Current Research in Microbiology and Biotechnology Vol. 2, No. 6 (2014): 495-500 Research Article Open Access

ISSN: 2320-2246

The Rhizosphere Effect on the Bacterial Genera Associated with Crude Oil Polluted Soil Ecosystem Kelechi Mary Ukaegbu-Obi1* and Chiaka Mbakwem-Aniebo2 1 2

Department of Microbiology, Michael Okpara University of Agriculture, Umudike, P. M. B. 7267, Abia State, Nigeria. Department of Microbiology, University of Port Harcourt, Port Harcourt,Rivers State, Nigeria.

* Corresponding author: Kelechi M. Ukaegbu-Obi; email: [email protected] Received: 01 October 2014

ABSTRACT

Accepted: 21 October 2014

Online: 01 November 2014

The rhizo-bacterial genera associated with these plants Cyperus amabalis, Desmodium triflorum, Phaseolus sp., Solenstemon sp., Mariscus sp were isolated, enumerated and studied. Statistical analysis showed no significant difference (P>0.05) between the rhizosphere and non-rhizosphere of total culturable heterotrophic and hydrocarbon-utilizing bacterial counts in both polluted and unpolluted soils. The rhizosphere effect values of the above named plants were determined. All the plants exhibited positive rhizosphere effects on the rhizo-bacteria. The rhizosphere effect ratio of the hydrocarbon-utilizing bacteria showed significant difference between the polluted and unpolluted soils of Phaseolus sp. (P=0.022), Solenstemon sp. (P=0.012). The hydrocarbon utilizing bacteria isolated were identified as Acinetobacter, Alcaligenes, Arthrobacter, Corynebacterium, Flavobacterium, Pseudomonas and Serratia spp. All the isolates grew on petroleum hydrocarbon at different growth rates. Based on these results, the hydrocarbon utilisers isolated can serve as seeds for bioaugmentation during remediation of crude oil polluted soil while the plants may be employed in rhizoremediation of oil polluted soil.

Keywords: Rhizosphere effect, Bacterial genera, Oil spill, Soil ecosystem, Bioremediation, Hydrocarbons. INTRODUCTION

Worldwide increase in the use of petroleum and its products has led to severe contamination and ground water. In Nigeria, the exploration and exploitation practices and the breaking of oil pipes lead to incessant pollution especially in the Niger Delta area and Southern part of Nigeria [1]. These spills have the largest immediate and economic impact as they harm, to a large extent, the ecosystem more than just the isolated location [2]. The presence of petroleum hydrocarbons in the environment is of considerable public health and ecological concern, given their persistence, toxicity and ability to bio-accumulate [3]. These pollutions, have directly and indirectly led to human and environmental health risks. Thus, there is need for removal of these pollutants from the environment [4]. In the past two decades, the use of bioremediation has been growing, because of the better understanding of microbial processes in the soil. Especially sites polluted with polycyclic aromatic hydrocarbons (PAHs) are

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treated with some success, with Exxon Valdez oil spill as an important example [5]. The application of bioremediation capabilities of indigenous organisms to clean up pollutants is viable and has economic values [6].

Remediation of soils containing organic pollutants can be enhanced by plants by various processes [7, 8]. The use of plants to extract, sequester or detoxify pollutants is therefore known as phytoremediation [9,10]. Plants frequently do not possess complete metabolic degradation pathway for pollutants, and even more toxic by-products may be produced.

The area around plant roots, known as the rhizosphere contains higher populations, greater diversities and activities of microorganisms than soil with no plants [11]. This causes the emergence of a green technology which employs the symbiotic relationship between plants and their rhizo-microorganisms in the breakdown of contaminants to clean up the 495

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environment. This technique is referred to as rhizoremediation. The key mechanism of rhizoremediation is the rhizospheric effect enhancing biodegradation [12, 13].

In the case of crude oil which is quite complex in nature, a consortium of microbes is required for its breakdown into carbon (IV) oxide and water by oxidation. Compounds to be degraded are attacked within the crude oil mixture due to the ability of the individual microbes to grow well on the hydrocarbons. The wide array of microbial metabolic capabilities enables microbes to act as nature’s incinerators which remove or reduce pollutant concentrations to levels that are no longer dangerous to human health and the environment in general [14]. Crude oil also provides an excellent chemical environment in which cometabolism can occur because it contains a multitude of potential primary substrates. Soil microbial communities vary depending on soil physical and chemical properties, type and amount of plant cover and climate. It is well known that plants influence the biodiversity of bacteria in soils. Through the release of compounds such as amino acids, sugars and growth factors in plant root exudates, microbial activity and growth are stimulated [15]. Bacteria respond differently to these compounds, differences in the composition of root exudates can influence the type of bacteria present in the rhizosphere community. Although, all plants show a rhizosphere effect, the plant species can influence the type of bacteria that are present in the rhizosphere [16].

