Use of microbial electrodes for observation of microbiological nitrogen elimination process

Microbial electrodes for the determination of ammonia and the estimation of biochemical oxygen demand (BOD) were applied to the nitrogen elimination process. The dimensions of the nitrification and the denitrification vessels were 170 and 70 L, respectively. The wastewater used for the experiment was obtained from a fermentation factory and adjusted to 470-530 mg/L of total Kjeldahl nitrogen and 1700-3000 mg/L of BOD. The ammonia electrode was assembled with a membrane containing nitrifying bacteria and an oxygen probe. The BOD electrode was similarly constructed, except it used a membrane containing the yeast, Trichosporon cutaneum. A flow system was employed for the automatic measurement of samples every 30 min. The nitrification and denitrification rates of the activated sludge were measured to investigate the optimum conditions and evaluate the capacity of the plant. The various data obtained by the microbial electrodes allowed us to inspect the situation of the plant and estimate control paramerers such as nitrogen and BOD loadings. The average removals of ammonia nitrogen and total Kjeldahl nitrogen were 96% and 89%, respectively, during the experiment for period of 2 weeks.     

Mediated microbial biosensor using a novel yeast strain for wastewater BOD measurement

Two new yeast strains (SPT1 and SPT2) were isolated and immobilized on glassy carbon electrodes to form microbial biosensors for estimation of biochemical oxygen demand (BOD). Ferricyanide was proven to be the most efficient mediator to shuttle electrons from the redox center of reduced microbial enzymes to the electrode in the presence of excess glucose/glutamic acid (GGA). With a 3-fold greater metabolic assimilation capability and greater responses to various effluent samples, SPT1 was selected for sensor-BOD measurements. BOD estimations for the GGA standard resulted in an extended linear range: 2-100 mg/l. Response reproducibility was +/-10% for a GGA standard containing 10 mg BOD/l. For analysis of pulp mill effluents, the BOD detection limit was 2 mg/l with a response time of 5 min.     

Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Polyvinylferrocenium modified Pt electrode for the design of amperometric choline and acetylcholine enzyme electrodes

A simple method of enzyme immobilization was investigated, which is useful for development of enzyme electrodes based on polyvinylferrocenium perchlorate coated Pt electrode surface. Enzymes were incorporated into the polymer matrix via ion exchange process by immersing polyvinylferrocenium perchlorate coated Pt electrode in enzyme solution for several times. Choline and acetylcholine enzyme electrodes were developed by co-immobilizing choline oxidase and acetylcholinesterase in polyvinylferrocenium perchlorate matrix coated on a Pt electrode surface. The amperometric responses of the enzyme electrodes were measured at +0.70 V versus SCE, which was due to the electrooxidation of enzymatically produced H2O2. The effects of the thickness of the polymeric film, pH, temperature, substrate and enzyme concentrations on the response of the enzyme electrode were investigated. The optimum pH was found to be pH 7.4 at 25 degrees C. The steady-state current of these enzyme electrodes were reproducible within +/-5.0% of the relative error. Response time was found to be 30-50s and upper limit of the linear working portions was found to be 1.2mM choline and acetylcholine concentrations in which produced detectable currents were 1.0 x 10(-6)M substrate concentrations. The apparent Michaelis-Menten constant and the activation energy of this immobilized enzyme system were found to be 1.74 mM acetylcholine and 14.9 kJ mol(-1), respectively. The effects of interferents and stability of the enzyme electrodes were also investigated.     

Microbial electrode BOD sensors

Two different types of biochemical oxygen demand (BOD) sensors using microbial electrodes were prepared. First, a microbial electrode using the bacteria–collagen membrane and oxygen electrode was used for the determination of BOD. When the electrode was inserted in a sample solution containing glucose and glutamic acid (model waste water), the current of the electrode decreased markedly with time until a steady state was reached. A linear relationship was observed between the steady state current and the concentration of the standard solution containing glucose–glutamic acid or the BOD of the solution. The BOD of industrial waste waters can be estimated within 15 min by using the microbial electrode. No decrease in current output was observed over a ten day period. The reproducibility was determined using the same sample (10% of the standard solution) and was found to be 26.2 ± 2.0 μA (7.5% of the relative standard deviation). Next, a biofuel cell utilizing microbial electrode (immobilized Clostridium butyricum–platinum electrode) was applied to the estimation of the BOD of waste waters. The current of the biofuel cell was decreased markedly with time until a steady state was reached. The steady state current was in all cases attained within 30–40 min at 37°C. A linear relationship was obtained between the steady state current and BOD. The BOD of industrial waste waters can be estimated by using the biofuel cell. Relative error of the BOD estimation was within ±10%. The current output of the biofuel cell was almost constant for 30 days.     

