生物传感器快速测定BOD的研究

生化需氧量(biochemicaloxygendemand,BOD)是一种表征水体有机污染程度的综合指标,广泛应用于水体监测和废水处理厂的运行控制。由于BOD的标准测定方法需时5天,不能及时地反映水质状况和反馈处理信息,因此快速测定BOD的方法和仪器化研究近年来得到广泛的重视。利用生物传感器测定BOD是一种有效地快速测定废水中可生化降解有机物的方法。介绍生物传感器的工作原理及其生物敏感材料,讨论BOD传感器的性能参数以及BOD快速测定值(BODst)与标准BOD5值的一致性问题。对现阶段市场上常见的几种BOD快速测定仪进行简单的介绍,并对它们的性能进行比较 。     

A flow injection analysis system with encapsulated high-density <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells for rapid determination of biochemical oxygen demand

The biochemical oxygen demand (BOD) determination was studied using a novel flow injection analysis (FIA) system with encapsulated Saccharomyces cerevisiae cells and an oxygen electrode and was compared with conventional 5-day BOD tests. S. cerevisiae cells were packed in a calcium alginate capsule at a dry cell weight of 250 g/l of capsule core. The level of dissolved oxygen (DO) was reduced due to the enhanced respiratory activity of the microbial cells when the injected nutrient passed through the bioreactor. The decrease in DO (ΔDO) was intensified with the amount of microbial cells packed in the bioreactor. However, the specific ΔDO decreased as the amount of cells loaded in the bioreactor increased. The ΔDO value was dependent on the pH and temperature of the mobile phase and reached its maximum value at 35°C and pH 7–8. Also, ΔDO became larger at longer response times as the flow rate of the mobile phase decreased. The measurement of ΔDO was repeated more than six times consecutively using a 20-ppm standard glucose and glutamic acid solution, which confirmed the reproducibility with a standard deviation of 0.95%. A strong linear correlation between ΔDO and BOD was also observed. The 5-day BOD values of actual water and wastewater samples were in accordance with the BOD values obtained by this FIA method using encapsulated S. cerevisiae cells. Unlike the cell-immobilized bead system, there was no contamination of the bioreactor resulting from any leak of yeast cells from the sensor capsules during BOD measurements.     

Immobilized multi-species based biosensor for rapid biochemical oxygen demand measurement

To improve the practicability of rapid biochemical oxygen demand (BOD) method, we proposed a stable BOD sensor based on immobilizing multi-species BODseed for wastewater monitoring in the flow system. The activation time of the biofilm was greatly shortened for the biofilm prepared by BODseed in the organic-inorganic hybrid material. Some influence factors such as temperature, pH, and concentration of phosphate buffer solution (PBS) were investigated in detail in which high tolerance to environment was validated for the BOD sensor permitted a wide pH and PBS concentration ranges. The minimum detectable BOD was around 0.5 mg/l BOD under the optimized 1.0 mg/ml BODseed immobilized concentration. The as-prepared BOD sensor exhibited excellent stability and reproducibility for different samples. Furthermore, the as-prepared BOD biosensor displayed a notable advantage in indiscriminate biodegradation to different organic compounds and their mixture, similar to the character of conventional BOD(5) results. The results of the BOD sensor method are well agreed with those obtained from conventional BOD(5) method for wastewater samples. The proposed rapid BOD sensor method should be promising in practical application of wastewater monitoring.     

