Surface tension and toxicity changes during biodegradation of carbazole by newly isolated methylotrophic strain Methylobacterium sp. GPE1 Q3 Grzegorz Pasternak * , Barbara Ko1wzan Wroclaw University of Technology, Institute of Environmental Engineering, Wyb. Wyspia nskiego 27, 50-370 Wroclaw, Poland article info Article history: Received 31 January 2012 Received in revised form 21 June 2012 Accepted 19 July 2012 Available online xxx Keywords: Heterocycles Heterocyclic compound Azaarenes Biosurfactant Microtox Ecotoxicity abstract The novel Methylobacterium sp. GPE1 strain was isolated from a former gasworks site. GPE1 was able to utilise carbazole as a sole source of carbon and energy. Its ability to produce surface-active compounds was investigated by monitoring the biomass and surface tension. During the adaptation phase, the GPE1 cells cultured on carbazole (9.9 mg l 1 ) showed an unexpected change in the surface tension of the culture. The biodegradation process seemed to be initiated by the production of the biosurfactant. The appearance of an early log phase caused decrease of surface tension which reached its minimal value, equal to 61 mN m 1 . The later surface tension changes were corresponding to biomass concentration and reached the control level at the end of experiment. In the 20 day experimental period, GPE1 utilised 87% of carbazole, giving the following values: m ¼ 0.08 h 1 , Y xs ¼ 0.62 gC gC 1 . The MicrotoxÔ toxicity assay was conducted for centrifuged and filtered water solutions of cultures and abiotic samples. The samples after biodegradation revealed the increased toxicity determined by luminescence inhibition which increased from 15.2% to 33%. Although the biosurfactant produced by GPE1 was not identified, its relation with signal molecules, which are often present in Methylobacterium sp. representatives, is being discussed. Ó 2012 Published by Elsevier Ltd. 1. Introduction Tricyclic heteroaromatic compounds are widely represented in industry and nature. Carbazole is a nitrogen-containing heterocy- clic compound, with a symmetrical structure. It is the major N- heterocyclic compound present in coal carbonisation products. Its concentration in creosote reaches up to 1% (Nylund et al. 1992), and it is also present in coal tar, shale oils and crude oils (Nestler, 1974; Allard and Neilson, 1997). Carbazole is also used for the synthesis of pesticides, dyes, pharmaceuticals, detergents and other chemicals (Padoley et al. 2008). The toxicity of carbazole has not been well studied. According to Tsuda et al. (1982), carbazole induced tumours in mouse livers and the estimated TD 50 value was of 164 mg kg 1 day 1 . Another study showed that carbazole induced chromosomal aberrations in mouse bone marrow at concentrations equal to 25 mg kg 1 (Jha et al. 2002). Environmental pollution cases related to creosote works, gasworks or coke plant facilities have been reported often. The carbazole contamination in these reports varied from 0.2 mg l 1 in river sediment to 30 mg l 1 in groundwater (Huntley et al. 1993; Mueller et al. 1993). Carbazole could be also released into the environment due to the contact of impregnated wood with water, as reported by Rotard and Mailahn (1987). Carbazole is often used as a model compound for the biodeg- radation of nitrogen heterocycles, due to their role in NO x forma- tion, during thermal processes of coal and other fossil fuels. Several bacterial strains were found to utilise carbazole co-metabolically or as a single source of carbon and energy. The biodegradation of carbazole starts with the attack on lateral or angular positions by dioxygenases and leads to ring cleavage in the following steps (Seo et al. 2006; Xu et al. 2006). The final metabolites of carbazole degradation may include 2-hydroxypenta-2,4-diene and 2- aminobenzoate, which could be mineralised in the toluene and 2- aminobenzoate pathways (Sato et al. 1997). Carbazole-degrading strains belong mainly to gram-negative species, examples of which include: Xanthamonas sp. (Grosser et al. 1991), Sphingomonas sp. (Kilbane et al. 2002; Yang et al. 2009) and Pseudomonas sp. (Sato et al. 1997). Biodegradation tests have been frequently performed with solvents or surfactants, but there is a lack of knowledge available about the biosurfactant-producing strains, that are capable of uti- lising heterocyclic pollutants. Microbial surfactants change the surface tension of liquid and interfacial tension, thus enhancing the bioavailability of insoluble compounds. As bioavailability is * Corresponding author. Tel.: þ48 713202532. E-mail address: grzegorz.pasternak@pwr.wroc.pl (G. Pasternak). Contents lists available at SciVerse ScienceDirect International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod 0964-8305/$ e see front matter Ó 2012 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.ibiod.2012.07.021 International Biodeterioration & Biodegradation xxx (2012) 1e7 Please cite this article in press as: Pasternak, G., Ko1wzan, B., Surface tension and toxicity changes during biodegradation of carbazole by newly isolated methylotrophic strain Methylobacterium sp. GPE1, International Biodeterioration & Biodegradation (2012), http://dx.doi.org/10.1016/ j.ibiod.2012.07.021 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 INBI2907_proof ■ 4 October 2012 ■ 1/7