Effect of temperature and organic nutrients on the biodegradation of linear alkylbenzene sulfonate (LAS) during the composting of anaerobically digested sludge from a wastewater treatment plant

Waste Management 26 (2006) 1237–1245 www.elsevier.com/locate/wasman

Effect of temperature and organic nutrients on the biodegradation of linear alkylbenzene sulfonate (LAS) during the composting of anaerobically digested sludge from a wastewater treatment plant E. Sanz a, D. Prats a, M. Rodrı´guez

b,*

, A. Camacho

c

a Institute of Water and Environmental Science, University of Alicante, Spain Department of Chemical Engineering, University of Alicante, P.O. Box 99, 03080 Alicante, Spain Department of Microbiology and Ecology & Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Spain b

c

Accepted 28 September 2005 Available online 18 November 2005

Abstract Limits on the application of biosolids (anaerobically processed sludges from wastewater treatment plants) as fertilizers for the amendment of soil are becoming greater because of the accumulation of recalcitrant substances, making necessary the use of techniques that bring the concentration of xenobiotics to lower concentrations than those permitted. In general, the biosolids composting process is sufficient to reduce the usual concentration of linear alkylbenzene sulfonates (LAS) to low levels. In this work, an assessment is made on the effect of temperature in the capacity of enriched bacterial populations to biodegrade LAS, together with the influence that the available nutrients may have in the biodegradation of these compounds. The results show that the microbial metabolism of LAS was not observed in the thermophilic range. The optimum temperature for the biodegradation of LAS appears to be around 40 °C, this is, the lowest assayed here, and at this temperature the differences in the biodegradation of LAS among the nutritionally supplemented cultures are small. Ó 2005 Elsevier Ltd. All rights reserved.

1. Introduction Surfactants are an important source of chemical compounds from artificial origin that are liable to contaminate different ecosystems. The production of surfactants was around 18,000 tonnes in 1998, corresponding mainly to soap (50%); followed by linear alkylbenzene sulfonates (LAS), derivatives of alcohols, and the remaining percentage comprised other kinds of surfactants. Among the total amount of surfactants, 23% was consumed in Europe, 28% in the USA and Canada, 32% in Asia, 9% in the South American countries and the remaining 8% in other regions (Colin A. Houston Associates, 2000). The commercial product generally used as LAS is a mixture of different alkyl homologues (C10–C14) and different *

Corresponding author. Fax: +34 96 590 3826. E-mail address: [email protected] (M. Rodrı´guez).

0956-053X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.wasman.2005.09.016

isomers with a different position of the benzene group (Fig. 1). Experiments in the laboratory and in natural conditions indicate that LAS degrades at high rates in aerobic environments (Swisher, 1987). On the other hand, the elimination of 17–60% of LAS may occur when wastewater flows through the sewage system (Berna et al., 1998). In wastewater treatment plants, 10% or more of the LAS present in wastewater (Giger et al., 1989) is eliminated by adsorption/precipitation processes, together with suspended solids during the primary treatment, while the rest is almost completely biodegraded in the aerobic stage of the treatment process. Nevertheless, as a consequence of the low biodegradability of LAS under anaerobic conditions, the sludge that is stabilized by anaerobic processes shows concentrations of LAS as high as 3–30 mg/g of dry solid (Matthijs and De Henau, 1987; Ruiz et al., 1989; Prats et al., 1993).

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E. Sanz et al. / Waste Management 26 (2006) 1237–1245

CH 3 - [ CH 2 ] m -CH- [ CH 2 ] n -CH 3

n+m = 6 - 12 SO 3 Na

ZnSO4 Æ 7H2O, 0.31 g/l; Na2MoO4 Æ 2H2O, 0.03 g/l) according to Ellis et al. (2002). The organic compounds used to study the effect on its amendment on the degradation of LAS were peptone (Merck), yeast extract (Merck), and triptone soya broth (Oxoid), all of them of microbiological quality, as well as D-glucose (Panreac).

Fig. 1. Molecular structure of LAS.