Stopped-flow system with ozonizer for the estimation of low biochemical oxygen demand in environmental samples

The stopped-flow system with an ozonizer was developed to estimate low biochemical oxygen demand (BOD) in rivers. Rivers contain many biopersistent organic compounds such as humic acid, lignin, and gum arabic. Free radicals generated by self-decomposition of ozone were used as powerful oxidants to split organic compounds. Ozonysis of the samples was carried out by 42.4 g N⁻¹ m⁻³ ozone for 3 min at pH 7.0. Artificial wastewater (AWW) solutions were employed as standard solutions for the calibrations of the BOD sensor. At a BOD of 1 mg l⁻¹, the sensor response after ozonation was 1.6-fold higher than that before ozonation. The response time of the BOD sensor was only 5 min, being independent of the concentrations, and the lower detection limit was 0.5 mg l⁻¹ BOD. The degradations of lignin and tannic acid by ozonation were 54.1 and 42.3%, respectively. In the biosensor responses by ozonation, lignin, gum arabic, and surfactant increased by double or more compared with previous responses. BOD in rivers was estimated using the stopped-flow system. Environmental samples pretreated with ozone gave high responses to the biosensor that were similar to those of the conventional BOD₅ method. Accordingly, a good correlation between the sensor and the conventional BOD₅ was obtained (r = 0.989). The system has to evolve the highly sensitive BOD determination.     

Measurement of biodegradable substances using the salt-tolerant yeast Arxula adeninivorans for a microbial sensor immobilized with poly(carbamoyl)sulfonate (PCS). Part II: Application of the novel biosensor to real samples from coastal and island regions

A microbial sensor for rapid measurement of the amount of biodegradable substances based on the salt-tolerant yeast Arxula adeninivorans LS3 has been developed especially for coastal and island regions. Our parameter, the so-called sensorBOD, that is available after only a few minutes, agrees with the 5-day value for the biochemical oxygen demand (BOD5) very well. We have employed the Arxula sensor in the short-time estimation and supervision of the BOD of both domestic and industrial wastewater with high salinity. The novel sensor makes it possible to monitor the different types of wastewater rapidly without pretreatment, and it can be used for an active process control of sewage treatment works. Compared to a commercially available sensor, the novel sensor achieves better agreement between sensorBOD and BOD5 measurements with salt containing samples.     

Short-term BOD (BODst) as a parameter for on-line monitoring of biological treatment process. Part I. A novel design of BOD biosensor for easy renewal of bio-receptor

A novel design of a biochemical oxygen demand (BOD) biosensor has been developed for on-line monitoring of easily biodegradable organic compounds in aqueous samples. The biological recognition element of the sensor could be easily renewed by injecting new bacterial paste without disassembling the sensor system. The sensor measurements were carried out in the initial-rate mode using a flow injection (FI) system, resulting in 60 s for one sample analysis followed by a recovery time less than 10 min. The sensor performance achieved showed a wide detection linearity over the range of 5-700 mg BOD5.l(-1) and a generally good agreement between the BOD values estimated by the biosensor and the conventional 5-day test. Furthermore, the precision test was in the control range (i.e. repeatability < or = /+/-7.5%/, reproducibility < or = /+/-7.3%/). The sensor could be used over 1 week in continuous test, however, the best performance was found within the first 24 h where standard deviation of the sensor response was +/-2.4%. The design of the sensor allows easy and fast renewal of the cells used as sensing elements. Replacement of biological recognition element and calibration of the sensor responses can be performed in a rather simple procedure on a daily regular basis. By using a mixed culture as the bio-receptor, one gets a sensor that reacts to a wide range of substrates. The new sensor construction will thus allow fast and convenient replacement of the bio-receptor and on-line assay of a broad range of substrates. This makes the sensor being an interesting and promising candidate for on-line monitoring of biological treatment process.     

Disposable sensor for biochemical oxygen demand

 Disposable-type microbial sensors were prepared for the determination of biochemical oxygen demand (BOD). The yeast, Trichosporon cutaneum, was directly immobilized on the surface of miniature oxygen electrodes using an ultraviolet crosslinking resin (ENT-3400). The oxygen electrodes (15 mm× 2 mm×0.4 mm) were made on silicon substrates using micromachining techniques. They were Clark-type two-electrode systems with−1021 mV applied to the working electrode. Typical response times of the BOD sensors were in the range of 7–20 min. At 20°C, the sensors’ dynamic range was from 0 to 18 mg/l BOD when a glucose/glutamate BOD standard solution was used. The lower limit of detection was 0.2 mg/l BOD. This value was one order of magnitude lower than that of sensors previously reported. The sensors’ operational lifetime of 3 days was satisfactory for a disposable type. The sensors’ responses were reproducible to within 8% relative standard deviation. The BOD sensors’ were applied to untreated and treated waste waters from industrial effluents and municipal sewage. BOD values determined using these sensors correlated well with those determined by the conventional 5-day BOD determination method. Received: 22 December 1995/Received revision: 19 February 1996/Accepted: 17 March 1996     

