Denitrifikator Pseudomonas stutzeri – izolovanje, optimizacija bioprocesnih parametara i primena

Abstract

U okviru ove doktorske disertacije prikazan je postupak izolacije i karakterizacije denitrifikatora uz optimizaciju biotehnoloških uslova proizvodnje biomase i ispitivanje efikasnosti proizvedene biomase u pogledu biočišćenja različitih građevinskih materijala. U okviru istraživanja koja su obuhvaćena ovom doktorskom disertacijom, izolovan je nov soj denitrifikatora koji je pomoću metode MALDI TOF identifikovan kao Pseudomonas stutzeri. U svim eksperimentima karakteristike i performanse novoizolovanog soja P. stutzeri, interno označenog kao D1, su poređene sa referentnom kulturom P. stutzeri ATCC 17588, koji je model mikroorganizam na kome se izučava proces denitrifikacije. Primenom PCR metode utvrđeno je postojanje četiri ključna denitrifikujuća gena (napA, nirS, norB i nosZ) u genomu P. stutzeri D1 i referentnog soja, čime je pokazano da ispitivani sojevi imaju genetsku predispoziciju za izvođenje potpune aerobne denitrifikacije. Takođe, uz primenu Boks-Benkenovog eksperimentalnog dizajna i metode odzivne površine, definisan je sastav hranljive podloge za dobijanje maksimalnog sadržaja biomase P. stutzeri D1 (1 g/L glukoze, 3 g/l KNO3 i 4 g/L peptone) i P. stutzeri ATCC 17588 (2 g/L KNO3 i 4 g/L peptona). Osim toga, optimizovani su i odabrani bioprocesni parametri za proizvodnju biomase oba denitrifikatora – temperatura 30°C, pH vrednost kultivacionog medijuma 7 jedinica i veličina inokuluma 5% (v/v). Validacija optimizovanih parametara kultivacije izvršena je izvođenjem nove serije ogleda pri definisanim uslovima u laboratorijskim bioreaktorima ukupnih zapremina 3 L i 7 L, čime je pokazana stabilnost bioprocesa prilikom uvećanja zapremine kultivacione tečnosti uz ostvarenje maksimalnog sadržaja biomase od 9 log CFU/ml za oba ispitivana soja uz minimalno opterećenje otpadnog toka. Rezultati primene denitrifikatora u vidu bioaktivnog sistema (pulpa sa suspenzijom denitrifikatora) koji je nanošen na površinu veštački kontaminiranih model supstrata opeke ukazuju na visoku efikasnost uklanjanja nitrata i to 95 % u slučaju P. stutzeri D1 i 72% u slučaju referentnog soja. Ispitivanjem bioaktivnih sistema primenjenih na kontaminirane uzorke stenskog materijala konstatovano je da se najveća efikasnost postiže tokom prvih 24 h, kada je efikasnost bioaktivnog sistema sa P. stutzeri ATCC 17588 47,7%, a bioaktivnog sistema sa P. stutzeri D1 čak 89,2%. U okviru doktorske disertacije izolovanjem i karakterizacijom visoko efikasnog denitrifikatora P. stutzeri D1 i detaljnim prikazom postupka optimizacije bioprocesnih parametara i validacije optimizovanih bioprocesnih parametara u laboratorijskim bioreaktorima načinjeni su prvi koraci ka definisanju idejnog rešenja i uvećanju razmera predloženog bioprocesa. Takođe, visoka efikasnost odabranih denitrifikatora u postupcima biočišćenja različitih građevinskih materijala, koja je postignuta u laboratorijskim uslovima predstavlja dobar osnov za nastavak istraživanja u realnim uslovima, kako na građevinskim materijalima, tako i u tretmanima zemljišta i otpadnih voda.

The main goal of this PhD thesis is to establish the procedure of isolation and characterization of denitrifiers along with the optimization of biotechnological conditions for the biomass production and testing the efficiency of the produced biomass in terms of biocleaning of various building materials. Within the research involved in this PhD thesis, novel denitrifier strain was isolated and identified as Pseudomonas stutzeri by using MALDI TOF. In all experiments, the characteristics and performances of the newly isolated strain of P. stutzeri (internally denoted as D1) were compared with the reference strain P. stutzeri ATCC 17588, which is a model microorganism for studying the denitrification process. By using PCR method the presence of four key denitrification genes (napA, nirS, norB and nosZ) in genome of the both strains was established indicating their genetic predisposition to perform complete aerobic denitrification. Also, the composition of the cultivation medium for obtaining the maximum content of biomass of P. stutzeri D1 (1 g/L glucose, 3 g/l KNO3 and 4 g/L peptone) and P. stutzeri ATCC 17588 (2 g/L KNO3 and 4 g/L peptone) was detemined by employing Box-Bhenken experimental design and RSM. In addition, the selected bioprocess parameters were optimized for the production of biomass of the both denitrifiers – temperature 30 °C, pH value of the cultivation medium of 7 units and inoculum size of 5% (v/v). Validation of the optimized bioprocess parameters was carried out by performing new series of experiments under previously define conditions in laboratory bioreactors with total volumes of 3 L and 7 L. The results of validation showed the stability of bioprocess while increasing the volume of the cultivation broth with the achievment of a maximum biomass content og 9 log CFU/ml for the both strains with minimum load of waste stream. The results of the application of the denitrifiers as a part of bioactive system (poultice with a suspension of denitrifiers) used onto the artificially contaminated brick model supstrates indicate a high efficiency of nitrate removal, namely 95% in the case of P. stutzeri D1 and 72% in the case of reference strain. By examing bioactive systems applied to contaminated stone material it was found that the highest efficiency in nitrate removal was achieved during the first 24 h (47.7% for the reference strian and 89.2% for the P. stutzeri D1). The results obtained in this PhD thesis including the established procedure for isolation and characterization of denitrifiers and detailed presentation of the optimization and validation process of bioprocess parameters represent the first steps made towards defining the conceptual solution and scale up of the proposed bioprocess. Moreover, the high efficiency of selected denitrificators in the biological treatment of various building materials, achieved in laboratory conditions is a good basis for continuing research in real conditions on construction materials, as well as in soil and wastewater treatment.