Use of a biostimulant obtained from okara in the bioremediation of a soil polluted by used motor car oil

Elsevier Editorial System(tm) for Journal of Hazardous Materials Manuscript Draft Manuscript Number: HAZMAT-D-19-04377R2 Title: Use of a biostimulant obtained from okara in the bioremediation of a soil polluted by used motor car oil Article Type: Research Paper Keywords: okara biostimulant; used motor-car oil; polycyclic aromatic hydrocarbons; total heavy metals; soil dehydrogenase activity Corresponding Author: Dr. Manuel Tejada, Corresponding Author's Institution: First Author: Patricia Paneque Order of Authors: Patricia Paneque; Pablo Caballero; Juan Parrado; Isidoro Gómez; Manuel Tejada Abstract: In this manuscript we studied in the laboratory the bioremediation effects of a biostimulant obtained from okara by enzymatic hydrolysis processes in a soil polluted with used motor-car oil at a rate of 1% (w/w) over an 89-day period. The biostimulant was added to the soil 6 times during the incubation period at a rate of 2%. Dehydrogenase activity and the evolution of polycyclic aromatic hydrocarbons (PAHs) and pseudo total heavy metals in soil were studied. The successive applications of the biostimulant to the polluted soil gradually increased PAHs degradation during the experimental period. Thus, at the end of the experiment, the application of the biostimulant decreased the concentration of naphthalene in soil by 74%, while PAHs with 3, 4, 5 and 6 aromatic rings had been reduced by around 58%, 44%, 30% and 23%, respectively. This degradation is possibly due to the high number of low molecular weight peptides (<300 Da) in the biostimulant which are readily available for PAHs-tolerant soil microorganisms that accelerate the degradation of the said toxins. The concentration of heavy metals in the oil used was not very high and consequently the dehydrogenase activity was not negatively affected. Dear Editor, Again I remit to you the paper entitled “Use of a biostimulant obtained from okara in the bioremediation of a soil polluted by used motor car oil”·corrected and revised according to the reviewer comments for its possible publication in Journal of Hazardous Materials. In this respect, the new full paper has the following words: Manuscript: 4419 words Tables: 1515 words Yours sincerely Dr. Manuel Tejada *Cover Letter Few manuscripts describe the use of organic matter in the bioremediation of soils polluted with used motor-car oil characterised by its high content of polycyclic aromatic hydrocarbons and heavy metals. The aim of this work was to study under controlled laboratory conditions the bioremediation effect of a edaphic biostimulant obtained from okara enzymatic hydrolysis processes applied continuously for a period of 89 days in a soil polluted with used motor-car oil. The authors believe that this work is an advance in the study in the use of different organic compounds in the bioremediation of polluted soils. Novelty Statement (maximum limit:100 words) 1 Abstract In this manuscript we studied in the laboratory the bioremediation effects of a biostimulant obtained from okara by enzymatic hydrolysis processes in a soil polluted with used motor-car oil at a rate of 1% (w/w) over an 89-day period. The biostimulant was added to the soil 6 times during the incubation period at a rate of 2%. Dehydrogenase activity and the evolution of polycyclic aromatic hydrocarbons (PAHs) and pseudo total heavy metals in soil were studied. The successive applications of the biostimulant to the polluted soil gradually increased PAHs degradation during the experimental period. Thus, at the end of the experiment, the application of the biostimulant decreased the concentration of naphthalene in soil by 74%, while PAHs with 3, 4, 5 and 6 aromatic rings had been reduced by around 58%, 44%, 30% and 23%, respectively. This degradation is possibly due to the high number of low molecular weight peptides (<300 Da) in the biostimulant which are readily available for PAHs-tolerant soil microorganisms that accelerate the degradation of the said toxins. The concentration of heavy metals in the oil used was not very high and consequently the dehydrogenase activity was not negatively affected. *Abstract Reviewer #1: I don’t know why I recorded some grammatical errors. Even though the author claimed that the manuscript was edited by a native English speaker, I suggest to author to recheck the language for readability. The grammar of the manuscript has been reviewed again by a native English speaker. In this sense, the grammar changes made are indicated in the revised manuscript in red. We hope that all grammatical problems indicated by the reviewer will be definitively resolved. *Response to Reviewers 1 Use of a biostimulant obtained from okara in the bioremediation of a soil polluted by 1 used motor car oil 2 3 Patricia Panequea, Pablo Caballerob, Juan Parradob, Isidoro Gómeza, Manuel Tejadaa* 4 5 aGrupo de Investigación Edafología Ambiental, Departamento de Cristalografía, Mineralogía 6 y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, 7 Spain 8 bDepartamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de 9 Sevilla, C/Prof. García González 2, 41012 Sevilla, Spain 10 11 *Corresponding author 12 E-mail: [email protected] 13 Tel.: +34954486468 14 Fax: +34954486436 15 16 *Revised Manuscript Click here to view linked References 2 Abstract 17 In this manuscript we studied in the laboratory the bioremediation effects of a 18 biostimulant obtained from okara by enzymatic hydrolysis processes in a soil polluted with 19 used motor-car oil at