Microbial sensors microorganisms

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. 

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. 

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. 

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 ... 

However, up till now there has been a certain discrepancy between research and development of biosensors on the one hand and their practical application on the other. Only very few biosensors are commercially produced and used on a large scale, like the microbial BOD-sensor. This situation is mainly due to the insufficient stability of microorganisms in comparison to chemical and physical methods under conditions of practical applications. In some cases, microbial sensors also suffer from relatively poor selectivity and sensitivity. 

The sensor did not respond to volatile compounds such as methyl alcohol, formic acid, acetic acid, propionic acid, and other nutrients for microorganisms such as carbohydrates, amino acids, and ions. The selectivity of the microbial sensor for ethyl alcohol was satisfactory. 

Microbial Sensors for Complex Variables and for Characterization of Microorganisms... 

The applications of microbial sensors in medicine are limited because they are unsuitable for use in biological liquids, which are usually the culture medium for microorganisms. The growth in biomass deforms the biocatalytic matrix leading to a leak of cells and a contamination of the sample medium. Furthermore, microbial sensors contain a large number of enzymes and are not sufficiently specific for many biomedical analyses. 

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). 

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