EFFECTS OF SIMULATED CRUDE OIL CONTAMINATION ON THE PHYSICOCHEMICAL PROPERTIES AND BACTERIAL POPULATION OF RHIZOSPHERE OF SORGHUM VULGARE PERS.

ABSTRACT

This study has been undertaken to investigate the physicochemical properties and the bacterial population of the rhizosphere of Sorghum vulgare. In order to simulate spillage, 0.2, 0.9 and 5.0

% v/w concentrations of crude oil were used to contaminate soil sown with seeds of Sorghum vulgare, while the control had no crude oil contamination.  0ne hundred and twenty days after contamination, the physicochemical properties and bacterial population of the rhizosphere were analyzed using standard techniques.  Results showed that 5.0 % v/w of crude oil  used in this study caused significant (P<0.05) increase in soil pH, soil temperature, bulk density and total petroleum hydrocarbon   while moisture content, sand particle, exchangeable cations and total organic  matter  were  significantly  (P<  0.05)  reduced  when  compared  with  all  the  other treatments. These physicochemical conditions may suggest low fertility and could have been as a result of 5.0 % v/w concentration of crude oil used to simulate pollution.  Soil treated with 0.9 % v/w concentration of crude oil  gave highest increase in soil  electrical conductivity, silt particle, exchangeable bases such as Na+, Ca2+, Mg2+  and K+, total nitrogen and bacterial population when compared with all the other treatments and significant decrease in total petroleum hydrocarbon when compared with all the crude oil treated samples. This suggests that there was synergistic cooperation between roots of Sorghum vulgare and rhizospheric bacteria which may have facilitated removal of petroleum hydrocarbon and improved the physicochemical conditions and bacterial population of the soil treated with 0.9 % v/w concentration of crude oil. Hydrocarbon- utilizing bacteria isolated from the crude oil contaminated rhizosphere of Sorghum vulgare were Pseudomonas sp., Bacillus sp., Klebsiella sp. and Streptomyces sp.

CHAPTER ONE

INTRODUCTION

The inevitable and disastrous consequence of crude oil pollution for the biotic and abiotic components of the ecosystem has been a major source of concern to the government and people living in oil producing and industrialized countries. This had led to ethnic and regional crises in the Niger Delta region that generated significant tension between  them  and  the  multinational  oil  companies  operating  in  the  region  (Vidal,

2010).Crude oil exploration, production and transportation in the Niger Delta region have increased tremendously since its discovery in Nigeria in 1956 and has become a veritable source of economic growth and the main stay of the Nigerian economy (Okoh, 2006).The global scale of oil production  is staggering and its demand is in the order of 3.25 x 109 tones or 3.8 x 1012  liters per year and much of  it is transported  thousands of kilometers before it is used (Prince and Lessard, 2004).

Crude oil is a complex mixture of organic compounds including volatile aromatic fractions and less volatile aliphatic fractions. The main constituents of crude oil are the elements hydrogen (10 – 40%) and carbon (83 – 87%). Various types of crude oil contain small quantities of sulphur, nitrogen, oxygen and trace metals such as vanadium, nickel, iron and copper which are not usually found in refined petroleum (Atlas and Bartha,

1973). Individual chemical composition of each crude petroleum however, depends on its origin and location and has a unique mixture of molecules which defines its physical and chemical properties. Crude oil has been part of the biosphere for  millennia and has been used since ancient times in one form or the other and has risen in importance due to rise

in commercial aviation, invention of internal combustion engines and the increasing use of pesticides, fertilizers and plastics which are mostly made from oil (Okoh, 2006).

Soil is an extremely complex, dynamic and living medium, formed by mineral particles, organic matter, water, air and living organisms.  It establishes the interface between earth, air and water and performs many vital functions. The importance of soil for the survival of   plants has become apparent due to numerous services it renders, ranging from filtration of ground water, removal of pathogens, degradation of organics, recycling of nutrients on which agriculture thrives and provision of raw materials for industries which are of economic value. Human activities such as the production, transportation, storage and sometimes vandalization of oil facilities accidentally release large quantities of crude oil and its fractions to marine and terrestrial environments thereby posing a long term threat to the soil and the services it renders (Blum, 1997).

Crude oil is a fossil fuel derived from ancient fossilized organic material. The fossilization  processes  include  the  initial  process  of  diagenesis  and  the  final  or completion process called catagenesis. The initial process of diagenesis occurs at temperatures at which microbes partially degrade the biomass and result in dehydration, condensation, cyclisation and polymerization of the biomass. Subsequent burial under more sediments at higher temperature and pressure allows catagenesis to complete the transformation of the biomass to fossil fuel by thermal cracking and decarboxylation (Prince and Lessard, 2004).

