Microbial degradation of sodium lauryl sulfate ( SLS ) using bacterial consortium isolated from coastline of Alexandria city

Sodium lauryl sulfate (SLS) is an anionic surfactant that may easily find its way to seawater through domestic and industrial wastewater discharge. The current study was suggested to investigate its fate in relation to the ability of heterotrophic bacteria isolated from samples to degrade SLS surfactant. In addition, the different factors affecting the biodegradation process. Out of nine dominant marine bacteria, only three were able to degrade SLS successfully. They were characterized throughout morphological, biochemical and physiological features as; Enterobacter gergoviae, Enterobacter cloacae and Bacillus alvei. Our data confirmed that the biodegradability of consortium consisting of E. gergoviae, E . cloacae and B. alvei combination was more effective than that of the individuals. The increase in inoculum size, support the increase in the biodegradation. The 30 o C as an incubation temperature was the most effective temperature (98% biodegradation). The pH 9 was the optimum for the growth of consortium (E. gergoviae, E. cloacae and B. alvei) and consequently for biodegradation rate (96%). The pH level 7 was more or less near to optimum with biodegradation percentage (89.2%). Glucose as a carbon source and casein as a nitrogen source improved the biodegradation process to 91.9% and 90.3% respectively. The biodegradation percentages showed an inverse relationship between the increase in SLS mass and the extent of its degradation. Under these growth conditions a complete degradation of 1000 ppm SLS biomass was achieved with biodegradation percentage (45.5%). Such findings contribute to a better understanding the fate of the SLS in the aquatic environment.


INTRODUCTION
In recent years, surfactant-mediated bioremediation is a research focus . (1) Biodegradation of surfactants has been the subject of substantial research works since 1950s, when synthetic detergents came into wide spread use.
( Pseudomonas strains degrade only down to four or five ethoxy groups . (7)It is also likely that surfactant addition will over time alter the microbial population with overall positive or negative consequences on rates and extents of biodegradation. ( Biodegradation is most often performed by soil or aquatic microorganisms and leads to generation of water and carbon dioxide gas. Aliphatic alkyl sulfate compounds commonly represented by Sodium Lauryl Sulfate (SLS) are anionic surfactants that may easily find their way to the environment through domestic and industrial wastewater. ( They are characterized by their linear long chain sulfate ester structure which can be degraded by several bacterial strains through desulfation and further assimilation. (11,12)Some strains of Acinetobacter are known to be involved in the biodegradation of different pollutants such as biphenyl or chlorobiphenylaniline, phenol, benzoate, crude oil and acetonitrile. ( Acinetobacter calcoaceticus, a facultative anaerobic strain that was able to degrade more of the alkanes than aromatic fractions of crude oil.It grew well on n-alkane up to C 33 as well as, on the branched chain alkane but failed to grow on cycloalkanes oil. (14)It was investigated that Sodium Lauryl Sulphate (SLS) could be used for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant.

Chemicals
Sodium lauryl sulfate surfactant was purchased from Sigma chemicals, USA.The chemicals used for biochemical tests were of pure grade and purchased from Sigma chemicals, USA.

Media
Ingredients of media were all of analytical grade and were obtained from recognized chemical suppliers.The medium for biodegradation activity; a minimal medium (M9).

Isolation and purification of bacterial isolates
Seawater samples were collected in 500 ml sterile screw-caped bottles as described by (Austin, 1999). (18)Serial dilutions from 10 and API 20E strip system.

Analysis of Sodium Lauryl Sulfate
Samples of 50 ml were placed in a separating funnel; then, 5 ml of 10 -3 mol/l methylene blue (MB) solution and 5 ml of chloroform were added, and the mixture was shaken for 1 minute.The separating funnel was allowed to stand one minute and the separated chloroform phase was filtered; the absorbance of the chloroform solution was then measured at 654 nm using UV-Visible double beam Shimadzu spectrophotometer with glass cell length of a 10 mm (1 cm).Each analysis was repeated at least three times.

Biodegradation process of sodium lauryl sulfate
The culture medium broth was inoculated with single colonies that were previously isolated by an overnight growth of two consecutive shake flask cultures in nutrient broth followed by 3h recovery.
with some modifications appropriating our work.However, seven factors affecting this process were investigated separately as follows.

