Pseudomonas putida S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkylbenzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-n-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or 'H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of200 ,uM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.
The degradation of a commercial linear alkylbenzenesulfonate (LAS) surfactant was examined kinetically in a trickling filter, which allowed simultaneous chemical determinations in the aqueous phase (e.g., DOC) and in the gas phase (CO2). About 60% of the carbon applied as LAS was released as CO2, whereas 15% remained as DOC in the eluate of acclimated trickling filters. The biomass was analysed after the experiment, and it was found to have sorbed about 23 mg LAS/g of dry biomass; this represented about 3% of the LAS applied to the filter. The LAS and the eluates from the trickling filter were further analysed by HPLC and UV and IR spectrometry. The residual carbon from acclimated filters contained no LAS‐like material (HPLC), which was obviously subject to quantitative biotransformation. The residual material comprised >50 polar metabolites, some of whose UV spectra differed from that of LAS, and most or all of which were sulfonated. These nondegraded metabolites included carboxylated dialkyltetralinesulfonates and sulfophenylcarboxylates. These residual materials showed no detectable toxicity to algae or Daphnia, and did not significantly lower the surface tension of water.
The standard procedure to examine the biodegradability of a (group of) compound(s) in a trickling filter is a continuousflow system. In this test, nondegraded metabolites from a commercial linear alkylbenzenesulfonate (LAS) surfactant are detected (Kolbener, Baumann, Leisinger, and Cook; accompanying paper). This procedure has now been augmented by two phases in closed cycle to give a test for refractory organic carbon (ROC test). First, the concentration of nondegraded metabolites was increased by readdition of LAS to the solution being cycled through the filter. Second, the concentrated residues were further recycled till the net dissolved organic carbon (DOC) stabilised at a finite value and the net released CO, stabilised at about zero. The organic compounds remaining at this phase of the experiment were considered recalcitrant and could be examined by global (e.g., DOC) and specific (e.g., HPLC) assays. Four different commercial preparations of LAS were examined, as were the (4-su1fophenyl)undecane homologue (C, l-LAS), the (4-sulfophenyl)dodecane homologue (C ,,-LAS), and some related compounds. The four commercial LAS preparations contained different levels of impurities (2% to about 17%, according to the producers), which were largely dialkyltetralinesulfonates (DATS) and branched-chain alkylbenzenesulfonates (bABS). The refractory organic carbon (ROC) remaining after biodegradation varied from 3 to 14%. The results were a characteristic of the LAS under study and were independent of the source of the biomass used in the experiment. Residues were examined by HPLC, and 50 to 100 peaks were observed, which were usually characteristic of the LAS studied. No peak has been conclusively identified. We consider the recalcitrants to represent carboxylated DATS and carboxylated bABS.
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