2.2. The composting tunnel The biosolids generated during the anaerobic treatment of wastewater are problematic due to their low stability (high concentration of easily biodegradable compounds), concentration of pathogenic agents and concentration of xenobiotic contaminants, including LAS. On the other hand, the composting process is a biological aerobic treatment that is considered to be one of the biotechnological options for the elimination of undesirable compounds. When applying this biotechnological process to the biosolids produced in a wastewater treatment plant, the sludge is converted into a stable organic product and is suitable for agricultural use as a fertilizer and for soil amendment (Moreno-Ortego, 1997). The final product, which is named compost due to the high concentration of organic material, develops within a combination of mesophilic and thermophilic processes. In the composting process, the microbial community present in the sludge causes the degradation of the organic material. The microbial metabolism is limited by high temperatures (Neidhardt, 1989). In our work, the effect of temperature on the degradation of LAS was studied. To carry out this study, the thermal profile of the composting tunnel at the wastewater treatment plant (WWTP) in Aspe (Alicante) was first determined. Then, a series of experiments was designed that, by means of the Microbial Enrichment technique, attempts to evaluate the biodegradation activity on LAS. The experiments were performed with the microbial populations thermally acclimatized during the industrial process. In these experiments, the effect of the addition of nutrients on this degradation was also studied. 2. Materials and methods 2.1. Chemicals and culture media LAS was supplied by Petroquı´mica Espan˜ola S.A. This is a commercial mixture Na-LAS (P-550), formed by a combination of isomers C10 (12.1%), C11 (34.1%), C12 (30.6%), C13 (23.2%), with a molecular weight of 342.6 and a concentration of 15.3% (w/w). In the enrichment tests, a mineral basal medium was used, comprising inorganic compounds (K2HPO4, 3.5 g/l; KH2PO4, 1.5 g/l; NH4Cl, 0.5 g/l; NaCl, 0.5 g/l; Na2SO4, 0.14 g/l; MgCl2 Æ 6H2O, 0.15 g/l), to which an oligoelement solution was added to a concentration of 0.1% (v/v) (prepared with FeCl3 Æ 6H2O, 0.24 g/l; CoCl2 Æ 6H2O, 0.04 g/l; CuSO4 Æ 5H2O, 0.06 g/l; MnCl2 Æ 4H2O, 0.03 g/l;

The samples of biosolids used in this work were collected in Aspe (Alicante, Spain), at the composting plant of biosolids, which is located in the same enclosure as the wastewater treatment plant (WWTP). This plant processes sludge generated in its WWTP, as well as those from the nearby WWTP of Monte Orgegia (Alicante). In our study, only biosolids from the Monte Orgegia (Alicante) WWTP were used. These biosolids are characterized by a greater concentration of LAS than the Aspe biosolids, as a result of the anaerobic stabilizing treatment process to which the Monte Orgegia biosolids are submitted, since the biosolids of the Aspe WWTP are submitted to an aerobic stabilization process resulting in a higher degree of LAS biodegradation. LAS is only mineralized in aerobic conditions (Scho¨berl, 1989; Cook, 1998). The characterization of sludges from Monte Orgegia and Aspe is shown in Table 1, in which the great difference in the concentration of LAS can be observed (Prats et al., 1993). The experiment only used sludge from Monte Orgegia in WWTP composted in Aspe, in order to work with the most problematic sludge containing higher LAS concentrations. The final product of this industrial process is a compost with a minimal amount of LAS (Ruiz et al., 1989). The system used in the plant for sludge composting is based on a combination of parallel tunnels, each one of which can process one type or a mixture of different biosolids. Each tunnel in this facility is 3 m wide, 2 m high and 75 m long. The aeration is based on three air turbines with a capacity of 1.7 m3; which work 1 h/day, 5 days per week. The material composted in the composting facility is a mixture (1:1 w/w) of sewage sludge with bulking materials (sawdust and straw in a proportion of 3:1 v/v) which also serve to adjust the C to N ratio and moisture content, reducing the excess wet in sludges (almost 80% moisture content). During the process, homogenization of the mixture is done by a compost turner (Volteco, model 2030), Table 1 Characterization of the biosolids from both WWTPs to be composted in the biosolids composting plant

Density (kg/l) Total solids (g dry solids/100 g wet sludge) Volatile solids (g dry solids/100 g dry sludge) Organic carbon (g C/100 g dry sludge) Inorganic carbon (g C/100 g dry sludge) LAS (mg/g dry sludge)

Monte Orgegia

Aspe

0.96 21.5 62.5 36.0 13.25 67.9

1.10 16.7 65.4 32.6 <0.5 0.58

E. Sanz et al. / Waste Management 26 (2006) 1237–1245

which moves from one end of the tunnel to the other, thus allowing the mass to advance throughout the length of the tunnel (4 m/flip) three times a week by a plug-flow system. The capacity is 8 tons per load, three times a week. Residence time is 40 days. The compost production is almost 3400 kg/day.

