, a chemical treatment with 1-methyl-3-nitro-1-nitrosoguanidine, resulting in mutants RW1-N6 and RW1-N7, and a biological insertion mutagenesis with the mini-Tn5 transposon pBSL118, resulting in mutant RW1-M3. Southern blot analysis and PCR experiments confirmed a single insertion of the mini-Tn5 into one of the genes coding for the oxygenase component of the dibenzofuran 4,4a-dioxygenase system. The genetic stability of these mutants was examined after growth with complex medium under nonselective conditions. All three mutants failed to revert to wild-type metabolic functions.     

A metabolic model of the biological phosphorus removal process: II. Validation during start-up conditions

A metabolic model of the biological phosphorus removal process has been developed and validated previously for complex conversions during the process under anaerobic and aerobic conditions at different growth rates in sequencing batch reactors in steady state. For additional validation of the metabolic model, the model was applied to the dynamic conditions which occur during the start-up phase of the biological P removal in the presence and absence of non-polyP heterotrophic microorganisms. In a laboratory scale sequencing batch reactor, experiments were performed to examine the enrichment of the population with polyphosphate organisms during the start-up and the subsequent shift from non-polyP, heterotrophic organisms to polyP organisms in the sludge. The effect of different influent loading patterns for acetate and phosphate was studied. In these experiments, the maximal growth rate of the polyP organisms and the behavior of the internal storage compounds could be derived. The metabolic model was capable of describing the experimental results, without the need to adjust the kinetic or stoichiometric parameters obtained under steady state conditions. (c) 1995 John Wiley & Sons, Inc.     

Metabolomics: a tool for early detection of toxicological effects and an opportunity for biology based grouping of chemicals-from QSAR to QBAR

BASF has developed a Metabolomics database (MetaMap(?) Tox) containing approximately 500 data rich chemicals, agrochemicals and drugs. This metabolome-database has been built based upon 28-day studies in rats (adapted to OECD 407 guideline) with blood sampling and metabolic profiling after 7, 14 and 28 days of test substance treatment. Numerous metabolome patterns have been established for different toxicological targets (liver, kidney, thyroid, testes, blood, nervous system and endocrine system) which are specific for different toxicological modes of action. With these patterns early detection of toxicological effects and the underlying mechanism can now be obtained from routine studies. Early recognition of toxicological mode of action will help to develop new compounds with a more favourable toxicological profile and will also help to reduce the number of animal studies necessary to do so. Thus this technology contributes to animal welfare by means of reduction through refinement (2R), but also has potential as a replacement method by analyzing samples from in vitro studies. With respect to the REACH legislation for which a large number of animal studies will need to be performed, one of the most promising methods to reduce the number of animal experiments is grouping of chemicals and read-across to those which are data rich. So far mostly chemical similarity or QSAR models are driving the selection process of chemical grouping. However, "omics" technologies such as metabolomics may help to optimize the chemical grouping process by providing biologically based criteria for toxicological equivalence. "From QSAR to QBAR" (quantitative biological activity relationship).     

Bioluminescence of Vibrio fischeri in continuous culture: optimal conditions for stability and intensity of photoemission

Continuous cultivation of the bioluminescent bacterium, Vibrio fischeri NRRL-B-11177, in a fermenter provided a constant supply of bacteria in exponential growth phase. These bacteria may be used as stable bioindicators to measure perturbation of the metabolic state by physical and chemical agents. However, optimization of light emission necessitates careful choice of growth medium and culture operating conditions. Optimized conditions that supported long-term cultivation of V. fischeri NRRL-B-11177 with stable intense bioluminescence were: a dilution rate of 0.08-0.09h(-1), air supply of 600mlmin(-1), stirring of 300-350rpm at a constant incubation temperature within the range of 20 to 26 degrees C and pH7.8. These conditions were as successful in a 500-ml as in a 10-ml fermenter. This system provided a reliable long-term (more than 1 month) continuous culture facility for the reproducible measurement of perturbation of V. fischeri by monitoring changes in luminescence.     

