Biosensor-rivi erinevate reovete biokeemilise hapnikutarbe uurimiseks

A Thesis for applying for the degree of Doctor of Philosophy in Engineering Sciences.Väitekiri filosoofiadoktori kraadi taotlemiseks tehnikateaduse erialal.The basis of all life on Earth is the existence of clean water. The state of water must be constantly monitored to ensure that the water is clean. One critical parameter in accessing water purity is the content of organic pollutants. A universal parameter - biochemical oxygen demand (BOD) – is widely used to assess this. The BOD test determines the amount of biodegradable organic compounds in wastewater. The dissolved oxygen concentration in the test sample is measured at the beginning and end of the incubation period, and the BOD value is calculated from the difference. The incubation period during which the decomposed organic matter content is measured is 5 days according to the American standard (BOD5) and 7 days according to the Swedish standard (BOD7). Such a long incubation time is a severe disadvantage of this method, as, during this time, elevated BOD can have irreversible consequences for the water body. An alternative method for measuring BOD is to use biosensors that can estimate the BOD value of the sample in minutes. A biosensor is an analytical device consisting of a biological recognition element and physicochemical transducer. BOD biosensors use microorganisms as the biological recognition element and the dissolved oxygen sensor as the transducer. The accuracy of the biosensor depends mainly on the microorganisms used. Microorganisms with a broad substrate spectrum that use different types of organic compounds for their activities provide a better match to the BOD7 value. There are several other solutions for the selection of microorganisms. Mixed bacterial cultures have a broader substrate spectrum however, their composition changes over time and causes signal instability. One possibility, when it comes to approaching these issues, is to use different bacterial cultures in a sensor array in which different cultures are spatially separated from each other. The use of spatially separated pure microbial cultures makes it possible to prevent contamination between different sensors in the array while also ensuring a longer lifetime for the sensor. Different possibilities for constructing a BOD sensor array were investigated in this work. Microchips based on various technologies were first studied, and their suitability for electrochemical measurements was tested. The experiments showed that the screen-printed electrode array was suitable for the biosensor. The biosensor array was then constructed using the bacterial culture Pseudomonas putida Pc15, and its behaviour in different solutions was investigated. It was shown that the BOD of water samples could be estimated with the obtained biosensor array. In the future, it may be feasible to measure BOD values with the sensor array consisting of several membranes that have been modified with different pure bacterial cultures.Kogu elu alus maakeral on puhta veel olemasolu. Selleks, et tagada vee puhtus tuleb selle reostustaset pidevalt jälgida. Üks oluline parameeter vee puhtuse hindamisel on orgaanilise reostuse sisaldus. Selle hindamiseks kasutatakse laialdaselt universaalset parameetrit - biokeemiline hapnikutarve ehk BHT. BHT väljendab lahustunud hapniku hulka, mis kulub vees leiduva orgaanilise aine lagundamiseks ning anorgaaniliste ühendite oksüdeerimiseks teatud aja jooksul ja kontrollitud tingimustel. Selle leidmiseks mõõdetakse lahustunud hapniku kontsentratsioon uuritavas proovis inkubatsiooniperioodi alguses ja lõpus ning nende vahe alusel arvutatakse BHT väärtus. Inkubatsiooniperiood, mille vältel lagunenud orgaanilise aine sisaldust mõõdetakse on vastavalt Ameerika standardile 5 päeva (BHT5) ning Rootsi standardile 7 päeva (BHT7). Nii pikk inkubatsiooniaeg on antud meetodi puhul tõsiseks puuduseks, sest sellise aja jooksul võivad kõrgenenud BHT-l olla veekogule pöördumatud tagajärjed. Alternatiivse võimalusena kiiremaks BHT määramiseks on mikroorganismidel baseeruvad biosensorid, mis võimaldavad anda hinnangu proovi BHT kohta minutitega. Biosensor on analüütiline seade, mis koosneb bioloogilisest äratundmiselemendist ja signaali muundurist. BHT biosensorites kasutatakse bioloogilise äratundmissüsteemina mikroorganisme ja muundurina lahustunud hapniku andurit. Biosensori mõõtetäpsus sõltub suuresti kasutatud mikroorganismidest. Parema kokkulangevuse BHT7 väärtusega tagavad laia substraadispektriga mikroorganismid, mis kasutavad oma elutegevuseks erinevaid tüüpe orgaanilisi ühendeid. Mikroorganismide valikuks on mitmeid erinevaid lahendusi. Kuigi mikroobide segude kasutamisega saavutatakse lai substraadispekter ja tundlik sensor, on antud sensorid lühikese kasutuseaga ja ebastabiilsed kuna mikroorganismide kooslus membraanis muutub pidevalt. Selle vältimiseks on võimalik kasutada puhaskultuuridel põhinevaid sensor-rivisid, mis koosnevad paljudest erinevatest biosensoritest ning kus erinevad mikroorganismide kultuurid on omavahel ruumiliselt eraldatud. Antud töös uuriti erinevaid võimalusi BHT sensor-rivi koostamiseks. Selleks uuriti esmalt erinevatel tehnoloogiatel põhinevaid mikrokiipe ning iseloomustati nende sobivust teostades erinevaid elektrokeemilisi mõõtmisi. Katsete tulemusena osutus sobivaks siiditrüki meetodil valmistatud sensor-rivi kiip. Järgnevalt koostati biosensor rivi kasutades bakterikultuuri Pseudomonas putida Pc15 ja uuriti selle käitumist erinevates lahustes. Doktoritöö tulemusena leiti, et antud põhimõtet on võimalik kasutada sensor-rivide valmistamisel ning saadud biosensor-rividega on võimalik hinnata veeproovide BHT-d. Edasistes uuringutes tuleks uurida erinevate, ruumiliselt eraldatud bakterkultuuride samaaegset kasutamist sensor-rivis.Publication of this thesis is supported by the Estonian University of Life Sciences. This study has been supported by Doctoral School of Energy and Geotechnology III (Estonian University of Life Sciences ASTRA project “Value-chain based bio-economy”), European Regional Development Fund and Archimedes Foundation