Distribution, Biochemical Properties and Genetic Relatedness of Endophytic Bacteria of Wet land Plants from Petroleum-Contaminated Sites of the Niger Delta, Nigeria

Microbe-assisted phytoremediation is a recent application of bioremediation with much prospects. The genetic relatedness of culturable endophytic bacteria of wetland plants growing on a six month-old and twelve month-old petroleum-contaminated sites, and an uncontaminated site in Bayelsa State of the Niger Delta Region, Nigeria were compared. Most of the endophyte species isolated from the roots, stems and leaves were common to all the sites and belong to the phyla Proteobacteria, Bacteroidetes Firmicutes, Actinobacteria, Chloroflexi and Actinomicrobia, with the γ-Proteobacteria dominating. Pseudomonas was the most prevalent species in all three sites, but higher in the petroleum contaminated sites. Biochemical properties (API 20E) of the common isolates; Pseudomonas spp.Chryseobacterium indologenes,Bacillus and Proteusvaried with sites while only Providencia rettgeri peculiar to the petroleumcontaminated sites showed the same properties. 16S rRNA PCR-DNA fragments of forty-five species of the isolates (15 from each site) were characterized using RFLP and MspI restriction enzyme and a genetic distance tree of the restriction patterns drawn. The percentage of similarity in the genetic relatedness of isolates ranged from 11.1 – 100%. The genetic tree analysis of the 45 species of identified bacteria revealed 3 major clusters with 17 DNA fingerprinting patterns. Pseudomonas species of the root and leaves of the six month-old petroleum-contaminated site and uncontaminated site were seen to cluster together irrespective of date of isolation. The endophytes may play a role in the in situ degradation of the petroleum hydrocarbon of the sites. Keywords— Endophytic bacteria, Petroleum, Phytoremediation, Wetlands, Wetland plants.


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Page | 156 resident plants cannot. The efficiency of phytoremediation is attributed to the presence and activity of plant associated microorganisms carrying genes for the degradation process [11]. The use of genes from endophytes in enhancing the efficiency of remediation in genetically engineered plants has continued to be explored [9,12,13,14,15,16]. Inoculating plants with endophytic bacteria have been shown to improve phytoremediation of petroleum [16,17]. Wetland or aquatic plants are used as food, in water quality assessment and as in-situ biomonitors and bioremediators [18]. They are frequently used in wastewater treatment in agricultural landfill and urban storm water runoff, to remove heavy metals and toxic organics from acid mine drainage, and as nutrients [19,20]. Aquatic plants are used in waste treatmentbecause they accumulate many pollutants and toxic substances efficiently due to their non-complex growth requirements and fast growth rates [20] Endophytes in wetland plants may be able to remove organic pollutants from wetlands . However, little investigation into the distribution and functions of endophytic bacteria of wetland plants is recorded. The occurrence of Gram-positive and Gram-negative endophytic bacteria ofaquatic plants; Phragmitescommunis, Nymphaeatetragona, Najas marinaandPotamogetoncrispuswere reported in which some of the organisms isolated degraded naphthalene and pesticides and in addition, showed potential to dissolve insoluble phosphate [5].
The study was carried out to investigate and compare the occurrence, distribution and biochemical properties of culturable endophytes in wetland plants of both petroleumcontaminated and uncontaminated soilsin wetlands of Bayelsa State, Nigeria; an oil rich region of the Niger Delta.

