Anaerobic degradation of linear alkylbenzene sulfonate


  • Anders S. Mogensen,

    1. Environmental Microbiology and Biotechnology Group, BioCentrum-DTU, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark
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  • Frank Haagensen,

    1. Environmental Microbiology and Biotechnology Group, BioCentrum-DTU, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark
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  • Birgitte K. Ahring

    Corresponding author
    1. Environmental Microbiology and Biotechnology Group, BioCentrum-DTU, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark
    • Environmental Microbiology and Biotechnology Group, BioCentrum-DTU, Building 227, The Technical University of Denmark, DK-2800 Lyngby, Denmark
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  • Presented at the Organic Soil Contaminants Meeting, SETAC Europe, Copenhagen, Denmark, September 2–5, 2001.


Linear alkylbenzene sulfonate (LAS) found in wastewater is removed in the wastewater treatment facilities by sorption and aerobic biodegradation. The anaerobic digestion of sewage sludge has not been shown to contribute to the removal. The concentration of LAS based on dry matter typically increases during anaerobic stabilization due to transformation of easily degradable organic matter. Hence, LAS is regarded as resistant to biodegradation under anaerobic conditions. We present data from a lab-scale semi-continuously stirred tank reactor (CSTR) spiked with linear dodecylbenzene sulfonate (C12 LAS), which show that C12 LAS was biodegradable under methanogenic conditions. Sorption of C12 LAS on sewage sludge was described with a Freundlich isotherm. The C12 LAS sorption was determined with different concentrations of total solids (TS). In the semi-continuously stirred tank reactor, 18% of the added C12 LAS was bioavailable and 20% was biotransformed when spiking with 100 mg/L of C12 LAS and a TS concentration of 14.2 mg/L. Enhanced bioavailability of C12 LAS was obtained in an upflow anaerobic sludge blanket (UASB) reactor inoculated with granular sludge and sewage sludge. Biodegradation under thermophilic conditions was 37% with LAS as sole carbon source. Benzaldehyde was produced in the UASB reactor during LAS transformation.


Surfactants are widely used for both domestic and industrial purposes as cleaning, emulsifying, and wetting agents, and a very large number of different surfactants are in commercial use. Linear alkylbenzene sulfonate (LAS) is one of the most widely used sulfonated hydrocarbons with an annual production of approximately 1 × 106 t/year in the United States out of a worldwide detergent production of 15 × 106 t/year. Linear alkylbenzene sulfonate is a mixture of related isomers and homologués consisting of a para-sulfonated benzene molecule with an alkyl chain attached to any position but the terminal [1].

Many surfactants have a complex molecular structure consisting of hydrophilic and the hydrophobic ends. Anaerobic degradation requires a consortium of bacteria that act on different parts of the molecule. For example, the biodegradation of linear alkylbenzene sulfonate requires alteration of an alkyl chain, a benzene ring, and a sulfonate linkage.

A large fraction of the LAS received at a sewage treatment facility is associated with suspended solids [2–4] and thus escapes aerobic treatment. Anaerobic treatment of primary and secondary sludge is conducted at many wastewater treatment plants as a stabilization process with reduction of the organic matter content. Commonly, LAS is found to severely impede methane production and persist during anaerobic sludge treatment [5,6]. Due to this persistence, LAS has been considered inaccessible to biodegradation without the presence of molecular oxygen [6,7]. Biodegradation of LAS under anaerobic conditions is rarely reported and only under certain conditions, e.g., in sulfate-limited environments where LAS is the only source of sulfur [8].

Biodegradation of organic contaminants not only requires an appropriate number of microorganisms and convenient growth conditions, but the compound also has to be available to microorganisms, which is a key problem in, e.g., bioremediation soil contaminated with polycyclic aromatic hydrocarbons [9]. With time, organic contaminants in soil become more resistant to extraction as they are entrapped in macro-molecular humus substances [10]. Biodegradation of adsorbed contaminants can be a function of the mass transfer rates (from adsorbed to the aqueous phase) rather than the biodegradation rates [11].

Few studies have been conducted with chemicals other than polycyclic aromatic hydrocarbons, but likely the same pattern is valid for other chemicals that sorb to organic matter, and it could be speculated that recalcitrance of LAS during anaerobic digestion of sewage sludge is due to decreased availability to anaerobic bacteria.