Effect of pyruvate and oleate on respiration of frog skeletal and heart muscle.

The authors studied the effect of pyruvate and oleate on O2 consumption of the frog sartorius and heart ventricle. 10 mM pyruvate raised O2 consumption of both tissues by over 100%, but only in the winter. Raised O2 consumption was only partly associated with glycogen synthesis from pyruvate. 0.1 mM oleate reduced O2 consumption in both tissues. A marked drop in O2 consumption was observed in the ventricle (up to 50%). Lecithin had a similar effect on O2 consumption. The addition of pyruvate plus oleate led to an 8-fold increase in O2 consumption of the ventricle, i.e. to maximum oxidation capacity of the tissue, but the addition of lecithin inhibited the pyruvate-induced increase in O2 consumption. It is assumed that both pyruvate and oleate influence resting metabolism in a specific manner which cannot be attributed solely to raised availability of substrate for resting energy metabolism requirements.     

Isolation of a microbial consortium from activated sludge for the biological treatment of food waste

The bacterial community in the activated sludge of a local wastewater treatment plant was studied in an effort to understand and exploit the metabolic versatility of microorganisms for the efficient biological treatment of food waste. Microorganisms capable of and efficient in degrading domestic food waste were screened based on their ability to produce areas of clearing on selective media containing protein, fat, cellulose and starch. Nine microbial species belonging to the genera Flavobacterium, Pseudomonas, Micrococcus, Aeromonas, Xanthomonas, Vibrio and Sphingomonas were found to degrade all components of food waste. These bacteria were added to domestic wastewater and shown to cause a 60% reduction in the biochemical oxygen demand (BOD) level of wastewater compared to a control in which no microorganisms were added. The ability of the microbial consortium to degrade domestic wastewater as evidenced by the decrease in BOD levels suggests its potential for use in the biological treatment of food waste.     

Glucose oxidase-polypyrrole electrodes synthesized in p-toluenesulfonic acid and sodium p-toluenesulfonate

Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0-1.0 mM glucose substrate for both electrodes, I(max) value of Pt/PPy-GOD(NapTS) electrode was approximately twice as high as that of Pt/PPy-GOD(pTSA) electrode as 25.4 and 14.2 microA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectrophotometrically using glucose kit. Results revealed that Pt/PPy-GOD(NapTS) electrode exhibited better biosensor response.     

Monitoring the biodegradability of bisphenol A and its metabolic intermediates by manometric respirometry tests

As a result of its wide usage in the production of polycarbonate plastics and epoxy resins, bisphenol A (BPA) is commonly detected in wastewaters. Recently, BPA became a major concern because its adverse effects as an endocrine disruptor. In this work, the biodegradation kinetics of BPA and its metabolic intermediates 4-hydroxyacetophenone (4HAP), 4-hydroxybenzaldehyde (4HB) and 4-hydroxybenzoic acid (4HBA) by BPA-acclimated activated sludge was studied using manometric respirometry (BOD) tests. For all the tested compounds, BOD curves exhibited two phases. In the first one, a fast increase of the oxygen consumption (OC) due to the active oxidation of the substrate was obtained. Then, when the substrate was exhausted, the endogenous respiration produced a slower increase of OC. A standard Monod-model with biomass growth was used to represent the OC profiles as a function of time. For all the tested compounds, a good agreement between the proposed model and the experimental data was obtained. According to their biodegradation rates, the tested compounds can be ordered as follows: BPA < 4HAP ? 4HB < 4HBA. Because the oxidation rate of BPA by BPA-acclimated activated sludge limits the rate of the whole biodegradation pathway, the accumulation of metabolic intermediates 4HAP, 4HB, and 4HBA would be negligible. To calculate the dissolved oxygen (DO) concentration (C) during the BOD tests, the oxygen transfer coefficient (k_La) of the BOD bottles was obtained using the sulfite method. Finally, a simple procedure to calculate the minimum DO concentration (C_min) based on BOD data was developed. Calculation results demonstrated that under the tested conditions, C values were high enough as not to be the limiting substrate for the microbial growth.     

