Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates

A plate assay based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of different dye-labelled polysaccharides as substrates, provides a specific, reliable and rapid simultaneous detection of corresponding polysaccharide-degrading microorganisms. It has potential for increasing the efficacy of screening of microorganisms, utilizing different polysaccharides, in large numbers of natural samples. Diversely colored insoluble forms of amylose, xylan and hydroxyethyl-cellulose (HE-cellulose) were prepared as chromogenic substrates by using the cross-linking reagent 1,4-butanediol diglycidyl ether and the dyes Brilliant Red 3B-A, Cibacron Blue 3GA and Reactive Orange 14. Using the method, the bacteria with amylase or xylanase or cellulase or a combination of these activities were screened from soil and sludge samples, selected and identified according to 16S rDNA sequencing.     

A mathematical model for the continuous culture of microorganisms utilizing inhibitory substrates

A mathematical model is presented for both batch and continuous cultures of microorganisms utilizing inhibitory substrates. The key feature of the model is the use of a inhibition function to relate substrate concentration and specific growth rate. Simulation studies show that the primary result of inhibition by substrate in a batch culture is an increase in the lag time whereas in continuous culture inhibition by substrate may result in process instability. The model should be of value in investigations of the stability of biological processes used for the treatment of certain industrial wastes such as those containing phenols, thiocyanates, nitrates, ammonia, volatile acids, etc., which are known to be inhibitory to many of the organisms metabolizing them.     

Kinetic analysis of a tod-lux bacterial reporter for toluene degradation and trichloroethylene cometabolism

Kinetics of toluene and trichloroethylene (TCE) degradation and bioluminescence from the bioreporter Pseudomonas putida B2 and TVA8 were investigated utilizing batch and continuous culture, respectively. Degradation was modeled using a Michaelis-Menten expression for the competition of two substrates for a single enzyme system, and bioluminescence was modeled assuming a luciferase enzyme saturational dependence on toluene as the inducer and growth substrate. During the batch experiments, bioluminescence increased at approximately 90 namp/min for initial toluene concentrations of 10 to 50 mg/L, but more slowly at higher toluene concentrations, suggesting maximum promoter induction at below 10 mg/L and toxic effects above 50 mg/L toluene. TCE degradation did not occur until toluene depletion, presumably due to competition between toluene and TCE for the toluene dioxygenase enzyme. During continuous culture, bioluminescence transiently increased, then gradually decreased in response to increasing step changes in toluene feed concentration. Bioluminescence in the CSTR appeared to be limited by growth substrate and/or inducer.     

Aerobic degradation of toluene in a closed chemostat with and without a head space outlet

The present paper describes the continuous aerobic cultivation of a Pseudomonas strain with toluene as the substrate in a closed chemostat with oxygen or air as the gas phase. Due to the constant supply of a nitrogen-saturated aqueous medium, nitrogen passes from the liquid phase of the chemostat into the gas phase (head space). This results in an increasing nitrogen content (asymptotic approach to 100%). The concomitant decrease in the partial pressure of the oxygen in the gas phase finally leads to an oxygen limitation for the bacteria in the medium and an incomplete toluene degradation. The critical nitrogen content of the gas phase at which oxygen limitation begins depends on the toluene concentration in the incoming medium. However, when the gas is continuously removed from the head space, the nitrogen content reaches a steady-state value of less than 100%, depending on the flow rate of the outgoing gas. The oxygen limitation and the associated incomplete toluene degradation can be prevented in this way. The method of gas removal from the head space to avoid oxygen limitation is also applicable when the reactor is supplied with air instead of oxygen. Waste waters contaminated with highly volatile pollutants can thus be biologically decontaminated under aerobic conditions, without shifting the pollution problem from the liquid to the gas phase.     

Modification of A-stat for the characterization of microorganisms

Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics. The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter. Physiological studies of Saccharomyces sp. and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch. The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates. Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.     

A theoretical derivation of the Contois equation for kinetic modeling of the microbial degradation of insoluble substrates

Although developed as an empirical model to describe microbial growth on soluble substrates, the Contois equation has been widely accepted for kinetic modeling of insoluble substrate degradation. Yet, the mechanistic basis underlining these successful applications remains unanswered. Unlike soluble substrates that mainly cultivate suspended cultures, microbes cultivated on insoluble substrates have the populations that attach to the substrate surface or remain suspended in the bulk solution, while those attached usually grow faster than those suspended due to their proximity to food resources. This imbalanced growth provides a plausible explanation to the inverse relationship between microbial concentration and their specific growth rate as conveyed in the Contois equation. Based on a theoretical derivation, this study revealed that the Contois equation holds true only when attached microbes substantially obstruct the access of food to their suspended counterparts. On the other hand, when plentiful insoluble substrate surfaces are exposed for cell attachment, the Contois equation will be reduced back to the classic Monod equation.     