Effect of biodegradability of the individuals and consortium:
To

Effect of the pH level:
To test the effect of pH level, the minimal M9 medium with SLS at a concentration of 100 ppm as sole carbon source was utilized.
The pH of flasks was adjusted at 5, 7 and 9, respectively, then all treatments were inoculated with consortium (E.gergoviae & E. cloacae & B. alvei).

Effect of different carbon sources:
To

Effect of surfactant concentrations on the biodegradation rate:
To examine the effect of surfactant concentrations on the biodegradation rate, five different concentrations of SLS (200, 400, 600, 800 and 1000 ppm) were applied.
The effects of surfactant biomass either on the growth of a mixed consortium in Nutrient Broth medium (NB medium) were determined by absorbance and then dry weight .

Results and discussion:
Several investigations were carried out to determine the degradation of surfactants by bacteria isolated from marine, other aquatic resources, and even wastewater.The biodegradability was observed under aerobic and anaerobic conditions.

Physico-chemical analysis and SLS concentrations of seawater samples
Physico-chemical analysis and SLS concentrations of the collected seawater samples from different sampling positions along the coastline of Alexandria city during 2009 were estimated (

Isolation of dominant bacteria capable of degrading SLS:
Only six dominant bacterial isolates growing on SLS as a sole carbon source were obtained from seawater samples (Table 3). .
In a study of linear alkylbenzene sulfonated . The opportunity . Swindoll and Aelion (47) also found that several types of organisms may be required to degrade some xenobiotic compounds sequentially.As each species may have its own particular nutrient requirements, a Ibrahim et al., 199 number of nutrients may be influencing metabolism by a heterogeneous population at any given time.Therefore, the concept of a single limiting nutrient may not be applicable to heterogeneous microbial populations.alvei, for SLS biodegradation, four ratios were examined.The results shown in Figure 2 reveal that the increase in inoculum size is accompanied with the increase in biodegradation.The ratio of 2.0 % was the most effective inoculum size in the biodegradation (95.9 %), while the lowest effective inoculum size towards biodegradation was the 0.5 % (81.2 %).

Effect of the incubation temperature on SLS biodegradation:
The consortium of E. gergoviae, E. cloacae  SDS.In addition, the data of Khleifat On the contrary, Hosseini et al. (43) found that mixed culture of the two isolates; Acinetobacter johnsoni and Pseudomonas beteli did not significantly increase SDS utilization, (97.6% biodegradation).

Effect of the pH level on SLS biodegradation:
A range of pH buffers 5-9 was used to examine the effect of mixed culture pH on the biodegradation process.From data shown in observed that all of the carbon sources being tested caused an increase in the LAS biodegradation rate.Sucrose, maltose and glucose were better carbon sources, although all carbon sources being tested produced better and more complete degradation activity than the positive control (without C-sources).
found that all nitrogen sources being tested produced the greatest rate of LAS degradation, which completed within 48 h.Khleifat (16) discussed that the nature of the carbon and nitrogen sources may affect both growth biomass and enzymes involved in LAS degradation.Based on our data, we may share him the same conclusion.In contrast, such supplementation of the mixed culture with carbon and nitrogen nutrients had adverse effects on the induction of SDS degradation .

Effect of different concentrations of SLS on the biodegradation rate:
Such effect was studied using several observed that increased surfactant (XP-100) concentration did not inhibit its biodegradation over the period studied

16 )
The presence of SLS in the culture medium with ndecane as main source of carbon and energy accelerated the growth of Pseudomonas C12B.SLS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated.Recent studies discussed association of the capacity of SLS to induce decanemineralization system in Pseudomonas C12B or with the ability of SLS to stimulate the surface attachment of competent bacteria resulting in close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.Two different bacterial consortia were isolated by Khleifat (from wastewater treatment plant to be used in the biodegradation of Sodium Lauryl Ether Sulfate (SLES).The two consortia consisted of three members.Acinetobacter calcoacetiacus and Klebsiella oxytoca in one co-culture (A-K) and Serratia odorifera in the second co-culture (S-A), which contains Acinetobacter calcoacetiacus as well.The current study investigated the ability of heterotrophic bacteria isolated from the Alexandria coastline seawaters samples to degrade SLS surfactant.In addition, the different effective factors affecting the biodegradation process were estimated.

