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Tropical Journal of Applied Natural Sciences Trop. J. Appl. Nat. Sci., 2(3): 39-51 (2019)
ISSN: 2449-2043
https://doi.org/10.25240/TJANS.2019.2.3.06
Available online: https://tjansonline.com
Organic and Inorganic Nutrients Mediated Enhanced
Bioremediation of Diesel Contaminated Soil
Uba, B.O.1*, Okoye, E.L.2, Ebodi-Henry, J.N.1, and Okoye, W.K.1
1Department of Microbiology, Chukwuemeka Odumegwu Ojukwu University, P.M.B.02 Uli Campus, Anambra State, Nigeria.
2Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, P.M.B. 5025 Awka, Anambra State, Nigeria.
*Corresponding author E-mail address: [email protected]; Tel. +2348069693773, +2347067562127
1. INTRODUCTION
rude oil and natural gas are the main sources of foreign exchange to the Nigerian economy. These sources contribute to
as much as 95 % to Nigeria’s budgetary expenditures. Oil and natural gas are found in the geological structures
underlying mangrove and associated coastal ecosystems of the Niger Delta (Ezekoye et al. 2017). Spillage of used
motor oils such as engine oil, diesel or jet fuel contaminates the natural environment with hydrocarbon. Hydrocarbon
contamination of the air, soil, and fresh water especially by polycyclic aromatic hydrocarbon (PAHs) has attracted public
attention because majority of PAHs are toxic, mutagenic, and carcinogenic (Omoni et al. 2015). Contamination of soil by oil
spills is a wide spread environmental problem that often requires cleaning up of the contaminated sites. This is because
petroleum hydrocarbons in soils adversely affect the germination and growth of plants in soils. There has been extensive
research to invent and improve methods for remediating polluted soils and water (Ogbo et al. 2010).
Efforts to remediate the negative impact of hydrocarbon pollution on the water and soil has resulted in several devices such as
Remediation by Enhanced Natural Attenuation (RENA) which involves many techniques including land farming by
biostimulation or bioaugmentation of soil biota with commercially available micro flora (Ezekoye et al. 2017). Bioremediation
technology which gives much hope on the restoration of polluted mangrove swamps is being utilized for the degradation of
crude oil in soil matrix by using microorganisms, to transform the petroleum hydrocarbons into less toxic compounds. This is
achieved by the help of bacteria, fungi, algae that produce enzymes capable of degrading harmful organic compounds (Orji et
C
ABSTRACT
The enhanced bioremediation of diesel contaminated soil as mediated by organic
and inorganic nutrients was evaluated in this study. The method employed for the
physicochemical analysis include determinations of pH, conductivity, temperature,
moisture content, nitrate, phosphate, total organic carbon and total petroleum
hydrocarbon while total heterotrophic bacterial count and total culturable
hydrocarbon utilizing bacterial count were employed for the microbiological
analysis during the 56th days experimental period. The results revealed that all the
five treatments of the polluted garden soil had slightly acidity to slightly alkaline
pH, decreased in conductivity, moderate temperature range, low to high moisture
contents, decreased trends of nitrate, phosphate and total organic carbon levels. It
was observed that as the TPH of the diesel decreased during the 56th days study
period, the population of the hydrocarbon utilizing bacterial isolates increased
significantly (P < 0.05) in all the treated options with the highest reduction observed
in the order: NaNO3 > poultry waste > NH4Cl > cow dung amended set ups in
comparison to the unamended control set up. Thus, the application of organic (cow
dung and poultry wastes) and the inorganic (NH4Cl and NaNO3) nutrients has
shown promising potentials in bioremediation of diesel polluted soil and pilot scale
study is therefore recommended.
Original Research Article
Received: 14th Jan., 2019.
Accepted: 6th Feb, 2019.
Published: 11th Feb., 2019.
Keywords:
Biofertilizers,
bioremediation,
contamination, diesel,
hydrocarbon utilizing
bacteria, inorganic
nutrients
40
al. 2012a). Biostimulation is the process of providing microbial communities with a favorable environment in which they can
effectively degrade contaminants and in most cases involves the provision of rate – limiting resources like nitrogen, phosphorus
and oxygen (usually by tilling to aerate the soil) to speed up the bioremediation process (Ezekoye et al. 2017).
