Evaluation of a FIA operated amperometric bacterial biosensor, based on Pseudomonas putida F1 for the detection of benzene, toluene, ethylbenzene and xylenes (BETEX)

Recently, the development and optimisation of a flow injection analysis (FIA) operated bacterial biosensor based on the aerobic catabolism of Pseudomonas putida ML2 was reported in the literature (Layon Y.H. e al. 2004a; Lanyon Y.H. e al. 2004b). Adapted from these reports, it was investigated whether operating parameters and procedures of the benzene biosensor could be directly applied to a new system based on a different bacterial strain for the detection of the whole benzene, toluene, ethylbenzene and xylenes range. Cells of the investigated bacterial strain, Pseudomonas putida F1, were immobilised between two cellulose acetate membranes and fixed onto a Clark dissolved oxygen electrode. P. putida F1 aerobically degrades benzene, toluene and ethyl-benzene (Gibson D.T. e al. 1968). The BTE biosensor in kinetic mode (Flow Injection Analysis) displayed a linear range of 0.02 – 0.14 mM benzene (response time: 5 min, base line recovery time: 15 min), 0.05 – 0.2 mM toluene (response time: 8 min, base line recovery time: 20 min), and 0.1 – 0.2 mM ethylbenzene (response time 12 min, base line recovery time: 30 min), respectively. Due to the differences in sensitivity, response and baseline recovery times for BTE, it was possible to differentiate each compound in mixtures of these VOCs. No response for Xylenes could be obtained since they cannot be completely metabolised by this bacterial strain. However, it was reported that the range of compounds degradable by P. putida F1 can possibly be expanded by cultivating the cells on different carbon sources (Choi E.N. e al. 2003). The sensor showed good intraand inter-assay reproducibility, and all obtained results were comparable with those reported in the literature. The demonstrated reproducibility and the simplicity and ease of use as well as the portability for in situ measurements, the biosensor could be suitable as a reliable initial warning device for elevated BTE levels in indoor and outdoor environments. INTRODUCTION The emissions of volatile organic compounds (VOCs) in the atmosphere represent one of the major causes to the air quality deterioration and environmental pollution. Among these VOCs, benzene, toluene, ethylbenzene, and xylenes (BTEX hereafter) are produced in huge amounts and are used in fuels, as solvents, and as starting materials for the production of plastics, synthetic fibers, and pesticides (Budavari, 1996). At room temperature and atmospheric pressure, these compounds are sufficiently vaporised to pose a significant health hazard to humans (Fruscella William 2002). A large amount of BTEX is therefore released into the atmosphere during manufacture, transportation, use, and disposal every year. BTEX vapors are corrosive and toxic substances and are on the EPA Priority Pollutant List (EPA, 1996), and in the top 100 chemicals on the Priority List of Hazardous Substances published by the Agency for Toxic Substances and Disease Registry (ATSDR, 2003). Exposure to BTEX primarily occurs via inhalation (WHO 2002); hence individuals working in industries producing or using these petrochemicals are exposed to the highest levels of these toxic aromatic hydrocarbons. Acute exposure to high levels of BTEX has been associated with skin and sensory irritation, central nervous system depression, and effects on the respiratory system. Prolonged exposure has similar effects and is additionally adversely affecting the kidney, liver and blood systems (Fruscella William 2002, Ozokwelu Dickson E. 2000, Cannella William J. 2000). Due to the proven different degrees of toxicity, permissible exposure levels for each single BTEX-compound were established. The current European permissible exposure levels for benzene, toluene, ethyl-benzene and xylenes are reported in Table 1. The conventional method for monitoring human BTEX exposure in the workplace includes the trapping of BTEX vapours on charcoal adsorption tubes, desorbtion with an appropriate solvent (e.g. carbon disulfide) and subsequent determination of the VOCs employing gas chromatography (WHO 2002, Healt and Safety Executive 1997). Classical chromatographic methods, such as GC-analysis, provide high accuracy and precision. These techniques however, are often time-consuming, require highly sophisticated equipment, are expensive, and are usually lab based. For the routinely performed detection of