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Title: Development of high-throughput biosensors for multi-sample determination of biochemical oxygen demand (BOD) based on optical detection of oxygen
Authors: Kwok, Nga-yan
Degree: Ph.D.
Issue Date: 2005
Abstract: Biochemical oxygen demand (BOD) is one of the most important and frequently used parameters for estimating the level of organic pollutants in water. Microbial BOD sensors have received much attention as they provide fast, portable and cost-effective methods for environmental monitoring and rapid determination of wastewater quality in wastewater treatment plants. In this project, we aim at developing a high-throughput multi-sample BOD sensor for BOD determination of a large number of wastewater samples based on the optical sensing of dissolved oxygen concentration. Isolation, identification and screening for new microorganisms with high organic assimilation ability will contribute to the improvement of BOD sensors. More than 500 single species have been isolated from activated sludge samples in Hong Kong, of which 13 species with reasonably good assimilation ability were identified by the MIDI Sherlock Microbial Identification System using cellular fatty acid and 16S ribosomal RNA (rRNA) analysis. The assimilation activities (degradation power and degradation rate) of the isolated bacteria were investigated by monitoring the respiration rate using an oxygen optrode. A particular strain of Stenotrophomonas maltophilia was identified to give excellent assimilation ability for glucose-glutamic acid (GGA) solution and Organisation for Economic Co-operation and Development (OECD) synthetic wastewater. This particular strain is a rod-shaped, gram-negative species and was used for the construction of the BOD sensor. Two prototype optical biosensors for multi-sample BOD determination have been constructed based upon the optical sensing of change in dissolved oxygen concentration by the luminescent metal complex Ru[(dpp)3]2+ (dpp = 4,7-diphenyl-1,10-phenanthroline). In the first prototype BOD sensor, activated sludge and Bacillus subtilis strain 1A304 (f105MU331) were immobilized on oxygen sensing films on the bottom of glass sample vials. The oxygen sensing films were prepared by adsorbing [Ru(dpp)3](ClO4)2 onto high surface area hydrophilic fumed silica and dispersing the ruthenium-loaded silica in silicone rubber support. The microorganisms were immobilized in a sol-gel derived material of silica and poly(vinyl alcohol)-grafted-poly(4-vinylpyridine) (PVA-g-PVP) copolymer. The rate of change of oxygen concentration as recorded by the oxygen sensor increased linearly with the BOD loading (GGA standard) of the water sample up to about 25 mg l-1 for B. subtilis and 60 mg l-1 for activated sludge, which can be used to estimate the BOD content in water samples. This BOD biosensor was found to retain about 77 % of its initial activity for activated sludge and 88% for B. subtilis after stored at 4 C for 30 days. Good agreement was achieved between the results of the BOD measurement by the activated sludge biosensor and those obtained from conventional BOD5 method for water samples. However, only fairly good agreement was obtained by the B. subtilis biosensor. The results show that the B. subtilis biosensor may be suitable for wastewater with low BOD levels, as the microbes cannot assimilate a wide enough range of organic substrates. Sol-gel is a suitable matrix for fabrication of oxygen sensors due to its high oxygen permeability, thermal stability, photostability and chemical inertness. A simple method to prepare oxygen sensing films by entrapping the ruthenium dye, [Ru(dpp)3]Cl2, into organically modified silicate (ormosil) has been presented. The ormosil oxygen sensing film was prepared via acid catalyzed hydrolysis and condensation of tetramethoxysilane (TMOS) with the organosilicate precursor, dimethyldimethoxysilane (DiMe-DMOS). The quenching ratio, quenching time and recovery time of this ormosil sensing film are superior over other oxygen sensing films. The results showed that ormosil films made from DiMe-DMOS : TMOS volume ratio of 1.7 : 1.0 (v/v) are crack-free and can be used as high sensitivity oxygen sensors that have linear Stern-Volmer plot and long-term stability. This ormosil sensing films were therefore utilized for the construction of the second prototype BOD biosensor. The second prototype BOD biosensor was constructed using ormosil oxygen sensing films and Stenotrophomonas maltophilia immobilized in an ormosil polymer hybrid material of tetramethoxysilane (TMOS), methyltrimethoxysilane (MTMS) and poly(vinyl alcohol) (PVA). This BOD biosensor has a wide range of BOD loading (linearly up to about 60 mg l-1 GGA standard), longer lifetime, higher stability and better correlation of low BOD5 values (especially for GGA solutions) as compared to the first prototype biosensor. Good agreement was achieved between the results of the BOD measurement by the immobilized Stenotrophomonas maltophilia biosensor and those obtained from conventional BOD5 method for water samples. The particular strain Stenotrophomonas maltophilia isolated from activated sludge is shown to be suitable for BOD biosensor fabrication. Attempts were made to construct BOD biosensors by immobilizing Stenotrophomonas maltophilia in imidazolium-based ionic liquids or sol-gel/ionic liquid composites. The ionic liquid retains water which is necessary for the survival of the microorganisms. The performance of the biosensor, however, was limited by the slow diffusion of substrates in ionic liquids. Addition of sol-gel to ionic liquids to give sol-gel/ionic liquid composites can enhance the diffusion of oxygen and organics, but the performance of the resulting biosensor is still inferior to those with microbial films prepared from sol-gel/polymer composites. However, it is anticipated that the sol-gel/ionic liquid film can be further improved through manipulation of its microstructure.
Subjects: Hong Kong Polytechnic University -- Dissertations
Biochemical oxygen demand
Organic thin films
Pages: xxxiv, 315 leaves : ill. (some col.) ; 30 cm
Appears in Collections:Thesis

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