Development and characterization of a novel immobilized microbial membrane for rapid determination of biochemical oxygen demand load in industrial waste-waters

The rapid determination of waste-water quality of waste-water treatment plants in terms of pollutional strength, i.e. biochemical oxygen demand (BOD) is difficult or even impossible using the chemical determination method. The present study reports the determination of BOD within minutes using microbial BOD sensors, as compared to the 5-day determination using the conventional method. Multiple criteria establish the basis for the development of a BOD biosensor useful for rapid and reliable BOD estimation in industrial waste-waters. Of these, preparation of a suitable novel immobilized microbial membrane used in conjunction with an apt transducer is discussed. As a result, a microbial biosensor based on a formulated, synergistic, pre-tested microbial consortium has been developed for the measurement of BOD load of various industrial waste-waters. The sensor showed maximum response in terms of current difference, when a cell concentration of 2.25 x 10(10) CFU, harvested in their log phase of growth were utilized for microbial membrane construction. The sensor showed a stability of 180 days when the prepared membranes were stored at a temperature of 4 degrees C in 50 mM phosphate buffer of pH 6.8. The reusability of the immobilized membranes was up to 200 cycles without appreciable loss of their response characteristics. A linear relationship between the current change and a glucose-glutamic acid (GAA) concentration up to 60 mg l(-1) was observed (r=0.999). The lower detection limit was 1.0 mg l(-1) BOD. The sensor response was reproducible within +/-5% of the mean in a series of ten samples having 44 mg l(-1) BOD using standard a GGA solution. When used for the BOD estimation of industrial waste-waters, a relatively good agreement was found between the two methods, i.e. 5-day BOD and that measured by the developed microbial sensor.     

Novel BOD optical fiber biosensor based on co-immobilized microorganisms in ormosils matrix

A biochemical oxygen demand (BOD) sensor has been developed, which is based on an immobilized mixed culture of microorganisms combined with a dissolved oxygen (DO) optical fiber. The sensing film for BOD measurement consists of an organically-modified silicate (ORMOSIL) film embedded with tri(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) perchlorate and three kinds of seawater microorganisms immobilized on a polyvinyl alcohol sol-gel matrix. The BOD measurements were carried out in the kinetic mode inside a light-proof cell and with constant temperature. Measurements were taken for 3 min followed by 10 min recovery time in 10 mg/L glucose/glutamate (GGA) BOD standard solution, and the range of determination was from 0.2 to 40 mg/L GGA. The effects of temperature, pH and sodium chloride concentration on the BOD sensing films were studied. BOD values estimated by this optical BOD sensing film correlate well with those determined by the conventional BOD5 method for seawater samples.     

Designing an amperometric thick-film microbial BOD sensor

Thick film oxygen electrodes manufactured by screen print method have been used as a transducer for a biochemical oxygen demand (BOD) sensor. The kinetics of the immobilized yeast, Arxula adeninivorans (Arxula) has been studied. The apparent KM of immobilized Arxula (> 100 microM) is higher than free cells of Arxula (70 microM). The increase in KM caused by the effect of immobilization extends the linear range of the sensor. End-point measurement and quasi-kinetic measurement have been studied comparatively as measurement procedures with a good correlation. The Vmax for end-point measurement is 790.7 microM/s and that for quasi-kinetic measurement is 537.3 microM/s. The limit of detection is calculated 1.24 mg/l BOD. Using the quasi-kinetic measurement, instead of end-point measurements, the measuring time can be reduced from 5-30 min to 100 s. The sensor layer thickness or increase in the layer of covering gel can increase the KM that is accompanied with the extension of the linear range of the sensor. Nevertheless, increase in the layer of covering gel will not increase the saturation signal. Domestic wastewater was checked by the thick film BOD sensor and the results are satisfactory.     

