Ethene as an auxiliary substrate for the cooxidation of cis-1,2-dichloroethene and vinyl chloride

Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3–20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade cDCE (400 μmol l^–1) or vinyl chloride (100 μmol l^–1) in the presence of ethene (≤ 80 μmol l^–1 and ≤ 210 μmol l^–1, respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l^–1 in the liquid phase). Degradation of cDCE (200 μmol l^–1) was not affected by the presence of trichloroethene (100 μmol l^–1) or tetrachloroethene (100 μmol l^–1). Transformation yields (T_y, defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml^–1) was unaffected by the transformation of ≤ 200 μmol l^–1 cDCE in 300 min. Received: 9 March 1999 / Accepted: 21 July 1999     

Glucose as a substrate in recombinant strain fermentation technology

Summary Glucose supplements to complex growth media of Escherichia coli affect the production of a recombinant model protein under the control of a temperature-sensitive expression system. The bacterial Crabtree effect, which occurs in the presence of glucose under aerobic conditions, not only represses the formation of citric acid cycle enzymes, but also represses the formation of the plasmid-encoded product even though the synthesis of this protein is under the control of the temperature-inducible lambda P _R-promoter/cl857-repressor expression system. When the recombinant E. coli is grown at a moderate temperature (35° C) with protein hydrolysate and glucose as substrates, a biphasic growth and production pattern is observed. In the first phase, the cells grow with a high specific growth rate, utilizing glucose and forming glutamate as a byproduct. The intracellular level of recombinant protein is very low in this phase. Later, glutamate is consumed, indicating an active citric acid cycle. The degradation of glutamate is accompanied by the intracellular accumulation of high amounts of recombinant protein.     

Formate as an auxiliary substrate for glucose-limited cultivation of Penicillium chrysogenum: impact on penicillin G production and biomass yield

Production of beta-lactams by the filamentous fungus Penicillium chrysogenum requires a substantial input of ATP. During glucose-limited growth, this ATP is derived from glucose dissimilation, which reduces the product yield on glucose. The present study has investigated whether penicillin G yields on glucose can be enhanced by cofeeding of an auxiliary substrate that acts as an energy source but not as a carbon substrate. As a model system, a high-producing industrial strain of P. chrysogenum was grown in chemostat cultures on mixed substrates containing different molar ratios of formate and glucose. Up to a formate-to-glucose ratio of 4.5 mol.mol(-1), an increasing rate of formate oxidation via a cytosolic NAD(+)-dependent formate dehydrogenase increasingly replaced the dissimilatory flow of glucose. This resulted in increased biomass yields on glucose. Since at these formate-to-glucose ratios the specific penicillin G production rate remained constant, the volumetric productivity increased. Metabolic modeling studies indicated that formate transport in P. chrysogenum does not require an input of free energy. At formate-to-glucose ratios above 4.5 mol.mol(-1), the residual formate concentrations in the cultures increased, probably due to kinetic constraints in the formate-oxidizing system. The accumulation of formate coincided with a loss of the coupling between formate oxidation and the production of biomass and penicillin G. These results demonstrate that, in principle, mixed-substrate feeding can be used to increase the yield on a carbon source of assimilatory products such as beta-lactams.     

The auxiliary substrate concept — an approach for overcoming limits of microbial performances

In biotechnological processes, fundamental performances of microorganisms are used. The economy of these processes is essentially determined by the efficiency, velocity (productivity) and quality of the products. Therefore it is a permanent task and challenge for basic and biotechnological research to seek out measures for improving the actually attained parameters. The auxiliary substrate concept supplics an approach. It is based on the fact that chemo-organo-heterotrophic substrates differ in the carbon: energy ratio, thus, growth yield is limited in energy and/or reducing power. It says that, by simultaneous utilization of physiologically similar substrates (mixed substrates), the growth yield increases. The substrates are to combine in such a way that with their simultaneous utilization a minimum of carbon is dissimilated merely for the purpose of the generation of biologically useful energy and/or reducing power. Since all chemo-organo-heterotrophic substrates are more or less energy-deficient, an increase in growth efficiency can be expected if the individual substrates of the mixture are assimilated more efficiently than the respective substrates alone. This may result, for instance, from an immediate assimilation of a substrate (according to the “manner of finished part construction”). An increased growth rate is rather the rule than the exception in mixed substrate utilization. In product syntheses the substrates are, depending on the concrete product and metabolic pathway, either energy-excess or energy-excess or energy-deficient. or, in other words, the processes are energy-generating or energy-consuming, respectively. If this is responsible for discrepancies between the possible yields determined by the carbon metabolism and the experimentally obtained yields, the discrepancies should be able to be decreased and the yields increased by mixing substrates. The substrates are to choose and combine so that, due to simultaneous utilization, the product formation process becomes energy neutral. As a rule, the enhanced efficiency is accompanied by an increased velocity. This does not only apply to syntheses, but also to degradation (and detoxification) reactions. Even supposedly inert compounds or persistent substances can be activated by simultaneous (co-)metabolization of another (an auxiliary substrate, victim substrate or co-substrate) and converted at a considerable rate. It is of interest for syntheses of products but in particular for degradation and decontamination of harmful and waste products in the environment that the residual concentrations of the substrates are smaller than those achieved if the compounds of a mixture are metabolized separately. The auxiliary substrate concept has proven to be fruitful, both for theoretical and practical questions. It was practically already being used before it was formulated (mixed substrate utilization, cometabolism). However, an abundance of regulatory and energetic aspects are waiting to be investigated in more detail.     

