DEPARTMENT OF SCIENCE LABORATORY TECHNOLOGY

FACULTY OF LIFE SCIENCES

UNIVERSITY OF BENIN

BENIN CITY

MCB 625 (RESEARCH PROJECT)

TOPIC:

MICROBIOLOGICAL ASSESMENT OF RHYZOSPHERIC SOIL OF SOME

EDIBLE PULSES GROWN ON OIL POLLUTED SOIL

SULE RACHEAL FUNMILAYO

LSC0711960
 ABSTRACT

This study was carried out to assess the microbial composition of

rhizospheric soils obtained from oil-polluted soil on which was sown, some
and bulk selected legumes, the present study however examine the
legumes for specificity. Sundried top soil was mixed with waste engine oil at
the rate of 5% w/w, and left to attenuate for one month. Seeds were sown
into each bucket at the rate of 3 seeds per hole and at a depth of 3cm, in a
triangular form then thinned down to 6 seedlings per bucket after seedlings
had attained a 2-leaf stage The pulses sown were soy bean, cow pea (Ife
brown), cow pea (Benin local), common bean (white), pigeon pea. The
result showed that some pulses were negatively affected by waste engine
oil, but pigeon pea (cejanus cajan) was not affected by WEO. Plants were
observed to have continues growth except the pulses sown on oil polluted
soil which became stunted then dies gradually after 35th DAP.
 CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF STUDY

In modern economies, various types of activity, including agriculture,

industries and transportation, release large amounts of wastes and new
types of pollutants. Soil, air and water have traditionally been used as sites
for the disposal of all these wastes. Abundant release of organic and
inorganic compounds into the environment occurs each year as a result of
human activities. The introduction of contamination into the natural
environment which causes adverse change in environment is pollution.
Pollution from the petroleum industry has been of serious concern to the
world at large.

The petroleum industry has created economic boom for Nigeria and at the
same time led to environmental and socio-economic problems. The result
of increase in population per day brings about the ever increase in the
demand for petroleum and its products, which apparently constitutes a
source of environmental population (Raven et al., 1993) as a result of oil
exploration, refining and marketing operations, they have been many
incident of oil spillage on both aquatic and terrestrial environment resulting
in environmental or soil pollution.

Engine oil is a major product of petroleum that helps in reducing friction

between engine surfaces and is produced by vacuum distillation of crude
oil. Petroleum contains chemical additives like amines, Phenol Benzene, Ca,
Zn, and Pb. Some of the WEO may contain foreign substances still as
synthetic Polly-chlorinated biphenyl. New lubricating oil contained only low
concentration of pollyaromatic hydrocarbon (PAH). While engine oil
collected from automobile which as caused up to 3,000km contained
considerably higher concentration of PAH (Wang et al. 2002). PAH have
very low water solubility and often tightly bound to soil particles. Metals
present in WEO are not necessarily the same as those present in the
unused lubricate.
Nigeria accounts for more than 87 million litres of WEO annually
(Anonymous, 1985).

Operations of motor mechanics involve extensive use of crude oil-based

products. One way in which motor mechanics uses engine oil is by Servicing
of motor vehicles which involves emptying of used lubricating oils, filling of
new ones and extensive cleaning of motor parts with gasoline.

Many motor mechanic workshop and workshops of other artisans uses

engine oil and disposes the used engine oil which is generally called Waste
Engine Oil (WEO) which occurs in urban and rural areas, in bushed and
open plots, gardens, and farm lands. Such areas are constantly under threat
of pollution, the plots of lands are used temporally as workshops while
awaiting their full development (Anolefo and Vwioko, 2001; Dede et al.,
2003). Most motor mechanics involve emptying of used lubricating oil and
filling of new ones and extensive cleaning of cars, vehicles, lorries and other
machines with engine oil and disposing of the WEO into these vacant plots,
farm gutters and wanted drains by those mechanics is an environmental
risks considering the water table and shallow borehole dug to get water for
domestic used in most part of Nigeria (Odjegba and Sadiq, 2002).

Atunya (1987) reported that pollution due to waste engine oil (WEO) is
more widespread and more diverstating than crude oil pollution. This is
because it is a major source of pollution within communities and so
constitute risks to human health and plants which includes decrease of soil
microbial activity and fertility.

Some of the WEO may contain foreign substances such as synthetic poly-
chlorinated biphenyls which destroys farmlands with detrimental impact on
agricultural crops and causes instantaneous negative and often violent
reactions with demand for compensations by the communities in the oil-
producing areas such as occur in the Nigerian Niger Delta.

Using plants to store, remove, degrade and metabolize environmental

contaminant including metals, hydrocarbons and other toxic organic
compounds is a bioprocess called. Phytoremediaton is an alternative to
another technology, including incinerators and chemical treatment. It is
well-suited at sites with shallow contamination of organic or metal
pollutants, for use at very large field sites, where other methods of
remediation may not cost-effective or practicable (Abedi-Koupai and
Afyuni, 2003; Abedi-Koupai, 2003) and it is practicable at sites with low
concentrations of petroleum contaminants. Interaction between
microorganisms associated with plants and plants is known the main
features of this phenomenon. There are three primary mechanisms by
which plants and microorganisms remediate petroleum–contaminated soil
and ground water.

These include degradation, containment and transfer of the hydrocarbons

from soil to atmosphere (Sims and Overcash, 1983; Cunningham et al.,
1996; Siciliano and Germida, 1998a, b). Several studies serve as examples of
rhizosphere effect in the phytoremediation of petroleum hydrocarbons.
Gunther et al. (1996) found higher microbial numbers and activity coupled
with increased degradation in hydrocarbon-contaminated soil planted to
ryegrass compared to un-planted soil. They suggested that plant roots
stimulated the microbial growth, which enhanced the degradation of
hydrocarbon mixture. Plants provide root exudates of carbon, energy,
nutrients, enzymes and sometimes oxygen to microbial populations in the
rhizosphere (Cunningham et al., 1996).

This plant-induced enhancement of the microbial population is referred to

as the rhizosphere effect (Atlas and Bartha, 1998), which is believed to
result in enhanced degradation of organic contaminants in the rhizosphere.