a rate of 1% (w/w) over an 89-day period. The biostimulant was added 20 to the soil 6 times during the incubation period at a rate of 2%. Dehydrogenase activity and 21 the evolution of polycyclic aromatic hydrocarbons (PAHs) and pseudo total heavy metals in 22 soil were studied. The successive applications of the biostimulant to the polluted soil 23 gradually increased PAHs degradation during the experimental period. Thus, at the end of the 24 experiment, the application of the biostimulant decreased the concentration of naphthalene in 25 soil by 74%, while PAHs with 3, 4, 5 and 6 aromatic rings had been reduced by around 58%, 26 44%, 30% and 23%, respectively. This degradation is possibly due to the high number of low 27 molecular weight peptides (<300 Da) in the biostimulant which are readily available for 28 PAHs-tolerant soil microorganisms that accelerate the degradation of the said toxins. The 29 concentration of heavy metals in the oil used was not very high and consequently the 30 dehydrogenase activity was not negatively affected. 31 32 Keywords: okara biostimulant; used motor-car oil; polycyclic aromatic hydrocarbons; total 33 heavy metals; soil dehydrogenase activity 34 35 1. Introduction 36 At present the use of lubricating oils is widespread because they are necessary for the 37 correct working of motor vehicle engines and other machinery such as pumps, hydraulic 38 motors, compressors and electrical transformers [1]. Due to the physical and chemical 39 reactions that occur during use, used motor oil is characterised by its high content of aromatic 40 and aliphatic hydrocarbons whose chain lengths range from C15 to C50 and heavy metals that 41 3 could contribute to the risk of chronic diseases, including mutagenicity and carcinogenicity 42 [1-3]. 43 As a result of their widespread use, terrestrial ecosystems are becoming increasingly 44 contaminated by such lubricants. The bioremediation of soils contaminated with used motor 45 oils is, therefore, an efficient and effective environmental technique for accelerating the 46 cleaning processes [2, 4]. 47 In recent years, applying organic biostimulants obtained from sewage sludge, chicken 48 feathers, wheat condensed distiller’s soluble enzymatic hydrolysate, rice bran extract, carob 49 germ enzymatic extract, etc. by enzymatic hydrolysis processes to soils polluted by organic 50 xenobiotics has been a widely-used environmental technique for accelerating the degradation 51 of the said xenobiotics in soil. The low molecular weight proteins, found in large quantities in 52 these biostimulants, are absorbed by the xenobiotics-tolerant microorganisms, increasing both 53 their proliferation and the biochemical activity of the soil, consequently accelerating the 54 degradation of the toxins in the soil [5-8]. 55 Okara is a by-product obtained from the manufacture of soy milk, tofu and their 56 derivatives. It has both a high fibre (56%) and protein (29%) content. Orts et al. [9] obtained a 57 new biostimulant from okara by enzymatic hydrolysis processes using the pH-stat technique 58 in order to remedy a soil polluted with chlorpyrifos. Eighty days after applying the 59 biostimulant to polluted soil, the insecticide had been completely degraded. Consequently, 60 this biostimulant obtained from okara could be of great use for remedying a soil contaminated 61 by motor oil with a high content of polycyclic aromatic hydrocarbons (PAHs). 62 It should be noted, however, that the effectiveness of these biostimulants obtained by 63 enzymatic hydrolysis processes has been verified after only a single application to polluted 64 soil. It is not known what the behaviour of organic xenobiotics in the polluted soil would be 65 after repeated applications of these biostimulants. 66 4 There is an abundant literature indicating that soil biological parameters evolve faster 67 than physical and chemical parameters [6, 10-12]. There is also a lot of information on how 68 PAHs and heavy metals negatively affect soil enzymatic activities, as well as affecting its 69 microbial population [8, 11, 13, 14]. For this reason, a study of these biological properties 70 would be very useful in order to verify the bioremediation capacity of organic matter in 71 polluted soils. 72 According to Kaczynski et al. [15] and Liu et al. [16], the oxidation-reduction 73 enzymes found in soil play a fundamental role in degrading contaminants, transforming 74 organic matter and in maintaining the metabolism of microorganisms. In this respect, 75 dehydrogenase activity (EC 1.1.1) is an essential intracellular oxidoreductase enzyme in 76 living cells. It participates in redox reactions, it promotes the dehydrogenation of soil organic 77 matter and transmits energy to hydrogen acceptors through the respiratory chain [17]. Due to 78 the above, this enzyme is now used as a soil contamination status bioindicator [18, 19, 20]. 79 The objective of this study, therefore, was to verify the bioremediation efficiency of a 80 biostimulant, obtained from okara by enzymatic hydrolysis processes, in a soil polluted by 81 used motor oil after repeated applications of the said biostimulant to the polluted soil and its 82 influence on the soil dehydrogenase activity. 83 84 2. Material and methods 85 2.1. Soil, biostimulant and motor oil characteristics 86 The soil used in the study was classified as Calcaric Regosol [21], with 574 ± 31g kg-1 87 sand, 148 ± 35 g kg-1 silt, and 278 ± 24 g kg-1 clay. Soil pH was 7.9 ± 0.1, 16.4 ± 1.3 gkg-1 88 organic matter, and 0.98 ± 0.05 g kg-1 N. The methodology used for determining the soil 89 parameters is described in Tejada et al. [7] and Rodríguez-Morgado et al. [22]. 