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Survival Trend of Lead Solubilizing Strains of Pseudomonas Species in Lead Polluted Soil Samples | OMICS International
ISSN: 2155-6199
Journal of Bioremediation & Biodegradation

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Survival Trend of Lead Solubilizing Strains of Pseudomonas Species in Lead Polluted Soil Samples

Sanuth Hassan A*, Fagade Obasola E and Ogunjobi Adeniyi A
Environmental Microbiology and Biotechnology Laboratory, Department of Microbiology, University of Ibadan, Nigeria
Corresponding Author : Dr. Sanuth Hassan A
Environmental Microbiology and Biotechnology Laboratory
Department of Microbiology
University of Ibadan, Nigeria
Tel: +2348028543040
E-mail: [email protected]
Received June 22, 2011; Accepted August 03, 2011; Published August 05, 2011
Citation: Sanuth HA, Fagade OE, Ogunjobi AA (2011) Survival Trend of Lead Solubilizing Strains of Pseudomonas Species in Lead Polluted Soil Samples. J Bioremed Biodegrad 2:123. doi:10.4172/2155-6199.1000123
Copyright: © 2011 Hassan SA. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

The survival of three Lead (Pb) solubilising strains of Pseudomonas species (B6, D4, and E4), with Minimum Inhibitory Concentration (MIC) ability of Lead (Pb) greater than 500 mg/l were monitored when seeded into two different lead polluted soil samples. The bacterial population was studied over Ninety days in sterilized and unsterilized polluted soil samples of different textures. The outcome revealed a general gradual decrease in the bacterial population of the isolates ranges from 22.0% to 56.6% over the 90 days but with higher survival strength in the sterilized soil samples than un-sterilized samples. Pseudomonas strain B6 shows percentage reduction value of 22.9% and 33.3% respectively in sterilized silty-clay polluted soil samples and loamy polluted soil samples, strain D4 shows 25.5% survival in silt-clay and 56% in loamy soil samples while E4 shows 35.5% and 22.0% population reduction in the two respective soil samples. This study reports the essential of adequate knowledge of physicochemical characteristics of soil environment as well as necessary biotic factors for survival of any isolates intended to be used in bioaugmentation for the remediation of polluted soils.