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and 40, 45, 47 and 50 °C for the zone at 2/3 of the tunnel (with lower temperature). These temperatures represent moderately high thermophilic conditions (1/3 of the tunnel), and the medium-low thermophilic conditions (2/3 of the tunnel). 2.4. Analytical and experimental procedure

2.3. Sampling in the composting tunnel and determination of the temperature profile The temperature along the composting tunnel at the composting plant in Aspe was monitored. This parameter, apart from being used to control the process, is also useful for the occasional activation of the aeration valves in the event that temperature rises above 70 °C. The activation of the aeration valves creates vertical thermal differences along the vertical profile of the composting material. The temperature of 70 °C is that which should be attained and maintained to obtain an adequate final product. Measurement of the temperature in the composting tunnel was carried out several times and in several places at a depth of 1 m, after 1 day without mixing, to allow the normal temperature profiles of the process to stabilize. Furthermore, during mixing, water was added to compensate the loss by evaporation, this process introducing thermal variations in the composting material which are not associated with the self thermal evolution of the process. Fig. 2 shows the temperature profile versus the distance from the beginning of the tunnel. In this figure two thermally differentiated zones appear (the distances representative of these zones are 1/3 and 2/3 of the total tunnel length) both belonging to the thermophilic range, with an average temperature of 57 °C along the tunnel. Compost samples used in the different enrichment tests were taken at 1/3 and 2/3 of the tunnel length. Samples were obtained at different depths, representatives of the temperatures to be studied of 65 and 55 °C for the zone at 1/3 of the tunnel (with higher average temperature)

70 65 60

Temperature (˚ C)

55 50 45 40 35 30 25

Experiments on the biodegradation of LAS were carried out by tests performed at different temperatures (40, 45, 47, 50, 55 and 65 °C). For each temperature, each of the tests analyzed contained, if any, only a particular type of nutritional additive (peptone, triptone soy broth, glucose or yeast extract). All of the nutritional substances were tested at each temperature. Microbial growth of the different biological liquid crops was studied by semiquantitative microscopic observations with a Carl Zeiss Axioscop 2 optical microscope, using phase contrast and dark field. The experimental sets were contrasted with negative abiotic controls prepared as their homologues and sterilized at the start of the incubations by humid heat (121 °C, 15 min). The tests were carried out for an initial volume of an aqueous solution of 100 ml prepared in 250 ml Erlenmeyer flasks, with an initial concentration of 20 mg/l of LAS. Liquid cultures were introduced into flasks and shaken for the homogenization of LAS, and incubated in darkness at the different test temperatures. The biodegradation assays were carried out in aqueous matrices with a ratio between the liquid solution and the sewage biosolids of 100:1. The concentration of LAS and the microbial growth were measured daily. The concentration of LAS was determined using a high performance liquid chromatograph (Agilent 1100 Series) with a UV detector at 225 nm. As stationary phase, a LiChrosorb RP-18 5 lm column was used, with a mobile phase consisting of a solution of methanol/water (80/20, 0.5 M in NaClO4), with a flow of 1 ml/min. Calibration with LAS standards were done periodically in distilled water with the following concentrations: 0, 2.5, 5, 10, 15, 20 and 25 mg/l. Microbial growth was measured semiquantitatively by optical microscopy in the 1R enrichments, while in the 2R enrichments these measurements were carried out spectrophotometrically by measuring the optical density at 600 nm. With respect to the calculation of the kinetic parameters, no lag phase was detected for the biodegradation of LAS, or if so, it was very short (less than 24 h). A first order kinetic model was used to predict the rate of LAS biodegradation as C ¼ C 0 expðK  tÞ, in which C0 is the initial concentration and K is the first order kinetic constant.

20

2.5. Microbial enrichment method

15 0

10

20

30

40

50

60

70

80

Distance (m)

Fig. 2. Thermal profile of the composting tunnel from which samples for the assays were obtained.