2.1
Sampling Location and Collection A random sampling of wetland plants growing in three freshwater soil locations ( Fig. 1) were conducted. The first location is a six month-old petroleum contaminated soil at Oloibiri Oil field, Ogbia LGA, Latitude 4. Sample Processing for Culturable Endophytic Bacteria After the removal of rhizosphere soil, plant samples from each site were rinsed with sterilized water to wash out sediments. The roots, stems and leaves of each plant species were separated to give three subsamples which were cut into 1cm pieces. Each subsample was washed in sterilized water for 5 minutes, then surface-sterilized with a solution containing 5% active chloride (w/v, added as a NaOCl solution) for 3 minutes and 70% ethanol for 1 min. This was then followed by rinsing 4 to 5 times in sterile deionized water. The plant subsets were further cut into 0.3 -0.5 cm pieces with sterile blades and ground in a mortar using a glass rod plus 0.1ml sterile deionized water. To ensure that there was no cross contamination of plant parts during processing, the glass rod and mortar were surface sterilized using sterile cotton wool dipped in 70% ethanol. Each plant slurry of the roots, stems and leaves was spread onto Luria-Bertani (LB) agar plates in triplicate and incubated at 25 o C for 24 -48 hours after which isolates were identified and stored at -70 o C in 20% glycerol. For sterility check, 100µl of the last rinse of each subsample in sterile deionized water was plated out on LB agar and incubated at 20 o C for 24 -48 hours [5,21]. No growth indicated the absence of contaminants (rhizosphere and phyllosphere bacteria).

Identification and Characterization of Isolates
Growth characteristics ofpure cultures of the isolates on Luria Bertani (LB) agar were observed after incubation at 25 o C for 24 -48 hours. Characteristics of colonies observed include colour, margin, elevation, consistency, opacity and approximate size. Growth and lactose fermentation on MacConkey agar was noted. The API 20 E identification kit (Biomereux, France) was used for the identification of members of the Enterobacteriaceae and other Gram negative bacteria based on 21 different biochemical tests and a database. The test strip contained tests for betagalactosidase, arginine dihydrolase, lysine and ornithine decarboxylases, citrate utilization, hydrogen sulphide production, urea hydrolysis, deaminase, indole and acetoin production (Voges-Proskauer), gelatinase and sugar fermentation tests. Gram positive isolates were also identified using some of the biochemical tests mentioned above in addition to sugar fermentation tests. Characterization of strains were done depending on their morphology on gram staining and biochemical tests. buffer solution containing50mM of Tris, 5mM EDTA, 50mM of NaCl, plus the addition of acetic acid to pH 8.0 was prepared and dispensed into Eppendorf tubes. Fresh colonies of the isolates were added and vortexed for complete mixing. 50mM of Tris, 25 % sucrose and 1mg/ml lysozyme were added and the mixture vortexed. Addition of 5 % SDS and 50mM of Tris was followed by inverting the Eppendorf tubes 3-5 times after which the lysates were incubated at 56 0 C for 1hr. The DNA extraction from the lysates involved the addition of 500 μl phenol/chloroform/isoamyl alcohol in ratio 25:24:1. Ultracentrifugation was carried out at 14,000rpm for 15mins and the supernatant dispensed into new Eppendorf tubes. The phenol/chloroform/isoamyl alcohol procedure was repeated and the supernatant transferred again into another set of Eppendorf tubes. The addition of 200 µl of 3M sodium acetate (pH 6.0 through the addition of glacier acetic acid) and 1ml of absolute ethanol was followed by vortexing, and the mixture allowed to precipitate on ice overnight. This was followed by centrifugation at 30,000rpm for 30mins and the supernatant discarded. The precipitate (chromosomal DNA) was washed twice with 70 % ethanol, centrifuged at 14,000rpm for 5 mins and air-dried after which sterile distilled water was added and kept below freezing temperature before further analysis [23] 2.4.2 Polymerase chain reaction probe for 16s RNA All primers were purchased from Jena Bioscience GmbH, (Jena, Germany) and were dissolved according to manufacturer's instructions. The primer used was universal 16S rRNA gene primers: 8f: 5'-AGA GTT TGA TCC TGG CTC AG-3' and 1492r: 5'-GGT TAC CTT GTT ACG ACTT-3.The PCR mixture (50 µl) contained 5 µl of 10× PCR buffer with 15 mmol l-1 MgCl2 (Takara), 200 µmol l-1 of each deoxynucleotide triphosphate (Takara), 10 pmol of each primer (Applied Biosystems), 1.5 units of TaqDNA polymerase (Takara) and 1 µl of DNA template. The mixture was vortexed for proper mixing. The PCR was performed in a thermocycler, with a thermal profile of 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 45 s and extension at 72°C for Construction of a genetic distance tree A similarity matrix of the strains and the related bacterial 16S rRNA was constructed by the neighbor-joining method using computational phylogeny inference package (PHYLIP, Washington DC, USA). Informative DNA fragments, derived from restriction endonuclease digestion longer than 100 bp, were scored for their presence or absence. The similarity and divergence were calculated. The similarity matrix indices and genetic distance trees were constructed based on the RFLP data from the 16S rRNA using DICE and UPGMA (Unweighted Pair Group Method with Arithmetic Mean) clustering methods.

