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Surfactants are synthetic organic chemicals that are formulated to have cleansing or solubilisation properties. With the development of the industrial economy and increase in population density, surfactants have become one of the most widely disseminated xenobiotics to enter the aquatic environment, creating a serious environmental problem. Their toxicities to organisms have been demonstrated previously. The main objective of this study was to isolate and characterize local bacteria with the potential to degrade Sodium Dodecyl Sulphate (SDS), a widely used anionic surfactant. Screening was carried out by the conventional enrichment culture technique and the isolate was tentatively identified as Pseudomonas aeruginosa sp. using BiologTM GN plates and partial 16S rDNA phylogeny. The optimal growth conditions in minimal medium and for degradation of SDS by Pseudomonas aeruginosa sp. were at 30Â°C and at pH 6.5 using phosphate buffer system. Sodium nitrate; at 8.0gL-1 was found to be the best nitrogen source. The isolated strain exhibited optimum growth at SDS concentration of 1gL-1 but can tolerate up to 14gL-1 SDS, indicating that this isolate was able to survive in a relatively high concentration of SDS. 100% of 1.0gL-1 SDS was completely degraded after 5 and 2 days of incubation before and after optimization respectively.
Keywords: SDS, biodegradation, MBAS assay, Pseudomonas aeruginosa sp.
Anionic surfactants are groups of xenobiotic compounds that contain either sulfonated or ester sulfate groups (Linfield, 1976, Karsa, 1987) which are widely used ingredients in several industrial products such as detergents and cosmetics (Swisher, 1987). Because of their large consumption worldwide, anionic surfactants have the potential for wide disposal into aquatic and terrestrial environments (Schulz et al., 2000)
Large consumption of anionic surfactants in the world together with their adverse effects on living organisms makes these chemical one of the major environmental concerns. Possible toxic effects of anionic surfactants have been studied by many researchers. Lewis (1991) reported that the chronic and sublethal toxicities of surfactants to aquatic animal usually occurs at concentrations greater than 0.1mg/L. Singh et al., (2002) found cytotoxic effects of 7 surfactants with different charges on four fish and two invertebrate species. Similarly, Mori et al., (2002) reported the cytotoxic effects of 8 surfactants of different charges (two cationic surfactants, three anionic surfactants, one amphiphatic surfactant and two non-ionic surfactants) on fathead minnow (FHM)-sp cell line, a cell line in suspension culture from fish.
One of the major xenobiotic anionic surfactants that have large-scale industrial applications and thus broad environmental release is Sodium Dodecyl Sullphate (SDS). It is mainly used in industrial cleaners and household detergents. It is also widely used in other industries as emulsifiers, dissertators, synergists in the pharmaceutical field, as auxiliaries in textile and fibre production, as well as in the plastic, paint, leather, photographic and metal industries (Piorr, 1987). This surfactant is mostly discarded through domestic or industrial wastewater.
Biodegradation of SDS ensures low concentration in the environment. However, under natural conditions, degradation rate of SDS is too slow and inadequate to allow for complete removal of the anionic surfactant. Hence, appreciable amounts of SDS are being detected in both surface and water extracts. The aim of this work was to isolate and to screen SDS-degrading bacteria that are able to degrade high concentrations of SDS rapidly and to optimize various conditions for enhancing the biodegradation of SDS by the selected bacteria. A bacterial isolate which was completely inhibited at 10gL-1 SDS has been reported to degrade 100% of SDS after an incubation period of 10 days. In this work, we report on the isolation and characterization of a local SDS-degrading bacterium that could tolerate up to 14gL-1 SDS and was able to degrade up to 100% of 1g/L SDS in 2 days after optimization of various conditions.