In order to characterize the capacity of the microbial communities living in the composting material to aerobically biodegrade LAS at different temperatures, a micro-

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E. Sanz et al. / Waste Management 26 (2006) 1237–1245

bial enrichment method was followed. This method is a traditional microbiological technique (Madigan et al., 1999). The method consisted of preparing an inoculum from composting material sampled from 1/3 or 2/3 of the tunnel length (starting with 1 g dry weight) that was diluted and transferred into fresh culture broth (5% dilution for the second enrichment). As many reinoculations were done as batches to be inoculated in the following microbial enrichment step. The culture broth consisted of complete mineral medium (mineral basal medium plus oligoelement solution) and LAS. In the first enrichments (1R), the incubation lasted for 24 h. This first-step enrichment enabled the micro-organisms contained in the composting material to adapt to the liquid phase, as well to spread them and generate a homogeneous nutrient solution. The second enrichment sets (2R) lasted for 14 days, during which measurements of the LAS concentration and population density were made. Both, the first and the second enrichment had the same amount of LAS (20 mg/l). In a combination of tests under the same conditions as those described previously, we also evaluated the effect of the presence, in the second enrichment, of complex organic compounds (Konopka et al., 1999), particularly of peptone (to obtain a wide spectrum of peptides of different molecular weights), yeast extract (as a good source of amino acids and vitamins), triptone soy broth (with a high content of carbohydrate), and glucose (as a source of carbon and energy widely used by micro-organisms). This modification of the culture medium was done by the addition of 500 mg/l of one of the compounds to one of the sets in the second re-inoculation (2R), in such a way that each batch contained only one compound of those above mentioned, with an additional set of inoculum without nutritional addition as biotic controls. The addition of nutritional supplements was carried out to check whether the addition of any one nutritional supplement could favour or activate the biodegradation of LAS. 2.6. Experiments to check the degradation of LAS under different conditions The removal of the LAS in sewage sludge under mesophilic temperatures is well known, and sufficient bibliography exists in this respect that supports its biodegradation even up to the mineralization in aerobic conditions (Scho¨berl, 1989; Cook, 1998). However, biodegradation of LAS at high temperatures, such as those to be found in industrial processes such as the treatment of biosolids from the WWTP, has not been well studied. Consequently, this study aims to develop the knowledge on the biodegradation of LAS in the thermophilic range. Studies with thermophilic bacteria have shown that these have greater nutritional requirements for the biodegradation of degradable compounds, which may be accomplished by the addition to the culture medium of organic

compounds such as peptone or yeast extract (Konopka et al., 1999; Lapara and Alleman, 1999). In our work, three types of tests were done with samples from the composting plant in order to evaluate the thermal effect on the biodegradation of LAS under thermophilic (65 °C) to mesophilic (40 °C) conditions. The first series of tests (Group I) were based on simple microbial enrichments (without nutritional additions) in which a dilution effect of nutrients occurred. The second series of tests (Group II) was based on nutritionally supplemented microbial enrichments, as an alternative for the correction of the effect of dilution of the nutrients, typical of the microbial enrichment technique employed. The third series of tests (Group III) consisted in some nutritionally supplemented microbial enrichments with reduction of temperature during the incubation process (47 ! 45 ! 40 °C). In Group I, the tests where only microbial enrichment was carried out (Fig. 3), the assay started from a series of inocula from samples obtained at 1/3 and 2/3 of the composting tunnel length. In the first enrichment (1R), these cultures were incubated at temperatures mimicking those of the sampling site (65 and 55 °C for inocula from 1/3, and at 50, 47 and 45 °C for those collected at 2/3 of the tunnel length). In the second enrichment (2R), the respective temperatures of 1R were maintained except for the set of 1R at 45 °C, which was divided into 2 batches at the beginning of the incubation, one of which was incubated at the same temperature (45 °C) whereas the temperature of incubation for the second one was 40 °C. The number of samples taken for the incubations at 65, 55 and 50 °C was 10 in 1R. From each one of these series of inoculations, 4 sub-series were generated for 2R. One of the quadruples of 2R (corresponding to an inoculum for the first enrichment, 1R) was sterilized as abiotic control. The number of samples taken for the incubations at 47 °C was 8 in 1R and 4 samples for 45 °C. From each one of these series of inoculations in 1R, 2 sub-series were generated for 2R. One of the duplicates of 2R was sterilized as abiotic control. The combination of tests by nutritionally supplemented microbial enrichment (Group II, Fig. 3) only differs from Group I by the addition of organic nutrients in phase 2R. The experimental development was the same as for the former. The number of replicates obtained in 1R was lower than in Group I tests, because the diversification due to the nutrient amendment generated a greater number of tests in phase 2R. The number of batch replicates in phase 1R was 3 for each temperature of incubation (65, 55, 50 and 47 °C). From these, 6 sub-samples were obtained from each one, to generate the 6 incubation conditions for phase 2R (four treatments with the 4 different nutrient additions, one without nutrient amendment and an abiotic control which was sterilized prior to incubation). For the temperature of 45 °C the assays started with only 1 sample incubated in 1R, which resulted in a subdivision into 12 sub-samples in 2R, 6 of which were incubated at 45 °C and the rest at 40 °C, following the same pattern of