A Procedure for gluconic acid synthesis with bacteria made continuous by means of an auxiliary substrate

A method is introduced which makes a continuous oxidation of glucose to glucose acid possible. This method is based on the auxiliary-substrate concept and co-metabolism, respectively. Micro-organisms (e.g. Acinetobacter calcoaceticus), which cannot assimilate glucose, but merely oxidize it, are grown continuously on a heterotrophic substrate (e.g. acetate). While growing they simultaneously synthesize gluconic acid. The productivity of the gluconic acid synthesis with a given strain depends on the dilution rate and the mixing proportion. Since growth and product synthesis are closely connected and growth yield is very much higher due to an auxiliary substrate effect in the presence of glucose than on the heterotrophic substrate alone, this method is suitable for SCP production as well. The productivity of gluconic acid production is controlled at a certain dilution rate by the mixing proportion of the growth substrate and glucose.     

AMPA receptors commandeer an ancient cargo exporter for use as an auxiliary subunit for signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling.     

Glucose as an energy donor in acetate growing Acinetobacter calcoaceticus

Since glucose can be oxidized but not assimilated by Acinetobacter calcoaceticus 69-V the question arose whether energy generated by glucose oxidation can help incorporate carbon from heterotrophic substrates and, if so, what the efficiency of ATP production is like. For this reason this species was grown in the chemostat on acetate. After having reached steady state conditions an increasing concentration of glucose was added. This led to an increase in the biomass level from about 0.4 g/g for growth on acetate alone to 0.6–0.65 g/g in the presence of glucose, independently of either the growth rate or the steepness of the glucose gradient used. This upper value approximates about the limit of the carbon conversion efficiency calculated for non-glycolytic substrates. Glucose was almost exclusively oxidized to gluconic acid, 2- and 5-ketogluconates, and pentose 5-phosphates were found only in traces. These results demonstrate that glucose functions as an additional energy source in Acinetobacter calcoaceticus 69-V. From the transient behaviour of biomass increase and the mixing proportion at which the maximum growth yield on acetate in the presence of glucose was obtained it followed that two mol of ATP must have been generated per mol of glucose oxidized. This property is discussed in terms of coupling glucose dehydrogenase with the respiratory chain.Abbreviations G _ox glucose oxidized to gluconic acid - G _t amount of glucose necessary for complete substitution of S _d - S _o inlet concentration of the limiting carbon substrate - S _a and S _d assimilated and dissimilated part respectively of the carbon substrate - PQQ pyrrolo-quinoline-quinone - V _ATP ^Ac ATP gain from complete oxidation to CO₂ of acetate (P/O=2) - V _ATP ^Glc ATP gain from oxidation of glucose to gluconic acid     

Hydrogen peroxide as an alternate substrate for the oxygen-evolving complex

Photosystem II reaction centers evolve O2 in the dark when H2O2 is added as a substrate. Although some of this activity can be attributed to catalase, as much as 75% of the activity was not affected by the addition of 1 mM KCN. Several lines of evidence demonstrate that this KCN-insensitive O2 evolution from H2O2 in the dark is catalyzed by the cycling of S states in the oxygen-evolving complex including: inactivation of H2O2-mediated O2 evolution by Ca/EDTA washing; susceptibility of the activity to inhibition by amines like ammonia and Tris; inhibition by CCCP which is known to accelerate the rate of deactivation of the S2 state and; a direct dependence of the rate of O2 evolution on the presence of calcium and chloride.     