1.1       CRUDE OIL POLLUTION OF THE ECOSYSTEM

The release of crude oil and its fractions into the natural environment has adverse ecological impacts on both terrestrial and aquatic ecosystems. In Nigeria, it has been  reported that, annually an estimated quantity of 2,105,393 barrels of oil was spilled on land, coastal and offshore marine in the Niger Delta region between 1976 and 1990 (Kontagora, 1991). The impact of oil exploration and exploitation on the environment is one of the inevitable consequences of industrialization and economic development in Nigeria (Osuji and Onojake, 2006). In aquatic ecosystem, crude oil floats  and  blocks  out  sunlight,  thus  initiating  the  death  of  phytoplanktons  and seaweeds, which are at the base of the aquatic food chain, thereby starving organisms that depend on them. Crude oil also has become one of the most frequently detected underground water pollutant caused by leakages from underground tanks, pipelines and other components of crude oil distribution (Kharoune et al., 2001; Gwendoline,

2010).   Soil  soaked   with   crude  oil  loses   fertility   and   initiates   environmental degradation and ecological succession. Crude oil pollution changes the composition of soil microorganisms and alters the physicochemical properties of the soil rhizosphere which     affects plant growth and development (Gesinde et al., 2008; Ebere et al.,

2010).

1.2       THE PHYSICOCHEMICAL PROPERTIES OF RHIZOSPHERE

The physicochemical properties of   rhizosphere are the physical and chemical characteristics of the soil surrounding plant roots, which differ from those of the bulk soil. Such properties include soil texture, soil porosity, bulk density, cation exchange

capacity,  mineral  composition,  soil  organic  matter,  soil  pH,  soil  water  or  moisture content  (Luthy et al., 1997; Gogoi et al., 2003). Densification of soil particles due to crude oil contamination gives rise to compaction, formation of organic ligands and binding of clay particles. This leads to increase in soil bulk density, low soil porosity and low  soil  moisture  content  (Xu  and  Johnson,  1995).  Moisture  level  affects  soil respiration,  nutrient  transport  and  availability  and  hence  limits  the  metabolism  and growth of microorganisms in the soil (Smith et al., 1998).   The growth rate of microorganisms on crude oil contaminated soil is limited by the availability of nutrients such as nitrogen and phosphorous (Pritchard and Costa, 1991). The rate of microbial growth depends on soil pH.   Soil pH is a critical factor for microbial growth and survival. Different microbial strains exhibit their maximum growth potential in a limited pH  range.  Soil  pH  value  near  neutral  is  suitable  for  growth  of  diverse  bacterial population (Barua et al., 2011).

1.3                   RHIZOSPHERE MICROFLORA

Rhizosphere is the zone of the soil surrounding the root of plants where the biological, physical and chemical properties of the soil are greatly influenced by the roots (Frick et al., 1999). It is the soil matrix and can be described as the longitudinal and radial gradients occurring with expanding root growth, nutrients and water uptake, exudation and subsequent microbial growth (Uren, 2000). The environment of plant rhizosphere is the most favorable microhabitat for microorganisms compared to the surrounding bulk soil (Bias et al., 2006). The rhizosphere has been reported to harbor more oil utilizing bacteria than adjacent non rhizospheric soil (Sorkhoh et al., 2010). Microorganisms  commonly  associated  with  the  rhizosphere  are  Pseudomonas  sp.,

Bacillus sp., Sphingomonas sp., Streptomyces sp., Micrococcus sp., Aspergillus sp.and

Penicillum sp.(Radwan et al., 1998; Obire et al., 2008).

1.4       BIOREMEDIATION OF CONTAMINATED SOIL

Biomediation is the use of living organisms to manage or remediate polluted soil (Bossert and Bartha, 1984).    Bioremediation is the elimination, attenuation or transformation  of  polluting  or  contaminating  substances  by  the  use  of  biological processes (Wenzel, 2009).   Biomremediation technologies include land farming, bioreactor, composting, landfilling, biopilling, biostimulation, bioaugmentation and phytoremediation (Siciliano and Germida, 1998).

1.4.1 LAND FARMING

Land farming is a waste disposal technology for handling hazardous chemical wastes. It involves  simultaneous  treatment  and  disposal.  Land  farming  is  a  biotechnology application that uses soil aerobic microorganisms to degrade petroleum hydrocarbons and their derivatives to carbon dioxide and water or other less toxic intermediaries (USEPA,

1990).

