UNIVERSITY OF EAST SARAJEVO FACULTY OF TECHNOLOGY ZVORNIK PROCEEDINGS V INTERNATIONAL CONGRESS

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2 UNIVERSITY OF EAST SARAJEVO FACULTY OF TECHNOLOGY ZVORNIK PROCEEDINGS V INTERNATIONAL CONGRESS ENGINEERING, ENVIRONMENT AND MATERIALS IN PROCESSING INDUSTRY UNDER AUSPICIES OF THE MINISTRY OF SCIENCE AND TECHNOLOGY OF REPUBLIC OF SRPSKA THE ACADEMY OF SCIENCE AND ART OF REPUBLIC OF SRPSKA JAHORINA, March 15 th -17 th 2017 REPUBLIC OF SRPSKA BOSNIA AND HERZEGOVINA

3 PUBLISHER FACULTY OF TECHNOLOGY Karakaj 34a, Zvornik Republika Srpska, BiH Telefon: Fax: web: FOR PUBLISHER: Ph.D. Miladin Gligorić, dean ORGANIZING COMMITTEE/ORGANIZACIONI ODBOR: Ph.D. Miladin Gligorić, president, Ph.D. Aleksandar Došić, secretary, Ph.D. Milovan Jotanović, Ph.D. Miomir Pavlović, Ph.D. Radoslav Grujić, Ph.D. Dragica Lazić, Ph.D. Goran Tadić, Ph.D. Milorad Tomić, Ph.D. Mitar Perušić, Ph.D. Ljubica Vasiljević, Ph.D. Vaso Novaković, Ph.D. Dragan Vujadinović, Dipl. ing. Novo Škrebić, M.Sc. Mirjana Beribaka, M.Sc. Danijela Rajić, M.Sc. Marija Riđošić, M.Sc. Stefan Pavlović SCIENTIFIC AND PROGRAMME COMMITTEE: Ph.D. Todor Vasiljević, Australia, Ph.D. Ivan Krostev, Bulgaria, Ph.D. Milovan Jotanović, Bosnia and Herzegovina, Ph.D. Miomir Pavlović, Bosnia and Herzegovina, Ph.D. Miladin Gligorić, Bosnia and Herzegovina, Ph.D. Jovan Đuković, Bosnia and Herzegovina, Ph.D. Radoslav Grujić, Bosnia and Herzegovina, Ph.D. Dragan Tošković, Bosnia and Herzegovina, Ph.D. Dragica Lazić, Bosnia and Herzegovina, Ph.D. Živan Živković, Bosnia and Herzegovina, Academician Dragoljub Mirjanić, Bosnia and Herzegovina, Ph.D. Siniša Moljević, Bosnia and Herzegovina, Ph.D. Ljiljana Vukić, Bosnia and Herzegovina, Ph.D. Jasmin Komić, Bosnia and Herzegovina, Ph.D. Dane Malešević, Bosnia and Herzegovina, Ph.D. Midhat Suljkanović, Bosnia and Herzegovina, Ph.D. Mira Vukčević, Crna Gora, Ph.D. Darko Vuksanović, Crna Gora, Ph.D. ing. Srećko Stopić, Germany, Ph.D. Milan Sak-Bosnar, Croatia, Ph.D. Gyuala Vatai, Mađarska, Ph.D. Svetozar Hadži Jordanov, Macedonia, Ph.D. Kiril Lisičkov, Macedonia, Ph.D. Vineta Srebrenkoska, Macedonia, Ph.D. Jurij Krope, Slovenia, Ph.D. Andrzej Kowal, Poland, Ph.D. Magdalena Parlinska-Wojtan, Poland, Ph.D. Časlav Lačnjevac, Serbia, Ph.D. Milan Antonijević, Serbia, Ph.D. Đorđe Janaćković, Serbia, Ph.D. Branko Bugarski, Serbia, Ph.D. Ivan Juranić, Serbia, Ph.D. Božo Dalmacija, Serbia, Ph.D. Sonja Đilas, Serbia, Ph.D. Đorđe Okanović, Serbia EDITORIAL BOARD: Ph.D. Miladin Gligorić Ph.D. Aleksandar Došić Ph.D. Dragan Vujadinović TECHNICAL EDITORS: M.Sc. Mirjana Beribaka, M.Sc. Danijela Rajić, M.Sc. Marija Riđošić, M.Sc. Stefan Pavlović AREA: ENGINEERING, ENVIRONMENT AND MATERIALS IN PROCESSING INDUSTRY PUBLISHED: PRINT: Eurografika Zvornik CIRCULATION: 200 copies ISBN: The authors have full responsibility for the originality and content of their own papers

4 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry UDK 504.5: Scientific paper EX SITU BIOREMEDIATION AS CIRCULAR ECONOMY: MICROBIAL MINERALIZATION OF WASTE HEAVY REZIDUAL OIL FUEL (MAZUT) FROM BELGRADE (SERBIA) POWER PLANTS AND ITS REUSE FOR LANDFILL STABILIZATION Srđan Miletić 1,2, Gordana Gojgić-Cvijović 1, Vladimir Beškoski 3, Jelena Avdalović 1, Mila Ilić 1, Jelena Milić 1, Miroslav M. Vrvić 2,3 1 Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, Belgrade, Serbia. srdjan@chem.bg.ac.rs 2 BREM GROUP Ltd., Oslobođenja 39b, Belgrade, Serbia 3 Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Abstract In our work we have studied the process of bioremediation of waste heavy residual oil fuel (mazut) from the site of the Belgrade heating plants. Bioremediation