Der substratlimitierte pH-Auxostat – Eine neue Methode zur kontinuierlichen Kultur

A method for continuous cultivation of microorganisms is demonstrated, the substrate limited pH-auxostat. The limiting substrate only is added with constant velocity. In this culture the cells grow with high utilization of the limiting substrate and with the highest specific growth rate possible at the given conditions. Yield coefficients and dilution rates of stable K⁺-limited steady states in yeast cultures with different pH-values and biomass concentrations were measured.     

Kinetic model for microbial uptake of insoluble solid-state substrate

A kinetic model for anaerobic digestion of insoluble solid-state substrates was developed. Rate equations for cell growth and substrate consumption were derived based on the assumption that the microorganisms assimilate the substrate mainly at the point of contact where they grow. The model emphasizes effects of substrate particle size, organic loading, and cell concentration on the rates of cell growth and substrate utilization. Batch digestion of a stearic acid emulsion with a mean particle size of 2.0 mum and a biological sludge was conducted at 30 and 37 degrees C to verify the proposed model. Agreement between the experimental and calculated results indicated the validity of the model for describing the microbial degradation of insoluble solid-state substrates. Further examinationof the model revealed that with low cell substrate affinity or at low cell concentration, it coincided with a Michaelis-Menten type kinetics in which the effect of particle size was taken into consideration.     

Continuous assay of protein tyrosine phosphatases based on fluorescence resonance energy transfer

An assay method that continuously measures the protein tyrosine phosphatase (PTP)-catalyzed dephosphorylation reaction based on fluorescence resonance energy transfer (FRET) was developed as an improvement of our previously reported discontinuous version [M. Nishikata, K. Suzuki, Y. Yoshimura, Y. Deyama, A. Matsumoto, Biochem. J. 343 (1999) 385-391]. The assay uses oligopeptide substrates that contain (7-methoxycoumarin-4-yl)acetyl (Mca) group as a fluorescence donor and 2,4-dinitrophenyl (DNP) group as a fluorescence acceptor, in addition to a phosphotyrosine residue located between these two groups. In the assay, a PTP solution is added to a buffer solution containing a FRET substrate and chymotrypsin. The PTP-catalyzed dephosphorylation of the substrate and subsequent chymotryptic cleavage of the dephosphorylated substrate results in a disruption of FRET, thereby increasing Mca fluorescence. In this study, we used FRET substrates that are much more susceptible to chymotryptic cleavage after dephosphorylation than the substrate used in our discontinuous assay, thus enabling the continuous assay without significant PTP inactivation by chymotrypsin. The rate of fluorescence increase strictly reflected the rate of dephosphorylation at appropriate chymotrypsin concentrations. Since the continuous assay allows the measurement of initial rate of dephosphorylation reaction, kinetic parameters for the dephosphorylation reactions of FRET substrates by Yersinia, T-cell and LAR PTPs were determined. The continuous assay was compatible with the measurement of very low PTP activity in a crude enzyme preparation and was comparable in sensitivity to assays that use radiolabeled substrates.     

A method for determining the CO2 released by resting cells of microorganisms during the oxidation of hydrocarbons

A method is proposed for determining the oxidation of hydrocarbon substrates by microorganisms. The method is based on measuring the evolution of CO2 into the medium when the substrate is oxidized by washed microbial cells adsorbed on a spongy surface and incubated in a closed space into which CO2-free air is passed.     