Keywords
Bioaugmentation; Survival; Pseudomonas species; Lead (Pb); Polluted soils
Introduction
Bioremediation of polluted soils through the introduction of exogenous microbial isolates into the polluted environment (bioaugmentation) has gain increasing interest as an alternative method to biostimulation in situation where the indigenous species cannot cope with their environmental pollutants. However, many factors including predation, competition with indigenous microorganisms, nutrient availability, physicochemical parameters and other environmental factors necessary for growth has been found to influence the survival of bioaugmented microbes as well as their bioremediation efficiencies [1-3].
Many of the previous studies reported the use of activated contaminated soils or sludge as inoculants for bioaugmentation [3-5] but the fair of transferring more hazardous contaminants through the activated soil or sludge is also seen as a concern hence, the report of possible usage of uncontaminated soil with potential contaminant degrader as inoculants [6]. Although this technique has been seen as a potential alternative to address the use of activated sludge, but the direct use of harvested microbial cells could be a better option in that one is sure of the organism being introduced into the environment. The key to the success of the direct microbial method is the survival of introduced bacteria [2,7] but poor survival and remediation by augmented microorganisms has been reported [8]. This observation is provoking more research into the fate of microorganisms in contaminated environments.
Lead (Pb) has been recognized as one of the most hazardous heavy metal among environmental pollutants. Its irregular inputs through the mining and smelting activities, combustion of leaded gasoline, land application of sewage sludge, battery disposal and Pb-bearing products result in the high concentrations of Lead (Pb) in soils [9]. Conversely, little information is available on the application of microbial cells as inoculum to augment the remediation of lead (Pb) contaminated soils.
In this study therefore, two texturally different lead (Pb) polluted soil sample, sterilized and un-sterilized were bioaugmented with isolated lead solubilizing strains of Pseudomonas species [10] and compared their survival trend in relation to the texture and sterilization. To achieve a better understanding of the conditions that can be used to sustain high population density of the isolates in the introduced soil environment.
Materials and Methods
Bacterial strains
A total of fourteen (14) lead solubilizing strains of Pseudomonas isolated from soils of a battery manufacturing plant in Ibadan [10] were rescreened for lead solubilising potentials [11]. The best three isolates (B6, D4 and E4) with maximum solubilizing ability and minimum inhibitory concentration (MIC) of more than 500 mgl-1 of lead sulphide (PbS) were selected for the study. Their growth curve in lead culture medium and the effect of inorganic nutrient supplement their growth was studied [11].
Reconfirmation of lead solubilizing ability
The Solubilizing ability of the strains was reconfirmed using the agar plate method [11] 15 ml nutrient agar was mixed with 50 mg of sterile lead sulphide (PbS) and was allowed to solidify. Exactly 15 ml of the same medium without PbS was added to form another layer on the plates and allowed to solidify after which the plates were inoculated by streaking with pure cultures of the bacterial strains and incubated at 37°C for 48 hours. Solubilizing ability of the bacteria strains was detected by brown pigmentation of the bacteria cells and graded to the degree of pigmentation.
Minimum Inhibitory Concentration (MIC)
The Minimum inhibitory concentration of the strains was reconfirmed on PbS Agar at varied concentrations of Lead (PbS) ranges from 300mg to 1000mg to determine the highest tolerable concentration for growth.
Soil samples and characterization
Soil samples were collected from the dump sites of two battery recycling companies in Ibadan, Nigeria. Samples were taken to the Environmental Microbiology laboratory, University of Ibadan for study. The soil particles size and texture were determined using graduated sieves [12], the moisture content was determined by oven drying at 105°C to constant weight while the pH of the soil in deionised water was also determined with the aid of pH meter (Model: Hanna Instruments HI 2210). Soil digestion for metal analysis was carried out using the Nitric acid method [13]. Exactly 15 ml of concentrated HNO3 was added to one gram of dried, sieved and grounded soil sample in a 100 ml beaker and allow evaporating to approximately 1ml on a heating plate at 180°C. After cooling, 5 ml of concentrated HCl was added and digestions continue at 120°C until effervescence ceased. Digests were filtered and made up to 20ml with deionised water and the lead concentration was determined by Atomic Absorption Spectrophotometer (AAS), Perkin Elmer Analyst 200.
Cultivation of bacterial isolates and bioaugmentation
Bacterial strains were cultivated in Nutrient broth and cells were harvested by centrifugation, washed and suspended in normal saline. About 50 ml of suspension of each strain were sprayed on 1kg of the differently treated soils and mixed in a plastic microcosm of 6cm x 6cm x 3cm in the laboratory. The inoculums density were determined using plate count technique while an approximate moisture content of 20% was maintained by addition of sterilized water at appropriate intervals.
Survival monitoring
Ten grams of the soils were routinely sampled at fifteen days intervals, serially diluted and appropriate diluents were inoculated into Kings A medium using pour plate technique and incubated at 37°C for 48hr after which the colonies on the plates were counted. All experiments were carried out in replicates.
Results and Discussion
Confirmation of lead solubilizing ability of the strains
The results of the lead solubilization potential of screened Pseudomonas strains are shown in (Figure 1). Bacteria capable of solubilizing PbS were detected by brown pigmented colour of the colonies. Out of the fourteen lead solubilizing strains tested, six showed good solubilizing ability with ratings ranges from 2 to 3.
Minimum Inhibitory Concentration (MIC)
Eight out of the fourteen Pseudomonas strains were found to grow at PbS concentration above 500mgl-1. However, three Pseudomonas strains (B6, D4, E4) exhibited the highest survival with growth above concentration of 800mgl-1 of PbS in nutrient agar (table 1).
Soil analysis
The physico-chemical and microbiological characteristics of the soil samples are shown in (Table 2). Soil sample A has a pH value of 5.7 and Lead (Pb) concentration of 2160 ppm while Soil sample B has pH value of 6.6 and Lead (Pb) concentration of 1367 ppm. The soil samples A and B were texturally classified as Silty clay and Sandy loam respectively based on their particle sizes. The Pseudomonas counts were 7.0 x 103 CFU/g and 11 x 103 CFU/g for soil sample A and B respectively.
Inoculum density
The inoculums density of the strains were 2.04 x 1010, 2.8 x 1010 and 1.94 x 1010 CFU/ml for strains B6, D4 and E4 respectively.
Survival pattern
The fate of the Pseudomonas strains B6, D4 and E4 after inoculation into the two Lead polluted soils showed a similar survival pattern in relation to each other (Figures 2, 3). The population density (Table 3) decreased generally over 90 days with percentage reduction in growth of 22.9%, 25.5% and 35.5% respectively in the sterilized silty-clay soil sample (Soil A) and 33.3%, 56.6% and 22.0% reduction in the loamy soil (Soil B). However, the growth of the strains in the corresponding un-sterilized soil as shown in (Table 3) revealed growth reduction percentages of 45.4%, 52.7% and 54.4% for the strains B6, D4 and E4 respectively in the silty-clay soil. Furthermore, reduction growths of 55.4%, 65% and 30.5% was also observed in the loamy soil sample. This observation which is similar to the reports of previous studies [3,7,14] may be due to the toxic effect of the Lead metal on the strains and/or that the substrate in the soil is not bio-available to the organisms.
The influence of the soil condition on the survival pattern as observed in the trend of the strains in sterilized and unsterilized soil samples (Figures 2, 3) showed that the strains relatively survived better in sterilized soil than un-sterilized soil samples. However, the survival of the Pseudomonas strains in relation to the soil type indicate better survival trend of strains in Silt clay (Soil sample A) than Sandy loam Soil (Soil Sample B) as measured by viable count after 90 days of incubation [15] in similar study observed a higher growth density of bacterial population in silt soil. The survival trend observed with respect to the soil treatment (sterilized and unsterilized soil) could be due to the absence of competition, bacteriorvory and predation in the sterilized soils as a result of the elimination of indigenous organisms in the process of sterilization. Similarly, the relative survival in silt clay soil type than the sandy loam at low moisture content may be as a result of the ability of the clay soils to retain enough water that support microbial growth than the sandy loam soil. In addition, clays may protect bacteria from predation by protozoans by increasing the number of protective microhabitats available to bacteria in soil [14].
This study therefore, shows that to maintain bacteria in an alien soil environment for in situ bioremediation, their survivability will depend on their ability to cope with the new stress including other microorganisms and predators presence in the new environment. And that soil nutrients or the targeted substrate must be bio- available to the incoming bacteria. Hence, the population dynamics, physiological as well as physico-chemical factors of any polluted ecosystem must be carefully investigated before the bioagumentation of the system in other to achieve a successful bioremediation on field. Hence this study emphasises the prerequisite of laboratory experimentation as a guide to a successful field operation.
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