Secondary Substrate Research Articles

Modeling cometabolic biodegradation of organic compounds in biofilms

A dynamic model that describes the biodegradation of secondary substrates through oxygenase reactions in biofilms has been developed. The model incorporates intracellular reducing power, in the form of NADH, as a link between the rate of utilization of primary substrates and the biodegradation of secondary substrates. The reaction kinetics were integrated into a diffusive biofilm model with internal and external mass transfer limitations and then combined into a continuous flow reactor model. Preliminary evaluation of the model has demonstrated that mass transfer limitations substantively reduced the removal of the secondary substrate as compared to a suspended growth reactor with an equivalent mass of biomass. A sensitivity analysts revealed that model predictions of the removal of secondary substrate were highly sensitive to the parameters describing the availability of reducing power and to the compound-specific biodegradation kinetic parameters.

A proposed transient model for cometabolism in biofilm systems.

A dynamic model was developed to describe the behaviour of primary and secondary substrates in a biofilm reactor. The model incorporates structured kinetics to describe the generation and consumption of reducing power in the catabolic and respiratory subsystems, respectively. Secondary substrate transformation through oxygenolytic or reductive mechanisms can be modelled under either single or dual limitation of the electron donor and electron acceptor substrates. An example simulation of a theoretical biofilm system was performed.

Ascorbic Acid-Dependent Cytoprotection of Ovarian Cells by Leukocyte and Nonleukocyte Peroxidases

Luteal cells contain high levels of ascorbic acid that is secreted by stimulation with agents like luteinizing hormone (LH) and prostaglandin F 2α (PGF 2α). One role for interstitial ascorbic acid, we propose, may be the detoxification of H 2O 2 by regeneration of catalytically active peroxidase. By serving as a preferred secondary substrate, ascorbic acid regenerates the catalytically active peroxidase that is inhibited irreversibly by H 2O 2 alone. To test this hypothesis, luteal cells were incubated in the absence and presence of peroxidases and H 2O 2, and the maximal cyclic AMP and steroidogenic response to LH was examined. In luteal cells, H 2O 2 is known to severely inhibit LH-sensitive cyclic AMP accumulation and steroidogenesis, and the addition of lactoperoxidase, myeloperoxidase, eosinophil peroxidase, or ascorbic acid (1 mM) alone had no effect on these responses to H 2O 2. However, co-incubation of ascorbic acid and the peroxidases completely reversed the inhibition of cyclic AMP accumulation and steroidogenesis produced by H 2O 2. These findings and the results that show direct oxidation of ascorbic acid in the presence of peroxidase and H 2O 2, but not with H 2O 2 alone, support the conclusion that ascorbic acid released from cells may detoxify H 2O 2 by regenerating the catalytically active state of peroxidases.

Protein memory through altered folding mediated by intramolecular chaperones.

The 77-residue propeptide of subtilisin acts as an intramolecular chaperone that organizes the correct folding of its own protease domain. Similar folding mechanisms are used by several prokaryotic and eukaryotic proteins, including prohormone-convertases. Here we show that the intramolecular chaperone of subtilisin facilitates folding by acting as a template for its protease domain, although it does not form part of that domain. Subtilisin E folded by an intramolecular chaperone with an Ile(-48)-to-Val mutation acquires an 'altered' enzymatically active conformation that differs from wild-type subtilisin E. Although both the altered and wild-type subtilisins have identical amino-acid sequences, as determined by amino-terminal sequencing and mass spectrometry, they bind their cognate intramolecular chaperones with 4.5-fold greater affinity than non-cognate intramolecular chaperones, when added in trans. The two subtilisins also have different secondary structures, thermostability and substrate specificities. Our results indicate that an identical polypeptide can fold into an altered conformation through a mutated intramolecular chaperone and maintains memory of the folding process. Such a phenomenon, which we term 'protein memory', may be important in investigations of protein folding.

