Microbial biosensors

In comparison to enzyme-based biosensors, microbial biosensors show lower analyte selectivity, slightly slower response times, but often much better stability. Microbial biosensor determination of analytes such as amino acids, alcohol, and lactate show sensor stability over several days, whilst enzyme-based sensors may have operational lives of only 2-24 h. 

Jia J., Tang M., Chen X., Qi L., Dong S., Co-immobilized microbial biosensor for BOD estimation based on sol-gel derived composite material, Biosens. Bioelectr. 2003 18 1023-1029. 

A. Mulchandani, I. Kaneva, and W. Chen, Biosensor for direct determination of organophosphate nerve agents using recombinant Escherichia coli with surface-expressed organophosphorus hydrolase. 2. Fiber-optic microbial biosensor. Anal. Chem. 70, 5042-5046 (1998). 

The fouling and microbial contamination of the surface during operation are main problems in the long-term use of enzyme biosensors. Wang et al. [121] reported for the first time a screen-printed electrode modified with GOD/HRP that was stable up... 

The sol-gel-entrapped microbial cells have shown excellent tolerance to different alcohols [99], The immobilized E. coli cells followed the Michaelis-Menten equation when quantified with the (3-glucosidase activity via the hydrolysis of 4-nitrophenyl-(3-D-galactopyranosdie [142], The sol-gel matrices doped with gelatin prevented the cell lysis, which usually occurs during the initial gelation process [143], Microorganisms are now widely used in the biosorption of different pollutants and toxicants. Bacillus sphaericus JG-A12 isolated from uranium mining water has been entrapped in aqueous silica nanosol for the accumulation of copper and uranium [144], Premkumar et al. [145] immobilized recombinant luminous bacteria into TEOS sol-gel to study the effect of sol-gel conditions on the cell response (luminescence). The entrapped and free cells showed almost the same intensity of luminescence (little lower), but the entrapped cells were more stable than the free cells (4 weeks at 4°C). This kind of stable cell could be employed in biosensors in the near future. 

Watts, H. Lowe, C. Pollard Knight, D., Optical biosensor for monitoring microbial cells, Anal. Chem. 1994, 66, 2465 2470... 

Some new developments are also proposed such as a system based on the use of electrochemically active bacteria in combination with a microbial fuel cell [34], giving good responses over 60 days, or a biosensor developed for fast... 

Currently, a large spectrum of microbial biosensors have been developed that enable the monitoring of pollutants by measuring light, fluorescence, colour or electric current and electrochemical signal [60]. A recent study [19] shows that whole-cell biosensors based on the detection of changes in gene... 

Biosensors based on microbial immobilization have also been used for food applications, e.g., the inexpensive and rapid high-throughput bacterial biosensor developed by Virolainen et al. for rapid detection of tetracyclines and their 4-epimer derivatives in poultry meat [188, 189]. 

Wood, K.V. Gruber, M.G. Transduction in microbial biosensors using multiplexed bioluminescence. Biosens. Bioelectron. 1996, II, 207 -214. 

Table 3.2 lists the most salient microbial sensors reported to date, together with the type of immobilized microorganism and measurement used, and the response time and dynamic range achieved in each instance. As can be seen, most of these biosensors rely on amperometric measurements. Some of them are described in detail below. 

Biochemical oxygen demand (BOD) is one of the most widely determined parameters in managing organic pollution. The conventional BOD test includes a 5-day incubation period, so a more expeditious and reproducible method for assessment of this parameter is required. Trichosporon cutaneum, a microorganism formerly used in waste water treatment, has also been employed to construct a BOD biosensor. The dynamic system where the sensor was implemented consisted of a 0.1 M phosphate buffer at pH 7 saturated with dissolved oxygen which was transferred to a flow-cell at a rate of 1 mL/min. When the current reached a steady-state value, a sample was injected into the flow-cell at 0.2 mL/min. The steady-state current was found to be dependent on the BOD of the sample solution. After the sample was flushed from the flow-cell, the current of the microbial sensor gradually returned to its initial level. The response time of microbial sensors depends on the nature of the sample solution concerned. A linear relationship was foimd between the current difference (i.e. that between the initial and final steady-state currents) and the 5-day BOD assay of the standard solution up to 60 mg/L. The minimum measurable BOD was 3 mg/L. The current was reproducible within 6% of the relative error when a BOD of 40 mg/L was used over 10 experiments [128]. 

In principle, there are two possible ways to measure this effect. First, there is the end-point measurement (steady-state mode), where the difference is calculated between the initial current of the endogenous respiration and the resulting current of the altered respiration, which is influenced by the tested substances. Second, by kinetic measurement the decrease or the acceleration, respectively, of the respiration with time is calculated from the first derivative of the currenttime curve. The first procedure has been most frequently used in microbial sensors. These biosensors with a relatively high concentration of biomass have a longer response time than that of enzyme sensors. Response times of comparable magnitude to those of enzyme sensors are reached only with kinetically controlled sensors. 

For these reasons, microbial sensors are less suitable for the determination of individual analytes. However, some practical apphcations for biosensors based on enzymes or antibodies for the specific determination of environmentally relevant compounds can be expected soon [11]. Furthermore, in some cases defined specific metabolic pathways in microorganisms are used, leading to microbial sensors for more selective analysis for those environmental pollutants which cannot be measured by the use of simple enzyme reactions, e.g., aromatic compounds and heavy metals. In this context it is also important to mention the aspect of bio availability, a parameter which is included by the measuring procedure of microbial sensors as an integral effect. 

For all these reasons, a microbial BOD-sensor needs to be calibrated before it can be used as a biochemical activity test. Only after a foregoing calibration procedure does a BOD-sensor finally reveal results which are both reliable and comparable to the conventional BOD5 method. Some consideration concerning the calibration of biosensors are given in Sect. 3.2.1.6. 

The selection of appropriate microorganisms is a possible way to improve the correlation between BOD and BODj [16,53]. The prerequisite for the use of microorganisms for BOD-sensors is a wide substrate spectrum. Therefore several samples of activated sludge from different wastewater plants were investigated [ 13,14]. One problem with an activated sludge based biosensor is the variability of sensor response with time. These BOD-sensors with an undefined variety of microbial species revealed no reproducible results. For that reason, BOD-sensors were developed using various types of defined cultures of microorganisms (Table 1). 

Table 3. Comparison of BOD-values estimated by microbial sensor containing Arxula ade-ninivorans and a commercial biosensor containing Issatchenkia orentalis and Rhodococcus erythropolis with BOD determined by the five day method for various domestic wastewater samples [19] ...

Microbial BOD-sensor Cell-biosensor Mycelia-biosensor ... 

The improvement of the correlation of sensorBOD and BODj can also be achieved by incubating the biosensor for some hours in this wastewater sample, which has to be measured [53]. This allows the induction of all of the microorganisms required metabolic degradation systems [65]. As shown in Table 5, preincubated microbial sensors and the conventional BODj method revealed similar results. 

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