Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities M Vin ˜as, M Grifoll, J Sabate ´ and AM Solanas Department of Microbiology, University of Barcelona, Diagonal 645, E - 08028 Barcelona, Spain Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [ PAHs ]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% ( F1AA ), 48% ( TD ) and 34% ( AM ), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n - alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% ( AM ), 11% ( TD ) and 7% ( F1AA ). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500 - fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252 – 260 DOI: 10.1038/sj/jim/7000236 Keywords: microbial consortia; crude oil; hydrocarbons; biodegradation; bioaugmentation Introduction Microbial degradation by natural populations, the primary process in natural decontamination [ 22 ], can be enhanced by bioremedia- tion technologies [ 3 ]. Petroleum products contain thousands of individual hydro- carbons and related compounds [ 23 ]. Their main components are usually subdivided into saturates ( n - and branched - chain alkanes and cycloparaffins ) and aromatics ( mono - , di - , and polynuclear aromatic compounds [ PAHs ] containing alkyl side chains and/or fused cycloparaffin rings ). The less abundant resins and asphal- tenes consist of more polar compounds, containing heterocycles, oxygenated hydrocarbons and aggregates with high molecular weight. Given the complexity of oil products, a combination of bacterial strains with broad enzymatic capabilities will be required to achieve extensive degradation. However, most of the crude oil degradation studies reported in the literature have been carried out with single or mixed bacterial strains isolated because of their ability to grow in mineral media with crude oil as the only carbon source [ 4,21,26 ]. Since alkanes are the most abundant compounds, these isolates are usually alkane degraders that, in some cases, are able to oxidize selectively the alkyl chains of certain alkylated PAHs, which are common in crude oils [ 11]. Our previous results using Pseudomo- nas sp. F21, isolated in a mineral medium with Arabian crude oil, corroborate this observation. Strain F21, which degrades all the n - alkanes and branched alkanes of low molecular weight, also causes selective depletion of methylated naphthalenes, phenanthrenes, chrysenes and pyrenes [ 4,26 ]. Nevertheless, since Pseudomonas sp. F21 is not able to utilize nonsubstituted PAHs (unpublished results , A.M. Solanas ), depletion of this type of compound is probably due to oxidation of the alkylic chains, as described for other alkane degraders [ 11 ]. Oil - degrading mixed cultures can be constructed either by combining a number of strains with known complementary degradative capabilities ( defined consortia ) [ 13,14,19 ] or by direct enrichment procedures ( nondefined consortia ) [ 5,27,28 ]. The first approach is well defined and easily repeatable, but has certain disadvantages. First, since any one bacterial strain degrades only a limited number of crude oil components [ 20 ], in order to achieve wide and extensive crude oil biodegradation a consortium with many strains will be needed. Alkane degraders reported in the literature usually do not break aromatic rings, and PAH degraders do not grow on alkanes [12]. Recently, a few strains capable of degrading high molecular weight PAHs and alkanes have been reported [ 9 ], but this does not seem to be a general feature. Aromatic degraders use either monoaromatic hydrocarbons, hydro- carbons with two to three rings or hydrocarbons with three to four rings [ 15 ]. Furthermore, there is little information about strains that grow on alkylated PAHs with three or more rings [16,25 ], very common in crude oils. Degradation of the large portion of crude oil consisting of nonresolved nonidentified compounds has not been addressed. Another consideration in relation to defined consortia is the possible formation of toxic intermediate metabolites [ 7 ]. It has been reported that during the degradation of mixtures of hydro- carbons by selected strains, a number of intermediates of unknown persistence and toxicity may accumulate as a result of cometab- olism [ 17 ]. Enrichment procedures with selected oil products or with some specific components can provide nondefined metabolically speci- alized microbial consortia. The result is a microbial population naturally selected by its metabolic cooperation in the degradation of Correspondence: Dr Anna Maria Solanas, Department of Microbiology, University of Barcelona, Diagonal 645, E - 08028 Barcelona, Spain Received 12 July 2001; accepted 11 November 2001 Journal of Industrial Microbiology & Biotechnology (2002) 28, 252 – 260 D 2002 Nature Publishing Group All rights reserved 1367-5435/02 $25.00 www.nature.com / jim