Title (srp)

Karakterizacija biofilmova i procena njihove efikasnosti u uklanjanju polutanata u biološkom tretmanu otpadnih voda

Author

Grujić, Sandra, 1989-

Contributor

Ostojić, Aleksandar, 1960-
Radojević, Ivana, 1973-
Teodorović, Ivana, 1971-
Kerkez, Đurđa, 1986-

Description (srp)

U ovom rukopisu ispitana je i utvrđena otpornost identifikovanih mikroorganizama koji su izolovani iz dva različita tipa otpadnih voda. Testirana je njihova sposobnost da formiraju pojedinačne i mešovite biofilmove, kao i njihova otpornost i biosorpciona sposobnost prema izabranim polutantima. Izabrani polutanti su rastvori teških metala: Pb2+, Cu2+, Cd2+, Zn2+, Hg2+ i Ni2+, i pet osnovnih boja iz automobilske industrije. Otpornost, a za neke mikroorganizme i biosorpciona efikasnost, je testirana i u planktonskoj formi. Izvršena je selekcija mikroorganizama prema sposobnosti da uspešno formiraju biofilmove i njihovoj otpornosti i biosorpciji prema izabranim polutantima. Dodatna selekcija je izvršena u korist najotpornijih biofilmova i tražena je idealna kombinacijamikroorganizama koji u okviru biofilma uspostavljaju sinergističke odnose i formiraju mešoviti biofilm sa velikom otpornošću i sposobnošću biosorpcije ka izabranim polutantima. Primenjena metodologija za selekciju najuspešnijih mešovitih biofilmova u uklanjanju teških metala je testirana i na laboratorijskim sojevima iz kolekcije sa Prirodnomatematičkog fakulteta Univerziteta u Kragujevcu i dobijeni rezultati su upoređeni. Isti princip u testiranju je primenjen i na izolatima iz otpadnih voda automobilske industrije za uklanjanje komercijalnih autoindustrijskih boja.Najveći broj izolovanih i identifikovanih mikroorganizama iz postrojenja Sistema za prečišćavanje otpadnih voda „Cvetojevac“ su pokazali sposobnost formiranja biofilma. Mikroorganizmi sa najboljom produkcijom biofilma (Enterobacter cloacae PMFKG-CV3, Serratia odorifera PMFKG-CV7, Saccharomyces cerevisiae PMFKG-CV10 i Klebsiella oxytocaPMFKG-CV4) su pokazali i najveću otpornost na testirane metale i oni su dalje testirani i na formiranje mešovitih biofilmova. Mešoviti biofilm koga čine K. oxytoca/S. odorifera/S. cerevisiae pokazao je najveću otpornost u prisustvu svih testiranih metala. Bolju sposobnost uklanjanja teških metala iz rastvora pokazali su mešoviti biofilmovi u odnosu na individualne. Iz rastvora je od metala najbolje uklanjan bakar. Kadmijum, olovo i živu najbolje uklanja biofilm E. cloacae/K. oxytoca/S. odorifera (uklonjeno 99.03%, 99.14% i 99.03%). Za uklanjanje nikla su najbolji rezultati zabeleženi za individualni biofilm K. oxytoca. Biofilm K. oxytoca/S. odorifera/S. cerevisiae je pokazao najbolje rezultate za uklanjanje bakra (99.19%), dok je za uklanjanje cinka, najbolje rezultate pokazao biofilm E. cloacae (98.06%).U planktonskoj formi najveću otpornost prema teškim metalima pokazala su dva soja Escherichia coli PMFKG-CV1 i CV2. Olovo najmanje utiče na mikroorganizme u planktonskoj formi, dok bakar i živa najviše utiču, sa izuzetkom izolata Candida utilis PMFKG-CV11.Na sličan način testirani su laboratorijski izolati E. coli LM1, Rhodotorula mucilaginosa i Saccharomyces bulardii iz kolekcije Prirodno-matematičkog fakulteta Univerziteta u Kragujevcu i rezultati su upoređeni. Testirana je otpornost biofilma i planktonskih ćelija prema teškim metalima. S. bulardii nije imao