Abstract
Nowadays, the increase of the unconventional oil deposit exploitation and the amount of oil sands process-affected waters (OSPW) in tailing ponds emerges the importance of developing bio-monitoring strategies for the restoration of these habitats. The major constituents of such deposits are naphthenic acids (NAs), emerging contaminant mixtures with toxic and recalcitrant properties. With the aim of developing bio-monitoring strategies based on culture-independent approach, we identified genes coding for enzymes involved in NA degradation from Rhodococcus opacus R7 genome, after the evaluation of its ability to mineralize model NAs. R. opacus R7 whole-genome analysis unveiled the presence of pobA and chcpca gene clusters putatively involved in NAs degradation. Gene expression analysis demonstrated the specific induction of R7 aliA1 gene, encoding for a long-chain-fatty-acid-CoA ligase, in the presence of cyclohexanecarboxylic acid (CHCA) and hexanoic acid (HA), selected as representative compounds for alicyclic and linear NAs, respectively. Therefore, aliA1 gene was selected as a molecular marker to monitor the biodegradative potential of slurry-phase sand microcosms in different conditions: spiked with CHCA, in the presence of R. opacus R7, the autochthonous microbial community, and combining these factors. Results revealed that the aliA1-targeting culture-independent approach could be a useful method for bio-monitoring of NA degradation in a model laboratory system.
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11 February 2020
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References
Alberta Energy Regulator (AER), Directive 085 (2017) Fluid tailings management for oil sands mining projects. https://www.aer.ca/documents/directives/Directive085.pdf. Accessed 10 March 2019
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwardset RA, Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Wilke A, Zagnitko O (2008) The RAST server: rapid annotations using subsystems technology. BMC Genomics 9:75. https://doi.org/10.1186/1471-2164-9-75
Biryukova OV, Fedorak PM, Quideau SA (2007) Biodegradation of naphthenic acids by rhizosphere microorganisms. Chemosphere 67:2058–2064. https://doi.org/10.1016/j.chemosphere.2006.11.063
Blakley ER (1974) The microbial degradation of cyclohexanecarboxylic acid: a pathway involving aromatization to form p-hydroxybenzoic acid. Can J Microbiol 20:1297–1306. https://doi.org/10.1139/m74-202
Blakley ER (1978) The microbial degradation of cyclohexanecarboxylic acid by a b-oxidation pathway with simultaneous induction to the utilization of benzoate. Can J Microbiol 24:847–855. https://doi.org/10.1139/m78-141
Blakley ER, Papish B (1982) The metabolism of cyclohexanecarboxylic acid and 3-cyclohexenecarboxylic acid by Pseudomonas putida. Can J Microbiol 28:1324–1329
Brown LD, Ulrich AC (2015) Oil sands naphthenic acids: a review of properties, measurement, and treatment. Chemosphere 127:276–290. https://doi.org/10.1016/j.chemosphere.2015.02.003
Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP (2016) DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods 13:581–583. https://doi.org/10.1038/nmeth.3869
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Peña AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336. https://doi.org/10.1038/nmeth.f.303
Cappelletti M, Fedi S, Zampolli J, Di Canito A, D'Ursi P, Orro A, Viti C, Milanesi L, Zannoni D, Di Gennaro P (2016) Phenotype microarray analysis may unravel genetic determinants of the stress response by Rhodococcus aetherivorans BCP1 and Rhodococcus opacus R7. Res Microbiol 167:766–773. https://doi.org/10.1016/j.resmic.2016.06.008
Cappelletti M, Fedi S, Zannoni D (2019a) Degradation of alkanes in Rhodococcus. In: Alvarez HM (ed) Biology of Rhodococcus. Microbiology Monographs. Springer, Cham, pp 137–171. https://doi.org/10.1007/978-3-030-11461-9_6