( 17 )
It was used to test the consumption of SLS surfactant as sole carbon source.The composition of this medium involved 3.0 Na 2 HPO 4 , 1.5 KH 2 PO 4 , 1.0 NH 4 Cl and NaCl that were dissolved in 500 ml of de-ionized water.The medium pH was adjusted to 7.4 before the addition of 0.24 MgSO 4 , 0.05 CaCl 2 .6H 2 O and 0.05 FeCl 3 .6H 2 O.After autoclaving, 0.001thiamine-HCl was dissolved in 2 ml-de-ionized water and sterilized by Millipore filter then supplemented to the minimal medium.

2. 6 . 6 .
investigate the effects of different carbon sources, glucose, sucrose and molass were supplemented individually for each nutrient at final concentration of 0.2% as an additive to the minimal M9 medium.SLS at a concentration of 100 ppm were utilized in this minimal medium.All treatments were inoculated with consortium (E.gergoviae & E. cloacae & B. alvei).Effect of different nitrogen sources: To investigate the effects of different nitrogen sources, yeast extracts, casein, ammonium chloride and tryptone were supplemented individually for each nutrient at final concentration of 0.2% as an additive to the minimal M9 medium.SLS at a concentration of 100 ppm were utilized in this minimal medium.All treatments were inoculated with consortium (E.gergoviae & E. cloacae & B. alvei).

( 32 )( 18 )
In addition, the bacterial community structure can change dramatically within weeks and may form blooms of bacteria. (33)The factors controlling the distribution of bacteria in marine environments are complex and vastly different from those experienced by terrestrial bacteria.Several studies have concluded that, the marine microbial communities are influenced by environmental variables such as temperature, nutrient availability, salinity, length of day, algal bloom and hydrostatic pressure. (25-28)In order to obtain the highest recovery of aerobic heterotrophic marine bacteria, sea water nutrient agar was selected.Various microbiological media were used to evaluate the microbial quality of seafood and strongly suggested the use of marine agar.However, the highest count observed along coastline of Alexandria city appears to be a result of combination of continuous effluent input and hydrographic dynamics which affect in situ microbial community.These sites experience varieties of human interference (domestic/industrial/ fishing).Dispersion and dilution of industrial and/or domestic wastes create a favorable situation for bacteria and other microbial heterotrophs.
Three of these isolates (MH1, MH2 and MH3) were isolated from the Eastern Harbour station.From Abu Qir Bay station; MH4 and MH5 were isolated.The bacterial isolate MH6 was of Western Harbour.Al Dekhaila Harbour had two isolates coded; MH8 and MH9.Al Max Bay had the isolate MH7 as dominant one.Bacteria capable of degrading SLS were examined to degrade SLS individually.The biodegradation percentage showed that three isolates coded as; MH3, MH5 and MH6 had the ability for SLS biodegradation as individuals with percentages of 46, 27.3 and 26.7%, respectively (Figure 1).These bacterial isolates were selected for furthermore biodegradation in the optimization step.Microorganisms have evolved different natural methods of uptake of hydrophobic compounds, in general reflected by their capacity to generate hydrophobic cell surfaces or to secrete biosurfactants into the surrounding medium

(
LAS) biodegradation by bacterial culturesoriginating from an estuarine site (Krka river estuary) it was found that the rate of biodegradation depended on the origin of the culture, temperature and the structure of the alkylbenzene group.