1.4.2    BIOREACTOR

This is a periodic treatment of contaminated groundwater or industrial effluent, using an engineered bioprocess. Bioreclamation of soil contaminated by petroleum hydrocarbons have been carried out, using bioreactors such as the conventional suspended growth sequencing batch reactors (SBRs), sequencing batch biofilm reactors (SBBRs) and soil slurry sequencing batch reactors (SS-SBRs). These bioreactors boost the population of

petroleum  degrading  microorganisms  and  thus  increate  bio-oxidation  rate  of  the pollutants (Irving and Ketchum, 1983).

1.4.3    COMPOSTING

This technology, which seems to operate more under thermophilic conditions, is used mainly for bioremediation of resistant (recalcitrants) chemicals and explosives such as

2,4, 6, trinitrotoluene (TNT).  The approach of this technology is mixing of the hazardous chemicals with the soil and the compostable materials before treatment (USEPA, 1990).

1.4.4    LAND FILLING

Land filling is an engineered and controlled treatment operation on land. The procedure is technically simple, less costly and easily managed.  Generally, the waste is spread out in layers (in a pit) and compacted down using either a tractor or landfill compactor.  The compacted  waste, which may  be about 2.4m thick,  is eventually  covered  with inert material (USEPA, 1994).

1.4.5    BIOPILING

This requires heaping or piling up of the pollutant in an enclosure such as tunnel or greenhouse structure or on top of a liner.  The “treatment’ heap can be aerated from time to time either by tilling or by forcing air through perforated pipes installed at the base of the heaps (Balba et al., 1991).

1.4.6    BIOSTIMULATION

This technology can be used alongside those already listed above but it is an important process that requires consideration on its merit. This innovative technology is employed for optimization of the environmental conditions in order to maximize the contaminant degrading potential of the native or indigenous bacteria (Atlas and Bartha, 1973).

1.4.7    BIOAUGUMENTATION

Seeding a contaminated environment with strains of bacteria that are tolerant (adapted) and capable of degrading a given contaminant and thus supplementing the natural  resident  microbiota,  has  proven  to  be  useful  in  bioremediation.  The  relative success of such adapted bacteria when added to the polluted site depends on many factors including competitive interactions with native bacteria, their rate of growth in the system as well as their tolerance to the physicochemical environment (Leahy and Colwell, 1990).

1.4.8    PHYTOREMEDIATION

Initially, bioremediation employed  microorganisms to degrade organic pollutants, but since the use of green plants was proposed for in situ soil remediation, phytoremediation has become an alternative topic of research and development (Salt et al., 1995). Phytoremediation appears attractive because in contrast to most other remediation technologies it is not invasive and, in principle delivers intact, biologically active soil (Wenzel, 2009).

The fundamental technologies applicable in phytoremediation of contaminated soil are phytostabilisation, phytoextraction, phytovolatilisation (rhizovolatilisation) and phytodegradation (rhizodegradation), (Salt et al.,   1995).

Phytostabilization is a contaminant process using plants often in combination with soil  additives  to  assist  plant  to  mechanically  stabilize  the  site  and  reduce  pollutant transfer to other ecosystem compartments and the food chain (Wenzel, 2009).

Phytoextraction is a removal process that takes advantage of unusual ability of some plants to hyper accumulate metals/metalloids in their shoots (Wenzel, 2009).

Phytovolatilisation (rhizovolatilization) is a removal process that employs metabolic capabilities of plants and rhizospheric microorganisms to transform pollutants into volatile compounds that are released into the atmosphere (Wilber, 1980).  Pollutant, toxicity, adverse soil conditions, water stress and nutrient deficiency are typical problems challenging the establishment of vegetation in contaminated sites (Tordoff et al.,2000).

Phytodegradation (rhizodegradation) refers to the use of metabolic capabilities of plants and rhizospheric microorganisms to degrade organic pollutants (Wenzel, 2009). Plants and microorganisms are involved, both directly and indirectly in the degradation of petroleum hydrocarbons into products that are less persistent in the environment than the parent compounds (Cunningham et al., 1996).  Low availability of pollutants is the main challenge in the rhizodegradation of field- contaminated and aged spiked soil (Wenzel,

2009). Limited pollutant bioavailability can be overcome by the design of plant microbial consortia that are capable of mobilizing pollutants by modification of rhizosphere pH (Siciliano and Germida, 1998).

1.4.9. RHIZODEGRADATION OF CRUDE OIL CONTAMINATED SOIL

The interaction between plants and microbial communities in the rhizosphere is exploited in the specific use of plants to enhance microbial degradation of organic compounds in the soil. The fibrous root structure of grasses is known to provide a large surface  area  for  colonization  by  microorganisms  than  the  taproot  system  (Atlas  and Bartha, 1993). Plant establishment during the process of phytoremediation follows many standard procedures that include adaptability of the plants to climatic conditions of the region, maximum root density and stress tolerance (Cuningham et al., 1996) and  these are not uncommon  with Sorghum  vulgare .