Cappelletti M, Zampolli J, Di Gennaro P, Zannoni D (2019b) Genomics of Rhodococcus. In: Alvarez HM (ed) Biology of Rhodococcus. Microbiology Monographs. Springer, Cham, pp 23–60 https://doi-org.proxy.unimib.it/10.1007/978-3-030-11461-9_2
Cavalca L, Colombo M, Larcher S, Gigliotti C, Collina E, Andreoni V (2002) Survival and naphthalene degrading activity of Rhodococcus sp. strain 1BN in soil microcosms. J Appl Microbiol 92:1058–1065. https://doi.org/10.1046/j.1365-2672.2002.01640.x
Chen S, Hu W, Xiao Y, Deng Y, Jia J, Hu M (2012) Degradation of 3-phenoxybenzoic acid by a Bacillus sp. PLoS One 7:e50456. https://doi.org/10.1371/journal.pone.0050456
Clemente JS, Fedorak PM (2005) A review of the occurrence, analyses, toxicity, and biodegradation of naphthenic acids. Chemosphere 60:585–600. https://doi.org/10.1016/j.chemosphere.2005.02.065
Clemente JS, MacKinnon MD, Fedorak PM (2004) Aerobic biodegradation of two commercial naphthenic acids preparations. Environ Sci Technol 38:1009–1016. https://doi.org/10.1021/es030543j
Del Rio LF, Hadwin AKM, Pinto LJ, MacKinnon MD, Moore MM (2006) Degradation of naphthenic acids by sediment micro-organisms. J Appl Microbiol 101:1049–1061. https://doi.org/10.1111/j.1365-2672.2006.03005.x
Demeter MA, Lemire J, George I, Yue G, Ceri H, Turner RJ (2014) Harnessing oil sands microbial communities for use in ex situ naphthenic acid bioremediation. Chemosphere 97C:78–85. https://doi.org/10.1016/j.chemosphere.2013.11.016
Demeter MA, Lemire JA, Yue G, Ceri H, Turner RJ (2015) Culturing oil sands microbes as mixed species communities enhances ex situ model naphthenic acid degradation. Front Microbiol 6:936. https://doi.org/10.3389/fmicb.2015.00936
DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, Huber T, Dalevi D, Hu P, Andersen GL (2006) Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol 72:5069–5072. https://doi.org/10.1128/AEM.03006-05
Di Canito A, Zampolli J, Orro A, D’Ursi P, Milanesi L, Sello G, Steinbüchel A, Di Gennaro P (2018) Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7. BMC Genomics 19:587. https://doi.org/10.1186/s12864-018-4965-6
Di Gennaro P, Rescalli E, Galli E, Guido S, Bestetti G (2001) Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from polycyclic aromatic hydrocarbon-contaminated soil. Res Microbiol 152:641–651. https://doi.org/10.1016/S0923-2508(01)01243-8
Di Gennaro P, Moreno B, Annoni E, García-Rodríguez S, Bestetti G, Benitez E (2009) Dynamic changes in bacterial community structure and in naphthalene dioxygenase expression in vermicompost-amended PAH-contaminated soils. J Hazard Mater 172:1464–1469. https://doi.org/10.1016/j.jhazmat.2009.08.013
Di Gennaro P, Terreni P, Masi G, Botti S, De Ferra F, Bestetti G (2010) Identification and characterization of genes involved in naphthalene degradation in Rhodococcus opacus R7. Appl Microbiol Biotechnol 87:297–308. https://doi.org/10.1007/s00253-010-2497-3
Di Gennaro P, Zampolli J, Presti I, Cappelletti M, D'Ursi P, Orro A, Mezzelani A, Milanesi L (2014) Genome sequence of Rhodococcus opacus strain R7, a biodegrader of mono- and polycyclic aromatic hydrocarbons. Genome Announc 2:e00827–e00814. https://doi.org/10.1128/genomeA.00827-14
Gröning JAD, Eulberg D, Tischler D, Kaschabek SR, Schlömann M (2014) Gene redundancy of two-component (chloro)phenol hydroxylases in Rhodococcus opacus 1CP. FEMS Microbiol Lett 361:68–75 https://doi-org.proxy.unimib.it/10.1111/1574-6968.12616
Han X, Scott AC, Fedorak PM, Batianeh M, Martin JW (2008) Influence of molecular structure on the biodegradability of naphthenic acids. Environ Sci Technol 42:1290–1295. https://doi.org/10.1021/es702220c