is performed on the projected biopile adding microbial consortium that was isolated from the pollutet site. We added nutrients (biostimulation) at biopile, and aeration is provided by periodic mixing. Biostimulation and inoculation of biopile for 150 days resulted in a decrease of total petroleum hydrocarbons (TPH) for about 80 times and increased content of humic acids that indicate the beginning of Soilification. This "primitive" soil was used as an overlay for the stabilization of municipal waste landfill after bioremediation. Keywords: bioremediation, heavy residual fuel oil, TPH, landfill Introduction One of the technology that has been increasingly used for the remediation of contaminated habitats, primarily soil, is bioremediation. Bioremediation is especially effective in the remediation of habitats polluted by oil and oil products, but is also used for the treatment of waste which has not yet entered the environment, and are increasingly being used for habitat polluted by heavy metals. One of the most effective types of bioremediation is the use of nonpathogenic microorganisms isolated from pollutants. Microorganisms that are naturally present on the contaminated site translate toxic substances in products that are non-toxic to humans and the environment. Although the bioremediation can be used by microorganisms that have been transferred from another contaminated or even unpolluted habitat, the best effect show those who are isolated at the site of contamination. Many components of pollutants can be decomposed only by joint operation of multiple strains of microorganisms - consortium. Experimental Preparation of bioremediation pile (biopile) and ex-situ bioremediation Biopile was made on a watertight asphalt surface of about 1500 m 2 with a slope of 1%. Biopile was consisted of a mixture containing the waste fuel oil, sawdust (as an additional source of 439

5 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry carbon and as a filler) and river sand, which was added due to increased porosity. In order to ensure homogeneity the biopile was stirred with a bulldozer and is aligned at the end of the tractor. Biopile final volume was 600 m 3. The optimum ratio of C : N : P : K (100 : 10 : 1 : 0,1) was achieved by spraying the dissolved ammonium nitrate, diammonium phosphate and potassium chloride throughout the biopile. During bioremediation bipile was wet, mixed and rotated every 15 days in order to achieve the necessary humidity and aeration. Biopile was reinoculated every 30 days with prepared consortium of microorganisms. After mixing biopile was covered with a plastic polyethylene foil to avoid direct impact of weather conditions. On the biopile was also added BioSolve CLEAR in a volume of 70 ml of the original solution per cubic meter. At the beginning of the experiment the control sample was separated of about 10 m 3 of biopile. Chemical and microbiological parameters of bioremediation were followed immediately after preparation of biopile and every 50 days over the next 150 days (marked with an S-50, S- 100 and S-150) [1]. Sampling For sampling we used soil auger (Eijkelkamp). Depending on the penetration of contamination into the soil, it was sampled about kg of samples. Soil of individual samples was homogenized and samples were packed in glass jars for the chemical determinations and in a sterile Petri dish for microbiological determination. Samples were transported to the laboratory at temperature of 4 C and then were stored in a cold chamber at the same temperature. Microbiological analyzes of all samples were made immediately and chemical and physicochemical within hours. Gravimetric determination