This research was conducted, therefore, to determine the amount of colony-forming bacteria present, to isolate and identify the hydrocarbon-utilizing bacterial genera associated with the rhizosphere of these plants: Cyperus amabalis, Desmodium triflorum, Phaseolus sp., Solenstemon sp., Mariscus sp., found in crude oil polluted soil in Odigiri and furthermore to determine the rhizosphere effect ratios of these plants on their rhizobacteria and to know the potential of the associated bacteria isolated to utilize crude oil.

MATERIALS AND METHODS

Study Site Polluted rhizosphere and non-rhizosphere soil samples were collected from crude oil polluted sites in Odigiri, Rivers State, Nigeria. These sites could be described as recovering ecosystems with few plants growing at these locations. Unpolluted rhizosphere and nonrhizosphere soil samples were collected from the same areas where there has been no known crude oil pollution which served as the control.

Sample Collection Plants of the same species were collected from the crude oil polluted and unpolluted sites in separately in marked sterile plastic bags. The plants were pulled out slowly to avoid breaking their roots. Non-rhizosphere (bulk) soil samples of crude oil polluted and unpolluted

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sites were collected at a distance of thirty centimeters (30cm) from the plants’ roots in marked sterile plastic bags and transported in an ice chest to the laboratory for analyses, after which the plants were taken to a Plant taxonomist for identification.

Sample Processing The roots were freed from adhering soil which is assumed to be the rhizosphere soil. One gram (1g) of the soil samples was aseptically added to different test tubes containing nine millilitres (9ml) of sterile distilled water. The test tubes were vigorously shaken to dislodge the microorganisms that might have adhered to the soil particles. The content of the tubes were serially diluted. From each dilution of 10-3 to 10-6, aliquots (0.1ml) of the serially diluted soil samples were plated on sterile Nutrient agar (NA) and Mineral Salt agar.

Isolation and Enumeration of Total Culturable Heterotrophic Bacteria and Hydrocarbon-Utilizing Bacteria Aliquots (0.1ml) of the serially diluted soil samples were plated out in duplicates on Nutrient agar (Oxoid) plates using spread plate method as described by Chikere et al. [17].The plates were incubated at 35oC for 24 to 48 hours for the total culturable heterotrophic bacteria count.

The modified mineral salt medium of Okpokwasili and Okorie [18] was used for the enumeration of the hydrocarbon-utilizing bacteria. It contained the following in g/l: NaCl 10.0g, MgSO4 0.42g, KCl 0.29g, KH2PO4 0.83g, NaPO4 1.25g, NANO3 0.42g, agar 15g and distilled water 1000ml. A sterile Whatman No.1 filter paper saturated with crude oil was aseptically placed on the inside cover of each Petri dish. The Petri dishes were incubated with the agar side up at 35 0C for 5 to 7 days. The crude oil saturated filter paper supplied the bacteria with the carbon and energy required through the vapour phase transfer of the hydrocarbon. After incubation, colonies where counted from duplicate plates and the mean counts recorded. Identification of Isolates The bacterial isolates were examined for colonial morphology, cell micro-morphology and biochemical characteristics. Tests employed included: Gram staining, Motility test, Catalase test, Citrate Utilization test, Indole test, Hydrogen Sulphide Production test, Methyl Red - Voges Proskauer test, Oxidase test, Sugar Fermentation test. Confirmatory identities of the bacteria were made using the Bergey’s Manual of Determinative Bacteriology [19]. Screen Test for Hydrocarbon-Utilization by Bacterial Isolates The bacterial isolates were tested for their ability to utilize crude oil using the turbidity method as described by Ibrahim et al [20]. The bacterial isolates were cultured in nutrient broth and incubated at 28+20C for 24 hours. Aliquot (0.1ml) of the young 496

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culture in nutrient broth grown was inoculated into each test tube containing 9.9ml of sterile mineral salt broth and 0.1ml of crude oil. A control test tube containing 9.9ml of sterile mineral salt broth plus 0.1ml of crude oil remained uninoculated. The tubes were incubated at room temperature for 7 days. The growth of the inocula was determined by visual observation of the mineral salt broth turbidity, as compared with the uninoculated control tube according to Okpokwasili and Okorie [18].

to analyse the polluted and unpolluted soil sample of each plant.

RESULTS AND DISCUSSION

The results of the enumeration of the total culturable heterotrophic bacterial counts of the polluted and unpolluted rhizosphere and bulk soil are shown in Figure 1.The total culturable heterotrophic bacteria counts for polluted rhizosphere ranged from 1.03 x 106cfu/g - 1.37 x 106 cfu/g, the unpolluted rhizosphere (control), ranged from 4.20x105 cfu/g - 7.55 x 105 cfu/g; the polluted non-rhizosphere, ranged from 2.56 x 105 cfu/g - 3.12 x 105cfu/g; the unpolluted nonrhizosphere, ranged from 3.10 x 105cfu/g - 4.12 x105 cfu/g.