90 11 the incubation period decreased progressively the soil PAHs concentration. Obviously, the 238 degradation was greater in those PAHs with a lower aromatic number. 239 In comparison with our experiment, Tejada et al. [6] and Rodriguez-Morgado et al. [8] 240 found a higher degradation percentage of phenanthrene, pyrene and benzo (a) pyrene after 241 applying to soil different edaphic biostimulants obtained by enzymatic hydrolysis processes 242 from rice bran, wheat condensed distiller’s soluble, sewage sludge and chicken feathers. In 243 our experiment the soil was polluted by a varied mixture of PAHs, presenting from 2 to 6 244 aromatic rings. Consequently, although applying the experimental biostimulants accelerated 245 degradation of those PAHs with fewer aromatic rings, the high concentration of PAHs with a 246 greater number of aromatic rings, mainly those with 5 and 6 aromatic rings, possibly 247 continued to exert a very toxic effect on soil microorganisms, as reflected by soil 248 dehydrogenase activity (Table 4). 249 Table 6 shows the evolution of the pseudo total heavy metals content in the 250 experimental treatments during the incubation period. The pseudo total heavy metals contents 251 in treatments C and C+BS were very similar on days 3 and 86 of the experiment, suggesting 252 that the pseudo total heavy metals concentration in the polluted and organically-amended soil 253 did not vary with respect to the non-amended and non-polluted soil. In the same way, and as 254 previously mentioned, the dehydrogenase activity in the C+BS treatment was stimulated when 255 compared to treatment C. 256 A large number of references indicate that a high concentration of heavy metals in the 257 soil solution can inhibit its dehydrogenase activity [11, 30, 31]. Our experiment measured the 258 pseudo total heavy metals content in soil, not merely the content of metals available. Since the 259 dehydrogenase activity was not inhibited in the C+BS treatment, we believe that the heavy 260 metals concentration in the biostimulant applied to the non-polluted did not exert any toxic 261 effect on the above enzymatic activity. 262 12 With respect to the C treatment, the concentration of pseudo total heavy metals in the 263 C+O treatment did not vary significantly during the incubation period. This suggests that, 264 possibly, the heavy metal contents in the experimental car motor oil used in our experiment 265 did not have any negative effects on the soil dehydrogenase activity. Therefore PAH content 266 alone would be the cause of soil toxicity. These results do not coincide with those obtained by 267 Dike et al. [1] and Wang et al. [32], who suggest that the toxic effect of used motor oil is 268 mainly a consequence of both the PAHs content and the heavy metals. 269 According to Ramadass et al. [3], the concentration of heavy metals in a used motor 270 oil is the result of wear and tear in the engine components and the heating and oxidation of the 271 lubricating oil. We believe that the concentration of heavy metals in the used motor oil 272 depends on how the motor has been used. In our experiment the oil was sourced from a 273 15000-km oil change performed on gasoline car engine. Such a short period of use may not be 274 sufficient for any great amount of heavy metals to be transferred from the engine to the oil. 275 Applying the biostimulant to the oil-polluted soil showed heavy metal values very 276 similar to those of treatment C, which suggests that applying the experimental biostimulants 277 to the polluted soil did not produce any significant change to its heavy metal content. 278 Applying the biostimulant to the non-polluted soil did not change the pH value (Table 279 7). The basic nature of the biostimulant, together with the soil pH value are sufficient reason 280 for a change in the pH of said soil not to occur. On the other hand, neither did applying the 281 biostimulant to the oil-polluted soil change the pH. These results suggest that the 282 concentration of heavy metals, acidic in character, is low in the experimental oil. In addition, 283 it must also be taken into account that the amount of oil applied to the soil was not very high, 284 so the heavy metal concentrations added to the soil was also low. Regarding the PAH 285 concentration in the oil, it should be noted that these chemical compounds do not have an 286 acidic character and, therefore, it also justifies the lack of change in soil pH. 287 13 4. Conclusions 288 It can be concluded that the toxic effect of used gasoline car engine oil is mainly due 289 to its content of polycyclic aromatic hydrocarbons of 2 to 6 aromatic rings. Since the 290 biostimulant has a high content of low molecular weight proteins which are easily assimilated 291 by such microorganisms, the successive applications of the biostimulant to the PAH-polluted 292 soil stimulated PAH-tolerant microorganisms. This accelerated the degradation of PAH in 293 soil, thus reducing its contaminating effect on the soil. The presence of hydrocarbons with a 294 high number of highly toxic aromatic rings did, however, slow down the degradation of those 295 polycyclic aromatic hydrocarbons with fewer aromatic rings. 296 Notwithstanding the above, and in order to establish with greater precision the 297 bioremedial effect of the said biostimulant, the current results justify further research into 298 using the biostimulant obtained from okara by enzymatic hydrolysis processes at other 299 concentrations and in different types of soils for remedying the effects of used gasoline car 300 engine oil on soil. 301 302 Acknowledgments 303 This work was supported by the Ministerio de Ciencia, Innovación y Universidades 304 (Spain), Plan Nacional I+D+i with reference RTI2018-097425-B-I00. 