The Missing Link in Linear Alkylbenzenesulfonate Surfactant Degradation: 4-Sulfoacetophenone as a Transient Intermediate in the Degradation of 3-(4-Sulfophenyl)Butyrate by Comamonas testosteroni KF-1

Biodegradation of the laundry surfactant linear alkylbenzenesulfonate (LAS) involves complex bacterial communities. The known heterotrophic community has two tiers. First, all LAS congeners are oxygenated and oxidized to about 50 sulfophenylcarboxylates (SPC). Second, the SPCs are mineralized. Comamonas testosteroni KF-1 mineralizes 3-(4-sulfophenyl)butyrate (3-C4-SPC). During growth of strain KF-1 with 3-C4-SPC, two transient intermediates were detected in the culture medium. One intermediate was identified as 4-sulfoacetophenone (SAP) (4-acetylbenzenesulfonate) by nuclear magnetic resonance (NMR). The other was 4-sulfophenol (SP). This information allowed us to postulate a degradation pathway that comprises the removal of an acetyl moiety from (derivatized) 3-C4-SPC, followed by a Baeyer-Villiger monooxygenation of SAP and subsequent ester cleavage to yield SP. Inducible NADPH-dependent SAP-oxygenase was detected in crude extracts of strain KF-1. The enzyme reaction involved transient formation of 4-sulfophenol acetate (SPAc), which was completely hydrolyzed to SP and acetate. SP was subject to NADH-dependent oxygenation in crude extract, and 4-sulfocatechol (SC) was subject to oxygenolytic ring cleavage. The first complete degradative pathway for an SPC can now be depicted with 3-C4-SPC: transport, ligation to a coenzyme A (CoA) ester, and manipulation to allow abstraction of acetyl-CoA to yield SAP, Baeyer-Villiger monooxygenation to SPAc, hydrolysis of the ester to acetate and SP, monooxygenation of SP to SC, the ortho ring-cleavage pathway with desulfonation, and sulfite oxidation.Linear alkylbenzenesulfonate (LAS) is the major synthetic laundry surfactant worldwide, with an annual production of 2.5 × 10⁶ tonnes, which in Germany means 3 g of LAS per person and day, or about 4% of the carbon entering the sewage works (e.g., see reference 15); hence, its degradation is important. European LAS surfactant is nominally a mixture of 20 congeners, each of which is a linear alkane (C₁₀-C₁₃) subterminally substituted with a 4-sulfophenyl moiety (15) (Fig. ​(Fig.1);1); 18 of these congeners are chiral.Open in a separate windowFIG. 1.Flow diagram of the primary degradation by P. lavamentivorans DS-1 of two LAS congeners to six SPCs, four of which are mineralized by C. testosteroni KF-1. None of the reactions or pathways indicated in strain DS-1 has been observed directly. None of the reactions in strain KF-1 has been observed directly, though 4-sulfophenol is a growth substrate (28): the major unknown in SPC degradation is the manipulation of the side chain.Mineralization of LAS has been known for 50 years (25), and the involvement of sulfophenylcarboxylates (SPCs) (Fig. ​(Fig.1)1) as intermediates in that process has been known for about 40 years (33). Recognition that the overall degradation of LAS involved communities of microorganisms developed 10 to 15 years ago (36), when routine high-performance liquid chromatography (HPLC) analysis of LAS and SPC in biological samples became available (16, 20). The coupling of HPLC technology to mass spectrometers has allowed the analysis of the transient SPC intermediates to be further improved (e.g., see references 7 and 19), such that we now have a comprehensive picture of some 50 SPC-like products (mostly chiral) formed from commercial LAS by the first-tier organism in an LAS-degrading bacterial community (27).A heterotrophic, bacterial, LAS-degrading community comprises two tiers. The first tier of organisms, which is so far represented only by Parvibaculum lavamentivorans DS-1 (6, 12, 27-29), converts all LAS congeners to about 50 SPCs (and related compounds, see below) and cell material derived from the acetyl coenzyme A (acetyl-CoA) released through β-oxidation from the alkane moiety. In principle, each LAS congener yields three products that are released by strain DS-1, an SPC, an α,β-unsaturated SPC (SPC-2H), and an SPC from the previous round of β-oxidation (SPC+2C) (Fig. ​(Fig.1)1) (27, 28); sulfophenyl-di-carboxylates (SPdCs) are generated by β-oxidation of both ends of the alkane chain, e.g., from the “centrally substituted” 4- and 5-C10-LAS congeners (Fig. ​(Fig.1)1) (27), but these products are not relevant in this work. The second tier of organisms degrades these SPCs (and SPC-2Hs and SPdCs) to cell material, CO₂, water, and sulfate (28). In this tier, many different organisms must be active because all known representatives have a narrow substrate spectrum of only 3 to 4 individual SPC-like compounds (28, 30; Results). The present work centers on Comamonas testosteroni KF-1, which utilizes four known SPC-like compounds (Fig. ​(Fig.1),1), 3-(4-sulfophenyl)butyrate (3-C4-SPC), 3-(4-sulfophenyl)-Δ2-enoylbutyrate (3-C4-SPC-2H), 3-(4-sulfophenyl)pentanoate (3-C5-SPC), and 3-(4-sulfophenyl)-Δ2-enoylpentanoate (3-C5-SPC-2H). This work is focused on the metabolism of 3-C4-SPC. Strain KF-1 utilizes both the (R)- and (S)-enantiomers of 3-C4-SPC (28), and this degradation may be enantioselective, resulting in different reaction rates (22), as observed for degradation of (R,S)-2-C4-SPC and (R,S)-4-C6-SPC by isolated Delftia acidovorans strains (28, 30).We now report that C. testosteroni KF-1 utilized 3-C4-SPC with transient excretion of two degradation intermediates, whose identification allowed us for the first time to draft a complete degradative pathway for an SPC.