III.
RESULTS Endophytic bacteria were isolated from the roots, stems and leaves of individual plant species picked from all three sites.  (TABLE 3). Combining all the isolates from the three sites, a total of 156 endophytes were isolated from all the plants with four genera common to all the sites (TABLE 4). Pseudomonas spp. had the highest frequency of occurrence of 20.51%, followed by Chryseobacterium indologenes (11.54%). Bacillus (10.26%) and Proteus (7.7%).In addition, Providencia rettgeri and Sphaerotilus natans were common to only the petroleum contaminated sites with a frequency of 10.26% and 1.92% respectively. There were more isolates of Providencia rettgeri, Bacillus spp., Proteus spp. and Sphaerotilus natans in site A than site B while Pseudomonas spp. and Chryseobacterium indologenes were more in site B than site A. Corynebacterium, Micrococcusand Aeromonas were found in site A only while Burkholderia, Serratia, Alcaligenes, Vibrio, Morganella and Type 0092 were in site B only.

Staphylococcus,
Myroides, Citrobacter youngae, Enterobacter cloaceae, Pastuerella pneumotropica and Microthrix parvicella were not isolated in both petroleum contaminated sites. Biochemical tests (API 20E) utilized to differentiate common organisms of the three sites is shown in TABLE 5.Pseudomonas spp.Chryseobacterium indologenes,Bacillus and Proteusoccurred in all three sites while Providencia rettgeri and Sphaerotilus natanswere of the petroleum-contaminated sites only.Only Providencia rettgeri of the contaminated sites and Ps. putida of the twelve month-old petroleum-contaminated site and the uncontaminated site exhibited the same biochemical characteristics. Some strains (total of 45) were selected from the different sites, source and their respective dates of isolation were subjected to DNA fingerprinting analysis using MspI restriction endonucleases (TABLE 6). The percentage of similarity in the genetic relatedness of isolates ranged from 11.1 -100% (TABLE 7). The genetic tree analysis of the 45 species of identified bacteriarevealed 3 major clusters with 17 DNA fingerprinting patterns (        Fig. 3 (a, b, c).Products of PCR-RFLP of isolated strains using MspI restriction endonuclease generated from 16S rRNA

IV. DISCUSSION
Aquatic macrophytes have long been used as biomonitors of environmental pollution and in the phytoremediation of wastewaters. Aquatic plants common to both petroleum-contaminated and uncontaminated sites were Commelina benghalensis, Chromoleana odorata and Asplilia africana. Ageratum conyzoides, Ipomoea involucrata and Kyllinga species (Kyllinga erecta and Kyllinga pumila) were common to the petroleum contaminated sites. These plants have also been found in both petroleum-contaminated and uncontaminated sites, and in the remediation of heavy metals [24]. Other plant species of the twelve-month old petroleum contaminated site were Fimbristylis littoralis, Sacciolepsis africana, Cyperus difformis, Solenostemon monostachyus and Echinochloa obtusiflora. Fimbristylis littoralis was not reported in petroleum polluted sites but as a common weed of the Niger Delta. It has the potential for enhanced phytoremediation of PAHs and heavy metals [25].The presence of these plants in a twelvemonth old contaminated site shows its survival and phytoremediation capabilities. Cyperus difformis shows the ability to survive in petroleum contaminated soil and to spring up after remediation. Solenostemon monostachyus, Echinochloa obtusiflora andother species of Echinochloa are potential phytoremediators. These plants could be able to remediate toxic metals since they are present in petroleum. This study is in agreement with past findings [24,26].Aquatic macrophytes of the noncontaminated sites include Diplazium sammatii Dissotis rotundifolia, Anielema sp., Panicum laxum, Scleria verrucosa, Cyathula prostrata and Costus sp.The nontolerance of Diplazium esculentum to chromium [27] and the absence of the genus in petroleum contaminated soils as reported in this study could indicate their inability to survive and grow in the presence of toxic substances, therefore, unsuitable for phytoremediation. Plants are known to harbour endophytes which are useful for the growth, development and maintaining functional activities of the plant. The association is beneficial for both the plants and the