Screening and Isolation of Bacterial Strains of Linear Alkylbenzene Sulfonate Tolerant for Industrial Purposes

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Published on International Journal of Health, Nursing, & Medicine
Publication Date: September, 2019

Oluwasola, P. O., Eniola, K. I. T. & Balogun O.B.
Joseph Ayo Babalola University, Ikeji-Arakeji, PMB 5006, Ilesa Osun State
Environmental and Public Health Research Laboratory, College of Natural Sciences

Journal Full Text PDF: Screening and Isolation of Bacterial Strains of Linear Alkylbenzene Sulfonate Tolerant for Industrial Purposes.

The responses of species of bacteria isolated from a segment of River Owena to linear alkylbenzenesulfonate (LAS) were assay in terms of ability of organisms to grow in the presence of varying concentration of LAS. A total of six (6) species were characterized and identified namely: Pseudomonasspp, Staphylococcus spp, (klebsiellaspp, Micrococcus spp, Escherichia coliandBacillusspp.The isolates varied in their responses to different concentrations of LAS and were categorized as LAS-sensitive bacteria (LAB), LAS-tolerant bacteria(LSB) and LAS-utilizing bacteria(LUB). Thirteen of the strains were tolerant to LAS (0.1% w/v), eight of the isolates showed the potential to degrade LAS and five of the isolates were sensitive to LAS (0.1% w/v). Their distribution and the pattern of their responses can be evaluated by assessing the river for detergent pollution. The potential of some of the resident bacteria to degrade LAS can be explored for bio-treatment of detergent polluted wastewater.

Keywords: Degradation potential, Owena, sensitivity, surfactants, wastewater treatment.

Detergents are cleansing products derived from synthetic chemicals and it have broad range of function and effectiveness to remove some microorganisms. The first detergent: alkyl naphthalene sulfonate, was establish and constitute in 1916 in Germany to avoid soap- curd in water (Kamaraj et al., 2008). By the early 1930s, fatty–alcohol based detergents were initiated and by 1946 the first detergent was establish in the United States (Manaham, 1993). Detergents gained prominence in1960s, when abnormal quantities of foam were noticed in streams in the United States. This led to a shift from the recalcitrant branched alkyl benzene sulfonate (ABS) to the biodegradable linear alkylbenzenesulfonate (LAS) (WHO, 1996). Subsequently, the problem of eutrophication led to reduction of sodium triployphosphate (STPP) in detergent and increase in amount of surfactant (Chen et al., 2008).
Since its introduction in the 1960s; linear alkylbenzenesulfonate (LAS) has been the most commonly used ionic surfactant in detergent formulations, because of its biodegradability (Gledhill, 1974; McAvoy et al., 1997; Britton, 1998; Cook and Hrsak, 2000 and OECD SIDS, 2005). The presence of LAS in many commonly used household detergents provides a wide variety of possible routes for it to get into the environment (Larson et al., 1995). However, the way in which LAS enters the environment varies from place to place. Eniola (1996) found that in Ilorin; the route of LAS into the water bodies is predominantly via discharge from a Soap and detergent industry. Domestic wastewater and discharge from waste treatment facilities have also been identified as possible routes into the environment.
Greiner and Six (1998) suggested that surfactant could have ecotoxicity at low concentrations. Studies by Akpata (1990) and Eniola and Olayemi (1999) reported decrease in population of viable heterotrophic bacteria in some streams following receipt of detergent bearing effluents. Eniola and Olayemi (2002) categorized bacteria isolated from some fresh water bodies, on the basis of interactions with detergent, into: detergent-sensitive bacteria (DSB), detergent-tolerant bacteria (DTB) and detergent-utilizing bacteria (DUB). Studies (Folker and Lander, 2000; HERA, 2004) have shown that LAS is toxic to aquatic biota at concentrations of more than 0.1mg l–1.
The extent to which a river can receive waste materials without some quality criteria that is exigently deteriorated referred to as its assimilative capacity (Gray, 2004). This depends on the ability of the water to make use of the natural ability of self-purification and get rid of toxic pollutants. Aerobic breakdown of organic materials by microorganisms is the most important processes in self-purification. Hence, any distortion of the natural species composition of a river portends serious consequence for the self-purification potential of such river(Alidina et al., 2004).
This research evaluates the response of bacteria, isolated froma segment of River Owena within Owena-Ijesa, Osun state Nigeria, to LAS. The response is estimated in the ability or failure to grow in the presence of varying concentration of the surfactant. The potential of the bacteria to grow with LAS as sole source of carbon, indicating ability to metabolize the surfactant was also assessed.

