International Journal of ISSN: 2573-2889 IJMBOA

Molecular Biology: Open Access
Research Article
Volume 2 Issue 4 - 2017
Isolation of Bacteria and Physicochemical Analyses of Petroleum-Products Contaminated Soil from NNPC/PPMC Depot, Benin City, Nigeria
Akeredolu DO* and Akinnibosun FI
Department of Microbiology, University of Benin, Nigeria
Received: April 18, 2017 | Published: June 28, 2017
#Corresponding author: Akeredolu DO, Department of Microbiology, University of Benin, Nigeria, Email:
Citation: Akeredolu D, Akinnibosun FI (2017) Isolation of Bacteria and Physicochemical Analyses of Petroleum-Products Contaminated Soil from NNPC/PPMC Depot, Benin City, Nigeria. Int J Mol Biol Open Access 2(4): 00028. DOI: 10.15406/ijmboa.2017.02.00028

Abstract

This study was aimed to evaluate the physico-chemical properties and presence of bacteria of from soil samples contaminated with different petroleum products which are petrol (PMS), diesel (AGO), pure kerosene (DPK) and mixed kerosene (DPK) obtained from the tank farm of NNPC/PPMC Depot in Benin City, Nigeria. The mean count for heterotrophic bacterial ranged from 1.83x 104 ±0.09 cfu/g soil contaminated with mixed DPK to 5.93x104±0.02 cfu/g for soil contaminated with PMS. Nine (9) bacterial isolates were characterized and identified; Bacillus subtilis, Micrococcus varians, Pseudomonas aeruginosa, Klebsiella aerogenes, Alcaligenes sp., Corynebacterium sp., Bacillus sp., Arthrobacter sp., and Pseudomonas sp. Bacillus sp. and Pseudomonas sp. were the most occurring bacteria isolates in the four soil samples. From the physico-chemical analysis, the pH value ranged from 6.50-6.70, electrical conductivity ranged from 14.20mhos/cm-32.50mhos/cm, moisture content from 10.20%-14.50%, carbon from 1.38%-2.97%, nitrogen from 2.37%-8.19%, 0.396%-0.950%, phosphate 1.95%-5.52% and total hydrocarbon from 125mg/kg-3,050mg/kg These results revealed that the presence of these bacteria under the stated soil condition can enhance bioremediation of petroleum contaminated soil.

Keywords: Bacteria; Physicochemical analyses; Petroleum-products; Contaminated soil

Introduction

Activities associated with petroleum exploration, development and production operations have local detrimental and significant impacts on the atmosphere, soils and sediments, surface and groundwater, marine environment, biological diversity and sustainability at terrestrial ecosystems in the Niger Delta [1]. Discharge of petroleum hydrocarbon and petroleum-derived waste, streams have caused environmental pollution, adverse human health effects, and detrimental impact on regional economy, socio-economy problem and degradation of host communities in the 9 oil-producing states in the Niger Delta region [1]. Recently, anthropogenic practices such as industrial activities, petroleum and petroleum derivatives (such as gasoline, diesel, and kerosene spills), and incomplete combustion of fossil fuels have caused an accumulation of petroleum hydrocarbons in the environment [2]. In fact, petroleum and derivatives have a major ecological impact on contaminated marine and terrestrial ecosystems [2]. Many important processes influence the destination of hydrocarbons in the environment. Among these are sorption, volatilization, abiotic transformation (chemical or photochemical), and biotransformation [3]. Biodegradation of oil contaminated soils, which exploits the ability of microorganisms to degrade and/or detoxify organic contamination, has been established as one of the efficient, economic, versatile and environmentally sound treatment [4].

 The presence of a high enzymatic capacity allows microbial communities to degrade complex hydrocarbons [5]. This capacity to modify or decompose certain pollutants, such as petroleum, summarizes the importance of enzymes in the bioremediation process. Their genetic diversity contributes to the metabolic versatility of microorganisms for the transformation of contaminants into less-toxic final products, which are then integrated into natural biogeochemical cycles [5]. However, appropriate environmental factors such as pH, available nitrogen and phosphorus, Organic matter, moisture and carbon content are essential for the performance of these organisms. The application of nutrients to oil contaminated site to stimulate the growth of naturally occurring hydrocarbon utilizing bacteria for bioremediation purpose, can greatly improve the rate of recovering of environments contaminated with petroleum products. Therefore this study was aimed to Isolation of Bacteria and Physicochemical Analyses of Petroleum-Products Contaminated Soil from NNPC/PPMC Depot, Benin City, Nigeria.

