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Effect of Temperature on the Biodegradation of Linear Alkylbenzene Sulfonate and Alcohol Ethoxylate Daniel Prats, Carmen López, Diana Vallejo, Pedro Varó, and Víctor M. León* Department of Chemical Engineering, Alicante University, Ctra. San Vicente del Raspeig s/n, San Vicente del Raspeig, Alicante, Spain

ABSTRACT: The effect of temperature on the biodegradation of linear alkylbenzene sulfonate (LAS) and alcohol ethoxylate (AE) was evaluated using method OECD 303 A, Confirmatory test (Husmann units). The experiments were performed using an initial surfactant concentration of 10 mg/L and working temperatures of 25, 15, and 9°C, keeping the biodegradation units inside a thermostatic chamber. In all cases, the removal of both surfactants tested, LAS and AE, was higher than 90%, regardless of the temperature used in the test. We observed that longer acclimation periods were needed by the microorganisms at lower temperatures. Paper no. S1488 in JSD 9, 69–75 (Qtr. 1, 2006). KEY WORDS: Activated sludge, alcohol ethoxylates, biodegradation, linear alkylbenzene sulfonate, temperature.

The widespread use of surfactants in many household and industrial products, together with the high volume consumed worldwide, justifies the abundant and frequent scientific scrutiny of the environmental safety characteristics of these products. Among the variety of surfactants used in the industry, anionic linear alkylbenzene sulfonates (LAS) and nonionic alcohol ethoxylates (AE) are the most relevant families, representing 27 and 22% of the total volume used in the European Union in 1998 (1). The behavior of these surfactants during wastewater treatment operations has been thoroughly studied (2–5). Biodegradation begins in the sewer system (5–7), with reported removals higher than 60% for LAS and between 28 and 58% for AE (5). The LAS concentration in raw sewage varies from 1 to 10 mg/L (8), reaching 32 mg/L in some isolated peak cases (7). Reported concentrations for AE are in the range of 1–4 mg/L (4,9). The elimination of LAS in wastewater treatment plants (WWTP) depends on the type of operation used; in plants operated with trickling filter systems, removals higher than *To whom correspondence should be addressed. E-mail: [email protected] Abbreviations: AE, alcohol ethoxylate; BiAS, bismuth active substances; cfu, colony-forming units; DO, dissolved oxygen; DOC, dissolved organic carbon; EO, ethylene oxide; HPLC, high-performance liquid chromatography; LAS, linear alkylbenzene sulfonate; MBAS, methylene-blue active substances; MLSS, mixed liquor suspended solids; NOEC, nonobserved effect concentration; OECD, Organisation for Economic Co-operation and Development; SRT, sludge retention time; WWTP, wastewater treatment plant(s). COPYRIGHT © 2006 BY AOCS PRESS

85% have been reported (7,10), whereas in plants using activated sludge systems, removals higher than 98% are widely obtained (2,5). Treatments based on trickling filters are reported to provide more variable degrees of removal (11). In the case of AE, the published information indicates removals higher than 98% (3,4,9). The degree of mineralization observed with LAS and AE in WWTP operating with activated sludge has been reported to be higher than 90% (12–14). LAS and AE are removed by biological processes (biodegradation) as well as by physical processes (sorption–precipitation) (2,3). The mass balance for LAS elimination according to different studies conducted at real environmental conditions (15) indicates that approximately 90% is biodegraded, 10% is removed by sorption onto the sludge, and less than 1% leaves the treatment plants with the treated water. Adsorption of LAS on activated sludge increases with the length of the alkyl chain (16), and it has been reported that the adsorption coefficient increases 2.8 times per methylene group (17). The physical removal of AE with primary sludge has been found to be between 6 and 60% of the total for C10EO9 and C16EO9, respectively. Physical removal is also influenced by the length of the ethoxy groups (3). Levels of AE on the sludge at 10°C were higher than those taken from the 20°C test, showing lower AE degradation efficiency during activated sludge sewage treatment at 10°C (14). The surfactants LAS and AE have been shown to be biodegradable in many laboratory tests. The mineralization of LAS based on dissolved organic carbon (DOC) measurements using continuous activated sludge tests [Organisation for Economic Co-operation and Development (OECD) 303 A] varies between 80 and 95%, whereas the results obtained following the protocol of inherent tests (OECD 302) are in the range of 95–98% (18). In the case of linear alcohol ethoxylates, their elimination is higher than 95% as measured by bismuth active substances (BiAS)/cobalt thiocyanate active substances (18), higher than 87% based on DOC (14,19) determination, and higher than 98% using specific analytical determinations (14). The extent and kinetics of LAS and AE biodegradation depend on the characteristics of the test product and the physicochemical and microbiological properties of the media used. Higher O2 concentrations and longer alkyl chains ensure higher biodegradation removal of LAS in untreated water systems (18). In general terms, an increase in the temperature JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