This study shows that only a minor fraction of C12 LAS was present in the water phase during anaerobic treatment of sewage sludge in a lab-scale semicontinuously stirred tank reactor (CSTR). The bioavailable part of C12 LAS in this sludge-fed reactor corresponded to the fraction that was bio-degraded. Enhanced bioavailability of LAS was achieved in a upflow anaerobic sludge blanket (UASB) reactor fed with mineral medium amended with a mixture of LAS homologues (C10 to C14). This resulted in improved anaerobic degradation of LAS. Furthermore, it was shown that benzaldehyde was formed during the treatment.


Sorption experiment

Two sorption tests to determine the bioavailable fraction of C12 LAS in sewage sludge were performed in batch series. Sorption of C12 LAS to sewage sludge as a function of the initial concentration of C12 LAS was tested in 500-ml batch vials. A sludge suspension of 200 ml was diluted with mineral medium [12], reaching a concentration of 6 g/L of total solid. The batch vials were autoclaved two consecutive times for 20 min at 140°C. Samples were taken after 48 h. The initial C12 LAS concentration varied from 50 to 500 mg/L.

Sorption of C12 LAS to sewage sludge as a function of the concentration of total solids was determined in a series of batch vials containing 3.3 to 40 g total solids (TS)/L of sterilized sludge in mineral medium as described above. The concentration of LAS was 100 mg C12 LAS/L in all batch vials.

CSTR reactor experiments

A mesophilic lab-scale semi-CSTR, RCSTR, was fed with a sludge suspension consisting of anaerobically digested sewage sludge spiked with C12 LAS in order to study LAS transformation.

CSTR inoculum and influent. The CSTR reactor was inoculated with anaerobically stabilized sewage sludge from a laboratory-scale CSTR reactor that digested a secondary sewage sludge suspension. The influent consisted of secondary sewage sludge diluted with mineral medium to reach a TS concentration of 20 g/L and spiked with C12 LAS to a final concentration of 100 mg/L if not stated otherwise. The C12 LAS was added from an aqueous stock solution. Prior to connection with the reactors, the sludge suspension was gassed with N2/CO2 (80/20% v/v) for 30 min. The influent container was closed, and a gas bag filled with N2/CO2 (80/20% v/v) was connected.

CSTR reactor and reactor operation. A reactor of 3.5 L of volume was kept at 37°C and fed semicontinuously, with a hydraulic retention time of 15 d. After a period of five retention times, the TS concentration to reactor RCSTR was decreased to 10 g TS/L. The C12 LAS concentration was not changed.

CSTR reactor sampling. The concentration of C12 LAS and TS was determined a minimum of three times weekly. Three aliquots were sampled and stored at −20°C until analysis.

UASB reactor experiments

Three UASB reactors (R55, Runamended, Rsterile) were fed with mineral medium with or without LAS amendment. The two control reactors (Runamended, Rsterile) were used to determine absorption of LAS, the background level of volatile fatty acids, and the background level of metabolites identified in reactor R55. All reactors were inoculated with granular sludge and were operated under thermophilic conditions with a hydraulic retention time of 12 h. The medium fed to reactor R55 and Rsterile contained 10 mg LAS/L. Control reactor Runamended was fed with medium that had not been amended with LAS. Control reactor Rsterile was autoclaved three consecutive times at 140°C for 30 min after inoculation in order to maximize sterile conditions.

Reactors. Two hundred-milliliter glass UASB reactors were used as described by Schmidt and Ahring [13]. Effluent was collected in a closed container fitted with a gas-tight aluminum bag. All reactors were kept in an incubator at 55°C. The reactors were continuously fed with a Watson-Marlow peristaltic pump. Reactor effluent was recirculated at a recirculation:influent ratio of 5:1 (v/v) in all reactors. The loading of LAS to R55 and Rsterile was 20 mg/L/d.

Inoculum. In all three UASB reactors, the inoculum consisted of 35 ml granules from a mesophilic UASB reactor-treating papermill effluent (Erbeek, The Netherlands) and 5 ml granular sludge from a lab-scale thermophilic UASB reactor previously fed with C12 LAS.

UASB reactor sampling. Influent samples were taken just before reactor inlet, and effluent samples were taken in sampling ports along the granular bed and in the upper section of the reactor above the granular bed. Samples were kept at −20°C prior to analysis.

Influent. Influent to R55 and Rsterile consisted of mineral medium spiked with 10 mg LAS/L. The mineral medium was composed of distilled water with addition of nutrients and vitamins according to Angelidaki et al. [12]. After mixing, the medium was autoclaved at 140°C for 90 min. A sterile sodium carbonate solution of 50 ml was added under sterile conditions (75 g/L of NaHCO3). The medium was gassed with N2/CO2 (80/20% by volume) for 30 min. To ensure methanogenic conditions, Na2S was added to a final concentration of 20 mM. After autoclaving, pH was adjusted to 7.0 by addition of NaOH or HCl.


