Determination Of Dehydrogenase Activity Research Articles

Express-Method for Determination of the Oxidizing Capacity of Activated Sludge and Its Biofilms in Pulp and Paper Mill

Microbial dehydrogenase activity can help to determine the oxidizing capacity of activated sludge. Here we propose an innovative and automated express-method based on rapid determination of dehydrogenase activity. The measurement is based on the rate of methylene blue reduction by living microbial cells in suspension. A single analysis takes 10 min. The method was adapted for biofilms immobilized on the floating carriers of industrial bioreactors and the kinetics of biological oxidation by activated sludge and biofilms was compared. New parameters were proposed to characterize the biological oxidation under low oxygen levels. The obtained make it possible to quickly determine the dehydrogenase activity of activated sludge and biofilms and promptly monitor the effectiveness of industrial biological wastewater treatment.

The Application of Inosine 5'-Monophosphate Dehydrogenase Activity Determination in Peripheral Blood Mononuclear Cells for Monitoring Mycophenolate Mofetil Therapy in Children with Nephrotic Syndrome.

In pediatric nephrotic syndrome, recommended mycophenolic acid (MPA) pharmacokinetics are higher than those for transplant recipients. In MPA therapeutic monitoring, inosine-5′-monophosphate dehydrogenase (IMPDH) activity may be useful. We modified the method established for renal transplant recipients and determined IMPDH activity in peripheral blood mononuclear cells (PBMCs) from healthy volunteers and children (4–16 years) with nephrotic syndrome treated with mycophenolate mofetil (MMF). From children, four blood samples were collected, and MPA concentrations were also determined. IMPDH activity was calculated using xanthosine monophosphate (XMP) normalized with adenosine monophosphate (AMP), both determined with the HPLC-UV method. The modified method was accurate, precise, and linear for AMP and XMP within 0.50–50.0 μmoL/L. Mean IMPDH activity in volunteers was 45.97 ± 6.24 µmoL·s−1·moL−1 AMP, whereas for children, the values were variable and amounted to 39.23 ± 27.40 µmoL·s−1·moL−1 AMP and 17.97 ± 15.24 µmoL·s−1·moL−1 AMP before the next MMF dose and 1 h afterward, respectively. The modified method may be applied to IMPDH activity determination in children with nephrotic syndrome treated with MMF. IMPDH activity should be determined after one thawing of PBMCs due to the change in AMP and XMP concentrations after subsequent thawing. For children, the lowest IMPDH activity was observed concomitantly with the highest MPA concentration.

Effect of spot burning of logging residues on the properties of mountain forest soils and the occurrence of ground beetles (Coleoptera, Carabidae)

We investigated the relationships between dehydrogenase activity and the physicochemical properties of mountain soils over three and five years from Norway spruce (Picea abies L. Karst) logging residue spot burning and the occurrence of epigeic carabid beetles. Six study sites were utilised, including 18 study plots (nine plots in a mixed coniferous mountain forest site and nine plots in a mixed broadleaf mountain forest site), with five replicate pitfall traps at each site located in southern Poland. Soil samples from the organic horizon were taken for pH, organic carbon, nitrogen, base cation content, acidity and dehydrogenase activity determination. Carabid beetles were monitored in weekly intervals during the period of July to August 2016. The burning of logging residues led to modified soil properties, especially the dehydrogenase activity. In all the tested variants, the activity was higher in soil samples after the burning in comparison to the control variants. We show no positive correlation between dehydrogenase activity and the number of carabid specimens. The preferences of dominant predatory hygrophilous carabids to acid habitats with weakly decomposed organic matter were proven. Simultaneously, the soil organic matter content was positively related to the carabid abundance. The significant impact of forest site conditions and the date of logging residue burning on the number of caught specimens were confirmed. In contrast, no relationships between the species richness, species diversity, mean individual biomass and spot burning effect were found. This work supports the recommendation of spruce fine woody debris utilisation by spot burning on mountain regions with rich habitats presenting moderate wet conditions and small land falls.