The regulation of pyruvate oxidation during membrane depolarization of rat brain synaptosomes.

Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.     

Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

Bilirubin oxidase from Bacillus pumilus: a promising enzyme for the elaboration of efficient cathodes in biofuel cells

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O(2) reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.     

Novel snowflake-like Pt-Pd bimetallic clusters on screen-printed gold nanofilm electrode for H2O2 and glucose sensing

Novel snowflake-like Pt-Pd bimetallic nanoclusters (Pt-PdBNC) were synthesized on a screen-printed gold nanofilm electrode (SPGFE) substrate by electrochemically reducing precursors with a new constant potential/multi-potential step deposition strategy. The electrocatalytic behavior of the modified electrode (SPGFE/Pt-PdBNC) towards H(2)O(2) was investigated. The results indicate that the as-prepared Pt-PdBNC significantly enhances the electrochemical reduction of H(2)O(2) in neutral media, exhibiting preferable electrocatalytic performance compared to Pt and Pd monometallic nanoclusters. Under optimum conditions, SPGFE/Pt-PdBNC offers linear responses for H(2)O(2) in the concentration range from 0.005 to 6 mM with an ultrahigh sensitivity of 804 mA M(-1) cm(-2) and excellent selectivity. Furthermore, glucose oxidase was immobilized on the Pt-PdBNC structure, and the fabricated biosensor presents favorable properties for glucose sensing.     

Regulation of citric acid cycle by calcium

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.     

Selective glucose detection based on the concept of electrochemical depletion of electroactive species in diffusion layer

A glucose detection approach based on the concept of electrochemical depletion of electroactive species in diffusion layer was established, using scanning electrochemical microscopy (SECM). By controlling the glucose oxidase (GOD) modified electrode (substrate electrode) at a proper potential of electrochemical oxidation of interfering electroactive species, i.e., ascorbic acid (AA), an interference-free microcircumstance was formed in the diffusion layer of the substrate electrode. Consequently, we could successfully sense hydrogen peroxide generated from an enzymatic reaction by locating a Pt ultramicroelectrode (UME) (tip electrode, 5 microm in radius) into the diffusion layer of the substrate electrode. Properties of this interference-removing approach based on electrochemical depletion were systematically investigated. Results showed that the interference-removing efficiency was significantly determined by the tip-substrate distance and substrate potential. When the tip-substrate distance was 11 microm (2.2 times of the tip electrode radius) and the substrate potential was 0.5 V, nearly 90% of AA (0.5 mM) could be depleted within 30s without consumption of H2O2. Under these conditions, 0.1 mM AA showed no influence on the detection of 0.5 mM glucose. The linear range of glucose detection is 0.01-1 mM with a detection limit (DL) of 0.005 mM (correlation coefficient is 0.9948). This research will open a new way for developing selective micro-biosensors.     

Performance Assessment of Full-Scale Wastewater Treatment Plants Based on Seasonal Variability of Microbial Communities via High-Throughput Sequencing

Microbial communities of activated sludge (AS) play a key role in the performance of wastewater treatment processes. However, seasonal variability of microbial population in varying AS-based processes has been poorly correlated with operation of full-scale wastewater treatment systems (WWTSs). In this paper, significant seasonal variability of AS microbial communities in eight WWTSs located in the city of Guangzhou were revealed in terms of 16S rRNA-based Miseq sequencing. Furthermore, variation redundancy analysis (RDA) demonstrated that the microbial community compositions closely correlated with WWTS operation parameters such as temperature, BOD, NH₄⁺-N and TN. Consequently, support vector regression models which reasonably predicted effluent BOD, SS and TN in WWTSs were established based on microbial community compositions. This work provided an alternative tool for rapid assessment on performance of full-scale wastewater treatment plants.     

