Novel BOD optical fiber biosensor based on co-immobilized microorganisms in ormosils matrix
Introduction
Biochemical oxygen demand (BOD) is one of the most widely used and important parameters for the estimation of water quality. The authorized method for BOD was adopted by the American Public Health Association Standard Methods Committee (APHA, 1989), which not only requires 5 days of tedious procedures, but also demands experience and skill to obtain reproducible results. Therefore, it is time-consuming and not suitable for in situ determinations or on-line measurements (JISC, 1989). Since Karube et al. (1997) first developed a rapid and reliable biosensor for BOD determination in 1977, several other kinds of microbial BOD sensor have been applied. (Qian and Tan, 1998, Tag et al., 2000, Kang et al., 2003, Chang et al., 2004, Rastogi et al., 2003). In most cases, the BOD sensors consist of a synthetic membrane using microorganisms as the biological recognition element, and a dissolved oxygen electrode (Clark electrode) as a transducer for the oxygen measurement. During the past decade, fiber optical chemical sensors for the determination of oxygen have appeared to be more attractive than conventional amperometric devices due to rapid performance, no oxygen consumption and less toxicity (Hartmann et al., 1998, Klimant and Wolfbeis, 1995, Chan et al., 1999, Amao et al., 2000). In the first report for BOD determination using a fiber optical microbial sensor (Preininger et al., 1994), a single microorganism, Trichosporon cutaneum, was selected and immobilized in polyvinyl alcohol (PVA) used for the BOD sensing membrane. Until now, many assimilative microorganisms have been applied and reported including: Arxulao adeninivorans LS3 (Riedel et al., 1998a, Riedel et al., 1998b, Gruendig et al., 2000, Lehmann et al., 1999), Bacillus cereus (Sun et al., 1992), Bacillus subtilis (Riedel et al., 1998a, Riedel et al., 1998b, Xie et al., 2003), Klebsiella oxytoca AS1(Ohki et al., 1994), Pseudomonas putida (Chee et al., 1999) Trichosporon cutaneum (Preininger et al., 1994, Murakami et al., 1998), Trichosporon brassicae (Xie et al., 2003), and yeast (Li et al., 2004, Chen et al., 2002). The BOD sensor is highly capable of analyzing a sample of complex constituents with relatively low selectivity. The sensor should respond to multiple biodegradable organic solutes in the sample, and give results comparable to those obtained using the conventional BOD methods. Since each microbial species has its metabolic deficiencies, a single BOD film-immobilized microorganism is only able to respond to limited organic solutes. Therefore, biosensors with mixed microorganisms (including activated sludge) immobilized within a single membrane onto an oxygen sensor were developed (Riedel et al., 1998a, Riedel et al., 1998b, Liu et al., 2000, Tan et al., 1993, KÖnig et al., 2000, Jia et al., 2003).
The immobilization method for binding microorganisms is another vital step for optical biosensor production. Silicones, Teflon®, plasticized polyvinyl chloride, cellulose and polyvinyl acetate are considered to be appropriate polymer materials for immobilization. Compared with many other organic polymers, organically-modified silicates (ormosils) are assimilated into silicon-oxide networks; these have been attracting great interest in recent years as novel materials for optical oxygen sensors (Chen et al., 2002, Brinker and Scherer, 1990, Walcarius, 2001). They have proven to be a better solid matrix to bind the microorganisms and at the same time maximally maintain their activity. Since PVA and silica associate well with interactions such as a hydrogen-bonding (Nakane et al., 1999), ormosils-PVA material has excellent biocompatibility that can immobilize biomolecules for fabricate biosensors. Dai et al. (2004) reported an effective and useful BOD sensor, in which a sol–gel acted as the immobilizing material to immobilize yeast in the sol–gel host matrix on an oxygen optical sensing film.
Although most of the BOD sensors previously reported have been designed for long-term use, and even commercialized, it seems that the performance of these sensors is still not satisfactory. Their limitations, such as (a) the depletion of oxygen occurring during BOD measurement using a sensor based on the Clark electrode; and (b) the microorganisms immobilized in current BOD sensors are not able to be applied in the samples containing a higher concentration of salt such as seawater. In this work, ormosil-PVA was used as a matrix to immobilize Bacillus licheniformis, Dietzia maris and Marinobacter marinus from seawater. A series of experiments characterizing the sensor were carried out including response time, reproducibility, linear range and the effects of temperature, pH and the sodium chloride concentration in the sample. The aim of the present study is to develop a biosensor for rapid and stable in situ determination of the BOD in seawater.
Section snippets
Chemicals and instrumentation
Tetramethoxysilane (TMOS) and polyvinyl acetate (PVA) were purchased from Aldrich (Milwaukee, WI, USA). Dimethyldimethoxysilane (DiMe-DMOS) was purchased from Fluka AG (Buchs, Switzerland). The [Ru(Ph2phen)3](ClO4)2 [Ph2phen = 4,7-diphenyl-1,10-phenanthroline, Ru(dpp)32+] used as the oxygen-sensing indicator was synthesized and purified in the laboratory of the Department of Applied Biology and Chemical Technology, Hong Kong Polytechnic University. Glucose/glutamate (GGA) solution was prepared by
Microstructure of ormosils
Typically, TMOS, DiMe-DMOS and 0.01 mol/L HCl (1:1.2:2, v/v) were selected for the preparation of the ormosils oxygen-sensing films. The color of the ormosils obtained was transparent and the texture rubbery. The percent of pores with sizes ranging from 0.8 to 2.0 nm for the ormosil film was about 47.8% (vol.%, same as below, Chen et al., 2004). Calculation by HYPERCHEM software showed the molecular size of Ru(dpp)32+ reagent had a diameter of about 2.0 nm. It indicates that the ormosils film
Conclusions
We have presented a novel optical BOD sensing film immobilizing sieved bacteria from seawater. In a matrix based on PVA-ormosils derived composite materials, the co-immobilized microorganisms can keep their activity even if they are kept up to one year at 4 °C. The stored sensing film can be employed for BOD measurement after 1 day's reactivation. The optimum response of the BOD sensing film was obtained at pH 7.9 and 35 °C. The minimum measurable BOD was 0.2 mg/L, and the response time was only 3
Acknowledgements
This research work was financially supported by the Natural Scientific Foundation of Fujian (D0410001) and the National High Technical Development Project (863 project) Foundations (2001AA635100, 2003AA635100), which are gratefully acknowledged. We express our sincere thanks to Miss Peiwei Li, Department of Biology, Indiana University, USA, for her kind revisions.
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