Study of Biological Properties of Effective Pesticides Destructor Pseudomonas Putida P2

A promising approach for remediation of contaminated soils is the use of biological products based on microorganisms-destructors. Of particular interest are the strains that can decompose several types of pollutants. Potential strains-destructors should be not only effective but also adapted for certain soil and climatic conditions. In addition, when introducing a significant amount of biological agents into the ecosystem, the lack of toxicological and pathogenic effects on plants, animals, and humans is particularly important. The object of the study is the destructor strain of sim-triazine herbicide of prometrin Pseudomonas putida P2. The study explores the ability of strain to use various pesticides as the only carbon source, the effect of bacterial culture on seed germination, biotesting of strain on toxicity using earthworms. Besides, the study presents the phytopathogenic factors and enzymes causing bacteria invasiveness into animals and humans. The determination of dehydrogenase activity of prometrin destructor strain shows that this strain can also use active ingredients of pesticide malathion, HCH, and 4,4-DDT as only sole carbon source. It is also established that the studied strain Pseudomonas putida P2 increases the germination of seeds of the variety Saratovskaya 70 and carrots of the variety Samson and does not possess pathogenicity factors. The strain biotesting on the earthworms revealed no acute toxicity of the culture. The analysis of data obtained from the model pollution shows that the introduction of the destructor of Prometrium Pseudomonas putida P2 into the soil can create favorable conditions for vital activity and reproduction of earthworms.

Discussion on methods of soil dehydrogenase determination

In this paper, we discuss the influence of different factors on the measured values of dehydrogenase activity. We focus on the incubation time of the sample and optimal 2,3,5-triphenyl-tetrazolium chloride concentration. We provide a comparison between results obtained from three methods: Casida method, standard method, and optimization method. Some disadvantages of traditional methods were critically discussed. We showed that the results of dehydrogenase activity determination strongly depend on the method used. To minimize these discrepancies, the increase in TPF concentration should be described with the kinetic model which allows determining tangent activity. Michaelis–Menten kinetics can be used to describe the relationship between TTC concentration and tangent activities. We suggest using the value of 4 km as the optimal TTC concentration.

Determination of Dehydrogenase Activities Involved in D-Glucose Oxidation in Gluconobacter and Acetobacter Strains.

Acetic acid bacteria (AAB) are known for rapid and incomplete oxidation of an extensively variety of alcohols and carbohydrates, resulting in the accumulation of organic acids as the final products. These oxidative fermentations in AAB are catalyzed by PQQ- or FAD- dependent membrane-bound dehydrogenases. In the present study, the enzyme activity of the membrane-bound dehydrogenases [membrane-bound PQQ-glucose dehydrogenase (mGDH), D-gluconate dehydrogenase (GADH) and membrane-bound glycerol dehydrogenase (GLDH)] involved in the oxidation of D-glucose and D-gluconic acid (GA) was determined in six strains of three different species of AAB (three natural and three type strains). Moreover, the effect of these activities on the production of related metabolites [GA, 2-keto-D-gluconic acid (2KGA) and 5-keto-D-gluconic acid (5KGA)] was analyzed. The natural strains belonging to Gluconobacter showed a high mGDH activity and low activity in GADH and GLDH, whereas the Acetobacter malorum strain presented low activity in the three enzymes. Nevertheless, no correlation was observed between the activity of these enzymes and the concentration of the corresponding metabolites. In fact, all the tested strains were able to oxidize D-glucose to GA, being maximal at the late exponential phase of the AAB growth (24 h), which coincided with D-glucose exhaustion and the maximum mGDH activity. Instead, only some of the tested strains were capable of producing 2KGA and/or 5KGA. In the case of Gluconobacter oxydans strains, no 2KGA production was detected which is related to the absence of GADH activity after 24 h, while in the remaining strains, detection of GADH activity after 24 h resulted in a high accumulation of 2KGA. Therefore, it is possible to choose the best strain depending on the desired product composition. Moreover, the sequences of these genes were used to construct phylogenetic trees. According to the sequence of gcd, gene coding for mGDH, Acetobacter and Komagataeibacter were phylogenetically more closely related each other than with Gluconobacter.

Electrochemical Treatment of Biotreated Landfill Leachate Using a Porous Carbon Nanotube-Containing Cathode: Performance, Toxicity Reduction, and Biodegradability Enhancement

Abstract The electrochemical treatment of biotreated landfill leachate was carried out using a Ti/SnO2-Sb2O5-IrO2 anode and a porous carbon nanotube-containing cathode. The ideal pollutant removal has been achieved by the electrochemical treatment evaluated by the decay of total organic carbon (TOC), chemical oxygen demand (COD), ammonia nitrogen (NH3-N), and total nitrogen (TN). The influences of cathodic potential and external addition of Fe2+ on TOC decay evidenced that both electro-Fenton oxidation and anodic oxidation were accounted for pollutant degradation. Cl− can play an important role in the removal of NH3-N and TN. The determination of dehydrogenase activity and BOD5/COD showed that the leachate toxicity became weaker and the biodegradability was enhanced after the electrochemical treatment. These results suggest that the electrochemical process may present a promising alternative for the advanced treatment of biotreated landfill leachate.