He Z, Gentry TJ, Schadt CW, Wu L, Liebich J, Chong SC, Huang Z, Wu W, Gu B, Criddle C, Zhou J (2007) GeoChip: a comprehensive microarray for investigating biogeochemical, ecological and environmental processes. ISME J 1:67–77. https://doi.org/10.1038/ismej.2007.2
Holowenko FM, MacKinnon MD, Fedorak PM (2001) Naphthenic acids and surrogate naphthenic acids in methanogenic microcosms. Water Res 35:2595–2606. https://doi.org/10.1016/S0043-1354(00)00558-3
Holowenko FM, MacKinnon MD, Fedorak PM (2002) Characterization of naphthenic acids in oil sands wastewaters by gas chromatography-mass spectrometry. Water Res 36:2843–2855. https://doi.org/10.1016/S0043-1354(01)00492-4
Iino T, Wang Y, Miyauchi K, Kasai D, Masai E, Fujii T, Fujii T, Ogawa N, Fukuda M (2012) Specific gene responses of Rhodococcus jostii RHA1 during growth in soil. Appl Environ Microbiol 78:6954–6962. https://doi.org/10.1128/AEM.00164-12
Iwaki H, Saji H, Abe K, Hasegawa Y (2005) Cloning and sequence analysis of the 4-hydroxybenzoate 3-hydroxylase gene from a cyclohexanecarboxylate-degrading Gram-positive bacterium, “Corynebacterium cyclohexanicum” strain ATCC 51369. Microbes Environ 20:144–150. https://doi.org/10.1264/jsme2.20.144
Johnson RJ, Smith BE, Sutton PA, McGenity TJ, Rowland SJ, Whitby C (2011) Microbial biodegradation of aromatic alkanoic naphthenic acids is affected by the degree of alkyl side chain branching. ISME J 5:486–496. https://doi.org/10.1038/ismej.2010.146
Johnson RJ, Smith BE, Rowland SJ, Whitby C (2013) Biodegradation of alkyl branched aromatic alkanoic naphthenic acids by Pseudomonas putida KT2440. Int Biodeterior Biodegradation 81:3–8. https://doi.org/10.1016/j.ibiod.2011.11.008
Kannel PR, Gan TY (2012) Naphthenic acids degradation and toxicity mitigation in tailings wastewater systems and aquatic environments: a review. J Environ Sci Health A Tox Hazard Subst Environ Eng 47:1–21. https://doi.org/10.1080/10934529.2012.629574
Kinley CM, Gaspari DP, McQueen AD, Rodgers JHJ, Castle JW, Friesen V (2016) Effects of environmental conditions on aerobic degradation of a commercial naphthenic acid. Chemosphere 161:491–500. https://doi.org/10.1016/j.chemosphere.2016.07.050
Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO (2013) Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res 41:e1. https://doi.org/10.1093/nar/gks808
Koma D, Sakashita Y, Kubota K, Fujii Y, Hasumi F, Chung SY, Kubo M (2004) Degradation pathways of cyclic alkanes in Rhodococcus sp. NDKK48. Appl Microbiol Biotechnol 66:92–99. https://doi.org/10.1007/s00253-004-1623-5
Lai JL, Pinto WS, Kiehlmann E, Bendell-Young LI, Moore MM (1996) Factors that affect the degradation of naphthenic acids in oil sands wastewater by indigenous microbial communities. Environ Toxicol Chem 15:1482–1491. https://doi.org/10.1002/etc.5620150909
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2016) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res 45(D1):D200–D203. https://doi.org/10.1093/nar/gkw1129
Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet journal 17:10–12. https://doi.org/10.14806/ej.17.1.200
McDonald D, Price MN, Goodrich J, Nawrocki EP, DeSantis TZ, Probst A, Andersen GL, Knight R, Hugenholtz P (2012) An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and archaea. ISME J 6:610–618. https://doi.org/10.1038/ismej.2011.139
McNeill J (2018) Oilsands tailing ponds are a nasty challenge that can’t be ignored. Calgary Herald. https://calgaryherald.com/opinion/columnists/mcneill-oilsands-tailing-ponds-are-a-nasty-challenge-that-cant-be-ignored. Accessed 2 Jan 2019
Mossop GD (1980) Geology of the Athabasca oil sands. Science 207:145–152. https://doi.org/10.1126/science.207.4427.145
Orro A, Cappelletti M, D’Ursi P, Milanesi L, Di Canito A, Zampolli J, Collina E, Decorosi F, Viti C, Fedi S, Presentato A, Zannoni D, Di Gennaro P (2015) Genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: genetic determinants and metabolic abilities with environmental relevance. PLoS One 10:e0139467. https://doi.org/10.1371/journal.pone.0139467