2.1 Study Area
The study involved a segment of River Owena within Owena-Ijesa (7°37′N, 5°18′E), a community along the Akure-Ilesa highway. River Owena in its course flows through a number of rural agrarian communities. The river flows at the outskirt of Bolorunduro; an agrarian community, where it used for washing of clothes and other things. it flows from Bolorunduro through farmland and into Owena-Ijesa. It is the major river in Owena-Ijesa; where it forms a natural boundary between Osun state and Ondo state dividing the community into two. The segment of the river within Owena-Ijesa runs behind residential areas, across the highway and through farmlands. The river receives largely domestic waste and wastewater from car washes located on the bank of the river. In the dry season, the river serves as source of water used for irrigation farming along the banks of the river before the residential areas downstream. The river also serves as source of raw water to a water treatment facility at Owena-Ijesa.

2.2 Sampling Points and Sample Collection
Four sampling point were selected on the segment of the river within Owena-Ijesa. The sampling points are: around residential areas where the river enters into Owena Ijesa designated OWN1 (Plate 1); where the river flows under the Akure-Ilesa highway designated OWN2 (Plate 2); where the river can flow through areas used for irrigation farming during dry season designated OWN3 (Plate 3); around residential areas as the river can flow away from Owena-Ijesa designated OWN4 (Plate 4). Samples were collected from the river following the procedure described by WHO (1997).The sample were carried in an improvised ice chest and taken to the laboratory for analysis; no sample was allowed to stand for more than 1 hour.

2.3 Physicochemical and Bacteriological Analyses
The pH, suspended solid and dissolved oxygen contents of the water samples from the river were determined, as described by ASTM (1985). The total heterotrophic bacterial counts were ddetermined using pour-plate method (Eniola and Olayemi 1999). Bacteria that could be tolerant to 0.1% (w/v) of LAS surfactant. Microorganisms were isolated and enumerated from water samples by plating 1ml of the 10–4 dilution of the river water samples was dispense to LAS enriched-nutrient agar (0.1% w/v pure LAS) from 10–1 to 10–10 dilution. The plates were incubated at 37°C for 18-224hours. Uninoculated plates of the media were incubated to serve as controls. Isolates were purified, characterized and identified, according to Holt et al. (1997). A 0.1% (w/v) stock LAS-solution was made and varying concentrations (10%, 25%, 50% and 100%) prepared from it. The bacterial isolated were screened for sensitivity to the different concentrations of LAS using the well method of media diffusion technique. Data obtained were subjected to statistical analysis, using the Analysis of Variance and Pearson product-moment correlation test.

Plate 1: OWN1: around residential areas as the river emerges into Owena-Ijesa

Plate 2: OWN2; where the river moves under the Akure-Ilesa highway

Plate 3:OWN3, where the river ml through areas used for irrigation farming during dry season

3. Results
3.1 Bacterial Load of River Owena
The pH ranged between 7.20 and 8.55, and the mean dissolved oxygen content ranged between 6.50 and 15.00 mgl–1. The suspended solid contents ranged between 1.85 and 4.00mgl–1 (Table 1). The total heterotrophic bacterial counts varied between 80 and 120 104cfuml–1. The population of bacteria that is resistant to 0.1mgl–1 of LAS (designated LTB) ranged between 25 and 65 104cfuml–1 (Figure 1). Eighteen bacterial strains were encountered, characterized and identified to belong to six (6) species namely: Pseudomonas spp, Staphylococcus spp, Klebsiella spp, Micrococcus spp, Escherichia coli and Bacillus spp. Their distribution along the segment of the river studied is shown in Table 2.

3.2 LAS sensitivity of the bacterial isolates
The bacterial strains varied in their sensitivity to different concentration of LAS. Pseudomonass spp, was the most tolerant of LAS (zone of inhibition of 11mm), it is followed by E. coil (12mm) and then Klebsiella spp. (13mm) while the most sensitive was Bacillus spp. (20mm). The sensitivity of the strains to different concentration of LAS is show in Figure 2. Bacillus spp showed the highest zone of inhibition of 20mm, 19mm, 18mm, 17mm and 15mm for the 100%, 50%, 25% and 10% of Linear Alkyl benzene sulphonate respectively while Pseudomonas spp showed the lowest zone of inhibition of 11mm, 10mm, 9mm, 7mm and 6mm for the 100%, 50% 25%, and 10% of Linear Alkyl benzene sulphonate respectively. Thirteen of the bacterial strains (72%) could withstand LAS that is Linear alkyl benzene suphonate Resistant Bacteria (LRB) while five could not survive with Linear alkyl benzene suphonate Susceptible Bacteria (LSB). The LRB constituted 73–90% of heterotrophic bacteria population in the effluent and 22% of heterotrophic bacteria population were present in effluent free water The response status of the various bacterial species is shown in Table 3.