Biodegradation kinetics and fate modelling of pentachlorophenol in bioaugmented activated sludge reactors

The growth and biodegradation kinetics of Mycobacterium chlorophenolicus comb nov. was studied in a series of batch experiments in the laboratory to identify and quantify the significant factors for its bioreactor performance. A mathematical model was formulated taking into account the degradation of primary and secondary substrate, as well as the growth of nonspecific and specific biomass. Using model parameters determined by the batch tests, the model was validated by simulations of a continuous flow experiment with bioaugmentation of the PCP-degrading culture in 2 activated sludge systems. A novel technique using immunofluorescence microscopy for quantifying the pure culture made it possible to evaluate the specific PCP degradation rates related to the PCP-degrading culture. The comparisons showed an excellent agreement for the total PCP removal, although there were some over- and underestimations of sorption and biodegradation as individual removal processes. The specific first order biodegradation rate constants were about 3 times higher in the bioaugmented systems that in batch experiments with the added culture alone. Even rough estimates for specific biodegradation rate constants may give meaningful predictions of the fate of specific organic compounds in wastewater treatment processes. For practical engineering and management purposes this may in many cases be adequate. The study highlighted the importance of reporting operating conditions in experiments from which biodegradation kinetic parameters such as Y, μ m and K S were derived.

Simulation of diauxic production of cephalosporin C by Cephalosporium acremonium: lag model for fed-batch fermentation.

We extend a previously reported model (Chu, W.B.; Constantinides, A. Biotechnol. Bioeng. 1988, 32, 277-288) for the batch fermentation of cephalosporin C under the diauxic growth of Cephalosporium acremonium on glucose and sucrose to a fed-batch system. For this purpose, a novel lag model is proposed for diauxie, which has two functional forms, each embodying the dependence of lag on total cell mass and secondary substrate concentration. This lag model is applicable for batch simulations for arbitrary initial glucose and sucrose concentrations. We used the previously reported batch data to perform locally optimized fed-batch simulations. When applied to fed-batch fermentations, multiple lag times were accounted for. These studies showed that fed-batch fermentations (under the restriction that cell mass concentration did not exceed 25 g/L) could be more productive than simple batch runs. A representative result for a glucose-pulse fed-batch run at optimal cephalosporin production is a productivity of 4.22 mg of cephalosporin C/(L.h) and a yield of 9.25 mg of cephalosporin C/g of total sugar used.

A model that links growth and secondary metabolite production in plant cell suspension cultures

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. (c) 1995 John Wiley & Sons, Inc.

Effect of secondary substrate on associated small crustaceans in a brackish lagoon

The influence of substrate provided by sessile organisms on epifaunal small crustaceans was investigated in a brackish lagoon in Sendai, Japan. The relative abundances of small crustaceans were compared between two sites at the same tidal height on concrete structures where the species composition of sessile organisms was different. In addition, the species composition of small crustaceans was studied as a function of three experimental substrates. Finally, by manipulating settlement plates, the influences of some environmental factors on the sessile organisms as well as on species interactions were studied. The field investigation and experiment showed that the secondary substrate created by sessile organisms caused the spatial and temporal changes in the most abundant species of small crustaceans. Moreover, some environmental factors and earlier colonists have inhibitory and/or facilitative effects on the colonizations of barnacles and oysters which produced site differences of secondary substrate. In addition, oyster growth over barnacles indicated the competitive superiority of the oyster over barnacles after their colonization. These results indicate that the secondary substrate, especially its physical structure, is an important factor which directly determines the species composition of associated small crustaceans. Environmental factors and interactions among sessile species have an indirect effect on species composition of small crustaceans, because they produce changes in the secondary substrate.

Secondary Substrate Binding in Aspartic Proteinases: Contributions of Subsites S3 and S′2 to kcat

The kinetic parameters kcat and Km were determined at 25°C and pH 5.5 for endothiapepsin and rhizopuspepsin acting on the series of substrates Ac-Alam-Lys-Nph-Alan-amide where Nph is p-nitrophenylalanine, and m and n equal 0-4. Kinetic parameters were also determined at 25°C and pH 3.5 for pig pepsin acting on the series of substrates Ac-Alam-Phe-Nph-Argn-Alan-amide, where m equals 0 to 2 and n equals 0 or 1, and another series based on -Ile-Glu-Phe-Nph-Arg-, which is the core of a series of peptides designed by Dunn et al. (1986, Biochem. J. 237, 899—906). Km values were found to be largely independent of increases in the chain length of the substrates whereas kcat values showed large increases with increasing chain length. With endothiapepsin and rhizopuspepsin the largest increases (between 13-fold and over 150-fold) were obtained when alanine residues were added in positions P3 and P′2 and thus are similar to those observed previously with penicillopepsin. Additions of alanines to positions P2, P′3, and P′4 gave much smaller increases. In the case of pig pepsin the results were not as clearcut. Whereas the largest increases were seen for positions P3 and P′2 only with some of the peptides, the increases were strongly dependent on the length of the peptides. With some of the longer peptides the increases were comparable to those seen for positions P2 and P′3. Thus, whereas the addition of two alanines in position P3 to the dipeptide Ac-Phe-Nph-amide caused a large proportional increase in kcat, the increase was much smaller when the addition was made to the homologous tetrapeptide and even smaller when made to the pentapeptide Ac-Ala-Phe-Nph-Arg-Ala-amide. Additions of arginine in position P′2 gave large increases in kcat for all three peptides of the series.