Materials and Methods

Soil samples contaminated with petroleum products (diesel, petrol, pure and mixed kerosene) were collected from tank draining points from tank farm at NNPC/PPMC Benin deport. Samples were collected at different point from the tank farm into polyethylene bags which were duly labelled and transported to the laboratory. All glassware used were thoroughly washed, air dried and sterilized using an autoclave at 121oC for 15 min.

Physic-chemical analysis of soil samples

The pH was determined electrometrically by suspending the soil in 1: 2 (soil: 0.01M CaCl2) mixture. The suspension of the soil was allowed to stand for 30 minutes with occasional stirring and the pH measured with a pH meter [6]. Moisture an aluminium dish was pre weighed (W1) using a sensitive weigh balance (State Model). Ten (10) grams of the fresh soil sample was transferred to the dish and weight of both the dish and soil was noted (W2). The dish containing the soil sample was placed in a hot air oven (State Model) at 1300C and dried to obtain a constant weight for 24 hours. The dish was immediately transferred to a desiccator and allowed to cool for 30 minutes. The resultant weight was taken (W3). The moisture content was calculated and recorded as a percentage by weight of the respective soil sample.

Total organic carbon

The Total Organic Carbon was determined by the Walkley-Black titrimetric method. About 0.3 g of each soil samples were weighed into an Erlenmyer flask with 10 ml of K2CrO7 and 20 ml concentrationH2SO4 was added Figure 1. The mixture was gently swirled until soil and reagents were properly mixed and were allow to stand for 30 min, 100 ml of distilled water was added and the content titrated against standardized ferrous sulphate solution to a reddish brown end point using ferroin as the indicator. The total organic matter was calculated from the value obtained for the total organic carbon [7].

Figure 1: Total heterotrophic and hydrocarbon utilizing bacteria count (cfu/g).

 Total hydrocarbon content

Hydrocarbon-utilizing bacteria population was enumerated by spread plate technique [7] by inoculating 0.1 ml of aliquot onto sterile Mineral Salt Medium (MSM) plates with 100μl each of petroleum products viz; diesel (AGO), petrol (PMS), pure kerosene (DPK) and mixed kerosene (DPK). The petroleum products used was sterilized by filtering through Millipore filter, 0.45μ diameter and stored in sterile bottles. The petroleum products were used as the sole carbon source to isolate hydrocarbon-utilizing bacteria. The plates were incubated at 25°C for 3-5 days. Any isolate which grow on nutrient agar plates but failed to grow on mineral salt medium plate were confirmed as non-degraders. The isolates which grow on both the agar plates were confirmed as hydrocarbon utilizes. Total hydrocarbon content was calculated as described by [8].

Available phosphorus

One gramme (1.0 g) of soil was shaken for 5 minutes with 10 ml of extracting solution containing 0.03 N NH4F and 0.1 N HCl). The solution was filtered through Whatman filter paper and 3 ml of the filtrate was transferred into a test tube and 3ml of ammonium molybdate was added. Thereafter, 5 drops of mixture of boric acid, sodium sulphite and sodium sulphates were added. The Phosphorus content was determined calorimetrically [9].

Available nitrogen

One gramme (1.0 g) of the soil sample was placed into Kjedahl digestion flask. One table of a catalyst and 20 ml concentrated tetraoxo sulphate acid was added and the mixture was hand shaken to ensure mixing. At completion of digestion, 10 ml distilled water was added and the solution was filtered through a Whatman filter paper. Nitrogen was determined calorimetrically at 625 nm [9].

Total heterotrophic bacteria

THB population was enumerated by pour plate method. 1g of the sample was aseptically transferred into 100ml of physiological saline and transferred in a series of eight 10 fold serial dilution using physiological saline. 1ml of the aliquot from each of the dilution of 102,104,106 and 108 was inoculated by pour plate method onto Nutrient agar (NA) in duplicates. The plates were incubated aerobically at room temperature for 48-72 hrs. The resulting colonies were counted and recorded as colony forming units per gram (cfu/g) of soil sample [10].