The use of inorganic fertilizers as source of limiting nutrients has been extensively carried out. However, the use of inorganic
fertilizers is still challenged by the large cost of bioremediation and likely chance of eutrophication/algae bloom especially in
aquatic environments. It is worthy to state that a good remediation method must be environmentally friendly and affordable.
The use of organic nutrients such as chicken droppings, periwinkle shells, cow dung for the bioremediation of crude oil polluted
environments other than mangrove swamps have been previously reported in Nigeria (Ijah and Antai, 2003; Obire et al., 2008).
Ezekoye et al. (2017) reported that the application of poultry wastes especially non – sterile poultry wastes can effectively
enhance bioremediation of hydrocarbon impacted mangrove soil and this could be attributed to the presence of indigenous
hydrocarbon utilizing bacteria in non -sterile poultry wastes. Orji et al. (2012a) reported that the use of organic nutrient sources
such as cow dung has shown good promises in bioremediation of crude oil impacted Mangrove Swamps in the Niger Delta.
Ezekoye et al. (2015) reported that NPK fertilizer though expensive was more effective in the bioremediation of hydrocarbon
polluted soils compared with poultry droppings. Although, previous studies reported the use of both organic and inorganic
nutrients in the remediation of crude oil polluted soil, there is dearth of information regarding the remediation of diesel oil
polluted soil using organic and inorganic nutrients. In this study, we report the organic and inorganic nutrients mediated
enhanced remediation of diesel contaminated soil.
2. MATERIALS AND METHODS
2.1 Study Area
The soil was obtained from Chukwuemeka Odumegwu Ojukwu University (COOU), Uli Ihiala L.G.A. Anambra State. The
site was selected due to high level of pollution arising from the generator or Lister owned by the University.
2.2 Sample Collection
The composite soil sample was collected with a sterile spade into sterile plastic buckets which were cleaned with cotton wool
soaked in 70 % ethanol to ensure that aseptic conditions are met during sampling as described by Eziuzor and Okpokwasili
(2009). The soil was collected from four sampling points after excavation, which was then transported to Microbiology
Laboratory of Chukwuemeka Odumegwu Ojukwu University for preliminary physicochemical analysis and bioremediation
study. Co-ordinates of the sampling points were determined using Handheld Global Positioning System (GPS) (GPSMAP
76sc). The co-ordinates were: 05°46”08.1”N; 06°50”01.0”E (Station 1), 04º47”34.9”N; 006º58”24.9” E (Station 2),
04º47”34.8” N; 006º58”24.9” E (Station 3) and 04º47”36.0” N; 006º58”24.9” E (Station 4). The diesel was obtained from a
commercial petrol station along Owerri – Onitsha Expressway, Uli Anambra State, Nigeria.
2.3 Preparation of the Organic and Inorganic Nutrients
Cow dungs were collected from cow slaughter house located at Odumodu Market, Umunya and poultry droppings were
collected from Okoye Farm located at Nkpor both in Anambra State. Cow dungs and poultry droppings of 200 g each were
sun-dried for 3 days until moisture was driven off completely and was later stored for usage. The inorganic nutrients Triton X
and NH4Cl were purchased from chemical dealers at Head Bridge Market, Onitsha Anambra State, Nigeria. Physicochemical
and microbiological analyses were carried out before bioremediation study.
2.4 Soil Contamination and Baseline Study
About 150 ml of diesel was poured into the bucket containing 1500 g of soil. The polluted soil at this point was sampled for
baseline studies. Baseline study is the analysis of current situation to identify the starting point for a project. It is also use to
determine the level of impact expected and to enable the monitory of impacts after the development has occurred (Ezekoye et
al. 2015).
2.5 Study Design
The study was experimentally designed by adopting the method of Eziuzor and Okpokwasili, (2009) and the details are shown
in Table 1 below:
41
Table 1: Bioremediation design of the study
2.6 Bioremediation Experiment
These was carried out ex situ in the Microbiology Laboratory (COOU). One thousand, five hundreds grams (1500 g) of soil
was mixed with 150 ml of diesel and was prepared in 5 setups using plastic buckets and were left in the laboratory for 6 days.