of the Total Petroleum hydrocarbons (TPH) Total Petroleum hydrocarbons (TPH) were extracted according to Beškoski et al. [1] and by ISO (2004) [2] and gravimetric determined by DIN EN (2004) [3]. Determination of TPH at a gas chromatograph TPH was determined on a gas chromatograph Agilent 7890A with FID detector. Isolation and determination of humic acids Humic acids have been isolated according to the changed standard procedure [4] with a solution of sodium pyrophosphate, and deposited with hydrochloric acid. They were determined by gravimetric analysis. Microbiological methods A consortium of microorganisms was obtained from soil contaminated with mazut by enrichment in 200 ml volumes of mineral medium (10 vol.%) [5] containing mazut (2 g L 1 ) as the only energy and carbon source in Erlenmeyer flasks (1 L). The conditions for microbial growth, were as follows: temperature 28 C, aeration on a shaker (120 rpm), ph 7.0 (adjusted with 1M HCl or NaOH) and the duration of growth for 96 hours. Microbial populations of the flasks was used to inoculate the bioreactor (approximately 1% by volume), volume was 1000 L. The conditions for growing germs were as follows: a non-sterile conditions, temperature 25 C, aeration and mixing of the air pump, a ph of 7, the duration of growth: 48 hours. 440

6 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry Determination of the number of microorganisms The number of micro-organisms was determined by serial dilution [6,7]. Total chemoorganoheterotrophs grown colonies of mesophilic aerobic and facultative anaerobic bacteria, as well as total mesophilic anaerobic chemoorganoheterotrophs bacteria were counted after 48 hours. The yeast and mold spores were counted after 72 hours, whereas the colonies of microorganisms which decompose hydrocarbons counted after 7 to 8 days. The seeded agar plates were incubated at a temperature of 28 C. Results and Discussion The main parameters for determining the performance of the bioremediation was reduction of TPH, increasing of humic acid content and changes the number of microorganisms that as the main source of carbon use oil hydrocarbons (Table 1, Figures 1-2). Isolated humic acids were good quality compared to standard (Figure 3). Before the start of bioremediation the soil has been placed to the hazardous waste, and at the end of bioremediation as non-hazardous and inert. Therefore it was used as an overlay for the stabilization of municipal waste landfill. Table 1. Basic parameters to monitor the process of bioremediation Parameter Unit S-0 S-50 S-100 S-150 Humidity % 15,4 0,5 13,0 0,7 14,5 0,2 13,4 1,5 ph 7,3 7,5 7,3 7,5 7,2 7,6 7,1 7,3 Loss on ignition 9,9 1,1 6,9 1,6 6,7 0,2 6,1 0,7 Organic carbon 2,46 1,87 1,19 1,08 0,04 0,08 0,06 0,05 Inorganic carbon % 0,65 0,66 0,60 0,56 0,03 0,04 0,03 Total nitrogen 0,25 0,23 0,22 0,03 0,04 0,02 Humic acid content % 1,92 2,30 2,72 2,83 TPH g / kg 39,9 15,21 5,3 < 0,5 Biopile Total chemoorganoheteroprophs Microorganisms which decompose hydrocarbons Total chemoorganoheteroprophs Microorganisms which decompose hydrocarbons CFU / g 0,03 0,25 0,01 2,0 x ,2 x ,3 x ,0 x ,2 x ,5 x ,9 x ,0 x 10 6 % Control CFU / g 9,7 x ,2 x ,2 x ,8 x ,6 x ,8 x ,2 x ,3 x 10 4 %

7 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry Figure 1. The content of TPH at the beginning of bioremediation Figure 2. The content of TPH at the end of bioremediation 442