2.0 MATERIALS AND METHODS
2.1 Screening and isolation of SDS-degrading bacteria
Detergent-contaminated samples were collected from several locations in Malaysia such as from car wash outlets, drains, sludges and soil samples. One mL of water samples or 3g of solid samples were inoculated into 10mL sterile SDS minimal medium in a universal bottle. The cultures were incubated at room temperature (28Â°C) on an orbital shaker at 100rpm. SDS minimal medium contained the following (g/L): KH2PO4, 1.36; Na2HPO4, 1.388; KNO3, 0.5g; MgSO4, 0.01; CaCl2, 0.01; (NH4)2SO4, 7.7; SL4, 10mL. SL4 contained (g/L): EDTA, 0.5; FeSO4, 0.2; SL6, 100mL. SL6 contained ZnSO4.7H2O, 0.1g; MnCl2.4H2O, 0.03g; H3BO4, 0.3g; CoCl2.6H2O, 0.2g; CuCl2.2H2O, 0.01g; Na2MoO4.2H2O, 0.03g. The medium was supplemented with filter-sterilized SDS as a carbon source with a final concentration of 1.0 g L-1 (Dhouib et al., 2003). The isolates that were able to survive in minimal medium containing 1g/L SDS were chosen for further screening.
2.2 Determination of residual SDS using Methylene Blue Active Substances (MBAS) assay
The concentrations of residual SDS were determined by a modified procedure of Jurado et al., (2006). Samples were diluted in the range of 0.5mg/L to 2.0mg/L final concentration of SDS using distilled water to a final volume of 5mL for each sample. 200ÂµL of 50mM sodium tetraborate pH 10.5 was added into 5mL of samples followed by the addition of 100 ÂµL of slightly acidic methylene blue reagent. This solution was mixed using a mixer. Four mL of chloroform was added and stirred vigorously for 30seconds and the mixture was let to separate and settle down for 5 minutes. The chloroform layer was separated using separating funnel and the absorbance was read at 650nm using glass cuvette against blank chloroform.
2.3 Genomic DNA isolation and sequencing of 16S rRNA
Extraction of genomic DNA from bacterial cells was carried out using DNeasyR Blood & Tissue Kit supplied by Qiagen according to manufacturers' recommodation. PCR amplification was performed using BiometraR T-gradient thermocycler. The PCR mixture contained 0.5ÂµL of deoxynucleotide triphosphates, 2ÂµL of PCR buffer, 2ÂµL of MgCl2, 0.5ÂµL of 100ÂµM forward and reverse primer, 0.5ÂµL of Taq DNA polymerase, 1ÂµL of template DNA and 43ÂµL of nuclease-free water. The 16S rDNA gene from the genomic DNA was amplified by PCR using as following forward and reverse primers of 16S rDNA respectively; 5'-AGAGTTTGATCATGGCTCAG-3' and 5'-ACGGTTACCTTGTTACGACTT-3'. PCR was performed under the following conditions: initial denaturation at 94 Â°C for 4 seconds, followed by 94 Â°C for 30 seconds, 56 Â°C for 30 seconds, and 2 steps of 34 cycles of 72 Â°C for 4 seconds and a final extension at 72 Â°C for 4 minutes. Bases were compared with the database in GenBank at http://www.ncbi.nlm.nih.gov/BLAST/.
2.4 Phylogenetic analysis
The Phylogenetic position of the SDS degrading bacteria isolated in this study was determined by sequencing analysis of PCR-amplified bacterial small subunit (16S) rRNA gene . The nucleotide sequences from the gene was aligned using CLUSTAL W, version 1.6 (Thompson et al 1994). A multiple alignment of 20 16S rRNA gene sequences that closely matches isolate D were retrieved from the GenBank. Construction of the phylogenetic tree was carried out using PHYLIP, version 3.573 (J. Q. Felsenstein, PHYLIP-phylogeny inference package, version 3.573, Department of Genetics, University of Washington, Seattle, WA. (http://evolution.genetics.washington.edu/phylip.html), with Streptococcus oralis strain CIP 103216 as outgroup in the cladogram. DNADIST algorithm program was used to compute the evolutionary distance matrices for the neighbor joining/UPGMA method. The model of nucleotide substitution is those of LogDet. The neighbour-joining method of Saitou and Nei (1987) was used to deduce the phylogenetic tree. Bootstrap analyses with 1000 resamplings were performed with the SEQBOOT program in the PHYLIP package to obtain confidence estimates for the phylogenetic tree topologies (Felsenstein, 1985). Majority rule (50%) consensus trees were constructed using the CONSENSE program (Margush and McMorris, 1981) and the tree was viewed using TreeView (Page, 1996).