BTEX in air, alternative, faster, more economical and portable in-situ detection devices, such as biosensors, would therefore be desirable. Biosensors incorporate a biological sensing element (e.g. microorganisms), which is either intimately connected to or integrated within a suitable transducing system (Turner A.P.F. e al. 2000). The sensing element specifically recognises the species under investigation; the transducer quantitatively converts the biochemical signal into an electronic signal that can be suitably processed and converted to an output. Therefore, analytes may be detected by using the assimilation capacity of the microorganism as an index of the respiration activity or of the metabolic activity (Mulchandani A., Rogers K.R. 1998). Such microbial biosensors based on respiratory activity have had several applications in environmental monitoring over the past years (Tan H.M. e al. 1994, D’Souza S.F. 2001). Recently, a FIA operated amperometric bacterial biosensor, based on Pseudomonas putida ML2, for detection of benzene in workplace air was reported by Lanyon et al. (Lanyon Y.H. e al. 2004a, Lanyon Y.H. e al. 2004b). This sensor was constructed by immobilising the bacterial cells between two cellulose acetate membranes, and subsequent fixation of the membranes onto an amperometric Clark type dissolved oxygen electrode. Starting from the reports by Lanyon et al. (Lanyon Y.H. e al. 2004a, Lanyon Y.H. e al. 2004b), the applicability of an amperometric bacterial biosensor for the detection of the whole BTEX range in air was investigated in this work. The sensor was based on the same principles as the aforementioned benzene-sensor, but utilising a different bacterial strain, Pseudomonas putida F1. Pseudomonas putida F1 (PpF1) is a fluorescent soil bacterium that can assimilate toluene, benzene, ethylbenzene, phenol, and other aromatics as sole carbon and energy sources (Gibson D.T. et al., 1968). Like other pseudomonads, many of its induced enzymes are nonspecific and its metabolic pathways contain a high degree of convergence allowing for the efficient utilization of a wide range of growth substrates (Hutchinson and Robinson, 1988). The enzymic pathway responsible for converting these aromatic hydrocarbons to TCA cycle intermediates is called the toluene degradation (tod) pathway (Finette et al., 1984; Gibson et al., 1990). The tod pathway consists of several enzymic reactions; for example (see Scheme 1), toluene is first transformed into cis-toluene dihydrodiol through the insertion of a molecule of oxygen catalyzed by a multicomponent toluene dioxygenase (Finette et al., 1984; Gibson et al., 1990; Yeh et al., 1977). cis-Toluene dihydrodiol is then dehydrogenated to form 3methylcatechol, which is cleaved at the meta position by insertion of a second dioxygen molecule (Scheme 1). Then the reaction products are converted to tricarboxylic acid cycle (TCA cycle) intermediates (Lau et al., 1994; Zylstra and Gibson, 1989). Benzene, toluene and ethylbenzene can be degraded by P. putida F1 via the same pathway (Scheme 1). Further studies on the substrate specificity of the tod pathway have revealed that n-propylbenzene, n-butylbenzene, cumene and biphenyl are degraded to only ring-fission dead-end products whereas p-xylene is only converted to 2,6-dimethylcatechol, (Cho et al., 2000; Gibson et al., 1974, Yu et al., 2001), revealing the limitations of the corresponding enzymes in channeling substrates into TCA cycle intermediates (Furukawa et al., 1993). Scheme 1: Pseudomonas putida F1 catabolyc pathways for benzene, toluene, ethylbenzene and p-xylene. During the aerobic assimilation of BTE, an increase in respiration rate and corresponding oxygen consumption mainly due to the dioxygenases catalysis occurs and can be used as basis for the detection of these VOCs in solution. Xylenes, the last member of the BTEX range, can so far not be degraded by this bacterial strain. However, Choi et al. (Choi E.N. e al. 2003) reported that the range of compounds which can be metabolised by Pseudomonas putida F1 can possibly be expanded by cultivating the cells with different aromatic hydrocarbons (e.g xylenes) as growth-additives. Pre-existing metabolic pathways can be altered by natural adaptation of the tod-degradation pathway and hence, the novel metabolic capabilities of the bacterial cells might be subsequently exploited in a biosensor. A sensing device based on this principle might therefore be employed as an early warning device for the presence of toxic VOCs in air.