Glucose oxidase-polypyrrole electrodes synthesized in <Emphasis Type="Italic">p</Emphasis>-toluenesulfonic acid and sodium <Emphasis Type="Italic">p</Emphasis>-toluenesulfonate

Amperometric glucose biosensors have been developed based on entrapment on platinum (Pt) electrode using cyclic voltammetry technique in glucose oxidase (GOD) and pyrrole containing p-toluenesulfonic acid (pTSA) or sodium p-toluenesulfonate (NapTS) as supporting electrolyte solutions. Both of electrolyte solutions were suitable media for the formation and deposition of polypyrrole-GOD (PPy-GOD) layers on Pt substrate. Pt/PPy-GOD electrodes brought about in different morphological properties as well as different electrochemical and biochemical response. The highest responses obtained in pTSA and NapTS electrolytes were observed at pH of 4.5 and 7.0 for Pt/PPy-GOD electrodes, respectively. While linearity was observed between 0.0–1.0 mM glucose substrate for both electrodes, I _max value of Pt/PPy-GOD_NapTS electrode was approximately twice as high as that of Pt/PPy-GOD_pTSA electrode as 25.4 and 14.2 μA, respectively. Five commercial drinks were tested with enzyme electrodes and compared with results obtained spectropho-tometrically using glucose kit. Results revealed that Pt/PPy-GOD_NapTS electrode exhibited better biosensor response.     

Stoichiometry and kinetics of poly-beta-hydroxybutyrate metabolism in aerobic, slow growing, activated sludge cultures

This paper discusses the poly-beta-hydroxybutyrate (PHB) metabolism in aerobic, slow growing, activated sludge cultures, based on experimental data and on a metabolic model. The dynamic conditions which occur in activated sludge processes were simulated in a 2-L sequencing batch reactor (SBR) by subjecting a mixed microbial population to successive periods of external substrate availability (feast period) and no external substrate availability (famine period). Under these conditions intracellular storage and consumption of PHB was observed. It appeared that in the feast period, 66% to almost 100% of the substrate consumed is used for storage of PHB, the remainder is used for growth and maintenance processes. Furthermore, it appeared that at high sludge retention time (SRT) the growth rate in the feast and famine periods was the same. With decreasing SRT the growth rate in the feast period increased relative to the growth rate in the famine period. Acetate consumption and PHB production in the feast period both proceeded with a zero-order rate in acetate and PHB concentration respectively. PHB consumption in the famine period could best be described kinetically with a nth-order degradation equation in PHB concentration. The obtained results are discussed in the context of the general activated sludge models.     

Biochemical Studies on Accelerated Treatment of Thiocyanate by Activated Sludge using Growth Factors such as Pyruvate

S ummary . Micro-organisms in activated sludge oxidizing thiocyanate behave in a similar way to thiobacilli. Organic compounds such as pyruvate stimulate thiocyanate oxidation and in so doing become incorporated into cellular material; such metabolites are not oxidized to produce energy. The presence of thiocyanate in batch cultures accelerated pyruvate uptake. The carbon of thiocyanate is recovered mainly as CO₂; carbonate becomes incorporated into cellular protein, but less so in sludges previously treated with pyruvate. TCA cycle enzymes are not altered appreciably after pyruvate is added to activated sludge.     

Effects of dried distillers' grain on fecal prevalence and growth of Escherichia coli O157 in batch culture fermentations from cattle

Distillers' grains (DG), a by-product of ethanol production, are fed to cattle. Associations between Escherichia coli O157 prevalence and feeding of DG were investigated in feedlot cattle (n = 379) given one of three diets: steam-flaked corn (SFC) and 15% corn silage with 0 or 25% dried distillers' grains (DDG) or SFC with 5% corn silage and 25% DDG. Ten fecal samples were collected from each pen weekly for 12 weeks to isolate E. coli O157. Cattle fed 25% DDG with 5 or 15% silage had a higher (P = 0.01) prevalence of E. coli O157 than cattle fed a diet without DDG. Batch culture ruminal or fecal microbial fermentations were conducted to evaluate the effect of DDG on E. coli O157 growth. The first study utilized microbial inocula from steers fed SFC or dry-rolled corn with 0 or 25% DDG and included their diet as the substrate. Ruminal microbial fermentations from steers fed DDG had higher E. coli O157 contents than ruminal microbial fermentations from steers fed no DDG (P < 0.05) when no substrate was included. Fecal fermentations showed no DDG effect on E. coli O157 growth. In the second study with DDG as a substrate, ruminal fermentations with 0.5 g DDG had higher (P < 0.01) E. coli O157 concentrations at 24 h than ruminal fermentations with 0, 1, or 2 g DDG. In fecal fermentations, 2 g DDG resulted in a higher concentration (P < 0.05) at 24 h than 0, 0.5, or 1 g DDG. The results indicate that there is a positive association between DDG and E. coli O157 in cattle, and the findings should have important ramifications for food safety.