{Oxidized humic acids from the soil of heat power plant} J. Serb. Chem. Soc. 85 (3) 421–426 (2020) UDC 504.53.05+620.4:547.992.2+579.6 JSCS–5311 Short communication 421 SHORT COMMUNICATION Oxidized humic acids from the soil of heat power plant SRĐAN B. MILETIĆ1#, JELENA AVDALOVIĆ1#, JELENA MILIĆ1#, MILA ILIĆ1, ALEKSANDRA ŽERAĐANIN1#, KRISTINA JOKSIMOVIĆ2# and SNEŽANA SPASIĆ1*# 1University of Belgrade, National Institute for Chemistry, Technology and Metallurgy, Serbia and 2University of Belgrade, Innovation Center of the Faculty of Chemistry, Serbia (Received 27 July, revised and accepted 9 September 2019) Abstract: Humic acids isolated from the soil of a heat power plant (HA-E) con- taminated with oil were analyzed by Fourier transform infrared spectroscopy (FTIR). In comparison with a humic acids standard (HA-S), a lack of an int- ense broad band of the stretching vibrations of hydrogen-bonded hydroxyl groups (3600–3200 cm-1) is evident. The HA-E spectra have a peak at 1649 cm-1, which could belong to carbonyl groups. HA-E are heavily oxidized and among the isolated microorganisms, Achromobacter denitrificans may be res- ponsible for such intensive oxidation of HA-E. To the phylogenetically diverse nitrate-reducing microorganisms that have the capacity to utilize reduced HA as electron donors in soils, A. denitrificans can be added. Keywords: humic acid; microorganism; FTIR; contaminated soil. INTRODUCTION Structurally diverse, humic acids (HA) contain numerous functional moi- eties, including carboxylic acid, ketone, quinone, and phenolic/alcoholic hyd- roxyl groups. HA may be of particular relevance to inherent soil denitrification.1 HA are recalcitrant and degrade relatively slowly. However, reduced redox-act- ive functional groups within HA can be readily oxidized as electron donors for bacterial respiration.1,2 The hydroquinone content of reduced HA are considered important humus-borne electron donors of this type, and microbial oxidation of hydroquinones to the corresponding quinones has been demonstrated to support nitrate, perchlorate, arsenate, and selenate reduction.1–7 Although nitrate-depen- dent humic acid-oxidizing bacteria (NHOx) are common in the environment,2 their prevalence and metabolic activity in soils are still not well known. * Corresponding author. E-mail: svujin@chem.bg.ac.rs # Serbian Chemical Society member. https://doi.org/10.2298/JSC190726099M ________________________________________________________________________________________________________________________ (CC) 2020 SCS. Available on line at www.shd.org.rs/JSCS/ 422 MILETIĆ et al. It was decided to isolate HA-E to determine the number of presented micro- organsims by the serial dilutions method on agar plates, to identify microorg- anisms and to compare the FTIR spectra of humic acid standard with the FTIR spectra of the humic acid isolated from samples of soil contaminated with oil from a heat power plant. EXPERIMENTAL The number of microorganisms The number of microorganisms present was determined by the serial dilutions method on agar plates at 28 °C. Several types of media were used: • nutrient agar for total chemoorganoheterotrophs (NA), • malt agar for yeasts and molds (SA), • mineral base medium for hydrocarbon degraders (UG)8 with D2 diesel fuel9 and • nutrient agar for anaerobes (HAG). Identification of microorganisms using API tests Analytical profile index (API) tests were realized using the “BioMerieux Industry” manual. The API 20 NE, Rapid 20E and API Coryne systems were used for the detection of the microorganisms. Isolation of humic acids Following the standard procedure, the humic acids were isolated from soil contaminated with oil from the heat power plant with an alkaline sodium pyrophosphate solution (ISO 5073:1999). The HA-E was precipitated with hydrochloric acid. FTIR FTIR spectra were obtained in solid state using the attenuated total reflectance (ATR) sampling technique on Thermo-Nicolet 6700 spectrophotometer (Thermo Fisher Scientific, USA) at wave numbers between 400 and 4000 cm-1 with a spectral resolution of 4.0 cm-1 in the transmission mode. The spectra were analyzed using Omnic 7.3 software. RESULTS AND DISCUSSION The number of microorganisms The obtained results (Table I) show that the number of UG microorganisms was high in comparison with other types of microorganisms, which could indi- cate a high level of oil pollution of the soil. TABLE I. Number of microorganisms Media Number of microorganisms, CFU g-1 NA 4.3×106 SA 2.6×103 UG 3.5×104 HAG 1.8×102 ________________________________________________________________________________________________________________________ (CC) 2020 SCS. Available on line at www.shd.org.rs/JSCS/ HUMIC ACIDS FROM THE SOIL 423 Identification of microorganisms using API tests Several microorganisms, such as Pseudomonas putida, Pseudomonas aeru- ginosa, Achromobacter