The application of a novel heat flux calorimeter for studying growth of Escherichia coli W in aerobic batch culture

The modification and principle of a novel heat flux calorimeter for the in situ, on-line measurement of the heat generated during microbial growth is described. Data concerning the physical characterization of the calorimeter as a fermentor, including stability and sensitivity of the heat signal, are presented. The calorimeter has been successfully applied to the study of the aerobic batch culture of Escherichia coli W on glucose under carbon and nitrogen limitation. A direct correlation between growth and heat evolution was obtained. Quantitative analysis of the data suggests that the new calorimetric technique could be used for monitoring growth and specific metabolic events, for convenient medium optimization, and as a basis for a novel fermentation process control system.     

Methods for prevention of mass development of the cyanobacterium Microcystis aeruginosa Kutz emend. Elenk. in aquatic ecosystems

Methods for prevention of mass development of the cyanobacterium Microcystis aeruginosa Kutz emend. Elenk. in continental water bodies and industrial water supply systems are reviewed. The physicochemical, chemical, and biological methods for prevention of M. aeruginosa development in water bodies and water supply systems are considered; examples of successful inhibition of M. aeruginosa growth in laboratory experiments are demonstrated. The scientific problems are outlined that are to be solved for perfecting techniques for prevention of M. aeruginosa mass development in open water bodies and in closed water supply systems.     

Online NMR for monitoring biocatalysed reactions

Monitoring biocatalysed reactions and metabolic pathways using NMR spectroscopy is of growing interest. As a non-invasive analytical method providing simultaneous information about intracellular and extracellular constituents, it is superior to other analytical techniques and has a wide range of applications: kinetics and stoichiometrics of metabolic events, metabolic fluxes and enzyme activities can be detected in situ or after taking a sample from the biotransformation mixture. New NMR pulse sequences provide even more valuable experiments in these fields. Research topics range from the monitoring of polymer formation to fermentations producing beverages or antibiotics. Routine monitoring of industrial fermentations by NMR seems to be imminent.     

Thermodynamics of natural selection II: Chemical Carnot cycles

This is the second in a series of three papers devoted to energy flow and entropy changes in chemical and biological processes, and to their relations to the thermodynamics of computation. In the first paper of the series, it was shown that a general-form dimensional argument from the second law of thermodynamics captures a number of scaling relations governing growth and development across many domains of life. It was also argued that models of physiology based on reversible transformations provide sensible approximations within which the second-law scaling is realized. This paper provides a formal basis for decomposing general cyclic, fixed-temperature chemical reactions, in terms of the chemical equivalent of Carnot's cycle for heat engines. It is shown that the second law relates the minimal chemical work required to perform a cycle to the Kullback-Leibler divergence produced in its chemical output ensemble from that of a Gibbs equilibrium. Reversible models of physiology are used to create reversible models of natural selection, which relate metabolic energy requirements to information gain under optimal conditions. When dissipation is added to models of selection, the second-law constraint is generalized to a relation between metabolic work and the combined energies of growth and maintenance.     

Development of efficient suicide mechanisms for biological containment of bacteria

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.     

Reconstruction of the Saccharopolyspora erythraea genome-scale model and its use for enhancing erythromycin production

Genome-scale metabolic reconstructions are routinely used for the analysis and design of metabolic engineering strategies for production of primary metabolites. The use of such reconstructions for metabolic engineering of antibiotic production is not common due to the lack of simple design algorithms in the absence of a cellular growth objective function. Here, we present the metabolic network reconstruction for the erythromycin producer Saccharopolyspora erythraea NRRL23338. The model was manually curated for primary and secondary metabolism pathways and consists of 1,482 reactions (2,075 genes) and 1,646 metabolites. As part of the model validation, we explored the potential benefits of supplying amino acids and identified five amino acids “compatible” with erythromycin production, whereby if glucose is supplemented with this amino acid on a carbon mole basis, the in silico model predicts that high erythromycin yield is possible without lowering biomass yield. Increased erythromycin titre was confirmed for four of the five amino acids, namely valine, isoleucine, threonine and proline. In bioreactor experiments, supplementation with 2.5?% carbon mole of valine increased the growth rate by 20?% and simultaneously the erythromycin yield on biomass by 50?%. The model presented here can be used as a framework for the future integration of high-throughput biological data sets in S. erythraea and ultimately to realise strain designs capable of increasing erythromycin production closer to the theoretical yield.     