Plants with a fibrous root structure and therefore greater root surface area
may enhance organic dissipation more than plants with simpler, less fibrous
systems (April and Sims, 1990)

1.2 RESEARCH PROBLEM

Soil microorganisms (flora and fauna) just like higher plants depends
entirely on soil for their nutrition, growth and activity. A number of factors
influence microbial population, distribution and their activity in the soil.
Some of these are soil fertility, soil moisture, soil temperature, soil
aeration, light, soil PH, organic matter, food and energy supply, nature of
soil and microbial associations. All these factors play a great role in
determining not only the number and type of organism but also their
activities. Variations in any one or more of these factors may lead to the
changes in the activity of the organisms which ultimately affect the soil
fertility level. This variation could be brought about by the presence of
contaminants which come from either deliberate or unconscious disposal of
waste engine oil in terrestrial environment.

Contamination of soil by crude oil could lead to a depression of microbial

density and activities even in case of relatively light contamination (Odu,
1972a,b).The extent of the effects depends on the original soil properties
and the plant exposed to contaminated soil. Hence, if leguminous plant is
planted on a crude oil-contaminated soil, the activities of the nitrogen-
fixing bacteria may be retarded. The oil also inhibits the action of the
enzyme “nitrogenise”, thereby disrupting the process of protein synthesis.
This would eventually result in inability of the plants to thrive and survive in
the affected ecosystem. However, in a survey of 15 oil-contaminated site,
Gadin and Syratt (1975) reported that leguminous plants were the
dominant flora; indicating a selective advantage of plants with a symbiotic
nitrogen fixing potential. Ile (1997) showed that a low (0.75%) level of oil in
soil improved the growth and nodule development of soya beans. This
improvement could be attributed to nitrogen fixation or the additional
nutrients released from the activities of the tolerant organisms. Assessing
microbial response to pollution stress may provide basic information for the
improvement of microbial activities in order to promote soil fertility and
plant growth. In the present study, microbial content of both rhizosphere
and bulk soils were used.

1.3 RHIZOSPHERE

Term "Rhizosphere" was introduced for the first time by the German
scientist Hiltner (1904) to denote that region of soil which is subjected to
the influence of plant roots. The concept of "Rhizosphere Phenomenon"
which shows the mutual interaction of roots and microorganisms was came
into existence with the work of Starkey et al (1929), Clark (1939) and
Rauath and Katznelson (1957).
It is the zone/region of soil immediately surrounding the plant roots
together with root surfaces. It is the region where soil and plant roots make
contact (plant-root interface), or it is the soil region subjected to influence
of plant roots and characterized by increased microbial activities (Hartmann
et al., 2008).

Roots serve many functions for a plant including anchorage and acquisition
of vital nutrients and water necessary for growth. The plant root-soil
interface is a dynamic region in which numerous biogeochemical processes
take place driven by the physical activity, and the diversity of chemicals
released by the plant root and mediated by soil microorganisms (McNear,
2013). In turn the processes occurring in this region control a host of
reactions regulating terrestrial carbon and other element cycling that
sustain plant growth and which have an enormous influence on plant and
microbial community function and structure which greatly influence a
variety of ecosystem level processes (van der Heijden et. al., 2008; Wardle,
2004; Berg and Smalla, 2009). Understanding and harnessing these
interactions for the sustainable production of food, fuel and fibre to
support a growing world population on a dwindling supply of arable land
will be the challenge of generations to come.

The great array of root–microbe interactions results in the development of

a dynamic environment known as the rhizosphere where microbial
communities also interact. The differing physical, chemical, and biological
properties of the root-associated soil are responsible for changes in
microbial diversity and for increased numbers and activity of
microorganisms in the rhizosphere micro environment (Kennedy, 1998).
Carbon fluxes are crucial determinants of rhizosphere function (Toal et al.,
2000). The release of root exudates and decaying plant material provide
sources of carbon compounds for the heterotrophic soil biota as either
growth substrates, structural material or signals for the root-associated
microbiota (Werner, 1998). Microbial activity in the rhizosphere affects
rooting patterns and the supply of available nutrients to plants, thereby
modifying the quality and quantity of root exudates (Bowen and Rovira,
1999; Gryndler, 2000; Barea, 2000).
1.3.1 TYPES OF RHIZOSPHERE INTERACTION

Two types of interactions in the rhizosphere are recognized;

Those based on dead plant material (the detritus-based interactions) which

affect energy and nutrient flows and

Those based on living plant roots.

Both types of interactions are relevant to both agronomy and ecology.

Broadly, there are three separate, but interacting, components recognized

in the rhizosphere. These are the rhizosphere (soil), the rhizoplane, and the
root itself. The rhizosphere is the zone of soil influenced by roots through
the release of substrates that affect microbial activity. The rhizoplane is the
root surface, including the strongly adhering soil particles. The root itself is
a part of the system, because certain micro- organisms, the endophytes, are
able to colonize root tissues (Kennedy, 1998; Bowen and Rovira, 1999).
Microbial colonization of the rhizoplane and/or root tissues is known as
root colonization, whereas the colonization of the adjacent volume of soil
under the influence of the root is known as rhizosphere colonization
(Kloepper et al., 1991; Kloepper, 1994).

1.3.2 CLASSIFICATION OF MICROORGANISMS IN THE RHIZOSPHERE

The composition of microflora of any habitat (soil/rhizosphere) is governed

by the biological equilibrium created by the associations and interactions of
all individuals found in the community. In soil and rhizosphere region, many
microorganisms live in close proximity and their interactions with each
other may be associative or antagonistic.

Associative interactions / activities in rhizosphere: The dependence of

one microorganism upon another for extra-cellular products (eg. amino
acids & growth promoting substances) can be regarded as an associative
activity / effect in rhizosphere. There is an increase in the exudation of
amino acids, organic acids and monosaccharide by plant roots in the
presence of microorganisms. Gibberellins and gibberellin- like substances
are known to be produced by bacterial genera viz Azotobacter,
Arthrobacter, Pseudomonas, and Agrobacterium which are commonly
found in the rhizosphere. Microorganisms also influence root hair
development, mucilage secretion and lateral root development. Fungi
inhabiting the root surface facilitate the absorption of nutrient by the roots.

Mycorrhiza is one of the best known associative / symbiotic interactions

which exist between the roots of higher plants and fungi. This mycorrhizal
association has been found to improve plant growth through better uptake
of phosphorus and zinc from soil, suppression of root pathogenic fungi and
nematodes. Another example is association between the bacterium
Rhizobium and roots of legumes and Azospirillum with cereal crops (wheat,
rye, bajara, maize etc).