Characterization and identification of isolates

The enumerated bacteria were isolated and stored in NA slants at 4°C for further identification. Primary identification was done on the basis of colony and cell morphology and Gram staining. Colonial examination of the isolates was carried out to determine the type of shape, elevation and pigmentation pattern they exhibited [11]. Secondary identification is carried out by performing a series of Biochemical tests [12].

Results and Discussion

The physicochemical parameters are show in Table 1. The pH of pure dual purpose kerosene (DPK) is slightly higher (6.7) than that of premium motor spirit(PMS), automobile oil and gas (AGO), and mixed dual purpose kerosene (DPK) which are 6.60, 6.50, and 6.60 respectively. The conductivity of pure DPK is higher (32.50mhos/cm) compared to the PMS, AGO and Mixed DPK. The moisture content of pure DPK was more with 14.50% while that of PMA AGO, and Mixed DPK were 10.20%, 10.80% and 10.20% respectively. Total Carbon content was higher in AGO (4.75%) compared to PMS, Mixed DPK and Pure DPK which have 2.19%, 1.38% and 2.97% respectively. Total organic matter, Nitrogen and Phosphate (8.19%, 0.95% and 5.52% respectively) were also higher in AGO. The total heterotrophic bacteria were higher in nutrient agar plates than in MSM which was spread with 0.1ml of petroleum products. The total heterotrophic bacteria count is shown in Table 2 & Table 3. The population density of heterotrophic bacteria in soil sample contaminated with PMS was 5.93×104 ±0.20 which was highest compared to the soil contaminated with AGO, pure DPK and mixed DPK with population densities of 3.53×104 ±0.15, 2.77x104 ± 0.24 and 1.83x104±0.09 respectively [13-17].

Parameters

PMS

AGO

Mixed DPK

Pure DPK

pH

6.6

6.5

6.6

6.7

Conductivity (mhos/cm)

14.6

14.2

15.1

32.5

Moisture (%)

10.2

10.8

10.2

14.5

Carbon (%)

2.19

4.75

1.38

2.97

Organic matter (%)

3.78

8.19

2.37

5.12

Nitrogen (%)

0.652

0.95

0.396

0.717

Phosphate (%)

3.71

5.52

1.95

4.03

THC (mg/kg)

1,860

3,050

125

1,040

Table 1: Physicochemical parameters of the soil samples contaminated with petroleum products.

Samples

Viable cell Count (cfu/ml)

Pure DPK

2.77×104 ±0.24

Mixed DPK

1.83×104 ±0.09

AGO

3.53×104 ±0.15

PMS

5.93×104±0.20

Table 2: Total Heterotrophic Bacterial Count (cfu/ml).

Where DPK-dual purpose kerosene, AGO-automobile oil and gas and PMS- premium motor spirit

Characteristics

1

2

3

4

5

6

7

8

9

Cultural Morphology

Elevation

Flat

Convex

Low convex

Convex

Convex

Low Convex

Low Convex

Low Convex

Low Convex

Margin

Serrated

Entire

Entire

Smooth

Entire

Entire

Entire

Entire

Entire

Colour

Cream

Orange

Green

Cream

Cream

Cream

Cream

Cream

Cream

Shape

Irregular

Circular

Circular

Circular

Circular

Circular

Circular

Circular

Circular

Morphological

Gram stain

+

+

-

-

-

+

+

+

-

Spore staining

+

-

-

-

-

-

-

-

-

Cell type

Rod

Cocci

Rod

Rod

Rod

Rod

Rod

Rod

Rod

Cell Arrangement

Chains

Single

Single

Single

Single

Single

Chain

Single

Single

Biochemical

Catalase

+

+

+

+

+

+

+

+

+

Oxidase

ND

ND

+

-

+

-

-

ND

+

Coagulase

ND

-

ND

ND

ND

ND

-

ND

ND

Urease

+

+

-

+

-

+

-

+

-

Indole

ND

-

-

-

-

-

-

-

-

Citrate

+

+

+

+

+

+

+

+

+

Glucose

+

+

+

+

+

+

+

-

+

Lactose

-

-

-

-

-

-

-

-

-

Table 3: Cultural, Morphological and Biochemical Characteristics of Bacteria Isolates.
Likely Identifiable organisms.