E. Sanz et al. / Waste Management 26 (2006) 1237–1245

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Fig. 3. Scheme of the experimental design.

nutrient addition described above for tests incubated at higher temperatures. The combination of tests of nutritionally supplemented microbial enrichment and temperature reduction or Group III of experiments (Fig. 3) comprises of 2 types of tests (IIIA and III-B) that followed the general test scheme as Group II. Both tests started from 1 replicate prepared from samples taken at 2/3 of the composting tunnel length at 47 °C. The incubation temperature at phase 1R was 47 °C. After 1R, this was divided into 6 sub-samples in 2R, to which the various nutritional compounds were added when necessary, resulting in the same experimental design as for phase 2R for Group II. Sub-samples of Group III-A were incubated for 3 days at 47 °C, after that for 3 days at 45 °C, and finally for 4 days at 40 °C. Subsamples of Group III-B were incubated for 7 days at 47 °C, after which each one of the samples was subdivided and each batch brought directly to 45 or 40 °C for 5 more days; those which followed further incubation at 45 °C were amended again with the corresponding nutrients after 2 days of restarting the incubation at 45 °C.

3. Results and discussion The concentration of LAS in the experiments of Groups I and II incubated at 65, 55, 50 and 47 °C in 2R showed no significant variation after 14 days, which means that the samples did not show substantial surfactant biodegradation after incubation. As an example, Fig. 4 shows the results of the incubations at 65 °C of Group I. The rest of the assays at thermophilic temperatures presented a similar pattern than those incubated at 65 °C, without LAS degradation (data not shown). No significant differences were found among the different conditions of incubations (nutrient amendment) for Group II of assays, except that the log phase, as well as both the stationary and dead phases, were shorter in the nutrient amended assays (data not shown). These results concerning incubation at thermophilic temperatures indicate that these are not suitable for the biodegradation of LAS, at least when using the microbial growth method tested. In this method of bacterial growth, we considered the dilution factor of the nutrients characteristic of

E. Sanz et al. / Waste Management 26 (2006) 1237–1245 22 20

LAS concentration (mg/l)

18

Inoculum at 65 °C Inoculum at 45 °C Inoculum at 40 °C Abiotic control

16 14 12 10 8 6 4 2 0 0

2

4

6

8

10

12

14

16

Time (days)

Fig. 4. Time course of LAS concentration for Group I of experiments. Mean values are shown and bars show maximal and minimal values.

the method itself (in the tests with Group I), as well as the addition of nutritional supplements (in Group II). These results contrast with those obtained by other researchers (Konopka et al., 1999) who obtained significant biodegradation of LAS at 53 and 62 °C, when inoculating active sludge on synthetic wastewater and operating with continuous flow bioreactors. These authors did, however, use the measurement of microbial respiration as indicative of the biodegradation of LAS, which does not necessarily imply that LAS biodegradation was occurring, whereas in our study we directly determined LAS biodegradation by directly measuring its concentrations by HPLC, which gives confidence to the fact that LAS was not degraded in our case. The content of suspended solids in the experimental erlenmeyer flask in the assays of second enrichment (2R) was very low, because the first enrichment served to mix and resuspend nutrients and micro-organisms from the sampled inoculum in the composting tunnel. The amount of LAS able to pass to the second enrichment adsorbed/ precipitated with the solids in suspension was consequently low, as shown by the initial concentration values of LAS in these crops (2R), which were very close to the amount of 20 mg/l added to each batch. The microbial enrichment technique used here, a traditional microbiological technique (Denger et al., 1997a,b) is used here as previous assays for more sophisticated assays (molecular procedures), which would then be used once evidence occurs of the existence of microbial populations that are capable of development at the expense of the studied compounds and under particular environmental conditions (Wang and Barlaz, 1998; Schleheck et al., 2000; Ellis et al., 2002). The results obtained at incubation temperatures of 45 and 40 °C within Group I of assays showed that, contrastingly, biodegradation of LAS took place at these temperatures. The consumption of LAS was high at 45 °C (average 77.5% in the reduction of LAS concentration);