305 306 References 307 [1] B.U. Dike, B.C. Okoro, N.N. Nwakwasi, K.C. Agbo, Remeadiation of used motor engine 308 oil contaminated soil: A soil washing treatment approach,J. Civil Environ. Eng. 3 309 (2013) 129. 310 [2] T. Mandri, J. Lin, Isolation and characterization of engine oil degrading indigenous 311 microorganisms in Kwazulu-Natal,Afr. J. Biotechnol. 6 (2007) 23-27. 312 14 [3] K. Ramadass, M. Megharaj, K. Venkateswarlu, R. Naidu, Ecological implications of 313 motor oil pollution: Earthworm survival and soil health,Soil Biol. Biochem. 85 (2015) 314 72-81. 315 [4] E. Dindar, F.O. TopaçŞağban, H.S. Başkaya, Biodegradation of used engine oil in a 316 wastewater sludge-amended agricultural soil, Turk. J. Agric. For. 40 (2016) 631-641. 317 [5] M. Tejada, A.M. Garcia-Martinez, I. Gomez, J. Parrado, Application of MCPA herbicide 318 on soils amended with biostimulants: Short-time effects on soil biological 319 properties,Chemosphere 80 (2010) 1088-1094. 320 [6] M. Tejada, C. Benitez, J. Parrado, Application of biostimulants in benzo(a)pyrene polluted 321 soils: Short-time effects on soil biochemical properties,Appl. Soil Ecol. 50 (2011a) 21-322 26. 323 [7] M. Tejada, B. Rodríguez-Morgado, I. Gómez, J. Parrado, Degradation of chlorpyrifos 324 using different biostimulants/ biofertilizers: Effects on soil biochemical properties and 325 microbial community, Appl. Soil Ecol. 84 (2014) 158-165. 326 [8] B. Rodríguez-Morgado, I. Gómez, J. Parrado, C. García, T. Hernández, M. 327 Tejada,Accelerated degradation of PAHs using edaphic biostimulantsobtained from 328 sewage sludge and chicken feathers,J. Hazard. Mater. 300 (2015a) 235-242. 329 [9] A. Orts, S. Cabrera, I. Gómez, J. Parrado, B. Rodríguez-Morgado, M. Tejada, Use of 330 okara in the bioremediation of chlorpyrifos in soil: Effects on soil biochemical 331 properties, Appl. Soil Ecol. 121 (2017) 172-176. 332 [10] M.C. Zabaloy, J.L. Garland, M.A. Gómez, An integrated approach to evaluate the 333 impacts of the herbicides glyphosate 2,4-D and metsulfuron-methyl on soil microbial 334 communities in the Pampas region, Argentina, Appl. Soil Ecol. 40 (2008) 1-12. 335 15 [11] M. Tejada, J. Parrado, T. Hernández, C. García, The biochemical response to different Cr 336 and Cd concentrations in soils amended with organic wastes,J. Hazard. Mater. 185 337 (2011b) 204-211. 338 [12] M. Tejada, E. Morillo, I. Gómez, F. Madrid, T. Undabeytia, Effect of controlled release 339 formulations of diuron and alachlorherbicides on the biochemical activity of 340 agricultural soils,J. Hazard. Mater. 322 (2017) 334-347. 341 [13] P. Thavamani, S. Malik, M. Beer, M. Megharaj, R. Naidu, Microbial activity and 342 diversity in long-term mixed contaminated soils with respect to polyaromatic 343 hydrocarbons and heavy metals, J. Environ. Manage. 99 (2012) 10-17. 344 [14] P. Zeng, Z. Guo, X. Xiao, C. Peng, Dynamic response of enzymatic activity and 345 microbial community structurein metal(loid)-contaminated soil with tree-herb 346 intercropping, Geoderma 345 (2019) 5-16. 347 [15] P. Kaczynski, B. Lozowicka, I. Hrynko, E. Wolejko, Behaviour of mesotrione in maize 348 and soil system and its influence on soil dehydrogenase activity, Sci. Total Environ. 349 571, 1079-1088. 350 [16] Y. Liu, G. Zeng, H. Zhong, Z. Wang, M. Liu, G. Cheng, X. Yang, S.J. Liu, Effect of 351 rhamnolipid solubilization on hexadecane bioavailability: enhancement or reduction?, 352 J. Hazard. Mater. 322 (2017) 394-401. 353 [17] S. Visser, D. Parkinson, Soil biological criteria as indicators of soil quality: soil 354 microorganisms, Am. J. Altern. Agric. 7, 33. 355 [18] A. Wolińska, Z. Stępniewska, Dehydrogenase activity in the soil environment. In 356 dehydrogenases, Cabuto, R.A. (Ed.), Intech, 2012, pp. 183-210. 357 [19] A. Lipińska, J.Kucharski, J. Wyszkowska, The effect of polycyclic aromatic 358 hydrocarbons on the structure of organotrophic bacteria and dehydrogenase activity in 359 soil, Polycycl. Aromat. Comp. 34 (2014) 35-53. 360 16 [20] X. Tan, Z. Wang, G. Lu, W. He, G. Wei, F. Huang, X. Xu, W. Shen, Kinetics of soil 361 dehydrogenase in response to exogenous Cd toxicity, J. Hazard. Mater. 329 (2017) 362 299-309. 363 [21] FAO, Carte mondiale des sols. Légende revise, FAO, 1989. 364 [22] B. Rodríguez-Morgado, I. Gómez, J. Parrado, M. Tejada, Behaviour of oxyfluorfen in 365 soils amended with edaphic biostimulants/biofertilizers obtained from sewage sludge 366 and chicken feathers. Effects on soil biological properties, Environ. Sci. Pollut. Res. 367 21 (2014) 11027-11035. 368 [23] J.T. Trevors, C.I. Mayfield, W.E. Inniss, Measurement of electron transport system 369 (ETS) activity in soil, Microb. Ecol. 8 (1982) 163-168. 370 [24] C. García, T. Hernández, F. Costa, B. Ceccanti, G. MasciandaroThe dehydrogenase 371 activity of soils an ecological marker in processes of perturbed system regeneration, J. 372 Gallardo-Lancho (Ed.), Proceedings of the XI International Symposium of 373 Environmental Biogeochemistry, Salamanca, Spain (1993), pp. 89-100 374 [25] B. Rodriguez-Morgado, I. Gomez, J. Parrado, A.M. Garcia-Martinez, C. Aragon, M. 375 Tejada, Obtaining edaphic biostimulants/biofertilizers from different sewage sludges. 376 Effects on soil biological properties, Environ. Technol. 36 (2015b) 2217-2226. 