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European Journal of Applied Sciences – Vol. 12, No. 3

Publication Date: June 25, 2024

DOI:10.14738/aivp.123.17066.

Olukunle, O. F., Abe, A. S., Fajoyegbe, E., Akinde, S. B., Kolawole, A. O., & Falese, B. A. (2024). Characterization of Novel Bacterial

Strains Associated with Egina Oil Field Deep-Sea, Port Harcourt, Rivers State, Nigeria. European Journal of Applied Sciences, Vol -

12(3). 398-410.

Services for Science and Education – United Kingdom

Characterization of Novel Bacterial Strains Associated with Egina

Oil Field Deep-Sea, Port Harcourt, Rivers State, Nigeria

Olukunle, O. F.

Department of Biotechnology,

Federal University of Technology, P. M. B. 704, Akure, Nigeria

Abe, A. S.

Department of Microbiology,

Federal University of Technology, P. M. B. 704, Akure, Nigeria

Fajoyegbe, E.

INQABA Biotechnology, Ibadan, Oyo State, Nigeria

Akinde, S. B.

Department of Microbiology, Faculty of Basic and Applied Sciences,

Osun State University, Osogbo, Nigeria & Multidisciplinary Research

Laboratory, Osun State University, Osogbo, Nigeria

Kolawole, A. O.

Department of Biochemistry,

Federal University of Technology, P. M. B. 704, Akure, Nigeria

Falese, B. A.

Department of Biochemistry,

Federal University of Technology, P. M. B. 704, Akure, Nigeria

ABSTRACT

The deep-sea environment has rich microbial resources, which are very much

underexploited and are of economic, industrial and medical importance. The aim

of this study is to isolate and characterize novel bacterial strains from EGINA oil

field deep-sea, Port Harcourt, Rivers State. Sediment and water samples were

collected with the aid of carousal and soil auger; and cultivated on both nutrient

agar and actinomycetes isolation agar. Serial dilution and plate methods were

used to determine the total heterotrophic bacterial counts. A total of 170 bacterial

strains, showing distinct morphologies, were isolated from both water and

sediment samples collected at various location at EGINA off-shore. These strains

were then categorized into 49 groups, collectively across all the stations based on

standard colony morphological characteristics and were isolated and identified by

conventional and molecular methods. Station AGE S4 had the highest

heterotrophic bacterial counts of 32 with dilution factor of x106 while the least

was from N3, having 11 heterotrophic bacterial counts with dilution factor x104.

The mean heterotrophic bacterial counts for water and sediment samples

exhibited the highest values at station EGINA-NS-TOP (50) and DC6-W1 (47) with

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399

Olukunle, O. F., Abe, A. S., Fajoyegbe, E., Akinde, S. B., Kolawole, A. O., & Falese, B. A. (2024). Characterization of Novel Bacterial Strains Associated

with Egina Oil Field Deep-Sea, Port Harcourt, Rivers State, Nigeria. European Journal of Applied Sciences, Vol - 12(3). 398-410.

URL: http://dx.doi.org/10.14738/aivp.123.17066

dilution factor x104 while the lowest heterotrophic bacterial counts were 30 for

EGINA-MINI-MID and 105 for both DC4-E1 and N3 with dilution factor x102

respectively. Bacillus strains were the most predominant strain isolated with

66.6% frequency of occurrence, some of the Bacillus strains include Bacillus

velezensis OOKAAOFFA-1, Bacillus tequilensis OOKAAOFFA-34 and Bacillus subtilis

OOKAAOFFA-24; likewise, Enterococcus gallinarum OOKAAOFFA-49, Brevibacillus

halotolerans OOKAAOFFA-36, Stutzerimonas stutzeri OOKAAOFFA-6, Arthrobacter

globiformis strain OOKAAOFFA-33 among others were also present in the deep

sea. The closest type relationship was seen with the Bacillus, Brevibacillus and

Enterobacter strains with branch support of 100% within a clade which also

included Bacillus xiamenensis, Bacillus aerius, Bacillus stratosphericus,

Arthrobacter globiformis, Enterobacter bugandensis, Enterobacter mori,

Brevibacillus formosus, and Brevibacillus halotolerans strains OOKAAOFFA. This

research has contributed to the understanding of deep-sea microbial diversity and

ecology. The novel bacterial strains obtained from this research can be further

exploited for their potentials.

Keywords: Deep sea, EGINA off shore, Bacterial strains, Molecular identification, Bacillus

sp.