Antagonistic interactions / activities in rhizosphere: The biochemical

qualities of root exudates and the presence of antagonistic microorganisms,
plays important role in encouraging or inhibiting the soil borne plant
pathogens in the rhizosphere region. Several mutualistic, communalistic,
competitive and antagonistic interactions exist in the rhizosphere. The
number and qualities of antagonistic microorganisms in the rhizosphere
could be increased through artificial means such as fertilizer application,
organic amendments, foliar spraying of chemicals etc.

1.4 MECHANISM OF RHIZOSPHERE – MICROBIAL INTERACTION

Rhizodeposits make the rhizosphere a desirable niche for microbial

communities to proliferate. Nutrient availability for microbial growth is
higher in the rhizosphere compared to the bulk soil; however, many
microbes are competing for these nutrients, some more successfully than
others. The plant rhizosphere -microbe relationships that have received the
most attention include those of Rhizobia bacteria and their symbiotic plant
partners, mychorrhizal fungi associations, and beneficial plant growth
promoting rhizobacteria (PGPR).

Roots exert a tremendous amount of pressure (>7kg/cm 2 or ~100 psi) at the

growing root tips in order to push their way through the soil. Helping to
lubricate and protect the root during growth, root cap and epidermal cells
secrete mucilage, a viscous, high molecular weight, insoluble,
polysaccharide-rich material. Beyond lubrication, the mucilage also
provides protection from desiccation, assists in nutrient acquisition, and
most notably binds soil particles together forming aggregates which
improve soil quality by increasing water infiltration and aeration. Also
serving to protect the root tip, cells lining and capping the root meristem
are programmed to release (slough-off) which helps to reduce frictional
forces that would otherwise damage the root (Bengough and McKenzie,
1997). Interestingly, the cells that are sloughed off continue to function and
secrete mucilage for several days and have been shown to attract beneficial
microorganisms, serve as "bait" for root pathogens, and sequester toxic
metals (e.g. Al3+) (Hawes et al., 2000).

Root exudates include both secretions (including mucilage) which are

actively released from the root and diffusates which are passively released
due to osmotic differences between soil solution and the cell or lysates
from autolysis of epidermal and cortical cells (Figure 4). The organic
compounds released through these processes can be further divided into
high and low molecular weight (HMW and LMW, respectively). By weight,
the HMW compounds which are those complex molecules that are not
easily used by microorganisms (e.g. mucilage, cellulose) make up the
majority of C released from the root; however, the LMW compounds are
more diverse and thus have a wider array of known or potential functions.
The list of specific LMW compounds released from roots is very long, but
can generally be categorized into organic acids, amino acids, proteins,
sugar, phenolics and other secondary metabolites which are generally more
easily used by microorganisms. The cocktail of chemicals released is
influenced by plant species, edaphic and climactic conditions which
together shape and are shaped by the microbial community within the
rhizosphere. There is still very little known about the role that a majority of
the LMW compounds play in influencing rhizosphere processes. A growing
body of literature is beginning to lift the veil on the many functions of root
exudates as a means of acquiring nutrients (e.g. acquisition of Fe and P),
agents of invasiveness (i.e. allelopathy) or as chemical signals to attract
symbiotic partners (chemotaxis) (e.g. rhizobia and legumes) or the
promotion of beneficial microbial colonization on root surfaces (e.g. Bacillus
subtilis, Pseudomonas florescence) (Bais, Park et al. 2004).

Plants respond to nutrient deficiency by altering root morphology,

recruiting the help of microorganisms and changing the chemical
environment of the rhizosphere. Components in root exudates assist plants
in accessing nutrients by acidifying or changing the redox conditions within
the rhizosphere or directly chelating with the nutrient. The present study
however examined the legumes for specificity.

1.5 PULSES OR LEGUMES

Pulses are dry seed of leguminous plants which are distinguished from
leguminous oil seed, by their low fat content and they contain little or no
cholesterol. Legumes are plants that produces nitrogen fixing root or stem
nodules which forms symbiotic association with Rhizobium. They include
beans, peas, clovers and soybean (Harrison, 2003).

1.5.1 SOME TYPES OF PULSES

GLYCINE MAX (Soya Bean)

Glycine max is the botanical name of soya beans widely grown for its edible
bean which has numerous uses. It belongs to the family Fabaceae. It is an
annual crop which grows to a height of twenty centimetres to two meters.
Remarkably, seeds such as soybean containing very high levels of protein
can undergo desiccation, yet survive and revive after water absorption.

CAJANUS CAJAN (Pigeon Pea)

Cajanus cajan is the botanical name of pigeon pea is a perennial leguminous

shrub that lives about one to five years. They are usually 2.5 to 10 cm long
and almost 3.5 cm wide, with nothing more than 0.1 to 0.2 stem girths from
germination period to 2 month after planting. Pigeon pea has taproots
which are tetrarch thin and grow deep underground. The deep roots help in
improving infiltration of water in to the soil. It is reported to have a wide
adaptability to different climate and soil (Troedson et al., 1990). Pigeon pea
remains one of the most drought tolerant legumes (Valenzuela and Smith
2002) and is often the only crop that gives some grain yield during dry spells
when other legumes such as field bean will have wilted and perhaps dried
up. The nitrogen fixing ability of pigeon pea is desirable for environmentally
sustainable agricultural production (Peoples et al., 1995).

VIGNA UNGUICULATA (Cowpea)

Cowpea (Vigna unguiculata), also known as black eye peas is a legume of

African origin that is useful as a rotational cover crop to help meet a cash
crops nitrogen need, to control erosion, and to improve soil properties.

Cowpea belongs to the Fabaceae family. Cowpea is a summer annual

legume with trifoliate leaves. Cowpea grows rapidly, reaching a height of
48-61cm when grown under favorable condition. Most root growth usually
occurs within the top soil layer but in time of drought cowpea can grow a
taproot as long as it feet to reach moisture deeper in the soil profile.

PHASEOLUS VULGARIS (Common Bean)

Also known as common bean (white), Phaseolus vulgaris is an important

leguminous crop of great nutritional value. It is nicknamed as a poor man’s
protein due to its potential role in the daily diet selling of the poor where
expensive animal protein cannot be afforded. It belongs to the family
Fabaceae. Phaseolus vulgaris is a dicotyledon and acquire its nitrogen
through an association with rhizobia, a species of nitrogen fixing bacteria.