The substrate analog bromopyruvate as a substrate, an inhibitor and an alkylating agent of malic enzyme of pigeon liver

Bromopyruvate is an alkylating agent of pigeon liver malic enzyme (malate dehydrogenase (decarboxylating), EC 1.1.1.40). It combines first with the enzyme to give an enzyme-bromopyruvate complex, then reacts with a proximal -SH group, resulting in the formation of a pyruvate derivative. Bromopyruvate is also a substrate for the reductase partial reaction, and a non-competitive inhibitor of L-malate in the overall oxidative decarboxylase reaction catalyzed by this enzyme. Modification of the -SH group by this compound is accompanied by concomitant loss of both oxidative decarboxylase activity and reductase activity on bromopyruvate. Inactivation of the overall activity is partially prevented by NADP⁺ or NADPH, singly or in combination with L-malate.     

Alginate as an auxiliary bacterial membrane: binding of membrane-active peptides by polysaccharides.

The chronicity of Pseudomonas aeruginosa infections in cystic fibrosis (CF) patients is characterized by overproduction of the exopolysaccharide alginate, in which biofilm bacteria are embedded. Alginate apparently contributes to the antibiotic resistance of bacteria in this form by acting as a diffusion barrier to positively charged antimicrobial agents. We have been investigating cationic antimicrobial peptides (CAPs) (prototypic sequence: KKAAAXAAAAAXAAWAAXAAAKKKK-NH(2), where X is any of the 20 commonly occurring amino acids) that were originally designed as transmembrane mimetic peptides. Peptides of this group above a specific hydrophobicity threshold insert spontaneously into membranes and have antibacterial activity at micromolar concentrations. While investigating the molecular basis of biofilm resistance to peptides, we found that the anionic alginate polysaccharide induces conformational changes in the most hydrophobic of these peptides typically associated with insertion of such peptides into membrane environments [Chan et al., J. Biol. Chem. (2004) vol. 279, pp. 38749-38754]. Through a combination of experiments measuring release of the fluorescent dye calcein from phospholipid vesicles, peptide interactions with vesicles in the presence and absence of alginate, and affinity of peptides for alginate as a function of net peptide core hydrophobicity, we show here that alginate offers a microenvironment that provides a protective mechanism for the encased bacteria by both binding and promoting the self-association of the CAPs. The overall results indicate that hydrophilic alginate polymers contain a significant hydrophobic compartment, and behave as an 'auxiliary membrane' for bacteria, thus identifying a unique protective role for biofilm exopolysaccharide matrices.     

Glucose stimulates the tyrosine phosphorylation of Crk-associated substrate in pancreatic beta-cells

Several years ago, we demonstrated that glucose induced tyrosine phosphorylation of a 125-kDa protein (p125) in pancreatic beta-cells (Konrad, R. J., Dean, R. M., Young, R. A., Bilings, P. C., and Wolf, B. A. (1996) J. Biol. Chem. 271, 24179-24186). Glucose induced p125 tyrosine phosphorylation in beta-TC3 insulinoma cells, beta-HC9 cells, and in freshly isolated rat islets, whereas increased tyrosine phosphorylation was not observed with other fuel secretagogues. Initial efforts to identify p125 were unsuccessful, so a new approach was taken. The protein was purified from betaTC6,F7 cells via an immunodepletion method. After electrophoresis and colloidal Coomassie Blue staining, the area of the gel corresponding to p125 was excised and subjected to tryptic digestion. Afterward, mass spectrometry was performed and the presence of Crk-associated substrate (Cas) was detected. Commercially available antibodies against Cas were obtained and tested directly in beta-cells, confirming glucose-induced tyrosine phosphorylation of Cas. Further experiments demonstrated that in beta-cells the glucose-induced increase in Cas tyrosine phosphorylation occurs immediately and is not accompanied by increased focal adhesion kinase tyrosine phosphorylation. Finally, it is also demonstrated via Western blotting that Cas is present in normal isolated rat islets. Together, these results show that the identity of the previously described p125 beta-cell protein is Cas and that Cas undergoes rapid glucose-induced tyrosine phosphorylation in beta-cells.     

Glucose as a mitogenic hormone

Glucose stimulates insulin secretion in β cells, which sense glucose concentrations through signals derived from glycolytic metabolism. In this issue of Cell Metabolism, Dor and colleagues (Porat et al., 2011) combine genetic, transplantation, and pharmacologic approaches to show that glucose also stimulates β cell proliferation through similar metabolic signals, providing a new framework to develop therapeutics for diabetes.     