sposobnost da formira biofilm, a u planktonskoj formi je pokazao otpornost jedino u prisustvu Pb2+ i Zn2+, pa je isključen iz istraživanja. Planktonske ćelije R. mucilaginosa su najotpornije u prisustvu Cu2+, Cd2+ i Zn2+, dok je ova vrsta značajno otporna u formi biofilma u prisustvu svih testiranih metala. R. mucilaginosa pokazuje i sposobnost formiranja mešovitog biofilma sa E. coli LM1 i taj mešoviti biofilm je otporniji na uticaj teških metala od individualnih. Rezultati su potvrđeni fluorescentom mikroskopijom. Ovi individualni biofilmovi, kao i mešani biofilm, pokazuju izuzetno veliki biosorpcioni potencijal. Količina apsorbovanih metala kod individualnih biofilmova je za nijansu manja u odnosu na mešoviti. Napravljen je i mešoviti biofilm od izolata iz otpadne vode sa najboljim karakteristikama E. cloacae i R. mucilaginosa koji nije ispoljio očekivano veću otpornost ka testiranim metalima. Iste metode primenjene su kod testiranja za uklanjanje komercijalnih autoindustrijskih boja. Testirani mikroorganizmi za ovu svrhu su izolati otpadnih voda iz postrojenja za prečišćavanje u Kompaniji FCA Srbija d.o.o. Kragujevac. Veći broj identifikovanih izolata uspešno formira biofilmove. Mikroorganizmima sa najboljom produkcijom biofilma (Escherichia coli PMFKG-F1, PMFKG-F2, Proteus vulgaris PMFKG-F3, P.mirabilis PMFKG-F4, Saccharomyces cerevisiae PMFKG-F6, PMFKG-F7, Rhodotorula mucilaginosa PMFKG-F8, Candida utilis PMFKG-F9, Cladosporium cladosporioides PMFKG-F-11, Penicillium variotii PMFKG-F12, P. chrisogenum PMFKG-F13 i P. expansum PMFKG-F14) je određen stepen otpornosti prema autobojama. Svi testirani biofilmovi su pokazali znatno veću otpornost u prisustvu svih testiranih supstanci u odnosu na planktonske ćelije. Rezultati su potvrđeni fluorescentom mikroskopijom. Svi testirani biofilmovi pokazali su slabe enzimske aktivnosti čiji su rezultati dobijeni ispitivanjem sinteze degradacionih enzima, kroz spektrofotometrijsko određivanje koncentracije proteinaza, aktivnosti alkalne fosfataze i lipaze. Oba soja E. coli pokazuju najbolju alkalno fosfataznu aktivnost u prisustvu A-F107137-MF, pa se može pretpostaviti da bi mogle da učestvuju u razlaganju masnoća u otpadnim vodama. R. mucilaginosa pokazuje najveću alkalno fosfataznu aktivnost u prisustvu A-F107119-CN, kao i C. cladosporioides. P. expansum ima najveću alkalno fosfataznu aktivnost (ß-glicerofosfat) u prisustvu A-F107107-FM. Dobre proteazne aktivnosti zabeležene su za C. utilis i oba soja E. coli.U prisustvu A-F107117-CP jedino je mešoviti biofilm pokazao kiselo invertaznu aktivnost. Ostale enzimske aktivnosti su bile slabe ili ih nije ni bilo.Istraživanje je utvrdilo da različite vrste autohtonih mikroorganizama izolovanih iz otpadnih voda u formi pojedinačnog i/ili mešovitih biofilmova imaju značajno veći potencijal od planktonskog oblika iste vrste u otklanjanju različitih organskih ili neorganskih polutanata. Iz tog razloga mogu da posluže kao adekvatni biosorbenti za prečišćavanje otpadnih voda različitog porekla. Pokazano je da korišćena metodologija ima potencijal da se primeni na različitm otpadnim vodama, ali i da određeni izolati iz prirode,koji nisu autohtono sa mesta zagađenja testiranih supstanci, takođe mogu efikasno poslužiti u iste svrhe. Sve navedeno ukazuje na potencijal za dalju primenu u bioremedijaciji otpadnih voda.