8 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry Figure 3. FTIR spectra of comparative display standard and extracted humic acid Since the initial concentration of the bacteria that use oil carbohydrates was 10 4 CFU /g, and in other experiments was determined that when the population of the bacteria is less than 10 5 CFU/g there isn't a significant degree of bioremediacionih process. Therefore, it was necessary to increase the number of those bacteria [8]. The zymogenous cosortium of microorganisms was isolated and amplified. Biopile was reinoculated every 30 days. If we compare the value of the number of microbes that break down hydrocarbons in biopile and in control it can be observed that the inoculation and re-inoculation accompanied by biostimulation and aeration contributed to maintaining the necessary number of microorganisms that break down hydrocarbons originating from petroleum and it reached up to 99% of total chemoorganoheterotrophs compared to the control (where the increase reached the highest value up to 9%). Towards the end of bioremediation the number of microorganisms that break down hydrocarbons are reduced in comparison to the total number of chemoorganoheterotrophs to 25%. According to published papers biodiversity of contaminated soil was decreased due to the increase of the number of microorganisms that break down hydrocarbons originating from petroleum and as a result of the toxic effects of contaminants [9]. A clear indicator of bioremdiation process is the decreasing of the TPH value from 39.9 g / kg at the beginning of bioremediation to <0.5 g / kg TPH at the end of bioremediation (about 99% of total petroleum hydrocarbons was degraded during this period, Figures 1-2). Degradation was the fastest in the first 50 days. There was a correlation between the number of microorganisms that use oil hydrocarbon as the only source of carbon and decrease of TPH in the first 100 days. Proportion of those microorganisms in the number of total chemoorganoheterotrophs in the first 50 days increased from 4 to 68%, while the TPH was reduced to 40% of the initial value. From 50 to 100 days, the number of micro-organisms that break down hydrocarbons originating from petroleum is further increased to 76%, and TPH was reduced to 13% of the initial value. In the last 50 days, the proportion of micro-organisms originating from oil has been reduced to only 25%, while the TPH in the same period decreased to about 1% of the initial value. 443

9 V međunarodni kongres Inženjerstvo, ekologija i materijali u procesnoj industriji V International Congress Engineering, Environment and Materials in Processing Industry Conclusion Bioremediation proved to be a successful technology for circular economy, since microorganisms origin from waste degraded waste heavy rezidual oil fuel into non-hazardous and inert material. At the same time, the content of humic acid, which are indicatiors for fertile land, were increased. At the end this land was used as an overlay for the stabilization of municipal waste landfill. Acknowledgement This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under Grant No. III Literature [1] Beškoski, V.P.; Gojgić-Cvijović, G.; Milić, J.; Ilić, M.; Miletić, S.; Šolević, T.; Vrvić, M.M. (2011) Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil) A field experiment. Chemosphere 83, 34 [2] ISO 16703, Soil Quality Determination of Content of Hydrocarbon in the Range C10 to C40 by Gas Chromatography, Geneva [3] DIN EN 14345, Characterization of Waste. Determination of Hydrocarbon Content by Gravimetry. DIN, Berlin [4] ISO 5073, Brown coals and lignites Determination of humic acids, Geneva [5] Löser, C.; Seidel, H.; Zehnsdorf, A.; Stottmeister, U. (1998) Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions. Applied Microbiology and Biotechnology 49, 631 [6] Gojgić-Cvijović, G.; Vrvić, M.M. (2003) Praktikum za mikrobiološku hemiju, Hemijski fakultet, Univerzitet u Beogradu [7] Collins, C.H.; Lyne, P.M.; Grange, J.M.; Falkinham, J.O. (2004) Microbiological Methods, Arnold, London [8] Forsyth, J.V.; Tsao, Y.M.; Bleam, R.D. (1995) Bioaugmentation for Site Remediation, R.E. Hinchee, J. Fredrickson, B.C. Alleman, Eds., Battelle Press, Columbus, 1 [9] Katsivela, E.; Moore, E.R.B.; Maroukli, D.; Strömpl, C.; Pieper, D.; Kalogerakis, N. (2005) Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites. Biodegradation 16(2),