2.5 SDS biodegradation and growth optimization of Pseudomonas aeruginosa sp.
Growth and SDS degradation conditions of the selected isolate were optimized using several parameters. These included the effects of different nitrogen sources, nitrogen source concentrations, pH, and temperature and SDS concentration as a sole source of carbon.
3.0 RESULTS AND DISCUSSIONS
3.1 Screening and isolation of SDS-degrading bacteria
Several enrichments which showed SDS degrading activity were obtained from wash outlets, drains, sludges and soil samples. Four different isolates were chosen based on their ability to grow in BSM containing 1g/L SDS as a sole source of carbon. The degradation of SDS was measured daily using Methylene Blue Active Substances (MBAS) assay. From the MBAS results obtained (Figure 1), isolate D gave the highest degradation with 100% of SDS being degraded after 5 days of incubation compared to the other isolates. Isolates I, E and K showed lower degradation of SDS with only 45%, 32% and 30% degradation respectively after 5 days. Control using uninoculated medium showed no degradation during the same period of time.
Figure 1: Degradation of 1g/L SDS by several isolates. Data represents means Â± SD, n=3
3.2 Identification of the isolate
Identification of isolate D was carried out using several identification techniques which included Gram staining, BiologTM identification system and 16S rRNA analysis. Isolate D was identified as a Gram negative bacterium. Under magnification, the bacterium appears rod shaped and gave positive results with the catalase as well as oxidase tests. Using BiologTM GN2, this isolate was identified as Pseudomonas aeruginosa with 99% of probability after incubation of 24 hours. The results obtained from the BiologTM Identification system showed that isolate SA28a(i) was similar to four genus of Acinetobacter with 0.302 similarity. A moderate bootstrap value (39%) links isolate SA28a(i) to Acinetobacter sp. PRGB16 [EF195346] indicating a moderate phylogenetic relationship (Figure 2). The strain is further grouped to the clade harboring different species of Acinetobacter such as Acinetobacter sp. PRGB 15 [EF195345]. Isolate SA28a(i) is tentatively identified as Acinetobacter sp. strain AQ5NOL 1.
16S rRNA analysis of isolate D also indicated that this isolate was similar to Pseudomonas aeruginosa sp. Previous studies have also shown that Pseudomonas aeruginosa is a good SDS degrader, but few studies have been reported on characterizing this isolate to enhance the biodegradation of SDS. Other surfactant-degrading bacteria that have been reported by previous studies included a consortium of Acinetobacter calcoaceticus and Pantoea agglomerans (Abboud et al., 2007), Citrobacter braakii (Dhouib et al., 2003), Comamonas terrigena strain N3H (Roig et al., 1998), Rhodococcus ruber DSM 44541 (Pogorevc et al., 2002), Saccharomyces carlsbergenesis (Walsh and Malone, 1995), Saccharomyces cerevisiae (Sirisattha et al., 2004) and yeast (Abd-Allah and Srorr, 1998).
Figure 2: A phylogram (neighbour-joining method) showing genetic relationship between Isolate D and other related reference microorganisms based on the 16S rRNA gene sequence analysis. Species names are followed by the accession numbers of their 16S rRNA sequences. The numbers at branching points or nodes refer to bootstrap values, based on 1000 re-samplings. Scale bar represents 100 nucleotide substitutions. Streptococcus oralis as an outgroup.
