Sensor microbial

Microbial sensors offer a number of assets, namely (a) they are less sensitive to inhibition by solutes and more tolerant to suboptimal pH and temperature values than are enzyme electrodes b) they have longer lifetimes than enzymes and (c) they are less expensive than enzyme electrodes as they require no active enzyme to be isolated. On the other hand, they lag behind enzyme electrodes in a few other respects thus, (a) some have longer response times than their enzyme counterparts b) baseline restoration after measurement typically takes longer and (c) cells contain many enzymes and due care must be exercised to ensure adequate selectivity e.g. by optimizing the storage conditions or using specific enzyme reactions) —some mutant microorganisms lack certain enzymes. 

Microbial sensors usually possess long lifetimes, yet proper maintenance is mandatory. Thus, the overall activity of immobilized microorganisms should be kept as constant as possible. For this purpose, sensors should be stored in phosphate buffer containing no nutrients at 4°C in order to avoid microorganism growth in the membrane. If the sensor activity diminishes for some reason, then the sensor should be dipped into a nutrient medium... 

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]. 

A novel bioassay for nystatin based on the use of a microbial sensor was recently reported. Nystatin is believed to bind to the steron present in the membranes of sensitive cells, leading to the formation of pores. The subsequent death of the microorganism is preceded by leakage of cellular materials. Microbial death can be detected by means of an oxygen electrode. 

One of the pitfalls of microbial sensors, viz. their low selectivity, can be overcome by combining cells with an immobilized enzyme. Thus, creatinine deaminase (CDA, EC 3.5.4.21) hydrolyses creatinine to N-methylhydantoin and ammonium ion, the ammonia produced being successively oxidized to nitrite and nitrate ion by nitrifying bacteria. These bacteria have not yet been characterized but are known to be a mixed culture of Nitrosomonas sp. and Nitrobacter sp. The reaction sequence involved is as follows ... 

Microbial Sensors on a Respiratory Basis for Wastewater Monitoring... 

Fig. 2. Physiological responses of a respiratory-based microbial sensor...

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. 

Without doubt, the favored field of application of microbial sensors is the measurement of complex effects like sum parameters. The difficulties involved in analyzing the numerous substances that are present in wastewater samples make sum parameters an indispensable part of the wastewater monitoring systems. Additionally, sum parameters often allow a better evaluation of the status of the environment than the determination of the concentration of individual substances. Examples for complex parameters are the sum of biodegradable or bioavailable compounds and toxicity (BOD, ADOC). 

A more rapid estimation of BOD is possible by using a microbial sensor. The first report of such a microbial BOD sensor was published in 1977 [13]. Meanwhile some BOD-sensor systems are commercially available (see review in [11]). Table 1 gives an overview of the hitherto described BOD sensors including the manufacturers. 

Eor the estimation of the suitability of microbial sensors for practical use, a sound evaluation of the applicability and the limitations of the method is necessary. As an example, the correlation to the conventional five-day test has to be investigated carefully. Repeatedly it has been described that the sensorBOD values are not in all cases identical to those of the BOD5 [11] (see Tables 3 and 4). 

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] ...

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. 

Due to their short time of exposure to the sample, microbial sensors are generally not able to detect polymers. The time required for the microbial degradation of proteins, starch, or cellulose normally is considerable longer than the usually applied measuring time. This is one of the main reasons why the sensorBOD value of wastewater is in most cases lower compared to the BOD5 method. 

Konig et al. [80-84] demonstrated that microbial sensors are suitable for the summary quantification of nitrifiable compounds (see also Sect. 3.3.1) as well as for the detection of nitrification inhibiting effects. Such biosensors, which contain a mixed population of the nitrifying bacteria Nitrosomonas sp. and Nitrobacter sp., exhibit a specific supplementary metabolic capacity. This enables the amperometric determination of ammonia according the following scheme of nitrification ... 

Moreover, a substantial advantage of this microbial sensor over other existing tests for measuring the inhibition of nitrification is the very short measuring... 

In addition to the summary registration of nitrifiable substances, it is also possible to quantify N-compounds selectively by using microbial sensors. Microbial sensors for the monitoring of ammonium ions, ammonia, nitrite, nitrate and urea have been described (see Table 9). Nitrifiers are generally used, but not only (see also Sect. 3.2.4). 

The ability of the chemolithoautotrophic bacteria Thiobacillus ferrooxidans to oxidize Fe has already been utilized for construction of a microbial sensor for the determination of iron [101]. The limit of determination of this biosensor is 60 pmol 1" with a response time ranging from 30 s to 5 min, depending on the Fe +-concentration in the sample. 

Fig. 6. Schematic design of a microbial sensor containing genetically manipulated yeast cell for detection of...

The monitoring of cyanide with microbial sensor is possible in two ways. The first principle is based on the inhibition of respiration of Saccharomyces cervisiae by cyanide [102, 103]. This sensor showed a linear response in the range 0-15 pmol 1 by a response time of approximately 10 min and a stability of 9 days. Another method for the determination of cyanide is enabled by the use of cyanide-degrading microorganisms such as Pseudomonas fluorescens [1041. This bacterium specifically oxidizes cyanide by consuming oxygen ... 

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