Ramularia mali strains isolated from petroleum product-contaminated soil are capable to grow on multiple aromatic compounds




Abstract. Tan WA, Teja HS, Stephane. 2020. Ramularia mali strains isolated from petroleum product-contaminated soil are capable to grow on multiple aromatic compounds. Biodiversitas 21: 3590-3595.  Aromatic compounds are present as a complex mixture in the environment, some of which are toxic and carcinogenic in humans and animals. In this study, we reported the first instance of arthroconidial yeast growth on more than one aromatic compound. Yeasts were isolated from soil surrounding gas stations and screened for growth on phenol, followed by qualitative growth testing on chlorobenzene, aniline, toluene, 2-nitrotoluene, benzoate, and naphthalene. Pure isolates identity and phylogeny were determined based on the ITS region. All isolates grew on phenol and benzoate, but none grew on naphthalene. Aside from the inability to grow on naphthalene, SR8 grew on other tested aromatics, while SR3 showed similar growth ability except on 2-nitrotoluene. Growth on 2-nitrotoluene was observed for PPS3, PB4, SR1, SR6, and SR8, a phenotype that has yet been reported in yeasts thus far. All isolates shared 99.81-100% similarity and were phylogenetically clustered with the arthroconidial yeast Ramularia mali CBS 129581, despite that some of them were morphologically different and had varying capability to grow on tested aromatics. Therefore, ITS sequences cannot be used to differentiate Ramularia mali up to the strain level. Overall, we demonstrated the diverse potential of yeast strains in reducing aromatic compound contamination.


Al-Khalid T, El-Naas MH. 2012. Aerobic biodegradation of phenols: a comprehensive review. Crit Rev Environ Sci Technol 42:1631-1690. https://doi.10.1080/10643389.2011.569872.
Ashliha IN, Alami NH. 2014. Karakterisasi khamir dari pulau Poteran Madura. J Sains Seni Pomits 3(2):49-52. https://doi.10.12962/j23373520.v3i2.6869.
Bakhshi M. 2018. Molecular phylogeny and morphology of four Ramularia species from Iran along with a checklist of ramularia-like taxa. Rostaniha. 19(2):92-112. https://doi.10.22092/BOTANY.2019.124392.1133.
Boyd DR, Sharma ND. Allen CCR. 2001. Aromatic dioxygenases: molecular biocatalysis and applications. Curr Opin Biotechnol 12(6):564-573. https://doi.10.1016/S0958-1669(01)00264-6.
Cong N, Thi L, Mai N, Thi C, Thanh VT, Nga LP, Minh NN. 2014. Application of a biofilm formed by a mixture of yeasts isolated in Vietnam to degrade aromatic hydrocarbon polluted wastewater collected from petroleum storage. Water Sci Technol 70(2):329-36. https://doi.org/10.2166/wst.2014.233.
Fialova A, Boschke E, Bley T. 2004. Rapid monitoring of the biodegradation of phenol-like compounds by the yeast Candida maltosa using BOD measurements. Int Biodeter Biodegrad 54(1):69-76. https://doi.10.1016/j.ibiod.2004.02.004.
Gami AA, Shukor MY, Khalil KA, Dahalan FA, Khalid A, Ahmad SA. 2014. Phenol and phenolic compounds toxicity. J Environ Microbiol Toxicol 2(1):11-23.
Haigler BE, Wallace WH, Spain JC. 1994. Biodegradation of 2-nitrotoluene by Pseudomonas sp strain JS42. Appl Environ Microbiol. 60(9):3466-3469.
Heras-Vazquez L, Mingorance-Cazorla L, Clemente-Jimenez JM, Rodriguez-Vico F. 2003. Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8 S rRNA gene and the two internal transcribed spacers. FEMS Yeast Res 3(1):3-9. https://doi.10.1016/S1567-1356(02)00134-4.
Karimia M, Hassanshahian M. 2016. Isolation and characterization of phenol degrading yeasts from wastewater in the coking plant of Zarand, Kerman. Braz J Microbiol 47(1):18–24. https://doi.10.1016/j.bjm.2015.11.032
Kulbat K. 2016. The role of phenolic compounds in plant resistance. Biotechnol Food Sci 80(2):97-108.
Kumar S, Stecher G, Li M, Knyaz C, Tamura K. 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 35(6):1547-1549. https://doi.10.1093/molbev/msy096.
Lõoke M, Kristjuhan K, Kristjuhan A. 2011. Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50(5):325-328. https://doi.10.2144/000113672.
Mulla SI, Hoskeri RS, Shouche YS, Ninnekar HZ. 2011. Biodegradation of 2-nitrotoluene by Micrococcus sp strain SMN-1. Biodegradation 22(1):95-102. https://doi.10.1007/s10532-010-9379-3.
Rabinovitch-Deere CA, Parales RE. 2012. Three types of taxis used in the response of Acidovorax sp. strain JS42 to 2-nitrotoluene. Appl Environ Microbiol 78(7):2306-2315. https://doi.10.1128/AEM.07183-11.
Resnick SM, Lee K, Gibson DT. 1996. Diverse reaction catalyzed by naphthalene dioxygenase from Pseudomonas sp. Strain NCIB 9816. J Ind Microbiol 17(5):438-457.
Salmeron-Alcocer A, Ruiz-Ordaz N, Juarez-Ramirez C, Galíndez-Mayer J. 2007. Continuous biodegradation of single and mixed chlorophenols by a mixed microbial culture constituted by Burkholderia sp., Microbacterium phyllosphaerae, and Candida tropicalis. Biochem Eng J 37(2):201-211. https://doi.10.1016/j.bej.2007.04.015.
Sanchez-García C, Alvarado-Capó Y, Acosta-Suárez M, Leiva-Mora M, Cruz-Martín M, Roque B. 2013. Quantification of phenols in lesions caused by Mycosphaerella fijiensis Morelet in ‘Cavendish naine’. Rev Proteccíon Veg 28(2):149-152.
Schülter R, Schauer F. 2017. Biotransformation and detoxification of environmental pollutants with aromatic structures by yeasts. In: Satyanarayana T, Kunze G, editor. Yeast diversity in human welfare. Singapore: Springer. https://doi.10.1007/978-981-10-2621-8_13.
Seo JS, Keum YS, Li QX. 2009. Bacterial degradation of aromatic compounds. Int J Environ Res Public Health 6(1):278-309. https://doi.10.3390/ijerph6010278.
Sridevi V, Chandana Lakshmi MV V, Manasa M, Sravani M. 2012. Metabolic pathways for the biodegradation of phenol. Int J Eng Sci Advan Technol 2(3):695–705.
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/10.1093/nar/22.22.4673
Videira SI, Groenewald JZ, Braun U, Shin HD, Crous PW. 2016. All that glitters is not Ramularia. Stud Mycol 83:49-163. https://doi.10.1016/j.simyco.2016.06.001.
Videira SIR, Groenewald JZ, Kolecka A, van Haren L, Boekhout T, Crous PW. 2015. Elucidating the Ramularia eucalypti species complex. Persoonia 34:50-64. https://doi.10.3767/003158515X685670.
Walmsley RM, Keenan P. 2012. The eukaryote alternative: advantages of using yeasts in place of bacteria in microbial biosensor development. Biotechnol Bioprocess Eng 5(6):387-394. https://doi.10.1007/BF02931936.
Wartmann T, Kunze G. 2000. Genetic transformation and biotechnological application of the yeast Arxula adeninivorans. Appl Microbiol Biotechnol 54:619-624. https://doi.10.1007/s002530000444.