Pelletier DA, Harwood CS (2000) 2-Hydroxycyclohexanecarboxyl coenzyme A dehydrogenase, an enzyme characteristic of the anaerobic benzoate degradation pathway used by Rhodopseudomonas palustris. J Bacteriol 182:2753–2760. https://doi.org/10.1128/JB.182.10.2753-2760.2000
Presentato A, Cappelletti M, Sansone A, Ferreri C, Piacenza E, Demeter MA, Crognale S, Petruccioli M, Milazzo G, Fedi S, Steinbüchel A, Turner RJ, Zannoni D (2018) Aerobic growth of Rhodococcus aetherivorans BCP1 using selected naphthenic acids as the sole carbon and energy sources. Front Microbiol 9:672 https://doi.org/10.3389/fmicb.2018.00672
Quagraine EK, Headley JV, Peterson HG (2005a) Is biodegradation of bitumen a source of recalcitrant naphthenic acid mixtures in oil sands tailing pond waters? J Environ Sci Health A 40:671–684. https://doi.org/10.1081/ESE-200046637
Quagraine EK, Peterson HG, Headley JV (2005b) In situ bioremediation of naphthenic acids contaminated tailing pond waters in the Athabasca oil sands region-demonstrated field studies and plausible options: a review. J Environ Sci Health A 40:685–722. https://doi.org/10.1081/ESE-200046649
Rho EM, Evans WC (1975) The aerobic metabolism of cyclohexanecarboxylic acid by Acinetobacter anitratum. Biochem J 148:11–15. https://doi.org/10.1042/bj1480011
Rontani JF, Bonin P (1992) Utilization of n-alkyl-substituted cyclohexanes by a marine Alcaligenes. Chemosphere 24:1441–1446. https://doi.org/10.1016/0045-6535(92)90266-T
Sawulski P, Clipson N, Doyle E (2014) Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil. Biodegradation 25:835–847. https://doi.org/10.1007/s10532-014-9703-4
Simkins S, Alexander M (1984) Model for mineralization kinetics with the variable of substrate concentration and population density. Appl Environ Microbiol 47:1299–1306
ST98 (2018) Alberta’s energy reserves and supply/demand outlook. Executive summary. https://www.aer.ca/documents/sts/ST98/ST98-2018_Executive_Summary.pdf. Accessed 10 March 2019
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680 https://doi-org.proxy.unimib.it/10.1093/nar/22.22.4673
Toor NS, Franz ED, Fedorak PM, MacKinnon MD, Liber K (2013) Degradation and aquatic toxicity of naphthenic acids in oil sands process-affected waters using simulated wetlands. Chemosphere 90:449–458. https://doi.org/10.1016/j.chemosphere.2012.07.059
van Nostrand JD, He Z, Zhou J (2012) Use of functional gene arrays for elucidating in situ biodegradation. Front Microbiol 3:339. https://doi.org/10.3389/fmicb.2012.00339
Wang X, Chen M, Xiao J, Hao L, Crowley DE, Zhang Z, Yu J, Huang N, Huo M, Wu J (2015) Genome sequence analysis of the naphthenic acid degrading and metal resistant bacterium Cupriavidus gilardii CR3. PLoS One 10:e0132881. https://doi.org/10.1371/journal.pone.0132881
Whitby C (2010) Chapter 3—microbial naphthenic acid degradation. In: Laskin AI, Sariaslani S, Gadd GM (eds) Advances in applied microbiology, USA, pp 93-125. https://doi.org/10.1016/s0065-2164(10)70003-4
Acknowledgments
We would like to thank our graduate student Giorgia Genini for support in the experimental work.
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This work was supported by NAZ-0298 2017 grant for basic research activities financed by MIUR.
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Zampolli, J., Di Canito, A., Cappelletti, M. et al. Biodegradation of naphthenic acids: identification of Rhodococcus opacus R7 genes as molecular markers for environmental monitoring and their application in slurry microcosms. Appl Microbiol Biotechnol 104, 2675–2689 (2020). https://doi.org/10.1007/s00253-020-10378-5
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DOI: https://doi.org/10.1007/s00253-020-10378-5