10 CIP - Каталогизација у публикацији Народна и универзитетска библиотека Републике Српске, Бања Лука (082)( ) 54(082)( ) 502/504(082)( ) INTERNATIONAL Congress "Engineering, Ecology and Materials in the Processing Industry" (5 ; 2017 ; Jahorina) Proceedings [Електронски извор] / V International Congress "Engineering, Ecology and Materials in the Processing Industry", Jahorina, March 15th-17th 2017 ; [editorial board Miladin Gligorić, Aleksandar Došić, Dragan Vujadinović]. - Zvornik : Faculty of Technology =Tehnološki fakultet, 2017 (Zvornik : Eurografika). - 1 elektronski optički disk (CD-ROM) : tekst, ilustr. ; 12 cm Sistemski zahtevi nisu navedeni. - Nasl. sa naslovnog ekrana. - Tiraž Bibliografija uz sve radove. - Abstract. - Registar. ISBN Faculty of Technology (Zvornik) COBISS.RS-ID

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12 EX SITU BIOREMEDIATION AS CIRCULAR ECONOMY: MICROBIAL MINERALIZATION OF WASTE HEAVY REZIDUAL OIL FUEL (MAZUT) FROM BELGRADE (SERBIA) POWER PLANTS AND ITS REUSE FOR LANDFILL STABILIZATION Srđan Miletić 1,2, Gordana Gojgić-Cvijović 1, Vladimir Beškoski 3, Jelena Avdalović 1, Mila Ilić 1, Jelena Milić 1, Miroslav M. Vrvić 2,3 1 Institute for Chemistry, Technology and Metallurgy, University of Belgrade, Njegoševa 12, Belgrade, Serbia. srdjan@chem.bg.ac.rs 2 BREM GROUP Ltd., Oslobođenja 39b, Belgrade, Serbia 3 Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Abstract In our work we have studied the process of bioremediation of waste heavy residual oil fuel (mazut) from the site of the Belgrade heating plants. Bioremediation is performed on the projected biopile adding microbial consortium that was isolated from the polluted site. We added nutrients (biostimulation) at biopile, and aeration is provided by periodic mixing. Results The main parameters for determining the performance of the bioremediation was reduction of TPH, increasing of humic acid content and changes the number of microorganisms that as the main source of carbon use oil hydrocarbons. Biostimulation and inoculation of biopile for 150 days resulted in a decrease of total petroleum hydrocarbons (TPH) for about 80 times and increased content of humic acids that indicate the beginning of Soilification. This "primitive" soil was used as an overlay for the stabilization of municipal waste landfill after bioremediation. Introduction One of the technology that has been increasingly used for the remediation of contaminated habitats, primarily soil, is bioremediation. Bioremediation is especially effective in the remediation of habitats polluted by oil and oil products., but is also used for the treatment of waste which has not yet entered the environment, and are increasingly being used for habitat polluted by heavy metals. One of the most effective types of bioremediation is the use of nonpathogenic microorganisms isolated from pollutants. Microorganisms that are naturally present on the contaminated site translate toxic substances in products that are non-toxic to humans and the environment. Although the bioremediation can be used by microorganisms that have been transferred from another contaminated or even unpolluted habitat, the best effect show those who are isolated at the site