1.7 JUSTIFICATION OF THE STUDY

Because of current public concerns about the side-effects of agrochemicals,
there is an increasing interest in improving the understanding of co-
operative activities among rhizosphere microbial populations and how
these might be applied to agriculture (kennedy, 1998; Bowen and Rovira,
1999; Barea et al., 2004; Lucy et al., 2004). Certain co-operative microbial
activities can be exploited as a low-input biotechnology, and form a basis
for a strategy to help sustainable, environmentally-friendly practices
fundamental to the stability and productivity of both agricultural systems
and natural ecosystems (Kennedy and Smith, 1995). An analysis of the co-
operative microbial activities known to affect plant development is the
general aim of this review.

1.8 LITERATURE REVIEW

A positive correlation between root biomass production and oil degradation

was found. The microbial communities and their degradative potential in
rhizosphere of alfalfa, reed and unplanted soil in response to bitumen
contamination of soil reduced the total number of microorganisms more
significantly (by 75%) in un-planted than in rhizosphere soil (by 42% and
7%) for reed and alfalfa, respectively (Muratova et al., 2003). They
indicated that the rhizosphere microflora of alfalfa was less inhibited by
hydrocarbon pollution and had a higher degradative potential than the
rhizosphere microflora of reed. Banks et al. (2003) indicated that the
presence and type of plants and level of contamination may greatly
influence microbial communities in plotted soils. Issoufi et al. (2006), while
studying seedling growth of 6 crop species in crude oil contaminated soils
reported that Zea mays and Glycine max seedlings show the greatest
potential to enhance remediation compared to the Meticago sativa, Lolium
perenne, Triticum aestivum and Vicia villosa.

Wolf et al. (2007) attribute rhizosphere effect to the very characteristics of

the roots. The contribution of the rhizosphere microbes in the degradation
of pollutants is referred to as rhizoremediation (Anderson et al., 1993;
Schwab et al., 1995). This emerging technology has been reviewed by a
number of authors (Salt et al., 1998; Schnoor 2002; Joner and Leyval,
2003a, b; Suresh and Ravi Shankar, 2004; Okoh, 2006).

Pigeon pea seedlings have been reported to possess greater potential in

enhancing remediation based upon percent emergence and plant biomass
production in contaminated soil (Issoufi et al., 2006).

Nichols et al. (1997) and Radwan et al. (1998) demonstrated increase in

hydrocarbon degrading microorganisms under the influence of
“rhizosphere effect” in the root zone of the plants which are utilized in the
phytoremediation of hydrocarbon-contaminated soil.

Vicia faba plants (broad beans) are suggested as tools for the
phytoremediation of oily desert areas because these can grow in poor soils
and tolerate crude oil concentrations up to 10% w/w (Radwan et al., 1998).
Their rhizospheres are rich in hydrocarbon utilizing microorganisms which
could affect oil attenuation in sand (Radwan et al., 2000). Co-inoculation of
Vicia faba plant roots with nodule bacteria and plant growth promoting
rhizobacteria enhanced plant growth and nitrogen fixation (Radwan et al.,
2005).

Phaseolus vulgaris has been selected as a good candidate for

phytoremediation because it can sustain growth in oil polluted soil (Nwoko
et al., 2007).

Ekpo and Nkanang (2010) investigated the nitrogen fixing capacity of

legumes cowpea (Vigna unguiculata) and groundnut (Arachis hypogea) and
their micrflora grown in diesel oil simulated utisol. Result revealed that
concentration as low as 1% v/w of diesel oil significantly affected the
densities of nitrogen fixing bacteria, bacteriods, actinomycetes and fungi
associated with the legumes. The heterotrophic bacteria count in the
rhizosphere of cowpea reduced from 2.46 ± 0.72 × 107 to 1.5 ± 0.37 ×
107cfu/g after growth duration of 12 weeks while it reduced from 3.4 ±
1.25 × 107 to 1.52 ± 0.36 × 107cfu/g for groundnut in the same growth
period. Nitrifying bacteria count reduced from 3.25 ± 1.19 × 104 to 4.5 ±
0.18 × 103cfu/g for cowpea and 3.43 ± 1.23 × 104 to 2.7 ± 0.21 × 103cfu/g
for groundnut. Bacteria’s count also significantly (P > 0.05) reduced from
3.85 ± 2.30 × 105cfu/g for the control treatment to 1.25 ± 2.23 × 105cfu/g
in 1% level of pollution with no bacteria formed in both 4 and 8% pollution
due to inhibition of nodule formation by the diesel oil. Significant reduction
(P>0.05) was also observed in fungal and actinomycetes counts. Generally,
organisms in the rhizosphere of groundnut exhibited more tolerance to
diesel oil pollution than those found in the rhizosphere of cowpea. This
study revealed that diesel oil adversely affected nitrogen fixing bacteria and
bacteriods and consequently the nitrogen fixation in the soil. ted the
phytoremediation of oil contaminated soil by some arid legume tree
species. They studied work the rhizosphere of Prosopis cineraria (L.) Druce,
Acacia senegal (L.) Willd and Acacia nilotica (L.) Willd. Ex. Del. plants were
tested for their abilities to stimulate the microbial degradation of soil
pollutants in desert soil contaminated with 2.5-2.6% crude petroleum oil.
The results showed that the roots of the three different plants were density
associated with total bacteria, fungi and oil-degrading microorganisms. This
is confirmed from the (R+/S+) ratios which ranged from 55.2-250.8 (for
total bacteria), 20 – 131.3 (for fungi) and 95.7-296.1 (for oil degraders).
Percentages of oil-degraders were higher in the rhizosphere soil of P.
cineraria (65.5%) as compared to the rhizosphere soil of A. senegal and A.
nilotica plants (22.5% and 20.2% respectively). The results of the
biodegradation of oil and its fractions showed that great reduction (26%) of
total petroleum hydrocarbons (TPHs) was observed in the rhizosphere soil
of P.cineraria as compared to 15.6% and 12.8% reduction in rhizosphere
soil of A. senegal and A. nilotica respectively. It was observed also that in
the polluted non-cultivated soil the TPHs were reduced by 8.2 -10.5% as a
result of biostimulation process only (addition of nutrients).The results also
showed that P.cineraria rhizosphere was able to reduce more of the
saturated (43.0%) and more of the aromatics (25.7%) fractions compared to
(35.2% and 7.9%) for A. senegal and (31.2% and 4.1%) for A. nilotica
rhizospheres. It is of interest to find that 5.3% of the hardly degradable
fraction resins were degraded in rhizosphere soil of P. cineraria. They
concluded from the study that P. cineraria provided successful
phytoremediation process of a contaminated desert soil as compared to
the other two legume trees.