Biodegradation kinetics of a mixture containing a primary substrate (phenol) and an inhibitory co-metabolite (4-chlorophenol).

Batch experiments on the simultaneous utilization of phenol (primary substrate) and 4-chlorophenol (cometabolic secondary substrate) demonstrated two critical substrate interactions. First, the cometabolic degradation of 4-chlorophenol was proportional to the rate of phenol oxidation, which provided the electrons for the initial monooxygenase reaction. Second, 4-chlorophenol inhibited the oxidation of the primary substrate, phenol. Modeling analyses of the degradation of phenol alone and of phenol and 4-chlorophenol together showed that the proportionality between phenol and 4-chlorophenol degradation rates averaged 0.1 mg 4-CP/mg phenol, which corresponds to 0.5% of the electrons generated by phenol oxidation being used as a cosubstrate for the monooxygenase reaction of 4-chlorophenol. In addition, modeling analyses suggest that 4-chlorophenol was a noncompetitive inhibitor of phenol oxidation for high phenol concentrations, but a competitive inhibitor for low phenol concentrations.

Endogenous Denitrification in Biofilm

The model on endogenous denitrification in a biofilm, previously presented by the authors, was verified well with bench scale experiments. Production of the secondary substrate due to lysis of microorganisms, which is necessary for the progress of endogenous denitrification, was experimentally proved. In order to investigate metabolic characteristics of the substrate, molecular weight distributions of the substrate on CODcr or carbohydrate bases were measured. The percentage of the secondary substrate with molecular weight less than 10,000, was more than 50% on both bases. The experimental results demonstrated that the production rate of the substrate can be expressed by the first order type expression with respect to the concentration of biomass. The specific constants for microorganisms lysis and maximum endogenous denitrification rate were determined (0.45 day−1 and 0.141 day−1, respectively).

The Effect of Oxidation-Reduction Potential on the Biotransformations of Chlorinated Hydrocarbons

Two batch experiments with acetate as the primary substrate and different combinations of chlorinated hydrocarbons as the secondary substrate were carried out to evaluate the effect of the redox potential of the environment on the biotransformations of chlorinated hydrocarbons. In both single and mixed contaminant(s) systems, biotransformations of 100 µg/L of tetrachloroethylene (PCE) and carbon tetrachloride (CT) were observed, but that of 1,1,1-trichloroethane(1,1,1-TCA) was not observed within 108 days. Chlorinated hydrocarbons acted as electron traps and scavenged the electrons when they underwent reductive dechlorination. Adequate activity of free available electrons is necessary for chlorinated hydrocarbons to undergo reductive dechlorination. The environment with low redox potential has relatively strong electron activity and therefore facilitates the biotransformation of the chlorinated hydrocarbons more readily. Disappearance of 17 to 62 % and 22 to 99.9 % of the original concentration of PCE and CT were observed when the redox potentials of the microcosms were ranged from 225 to -263 mV and 188 to -263 mV, respectively. The viable count of microorganisms determined by the epifluorescence technique showed that higher concentration of primary substrate produced more biomass than lower concentration of primary substrate did, but the DNA content of the microbes was not a good biochemical indicator for the biotransformability of the chlorinated hydrocarbons. It is concluded that oxidation-reduction potential is the major factor controlling the biotransformation efficiencies of chlorinated hydrocarbons. In the case of in-situ biorestoration, proper control of redox potential of the environment will give a good result of remediation of the groundwater contaminated with chlorinated hydrocarbons.

Solvent and secondary substrate isotope effects on the acid‐catalyzed ketonization of acetophenone enol in aqueous solution

AbstractThe enol of acetophenone was generated flash photolytically in aqueous solution by photosolvolysis of PhCBrCH2 and photohydration of PhCCH and PhCCD, and rates of its ketonization were measured in dilute perchloric acid solutions in H2O and D2O at 25°C. The rate constants so obtained provide the solvent isotope effects, kH+/kD+ = 5.02 ± 0.08, and the secondary isotope effect for deuterium substitution at the β‐position of the enol double bond, (kH/kD)β = 0.999 ± 0.014. Arguments are presented which show that these isotope effects requre a stepwise rather than a concerted mechanism for the ketonization reaction.