endophytes. Endophtye numbers of the plants varied with the plant species; 2-6 bacterial species were isolated from each plant and at least one species occurred in the root, stem and leaf [1]. The number of endophytes are said to decrease from their point of entry i.e. root region to the shoot and leaf [28]. In this study, more endophytes were found in the roots o f the plants from the petroleum-contaminated sites probably because of the increase in number of petroleum degraders in the rhizosphere of the plants. More endophytes are found in the root because these microorganisms from the soil colonize the root zone before entering the plant [29]. Nine genera of endophytes were isolated from the six month-old petroleum-contaminated soil (site A) most of which were Gram negative members of the Proteobacteria, particularly of the Class γ-proteobacteria; More bacterial genera were present in this site but their numbers were fewer compared to the petroleum-contaminated sites which suggests the proliferation of specific degraders of the hydrocarbon in these sites. Past studies on plant endophytic bacteria have reported the dominance of the Proteobacteria, particularly the γproteobacteria with Pseudomonas as the most frequently isolated, and a large number of the α-proteobacteria and β-proteobacteria, but fewer numbers of the Firmicutes, Actinobacteria and Bacteroidetes [7,30]. The dominance of the γ-proteobacteria is in agreement with the findings of this work but more Bacteroidetes and Firmicutes were isolated than β-proteobacteria. Endophytes ofsome aquatic plants were found to have γproteobacteriaparticularly, Pseudomonas spp.and Bacilli predominant [5] and others;Phragmitesaustralisand Ipomoea aquatica, had predominantly, γ-proteobacteria and mostly Pseudomonas spp.Acinetobacter spp., Stenotrophomonas spp. with less of Serratia spp. Chryseobacterium spp. and Erwinia spp. Of the fewer gram-positive bacteria the predominant strains were Bacillus spp., Paenibacillus spp., and Microbacteriumspp. [31]. Bacillus spp. were predominant in this study. Gram positive bacterial genera could be few in petroleum-contaminated sites but can actually dominate the bioremediation process considering their metabolic versatility [32]. The γ-Proteobacteria were the most cultured group of bacteria while the other groups differed slightly according to the location where plants were picked. Bacteroidetes was the second dominant phylum occurring more in the twelve month-old contaminated site followed by the Firmicutes occurring more in the six month-old site, Actinobacteria (absent in site B) and β-proteobacteria (more in site B) occurred in equal numbers while other filamentous bacteria (least found) were specific to the uncontaminated site. This suggests that the endophytes may be site specific and their diversity may be dependent on the species of plants present at the sites . Providencia rettgeri was present in only the petroleum-contaminated sites. This genus is also a reported soil and rhizosphere isolate of oil-polluted sites but not as an endophyte. Its presence as an endophyte of only the polluted sites is noteworthy.Providencia rettgeri is usually an important pathogen of insects and humans [33,34].Sphaerotilus natans, like other filamentous bacteria, are found in sewage and wastewater treatment plants , and occur in marshlands and ponds and water bodies .Thegenus Sphaerotilusconsist oforganoheterotrophic bacteria, but occurred in plants of the oil polluted sites only. This implies that the organisms also utilized the hydrocarbon as an energy source in the plants.Compartmentalization of the endophytes showed that the β-proteobacteria occurred mostly in the stem, the γ-proteobacteria, Firmicutes and Actinobacteria in the roots and Bacteroidetes in the roots and stems.Increasing petroleum hydrocarbon concentrations was shown to enhance the proliferation of Proteobacteria (γ-proteobacteria) in the root while βproteobacteria favored the stem [35,36]. Biochemicalcharacterization of common is olates showedChryseobacterium indologenesof all the sites were citrate utilizers, urea was broken down by the organism in the twelve month-old contaminated site and the uncontaminated site. This ability to produce urease would be related to the presence of urea (a protein of animal origin) in the environment. Gelatinasewas