70 D. PRATS ET AL.

will favor the metabolic processes and, consequently, the activity of microbial cultures up to a maximum of 30–40°C as demonstrated with AE (14) and LAS (18). However, the information on the effect of temperature on the biodegradation of AE and LAS is very scarce, and it is relevant to establish seasonal variations on the biodegradation of these extensively used surfactants. Some studies of the effects of temperature on biodegradation have been done separately for each surfactant type (14,18,20), although a comparison of both these important commercial surfactants using similar experimental conditions is lacking. In this work, the biodegradation of commercial LAS and AE was studied over a broad range of temperatures (9–25°C) and using the OECD activated sludge simulation test for both compounds at the same experimental conditions. Characterization of the surfactant portion associated with suspended material was also investigated.

MATERIALS AND METHODS Test chemicals. LAS with an average alkyl chain of 11.6 (CAS number: 68411-30-3) was supplied by PETRESA (San Roque, Spain). The homolog weight distributions were 12.1, 34.1, 30.6, and 23.2%, respectively, for C10LAS, C11LAS, C12LAS, and C13LAS. The nonionic surfactant used was an AE supplied by SASOL Italy (Milan, Italy; CAS number: 68131-39-5) with an average of 7.3 ethylene oxide (EO) groups and the following alcohol weight distributions: 2, 13, 57, and 28%, respectively, for C11, C12, C13, and C14. Biodegradation equipment. The protocol suggested by the OECD 303 A (Confirmatory test) method was used in our study. Five Husmann units were used in the study, each comprising a 3-L aeration vessel and a 2-L settling tank. Two units were used for LAS tests (units 1 and 2), two for AE tests (units 3 and 4), and the fifth one was a control (unit 5). The units were inoculated with a mixed inoculum (secondary effluent of a sewage treatment plant + soil + natural surface water) from the Alicante (Spain) area. The biodegradation units were operated with a hydraulic residence time of 6 h and a sludge retention time (SRT) of 10 d to simulate the conditions prevalent in WWTP (2), although the times were slightly longer than those reported in other studies (14,21). The most important parameters affecting the efficiency of biodegradation of surfactants in WWTP are the SRT and the temperature (22); consequently, a range of temperatures from 9 to 25°C were studied while maintaining the same SRT. Throughout the entire experiment, the biodegradation units were in operation inside a thermostatic chamber using the following working temperature pattern: 25, 15, and 9°C ± 0.5°C. Once the test at 25°C was completed, the temperature was progressively reduced at a rate of 1°C/d until it reached 15°C, the operating temperature of the subsequent test. After completing the test at 15°C, the same procedure described above was followed until a temperature of 9°C was reached. The sequence with the three test temperatures was run twice JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