Linear alkylbenzene sulfonate used in the UASB reactor experiment was a mixture of C10 to C13 alkanes substituted with 4-benzenesulphonic acid. The C10 to C13 homologs were found in 17, 33, 27, and 23% ratios, respectively. It was purchased from Sigma-Aldrich (Dorset, UK) and is termed LAS. Linear alkylbenzene sulfonate used in the CSTR reactor and sorption experiments was the sodium salt of 4-(2-dodecyl) benzene sulfonate (2-C12 LAS, CAS No. 25155-30-0) and was bought from RISOE National Laboratory (Roskilde, Denmark). It is termed C12 LAS.

Benzaldehyde was obtained from Merck Eurolab (Alberts-lund, Denmark). High-pressure liquid chromatography-grade dichloromethane and acetonitrile were purchased from Lab-scan (Dublin, Ireland). Sodium perchlorate was bought at Sigma (Milwaukee, WI, USA). Thirty-seven percent hydrochloric acid was purchased from Kebolab (Albertslund, Denmark).

Analytical procedures

The UASB reactor samples for LAS quantification were centrifuged at 14,000 rpm for 10 min prior to analysis. Quantification was conducted on a HP1100 high-pressure liquid chromatography system (Hewlett-Packard, Waldbronn, Germany) equipped with a C18 column (250 by 4.6 mm; particle size 5 μm) from Agilent (Nærum, Denmark) using ultraviolet detection (λ = 225 nm). The LAS homologs were separated using a mobile phase of 9.2 g/L of NaClO4 in Milli-Q® water (Millipore, Bedford, MA, USA) with an acetonitrile gradient (0–100% v/v) as described by Kertesz et al. [14].

Determination of total solids was done according to standard methods [15].

The C12 LAS in sludge samples from the CSTR reactor was extracted with alkaline methanol by vigorous shaking for 30 min. The methanolic samples were then filtered and analyzed by high-pressure liquid chromatography equipped with an RP-C18 column as described above. The mobile phase had a flow of 1.5 ml/min and was composed of 78% (v/v) methanol, 22% (v/v) Milli-Q water, and NaClO4 (9.18 g/L). Quantification of dissolved C12 LAS (i.e., C12 LAS in the water phase of sludge samples) was carried out as the quantification of C12 LAS in sludge samples but without extraction of the samples. The extraction of C12 LAS from sludge samples had an efficiency of 78%.

Metabolites in acidified effluent samples (pH = 2) from the UASB reactors were extracted with dichloromethane. Gas chromatographic separations of extracts were performed with a Hewlett Packard (Wilmington, DE, USA) Model 6890 gas chromatograph equipped with a HP-5 column. A 3 μl sample was injected in splitless mode with an injection temperature of 250°C. Helium was used as the carrier gas at 1.1-ml constant flow. The oven temperature program started at 2 min at 50°C, followed by a 20°C/min ramp to 310°C. This temperature was held for 10 min. The mass selective detection was performed using a Hewlett Packard Model 5973 mass selective detection in SCAN acquisition mode. The electron multiplier voltage was set to +400 V relative to the tune file parameter. The National Institute of Standards and Technology (Gaithersburg, MD, USA) Mass Spectral Search Program (nbs75k) was used for the library search.

Figure Fig. 1..

Linear dodecylbenzene sulfonate (C12 LAS) in the water phase and methanol extract of mixed sample in the continuously stirred tank reactor (RCSTR).


Sorption experiment

The results from the sorption experiment reveal that sorption of C12 LAS was dependent on the concentration of C12 LAS (the amount of LAS molecules relative to organic matter) and the TS concentration (the concentration of organic matter).

The relationship between sorbed and dissolved C12 LAS concentration at equilibrium (expressed as mg/kg TS and mg/L, respectively) could be represented by the Freundlich sorption isotherm. The Kd and 1/n were calculated to be 796 L/kg and 0.35, respectively. Hence, the Freundlich sorption isotherm was

equation image(1)

Where R2 is the square of slope determined by linear regression. Even though LAS has high water solubility, the hydrophobic alkyl chain sorbs to suspended particles in sludge, resulting in low amounts of LAS in the water phase. This susceptibility to sorption is in fact used for attachment to dirt particles in its function as a detergent. The specific LAS homologue and isomer influence the hydrophobicity as well as the organic content of the sludge [16]. Digested secondary sludge used in the experiment reported here has a lower content of organic matter. The Kd value would therefore be low compared with, e.g., values determined in undigested secondary sludge [17].