The physicochemical characteristics of the river segment were consistent with a typical fast flowing river. The pH range (7.20- 8.55) it shows that the river is effectively assimilating materials from both point and non point sources. Upstream of the study area, in Bolorunduro community, the river is used for domestic washings which involve use of detergents. Several Studies (WHO, 1996, Eniola and Olayemi, 2008) had reported that input of detergent waste results in increase in pH due to the alkaline nature of detergent. The pH conditions in the segment of the river are such that favour proliferation of heterotrophic bacterial. Most heterotrophic bacteria are known to thrive best at near neutral pH.The levels of dissolved oxygen in the river (6.50 and 15.00 mgl–1) were consistent with the flow pattern of the river; higher DO were found in areas where the river was fast flowing. The generally high DO also favour assimilation of any input of organic waste; which is largely agricultural wastes.
The suspended solid content ranged between 1.85 and 4.00mgl–1 (Table 1). The high suspended solid contents alongthe river provide surfaces onto which absorb (Westall et al., 1999). The presence of surfaces makes formation of biofilms a high possibility. The high heterotrophic bacterial count is consistent with the physicochemical characteristics of the river and input of domestic waste, especially around the residential areas; it varied between 8.0×105cfuml– and 1.20 106cfuml–1. The high population of heterotrophic bacteria found is similar to results obtained by Takada et al. (1997) for a detergent-polluted water body in Germany. Statistical analysis showed a negative correlation between surfactant concentration and bacterial population. The majority of the bacterial species were able to grow where LAS was presence. Most of them further showed tolerance to LAS, these were predominantly members of the family. Enterobacteriaceae. The eight (LRB) that showed potential to degrade LAS could have ability to degrade the surfactant exposure due to the surfactant over time. Spain and Van Veld (1983) reported that the ability of an organism to degrade some contaminants in wastewater Takada et al. (1997) attribute the large population of LAS degrading to consistent exposure of the organisms to chronic concentrations of the surfactant which they adapt and develop mechanism in degrading some contaminants present in wastewater. Hardman (1989) and Marchesi et al. (1991) had suggested that bacteria are capable of modifications and reorientation in response to environmental changes or alteration in substrate availability.
The large number and population of bacteria species demonstrating potential to degrade the surfactant which support the assertion that LAS is biodegradable. The pathway elucidated by Cook and Hrsak (2000) suggest that three tiers of organisms were involved. The first-tier organisms initiate the mechanism by oxidation of the alkyl chain to long chain sulphophenyl carboxylates (lc-SPCs).
The second-tier organisms convert the lc-SPC to short-chain sulphophneylcarboxylates (sc-SPC), while the third-tier organisms mineralize the sc-SPC via four sulfocatechol and ortho () ring cleavage. Cavalli et al. (1996) reported that the lc-SPCs and sc-SPCs are transient intermediates that are rapidly metabolized. The organisms isolated in this study had a potential to reduce LAS could belong to any of the three tiers. They play a role in the degradation of LAS and could be relied upon to undertake the biotreatment of detergent-bearing effluent.

Table 1. Physicochemical properties of a segment of River Owena

Figure 1. Population of Bacteria along a segment of River Owena
THBC- Total heterotrophic bacteria count
LTB- LAS-tolerant bacteria

Table 2. Distribution of Bacterial isolates along a segment of River Owena

Table 3. Status of Bacterial strains isolated along a segment of River Owena

The findings from this study show that the bacteria.
(1) Resident in the segment of the river responded differently to LAS.
(2) They can be categorized in three groups: LAS-sensitive bacteria (LSB), LAS-resistant bacteria (LTB) and LAS-utilizing bacteria (LUB).
(3) The pattern of their response is such that it can be used in a predictable manner to monitor the status of the river.
(4) The potential of some of the resident bacteria,desipacted LAS-utilizing bacteria(LUB), to utilize the surfactant present a possibility of bio-treatment of detergent effluent.