A comparative study of test methods for assessment of the biodegradability of chemicals in seawater—Screening tests and simulation tests

A comparative study has been performed on test methods for assessing the biodegradability of chemicals in seawater environments. A simple shake flask die-away test with natural seawater and 14C-labeled chemicals added in μg/liter concentrations is proposed as a “simulation” test. The analytical parameter used in this test is residual dissolved 14C activity. The performance of the simulation test has been compared with the performance of similar screening tests with dissolved organic carbon analysis and test compounds added in mg/liter concentrations to nutrient-enriched seawater. All chemicals investigated that passed the screening tests were also degradable in the simulation test and some results with simulation tests were positive; even screening tests were negative, while some compounds, including maleinhydrazide, known to be degradable in soil, remained undegraded in either type of test. Disappearance times after the ended lag time were smaller in screening tests than in simulation tests, but the rates of biodegradation cannot be meaningfully compared, as zero-order kinetics in combination with an exponentially growing population of degraders prevail in screening tests, while first-order kinetics and frequently a constant activity of degraders (cooxidation) prevail in simulation tests where the test material is a secondary substrate only. In screening tests, lag times are sometimes excessively long and highly variable. Whether the lag times could be decreased and their variability narrowed by supplementation with a cosubstrate (yeast extract) or by inoculation with seawater that had been preadapted to the test material was investigated. In most experiments such test modifications had no significant effect but in one experiment with 4-nitrophenol, inoculation with 1% preadapted seawater decreased the lag phase from >35 to 9 days.

Long-lived photoinduced charge separation in a redox system trapped in a sol–gel glass

A KEY feature in the mechanism of photosynthesis is the initial storage of a substantial fraction of the light energy in the form of a long-lived radical pair1. In attempts to mimic this process in artificial systems2–4, impressive progress has been made in increasing the efficiency of the charge-separation reaction between an excited photosensitizer and an appropriate electron acceptor, but prevention of the energy-wasting back-reaction to neutral species still constitutes a major challenge. The back-reaction limits the length of time during which charge separation (and thus energy storage) can be maintained. Here we report exceedingly long-lived (up to a few hours) photoinduced charge separation in an artificial photosynthetic system that does not require a secondary substrate to react with the charged species. Our system uses pyrene (Py*) as the photosensitized electron donor and N,N'-dimethy 1-4,4'-bipyridinium (methyl viologen, MV2+) as the electron acceptor, both immobilized in a porous sol–gel silica glass5. The redox reaction is carried out by the mediation of a third mobile charge carrier in the intrapore space6. The spatial separation between the donor and acceptor inhibits the back-reaction to produce the long lifetimes of the charge-separated pair.

Aldehyde dismutation catalyzed by pulmonary carbonyl reductase: kinetic studies of chloral hydrate metabolism to trichloroacetic acid and trichloroethanol

The kinetics of the NAD(P) +-linked aldehyde dismutation by pulmonary carbonyl reductase of guinea pig were studied using a highly hydrated substrate, chloral hydrate (CH). The enzyme irreversibly converted the substrate into trichloroacetic acid (TCA) and trichloroethanol (TCE) in the presence of the reduced or oxidized cofactors, of which NAD(P) + gave a higher reaction rate than did NAD(P)H, and the concentration ratios of the two products (TCA plus TCE) to CH utilized were 1:1. In the NAD(P) +-linked reaction TCA was the predominant product and its amount was compatible with that of TCE plus NAD(P)H produced, whereas in the NAD(P)H-linked reaction equal amounts of TCA and TCE were formed and the cofactor was little oxidized. These results suggest that the enzyme oxidized the hydrated aldehydes to TCA with NAD(P) + as the cofactor and reduced the unhydrated aldehyde to TCE with NAD(P)H. The steady-state kinetic measurements in the NADP +-linked CH oxidation were consistent with an ordered Bi Bi mechanism which is the same as that for the secondary alcohol oxidation by the enzyme. The dehydrogenase activity was inhibited competitively with respect to CH by a secondary alcohol substrate, propan-2-ol. The CH and propan-2-ol dehydrogenase activities were similarly inactivated by 2,4,6-trinitrobenzene-sulfonate, and NADP(H), several cofactor analogs and a cofactor-competitive inhibitor, Cibacron blue dye, protected against the inactivation, which suggest that lysine residues are essential for catalysis.