Description (srp)

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Description (eng)

In this manuscript, the resistance of identified microorganisms isolated from two different types of wastewater was examined and determined. Their ability to form single and mixed biofilms, as well as their resistance and biosorption ability to selected pollutants was tested. The selected pollutants was solutions of heavy metals: Pb2+, Cu2+, Cd2+, Zn2+, Hg2+ and Ni2+, and five basic paints from the automotive industry. Resistance, and for some microorganisms, biosorption efficiency, was also tested in planktonic form. The selection of microorganisms according to the ability to successfully form biofilms and their resistance and biosorption according to selected pollutants was performed. Additional selection was performed in favor of the most resistant biofilms and the ideal combination of microorganisms was sought, which within the biofilm establish synergistic relationships and form a mixed biofilm with high resistance and biosorption ability to selected pollutants. The applied methodology for the selection of the most successful mixed biofilms in the removal of heavy metals was tested on laboratory strains from the collection from the Faculty of Sciences, University of Kragujevac, and the obtained results was compared. The same principle in testing was applied to automotive industry wastewater isolates to remove commercial automotive paints.The largest number of isolated and identified microorganisms from the plant of the Wastewater Treatment System "Cvetojevac" showed the ability to form a biofilm. The microorganisms with the best biofilm production (Enterobacter cloacae PMFKG-CV3, Serratia odorifera PMFKG-CV7, Saccharomyces cerevisiae PMFKG-CV10 and Klebsiella oxytoca PMFKG-CV4) also showed the highest resistance to the tested metals and were further formed. Mixed biofilm made up of K. oxytoca / S. odorifera / S. cerevisiae showed the highest resistance in the presence of all tested metals. Mixed biofilms showed better ability to remove heavy metals from the solution compared to individual ones. Copper is best removed from the metal solution. Cadmium, lead and mercury was best removed by the biofilm E. cloacae / K. oxytoca / S. odorifera (removed 99.03%, 99.14% and 99.03%). For nickel removal, the best results were recorded for the individual biofilm of K. oxytoca. Biofilm K. oxytoca / S. odorifera / S. cerevisiae showed the best results for copper removal (99.19%), while for zinc removal, E. cloacae biofilm showed the best results (98.06%).In the planktonic form, the highest resistance to the heavy metals was shown by two strains of Escherichia coli PMFKG-CV1 and CV2. Lead has the least effect on microorganisms in planktonic form, while copper and mercury have the greatest effect, with the exception of the Candida utilisPMFKG-CV11 isolate.Laboratory isolates of E. coli LM1, Rhodotorula mucilaginosa and Saccharomyces bulardiifrom the collection of the Faculty of Science, University of Kragujevac were similarly tested and the results were compared. The resistance of biofilm and planktonic cells to heavy metals was tested. S. bulardii did not have the ability to form a biofilm, and in the planktonic form it showed resistance only in the presence of Pb2+ and Zn2+, so it was excluded from the research. Planktonic cells of R. mucilaginosa were most resistant in the presence of Cu2+, Cd2+ and Zn2+, while this species is significantly resistant in the form of biofilm in the presence of all tested metals. R. mucilaginosa also showed the ability to form a mixed biofilm with E. coli LM1 and this mixed biofilm was more resistant to the influence of heavy metals than individual ones. The results were confirmed by fluorescence microscopy. These individual biofilms, as well as the mixed biofilm, showed extremely high biosorption potential. The amount of absorbed metals in individual biofilms was slightly lower compared to mixed. The mixed biofilm of wastewater isolates with the best characteristics of E. cloacae and R. mucilaginosawas also made, which did not show the expected higher resistance to the tested metals.The same methods were applied in testing to remove commercial automotive paints. The microorganisms tested for this purpose was wastewater isolates from the treatment plant in the Company FCA Srbija d.o.o. Kragujevac. A large number of identified isolates successfully form biofilms.Microorganisms with the best biofilm production (Escherichia coli PMFKG-F1, PMFKG-F2, Proteus vulgaris PMFKG-F3, P. mirabilis PMFKG-F4, Saccharomyces cerevisiae PMFKG-F6, PMFKG-F7, Rhodotorula mucilaginosa PMFKG-F8, Cladosporium cladosporioides PMFKG-F-11, Penicilliumvariotii PMFKG-F12, P. chrisogenum PMFKG-F13 and P. expansum PMFKG-F14) has a certain degree of resistance to car paints. All tested biofilms showed significantly higher resistance in the presence of all tested substances compared to planktonic cells. The results were confirmed by fluorescence microscopy. All tested biofilms showed weak enzymatic activities, the results of which were obtained by examining the synthesis of degradation enzymes, through spectrophotometric determination of proteinase concentration, alkaline phosphatase and lipase activity. Both strains of E. coli show the best alkaline phosphatase activity in the presence of A-F107137-MF, so it can be assumed that they could participate in the decomposition of fats in wastewater. R. mucilaginosa shows the highest alkaline phosphatase activity in the presence of A-F107119-CN as well as C. cladosporioides. P. expansum has the highest alkaline phosphatase activity (ß-glycerophosphate) in the presence of A-F107107-FM.Good protease activities have been reported for C. utilis and both strains of E. coli. In the presence of A-F107117-CP, only the mixed biofilm showed acidic invertase activity. Other enzyme activities were weak or non-existent. The study found that different species of indigenous microorganisms isolated from wastewater in the form of single and / or mixed biofilms have significantly higher potential than the planktonic form of the same species in removing various organic or inorganic pollutants. For that reason, they can serve as adequate biosorbents for wastewater treatment of various origins. It has been shown that the methodology used has the potential to be applied to different wastewaters, but also that certain isolates from nature, which are not autochthonous from the place of contamination of the tested substances, can also effectively serve the same purposes. All of the above indicates the potential for further application in wastewater bioremediation.

Object languages

Serbian

Date

2021

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