The use of co-immobilization of Trichosporon cutaneum and Bacillus licheniformis for a BOD sensor

The microorganisms Trichosporon cutaneum and Bacillus licheniformis were used to develop a microbial biochemical oxygen demand (BOD) sensor. It was found that T. cutaneum gave a greater response to glucose, whereas B. licheniformis gave a better response to glutamic acid. Hence, co-immobilized T. cutaneum and B. licheniformis were used to construct a glucose and glutamic acid sensor with improved sensitivity and dynamic range. A membrane loading of T. cutaneum at 1.1x10(8 )cells ml(-1) cm(-2) and B. licheniformis at 2.2x10(8) cells ml(-1) cm(-2) gave the optimum result: a linear range up to 40 mg BOD l(-1) with a sensitivity of 5.84 nA mg(-1) BOD l. The optimized BOD sensor showed operation stability for 58 intermittent batch measurements, with a standard deviation of 0.0362 and a variance of 0.131 nA. The response time of the co-immobilized microbial BOD sensor was within 5-10 min by steady-state measurement and the detection limit was 0.5 mg BOD l(-1). The BOD sensor was insensitive to pH in the range of pH 6.8-7.2.     

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.     

A BOD monitoring disposable reactor with alginate-entrapped bacteria

Biochemical oxygen demand (BOD) is a measure of the amount of dissolved oxygen that is required for the biochemical oxidation of the organic compounds in 5 days. New biosensor-based methods have been conducted for a faster determination of BOD. In this study, a mathematical model to evaluate the feasibility of using a BOD sensor, based on disposable alginate-entrapped bacteria, for monitoring BOD in situ was applied. The model considers the influences of alginate bead size and bacterial concentration. The disposable biosensor can be adapted according to specific requirements depending on the organic load contained in the wastewater. Using Klein and Washausen parameter in a Lineweaver–Burk plot, the glucose diffusivity was calculated in 6.4 × 10⁻¹⁰ (m²/s) for beads of 1 mm in diameter and slight diffusion restrictions were observed (n = 0.85). Experimental results showed a correlation (p < 0.05) between the respirometric peak and the standard BOD test. The biosensor response was representative of BOD.     

Accuracy and repeatability of a pressure measurement system in the patellofemoral joint

The objective of this study was to assess how accurately and repeatably the Iscan system measures force and pressure in the natural patellofemoral joint. These measurements must be made to test widely held assumptions about the relationships between mechanics, pain and cartilage degeneration. We assessed the system's accuracy by using test rigs in a materials testing machine to apply known forces and force distributions across the sensor. The root mean squared error in measuring resultant force (for five trials at each of seven load levels) was 6.5±4.4% (mean±standard deviation over all trials at all load levels), while the absolute error was −5.5±5.6%. For force distribution, the root mean squared error (for five trials at each of five force distributions) was 0.86±0.58%, while the absolute error was −0.22±1.03%. We assessed the repeatability of the system's measurements of patellofemoral contact force, pressure and force distribution in four cadaver specimens loaded in continuous and static flexion. Variability in measurement (standard deviation expressed as a percentage of the mean) was 9.1% for resultant force measurements and 3.0% for force distribution measurements for static loads, and 7.3% for resultant force and 2.2% for force distribution measurements for continuous flexion. Cementing the sensor to the cartilage lowered readings of resultant force by 31±32% (mean±standard deviation), area by 24±13% and mean pressure by 9±34% (relative to the uncemented sensor). Maximum pressure measurement, however, was 24±43% higher in the cemented sensor than in the uncemented sensor. The results suggest that the sensor measures force distribution more accurately and repeatably than absolute force. A limitation of our work, however, is that the sensor must be cemented to the patellar articular surface to make the force distribution measurements, and our results suggest that this process reduces the accuracy of force, pressure and area measurements. Our results suggest that the Iscan system's pressure measurement accuracy and repeatability are comparable to that of Fuji Prescale film, but its advantages are that it is thinner than most Fuji Prescale film, it measures contact area more accurately and that it makes continuous measurements of force, pressure and area.     