Microbial Degradation of Pentachlorophenol and Lindane in Laboratory-Scale Activated Sludge Reactors

The microbial degradation of pentachlorophenol (PCP) and lindane in the activated sludge process was studied. 14 steady-state experimental runs were performed in single-stage activated sludge reactors under various operating conditions. PCP and lindane mixed with three other model compounds were added to the feed as secondary substrate, with synthetic sewage as primary substrate. Reactor concentrations (dissolved) of the model compounds were in the range of 1-200 µg/l. For PCP, the biodegradation increased with increasing solids retention time SRT. This indicates that degradation takes place by catabolic growth of a specific fraction of the biomass. At SRT > 8 days the 1st order biodegradation rate constant was about 2.5 × 10−3 1/(mg MLSS day) at a temperature of 15°C. For lindane, the reverse relation was observed. Increased degradation was observed with increasing degradation of primary substrate. This indicates the presence of other mechanisms such as co-metabolism or probably reductive dechlorination. In relation to upgrading of wastewater treatment plants to biological nutrient removal, xenobiotic compounds behaving like PCP are expected to be increasingly degraded. For compounds behaving like lindane, co-metabolic activity can be stimulated in plug-flow reactors rather than CSTR, and anaerobic zones in the activated sludge process may play an important role for the degradation.

Photodynamic action of sulphonated aluminium phthalocyanine (SALPC) on isolated rat pancreatic acini

The photodynamic action of SALPC has been investigated on dispersed, perifused, acini isolated from the rat pancreas. Stimulation of secretion was assessed by measuring amylase release and membrane permeabilization determined by the leakage of cytoplasmic lactate dehydrogenase (LDH) and by the efflux of 86Rb from preloaded acini. Light alone (>570 nm, ⩽ 18,400 lux), or SALPC (⩽ 1 μM) in the absence of light, had no effect on pancreatic acini but cellularly bound SALPC when illuminated caused a dose-dependent, light intensity-dependent and temperature-dependent release of amylase. Singlet oxygen generated by photon-activation of SALPC was measured by the formation of an imidazole adduct and bleaching of the secondary substrate, RNO. Whereas illumination caused a rapid increase in photodynamically-evoked pancreatic amylase release, the efflux of 86Rb and loss of cytosolic LDH were markedly delayed in onset: similar results were obtained with monochromatic laser light (633 nm). In contrast, the muscarinic agonist bethanechol evoked a rapid increase in amylase release but with an almost immediate efflux of 86Rb. Finally, electron microscopy confirmed that the structural integrity of the pancreatic acinar cells was maintained after the photodynamic action of SALPC. It is concluded that the stimulation of amylase secretion and membrane permeabilization by SALPC is due to the generation of singlet oxygen. However, the consistent difference between the time course of amylase secretion and membrane permeabilization makes it likely that an initial stage in photodynamic drug action involves oxidation of plasma membrane protein and activation of secretagogue receptors or the G-proteins and their effector systems.

An enzyme immunoassay for atractyloside, the nephrotoxin of Callilepis laureola (Impila)

Tubers of Callilepis laureola, a traditional remedy, contain an inhibitor of oxidative phosphorylation; atractyloside. A “competitive” ELISA was developed, using the antiserum produced to an atractyloside-protein conjugate. An ovalbumin-atractyloside conjugate was adsorbed to microtitre wells and plates incubated with sample (atractyloside or tuber extract) and antiserum. After successive incubation with secondary antibody-enzyme conjugate and substrate, the absorbance was read at 405nm. Antibody working dilution was low, but results, confirmed by thin layer chromatography, indicate the immunoassay has diagnostic potential.

Checkerboard immunoblotting (CBIB): an efficient, rapid, and sensitive method of assaying multiple antigen/antibody cross-reactivities

A simple technique, checkerboard immunoblotting (CBIB), is described, which facilitates the examination of multiple antigen/antibody interactions, conveniently and reproducibly, using minimal amounts of reactants. Antigens, immobilized on a solid-phase membrane in parallel lanes, are allowed to react with primary antibodies, applied in lanes perpendicular to the antigens, and the reactions are developed with appropriately labeled secondary antibody and substrate. Positive reactions, at the intersections of antigen/antibody lanes, are small squares, giving a checkerboard apperance to the blot. The results are easily read visually and presented in the form of a permanent record. CBIB has wide range of applications, including the screening of hybridomas for monoclonal antibody production. With the cholera toxin (CT)-related antigens used, homologous reactions were markedly stronger then heterologous reactions.