The Chemopreventive Effect of the Dietary Compound Kaempferol on the MCF-7 Human Breast Cancer Cell Line Is Dependent on Inhibition of Glucose Cellular Uptake

Our aim was to investigate the effect of several dietary polyphenols on glucose uptake by breast cancer cells. Uptake of 3H-deoxy-D-glucose (3H-DG) by MCF-7 cells was time-dependent, saturable, and inhibited by cytochalasin B plus phloridzin. In the short-term (26 min), myricetin, chrysin, genistein, resveratrol, kaempferol, and xanthohumol (10–100 µM) inhibited 3H-DG uptake. Kaempferol was found to be the most potent inhibitor of 3H-DG uptake [IC50 of 4 µM (1.6–9.8)], behaving as a mixed-type inhibitor. In the long-term (24 h), kaempferol (30 µM) was also able to inhibit 3H-DG uptake, associated with a 40% decrease in GLUT1 mRNA levels. Interestingly enough, kaempferol (100 µM) revealed antiproliferative (sulforhodamine B and 3H-thymidine incorporation assays) and cytotoxic (extracellular lactate dehydrogenase activity determination) properties, which were mimicked by low extracellular (1 mM) glucose conditions and reversed by high extracellular (20 mM) glucose conditions. Finally, exposure of cells to kaempferol (30 µM) induced an increase in extracellular lactate levels over time (to 731 ± 32% of control after a 24 h exposure), due to inhibition of MCT1-mediated lactate cellular uptake. In conclusion, kaempferol potently inhibits glucose uptake by MCF-7 cells, apparently by decreasing GLUT1-mediated glucose uptake. The antiproliferative and cytotoxic effect of kaempferol in these cells appears to be dependent on this effect.

Study on decaying characteristics of activated sludge from a circular plug-flow reactor using response surface methodology

Using pH values, temperature, and dissolved oxygen as the influencing factors, a decaying characteristics experiment of activated sludge was carried out by combining the LIVE/DEAD® Baclight technique with the 2,3,5-triphenyl tetrazolium chloride – dehydrogenase activity determination method. Using batch experiments, a response surface methodology was applied in the experimental design to determine the most important influential factor in the decay of activated sludge. The activated sludge mixed liquor for the experiment was generated in a laboratory-scale circular plug-flow reactor, which has already been approved for an invention patent. The analyzed results revealed that the most important influential factor in sludge activity decay is the pH, followed by temperature and then dissolved oxygen. After the decay experiment, 40.94–90.03% of sludge activity decay is caused by reduced cell activity, and the rest is due to cell death.

Aerobic and anaerobic microbial activities in the foreland of a receding glacier

The foreland soils of a receding glacier were investigated for both aerobic and anaerobic microbial activities. Estimation of microbial biomass via substrate induced respiration, and determination of dehydrogenase activity, dimethylsulfoxide reduction and ammonification were performed to describe soil microflora and its activity. These parameters distinctly increased along the chronosequence from the youngest to the oldest soil. Additionally, these properties showed remarkably higher values in the samples collected at the end of the growing season indicating distinct seasonal changes. The determination of methane emission by the three soils incubated at 10, 37 and 50 °C clearly pointed to higher methanogenic activities within the thermophilic temperature range. Our results also show that water saturation in order to simulate spring snow melt led to increased emissions of methane due to reduced oxygen availability. Methane release was generally low in the primary soil, slightly increased as soil formation proceeded and in all soils was distinctly higher at the end of the growing season. Potential methane consumption also increased along the foreland and during the year. Overall methanotrophic activity greatly exceeded methanogenic activity, and the net CH4 consumption distinctly increased along the chronosequence.

Characterization of Candida parapsilosis infection of an in vitro reconstituted human oral epithelium

Oral candidosis is a common problem in immunocompromised patients, and whilst Candida albicans is regarded as the principal cause of infection, other non-Candida albicans Candida (NCAC) species are increasingly being recognized as human pathogens. Relatively little is known about the virulence factors associated with NCAC species, and the aim of this study was to use a reconstituted human oral epithelium (RHOE) to examine epithelial infection withCandida parapsilosis. Strains originating from the oral and vaginal mucosa and from the urinary tract were all shown to colonize RHOE in a strain-dependent manner. Strain differences were found in the colonizing morphology and in the extent of invasion of the RHOE. Low invasion of RHOE was detected for strains after 12 h, whereas extensive tissue damage was evident after 24 h when assessed using histological examination and lactate dehydrogenase activity determination. Tissue damage was reduced in the presence of pepstatin A, although C. parapsilosis invasion of the tissue was not inhibited. Real-time polymerase chain reaction of secreted aspartyl proteinase (SAP) genes (SAPP1-3) showed that expression was strain dependent, with an increased expression generally occurring for Candida infecting RHOE compared with planktonic equivalents. In summary, C. parapsilosis was not highly invasive of RHOE but did induce significant tissue damage, which could relate to specific SAPgene expression.