臭氧对无土栽培营养液的消毒作用研究

采用稀释平板法和MPN法,测定了臭氧对无土栽培营养液的消毒杀菌作用。结果表明,随着臭氧浓度(时间)的增加,对微生物杀灭率明显上升;连续处理20min,可杀灭90%以上的细菌、真菌和藻类;臭氧间歇曝气消毒营养液,有效控制微生物的滋生,对莴苣根系无不良影响,增加产量36.8%。     

Selective colonization of insoluble substrates by human faecal bacteria

Insoluble plant polysaccharides and endogenous mucin are important energy sources for human colonic microorganisms. The object of this study was to determine whether or not specific communities colonize these substrates. Using faecal samples from four individuals as inocula for an anaerobic in vitro continuous flow system, the colonization of wheat bran, high amylose starch and porcine gastric mucin was examined. Recovered substrates were extensively washed and the remaining tightly attached bacterial communities were identified using polymerase chain reaction-amplified 16S rRNA gene sequences and fluorescent in situ hybridization. The substrate had a major influence on the species of attached bacteria detected. Sequences retrieved from bran were dominated by clostridial cluster XIVa bacteria, including uncultured relatives of Clostridium hathewayi, Eubacterium rectale and Roseburia species. Bacteroides species were also detected. The most abundant sequences recovered from starch were related to the cultured species Ruminococcus bromii, Bifidobacterium adolescentis, Bifidobacterium breve and E. rectale. The most commonly recovered sequences from mucin were from Bifidobacterium bifidum and uncultured bacteria related to Ruminococcus lactaris. This study suggests that a specific subset of bacteria is likely to be the primary colonizers of particular insoluble colonic substrates. For a given substrate, however, the primary colonizing species may vary between host individuals.     

Interaction of human leukocyte elastase with soluble and insoluble protein substrates. A practical kinetic approach

A progress-curve kinetic method was developed to investigate the interaction between human leukocyte elastase and macromolecular substrates, such as insoluble elastin and soluble plasma proteins. A fluorogenic, synthetic peptide (reporter substrate) was incubated in the presence of finely powdered elastin and enzyme under continuous stirring. The progress curves, which corresponded to the release of product from the reporter substrate, were very sensitive to the presence of various amounts of the macromolecular substrate. The kinetic parameters for the interaction between elastase and elastin were calculated using a pre-steady-state approach characteristic of slow-binding inhibitors. The interaction of elastase with the soluble protein substrates was studied with similar techniques, but formally treating the substrates as classical, fully competitive inhibitors. The adsorption of elastase on insoluble elastin was a time-dependent process consisting of at least three observable phases: The first step was a rapid formation of an encounter complex followed by a very slow step lasting several minutes, and the third step consisted of a steady-state release of products. On the contrary, elastase very rapidly formed productive complexes with bovine serum albumin and a human monoclonal immunoglobulin G. The progress-curve method was also suitable for analyzing the behavior of inhibitors in the presence of protein substrates. The kinetic parameters which characterize the interaction between elastase and protein substrates represent a practical tool to formulate hypotheses on the efficiency of inhibitors in vivo.     

A rapid electrochemical method for assimilation test of microorganisms

Summary A microbial electrode consisting of the immobilized microorganisms to be tested and an oxygen electrode was used to study the assimilation characteristics of microorganisms. When a sample solution containing a substrate was injected into the microbial sensor system, the current of the sensor markedly decreased with time if the microorganisms assimilated the substrate. On the other hand, no current decrease was observed if the microorganisms could not assimilate the substrate. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actinomycetes and activated sludges were tested with various substrates. The time required for a test was 30 min per substrate by the pulse method (sample injection period, 5 min). Good correlations were obtained between this electrochemical method and the conventional growth test. The fundamental differences between the two methods and the application of the electrochemical method are discussed.     

Physiology of Geobacter metallireducens under excess and limitation of electron donors. Part I. Batch cultivation with excess of carbon sources

For microorganisms that play an important role in bioremediation, the adaptation to swift changes in the availability of various substrates is a key for survival. The iron-reducing bacterium Geobacter metallireducens was hypothesized to repress utilization of less preferred substrates in the presence of high concentrations of easily degradable compounds. In our experiments, acetate and ethanol were preferred over benzoate, but benzoate was co-consumed with toluene and butyrate. To reveal overall physiological changes caused by different single substrates and a mixture of acetate plus benzoate, a nano-liquid chromatography–tandem mass spectrometry-based proteomic approach (nano-LC–MS/MS) was performed using label-free quantification. Significant differential expression during growth on different substrates was observed for 155 out of 1477 proteins. The benzoyl-CoA pathway was found to be subjected to incomplete repression during exponential growth on acetate in the presence of benzoate and on butyrate as a single substrate. Peripheral pathways of toluene, ethanol, and butyrate degradation were highly expressed only during growth on the corresponding substrates. However, low expression of these pathways was detected in all other tested conditions. Therefore, G. metallireducens seems to lack strong carbon catabolite repression under high substrate concentrations, which might be advantageous for survival in habitats rich in fatty acids and aromatic hydrocarbons.     