377 [26] A.R. Johnsen, L.Y. Wick, H. Harms, Principles of microbial PAH-degradation in 378 soil,Environ. Pollut.133 (2005) 71-84. 379 [27] E. Wolejko, U. Wydro, A. Jablońska-Trypuć, A. Butarewicz, T. Łoboda, The effect of 380 sewage sludge fertilization on the concentration of PAHs in urban soils, Environ. Poll. 381 232, 347-357. 382 [28] J.N. Edokpayi, J.O. Odiyo, O.E. Popoola, T.A.M. Msagati, Determination and 383 distribution of polycyclic aromatic hydrocarbons in rivers, sediments and wastewater 384 17 effluents in Vhembe district, South Africa,Int. J. Environ. Res. Public Health 13 385 (2016) 387. 386 [29] Y. Yang, N. Zhang, M. Xue, S.T. Lu, S. Tao, Effects of soil organic matter on the 387 development of the microbial polycyclic aromatic hydrocarbons (PAHs) degradation 388 potentials,Environ. Poll. 159, 591-595. 389 [30] M. Tejada, J.L. Moreno, M.T. Hernández, C. García, Soil amendments with organic 390 wastes reduce the toxicity of nickel to soil enzyme activities,Eur. J. Soil Biol. 44 391 (2008) 129-140. 392 [31] J. Chen, X. He, X. Zhang, X. Sun, J. Zheng, J. Zheng, Heavy metal pollution decreases 393 microbial abundance, diversity and activity particle-size fractions of a paddy soil, 394 FEMS Microbiol. Ecol. 87 (2014) 164-181. 395 [32] W. Wang, D. Lyons, N. Clark, M. Gautan, P. Norton, Emissions from nine heavy trucks 396 fueled by diesel and biodiesel blend without engine modification, Environ. Sci. 397 Technol. 34 (2000) 933-939. 398 399 Table 1 Chemical characteristics and protein molecular weight distribution (mean ± standard error, n=3) of okaraand biostimulant obtained by enzymatic hydrolysisprocesses okara BS pH ND 8.5 ± 0.2 Organic matter (g kg-1) 540 ± 19 445 ± 12 N (g kg-1) 61.1 ± 2.2 102 ± 13 P (g kg-1) 6.6 ± 1.4 5.3 ± 0.3 K (g kg-1) 9.4 ± 1.0 9.1 ± 0.5 S (g kg-1) 4.0 ± 1.3 24.6 ± 1.1 Ca (g kg-1) 1.6 ± 0.3 2.5 ± 0.4 Mg (g kg-1) 2.2 ± 0.7 27.8 ± 2.5 Fe (g kg-1) 63.6 ± 5.1 64.6 ± 2.7 Cu (mg kg-1) 10.7 ± 1.1 13.7 ± 1.3 Mn (mg kg-1) 31.0 ± 2.4 26.9 ± 1.4 Zn (mg kg-1) 27.8 ± 2.4 24.0 ± 1.4 Protein molecular weight distribution (Da) > 10000 90.7 ± 3.4 5,6 ± 1,6 10000 – 5000 1.3 ± 0.4 4.1 ± 1.1 5000 – 1000 1.6 ± 0.3 16.7 ± 3.1 1000 – 300 0.5 ± 0.1 17.5 ± 2.3 < 300 5.9 ± 1.6 56.1 ± 2,5 ND: non determined Table Table 2 Polycyclic aromatic hydrocarbons and total heavy metal contents (mean ± standard error, n=3) in motor oil Polycyclic aromatic hydrocarbons(mg kg-1) Heavy metals(mgkg-1) Naphthalene 494.0 ± 13.2 Fe 47.4 ± 5.1 Acenaphthylene 11.5 ± 2.4 Cu 6.6 ± 1.3 Acenaphtene 2.1 ± 0.8 Mn 1.2 ± 0.2 Fluorene 122 ± 2.6 Zn 7.6 ± 1.1 Phenantrene 106 ± 3.9 Ni 0.43 ± 0.05 Anthracene 29.4 ± 2.6 Cr 0.19 ± 0.03 Fluoranthrene 108.0 ±2.1 As 0.58 ± 0.1 Pyrene 73.3 ± 5.9 Cd 0.17 ± 0.06 Benzo(a)anthracene 32.6 ± 2.6 Pb 4.6 ± 1.1 Chrysene 50.5 ± 4.4 Al 63.4 ± 2.6 Benzo(b)fluoranthrene 41.4 ± 3.8 Benzo(k)fluoranthrene 18.6 ± 1.6 Benzo(a)pyrene 43.8 ± 4.5 Dibenzo(a, h)anthracene 6.9 ± 1.4 Indene(1, 2, 3 cd)pyrene 26.8 ± 3.6 Benzo(g, h, i)perylene 32.3 ± 2.2 Table 3 Detection limit and quantification limit per PAH Detection limit (µg kg) Quantification limit (µg kg) Naphthalene 0.0093 < 0.5 Acenaphthylene 0.0144 < 0.1 Acenaphtene 0.0046 < 0.1 Fluorene 0.0249 < 0.1 Phenantrene 0.0282 < 0.1 Anthracene 0.0315 < 0.1 Fluoranthrene 0.0300 < 0.1 Pyrene 0.0267 < 0.1 Benzo(a)anthracene 0.0063 < 0.1 Chrysene 0.0078 < 0.1 Benzo(b)fluoranthrene 0.0295 < 0.1 Benzo(k)fluoranthrene 0.0273 < 0.1 Benzo(a)pyrene 0.0087 < 0.1 Dibenzo(a, h)anthracene 0.0078 < 0.1 Indene(1, 2, 3 cd)pyrene 0.0129 < 0.1 Benzo(g, h, i)perylene 0.0099 < 0.1 1 Use of a biostimulant obtained from okara in the bioremediation of a soil polluted by 1 used motor car oil 2 3 Patricia Panequea, Pablo Caballerob, Juan Parradob, Isidoro Gómeza, Manuel Tejadaa* 4 5 aGrupo de Investigación Edafología Ambiental, Departamento de Cristalografía, Mineralogía 6 y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, 7 Spain 8 bDepartamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de 9 Sevilla, C/Prof. García González 2, 41012 Sevilla, Spain 10 11 *Corresponding author 12 E-mail: [email protected] 13 Tel.: +34954486468 14 Fax: +34954486436 15 16 Revised Manuscript (with marked changes) Click here to view linked References 2 Abstract 17 In this manuscript we studied in the laboratory the bioremediation effects of a 18 biostimulant obtained from okara by enzymatic hydrolysis processes in a soil polluted with 19 used motor-car oil at a rate of 1% (w/w) over an 89-day period. The biostimulant was added 20 to the soil 6 times during the incubation period at a rate of 2%. Dehydrogenase activity and 21 the evolution of polycyclic aromatic hydrocarbons (PAHs) and pseudo total heavy metals in 22 soil were studied. The successive applications of the biostimulant to the polluted soil 23 gradually increased PAHs degradation during the experimental period. Thus, at the end of the 24 experiment, the application of the biostimulant decreased the concentration of naphthalene in 25 soil by 74%, while PAHs with 3, 4, 5 and 6 aromatic rings had been reduced by around 58%, 26 44%, 30% and 23%, respectively. This degradation is possibly due to the high number of low 27 molecular weight peptides (<300 Da) in the biostimulant which are readily available for 28 PAHs-tolerant soil microorganisms that accelerate the degradation of the said toxins. The 29 concentration of heavy metals in the oil used was not very high and consequently the 30 dehydrogenase activity was not negatively affected. 