Determination of dehydrogenase activity in sediment of shallow lakes and its ecological significance

以武汉市浅水湖泊(月湖)为实验对象确定了沉积物脱氢酶活性(DHA)测定的最佳条件:以经甲醛灭菌的沉积物为对照,以氯化三苯基四氮唑为电子受体(0.4%),沉积物用量为0.5g,pH值为7.5,培养时间为3h.据上述条件测定的DHA比未优化条件下的相应值高3-5倍,且表现出与沉积物有机质含量更为显著的正相关关系.武汉东湖3个子湖共23个采样点沉积物样品的测试结果进一步证实了上述方法的可行性,同时初步揭示了湖泊沉积物脱氢酶反映微生物活性与有机质状态的生态学意义.;The samples were taken from the shallow urban lakes(Lake Yuehu) in Wuhan city to optimize the determination of dehydrogenase activity (DHA) in sediment, which gave the following suitable conditions: the sediment sterilized by formaldehyde was used as the control, the concentration of electron acceptor (2,3,5-triphenyl-2H-tetrazolium chloride) was 0.4%, the sediment amount used was 0.5g, pH value was 7.5 and the reaction time was 3h. Under these conditions, the DHA assayed was 3-5 times higher, and its positive relation to the content of organic matter in sediment was more significant, compared to those determined by the unmodified method. The data based on the 23 sites from 3 basins of Lake Donghu demonstrated the feasibility of the method. In the same time, the ecological significance of DHA was further shown in terms of the measurements of microbial activities and organic matter status in lake sediment

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

A standardised protocol for the quantification of lactate dehydrogenase activity in saliva

The literature about salivary lactate dehydrogenase (LDH) activity shows different results, conditioned by the diversity of methods used for sampling, handling and analysis. Objetives: The aim of this study was to design a protocol for quantifying salivary lactate dehydrogenase activity. Methods: Whole saliva samples were collected from 100 healthy volunteers by chewing a roll of cotton. Lactate dehydrogenase activity determinations were made with the Cobas Mira Plus II ® autoanalyser using the recommendations proposed by the International Federation of Clinical Chemistry (IFCC) and the French Society of Clinical Biology (SFBC). Samples were stored at room temperature (20–22 °C), in the refrigerator (4 °C) and freezer (−20 °C). The correction capacity of the autoanalyser was evaluated by making serial determinations in samples of saliva diluted in water. Results: The frozen samples, but not those stored in the refrigerator or at room temperature, showed an important degradation of lactate dehydrogenase activity. When the samples were diluted in water, the lactate dehydrogenase activity varied substantially with the International Federation of Clinical Chemistry method after correction, but not when using the French Society of Clinical Biology method. Conclusion: For salivary lactate dehydrogenase activity quantification we suggest that samples be stored at 4 °C and analyse with the French Society of Clinical Biology method.

Measurement of dehydrogenase activity in acid soils rich in organic matter

Dehydrogenase activity can be considered to be a good measure of microbial oxidative activity in soils. It is usually determined by measuring the amount of an artificial electron acceptor reduced by microbial activity, such as a soluble tetrazolium salt with a red colored reduced form (a formazan) that can be determined colorimetrically following extraction with a suitable solvent. In an earlier study of acid organic-matter rich forest soils of Galicia (N.W. Spain), measured dehydrogenase activities were low, at variance with respiratory activity data indicating high biological activity. To investigate the possibility that these low dehydrogenase activities were underestimated due to adsorption of the formazan, we have studied the interaction of six soils with iodonitotetrazolium formazan (INTF). At the same time, the capacities of two extractants, methanol and 1:1 dimethylformamide–ethanol (DMF-ethanol), to extract INTF were compared. The results show that INTF is adsorbed by the soils studied with an intensity that closely correlates with soil carbon content, and that dehydrogenase activity is thus underestimated to a different degree for each soil. A mixture of 1:1 DMF-ethanol was more effective than methanol in extracting INTF, thereby improving estimates of dehydrogenase activity. Correction for the effects of INTF adsorption could be achieved by using reference standards containing soil to construct a separate calibration curve for each soil. These standards were prepared by incorporating different concentrations of INTF with the soil under the same conditions used for determination of the dehydrogenase activity. The use of DMF-ethanol and reference standards containing soil is thus recommended for determination of dehydrogenase activity, at least in soils with similar properties to those studied here.