1.4.10  ROLE OF MICROORGANISMS IN RHIZODEGRADATION

Soil microorganisms are known to convert some organic and inorganic pollutants (hydrocarbon, arsenic, boron, antimony, selenium, tin, lead and mercury) to their volatile species  (Meyer  et al.,  2007).   This microbial  conversion  is usually considered  as  a detoxification  mechanism  by which  the  microorganism  decreases  the toxicity  of  the surrounding microenvironment (Wenzel, 2009).  Microorganisms can increase solubility and change speciation of metals/metalloids through the production of organic ligand via microbial  decomposition  of  soil  organic  matter  and  exudation  of  metabolites  and microbial siderophores that can complex cationic metals or desorb anionic species by ligand exchange (Gadd, 2004).  Beneficial interactions between phytoremediation crops and  bacteria  have  been  demonstrated  to  alleviate  pollutant  toxicity  and  nutrient deficiency.  Ectomycorrhizal associations can display considerable resistance against toxicity in the soil polluted with metal, organic compound and petroleum (Sarand et al.,

(1998). Microbial degradation of organic contaminants normally occurs as a result of microorganisms using the contaminant for their own growth and reproduction. (Cunningham et al., 1996). Another role played by microbes involves their ability to reduce the phytotoxicity of contaminants to the point where plants can grow in adverse soil   conditions,   thereby   stimulating   the   degradation   of   other,   non   phytotoxic contaminants (Siciliano and Germida, 1998). The defence of plants to contaminants may be supplemented through the external degradation of contaminants by micro organisms in the  rhizosphere  (Anderson  and  Coats,  1997).     Microorganisms  are  the  primary mechanism responsible for petrochemical degradation in phytoremediation efforts (Siciliano and Germida, 1998).

1.5 RHIZOSPHERIC  MICROORGANISMS  ASSOCIATED  WITH PHYTOREMEDIATION

A variety of microorganisms are reportedly involved in the degradation of petroleum hydrocarbons.   Bossert and Bartha (1984) reported the use of bacteria such as Pseudomonas sp., Arthrobacter sp., Bicaligenes sp., Combacteriu sp.,, Flavobacterium sp., Achromobacter sp., Micrococcus sp., Mycobacterium sp. and Norcardia sp., as the most actual bacterial species in the degradation of hydrocarbons in soil. Pseudomonas, Arthrobacter sp. and Achromobacter sp. often occur in greater numbers within the rhizosphere than in the bulk soil (Anderson and  Coats, 1997).  Soil fungi also play role in  the  degradation  of  petroleum  hydrocarbons.  Surtherland  (1992)  reported  that  a diversity   of   fungi   including    Aspergillus   ochraceus,   Cunninghamella    elegans,

Phanerochaete chrysosporium, Saccharomyces cerevisiae and Syncephalastrum racemosum can oxidize various polycyclic aromatic hydrocarbon.   Higher microbial numbers and increased degradation of hydrocarbon – contaminated soil were observed in contaminated soil plant with   ryegrass compared to unplanted soil (Gunther et al., 1996).

1.6 A BRIEF IN SORGHUM   VULGARE

Sorghum vulgare Pers. also known as guinea corn is a member of the grass family (Poaceae) under the class Angiospermeae and sub class Monocotyledonae. It is self pollinated, more drought and temperature resistant than maize, soybeans and wheat. It has deep fibrous root system, grows fast and can withstand conditions of stress which are important characteristics that make it useful in phytoremediation (Siciliano and Germida,

1998). S. vulgare is grown primarily in the semiarid tropics of Africa, India, China, South America, and stress- prone areas of United States (Subudhi and Nguyen, 2000). S. vulgare and other related Sorghum species can accumulate biomass very rapidly and attain heights greater than 2.5 m in less than 6 weeks (Subudhi and Nguyen, 2000).

SIGNIFICANCE OF THE STUDY

This study may help to foster good neighborliness, profitable dependability on nature’s rich ecosystem and create conducive environment for economic activities in polluted environments through efforts to remediate crude oil contaminated soil, assessing its effects on bacterial population and physicochemical properties of rhizosphere which affect the degradation potential of hydrocarbon-utilizing bacterial community. The result of the study will provide data for assessing the changes in bacterial population and physicochemical properties of soil rhizosphere in response to crude oil contamination. The data may be used to design effective remediation system and drive further improvement and innovation in the field of biotechnology.

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