31 32 Keywords: okara biostimulant; used motor-car oil; polycyclic aromatic hydrocarbons; total 33 heavy metals; soil dehydrogenase activity 34 35 1. Introduction 36 At present the use of lubricating oils is widespread because they are necessary for the 37 correct working of motor vehicle engines and other machinery such as pumps, hydraulic 38 motors, compressors and electrical transformers [1]. Due to the physical and chemical 39 reactions that occur during use, used motor oil is characterised by its high content of aromatic 40 and aliphatic hydrocarbons whose chain lengths range from C15 to C50 and heavy metals that 41 3 could contribute to the risk of chronic diseases, including mutagenicity and carcinogenicity 42 [1-3]. 43 As a result of their widespread use, terrestrial ecosystems are becoming increasingly 44 contaminated by such lubricants. The bioremediation of soils contaminated with used motor 45 oils is, therefore, an efficient and effective environmental technique for accelerating the 46 cleaning processes [2, 4]. 47 In recent years, applying organic biostimulants obtained from sewage sludge, chicken 48 feathers, wheat condensed distiller’s soluble enzymatic hydrolysate, rice bran extract, carob 49 germ enzymatic extract, etc. by enzymatic hydrolysis processes to soils polluted by organic 50 xenobiotics has been a widely-used environmental technique for accelerating the degradation 51 of the said xenobiotics in soil. The low molecular weight proteins, found in large quantities in 52 these biostimulants, are absorbed by the xenobiotics-tolerant microorganisms, increasing both 53 their proliferation and the biochemical activity of the soil, consequently accelerating the 54 degradation of the toxins in the soil [5-8]. 55 Okara is a by-product obtained from the manufacture of soy milk, tofu and their 56 derivatives. It has both a high fibre (56%) and protein (29%) content. Orts et al. [9] obtained a 57 new biostimulant from okara by enzymatic hydrolysis processes using the pH-stat technique 58 in order to remedy a soil polluted with chlorpyrifos. Eighty days after applying the 59 biostimulant to polluted soil, the insecticide had been completely degraded. Consequently, 60 this biostimulant obtained from okara could be of great use for remedying a soil contaminated 61 by motor oil with a high content of polycyclic aromatic hydrocarbons (PAHs). 62 It should be noted, however, that the effectiveness of these biostimulants obtained by 63 enzymatic hydrolysis processes has been verified after only a single application to polluted 64 soil. It is not known what the behaviour of organic xenobiotics in the polluted soil would be 65 after repeated applications of these biostimulants. 66 4 There is an abundant literature indicating that soil biological parameters evolve faster 67 than physical and chemical parameters [6, 10-12]. There is also a lot of information on how 68 PAHs and heavy metals negatively affect soil enzymatic activities, as well as affecting its 69 microbial population [8, 11, 13, 14]. For this reason, a study of these biological properties 70 would be very useful in order to verify the bioremediation capacity of organic matter in 71 polluted soils. 72 According to Kaczynski et al. [15] and Liu et al. [16], the oxidation-reduction 73 enzymes found in soil play a fundamental role in degrading contaminants, transforming 74 organic matter and in maintaining the metabolism of microorganisms. In this respect, 75 dehydrogenase activity (EC 1.1.1) is an essential intracellular oxidoreductase enzyme in 76 living cells. It participates in redox reactions, it promotes the dehydrogenation of soil organic 77 matter and transmits energy to hydrogen acceptors through the respiratory chain [17]. Due to 78 the above, this enzyme is now used as a soil contamination status bioindicator [18, 19, 20]. 79 The objective of this study, therefore, was to verify the bioremediation efficiency of a 80 biostimulant, obtained from okara by enzymatic hydrolysis processes, in a soil polluted by 81 used motor oil after repeated applications of the said biostimulant to the polluted soil and its 82 influence on the soil dehydrogenase activity. 83 84 2. Material and methods 85 2.1. Soil, biostimulant and motor oil characteristics 86 The soil used in the study was classified as Calcaric Regosol [21], with 574 ± 31g kg-1 87 sand, 148 ± 35 g kg-1 silt, and 278 ± 24 g kg-1 clay. Soil pH was 7.9 ± 0.1, 16.4 ± 1.3 gkg-1 88 organic matter, and 0.98 ± 0.05 g kg-1 N. The methodology used for determining the soil 89 parameters is described in Tejada et al. [7] and Rodríguez-Morgado et al. [22]. 90 5 The okara from which the new biostimulant was manufactured was provided by the 91 Soria Natural S.A company. The experimental biostimulant was obtained from the okara by 92 enzymatic hydrolysis using an endoprotease obtained by liquid fermentation of Bacillus 93 licheniformis ATCC 21415. Table 1 shows the chemical characteristics of both the okara and 94 the resulting biostimulant. The characteristics of this process of enzymatic hydrolysis are 95 detailed in Orts et al. [9]. The methodology used for determining organic matter, macro- and 96 micronutrients, and molecular protein mass distribution is described in Rodríguez-Morgado et 97 al. [22]. 