for each surfactant using different biodegradation units. During the first set of tests (experiment I), the maximum duration of each test was 6 wk as prescribed by the OECD (23). In the second set (experiment II), the units were allowed to operate for a longer period of time to improve the acclimation at low temperatures. Units 1 and 2 were used for the LAS tests for experiments I and II, whereas units 3 and 4 were used for AE experiments I and II. The five units were initially started with synthetic water (23), prepared with water from the municipal water supply system of Alicante and conditioned in the laboratory to adjust the water hardness level to 150 mg/L (as CaCO3). Once the biomass formed was determined to be stable, the surfactants were added to the units at a concentration of 10 mg/L. This concentration was in the same range or slightly higher than the reported surfactant levels in wastewater streams: 1–4 mg/L for AE (9), and 1–10 mg/L for LAS (8). The feed tank containing the surfactant solution was continuously agitated to homogenize it and avoid the accumulation of any surfactant in any zone. Analytical methods. The analyses performed during the study are summarized in Figure 1. The effluent samples were taken from a composite mixture representative of 24 h of operation. The samples for surfactant analysis were preserved with 3% (vol/vol) formalin (37% formaldehyde) at the time of collection. The samples for the determination of other parameters were preserved in the freezer at 4°C until they were analyzed (within 24 h after collection). The analyses corresponding to solids, dissolved oxygen (DO), and DOC were carried out to monitor the operation of the units during each test. The total solids, suspended solids, and volatile solids were determined by gravimetric analysis up to a constant weight at

FIG. 1. Summary of the analyses performed during the study. MBAS, methylene-blue active substances; HPLC-UV, high-performance liquid chromatography with ultraviolet detection; BiAS, bismuth active substances; LAS, linear alkylbenzene sulfonate.

71 TEMPERATURE EFFECT ON LAS AND AE BIODEGRADATION

105°C (total solids and suspended solids) and 550ºC (volatile solids) according to the American Public Health Association (APHA) method (24). The DO and the temperature in the biological reactors were determined using a specific electrode and temperature bore. The DOC content was determined by the difference between the total carbon content and the inorganic carbon content after filtration of samples through a 0.45-µm filter. For this purpose, a total organic carbon analyzer, Shimadzu TOC-5000A, based on catalytic combustion and a nondispersive detection method (Standard Method 5310B; Ref. 25), was used. LAS was analyzed using the nonspecific methylene-blue active substances (MBAS) method (26) as well as by specific high-performance liquid chromatography (HPLC) analysis (27). The nonionic surfactant was determined using the nonspecific BiAS method in accordance with the procedure of Wickbold (28,29), using the ethylene diamine tetraacetic acid modification as proposed by other authors for the quantification of nonionic surfactants (30). The percentage of MBAS or BiAS elimination was based on the determination of MBAS and BiAS in the influent and effluent streams of the Husmann units using the OECD 303A calculation criteria (23). The dynamics of the most important microbial groups (bacteria, protozoa, and metazoa) were also monitored. A weekly count of the viable total heterotrophic bacteria was done by means of the plate count bacterial culture technique on the mixed liquor of each unit. The abundance of the protozoan and metazoan microfauna was determined using a phase contrast microscopy technique with 10 and 40× oculars.

RESULTS AND DISCUSSION Physical chemical conditions of the tests. The tests were all conducted under aerobic conditions with an average DO concentration range between 4 and 6 mg/L. The average concentration of suspended solids in the mixed liquor during the three sets of tests is summarized in Table 1. The values at 15 and 25°C were slightly lower than those reported for sewage treatment plants operating with activated sludge (2) and were quite similar to values obtained in other laboratory tests (14,31). The mixed liquor suspended solids (MLSS) concentration in the blank unit was consistently lower than those in the units containing the test chemicals because of the lower input of organic matter. Also, the MLSS concentrations in the AE units were higher than in those with LAS at the three tested temperatures. The MLSS concentration increased at lower temperatures, and in most cases it approximately tripled when changing from 25 (0.7 g/L) to 9°C (2 g/L). The same effect was observed in both the blank and in the tested surfactant units. These results are in good agreement with previous observations in activated sludge reactors (31) when switching from 20 to 8°C. The use of mathematical models suggests that the rapid decrease in the hydrolysis rate with temperature is the primary explanation for this observation. This phenomenon allows the accumulation of organic particulate matter in the

TABLE 1 Averaged Mixed Liquor Suspended Solids (MLSS) Concentration and Dissolved Organic Carbon (DOC) Elimination During the Test Periods at 25, 15, and 9°C for Both Experimentsa T (°C)

Unit

n

MLSS (g/L)

DOC removal (%)