( 21 )
isolated several bacterial combinations from wastewater.They that a mixed culture of the facultative anaerobic bacteria Pantoea agglomerans and Acinetobacter calcoaceticus grown on NB was found most effective in breaking down the surfactants Linear Alkylbenzene Sulfonates (LAS) and Sodium Dodecyl Sulphate(SDS).Hosseini et al. (43) isolated two different bacteria from Tehran municipal activated sludge.They were identified as; Acinetobacter johnsoni and Pseudomonas beteli.They showed the ability to rapidly and actively degrade SDS as their sole source of carbon, with rate of 97.2% and 96.4% of the original SDS levels after 10 days of growth, respectively.In order to improve the efficiency of biodegradation for SLS surfactants, the effects of different conditions on this biodegradation process were further investigated.The isolates Enterobacter cloacae, Enterobacter gergoviae and Bacillus alvei were proved to be the most potent in the biodegradation of SLS surfactant.Therefore, they were selected for further study to maximize and evaluate the efficiency of the biodegradation process.Biodegradation process is influenced by several factors such as medium composition and medium optimization may result in a significant increase in the productivity.(41)Optimization of physicochemical conditions is inevitable in any bioprocess development and usually performed by varying the levels of one independent variable while, fixing other variables at a certain level.
synergism to develop exists in a consortium.Such interactive effects lead to more effective biodegradation than is possible by any individual microorganism.The biodegradation of LAS requires a four member consortium, three members of which oxidize the alkyl chain but synergism amongst the four members was essential for mineralization of the aromatic ring.

( 46 )O
In other studies, bacterial consortium consisted of two members, Pantoea agglomerans and Serratia odorifera 2, cells were grown evenly together in a minimal medium (M9) and nutrient broth (NB).The bacterial consortium was able to grow in the minimal medium containing LAS as the only carbon source.The percentage degradation of 200 ppm LAS by this bacterial consortium was better when cells were grown in NB (~70%) than in the M9 medium (36%).Also, the degradation ability by the bacterial consortium was very much higher than by its individual cells.An incubation temperature of 32 addition of different carbon and nitrogen sources all independently caused complete mineralization of 200 mg/L LAS within 48-72 h

showed
and B. alsvei completed the next experiments in optimization.Five temperature degrees were examined to detect the best one for SLS biodegradation.The results in Figure 2 Our results were in agreement with the work of Abbouda et al.

Figure 2 ,
Figure 2, the pH 9 was the optimum for the growth of consortium (E.gergoviae, E. cloacae and B. alvei) and consequently for

( 21 )3. 5 . 5 .
stated that a pH of 8.5 would be the optimum level for maximum LAS degradation by the combined bacteria.Effect of different carbon sources on SLS biodegradation: Various carbon sources including glucose, sucrose, and maltose were added separately at a fixed concentration of 0.2% to broth medium for growing consortium (E.gergoviae, E. cloacae and B. alvei).The biodegradation was clearly observed in presence of glucose and molase as carbon sources with biodegradation percentage 94.9 and 87.2, respectively (Figure 2).On the other side, the supplementation of sucrose did not improve the biodegradability of microbial SLS.Moreover, a positive control of mixed culture growing under similar conditions but lacking additional supplementation of carbon sources has failed to produce similar degree of biodegradation.Our results were parallel to that of Abbouda et al. (21) who found that the supplementation of either surfactant as sole carbon source to the mixed culture growing in minimal medium produced 10% degradation of SDS and 60% degradation of LAS, respectively.These data are discussed in terms of differences between the enzymatic induction of LAS and SDS bacterial biodegradation.In addition, Khleifat Figure 3, an inverse relationship between the increase in SLS mass and the extent of its degradation.Under these growth conditions a complete degradation of 1000 ppm SLS biomass was achieved with biodegradation percentage (45.5%).Our results were matched with that of Abbouda et al. (21) who observed an inverse relationship between the increase in LAS and SDS mass and the extent of its degradation.Moreover, they obtained that the mixed culture produced about 60% of LAS degradation when this surfactant was added at 300 ppm level compared with 10% degradation at 700 ppm surfactant supplementation.In contrast, higher ranges of SDS concentration (3000-8000 ppm) were degraded by the same mixed culture but still an inverse relationship existed between the increase in mass and the extent of degradation.On the contrary, Abd-Allah and Srorr (48)

Figure 2 :
Figure 2: Optimization of different variables affecting the biodegradation process of SLS

a= Initial SLS surfactant concentration was 100 ppm
Bull High Inst Public Health Vol.42 No.2[2012]

a= Initial SLS surfactant concentration was 100 ppm 3.5.2. Effect of the inoculum size on SLS biodegradation:
To detect the best inoculum size of the consortium of E. gergoviae, E .cloacae and B.