Quantification of proteome dynamics in Corynebacterium glutamicum by (15)N-labeling and selected reaction monitoring

Selected reaction monitoring allows quantitative measurements of proteins over several orders of magnitude in complex biological samples. Here we present a targeted approach for quantification of 19 enzymes from Corynebacterium glutamicum applying isotope dilution mass spectrometry coupled to high performance liquid chromatography (IDMS-LC-MS/MS). Investigations of protein dynamics upon growth on acetate and glucose as sole carbon source shows highly stable peptide amounts for enzymes of the central carbon metabolism during the transition phase and after substrate depletion. However significant adaptations of protein amounts are observed between both growth conditions well agreeing with known changes in metabolic fluxes. Time-resolved measurements of protein expression after metabolic switch from glycolytic to gluconeogenetic conditions reveal fast responses in protein synthesis rates for glyoxylate shunt enzymes.     

Methodology of chemical monitoring in the marine environment

Monitoring of the marine environment requires considerable resources. Effective programs providing information tailored to needs are essential. The design of monitoring programs should follow from a clear strategy, integrating informational needs, chemical, physical and biological processes, statistics, analytical aspects and specific (chemical or hydrological) features of the area to be monitored. A quality system should be devised and maintained. In this paper, monitoring strategy is discussed and critical aspects in the execution are dealt with. Examples from the recently revised Dutch monitoring program are given. Presented at the VI International Wadden Sea Symposium (Biologische Anstalt Helgoland, Wattenmeerstation Sylt, D-2282 List, FRG, 1–4 November 1988)     

Development of efficient suicide mechanisms for biological containment of bacteria.

To optimize plasmid containment, we have systematically investigated the factors that limit the killing efficiency of a suicide system based on the relF gene from Escherichia coli controlled by inducible lac promoters and placed on plasmids. In induction experiments with this suicide system, killing efficiency was unaffected by temperature and growth medium; there was no requirement for great promoter strength or high plasmid copy number. We could demonstrate that the factors limiting killing were the mutation rate of the suicide function and the reduced growth rate caused by a basal level of expression of the suicide gene during normal growth, which can give a selective growth advantage to cells with mutated suicide functions. The capacity of the plasmid-carried killing system to contain the plasmid was tested in transformation, transduction, and conjugational mobilization. The rate of plasmid transfer detected in these experiments seemed too high to provide adequate biological containment. As expected from the induction experiments, plasmids that escaped containment in these transfer experiments turned out to be mutated in the suicide function. With lac-induced suicide as a test, the efficiency of the system was improved by tightening the repression of the suicide gene, thereby preventing selection of cells mutated in the killing function. Reduction of the mutational inactivation rate of the suicide system by duplication of the suicide function augmented the efficiency of the suicide dramatically. These results permit the construction of extremely efficient biological containment systems.     

Metabolic networks evolve towards states of maximum entropy production

A metabolic network can be described by a set of elementary modes or pathways representing discrete metabolic states that support cell function. We have recently shown that in the most likely metabolic state the usage probability of individual elementary modes is distributed according to the Boltzmann distribution law while complying with the principle of maximum entropy production. To demonstrate that a metabolic network evolves towards such state we have carried out adaptive evolution experiments with Thermoanaerobacterium saccharolyticum operating with a reduced metabolic functionality based on a reduced set of elementary modes. In such reduced metabolic network metabolic fluxes can be conveniently computed from the measured metabolite secretion pattern. Over a time span of 300 generations the specific growth rate of the strain continuously increased together with a continuous increase in the rate of entropy production. We show that the rate of entropy production asymptotically approaches the maximum entropy production rate predicted from the state when the usage probability of individual elementary modes is distributed according to the Boltzmann distribution. Therefore, the outcome of evolution of a complex biological system can be predicted in highly quantitative terms using basic statistical mechanical principles.