Submersible microbial fuel cell sensor for monitoring microbial activity and BOD in groundwater: Focusing on impact of anodic biofilm on sensor applicability

A sensor, based on a submersible microbial fuel cell (SUMFC), was developed for in situ monitoring of microbial activity and biochemical oxygen demand (BOD) in groundwater. Presence or absence of a biofilm on the anode was a decisive factor for the applicability of the sensor. Fresh anode was required for application of the sensor for microbial activity measurement, while biofilm‐colonized anode was needed for utilizing the sensor for BOD content measurement. The current density of SUMFC sensor equipped with a biofilm‐colonized anode showed linear relationship with BOD content, to up to 250 mg/L (～233 ± 1 mA/m²), with a response time of <0.67 h. This sensor could, however, not measure microbial activity, as indicated by the indifferent current produced at varying active microorganisms concentration, which was expressed as microbial adenosine‐triphosphate (ATP) concentration. On the contrary, the current density (0.6 ± 0.1 to 12.4 ± 0.1 mA/m²) of the SUMFC sensor equipped with a fresh anode showed linear relationship, with active microorganism concentrations from 0 to 6.52 nmol‐ATP/L, while no correlation between the current and BOD was observed. It was found that temperature, pH, conductivity, and inorganic solid content were significantly affecting the sensitivity of the sensor. Lastly, the sensor was tested with real contaminated groundwater, where the microbial activity and BOD content could be detected in <3.1 h. The microbial activity and BOD concentration measured by SUMFC sensor fitted well with the one measured by the standard methods, with deviations ranging from 15% to 22% and 6% to 16%, respectively. The SUMFC sensor provides a new way for in situ and quantitative monitoring contaminants content and biological activity during bioremediation process in variety of anoxic aquifers. Biotechnol. Bioeng. 2011;108: 2339–2347. © 2011 Wiley Periodicals, Inc.     

MICREDOX--development of a ferricyanide-mediated rapid biochemical oxygen demand method using an immobilised Proteus vulgaris biocomponent

Biochemical oxygen demand (BOD) is an international regulatory environmental index for monitoring organic pollutants in wastewater and the current legislated standard test for BOD monitoring requires 5 days to complete (BOD5 test). We are developing a rapid microbial technique, MICREDOX, for measuring BOD by eliminating oxygen and, instead, quantifying an equivalent biochemical co-substrate demand, the co-substrate being a redox mediator. Elevated concentrations of Proteus vulgaris, either as free cells or immobilised in Lentikat disks, were incubated with an excess of redox mediator (potassium hexacyanoferrate(III)) and organic substrate for 1h at 37 degrees C without oxygen. The addition of substrate increased the catabolic activity of the microorganisms and the accumulation of reduced mediator, which was subsequently re-oxidised at a working electrode generating a current quantifiable by a coulometric transducer. The recorded currents were converted to their BOD5 equivalent with the only assumption being a fixed conversion of substrate and known stoichiometry. Measurements are reported both for the BOD5 calibration standard solution (150 mg l(-1) glucose, 150 mg l(-1) glutamic acid) and for filtered effluent sampled from a wastewater treatment plant. The inclusion of a highly soluble mediator in place of oxygen facilitated a high ferricyanide concentration in the incubation, which in turn permitted increased concentrations of microorganisms to be used. This substantially reduced the incubation time, from 5 days to 1h, for the biological oxidation of substrates equivalent to those observed using the standard BOD5 test. Stoichiometric conversion efficiencies for the oxidation of the standard substrate by P. vulgaris were typically 60% for free cells and 35-50% for immobilised cells.     