denitrificans, Pseudomonas sp., Bacillus cereus, Rhodo- coccus sp., Aeromonas hydrophila, etc., were identified. FTIR spectra In FTIR spectrum of HA-S, intense broad band of stretching vibrations of hydrogen bonded hydroxyl groups (3600–3200 cm–1) could be observed (Fig. 1). The standard has a very strong band at 1737 cm–1 that is assigned to C=O stretching of COOH group and a band at 1229 cm–1 that corresponds to in-plane bending vibrations of OH groups from COOH (Fig. 1). Fig. 1. FTIR of HA-S. Extracted humic acids (HA-E) lack OH groups and aliphatic chains com- pared to the standard HA, because peaks is absent in the 3000–3700 cm–1 and 2920–2850 cm–1 regions are absent. The peak at 1649 cm–1 could belong to car- bonyl group (Fig. 2). It was previously reported that HA promote zymogenous microbial consor- tium growth.10 HA influences microbial consortium growth via protection from oxidative stress, and this mechanism is highly significant in oxidative conditions (i.e., in the presence of free iron). A. denitrificans was identified from samples of ________________________________________________________________________________________________________________________ (CC) 2020 SCS. Available on line at www.shd.org.rs/JSCS/ 424 MILETIĆ et al. soil contaminated with oil from the heat power plant. It was shown that A. denit- rificans can induce biodegradation of sulfamethoxazole in a bacterial consortium and Leucobacter sp. GP.11 A. denitrificans efficiently utilizes 16 phthalate dies- ters and their downstream products through the protocatechuate 3,4-cleavage pathway, which makes A. denitrificans SP1 a very attractive candidate to be emp- loyed as an efficient biofactory in waste water treatment processes.12 A. denit- rificans strain SP1 efficiently remediates di(2-ethylhexyl) phthalate.13 Fig. 2. FTIR spectrum of HA-E. Nitrate-dependent HA oxidizing organisms isolated from agricultural soils were phylogenetically diverse and included members of the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria.14 CONCLUSIONS The present results indicate that HA from samples of soil contaminated with oil from a heat power plant are oxidatively degraded mainly by Achromobacter denitrificans. When the isolated HA was compared with Aldrich humic acid sodium salt (H16752) as standard, there were differences that may offer a new promising improvement in cleaning technologies in all environments, especially for soil (bioremediation) and high levels of pollution in water and air. Acknowledgment. This research is a part of Project III 43004 funded by the Ministry of Education, Science and Technological Development of the Republic of Serbia. The authors have declared no conflict of interest. ________________________________________________________________________________________________________________________ (CC) 2020 SCS. Available on line at www.shd.org.rs/JSCS/ HUMIC ACIDS FROM THE SOIL 425 И З В О Д ОКСИДОВАНЕ ХУМИНСКЕ КИСЕЛИНЕ ИЗ ТОПЛАНЕ СРЂАН Б. МИЛЕТИЋ1, ЈЕЛЕНА АВДАЛОВИЋ1, ЈЕЛЕНА МИЛИЋ1, МИЛА ИЛИЋ1, АЛЕКСАНДРА ЖЕРАЂАНИН1, КРИСТИНА ЈОКСИМОВИЋ2 и СНЕЖАНА СПАСИЋ1 Универзитет у Београду, Институт за хемију, технологију и металургију, Институт од националног значаја, Београд и 1Иновациони центар Хемијског факултета, Универзитет у Београду, Београд Хуминске киселине, изоловане из земљишта контаминираног нафтом из топлане (HA-E) је анализирано уз помоћ инфрацрвеног спектрометра (FTIR). У поређењу са стандардом хуминских киселина (HA-S) уочљив је недостатак интензивног пика вибра- ција водоничне везе из хидоксилне групе (3600–3200 cm-1). HA-E има пик на 1649 cm-1 који вероватно припада карбонилној групи. HA-E су веома оксидоване, а претпоставља се да су микроорганизми Achromobacter denitrificans за ову интензивну оксидацију HA-E. Они су филогенетски различити нитрат-редукујући микроорганизми који имају капа- цитет да редукују хуминске киселине где се понашају као електрон донори у земљишту. (Примљено 27. јула, ревидирано и прихваћено 9. септембра 2019) REFERENCES 1. D. R. Lovley, J. L. Fraga, J. D. Coates, E. L. Blunt-Harris, Environ. Microbiol. 1 (1999) 89 (https://doi.org/10.1046/j.1462-2920.1999.00009.x) 2. J. D. Coates, K. A. Cole, R. Chakraborty, S. M. O’Connor, L. A. Achenbach, Appl. Environ. Microbiol. 68 (2002) 2445 (https://doi.org/10.1128/aem.68.5.2445-2452.2002) 3. J. I. Van Trump, Y. Sun, J. D. Coates, Adv. Appl. Microbiol. 60 (2006) 55 (https://dx.doi.org/10.1016/S0065-2164(06)60003-8) 4. F. J. Stevenson, Humus chemistry: genesis, composition, reactions, Wiley, New York, 1994 (ISBN: 978-0-471-59474-1) 5. J. N. Boyer, P. M. Groffman, Soil Biol. Biochem. 28 (1996) 783 (https://dx.doi.org/10.1016/0038-0717(96)00015-6) 6. J. S. Gaffney, N. A. Marley, S. B. Clark, in Humic and fulvic acids – isolation, structure, and environmental role, Vol. 651, J. S. Gaffney, N. A. Marley, S. B. 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