25 25 25 25 25

Unit 1 (LAS) Unit 2 (LAS) Unit 3 (AE) Unit 4 (AE) Unit 5 (blank)

10 10 9 10 10

0.58 ± 0.17 0.85 ± 0.14 0.93 ± 0.15 0.95 ± 0.09 0.42 ± 0.15

97.8 ± 1.6 97.71 ± 1.54 96.28 ± 1.58 98.32 ± 1.45

15 15 15 15 15


8 11 11 10 10

0.46 ± 0.05 0.60 ± 0.09 1.69 ± 0.13 1.14 ± 0.15 0.65 ± 0.12

93.8 ± 3.1 93.8 ± 2.5 97.8 ± 2.1 97.9 ± 1.5

9 9 9 9 9


20 20 20 20 20

1.96 ± 0.43 2.05 ± 0.53 3.56 ± 0.33 2.70 ± 0.30 2.57 ± 0.34

94.66 ± 2.4 95.84 ± 1.5 96.12 ± 2.62 95.36 ± 2.52

a

n = Number of samples tested. LAS, linear alkylbenzene sulfonate; AE, alcohol ethoxylate.

mixed liquor and a resulting increase of volatile suspended solids under cold-weather conditions. General microbiological conditions of the test. The bacterial concentration during the acclimation period showed a considerable increase, corresponding to a better removal efficiency of the system. The overall bacterial population during the whole experiment showed only small variations not affected by changes in temperature. This evolution is in agreement with the observed degradation efficiency. An alteration of the microbial population (a decrease in diversity and different species) was detected in unit 2 of experiment II, and a new inoculum was used. The surfactant concentration in the influent liquor (10 mg/L) was, in all cases, below the reported LAS NOEC (nonobserved effect concentration) in sewage treatment plants (15), guaranteeing a lack of inhibition of biota when adding the tested surfactant. In the case of the LAS tests, the average populations, expressed as colony-forming units (cfu), were 107, 5 × 107, and 3 × 106 cfu/mL at 25, 15, and 9°C, respectively. When AE was tested, the concentrations were 5 × 106, 5 × 106, and 106 cfu/mL, respectively, at the same temperatures. In all cases, Pseudomonas was the most abundant genus during the degradation tests. General treatment efficiency (DOC). The operating conditions of the Husmann units were assessed based on the removal of DOC in the biodegradation units. For example, Figure 2 shows the DOC removal at the three tested temperatures during the biodegradation tests for LAS and AE using units 2 and 4. In all cases, during the tests (experiment II) where longer acclimation times were used to allow better acclimation of microbiota at low temperatures, the DOC removal was higher than 90%, as shown in Table 1. These results indicate that the operating conditions of the units were good, according to ISO recommendations (32), and were similar to those reported by other authors during AE biodegradation at 20°C JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

72 D. PRATS ET AL.

FIG. 3. Primary degradation of LAS (%), expressed as MBAS at 25°C for experiment I. For abbreviations see Figure 1.

FIG. 2. Dissolved organic carbon (DOC) removal during LAS and alcohol ethoxylate (AE) degradation in a continuous activated sludge plant. For other abbreviation see Figure 1.

(14). Also, the DOC removal levels were slightly higher than in our first set of experiments of shorter duration (30 d) performed according to OECD guidelines. LAS primary biodegradation: nonspecific method (MBAS). The LAS removal efficiency reached a maximum after the acclimation period, as shown in Figure 3, after which there was no significant change in LAS removal during the test phase. During the acclimation period, a special growth of microorganisms capable of degrading LAS took place, until the critical biomass required to efficiently degrade this compound was present. The results of the LAS primary biodegradation, expressed as MBAS removal, are summarized in Table 2. In all cases, the removal was higher than 90%, and there was no significant influence of operating temperature on the final removal of the surfactant. In some cases, there were minor differences JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