Toxicity screening of biodegradable polymers. II. Evaluation of cell culture test with medium extract

Cell culture testing with material extracts was applied to toxicity screening of some commercial degradable plastics: a plasticized cellulose acetate, an aliphatic polyester (Bionolle), polyhydroxybutyrate-co-hydroxyvalerate (Biopol), and polycaprolactone (TONE polymer). Cell culture medium with serum was used as extraction medium. Methods for the determination of morphology and viability of cells cultured in the extract were investigated. Phase-contrast light microscopy of cells, enhanced by neutral red staining, provides high-contrast images for qualitative evaluation of cell morphology and lysis. Compared to the determination of protein using the Bradford method and of neutral red uptake, the determination of dehydrogenase activity using 3-[4,5-dimethylthia-zol-2-yl]-2,5-diephenyl-tetrazolium bromide (MTT) is more sensitive and accurate. The relative MTT activity of cells cultured in fresh extracts indicate that TONE polymer (all shapes) and Bionolle (test bars and films) are comparable to materials currently used in the food industry (polyethylene terephthalate, atactic and isotactic polystyrene) with no toxic effects on cells.

Enzyme activities in Appalachian soils: 4. Dehydrogenase

Abstract Dehydrogenase enzymes significantly mediate biological oxidation of organic compounds in soil. Dehydrogenase activities were determined in surface and subsurface horizons of 14 major hill land soils of the Appalachian Region. Soil samples were stored air‐dry (AD) and field‐moist at 4°C prior to determination of dehydrogenase activities. Each soil type had its own level of dehydrogenase activity. The average dehydrogenase activities of surface and subsurface horizons stored at 4°C were 1.4 and 3.3 times higher than soil samples stored AD. Mean dehydrogenase activities of surface horizons were 5.3 and 2.2 times higher than subsurface horizons stored at AD and 4°C, respectively. Depending on storage method (AD or 4°C) and type of horizon (surface or subsurface). dehydrogenase activities were either positively or negatively related to soil moisture status, organic C, total N, soil texture, and forms of ? and S. Most of these relationships were nonsignificant. Dehydrogenase activities were positively ...

Scale-up and scale-down techniques for fermentations of polyene antibiotics

Our philosophy of successful biotechnology transfer to industrial scale covers the comparison of complex sets of microbiological, analytical and bioengineering data from cultivations in various scales and different geometries of mixing with laboratory findings. In particular, the availability of nutrients to producing microorganism should be understood, therefore for quick scaling-up procedure of polyene antibiotics produced by Streptomyces noursei we recommend to use physiological marker as total dehydrogenase activity determination. The utility of scale-down tests for identification of process fluctuation, validation of new substrate batches and simultaneous control of inoculum quality was proved.

Bioluminescent flow sensors: L-lactate dehydrogenase activity determination in serum.

The catalytic activity of serum L-lactate dehydrogenase (LDH), was determined by monitoring the NADH produced by LDH with bacterial bioluminescent enzymes immobilized on a nylon coil. The LDH reaction of L-lactate with NAD took place in a flow-through mixing coil that preceded the bioluminescent detector coil. The response was linear from 1 to 5000 U/l at 37 degrees C and from 3 to 2000 U/l at 25 degrees C. The intra- and inter-assay reproducibility (CV%) were less than 10% and recovery range was 92% to 110%. The results agreed well with those obtained with a spectrophotometric method.

Factor analysis of the relationships between several physico-chemical and microbiological characteristics of some Belgian agricultural soils

Twenty Belgian agricultural soils, 16 of which had been organically cultivated, were examined for their biochemical and microbiological properties. In spite of the very different nature of the characteristics studied, close relationships were found between soil respiration and glucose mineralization rates, biomass-C as measured by the fumigation-incubation method, and several enzyme activities, namely FDA-hydrolase, alkaline phosphatase and dehydrogenase. Correlation coefficients between urease activity and other biological measurements were always found to be endowed with a negative sign. Moreover, factor analysis of the data showed that some physico-chemical characteristics such as soil organic C, total N, clay content and CEC were closely related to most of the biological measurements, while pH and sand content were not. Two procedures for the determination of dehydrogenase activity and two methods of calculation of biomass-C were also compared.