Rates of dissolution and biodegradation of water-insoluble organic compounds.

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.     

Rates of dissolution and biodegradation of water-insoluble organic compounds

We conducted a study of the relationship between the dissolution rates of organic compounds that are sparingly soluble in water and the biodegradation of these compounds by mixed cultures of bacteria. The rates of dissolution of naphthalene and 4-chlorobiphenyl were directly related to their surface areas. The bacteria caused a decline in the concentration of the soluble substrate. The rate of bacterial growth fell abruptly when 4-chlorobiphenyl or naphthalene was no longer detectable in solution. The population continued to increase in media with different surface areas of insoluble 4-chlorobiphenyl, but the final counts were higher in media in which the surface areas of the substrate were larger. The rates of dissolution of palmitic acid, octadecane, di(2-ethylhexyl) phthalate, and 1-naphthyl N-methylcarbamate were determined in the absence of microorganisms. A mixed culture of microorganisms mineralized palmitic acid, di(2-ethylhexyl) phthalate, and Sevin (1-naphthyl N-methylcarbamate) at a logarithmic rate, but octadecane mineralization was linear. The rates of mineralization at the end of the active phase of the biodegradation were lower than the rate of dissolution of palmitic acid but higher than the rate of dissolution of octadecane in the uninoculated medium. We suggest that spontaneous dissolution rates are only one of the factors that govern the rates of biodegradation.     

Potentiometric measurements of carbon dioxide flux of submerged aquatic macrophytes in pH-statted natural waters

SUMMARY. 1, An apparatus has been described that is suitable for potentiometric measurement of carbon dioxide flux in photosynthesizing shoots of submerged aquatic macrophytes 2. The procedure, based on methods described by Tailing (1973) for measurement of phytoplankton photosynthesis, relies upon the continuous pH-statting of the solution surrounding the tissues. The pH of the solution is monitored by electrodes from a pH meter which is linked to an auto-titrator. The rise in pH during photosynthesis is then compensated tor by controlled, small titrant additions of CO₂-ennched solution (titrant water). This replaces the CO₂ removed by the tissues without affecting the total alkalinity of the solution. If the concentration of CO₂ in the titrant water, and the volume of titrant added arc known precisely, the CO₂ flux can be calculated. 3. Total alkalinity, total CO₂ and free-CO₂ acidity of the bathing solutions and titrant waters are estimated by Gran titrations and the pH: tilre-volume data pairs are analysed by computer to provide rapid data feed-back. A modification to Tailing's equation for calculation of F₁functions has been necessary for accurate calibration of the CO₂enriched tilrant water. 4. The photosynthesis cuvette, which is surrounded by a water-jacket, is approximately I dm³ in capacity and has six compartments for the shoots. An impeller at the base of the cuvette rapidly mixes and cycles the bathing solution and flushes it over the tissues. 5. Information on temperature, light flux density, oxygen concentration. pH and titre-volume is continuously recorded into a data-logger and is fed into a computer which is programmed for data analyses. 6. Results from a typical experiment show the system to be sound and the method has considerable potential, especially in the study of aquatic plant photosynthesis in natural waters.     

The dynamics of the macromolecular composition of biomass

The biomass composition of microorganisms depends on the growth conditions. This study explores whether a two-component model can explain how the elemental and macromolecular composition of the biomass of bacteria varies with the specific growth rate. The model describes the rates at which microorganisms assimilate substrates into reserves and utilize reserves for maintenance and growth. Crucial model assumptions are that biomass consists of reserves and structure and that each of these components has an invariant composition. The composition of biomass can vary when the ratio between reserves and structure varies. Literature data on the macromolecular composition of Escherichia coli, cultivated on various substrates, show that the protein, RNA and DNA content of biomass follow a distinctive trend when plotted as a function of the dry-weight-specific growth rate. This observation leads to the proposition that the macromolecular composition of E. coli depends directly on the growth rate, and only indirectly on the carbon- and energy-source used as substrate. We show that the variation of the macromolecular composition of E. coli over its entire range of growth rates can be described with invariant macromolecular compositions of the reserve and structural components of biomass. The model is also applied to our data on a succinate-limited continuous culture of Paracoccus denitrificans.