When the concentration of LAS was kept constant at 100 mg/L, an exponential relation between the fraction of C12 LAS sorbed to sludge and the TS concentration was found, i.e.,

equation image(2)

Where CTS is defined as the concentration of total solids. By increased addition of total solids, the concentration of C12 LAS in the water phase was lowered. It was found that as much as 96% of the spiked C12 LAS was sorbed at 40 g TS/L.

The results from the sorption experiment revealed that bioavailability of C12 LAS (i.e., C12 LAS in the water phase [18]) was dependent on the TS concentration and the C12 LAS concentration.

CSTR reactor experiment

The biodegradation of organic matter in CSTR reactors is typically 30% of volatile solids [19]. Degradation of organic contaminants can be much lower due to decreased bioavailability of the contaminants or type of organic waste, thus resulting in high outlet concentrations (mg/kg TS) following biodegradation in CSTR reactors [20]. As shown in Figure 1, the concentration of C12 LAS in reactor RCSTR in the water phase (the readily bioavailable C12 LAS) was lower than 4 mg/L. This was significantly lower than the total concentration (measured as mg/L).

The average influent and effluent C12 LAS concentration from the CSTR reactor experiment is summarized in Table 1. Throughout the experiment, the pH of the reactor was 7.5 ± 0.1. In order to increase the bioavailability of LAS in reactor RCSTR, the average influent TS concentration was lowered from 20.9 g TS/L in period A (days 50–72) to 11.4 g TS/L by using a more diluted sludge solution as reactor feed in period B (days 94–128). The transformation of C12 LAS was increased from 20% in period A to 28% in period B. According to Equation 2, the average TS concentration of 14.2 g/L in period A would result in a sorption of C12 LAS of 82%. In period B, the sorption decreased to 76% due to dilution of the influent sludge suspension. A comparison of the calculated bioavailable C12 LAS and the measured anaerobically transformed C12 LAS observed in the reactor experiment is presented in Figure 2. From this comparison, a connection between transformed and bioavailable C12 LAS was seen. This suggests that persistence of LAS during anaerobic digestion of sewage sludge is related with sorption of the compound.

Table Table 1.. Concentration of linear dodecylbenzene sulfonate (C12 LAS) in reactor influent, concentration of C12 LAS in the water fraction in reactor effluent, concentration of total solids (TS) in reactor influent and effluent, and transformation of C12 LAS. ± indicates standard deviation of measurements during the experimental period
  Period APeriod B
Experimental periodDays50–7294–128
C12 LAS in reactor influentmg C12 LAS/L104.3 ± 5.3109.4 ± 6.9
C12 LAS in reactor effluentmg C12 LAS/L83.5 ± 10.979.2 ± 7.9
C12 LAS in water fraction of effluentmg C12 LAS/L1.8 ± 0.092.8 ± 0.5
CTS in reactor influentg TS/L19.9 ± 0.911.4 ± 0.7
CTS in reactor effluentg TS/L14.2 ± 2.09.6 ± 0.6
C12 LAS transformation%2028
Figure Fig. 2..

Calculated amount of bioavailable linear dodecylbenzene sulfonate (C12 LAS) and the measured anaerobic transformation of C12 LAS in the continuously stirred tank reactor (RCSTR) in period A and period B.

UASB reactor experiment

The design of the UASB reactor allows treatment of waste-water low in suspended solids, such as certain industrial effluents. The biomass in the UASB reactor consists of bacteria in dense granules, which are retained within the reactor during operation. Hence, the UASB reactor would not be applicable in treatment of sewage sludge due to wash out of biomass. In the UASB reactor experiments reported here, a LAS-amended mineral medium without suspended solids was used. In opposition to the CSTR reactor fed with sewage sludge, the LAS mixture was the sole source of carbon and energy in the UASB reactor experiment.

In Table 2, the concentrations of LAS in reactor R55 and Rsterile effluents are given at selected time points. In the sterile reactor, LAS was only removed by sorption (no methane production was detected). A gradual increase in the concentration of LAS in the effluent stream was observed in both reactors. The concentration of LAS in the effluent of R55 stabilized at 6.3 mg/L. Thirty-seven percent of the LAS was biodegraded in the reactor with biologically active granular biomass at 432 h, compared with 10% removal in the reactor with sterilized granular biomass.

