Immobilised activated sludge based biosensor for biochemical oxygen demand measurement

A biochemical oxygen demand (BOD) sensor, based on an immobilised mixed culture of microorganisms in combination with a dissolved oxygen electrode, has been developed for the purpose of on-line monitoring of the biological treatment process for waste and wastewater. The sensor was designed for easy replacement of the biomembrane, thereby making it suitable for short-term use. The drawbacks of activated sludge based sensor, such as short sensor lifetime, were thereby circumvented. The sensor BOD measurements were carried out in the kinetic mode using a flow injection system, resulting in 25 s for one measurement followed by 4–8 min recovery time. Based on the results of normalised sensor responses, the OECD synthetic wastewater was considered to be a more suitable calibration solution in comparison with the GGA solution. Good agreement was achieved between the results of the sensor BOD measurement and those obtained from BOD₅ analysis of a wastewater sample from a food-processing factory. Reproducibility of responses using one sensor was below ±5.6% standard deviation. Reproducibility of responses using different sensors was within acceptable bias limits, viz. ±15% standard deviation.     

Development of photocatalytic biosensor for the evaluation of biochemical oxygen demand

The photocatalytic biosensor of flow system using semiconductor TiO2 was developed to evaluate biochemical oxygen demand (BOD) levels in river water. Photocatalysis of sample was carried out in a photoreactor with TiO2 and a 6W black-light blue fluorescent tube as light source. Sample from a photoreactor outlet was measured by an oxygen electrode with a biofilm. The sensor response of photocatalytic biosensor was between 5 and 10 min depending on concentration of biochemical in the samples. At BOD of 1 mgl-1, the sensor response increased 1.33-fold in comparison with that without photocatalysis. The degradation of tannic acid and humic acid with photocatalysis were 51.8 and 38.4%, respectively. Gum arabic and linear alkylbenzene sulfonate (LAS) were degraded a little, but gave the responses of more than double to the sensor. Free radicals yielded by photocatalysis in a photoreactor did not affect the sensor response because their lifetime is extremely short. Fairly good correlation (r=0.983) between the sensor method and the conventional method was obtained for test samples. This biosensor using photocatalytic pretreatment improved the sensitivity.     

Optical fiber biosensor for the determination of low biochemical oxygen demand

An optical fiber biosensor was developed for the evaluation of low Biochemical Oxygen Demand (BOD) values in river waters. Artificial wastewater (AWW) solution was employed as standards for the calibration of the BOD sensor. The response time of the sensor was 15 min, and the optimal BOD response was observed at 30 degrees C, pH 7.0. A linear relationship was obtained between the output voltage and BOD5 values, and the range of determination was 1-10 mg l(-1) BOD. The sensor response was almost not influenced by chloride ion up to 1000 mg l(-1), and also not affected by heavy metal ions (Fe3+, Cu2+, Mn2+, Cr3+, Zn2+). The BOD of river waters was estimated by using the optical fiber biosensor, and good correlation between the sensor and BOD5 test was obtained (r2 = 0.971).     

Continuous determination of biochemical oxygen demand using microbial fuel cell type biosensor

A mediator-less microbial fuel cell (MFC) was used as a biochemical oxygen demand (BOD) sensor in an amperometric mode for real-time wastewater monitoring. At a hydraulic retention time of 1.05 h, BOD values of up to 100 mg/l were measured based on a linear relationship, while higher BOD values were measured using a lower feeding rate. About 60 min was required to reach a new steady-state current after the MFCs had been fed with different strength artificial wastewaters (Aws). The current generated from the MFCs fed with AW with a BOD of 100 mg/l was compared to determine the repeatability, and the difference was less than 10%. When the MFC was starved, the original current value was regained with a varying recovery time depending on the length of the starvation. During starvation, the MFC generated a background level current, probably due to an endogenous metabolism.     

Characterization of microbial membranes used for the estimation of biochemical oxygen demand with a biosensor

Summary A bacterial mixed culture was immobilized in Millipore filters to construct microbial-membranes for BOD determination using an oxygen electrode. The biosensor response was best when 0.5 mg cells were immobilized per cm² of membrane, at 30°C, pH 7 and 0.05 M phosphate buffer. Reproducible microbial-membranes can be constructed and they can be stored for up to 20 days without appreciable loss of their response characteristics.     