Glucose enzyme electrode using cytochrome b(562) as an electron mediator

We demonstrate the construction of glucose sensors employing pyrroloquinoline quinone (PQQ) glucose dehydrogenase (PQQGDH) from Acinetobacter calcoaceticus and glucose oxidase (GOD) from Aspergillus nigar coupled with Escherichia coli soluble cytochrome b(562) (cyt b(562)) as electron acceptor. PQQGDH and GOD do not show direct electrochemical recycling of the prosthetic group at the electrode surface leading to a corresponding current signal. We constructed PQQGDH and GOD electrodes co-immobilized with 100-fold molar excess of cyt b(562) and investigated the electrochemical properties without synthetic electron mediators. PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes both responded well to glucose whereas no current increase was observed from the electrode immobilizing enzyme alone. The detection limits for the PQQGDH/cyt b(562) and GOD/cyt b(562) electrodes were 0.1 and 0.8 mM, respectively, and their linearity extended to over 2 and 9 mM, respectively. These results demonstrate that a sensor system can be constructed without a synthetic electron mediator by using a natural electron acceptor. Furthermore, we have demonstrated the potential application of cyt b(562) in direct electron transfer type sensor systems with oxidoreductases whose quaternary structure do not contain any electron transfer subunit.     

Genistein as an endogenous natural substrate of acidic peroxidases in lupin hypocotyls

Crude steam distillate from Ocimum gratissimum sprayed onto infection courts on detached cocoa pods moments after inoculation with Phytophthora palmivora completely inhibited the pathogen and blackpod lesion development on 75% of the infection courts. Disease suppression obtained with the extract was comparable to that obtained with a 2% Kocide 101 suspension. In the field, the O. gratissimum extract also suppressed lesion development although to a significantly lower (P = 0.05) extent in comparison with Kocide 101. Blackpod lesion expansion rates of 3.80, 3.56, 2.71 and 0.78 cm/day, respectively, were associated with pods treated in the field with C. citratus extract, tap water, O. gratissimum extract and 2% Kocide 101. The extract from Cymbopogon citratus was also ineffective on detached pods. Sporangia of P. palmivora from sporu-lating blackpod lesions on both detached and non-detached pods lost their infectivity within 1 h of treatment with the O. gratissimum extract. This effect was superior to that obtained with Kocide 101. Fungitoxicity of the extract on pods, however, was lost within 3 h of application. Thus, despite its in vivo effectiveness as an eradicant, the O. gratissimum extract, in its present form, has limited utility as a protectant fungicide.     

n-Dodecane as a substrate for nitrogen fixation by an alkane-utilizingAzospirillum sp.

Azospirillum sp. ANK-BI-11 was isolated from petroliferous soil. Glucose, nutrient broth, and sugar acids showed better growth thann-alkanes under aerobic conditions. The utilization of glucose was inhibited in the presence ofn-hexane. Microaerobically, succinic acid, pyruvic acid, and lactic acid were the best C-sources for acetylene reduction, whereas glucose was the best source for growth.n-Dodecane, a nonconventional C-source, also showed good response towards acetylene reduction, although growth was not so pronounced here as with glucose but was equal to that of Na-succinate. Optimum pH and temperature for acetylene reduction were between 7.0 and 8.0 and 30°C, respectively. Scanning electron microscopic studies revealed structural alteration in the shapes and sizes of the cells ofAzospirillum sp. when grown onn-hexane andn-dodecane compared with the cells grown on glucose.     

Coal fly ash as an amendment to container substrate for Spathiphyllum production

Coal fly ash, possessing alkalinity and containing some essential mineral elements, could be an alternative to lime amendment and a nutrient source of container substrates for ornamental plant growth. This study examined physiochemical properties of three fly ashes collected from Florida, Michigan, and North Carolina and container substrates formulated by incorporating commercial dolomite and the three fly ashes, respectively into a soilless basal substrate. The basal, dolomite- and fly ash-amended substrates were used to grow peace lily (Spathiphyllum Schott 'Ty's Pride'), a popular ornamental foliage plant, in 15-cm diameter containers in a shaded greenhouse. Electrical conductivities and pH of the substrates were monitored monthly. Plant canopy heights and widths, shoot fresh and dry weights were recorded five months after transplanting, and tissue nutrient contents were measured. Three fly ashes and the commercial dolomite were able to raise pH of the basal substrate from 3.8 to about 6.8. Canopy heights and widths as well as shoot fresh and dry weights of plants produced from fly ash-amended substrates were comparable to those produced from dolomite-amended substrate but significantly different from those produced from the basal substrate. On an average, five necrotic leaves appeared from plants produced in the basal substrate; however, less than one necrotic leaf occurred on plants produced in either dolomite- or fly ash-amended substrates. As a result, the quality grade of plants grown in the basal substrate was low, and plants were not marketable. Additionally, electrical conductivities of fly ash-amended substrates were consistently higher during the course of plant growth, suggesting that, in addition to neutralizing pH, the amended fly ashes provide nutrients for peace lily growth, which was confirmed by high nutrient contents in plant shoots. This study demonstrates that the three fly ashes can be alternatives to commercial dolomites used as amendments to soilless substrates for ornamental plant production. Utilization of fly ashes as container substrate amendments should represent a new market for the beneficial use of coal combustion byproducts.