On-line load monitoring of wastewaters with a respirographic microbial sensor

This paper demonstrates the functionality, laboratory testing and field application of a microbial sensor that is capable of monitoring the organic pollution extent of wastewaters both off-line in a laboratory and on-line in a wastewater treatment plant. The biosensor was first developed in the laboratory using synthetic wastewater and then applied to monitor the effluent of the unit. The basic working principle of the biosensor is based on the on-line measurement of CO2 concentration in the off gas produced during carbon compound degradation by microbial respiration activities. CO2 concentration under operation conditions (constant oxygen flow rate, residence time and pH) is closely related to the extent of organic pollution (biochemical oxygen demand, chemical oxygen demand). CO2 monitoring is carried out by an infrared spectrometer, whereas current organic pollution is determined off-line according to the conventional 5-day lasting BOD analysis. Off gas analysis of CO2 concentration strongly correlates with off-line biochemical oxygen demand measurements allowing continuous on-line monitoring of the organic load within a wastewater treatment plant. Thus, real time process control and operation become feasible.     

Improvement of a mediator-type biochemical oxygen demand sensor for on-site measurement.

We characterized a mediator-type biochemical oxygen demand (BOD) sensor with a three-electrode system using potassium ferricyanide (FC) and Pseudomonas fluorescens in our previous study. In the present study, we have utilized the advantages of a mediator-type biosensor, which does not require air-supply equipment for on-site measurements, and made a fully disposable sensor tip for a portable device. The tip consists of a two-electrode system with P. fluorescens immobilized on a cellulose acetate membrane and is packaged in polyester film to prevent it from drying out. By aeration with a 0.1 M NaCl solution of P. fluorescens (after growth), the sensor responses as well as their reproducibility and stability have been successfully improved. The responses increased more than seven times, and the calibration curve from 15 to 260 mg l(-1) also remained linear although the response decreased approximately half the original after at least 35 days in storage. The reproducibility of the sensor responses improved to 12.7% (average of relative standard deviations (RSDs)) in the calibration curve obtained by using the Organization for Economic Cooperation and Development synthetic sewage. Examination of real samples from three different sources showed that the BOD as determined by the sensor correlates well with the conventional 5-day BOD method (r(2)=0.982, 0.823, and 0.809). Consequently, the aeration process makes it possible to realize rapid, and in situ measurements without the long conditioning process that is generally required to activate the microorganisms immobilized on bio-films before use. Finally, we have designed a portable device that utilizes our disposable sensor tip.     

A novel BOD sensor based on bacterial luminescence

A reagent-type BOD sensor with a new principle employing a luminous bacterium, Photobacterium phosphoreum, was developed. The increased intensity of luminescence resulting from the cellular assimilation of organic compounds in wastewater was detected by a photodiode. The BOD response of the bacterial reagent could be obtained within 15 min with +/-7% error. The temperature condition for optimal BOD response was 18 degrees to 25 degrees C at pH 7 to 8, indicating that it is possible to measure BOD at room temperature without having to stabilize the temperature of the measuring system. For practical use, two procedures for long-term preservation of the bacterial reagent, vacuum drying method and freezing method, are suggested. The metabolic characteristics of employed luminous bacteria were investigated by comparing the BOD values for several pure organic substrates estimated by the BOD sensor with conventional 5-day BOD values. In comparison with the 5-day measurement for some wastewater samples, BOD values estimated by the sensor showed comparatively good agreement with those measured by the 5-day method. (c) 1993 Wiley & Sons, Inc.     

Novel BOD (biological oxygen demand) sensor using mediator-less microbial fuel cell

A microbial fuel cell type of biosensor was used to determine the biochemical oxygen demand (BOD) of wastewater. The biosensor gave a good correlation between the BOD value and the coulomb produced. The BOD sensor has been operated for over 5 years in a stable manner without any servicing. This is much longer that that of previously reported BOD biosensors.