1.9 AIMS AND OBJECTIVE

The aim of the research work is to affirming the effect of the microbial
activities of the crops grown on an oil polluted soil.

Specific objective of the present sturdy includes;

To ascertain the microbial activities of some selected pulse planted on an

oil polluted soil.

To investigate the effects of waste engine oil on the growth and survival of
the selected pulses.

MATERIALS AND METHODS

STUDY AREA

The site chosen for the research was a plot of land lying behind the Botanic
Garden near John Harris library Life Sciences Faculty, University of Benin,
Benin City, Nigeria, which lies within the rainforest ecological zone of
Midwestern Nigeria, with a mean annual rainfall of 1825 mm.

Collection and Preparation of Materials for the Experiment

Soil used in the present study was collected from an area measuring 50m x
50m marked on a farmland. Top soil (0-10cm), of pre-different
physicochemical property, was collected randomly from the marked plot in
the morning (7.00 am) and placed on polythene sheets that were spread on
an open platform and left in the sun until evening (5.00 pm) for drying.
Thereafter, 8kg soil each was placed into 30 large perforated 20-litre
buckets with 5 perforations made, at the bottom of each bucket, with a 2-
mm diameter nail.WEO was obtained as pooled sample from an auto-
mechanic workshop in Isihor, Ugbowo, Benin City that specialized in repair
of heavy duty trucks/vehicles.

Soil contamination using 140g of WEO in each Bucket

Soils in each bucket (8kg) were carefully poured out onto a flat platform
covered with cellophane. WEO was poured into the measured soil, and
thoroughly mixed, before taking back into each bucket.

Selection and Collection of pulses

The pulses were selected carefully by picking the seeds without any blemish
or crack on the seed, seed viability was further testes by floatation method.
The seed was collected from the market and was kept in a jar till the day of
planting.

Segregation into Treatment Buckets

The entire set up ( the polluted and non polluted ) was left in an open shade
for 1months, without mechanically disturbing the soil. Soil was carefully
irrigated every day until after 1 month which was later done twice a week
with 200 ml of water. The buckets were arranged into 3 replicates in each
species. 15 buckets of polluted soil and none polluted soil.

Seed propagation
 After one month, seeds were sown into each bucket at the rate of 3 seeds
per hole and at a depth of 3cm, in a triangular form then thinned down to 6
seedlings per bucket after seedlings had attained a 2-leaf stage.

Experimental Design

The experimental design chosen was the completely randomized design

(CRD) following assumption of homogeneity of the experimental plot in
use. As a result, treatments were randomized over the whole plot. Each
treatment consisted of 3 replicates. The treatment is in one concentration
of WEO in soil. The experiment was conducted for two approaches to
analyze WEO on polluted soil. Each buckets were properly labelled
according to a given specie name and replicate number, and were carefully
arranged according to its replicate.

Parameters Studied.

Number of sprouted seedlings

Plant height (cm)

Leaf number

Leaf area

Stem girth

Numbers of days of first flowering

Numbers of first pod formation.

Soil microbial analysis.

Some Growth and Yield Parameters Determined in Cowpea

Germination Experiments
Percentage emergence was calculated as the percentage of seeds that
sprouted above soil level of the 5 seeds originally sown per bucket. Heights
were also taken by aid of a transparent calibrated ruler at 9 days after
sowing (DAS). Dry weights were obtained after the stem is properly dried,
this is done by uprooting it carefully shaking off the sand attached to it.

Numbers of days of sprouting

This is the day it took each plant to germinate from the soils

Numbers of sprouted seedlings

This is the numbers of seeds sprouted out in each bucket.

Plant height:

This is the length of the main axils of the plant from stem level to the
meristematic` top of the plant, which was measured periodically with the
aid of a tape rule.

Leaf numbers:

The African yam bean plant is trifoliate, hence for every number of leave
counted; only trifoliate leaves were regarded. Measurements were
periodically repeated.

Leaf area:

Total leaflet area (surface area of a leaflet was calculated according to

Redford 1967), Leaf area= Maximum length x width x 0.75. This was
determining by aid of calibrated ruler.
Stem girth:

This was determining 1cm from the soil level. The width (i.e. the thickness)
was determine by using a venire calliper.

Measurement of Death of Cowpea Seedling

Total number of cowpea seedlings that turned yellow and eventually

became necrotic was determined. Survival percentage was also calculated
for seedlings at 2 weeks after sowing (WAS), being the percentage of
seedlings that survived at 2 WAS relative to total number of seedlings that
originally sprouted.

Number of Days to First Flowering: determined as number of days after

planting (DAP) taken when the first brightly coloured petals of emerges
from the floral bud (Ikhajiagbe, 2006).

CHARPTER 3
 RESULTS

INTERPRETATION OF TABLES

Table 1The numbers of days of sprouting of each selected edible

pulses.

Note : + dw2enotes sprouted seedling, while

Names of pulse 1st DAP 2nd DAP 3rd 4th 5th 6th 7th DAP
DAP DAP DAP DAP
Pol N. Poll N. P N. P N P N P N P N.
l po poll ol po oll . o .p o .p ol po
ll l ll p ll oll ll oll l ll
ol
l
Soya bean + + _ _ _ _ _ _ + +
(Glycin
Max)
Cow pea I.B + + + + _ _ _ _ _ _
(V.Uniguiculata)

Cow pea B.L + + + + + + + _ _ _ + +

( V.Unguiculata)
Common bean ( + + + + _ _ _ _ + _
Phaseolus
Vogaris )
Pigeon pea + + + + + +
( Cejanus Cajan)
_ denotes no sprouted seedlings.