produced by the organisms from the petroleum-contaminated sites. As with other strains of Ps. aeruginosa, L-arginine, citrate and gelatin were utilized by the organisms in the three sites, urea was hydrolysed by the strain in site B, again signifying the presence of the protein in the environment, while rhamnose was fermented in the strain occurring in the uncontaminated site and amylose fermented by that of the six month-old contaminated site. These sugar fermentation abilities could be site related. Pseudomonas sp. of site A and Ps. flourescens of site B (petroleumcontaminated) produced enzymes argininedihydrolase,gelatinase and utilized citrate, andcould be the same species. Both Ps. putida of the uncontaminated site and the twelve month-old contaminated site showed the same biochemical properties (L-arginine and citrate utilization). Proteus vulgaris group was peculiar to the six month-old contaminated site with its biochemical properties while Proteus mirabilis of same metabolic properties were at site B and C ( soils [37]. The same biochemical properties were shown by Providencia rettgeri of the petroleum-contaminated sites which suggests their genetic similarities. Sphaerotilus natans of site B produced beta galactosidase, giving it an added nutritional advantage over that of site A which did not. Biochemical properties of Bacillus species of the three sites differed. Species of the six month-old contaminated site showed a wide nutritional ability by fermenting sugars and utilizing L-arginine in addition to citrate, urea and production of enzyme deaminase (tryptophan) and gelatinase. The species of the twelve month-old site had limited biochemical properties showing only citrate utilization and gelatinase activity while Bacillus of the uncontaminated site produced indole in addition to citrate, urea, tryptophan and gelatin hydrolysis . Differences in biochemical properties of the endophytes could be attributed to conditions in the sites related to soil characteristics; structure and texture of soils, nutrients available, moisture and other environmental factors. These endophytes were isolated from different wetland plants occurring in different habitats and with specific abilities. All these factors could have contributed to the adaptability of the endophytes in them. Genetic analysis of the clonal relatedness of the bacterial species showed 100 % relatedness of Pseudomonas aeruginosa of the six month-old petroleum-contaminated site and the uncontaminated site, as well as Pseudomonas putida. The same species of organism was isolated at different times, location and in different plant compartments. This shows strong physiological similarities and probably, same abilities of the two as petroleum degraders. Site A and site C are located in the same region where oil was first discovered in Nigeria (Ogbia Local Government Area). Other members of the γproteobacteria also clustered together. The percentage of clonal relatedness ranged from 11.1 -100% genetic homology, which shows diverse distribution in the genetic components and capabilities of strains. The 16S rRNA generated data of DNA bands showed 100% genetic homology of same species and closely related bacterial group which indicated the possibility of possession of similar traits associated with bioremediation. On the other hand, the clustering of species of different phyla shows some similarities between the species but highlights the possible shortcomings of the PCR-RFLP method in phylogenetic analysis [38].

V.
CONCLUSION The present study has highlighted a variety of wetland plants and their accompanying endophytes with potentials for phytoremediation of petroleum polluted wetland soils.
A more precise molecular technique for the characterization of the endophytes will reveal the true diversity and functionality of the eendophytes as these were limited in the present work. The presence of these plants indicate the natural recovery process of the vegetation since they were tolerant to the initial impact of the oil. Majority of the above mentioned wetland plants have not been demonstrated in preliminary petroleum hydrocarbon bioremediation laboratory studies as such information would be required for further and more advanced studies on plant-microbe interactions, before a successful implementation of a bioremediation strategy.