between the surfactant removal of duplicates, because the degradation activity of microorganisms was slightly more efficient in some cases (e.g., unit 2 at 9°C), but there were no significant differences in the experimental conditions or design that could explain this result. The efficiency of reactor 2 in eliminating LAS was reduced significantly at 25°C (experiment II). This effect was probably a consequence of the aging of the sludge, as the reduction in the diversity of the microbial populations indicated. For this reason, we decided to reinoculate all the units to start the test at 15°C. A period of adaptation of the new inoculum to the test conditions was allowed before introducing the surfactant. Consequently, the efficiency of reactor 2 at 25°C (experiment II) was replaced by the corresponding data from experiment I in Table 2. The decrease in temperature had an effect in the acclimation periods of the Husmann units, which were 10, 20, and 40 d at 25, 15, and 9°C, respectively. Decreasing the temperature reduced the velocity of all the metabolic processes; consequently, a longer period was needed to reach the necessary biomass concentration. However, this effect is unlikely to occur in WWTP operating continuously except during start-up operations. The effect of temperature also has been observed in discontinuous feed reactors using seawater (33), where the reduction in temperature was reflected in the acclimation period, although it did not affect the final degree of LAS removal. TABLE 2 LAS Primary Biodegradation Determined as Methylene-Blue Active Substances During the Test Periods at 25, 15, and 9°C for Both Experimentsa T (°C) 25 25 15 15 9 9 a

Unit

Removal (%)

n

Unit 1 Unit 2 Unit 1 Unit 2 Unit 1 Unit 2

97.8 ± 1.7 92.8 ± 2.1b 87.8 ± 3.2 93.5 ± 2.4 91.0 ± 4.1 96.7 ± 0.1

10 10 15 15 20 20

n = Number of samples tested. Data from experiment I (reactor destabilized in experiment II). For abbreviation see Table 1.

b


FIG. 4. Primary degradation of LAS at 25, 15, and 9°C for experiment II, determined by a specific method (HPLC). For abbreviations see Figure 1.

FIG. 5. Evolution of AE removal (measured as BiAS) at 25, 15, and 9°C for experiment II. For abbreviations see Figures 1 and 2.

LAS primary biodegradation: specific analysis (HPLC). The evolution of LAS primary biodegradation determined by specific analysis (HPLC) at 25, 15, and 9°C is shown in Figure 4. Degradation was higher than 90% in all cases and at all temperatures. Consequently, it was apparent that the operating temperature of biodegradation units in the range of 9 to 25°C had no significant effect on the final removal of LAS. The results obtained for LAS removal were similar for the nonspecific and the specific analytical methods. The average concentration of LAS in the effluent was less than 0.6 mg/L in the dissolved phase (except for unit 2 at TABLE 3 Primary Biodegradation of LAS Determined by High-Performance Liquid Chromatography During the Test Periods at 25, 15, and 9°C Indicating the Percentage of Initial LAS Present in the Effluent That Is Associated with Particulate Materiala T (ºC) 25 25 15 15 9 9 a

Unit

n

Particulate LAS (%)

Primary degradation (%)


10 10 16 16 20 20

3.5 4.0 4.3 4.1 3.4 3.7

93.6 ± 0.9 95.5 ± 1.2b 91.7 ± 1.2 92.3 ± 1.4 92.9 ± 1.6 95.0 ± 1.6

n = Number of samples tested. Data from experiment I (reactor destabilized in experiment II). For abbreviation see Table 1.

b

25°C, which destabilized at the end of the experiment), and varied between 0.3 and 0.7 mg/L in the solids in suspension (Table 3). Therefore, the average elimination of LAS was always greater that 90%, even in the most adverse climatic conditions (9°C). The proportion of LAS associated with the particulate material was between 25 and 55% of the LAS present in the effluent, corresponding to between 3 and 4.5% of the total amount of LAS introduced into the system (Table 3). LAS homologs with longer alkyl chains exhibited a higher sorption capacity on sludge than the rest, as other authors have previously noted (16). The average distributions of LAS homologs on the suspended solids were 6.4 ± 3.5, 20.7 ± 3.4, 29.7 ± 2.9, and 43.2 ± 4.4 for C10LAS to C13LAS, respectively, and were similar for all tested temperatures, as shown by the low standard deviations. There was a preferential degradation of long-chain LAS homologs at the three tested temperatures. As an example, in the 9°C biodegradation test, the distributions of LAS homologs in the influent were 12.1, 34.1, 30.6, and 23.2% for C10LAS to C13LAS, respectively, and 32, 33, 24, and 11% for C10LAS to C13LAS, respectively, in the effluent. These results are in agreement with those obtained previously in real treatment plants and in CAS units (18,34). AE primary biodegradation: nonspecific method (BiAS). Figure 5 represents the results at 25, 15, and 9°C according to the BiAS method. The biodegradation of AE was, in all cases, JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006