98 The motor oil used was a sample of gasoline automobile engine oil taken when the 99 vehicle’s oil was changed at 15000 km. Table 2 shows the content of polycyclic aromatic 100 hydrocarbons and total heavy metals in the motor oil. 101 The PAHs were detected by a GC 7890B Agilent gas chromatograph connected to a 102 MS Triple Q Agilent 7000C mass detector. The analysis conditions were the following: 103 104 Column: DB-EUPAH 30X0.25X0.25 105 Injection: 2 μl 106 Column pressure: 15.7 psi 107 Constant flow: 1.2 ml min-1 108 Injection mode: Splitless 50 psi 109 Injection temperature: 310 °C 110 Initial temperature: 80 °C for 1 minute 111 Temperature ramp: 25 °C minute-1 to 200 °C for 8 minutes; 5 °C minute-1 to 305 °C for 9 112 minutes; 50 °C minute-1 to 330 °C for 3 minutes; (48.5 total minutes) 113 114 6 Table 3 shows the detection limit and quantification limit per each PAH. The internal 115 standards used in GC/MS study were: acenaphthene d8, chrysene d10, naphthalene d8, 116 perylene d12 and phenatrene d10. Recovery analyses were carried out in every treatment, 117 showing values of between 70 and 80%. 118 The concentration of heavy metals in the engine oil was determined following US 119 EPA method 3052. For this, 0.5 g of oil was digested in 9 ml of HNO3 and 3 ml of HCl using 120 a microwave digester. Digests were filtered through 0.45 mm Millipore filters and diluted 121 appropriately for analysis by a Thermo Elemental ICP-MS X7 Inductively Coupled Plasma 122 Mass Spectrometry system (Thermo Fisher, Cambridge, UK) with quadrupole mass analyser, 123 multichannel detector (Pulse Counting and Analogue Methods), auto sampler ASX-500 124 (CETAC, Omaha, NE, USA). The measurement accuracy (%) of each heavy metal was as 125 follows: Fe: 2.2; Cu: 5.2; Mn: 2.5 Zn: 3.4; Ni: 4.0; Cr: 4.7; As: 1.1; Cd: 3.9; Pb: 5.0; and Al: 126 3.1. 127 128 2.2. Experimental layout 129 Five hundred grams of dried, sieved (<2 mm) soil were preincubated at 25 °C for 7 130 days at 30-40 % of their holding capacity [5]. After this pre-incubation period, soil samples 131 were mixed with gasoline motor oil at a rate of 1% (w/w) in 1-L glass bottles., Motor oil was 132 dissolved in acetone as a carrier solvent at a concentration of 30 mL kg-1 according to 133 Ramadass et al. [3]. Acetone was also added to the non-polluted soils (control treatment). The 134 biostimulant was added to the soil 6 times at a rate of 2% once at the beginning of the 135 experiment and then on days 13, 27, 41, 55 and 69 during the incubation period. The 136 biostimulant used was liquid and was solubilised in distilled water (500 l ha−1) before 137 application. Distilled water was added to each soil to bring it to 60% of its water-holding 138 7 capacity. An unamended, polluted and an amended, non-polluted soil were used as controls. 139 The incubation treatments are detailed as follows: 140 141 1. C, control soil, non-polluted soil and non-organically amended 142 2. C+O, polluted soil with used motor oil and non-organically amended 143 3. C+BS, non-polluted soil and amended with the biostimulant 144 4. C+O+BS, polluted soil with used motor oil and amended with the biostimulant 145 146 Triplicate treatments were kept in semi-closed microcosms at 25 ± 1 °C for 50 days, 147 respectively. The moisture content was controlled gravimetrically and moisture loss was 148 replaced as necessary by distilled water. 149 150 2.3. Soil analysis 151 For each experimental treatment, dehydrogenase activity was measured at days 3, 13, 152 16, 27, 30, 41, 44, 55, 58, 69, 72 and 86 during incubation experiment. The soil samples from 153 days 3, 13, 27, 41, 55 and 69 were selected and analysed before applying the okara 154 biostimulant to the soil. 155 The method of Trevors et al. [23] as modified by García et al. [24] was used to 156 measure dehydrogenase activity, reducing 2-p-iodophenyl-3-p-nitro-phenyl-5-157 phenyltetrazolium chloride (INT) to iodonitrophenyl-formazan (INTF). This was measured in 158 a spectrophotometer at 490 nm. 159 PAHs were extracted from 30 g of soil. These samples were placed in small glass jars 160 with 10 g of anhydrous sodium sulphate in order to remove moisture from the soil samples. 161 50 ml of a dichloromethane/pentane 1/1 mixture were then added and the mix was shaken for 162 30 seconds. The samples were then sonicated for 10 minutes and filtered through anhydrous 163 8 sodium sulphate. The total volume extracted was concentrated in a rotary evaporator at a 164 temperature of 50 °C. This process was performed in triplicate. Finally, the volume of the 165 extract was adjusted to 1 mL with isooctane. 166 Once the PAHs had been extracted, they were measured according to the instruments 167 and conditions previously described. 168 At days 3 and 86 after the start of the incubation process, the pseudo total heavy metal 169 content in soil was determined, the method of determination being highly similar to that 170 described above. Using a microwave digester, 5 g of soil were digested in 9 mL of HNO3 and 171 3 mL of HCl. Digests were filtered through 0.45 mm Millipore filters and appropriately 172 diluted for posterior analysis using the Thermo Elemental ICP-MS X7 Inductively Coupled 173 Plasma Mass Spectrometry system. 174 175 2.4. Statistical analysis 176 With the data obtained from each soil analysis and using Statgraphics Plus 2.1 177 software package, a two-way analysis of variance (ANOVA) was performed with treatment 178 and sampling time as factors, followed by Tukey’s significant difference as a post hoc test, 179 considering a significance level of p < 0.05 throughout the study. For the ANOVA, triplicate 180 data were used for each treatment and for each day of incubation. 