Table 2 shows the number of days it took each sown seedlings to sprout
from the ground. In Glycin Max, the first seeds sprouted on the third DAP
while the second seed sprouted on the seventh DAP which appears on both
the polluted and non polluted soil. In Cow Pea (I.B), seedling germinated on
the third, forth, and the seventh DAP both on the polluted and non
polluted. In Cow Pea (B.L) the seeds were germinating at a low pace
throughout day two to seven DAP it had the highest germinated seedling
while soya bean has the lowest germinated seedlings. In Common Bean
seed sprouted on the third and fourth DAP, germinated seed was later
observed on the seventh DAP only on the polluted soil. In Pigeon Pea seeds
starts its germination on the fourth DAP through till on the seventh DAP
when no germinated seeds was observed.
 Graph from 3- 7

TABLE 3: The numbers of sprouted seedling of each selected edible

pulses.

Names of pulse 1st DAP 2nd DAP 3rd 4th 5th 6th 7th DAP TOTAL (%)
DAP DAP DAP DAP
Pol N. Poll N. P N. P N P N P N P N. POL N.PO
l po poll ol po ol . o .p o .p ol po
ll l ll l p ll oll ll ol l ll
L LL
ol l
l
4 3 1 1 44.4 44.44
4
Soya bean
(Glycin
Max)
Cow pea I.B 4 3 2 2 67 56
(V.Uniguiculata)

Cow pea B. l 2 2 1 2 2 1 1 1 1 78 56
( V.Unguiculata)
Common bean 2 2 2 2 1 56 44.44
(Phaseolus
Vogaris )
Pigeon pea 1 2 1 1 2 1 56 44.44
(Cejanus Cajan)
Table 3: shows the number of sprouted seedling of each selected edible
pulses. Germination basically occurs on the 3rd and 4th DAP only Cow pea
(B.L) germinated on the 2nd DAP. On the 3rd DAP, 4 soya bean, seeds sown
on polluted and 3 soya seeds sown on non polluted soil appeared, It was
also observed that after the 3rd DAP, soya bean did not germinate on 4 th, 5th,
and 6th, DAP then1 seed each later germinated on the seventh DAP both on
polluted and non polluted soil.. In Cow pea (I.B), 4 seeds germinated on the
polluted soil and 3 seeds germinated on the non polluted soil. 7 seeds have
germinated in Cow pea (B.L) 3 on polluted soil and 4 seeds on non polluted
soil. In common bean, 4 seeds has germinated 2 in polluted and 2 on non
polluted soil; pigeon pea had no sprouted seedling till this day.
 On the 4th to 7th DAP, soya bean shows no germinated seeds until the 7 th
DAP when 1 seed each sprouted on both polluted and non polluted soil so
the total percentage of soya bean seed germination is 44% both on polluted
and non polluted soil. Cow pea (I.B) had 2 germinated seeds each both on
polluted and non polluted only on the 4th DAP and it total seed percentage
seed germination is 67% in polluted and 56% in unpolluted soil. Cow pea
(B.L) which has the highest germinated seedlings in turn had the highest
percentage which was 78%. In common bean, 2 seedlings each sprouted on
4th DAP both in polluted and unpolluted soil then 1 seed later germinate on
the 7th DAP only on polluted soil. Pigeon pea start its germination on the 4 th
DAP with 1 seed on polluted and 2 seeds on unpolluted soil, then on the 5 th
DAP, 1 seedling each germinated both in polluted and unpolluted soil, later
on the 7th DAP, 2 seeds germinated in polluted and 1 seed on unpolluted
soil.

TABLE 4: Growth pattern in plant height (cm) of some selected

pluses .Some days after planting (DAP)

mes of pulse 5th Dap 8th 14th Dap 21st Dap 35th Dap 49th Dap 63th Dap 77th
Da Dap
p
Pol N. Poll N. Poll N. Poll N. Pol N Pol N. Poll N. Po N.
l poll poll poll poll l .p l poll pol ll po
oll l ll
2.2 2.2 12 11 23 12 24 30 25 31 27 38 28 39 30 50

ya bean (Glycin
ax)
w pea I.B 4 5 8 17 13 30+ 14. 31 16 35 18 37 19 40 21 69
Uniguiculata) 3

w pea B.L 2 4 4.7 3 8 20 11 28 8 33 8 39 10 42 19 67

Unguiculata)
mmon bean 2 4 8.4 16 13 29 17 28 21 35 26 39 28 41 30 63
haseolus Vogaris )
geon pea 1 2.2 5.7 5.7 15 15 19 19. 21 35 28 30 51 52 58 69
ejanus Cajan) 5
Table 4 shows the growth pattern in plant height (cm) of some selected
pulses DAP. In Glycine Max the height was 2.2cm high at the 5 th DAP which
 later grew to 30cm on the 77 th DAP on the polluted soil. While on the
unpolluted soil, the height was 2.2cm on the 5 th DAP then later became
above 50cm high on 77th DAP. For cow pea (I.B), the initial height on 5 th DAP
was 4cm high in polluted soil which grew with a steady growth rate until
21st DAP then remain stagnant in growth, but on unpolluted soil the initial
height was 5cm which attain a steady growth. In V.ungiuculata(Benin local)
the initial height on 5th DAP was 2cm then later grown to 24cm high on
polluted soil, while on non polluted soil, it was 4cm high initially which later
grew up to 69cm high. In Phaseolus Vagaris (common bean), the initial
height was 2cm on 5th DAP then later became 39cm on 77 th DAP on polluted
soil. While scheduled non polluted soil, the height was 4cm on 5 th DAP then
later grown to above 69cm height on the 77th DAP.

IN Cajanus Cajan, the height was 1cm on 5th DAP then later became 58cm
0n 77th DAP in polluted soil, while on the unpolluted soil, the height was
2.2cm high on 5th DAP and later grown to 69cm high on 77th DAP.

TABLE 5: The leaf numbers of some pulses

Names of pulse 5th DAP 8th DAP 14th 21sh DAP 35th DAP 49th DAP 63rd DAP 77th DAP
DAP
P N. P N. P N. Poll N. Poll N Pol N .p Poll N. P N.
o poll ol poll ol poll poll .p l oll poll ol poll
ll l l oll l

2 8 2 2 8 5 12 13 9.3 12 9.3 14 7.3 21 6 27

Soya bean
(Glycin
Max)
Cow pea I.B 3 8 2 2 5 9 11 27 11 33 8 42 8 2 49
( V.Uniguiculata
)

Cow pea B.L 3 8 2 2 6 8 0.3 27 1.3 36 0.6 45 49 54

(V.Unguiculata) 3 3
Common bean 2 10 2 5.3 6 10 7 21 10 2 1.2 22 8 28 8 32
(Phaseolus
 Vogaris )
Pigeon pea 4 2 2 5 8 12 21 15 32 20 44 26 49 5 54
(Cejanus Cajan) 1
Table 5 shows the leaf number of some selected pulses. Glycine Max
sprouted with two leafs each initially both on polluted and unpolluted
which later increased. Both Cow pea (I.B), (B.L) and common bean sprouted
with more than two leaves (trifoliate) while pigeon pea sprouted with two
tinny leaves.