although a higher removal percentage was achieved at 40 °C. At 45 °C, certain differences were found among the different assays as represented by the deviation bars for mean values in Fig. 4; this variability was not so marked at 40 °C due to the rapidity and high percentage of surfactant biodegradation. The results obtained both at 45 and 40 °C within Group II of assays also showed biodegradation of LAS in contrast to the rest of the temperatures tested with the same group of experiments. At 45 °C, assays without nutrient amendment (blank, with no nutritional additive) showed less biodegradation of LAS than the rest of the assays that were supplemented with nutritional additives (Fig. 5). In the tests carried out at 40 °C, we found an initial delay in the biodegradation of LAS in the non-amended tests compared to the rest of incubations (Fig. 5). Here, microscopic observations showed a higher bacterial population density in all batches with nutritional additives compared to those without additives. When comparing the final LAS concentrations in the Group I of the experiments (Table 2) in those cases where biodegradation of LAS took place (45 and 40 °C), a substantial increase in the rate of consumption of LAS was observed with the reduction of the incubation temperature from 45 to 40 °C. In Group II of the assays (Table 3) a huge increase of the consumption of LAS was also found when incubating at 40 °C compared to incubations at 45 °C, regardless of nutrient addition; nutrient amendment, however, almost duplicated the rates of biodegradation of LAS compared to those not supplemented. In the 2R tests belonging of Group III, neither in III-A (Fig. 6) nor in III-B (Fig. 7), was LAS biodegradation observed throughout the incubation at 47 °C, despite that microbial growth was confirmed by microscopic observations. Subsequent reduction in the incubation temperatures

22

Blank 45°C Glucose 45°C Soya 45°C Yeast 45°C Peptone 45°C Blank 40°C Glucose 40°C Soya 40°C Yeast 40°C Peptone 40°C

20 18

LAS concentration (mg/l)

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16 14 12 10 8 6 4 2 0 0

1

2

3

4

5

6

7

8

Time (days)

Fig. 5. Time course of LAS concentration in the second re-inoculation (2R) for samples incubated at 45 °C and 40 °C (blank: no addition of nutrients) for the Group II of experiments.

E. Sanz et al. / Waste Management 26 (2006) 1237–1245 Table 2 Kinetic data corresponding to Group 1 of the experiments in the second re-inoculation (2R)

[LAS] initial t (mg/l) [LAS] final (mg/l) K (d1) r2 LAS biodegradation (%)

47 °C

45 °C

40 °C

20

2R 45 °C

2R 40 °C

20 4.5 (2.6–6.2) 0.210 (0.135–0.299) 0.855 (0.734–0.957) 77.5 (69–87)

20 0.3 (0.3–0.3) 2.91 (2.6–3.22) 0.999 (0.999–0.999) 98.5 (98.5–98.5)

Blank Glucose Soya Yeast Peptone

15

LA S ( m g / L )

co

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Mean values are shown (maximum and minimum in brackets) for 45 and 40 °C (k: first order kinetic constant; [LAS] initial tco: initial concentration).

10

5

0 0

2

4

6

8

10

12

Time (days)

Fig. 6. Group III-A of experiments. Time course of LAS concentration in the second re-inoculation (2R) for samples incubated under a temperature sequence of 47 ! 45 ! 40 °C. Arrows show the moment at which the temperature was lowered from 47 to 45 °C and then, from 45 to 40 °C.

45/40 °C

Nutrients

22 20 18 16

LAS (mg/L)

following different patterns resulted in different results. In Group III-A, the reduction to 45 °C allowed the beginning of LAS biodegradation, whilst in Group III-B, additional addition of nutrients was required at 45 °C for the activation of LAS biodegradation or, alternatively, a higher reduction in the temperature to 40 °C. When the consumption of surfactant stopped at 45 °C in Group III-A of experiments, the reduction of the incubation temperature to 40 °C reactivated the consumption of LAS albeit very slowly, but it stopped once more after a short time. The low reactivation velocity at 40 °C could be due to the exhaustion of the medium and to the accumulation of metabolites (as happens in a closed system) that might affect the physiology of the microbiota. This appears to be corroborated by the biodegradation of LAS stopping after a short time, which impedes the total elimination of LAS. Nevertheless, and in comparison with the other tests with nutrient additions, those to which glucose was added showed greater reactivation at 40 °C, although this was lower than at their previous stage at 45 °C. In Group III-B of experiments, only a new addition of nutrients stimulated the biodegradation of LAS, after 2 days of apparent non-consumption of LAS when already transferred to 45 °C, in contrast with tests without nutrient amendment whose rate of consumption of surfactant was negligible at this temperature. From this it may be deduced that at 45 °C the molecules are biodegraded at some point of the microbial community development. On the other hand, no nutritional addition was necessary for the biodegradation of LAS to start for tests transferred to incubation at 40 °C, although they were started with the same conditions of nutritional exhaustion as their homologues at 45 °C. This could indicate that at 40 °C the enzymatic sys-