74 D. PRATS ET AL. TABLE 4 Averaged Primary Degradation of AE (measured as bismuth active substances) During the Test Periods as a Function of Temperaturea T (ºC) 25 25 15 15 9 9

Unit

Removal (%)

n


94.1 ± 2.3 98.4 ± 0.9 98.4 ± 0.7 98.7 ± 0.6 99.0 ± 0.4 99.1 ± 0.3

10 10 16 16 20 20

a

n = Number of samples tested. For abbreviation see Table 1.

higher than 90%, and the acclimation period was shorter than with LAS. As was explained previously for LAS, in some cases there were minor differences between the average removal of duplicates, probably because the degradation activity of microorganisms was more efficient (e.g., unit 4 at 25°C). The effect of temperature on the acclimation period of tests with AE was also less remarkable than with LAS. The average biodegradation results obtained in all tests are summarized in Table 4. As was found with LAS, the operating temperature of biodegradation units in the range of 9 to 25°C had no effect on the final removal of AE. The proportion of AE associated with the particulate material in the effluent was typically less than 0.8% of the total amount of AE introduced into the system. In general, the behavior in both test runs was similar for LAS and AE. The removal of both surfactants tested, LAS and AE, was higher than 90% at 25, 15, and 9°C. We observed that longer acclimation periods were needed by the microorganisms at lower temperatures (winter periods or high-latitude areas).

ACKNOWLEDGMENTS Support for this research was provided by the Spanish Inter-Ministerial Science and Technology Commission, project REN 20010754, “Optimization of the Process of Sludge Compostage: Reduction of LAS and Pathogenic Microorganisms”; and a project with ECOSOL (CEFIC, ECOSOL/1-02I), “Study of the Degradation of LAS and Non-ionic Surfactants at Low Temperature.”

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Daniel Prats received his Ph.D. in industrial chemistry from the University of Madrid (Spain) in 1981. At present, he is a professor in the Chemical Engineering Department at the University of Alicante (Spain) and is director of the Institute of Water and Environmental Sciences (University of Alicante). Carmen M. López graduated with a degree in chemistry from the University of Almería (Spain) in 1999, and received her M.S. degree in water management and treatment from the University of Alicante (Spain) in 2001. From 2002 to 2003, she collaborated with the Department of Chemical Engineering and with the Institute of Water and Environmental Sciences of the University of Alicante on several research projects. Diana Vallejo graduated with a degree in microbiology from the University of Los Andes (Colombia) in 2000, and received her M.S. degree in water management and treatment from the University of Alicante (Spain) in 2001. From 2002 to 2003, she collaborated with the Department of Chemical Engineering of the University of Alicante on several research projects. Pedro Varó holds a Ph.D. in chemical engineering from the University of Alicante (Spain). He worked in environmental health in the Valencian Health Service (1989–1994) and in the Environmental Service in the Valencian community (1995–2001), and was appointed technical director of the Health Public Laboratory of Alcoi. He has been a professor in the Chemical Engineering Department at the University of Alicante since 1997. Víctor M. León holds a Ph.D. in chemistry (University of Cádiz, Spain). He was awarded an FPI scholarship (1996–2000) from the University of Cádiz (Spain). From 2000 to 2001 he was an associate professor in the Physical Chemistry Department of the same university. At present, he is a professor in the Chemical Engineering Department at the University of Alicante (Spain).

[Received March 14, 2005; accepted September 12, 2005]

JOURNAL OF SURFACTANTS AND DETERGENTS, VOL. 9, QTR. 1, 2006