TABLE 6: The leaf area of some pulses

Names of pulse 5th DAP 8th DAP 14th DAP 21sh DAP 35th DAP 49th DAP 63rd DAP 77th DAP

Po N Poll N. Pol N .p Poll N. Pol N .p Pol N .p Pol N. Pol N.

ll . poll l oll poll l oll l oll l pol l pol
p l l
ol
l
Soya 6.7 7 9 9 9 29 9 28 7 28 7 30 30
bean(Glycin
Max)
Cow pea I.B 9.4 2.7 17 18 8 33 8 36 10 39 9 40 8 44
( V.Unguiculata)

Cow pea B.L 14 23 13 11 1.3 33 0.2 46 41 47 51

(V.Unguiculata)
Common bean 12 25 13 22 8.5 42 61 46 49 52 57
(Phaseolus
Vogaris)
Pigeon pea 6 54 59 7 14 3 14 4 15 5 18 5 20 7
(Cejanus Cajan)
Table 6 shows the leaf area of some selected edible pulses. Glycine Max
had 6.7lb leaf area in polluted soil on 8 th DAP which later increased to 9lb
on 14th DAP then later reduced to 7 on 48th DAP later died before 77th DAP,
but in unpolluted soil, the initial leaf area was 7 on 8 th DAP which increased
continually above 30lb on 77th DAP. For Cow pea (Ife Brown), it had 9.4lb
leaf area on 8th DAP which maintain the increment till 14 th DAP then
dropped to 8lb till death in polluted soil. While on unpolluted soil, had it
initial leaf area to be 27lb on 8th DAP which had continues increment till the
end of the experiment. For common beans, the initial leaf area was 12lb on
8th DAP in polluted soil which later die off, while in unpolluted soil the initial
leaf are was 25lb which had constant increment. For pigeon pea, the leaves
area kept increasing in both polluted and none polluted soil.

TABLE 7: The stem girth some pulses

Names of 5th 8th DAP 14th DAP 21sh DAP 35th DAP 49th DAP 63rd DAP 77th DAP
pulse DAP
P N Pol N. Pol N. Pol N. Pol N. Pol N. Pol N. Pol N.
o . l pol l pol l pol l poll l pol l pol l poll
ll p l l l l l
ol
l
Soya 0.1 0.2 0.3 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.2 0.3 0.3
bean(Glycin
Max)
Cow pea I.B 0.2 0.3 0.3 0.4 0.3 0.4 9.4 0.4 0.4 0.4 0.3 0.4 0.3 0.5
(V.uniguiculata
)

Cow pea B.L 0.2 0.2 0.2 0.2 0.5 0.5 0.2 0.4 0.5 0.6 0.6
( V.unguiculata
)
Common bean 0.3 0.3 0.3 0.3 3.6 0.5 0.6 0.6 0.6 0.7 0.7
(Phaseolus
Vogaris )
Pigeon pea 0.1 O.1 0.1 0.1 0.2 0.3 0.2 0.3 0.3 0.5 0.3 O.5 O.4 0.6
(Cejanus
Cajan)
Table 7: shows stem girth of the selected pulses. For Glycine Max, the initial
stem girth was 0.1cm in polluted soil on 8 th DAP which later died after 63rd
DAP, while in unpolluted soil, its initial stem girth was 0.1 then increased to
0.3cm on 77th DAP. For Cow pea, (Ife brown and Benin local) and common
beans stem girth were initially 0.2cm then had a continuous increase in
unpolluted soil. While in polluted soil, it increased a little, some died off
while some remain stagnant compare to Pigeon pea that have same
continues stem girth which ranges from 0.1 to 0.4 from 8 th DAP to 77th DAP
throughout the research work.

Table 8. The collation of the parameter taken at 77 th DAP

Names of pulse Num. of Plant height Leaf Num. Leaf area Stem girth
sprouted (cm) (cm)
seedlings

Poll N. Poll N. Poll N. Poll N. Poll N.

Poll Poll Poll Poll Poll

5 4 30 50 6 27 30 0.3

Soya bean (Glycin

Max)
Cow pea I.B 6 5 4 69 49 8 44 0.3 0.5
(V.Uniguiculata)

Cow pea B.L 7 7 19 67 54 51 0.6

( V.Unguiculata)
Common bean 5 4 30 63 8 32 57 0.7
( Phaseolus Vogaris )
Pigeon pea ( Cejanus 4 4 58 69 51 54 20 7 0.4 0.6
Cajan)
Table 8 shows the coparerism of all the parameters of five edible pulses
grown on OPS at 77th DAP. Cow pea (B.L) has the highest germinated
seedling both on polluted and unpolluted while pigeon pea has the lowest
sprouted seedlings. When compared to their stem girth, cow pea (B.L) later
died in oil polluted soil but pigeon pea grows continually because of its
wide adaptable capability to different climate and soil (Troedson et al.,
1990).