3.3 Optimization of Growth and SDS biodegradation by Pseudomonas aeruginosa sp.
3.3.1 The effects of SDS concentrations
The effect of SDS as a sole source of carbon was carried out using SDS concentrations ranging from 0g/L to 14g/L (Figure 3). The cellular growth of Pseudomonas aeruginosa sp. increased as the concentration of SDS increased until it reached the optimum concentration at 1.0g/L of SDS. High cellular growth was also observed at 0.8, 1.5 and 2.0g/L SDS. However, there is no significant difference for the growth of Pseudomonas aeruginosa sp. between 0.8g/L SDS and 2.0g/L SDS (p>0.05). The figure also shows that the isolate was able to grow up to concentrations of 14g/L SDS, indicating that this isolate was able to survive in a higher range of SDS concentrations compared to previous studies. Figure 4 shows the degradation of different concentration of SDS. The degradation time increased as the concentration of SDS was increased. The figure also shows that complete degradation of SDS concentration up to 1.0g/L occurred within 5 days. Higher concentrations take longer before being completely degraded. Surfactants have the ability to enhance the cellular permeability (Muller et al., 1999; LaMaza et al., 1991). This characteristic of surfactants will change the microorganisms' cell membrane integrity causing disruption in ion gradients and leakage of essential cell components (Brandt et al., 2001). In addition, anionic surfactants are able to alter the hydrophobicity of the cell membrane (Marchesi et al., 1994) and cause toxic effect to the organisms. SDS also may produce toxic effects on the bacterial cell by unfolding the functional protein. SDS has amphiphilic properties; therefore it is able to bind to the protein via interaction of sulfate group and positively charge amino acid side chains, and between the alkyl chain and hydrophobic side chains, causing protein unfolding (Wang et al., 1996; Yonath et al., 1977). The result from this work shows that this isolate required 1.0 g/L of SDS for optimum growth. Previous study showed that Citrobacter braakii grows optimally at surfactant concentration of 1.0 g/L (Dhouib et al., 2003). In another study, Pseudomonas Strain CL12B grows optimally when supplemented with 0.025 M SDS or 7.2 g/L (Payne and Feisal, 1963). In all cases, growth dramatically decreased at higher concentrations of SDS. Higher concentrations of SDS will unfold the protein by forming cylindrical micelles that are able to wrap themselves around the proteins and bind progressively more tightly in the transition state (Otzen 2002) before the major protein unfolding transition.
Figure 3: The effects of SDS concentration on the growth of Pseudomonas aeruginosa sp. Data represent means Â± SD, n=3.
Figure 4: Degradation of different concentration of SDS by Pseudomonas aeruginosa sp. The degradations were measured using MBAS assay.
3.3.2 The effects of nitrogen sources
Figure 5 shows the effect of different nitrogen sources on the growth of Pseudomonas aeruginosa sp. and their effect on the biodegradation of 1g/L SDS. Sodium nitrate was found to be the best nitrogen source among the nitrogen sources studied. The effect of different concentrations of sodium nitrate was carried out at a range of 0g/L to 9 g/L as shown at figure 6. The results show that Pseudomonas aeruginosa sp. required 8.0 g/L of sodium nitrate to obtain the optimum growth and optimum conditions for degradation of 1g/L SDS. The growth was reduced when the concentrations of the nitrogen source was further increased. In contrast, ammonium sulphate was reported to be the best nitrogen source for SDS degradation by Citrobacter braakii (Dhouib et al., 2003). Comamonas terrigena showed optimum growth with ammonium nitrate as the nitrogen source (Roig et al., 1998). Identification of the best nitrogen source and its optimum concentration for growth would help in designing effective bioremediation strategies for surfactant contamination (Fritsche and Hofrichter, 1999).