Table Table 2.. Concentration of linear alkylbenzene sulfonate (LAS) in the influent (Cin) and effluent (Cout) from the upflow anaerobic sludge blanket (UASB) reactors R55 and Rsterile
  Cout (mg/L)
Time (hours)Cin mg/LR55Rsterile
Figure Fig. 3..

Total ion chromatogram of reactor effluent from upflow anaerobic sludge blanket reactor R55.

Mass spectrometry analysis of effluent from reactor R55 and the two control reactors revealed production of compounds in R55 that were not found in the effluent from either the sterile, LAS-amended reactor, Rsterile, or the unamended reactor, Runamended. According to the total ion chromatogram (TIC) of an effluent sample from reactor R55, 14 peaks were observed (see Fig. 3). None of the peaks were observed in the TICs of the samples from the control reactors (chromatographs not reported).

At retention time 5.22 min, a peak with high intensity was observed. Figure 4 shows the mass spectrum of R55 effluent extract for this peak after extraction of background in the TIC. The abundant ions were 50, 77, 105, and 106. In Figure 4, the mass spectrum of a library search of benzaldehyde is also reported. The mass spectrum had a match quality of 0.97 with benzaldehyde. The search was conducted using the National Institute of Standards and Technology (Gaithersburg, MD, USA) spectral database.

Figure Fig. 4..

Mass spectrum of benzaldehyde from upflow anaerobic sludge blanket reactor R55 sample (upper part) and mass spectrum of library search of benzaldehyde (lower part).

Figure Fig. 5..

Extracted ion chromatogram of reactor effluent sample from upflow anaerobic sludge blanket reactor R55. See text for further details.

The single m/z values observed in the mass spectrum of benzaldehyde might also be found in the mass spectra of other peaks appearing in the TIC of the effluent sample. In order to confirm benzaldehyde as a metabolite in R55, an extracted ion chromatogram was drawn. For this chromatogram, the ions with m/z 50, 77, 105, and 106 were chosen. The extracted ion chromatogram shows the abundance of each selected ion during a selected time period by scanning the TIC for those ions only. In Figure 5, this is shown for 5.16 to 5.60 min. Only at retention times of 5.20 to 5.25 were all four ions observed mutually in the R55 effluent sample. These ions were not observed mutually at any retention time in the two control reactors. This confirmed that benzaldehyde was a metabolite in the anaerobic treatment of LAS.

Even though a connection between bioavailability in anaerobic reactors and biodegradation has been established, very little data is available concerning anaerobic LAS biodegradation. A pure culture or a consortium of microorganisms capable of degrading LAS has so far never been reported, though the study of anaerobic degradation of sulfonated compounds is receiving increased attention [21–24].

Surfactants are removed from wastewater by sorption to organic matter in wastewater treatment plants [3,25]. In the case of LAS, it is typically found to increase in concentration (when measured in amount of LAS per weight unit of total solids) after anaerobic sludge bioprocessing. This accumulation is a result of higher digestion of organic matter compared with the LAS degradation. The bioavailable concentration is, as shown by the CSTR experiments reported here, much lower compared with the concentration of total C12 LAS. Hence, it could be speculated that many unsuccessful attempts to demonstrate anaerobic LAS biodegradation have been due to lack of bioavailable LAS. In the UASB reactor experiment, the bioavailability was maximized due to lack of suspended solids in the feed. This increases the possibility of LAS degradation. This type of reactor is designed for wastewater with low amounts of suspended solids but has been proven efficient with wastewater high in dissolved organic matter and xenobiotic compounds [26,27]. Linear alkylbenzene sulfonates were sorbed to the granular biomass initially, but sorption ceased eventually in the sterile reactor. In the CSTR reactor (frequently used in treatment of sewage sludge and manure), the amount of bioavailable LAS was low. Therefore, biodegradation was hampered, and the degradation rate will depend on desorption, a phenomenon previously shown for polycyclic aromatic hydrocarbons [28]. The removal of LAS and formation of benzaldehyde in the UASB reactor illustrate that the degradation can occur under anaerobic conditions. The difference in degradation between the CSTR reactor and the UASB reactor supports the speculation that it is merely the bioavailable LAS that is transformed during digestion of sewage sludge and anaerobic treatment of LAS-enriched medium. With the high sorption potential of LAS in sewage sludge, only very limited amounts of LAS will be removed from sewage sludge during anaerobic treatment and stabilization of sludge.


This work was supported by the Danish Environmental Research Programme (Center for Sustainable Land Use and Management of Contaminants, Carbon, and Nitrogen).