INTRODUCTION

Marine ecosystem is the most significant known environment on this planet (Bull and Stach,

2007). More than 90% of the total marine ecosystem is designated as deep-sea characterized

with many distinct features (Subramani and Aalbersberg, 2012) that attribute for individual

species distribution (Fenical and Jensen, 2006) and an important reservoir for bioactive

molecule discovery. The vast expanses of the deep-sea constitute the largest continuous

ecosystems on earth. The ocean covers 70% of the total world’s surface and the majority of it

is below 1000 meters of depth (Skropeta, 2008). Deep-seas are the most extreme

environments on earth and has the highest richness in biodiversity, surpassing the rainforests

and the coral reef. Organisms inhabiting the deep-sea can cope with such harsh conditions in

the absence of light and under low percentage of oxygen and extremely high pressures,

requiring several adaptations in terms of biochemical and physiological processes (Skropeta,

2008). These special environment variables may lead to producing distinct chemical entities

with diverse biological activities. Certain areas like hydrothermal vents and cold seeps are

recognized as biodiversity hotspots, however, much of the deep-sea environment, including

abyssal plains, remains largely unexplored and understudied, as noted by Jorgensen and

Boetius (2007). The deep-sea is a unique and extreme environment and characterized by

extreme variations in available nutrients, lights, oxygen, concentration, pressure, salinity and

temperature. Hence, deep-sea bacteria have developed unique biochemical metabolic and

physiological capabilities, which not only ensure their survival in this habitat but also provide

potentials for the production of novel metabolites absent in terrestrial bacteria. (Parilli et al.,

2021). It is estimated that biological diversity is higher in marine ecosystem than tropical

rainforests (Valli et al., 2012). However, this marine ecosystem are still under explored for

marine bacterial strains which may have different characteristics from those of terrestrial

counterparts, thereby producing different types of bioactive compounds which may be novel

in nature Liao et al., 2016; Valli et al., 2012; Thenmozhi and Krishnan, 2011).

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European Journal of Applied Sciences (EJAS) Vol. 12, Issue 3, June-2024

Typically, the life forms inhabiting EGINA oil field deep-sea, face constraints due to limited

food availability, as these areas lack primary production and rely on organic matter supplied

from surface waters. The deep-sea, characterized by their vastness and challenging

accessibility, remain one of the least explored ecosystems on earth, despite their potential to

harbor significant diversity, as noted by Smith and colleagues (Smith et al., 2008). Marine

organisms possess significant potentials, due to their ability to produce secondary

metabolites, which can serve as lead compounds in drug discovery. Secondary metabolites

synthesized by these bacteria are of biotechnological and biomedical importance as they

include broad range of several therapeutic compounds such as antibiotics and antitumor

agents (Reddy et al., 2021). The potential of deep-sea Streptomyces as a source of new

antibacterial and anticancer was highlighted (Sivalingam et al., 2019). The marine

environment has actually proved to be a particularly rich reservoir for novel halogenated

compounds with biologically active properties (Tortorella et al., 2018). Some of the antitumor

drugs include anthracyclines (aclarubicin, daunomycin, and doxorubicin), peptides

(bleomycin and actinomycin D), aureolic acids (mithramycin), enediynes (neocarzinostatin),

antimetabolites (pentostatin), carzinophilin, and mitomycins (Olano et al., (2009a), 2009b;

Newman and Cragg, (2007). Zheng et al., (2005) reported on a strain of Pseudoalteromonas

piscicida producing antimicrobial alkaloids, while Lin et al., (2005) observed potential

apoptosis effects of marine bacterial metabolites on human HeLa cells. Additionally, marine

bacteria such as Streptomyces, Cyanobacteria, Bacillus, Pseudomonas, Halomonas, and

Sulfitobacter have demonstrated antimicrobial and anticancer activities. Despite the known

therapeutic potential of marine organisms against cancer, their rich biodiversity suggests

untapped opportunities for exploration. In this study, we focused on the isolation and

characterization of deep-sea bacteria.

MATERIALS AND METHODS

Study Site

The study site is EGINA. The EGINA field is located in the Oil Mining Lease (OML) 130

concessions in deep offshore Nigeria. The field belongs to Total Upstream Nigeria Limited

(TUPNI), a TOTAL Nigeria subsidiary, and is situated 200km south of Port Harcourt in water

depths ranging from 1150 to 1750m and some 20km from AKPO FPSO. Seawater and

sediment samples were collected within the geographic coordinates 572140.2 - 586170.2

Easting and 328981.3 - 352695.3 Northing in the EGINA field.

Sample Collection

The sample area was divided into various regions, from each of the region, 30 seawater

samples were collected with the aid of Sea-Bird Electronics (SBE) 32 Carousel water sampler;

and 30 sediment samples were also collected using stainless steel day grab sampler (Akinde

et al., 2012). These samples were collected inside sterile glass bottles and transported

immediately to the laboratory on ice and kept at 4±2 ̊C till further use.

Serial Dilution and Plating

Serial dilution was carried out on the water and sediment samples according to standard

methods. After serial dilution, 0.1 ml of each sample was separately plated on nutrient agar

and actinomycetes isolation media using pour plate techniques. After 24 hours, plates were