of contamination. Many components of pollutants can be decomposed only by joint operation of multiple strainsof microorganisms - consortium. Experimental Preparation of bioremediation pile (biopile) and ex-situ bioremediation Biopile was made on a watertight asphalt surface of about 1500 m 2 with a slope of 1%. Biopile was consisted of a mixture containing the waste fuel oil, sawdust (as an additional source of carbon and as a filler) and river sand, which was added due to increased porosity. In order to ensure homogeneity the biopile was stirred with a bulldozer and is aligned at the end of the tractor. Biopile final volume was 600 m 3. [1]. Determination of the Total Petroleum hydrocarbons (TPH) Basic parameters to monitor the process of bioremediation Parameter Unit S-0 S-50 S-100 S-150 Humidity % 15,4 0,5 13,0 0,7 14,5 0,2 13,4 1,5 ph 7,3 7,5 7,3 7,5 7,2 7,6 7,1 7,3 Loss on ignition 9,9 1,1 6,9 1,6 6,7 0,2 6,1 0,7 Organic carbon 2,46 0,04 1,87 0,08 1,19 0,06 1,08 0,05 % Inorganic carbon 0,65 0,03 0,66 0,04 0,60 0,03 0,56 0,03 Total nitrogen 0,25 0,03 0,23 0,04 0,22 0,02 0,25 0,01 Humic acid content % 1,92 2,30 2,72 2,83 TPH g / kg 39,9 15,21 5,3 < 0,5 Biopile Total chemoorganoheteroprophs 2,0 x ,2 x ,3 x ,0 x 10 6 Microorganisms which decompose hydrocarbons Total chemoorganoheteroprophs Microorganisms which decompose hydrocarbons CFU / g 7,2 x ,5 x ,9 x ,0 x 10 6 % Control CFU / g 9,7 x ,2 x ,2 x ,8 x ,6 x ,8 x ,2 x ,3 x 10 4 % The content of TPH Total Petroleum hydrocarbons (TPH) were extracted according to Beškoski et al. [1] and by ISO (2004) [2] and gravimetric determined by DIN EN (2004) [3]. Also, TPH was determined by a gas chromatograph. Microbiological methods A consortium of microorganisms was obtained from soil contaminated with mazut by enrichment in 200 ml volumes of mineral medium (10 vol.%) [4] containing mazut (2 g L 1 ) as the only energy and carbon source in Erlenmeyer flasks (1 L). Microbial populations of the flasks was used to inoculate the bioreactor (approximately 1% by volume), volume was 1000 L. The number of micro-organisms was determined by serial dilution [5,6]. At the beginning of bioremediation At the end of bioremediation Bioremediation of waste heavy residual oil fuel (mazut) from the site of the Belgrade heating plants FTIR spectra of comparative display standard and extracted humic acid Conclusion Bioremediation proved to be a successful technology for circular economy, since microorganisms origin from waste degraded waste heavy rezidual oil fuel into non-hazardous and inert material. At the same time, the content of humic acid, which are indicatiors for fertile land, were increased. At the end this land was used as an overlay for the stabilization of municipal waste landfill. Acknowledgement This research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia under Grant No. III Literature 1. Beškoski, V.P.; Gojgić-Cvijović, G.; Milić, J.; Ilić, M.; Miletić, S.; Šolević, T.; Vrvić, M.M. (2011) Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil) A field experiment. Chemosphere 83, ISO 16703, Soil Quality Determination of Content of Hydrocarbon in the Range C10 to C40 by Gas Chromatography, Geneva 3. DIN EN 14345, Characterization of Waste. Determination of Hydrocarbon Content by Gravimetry. DIN, Berlin 4. Löser, C.; Seidel, H.; Zehnsdorf, A.; Stottmeister, U. (1998) Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions. Applied Microbiology and Biotechnology 49, Gojgić-Cvijović, G.; Vrvić, M.M. (2003) Praktikum za mikrobiološku hemiju, Hemijski fakultet, Univerzitet u Beogradu 6. Collins, C.H.; Lyne, P.M.; Grange, J.M.; Falkinham, J.O. (2004) Microbiological Methods, Arnold, London