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.     

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     

Towards an on-line biochemical oxygen demand analyser

A simple, and effective tool to measure BOD with a 98% correlation to theoretical BOD values has been developed using a technique of continuous short term measurements of O₂ consumption rates. Measurement times are in the range of 5 to 10 h depending on the initial BOD, instead of 5 days required in the conventional BOD₅ analysis. A bioreactor containing calcium alginate entrapped activated sludge microorganisms was used with phenol and hydroxybutyric acid (HBA) as model substrates.     

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.     

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.     

A fast estimation of biochemical oxygen demand using microbial sensors

Summary Microbial amperometric sensors for biochemical oxygen demand (BOD) determination using Bacillus subtilis or Trichosporon cutaneum cells immobilized in polyvinylalcohol have been developed. These sensors allow BOD measurements with very short response times (15–30s), a level of precision of ±5% and an operation stability of 30 days. A linear range was obtained for a B. subtilis-based sensor up to 20 mg/l BOD and for a T. cutaneum-based sensor up to 100 mg/l BOD using a glucose/glutamic acid standard.     

Immobilized plant thylakoid membranes as a biosensor for herbicides

Summary Thylakoid membranes isolated from spinach leaves were used as the biological sensing material to elaborate a biosensor for the detection of small amount of the herbicides atrazine and diuron. Free and immobilized thylakoid membranes were compared for their responses to inhibition by herbicides by following the variation of the photocurrent. Immobilized thylakoid membranes were twice as sensitive to inhibition by herbicides than the native thylakoids.     

Application of biochemical oxygen demand (BOD) biosensor for optimization of biological carbon and nitrogen removal from synthetic wastewater in a sequencing batch reactor system

A bench scale reactor using a sequencing batch reactor process was used to evaluate the applicability of biosensors for the process optimization of biological carbon and nitrogen removal. A commercial biochemical oxygen demand (BOD) biosensor with a novel microbial membrane was used to determine the duration of each phase by measuring samples in real time in an SBR cycle with filling/anoxic-anaerobic/aerobic/sludge wasting/settling/withdrawal periods. Possible strategies to increase the efficiency for the biological removal of carbon and nitrogen from synthetic wastewater have been developed. The results show that application of a BOD biosensor enables estimation of organic carbon, in real time, allowing the optimization or reduction the SBR cycle time. Some typical consumption patterns for organic carbon in the non-aeration phase of a typical SBR operation were identified. The rate of decrease of BOD measured using a sensor BOD, was the highest in the initial glucose breakdown period and during denitrification. It then slowed down until a 'quiescent period' was observed, which may be considered as the commencement of the aeration period. Monitoring the BOD curve with a BOD biosensor allowed the reduction of the SBR cycle time, which leads to an increase in the removal efficiency. By reducing the cycle time from 8 to 4 h cycle, the removal efficiencies of nitrate, glucose, and phosphorus in a given time interval, were increased to nearly double, while the removal of nitrogen ammonium was increased by one-third.     

Disposable sensor for measuring the biochemical oxygen demand for nitrification and inhibition of nitrification in wastewater

A disposable-type microbial sensor was developed for the determination of both the biochemical oxygen demand for nitrification (N-BOD) and inhibiting effects on nitrifying bacteria. The sensor was based on the respiratory activity of nitrifying bacteria immobilized on a miniature oxygen electrode. Typical response times for measuring N-BOD of ammonium standard solutions as well as of wastewater samples were in the range of 6–12 min. A dynamic evaluation of the signals after a measuring time of 120 s also resulted in good reproducibility and sensitivity. A daily profile of a municipal sewage plant was recorded, comparing the biosensor data with two standard methods. For the measurement of nitrification-inhibiting effects a 120-s dynamic signal evaluation was preferred to a steady-state method because of the long recovery times resulting from extended exposure to inhibitors. However, steady-state measurement techniques allowed allylthiourea detection with a ten times higher sensitivity. Because of the advantages of this miniaturized electrode, e.g. short response time, simple measuring procedure and low costs of production, this sensor system is considered to be suitable for commercial application in environmental analysis. Received: 30 April 1998 / Received revision: 4 September 1998 / Accepted: 13 September 1998