*1= Bacillus subtilis *2= Micrococcus varians *3= Pseudomonas aeruginosa *4= Klebsiella aerogenes * 5= Alcaligenes sp. *6= Corynebacterium sp.          *7= Bacillus sp.*8= Arthrobacter sp.*9= Pseudomonas sp.

References

  1. Aniefiok IE, Udo JI, Margaret UI, Sunday WP (2013) Petroleum Exploration and Production: Past and Present Environmental Issues in the Nigeria’s Niger Delta. American Journal of Environmental Protection1(4): 78-90.
  2. Santos HF, Carmo FL, Paes JE, Rosado AS, Peixoto RS (2011) Bioremediation of mangroves impacted by petroleum. Water, Air, and Soil Pollution 216(4) 329-350.
  3. Korda A, Santas P, Tenente A, Santas R ( 1997) Petroleum hydrobcarbon bioremediation: sampling and analytical techniques, in situ treatments and commercial microorganisms currently used. Appl Microbiol Biotechnol 48(6): 677-686.
  4. Margesin R, Schinner F (1997) Bioremediation of diesel-oil contaminated alpine soils at low temperatures. Applied Microbiological Biotechnology 47(4): 462-468.
  5. Alexander M (1994) Biodegradation and Bioremediation. Academic Press, San Diego, Califonia, USA.
  6.  Kalra YP, Maynard DG (1991) Methods Manual for Forest soil and Plant Analysis. Minster of supply and services Edmonton, Canada, pp. 125.
  7. Udochukwu U, Omoghie EM, Chikezie CC, Udinyiwe OC (2014) The Role of Bacteria in the Mineralization of Diesel-Base Engine Oil. International Journal of Pharmaceutical Science Invention 3(7): 50-55.
  8. Emitiazi GZ, Etemadifar M, Tavassoli (2003) A novel nitrogen-fixing cellulitic bacterium associated with root of cam is a candidate for production of single cell protein. Biomass Bioenergy25: 423-426.
  9. Onyeonwu RO (2000) Manual for Waste/Wastewater, Soil/ Sediment, Plant and Fish analysis. MacGill Environmental Research Laboratory Manual, Benin City pp. 81.
  10. Chikere CB, Okpokwasili GC, Ichiakor O (2009) Characterization of hydrocarbon utilizing bacteria in tropical marine sediments. African Journal of Biotechnology 8(11): 2541-2544.
  11. Udochukwu U, Inetianbor J, Omorotionmwan FO, Okpuruka NS (2016) Quality of Restaurants in Lokoja Metropolis and Its Public Health Impact. American Journal of Microbiological Research 4(2): 51-55.
  12. Cheesbrough M (2010) Microscopical Techniques Used in Microbiology In: District Laboratory Practice in Tropical Countries Part2. Cambridge Low-Price Edition, Cambridge University Press, USA, pp. 35-70.
  13.  Amund OO, Adebiyi AG (1991) Effect of viscosity on the biodegradability of automotive lubricating oils. Tribol Intern 24(4): 235-237.
  14. Nwachukwu SCU (2001) Bioremediation of sterile agricultural soils polluted with crude petroleum by application of the soil bacterium, Pseudomonas putida, with inorganic nutrient supplementations. Curr Microbiol 42(2): 231-236.
  15. Adriano PM, de Arruda APGK, Dejanira FA, Bonotto DM (2007) Laboratory Study on the Bioremediation of Diesel Oil contaminated soil from a petrol station. Brazilian Journal of Microbiology 38(2): 346-353.
  16.  Yousefi KD, Khodadadi A, Ganjidoust H, Badkoubi A, Amoozegar MA (2009) Isolation and characterization of a novel native Bacillus strain capable of degrading diesel fuel. Int J Environ Sci Tech 6(3): 435-442.
  17. Jyothi K, Babu KS, Nancy CK, Kashyap A (2012) Identification and Isolation of Hydrocarbon Degrading Bacteria by Molecular Characterization. Helix (2): 105-111.
 
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