Blank 40 Glucose 40 Soya 40 Yeast 40 Peptone 40 Blank 50 Glucose 50 Soya 50 Yeast 50 Peptone 50

14 12 10 8 6 4 2 0 0

2

4

6

8

10

12

Time (days)

Fig. 7. Group III-B of experiments. Time course of LAS concentration in the second re-inoculation for samples incubated under a temperature sequence of 47 ! 45 and subsequent nutrient addition, or from 47 ! 40 °C. The first arrow shows the moment in which the temperature was lowered from 47 to 45 °C or to 40 °C; the second arrow shows the moment at which nutrients were added to the set of batches incubated at 45 °C.

Table 3 Kinetic data corresponding to Group II of experiments in the second inoculation (2R) incubated at 45 and 40 °C Test 45 °C

Blank (no additions) +Glucose +Soy broth +Yeast +Peptone

Test 40 °C

C0 (mg/l)

K (d1)

r2

Biodegradation (%)

C0 (mg/l)

K (d1)

r2

Biodegradation (%)

20.84 20.70 19.77 20.11 20.06

0.160 0.222 0.247 0.267 0.212

0.973 0.967 0.966 0.961 0.974

61.5 72.5 74.0 76.5 73.5

19.67 19.93 19.88 19.91 19.93

2.185 3.754 3.544 3.782 3.889

0.976 0.992 0.983 0.987 0.991

98.5 98.5 98.5 98.5 98.5

(C0: initial LAS concentration calculated using the first order kinetics equation.)

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Table 4 Kinetic data for Group III of experiments from the second nutritionally added microbial enrichment incubated at 45 °C and later transferred to 40 °C (k: first order kinetic constant; C0: initial LAS concentration calculated using the first order kinetics equation) Test 45 °C

Blank (no additions) +Glucose +Soy broth +Yeast +Peptone

Test 40 °C

C0 (mg/l)

K (d1)

r2

Biodegradation (%)

C0 (mg/l)

K (d1)

r2

Biodegradation (%)

20.89 20.94 20.40 21.37 20.48

0.355 0.132 0.495 0.827 0.522

0.851 0.698 0.758 0.856 0.745

52.5 27.3 58.2 71.6 60.1

10.16 16.79 8.86 6.12 8.77

0.121 0.595 0.206 0.182 0.264

0.948 0.954 0.945 0.948 0.980

70.6 87.4 80.4 85.6 85.2

Table 5 Kinetic data for the batches incubated at 40 and 45 °C after prior incubation for 1 week at 47 °C (k: first order kinetic constant; C0: initial LAS concentration calculated using the first order kinetics equation) Test 45 °C

Blank (no additions) +Glucose +Soy broth +Yeast +Peptone

Test 40 °C 1

C0 (mg/l)

K (d )

r

Biodegradation (%)

C0 (mg/l)

K (d1)

r2

Biodegradation (%)