Statistical Analysis The statistical tools – One-way Analysis of Variance (ANOVA) was used to analyse the data obtained from the plants while Independent Student’s t-test was used 6

5.8

Mean log TCHC (cfu/g)

5.6

5.4 Polluted Unpolluted 5.2

5

4.8

Rhizosphere

Mariscus sp.

Solenstemon sp.

Phaseolus sp.

Desmodium triflorum

Cyperus amabalis

Mariscus sp.

Solenstemon sp.

Phaseolus sp.

Desmodium triflorum

Cyperus amabalis

4.6

Non-Rhizosphere

Figure 1. Total heterotrophic bacterial counts of polluted and unpolluted rhizosphere and non-rhizosphere samples at Odigiri. 6

Mean log HUBC (cfu/g)

5

4

Polluted Unpolluted

3

2

1

Rhizosphere

Mariscus sp.

Solenstemon sp.

Phaseolus sp.

Desmodium triflorum

Cyperus amabalis

Mariscus sp.

Solenstemon sp.

Phaseolus sp.

Desmodium triflorum

Cyperus amabalis

0

Non-Rhizosphere

Figure 2. Hydrocarbon-utilizing bacterial counts of polluted and unpolluted rhizosphere and non-rhizosphere samples at Odigiri.

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The various counts of the hydrocarbon-utilizing bacteria are shown in Figure 2. The polluted rhizosphere ranged from 7.49 x 105 cfu/g - 1.05 x 106 cfu/g; the unpolluted rhizosphere ranged from 1.85 x 105 cfu/g - 3.38 x 105 cfu/g; the polluted nonrhizosphere, ranged from 1.02 x 105 cfu/g 1.32x105cfu/g; the unpolluted non-rhizosphere, ranged from 7.10x104cfu/g - 8.90 x 104 cfu/g. These values were obtained from the ratio of the mean counts in the rhizosphere (R) to the mean counts in the

non-rhizosphere (S) i.e. (R/S) for both the polluted and unpolluted soils. The rhizosphere effect ratio values show the effect of the plant roots on rhizo-bacteria. The results for the rhizosphere effect ratio of total culturable heterotrophic count and hydrocarbonutilizing bacteria are shown in the Figures 3 and 4. The crude oil utilization test as source of carbon and energy is presented in Table 1. The bacterial isolates were able to use crude oil at varied rates.

1.16

1.14

1.12

Mean log TCHC

1.1

1.08

Polluted

1.06

Unpolluted

1.04

1.02

1

0.98 Cyperus amabalis

Desmodium triflorum

Phaseolus sp.

Solenstemon sp.

Mariscus sp.

Rhizosphere Effect Ratio

Figure 3. Total heterotrophic bacterial counts for rhizosphere effect ratio for polluted and unpolluted soil at Odigiri 1.22

1.2

1.18

Mean log HUBC

1.16

1.14 Polluted Unpolluted

1.12

1.1

1.08

1.06

1.04

1.02 Cyperus amabalis

Desmodium triflorum

Phaseolus sp.

Solenstemon sp.

Mariscus sp.

Rhizosphere Effect Ratio

Figure 4. Hydrocarbon-utilizing bacterial counts for rhizosphere effect ratio for polluted and unpolluted soil at Odigiri

The extensive use of petroleum products leads to the contamination of almost all components of the environment [21]. On the other hand, increasing petroleum exploration, refining and other allied industrial activities in the Niger Delta have led to the

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wide scale contamination of most of its creeks, swamps, rivers and streams with hydrocarbons and dispersant products. The contamination of these habitats constitutes public health and socio-economic hazards [22]. 498

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In this study where the amount of colony-forming bacteria present were determined, the results show that total culturable heterotrophic bacterial counts were higher in the polluted rhizosphere than in the polluted non-rhizosphere of all the plants (Figure1). The total culturable heterotrophic counts were higher also in the rhizosphere than in the bulk soil. This agrees with the findings of Amadi et al [23], who observed high proliferation and metabolism of various microbial types in plant rhizosphere. Figure 1 also shows that the

mean counts of total culturable heterotrophic bacteria in the polluted non-rhizosphere were less than in the unpolluted non-rhizosphere of all the plants. It has however, been demonstrated that microbial communities can affect chemical pollutants, although the presence of chemical pollutants can also adversely affect the microbial community structure. John et al [24] reported that heavy pollution decreases the microbial indices while very light pollution serves as nutrient to the organism.