Table 9: Microbial counts for soils collected away from the plants’ rhizospheric soil region
Unpolluted soil Oil-polluted soil
Cowpe Comm Soyb Pigeon Cowpe Cowpe Comm Soybea Pigeo Cowp
a (IB) on ean pea a (BL) a (IB) on n n ea
bean bean (BL)
Clostridium sp + + + + + + - + + -
Sarcina sp + - + + - + + + + +
*Micrococcus varians - + - - + + + - + -
M. luteus + + - - + - + - + +
Bacillus pumilis + + - + - - - + + -
*B. subtilis + + + + - + - + + +
Enterobacter aerogenes - - + - + + + - - -
Pseudomonas. aeruginosa + - + - + - + - + +

Heterotrophic bacteria (x 105 cfu/g) 2.1 2.3 0.9 2.1 2.1 1.3 1.0 1.9 2.9 1.2
Hyd. Deg. bacteria (x 105 cfu/g) 1.8 1.3 0.2 1.3 1.3 0.8 0.6 0.8 1.6 0.6
% Hydrocarbon degraders 85.71 56.52 22.2 61.90 61.90 61.54 60.0 42.1 55.2 50.0

Aspergillus niger + + + + + + + + + +
A. fumigatus - + + + + + - + - +
Penicillium sp + + + - + + + - + -
Fusarium sp - + + - - + - - - +
Rhiszopus stolonifer + + + + + - - + - -
Mucor sp - + - - + - + + + +
Geotrichum sp + - + + + + + + - +
Trichoderma sp + + - - + + + - - -

Heterotrophic Fungi (x 105 cfu/g) 3.2 1.9 1.5 1.6 1.6 4.6 4.2 2.9 2.1 2.5
Hyd. deg. Fungi (x 105 cfu/g) 2.6 1.2 1.1 0.9 1.0 3.0 2.8 1.8 1.3 0.8
% Hydrocarbon degraders 81.25 63.16 73.3 56.3 62.5 65.22 66.67 62.07 61.9 32.0

Table 9: Shows the microbial count for soils collected from the plants’
rhizopheric soil region 3MAPP. Clostridium sp, sarcina sp, M. luteus, Bacillus
pumilis, B.subtilis, and pseudomonas aerogeinosa are present in Cow pea
(I.B) on unpolluted soil compared to the microorganisms found in Cow pea
(I.B) grown on oil polluted soil, M. luteus, Bacillus pumilis, and
pseudomonas aerogeinosa were not found but contains micrococcus
varians and Enterobacter aerogines. Also pigeon pea grown on oil polluted
soil has all the microorganism except Enterobacter aerogines but M. luteus
pseudomonas aerogeinosa, micrococcus varians, Enterobacter aerogines,j
are also not present in pigeon pea grown on unpolluted soil.

Heterotrophic bacteria (x105 cfu/g) ranges from 0.9 soya bean to 2.3
common beans on unpolluted soil while in oil polluted soil, the lowest was
1.0 in common bean to the highest 2.9 in soya bean. Also in hydrocarbon
degrading bacteria x105 cfu/g soya bean in unpolluted soil had the lowest
rang while on the oil polluted soil the lowest rang was 0.6 found in cow pea
(B.L) and common bean. % hydrocarbon degraders shows that soya beans
have the lowest range which is 22.2% in unpolluted soil when compared o
the pulses sown on oil polluted soil which was 42.11% in soya bean.

Heterotrophic fungi (x105 cfu/g ) gives soya beans as the lowest 1.5 in
unpolluted soil while in oil polluted soil, the count ranges from 2.1 in
pigeon pea to 4.6 in cow pea(I.B). The lowest % hydrocarbon degrader is
pigeon pea which is 56.25% in unpolluted soil while the oil polluted soil
shows that cow pea had the lowest rang 32.0.

Table 10: Microbial composition of rhizospheric soils collected from within plants’ rhizospheric
soil region

Cowpe Comm Soyb Pige Cow Cowpe Comm Pigeon Benin Cowpe
a (IB) on ean on pea a (IB) on local a (BL)
bean pea (BL) bean
Clostridium sp - - - + + + - + -
Sarcina sp + - - - - - - - -
*Micrococcus varians - + - - + - + - -
M. luteus + - - + + + + - -
Bacillus pumilis - - - + - + - - -
*B. subtilis + + + + - + - + +
Enterobacter aerogenes - - - - - - - - -
Pseudomonas. aeruginosa - - - + - + - - -

Heterotrophic bacteria (x 105 cfu/g) 3.6 5.4 1.6 2.5 3.7 2.5 1.8 3.8 1.6
Hyd. Deg. bacteria (x 105 cfu/g) 2.5 2.9 1.1 1.3 1.2 1.2 0.4 1.8 0.8
% Hydrocarbon degraders 69.44 53.7 68.8 52.0 32.4 48.0 22.22 47.37 50

Aspergillus niger + + + - - + + + +
A. fumigatus - - - - - + - - -
Penicillium sp + + + + + + + + -
Fusarium sp - - - + - - - - -
Rhiszopus stolonifer + + + - + + - - -
Mucor sp - + - + + - + + -
Geotrichum sp - - - + - + - - -
Trichoderma sp - - - - - - - - -

Heterotrophic Fungi (x 105 cfu/g) 4.5 2.5 2.0 2.3 1.8 4.8 6.0 1.5 2.3
Hyd. deg. Fungi (x 105 cfu/g) 2.5 1.7 0.9 1.5 0.9 2.6 3.2 1.0 1.1
% Hydrocarbon degraders 55.56 68.0 45.0 65.2 50.0 54.17 53.33 66.67 47.83

Table 10: Microbial composition of rhizosperic of OPS collected from within

plants’ rhizospheric soil 3MAPP.

Table 10 shows microbial composition of rhizospheric soils collected from

within plants’ 3mapp. The analysis shows that most of the bacteria and
fungi were not present in most of the pulses when compared with table 9.
Cow pea(B.L) have the lowest number of bacteria and fungi which is only
one each

that is B. subtilis bacteria and Aspergillus niger fungi then the pulse with the
highest numbers of bacteria and fungi was cow pea (I.B) which had
clostridium sp, M. luteus, Bacillus punmilis, B. subtilis, and Pseudomonas
aeruginosa ( bacteria) then Aspergillus niger, A.fumigatus, Penicillum sp,
Rhizopus stolonifer, and Geotrichum sp (fungi) these in oil was B.subtilis.
heterotrophic bacteria ranges from 1.6cfu/g soya bean to 5.4cfu/g in
common bean then the lowest hydrocarbon degraders in percentage was
cow pea 32.43% while the highest was in cow pea 69.44% in unpolluted soil
compare these with the oil polluted soils that has it lowest bacteria in
common bean which was 22.22% and it highest bacteria in cow pea (B.L)
50%. In fungi, the lowest percentage hydrocarbon degraders was found to
be in soya bean 45.0%, while the highest was in common bean 68.0 in
unpolluted soil when compared to the oil polluted soil, pigeon pea had the
highest percentage hydrocarbon degraders which was 66.67% and the
lowest was in cow pea (B.L) 47.83%.