20.0 22.1 21.7 22.4 21.9

0 0.076 0.063 0.070 0.062

0 0.950 0.992 0.972 0.997

0 18.7 17.7 17.9 16.9

20.4 20.6 19.7 19.8 20.4

0.230 0.284 0.346 0.318 0.306

0.950 0.973 0.965 0.979 0.978

59.6 69.5 74.5 73.0 71.8

2

tems required to biodegrade LAS are not bound to only one stage of the microbial consortium development. Tables 4 and 5 show the kinetic data for the experimental test Groups III-A y III-B, respectively, omitting the reference to 47 °C because of the inappreciable biodegradation of LAS at this temperature. Once again, a positive effect was produced on the biodegradation of LAS with the addition of nutritional supplements, in the same way as occurred when lowering the temperature of incubation from 45 to 40 °C. In all tests of the Group III of assays the consumption of LAS stopped at LAS concentrations greater than those found for Group II, which did not include pre-incubation at 47 °C. In particular, for Group III-A, an average reduction in the biodegradation of LAS of 25% and 18% at 45 and 40 °C, respectively, was found compared to these tests that were not previously incubated at 47 °C. On the other hand, for Group III-B, an even greater reduction of 75% and 30%, on average, for 45 and 40 °C, respectively, was obtained; furthermore, tests without nutrient amendment from this group were unable of degrading LAS at 45 °C. Therefore, it seems very possible that the thermal difference between 45 and 40 °C, as well as the lesser restrictions to biodegrade LAS at 40 °C as opposed to 45 °C, are an indication that there might be different microbial groups participating in the surfactant biodegrading process in the different conditions assayed. Further investigations on the composition of the microbial consortia responsible for LAS biodegradation at different temperatures are currently being conducted to explore this issue. Comparisons with our own tests shown in this manuscript and those of other researchers indicate that the optimum temperature for LAS biodegradation is around 40 °C, where the rate of consumption of LAS is extremely

rapid, as opposed to higher (45 °C) and lower (30 °C) temperatures (Lapara et al., 2000). 4. Conclusions The thermal profile of a composting tunnel in the WWTP in Aspe (Alicante) has been studied, and as a result it can be confirmed that longitudinally two thermally different zones exist; corresponding to the range of highmoderate thermophilic conditions and to moderate-low thermophilic conditions. By the application of the microbial enrichment technique on thermally acclimatized samples across the industrial process, and because of the maintenance of LAS concentrations at temperatures equal to or above 47 °C, we can conclude that biodegradation of LAS under thermophilic temperatures with the used technique does not occur, even when nutrients are added. In the thermophilic–mesophilic (45 °C) interface, biodegradative activity was observed on the LAS molecule, and greater rates of consumption were found when the culture media contained nutritional supplements. Similarly, incubation at 40 °C rapidly increased the consumption of LAS until the surfactant was totally exhausted, and this process was accelerated when nutrients were added to the culture medium. In spite of our results coming from laboratory studies, LAS can be consumed within the composting tunnel even though that average temperatures are usually on the range of thermophilic conditions as previously stated by other authors (Ruiz et al., 1989; Prats et al., 1993) who have studied the reduction in concentration throughout the process. This biodegradation is easily explained because in the industrial composting process all of the composting material does not suffer thermophilic conditions during all of the time, since the activity of aeration valves, the addition

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of water, as well as the mechanical homogenization of the composting material can reduce the temperature of portions of the material for enough time to allow aerobic degradation at temperatures close to the thermophilic–mesophilic interface. It becomes evident that the structure of the microbial community changes with temperature, thus microbial consortia appearing under thermophilic and mesophilic conditions are likely to be different and consequently different metabolic capacities may be exhibited, which could explain the results presented here. Nevertheless, it is also possible that our results could have shown different biodegradative behaviour of LAS by the same microbial consortia which might change their physiological features with temperature. These issues will be addressed with future studies using molecular techniques to identify the microbiota dominating each of these stages. Acknowledgements The authors are indebted to the personnel from the Aspe wastewater treatment plant for their valuable collaboration. This work was supported by the Spanish Ministry for Science and Technology contract REN 2001-0754/ TECNO. References Berna, J.L., Ferrer, J., Moreno, A., Ruiz, F., Prats, D., 1998. The fate of LAS in the environment. Tenside Surf. Det. 26, 102–107. Colin A. Houston Associates. 2000. Available from: http://www.colinhouston.com/Press_Releases/Press_Releases.htm. Cook, A.M., 1998. Sulfonated surfactants and related compounds: facets of their desulfonation by aerobic and anaerobic bacteria. Tenside Surf. Det. 35, 52–56. Denger, K., Laue, H., Cook, A.M., 1997a. Anaerobic taurine oxidation: a novel reaction by nitrate-reducing Alcaligenes sp. Microbiology 143, 1919–1924. Denger, K., Laue, H., Cook, A.M., 1997b. Thiosulfate as a metabolic product: the bacterial fermentation of taurine. Arch. Microbiol. 168, 297–301. Ellis, A.J., Hales, S.G., Ur-Rehman, N.G.A., White, G.F., 2002. Novel alkylsulfates required for biodegradation of the branched primary

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