Table 1. Utilization test of crude oil by the bacterial Isolates

ISOLATE CODE CRIO 1A CSIO 2B CSIO 3A CRIO 5A USIO 1B USIO 2A URIO 3A URIO 3A URIO 4B URIO 5B

GROWTH IN CRUDE OIL MEDIUM

+++ = Heavy growth

++ ++ ++ ++ ++ + + + ++ + ++ =Moderate Growth

The hydrocarbon-utilizing bacterial counts were higher in the polluted rhizosphere than in the unpolluted rhizosphere of all the plants (Figure 2). Reilley et al [25] showed that degradation of pyrene increased in the rhizosphere soil and that the highest pyrene mineralization rate was found when organic acids, typically found in the root exudates, were added to the soil. Statistically, there was no significant difference in mean bacterial counts between the polluted rhizosphere and unpolluted rhizosphere and amongst the plants (P>.05). It was also observed that the mean counts of the hydrocarbon-utilizing bacteria were higher in the polluted non-rhizosphere soil for all the plants than that of the total culturable heterotrophic counts (Figure 2). Leahy and Colwell [26] stated that the presence of the pollutants which serve as nutrient can alter the community structure of the soil through selection of pollutant degraders, thereby causing the numbers of microorganisms that can utilize the compound of interest to increase within the community.

The results obtained in this study reflect the positive rhizosphere effects of all the plants on the bacterial communities as indicated in the rhizosphere effect ratios (Figures 3 and 4). The rhizosphere effect ratio values [R/S] were more pronounced in polluted site than in unpolluted site. Walton et al [27] speculated that when a chemical stress is present in the soil, a plant may respond by increasing or changing exudation to the rhizosphere, which modifies the rhizosphere microflora composition or activity. As a result, the microbial community might increase the transformation rates of toxicant. Murotova et al [28] also explained that the success of phytoremediation of hydrocarbon contaminated soil is connected with

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BACTERIAL ISOLATE

Arthrobacter sp. Corynebacterium sp. Alcaligenes sp. Acinetobacter sp. Pseudomonas sp. Flavobacterium sp. Arthrobacter sp. Serratia sp. Pseudomonas sp. Corynebacterium sp. + =Minimal Growth

plants’ capacity to enhance microbial activity in the rhizosphere. The composition of the microbial population in the rhizosphere depends on the composition of the exudates as well as on the plant species, root type, plant age, soil type and history of soil [29]. It is known that bulk soil and rhizosphere microbial community structure is determined by the local native microbial community, impacted by the soil effects and vegetation [30]. The bacterial genera isolated from different rhizosphere and nonrhizosphere of polluted soils in this study were identified as Acinetobacter sp., Alcaligenes sp., Arthrobacter sp., Corynebacterium sp., Flavobacterium sp., Pseudomonas sp. and Serratia sp. Similar organisms have been reported by Antai; Ekhaise and Nkwelle [31,32]. The result of the hydrocarbon utilization test by the bacterial isolates presented in Table 1 showed that the isolates were able to utilize crude oil as their carbon and energy source. These bacterial isolates grew at different rates- heavily, moderately and minimally. The moderate growth of most of the bacterial isolates on the petroleum hydrocarbon may be as a result of each isolate not having a broad enzymatic capacity to degrade crude oil which is a complex hydrocarbon. Ghazali et al [33] reported that biodegradation of complex hydrocarbon usually requires the cooperation of more than a single species. This is particularly true for pollutants that are made up of many different compounds such as crude oil or petroleum where complete mineralization to carbon (IV) oxide and water is desired. Individual microorganism can metabolize only a limited range of hydrocarbon substrates, so assemblages of mixed populations with overall broad enzymatic capacities are required to bring the rate and extent of petroleum biodegradation further. Leahy and 499

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Colwell [26] observed that greater degradation of a mixed hydrocarbon substrate and South Louisiana crude oil occurred by sediment bacteria from an oilpolluted harbor than by same bacteria from a relatively unpolluted environment.

CONCLUSION

The biodegradation of petroleum hydrocarbons in the environment is a complex process, whose quantitative and qualitative aspects depend on the nature and amount of the oil or hydrocarbons present, the ambient and seasonal environmental conditions and the composition of autochthonous microbial community and their adaptive response to the presence of hydrocarbons.

The findings of this work depict that crude oil utilizing bacteria can be found in the rhizosphere of Cyperus amabalis, Desmondium triflorum, Phaseolus sp., Solenstemon sp., Mariscus sp. Qualitative differences in root exudates among these plants also induced the increase of bacterial population in the rhizosphere. The screen test for hydrocarbon utilization by the bacterial isolates showed that these isolates could be used as seeds for bioaugmentation during remediation of petroleum contaminated soil. On the basis of the findings in this study rhizoremediation is strongly considered as an alternative management option for remediation of oil polluted soil.

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