181 182 3. Results and discussion 183 The continuous application of the biostimulant to the non-polluted soil during the 184 experimental period caused an increase in dehydrogenase activity (Table 4). In other 185 experiments carried out by Rodriguez-Morgado et al. [8, 25] and Orts et al. [9], performing a 186 single application to the soil of biostimulants obtained from sewage sludge, chicken feathers 187 and okara by enzymatic hydrolysis processes, it was observed that the dehydrogenase activity 188 9 showed maximum stimulation during the first 7 days after application. The dehydrogenase 189 activity then declined progressively until reaching values similar to the non-organically 190 amended soil. According to these authors, the main reason for the behaviour of these 191 biostimulants is a consequence of the protein size distribution obtained in said organic 192 compounds, such behaviour highlighting the influence/high content of the low molecular 193 weight proteins (<300 Da), which are in the majority. The higher content of low molecular 194 weight proteins suggests that N is in a more readily-available form for soil organisms, 195 promoting a greater proliferation of soil microorganisms and, consequently, dehydrogenase 196 activity. 197 In our experiment, this increase in dehydrogenase activity at the end of the incubation 198 period may be due to the constant accumulation of high molecular weight proteins after the 199 continuous application of the experimental biostimulant, thus requiring more time for the 200 microorganisms to degrade them. Table 1 shows that the protein content of molecular weight 201 > 1000 Da is 26.4%, which may be responsible for stimulating soil microbial activity at the 202 end of the incubation period. 203 The application of motor oil to the soil showed a significant decrease (p <0.05) in 204 dehydrogenase activity during the whole experimental period (Table 4). At the end of the 205 incubation period and compared to C treatment, dehydrogenase activity decreased 206 significantly (p <0.05) by 62.5% in soils polluted with used motor oil. This inhibition in soil 207 dehydrogenase activity is possibly a consequence of adding different PAHs with 2 to 6 208 aromatic rings in the used motor oil. These results are in agreement with those obtained by 209 Tejada et al. [6], Rodríguez-Morgado et al. [8] and Lipińska et al. [19], who found that 210 dehydrogenase activity was inhibited in soils polluted by various PAHs due to their toxicity to 211 soil microorganisms. According to these authors, this toxic effect inhibits an abundant 212 population of soil microorganisms and, consequently, reduces soil dehydrogenase activity. 213 10 Table 5 shows the evolution of PAHs in soil during the experimental period after 214 polluting it with automobile motor oil. In non-organically amended soils, the concentration of 215 the PAHs remained highly similar throughout the incubation period. Applying the 216 experimental biostimulant to polluted soil increased PAH degradation. This degradation was 217 gradual throughout the experimental period and was greater among those hydrocarbons with 218 the lowest number of aromatic rings. Thus, at day 47 of the incubation period and in 219 comparison with the C+O treatment, the naphthalene concentration decreased significantly (p 220 <0.05) by 68.5% in the C+O+BS treatment, while the hydrocarbons with 3, 4, 5 and 6 221 aromatic rings decreased by values of around 43%, 33%, 25% and 16%, respectively. At the 222 end of the experimental period, and in comparison with the C+O treatment, naphthalene 223 concentration in the C+O+BS treatment had decreased significantly (p <0.05) by 74%, while 224 concentrations of hydrocarbons with 3, 4, 5 and 6 aromatic rings had decreased by around 225 58%, 44 %, 30% and 23%, respectively. 226 There is an abundant bibliography that suggests that PAH degradation in soil is a very 227 slow process carried out by microorganisms conditioned by the number of aromatic rings in 228 the hydrocarbon’s structure [8, 26, 27]. According to Edokpayi et al. [28], as the molecular 229 weight of the PAH increases, solubility decreases, increasing in turn environmental 230 persistence and toxicity. For this reason as the number of aromatic rings in its chemical 231 structure increases, PAH degradation decreases. 232 Applying organic matter to the soil accelerates PAH degradation [6, 8, 27, 29]. 233 According to these authors, soil microorganisms are responsible for PAH degradation. In this 234 sense, applying organic matter to soil contaminated by PAHs stimulates the PAH-tolerant 235 microorganisms, favouring PAH degradation and thus reducing their polluting effect on the 236 soil. In our experiment, successive applications of the experimental biostimulants throughout 237 17 effluents in Vhembe district, South Africa,Int. J. Environ. Res. Public Health 13 385 (2016) 387. 386 [29] Y. Yang, N. Zhang, M. Xue, S.T. Lu, S. Tao, Effects of soil organic matter on the 387 development of the microbial polycyclic aromatic hydrocarbons (PAHs) degradation 388 potentials,Environ. Poll. 159, 591-595. 389 [30] M. Tejada, J.L. Moreno, M.T. Hernández, C. García, Soil amendments with organic 390 wastes reduce the toxicity of nickel to soil enzyme activities,Eur. J. Soil Biol. 44 391 (2008) 129-140. 392 [31] J. Chen, X. He, X. Zhang, X. Sun, J. Zheng, J. Zheng, Heavy metal pollution decreases 393 microbial abundance, diversity and activity particle-size fractions of a paddy soil, 394 FEMS Microbiol. Ecol. 87 (2014) 164-181. 395 [32] W. Wang, D. Lyons, N. Clark, M. Gautan, P. Norton, Emissions from nine heavy trucks 396 fueled by diesel and biodiesel blend without engine modification, Environ. Sci. 397 Technol. 34 (2000) 933-939. 398 399 *Graphical Abstract The manuscript has no conflict of interest *Declaration of Interest Statement All authors have contributed to the manuscript in the same way *Author Contributions Section