Figure 5: The effect of different nitrogen sources on the cellular growth of Pseudomonas aeruginosa sp. and remaining SDS (O). Data represent means Â± SD, n=3
Figure 6: The effect of different concentration of sodium nitrate on the cellular growth (ï‚˜) of Pseudomonas aeruginosa sp., remaining SDS (O) and SDS concentration in abiotic control (â-Š). Data represent means Â± SD, n=3
3.3.3 The effects of pH
The effect of pH on the growth and degradation of SDS by isolate D was studied using an overlapping buffer system consisting of phosphate, borate and carbonate buffer with a pH range of 5.6 to 10.0 (figure 7 and 8). The optimum pH for growth and degradation of SDS by Pseudomonas aeruginosa sp. was at pH 6.5 using phosphate buffer. The growth of this isolate and the degradation rate was dramatically decreased at higher pH using borate and carbonate buffers. Compared to the previous studies, the optimum pH for Citrobacter braakii was at pH 7.0 (Dhouib et al., 2003), Delftia acisovorans strain SPB1 at pH 7.2 (Schulz et al., 2000), Comamonas terrigena strain N3H at pH 7.4 (Roig et al., 1998) consortium of Acinetobacter calcoaceticus and Pantoea agglomerans at pH 8.5 (Abboud et al., 2007) and pH 7.5 to 8.0 for Pseudomonas Strain C12B (Payne and Feisal, 1963). Optimization of pH is very important since pH will strongly affect the bacterial growth and it is very important in order to enhance the effectiveness of bioremediation during the bioaugmentation experiment.
Figure 7: The effects of pH on the cellular growth of Pseudomonas aeruginosa sp. using three overlapping buffers. Data represent means Â± SD, n=3.
Figure 8: The effects of pH on the degradation of SDS using three overlapping buffers. Data represent means Â± SD, n=3.
3.3.4 The effects of temperature
The effect of different temperatures on the SDS biodegradation and on the growth of this isolate in BSM containing 1g/L SDS was carried out at temperatures ranging from 20Â°C up to 45Â°C (Figure 9). Cellular growth and degradation of SDS gradually increased as the temperature increased until it reached an optimum temperature at 30Â°C. Growth and the ability of this isolate to degrade SDS decreased slowly after the optimum temperature until no growth and degradation was observed at 45Â°C. From previous published report, the optimum temperature for degradation of SDS by a consortium of Acinetobacter calcoaceticus and Pantoea agglomerans was between 30Â°C to 37Â°C (Abboud et al., 2007). Citrobacter braakii (Dhouib et al., 2003), Delftia acidovorans strain SPB1 (Schulz et al., 2000) and Pseudomonas Strain C12B (Payne and Feisal, 1963) degraded SDS optimally at 30oC. Comamonas terrigena strain N3H showed optimum growth at 28Â°C (Roig et al., 1998). Marchesi et al. (1997) reported that the lowest optimum temperature for the degradation of SDS by Pseudomonas sp. was at 25Â°C.
Figure 11: The effect of temperatures on the cellular growth (ï‚˜) of Pseudomonas aeruginosa sp., remaining SDS (O) and SDS concentration in abiotic control (â-Š). Data represent means Â± SD, n=3
3.4 Growth and SDS Degradation after optimization
Growth and the degradation of 1g/L SDS as a sole source of carbon by this isolate were studied using before and after optimization conditions as shown in figures 10(a) and figure 10(b) respectively. Complete degradation of 1g/L SDS before optimization took almost 5 days compared to the degradation under optimized conditions where degradation was completed after 2 days of incubation using minimal medium. The SDS content remains unchanged in uninoculated control (abiotic control) showing there are no elimination of SDS occurring. Degradation of 3g/L SDS using consortium of Acinetobacter calcoaceticus and Pantoea agglomerans studied by Abboud et al., (2007) was completed after 5 days of incubation in NB medium supplemented with SDS. However, in their work only 10% of 3g/L was successfully degraded after 6 days in minimal medium due to the limited supplementation compared to the NB medium (Abboud et al., 2007).
Figure 10: Percentage of remaining SDS (O), SDS concentration in abiotic control (â-Š) and cellular growth (ï‚˜) of Pseudomonas aeruginosa sp. before (a) and after (b) growth optimizations. Data represents mean Â± SEM, n=3.
Based on this study, Pseudomonas aeruginosa was shown to be among the best degrader in biodegradation of SDS. The optimal growth conditions for this strain and the optimal conditions for biodegradation of SDS have been established to be at 30Â°C, pH 6.5 using phosphate buffer system. Sodium nitrate; at 8.0gL-1 was found to be the best nitrogen source. The optimization procedures were proven to enhance the rate of SDS biodegradation. Immobilization studies are in progress to further enhance SDS biodegradation.