Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
2001-04-03
2004-03-23
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S041000, C435S042000, C435S162000, C435S170000, C435S177000, C435S252400, C435S253300, C435S260000, C435S261000
Reexamination Certificate
active
06709853
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a process for the preparation of cell beads BOD sensor useful for instant BOD estimation. The cell beads comprise of a formulated, synergistic, immobilized microbial consortium.
DESCRIPTION OF THE PRIOR ART
The problem of water pollution is increasing day by day with industrial development and urbanization. Many toxic and recalcitrant chemical compounds are being released in increasing amounts in the aquatic environment without proper treatment. The multitude of industries are by far the largest pollution creating units, whose discharges require a great degree of detoxification before they are released into the aquatic environment.
Before diverting the industrial effluents for treatment, their monitoring is crucial. Monitoring is an essential tool for waste-water management, for it details the ambient or background pollution level. Till date, classical chemical or spectrophotometric methods, requiring long reaction time and complicated procedures are used for monitoring of parameters/compounds of environmental interest such as Biochemical Oxygen Demand (BOD), heavy metals, pesticides, phenols, etc.
Amongst all these, BOD is a key parameter in the monitoring of water quality and treatment. BOD is a measure of the oxygen demand in a sample as a result of its organic content. BOD plays a very crucial role in assessing the pollutional strength of waste-waters, as high BOD means high organic content which further implies an increased consumption of oxygen to decompose these organics. BOD test is still carried out by the conventional method, which is very tedious and time consuming. The control of waste-water plants is difficult or even impossible using the classical determination method for BOD because of its high time consumption of five days.
Thus, there arises a need for determining the BOD values within minutes, which is possible only by using rapid BOD measuring devices. Sensors, which are devices that transduce a selective biochemical response into an electrical signal, can offer practical alternatives in environmental monitoring in terms of convenience, ability to reduce the reaction time and test system's complexity to a minimum and suitability for on-line applications.
For environmental monitoring, microbial biosensors are extensively used as compared to enzyme sensors. This is so because cell based sensors show a high degree of stability and are able to perform complex reaction sequences. Moreover, microorganisms employed in microbial biosensors are relatively easy to maintain in pure cultures, grow up and harvest at low cost, circumventing the costly extraction and purification procedures prior to use as biocatalysts as in the case of enzyme based sensors. In addition, BOD sensing by rapid devices cannot be achieved using enzyme-based sensors as BOD test is a sequence of complex reactions requiring an array of enzymes which are difficult to assemble at a time on a support. As evident from the literature, one report on whole cell based sensor for BOD estimation is available using single pure culture of microorganism (Su et al. 1986). The drawback of such developed cell beads sensor using single pure culture is that a single microbe is unable to assimilate all the organic matter present in the test sample; from either a single or a wide range of industries.
For solving the aforementioned problems, the applicants have realized that there exists a need to provide a process for the preparation of a defined synergistic microbial consortium immobilized in a suitable support i.e., agarose, useful for instant BOD estimation.
To overcome these problems, a number of bacterial strains have been used for the preparation of cell beads sensor individually as well as in combination. When the cell beads sensor using, single microorganism was used for instant BOD estimation they did not serve the said purpose. On the other hand, if the cell beads are prepared using combinations of microorganisms having synergistic effect and used for instant BOD estimation, it may yield reproducible and reliable results. Besides, the immobilization support also plays a crucial role in determining the response time and sensitivity of the sensor. Greater is the shelf life, stability and viability of the immobilized biocatalyst, better is the response.
In the present invention, cell beads sensor has been developed by immobilizing the formulated microbial consortium on a suitable support i.e., agarose under a hydrophobic condition, useful for instant BOD estimation. The support used for the immobilization in the present invention confers high stability, viability and negligible leaching to the biocatalyst i.e., microorganisms. This can be attributed to the fact that the biocatalysts immobilized in hydrophilic supports under hydrophobic conditions confer least leaching to the immobiliezed biocatalyst.
There is no prior art for the development of cell beads sensor using agarose as a support, for instant BOD estimation. On the contrary, there is only one report for the development of cell beads sensor for instant BOD estimat ion using supports other than agarose, such as &kgr;-carrageenan under hydrophilic conditions. The limitations of such developed sensor is that it is used only for the BOD estimation of glucose-glutamic acid (GGA) covening a very low range i.e., up to a concentration of 24 mg/l. The cell beads BOD sensor developed in the present invention can instantly estimate BOD of higher GGA concentrations (i.e., up to 300 mg/l), covering a wide range of synthetic samples (peptone, glucose, glutamic acid and citric acid) as well as a range of highly, moderately and low biodegradable industrial effluents.
The reproducibility of cell beads sensor can only be obtained if a defined microbial consortium is used as a biocatalyst, because sensors based on single organisms or microbial consortium isolated from activated sludge exhibit erroneous results. Further, they do not respond to higher GGA concentrations and a range of synthetic and industrial samples. Such specific microbial consortium based cell beads sensors may also find great applications in on-line monitoring of the degree of pollutional strength in a wide variety of industrial waste-waters within a very short time, which is very essential for combating the problem of water pollution. The said microbial consortium is capable of assimilating most of the organic matter present in different industrial effluents.
OBJECTS OF THE INVENTION
The main object of the present invention is to provide cell beads BOD sensor useful for instant BOD estimation.
Another object of the present invention is to provide a microbial consortium exerting a synergistic effect, capable of assimilating a variety of synthetic samples and industrial effluents.
Still another object of the present invention, is to provide a support for immobilization, which is non-toxic to the formulated microbial consortium and causes least physical and chemical damages to the microbial cells.
Yet another object of the present invention, is to provide a process for the preparation of cell beads useful for instant monitoring of BOD load of a wide range of synthetic samples as well as industrial effluents with low, moderate and high BOD load.
SUMMARY OF THE INVENTION
The present invention provides cell beads incorporating a formulated microbial consortium and a process for the preparation of the said cell beads useful for instant BOD estimation of a wide range of synthetic samples and industrial effluents with low, moderate and high BOD load.
DETAILED DESCRIPTION OF THE INVENTION
Accordingly the present invention provides bacteria namely
Aeromonas sobria, Enterobacter sakazaki, Pseudomonas aeruginosa
useful in the estimation of BOD, which have been deposited in the Center for Biochemical Technology, New Delhi, India, and either Deutsche Sammlung Von Mikroorganismen Und Zelikulturen GmbH Braunschweig, Germany, or the American Type Culture Collection, Manassas, Va. and given accession nos. CBTCC/Micro/13 (DSM 15063) having characteristics s
Kumar Rita
Rastogi Shikha
Saxena Tushya Kumar
Sharma Alka
Cooper & Dunham LLP
Council of Scientific & Industrial Research
Srivastava Kailash C.
Tate Christopher R.
White John P.
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