After contamination, 50 g of cow dungs, poultry droppings, ammonium chloride (NH4Cl) and sodium nitrate (NaNO3) were
added to the diesel polluted soil and the control was not amended either of the organic or inorganic nutrients and it was called
zero hour as described by Ezekoye et al. 2015. The samples containing nutrients and control were regularly turned using
different sterile spatula as well as moistened with 20 ml of sterile distilled water every 2 weeks. Samples were taken for
laboratory analysis at 2 weeks intervals on the 1st, 14th, 28th, 42nd, and 56th days (Romanus et al. 2015). The bioremediation of
diesel in the different experimental setups were studied as described below:
2.6.1 Physicochemical analysis
The pH, conductivity and temperature were measured using digital multimeter (DSS – 11A, China) by adopting the standard
method of AOAC (2012). The moisture contents were determined by dry weight procedure in oven (DHG- 9053AA, Life
Assurance Scientific, UK) using the standard method described by AOAC (2012).
2.6.2 Chemical analysis
The Brucine method stated by UNEP (2004), was employed for the measurement of nitrate content at 470 nm on
spectrophotometer (Astell, UV - Vis Grating, 752 W). Colorimetric method was employed for the estimation of phosphate
content as defined by UNEP (2004), measured spectrophotometrically at 660 nm and matched with identically prepared
standard (water). The colorimetric method of Nelson and Sommers (1975), was adopted for estimating the total organic carbon
(TOC) by titrating blank containing oxidant (potassium chromate) and sulphuric acid as against the sample and the titre value
was recorded. The spectrophometric method of of Adesodun and Mbagwu (2008), was used for determination of total petroleum
hydrocarbons (TPH) at 640 nm using N – Hexane as the extractive solvent.
2.6.3 Microbiological analysis
2.6.3.1 Enumeration of total heterotrophic bacteria count (THBC)
The spread plate method on nutrient agar was used in the enumeration of total heterotrophic bacteria at fourteen days interval.
A 10 - fold serial dilution of the soil samples were carried out by weighing 1 g each of soil samples into sterile test tubes
containing 9 ml of sterile physiological saline and diluted to 10-5. From each dilutions, 0.1 ml were pipetted and inoculated on
nutrient agar plates. However, a triplicate plating of each dilutions were employed. A sterile glass rod was used to spread the
inoculums over the media. The plates were incubated for 18 – 24 hours at a temperature of 37 oC. After which the emerging
colonies were counted. Colonies that formed during this incubation period were counted using this formula:
𝐍𝐨. 𝐨𝐟 𝐜𝐨𝐥𝐨𝐧𝐢𝐞𝐬 𝐱 𝐃𝐢𝐥𝐮𝐭𝐢𝐨𝐧 𝐟𝐚𝐜𝐭𝐨𝐫
𝐀𝐦𝐨𝐮𝐧𝐭 𝐮𝐬𝐞𝐝
Values were expressed as colony forming units per g (Cfu /g). Enumeration of total heterotrophic bacteria was carried out using
the stated procedures have been previously reported by Chikere et al. (2009).
2.6.3.2 Enumeration of total culturable hydrocarbon utilizing bacteria (TCHUB)
The modified method of Chikere and Chijioke-Osuji, (2006) was used to determine the total culturable hydrocarbon utilizing
bacteria on mineral salt agar containing: 0.04 g MgSO4. 7H2O, 0.03 g KCl, 0.09 g KH2PO4, 0.04 g NaNO3, 0.13 g K2HPO4,
Experimental setup Test experiment
Setup 1 (control) 1500 g of polluted soil + 150 ml of diesel + 20 ml of water
Setup 2 1500 g of polluted soil + 150 ml of diesel + 50 g of cow
dung + 20 ml of water
Setup 3 1500 g of polluted soil + 150 ml of diesel + 50 g of poultry dropping + 20
ml of water
Setup 4 1500 g of polluted soil + 150 ml of diesel + 50 g ammonium chloride +
20ml of water
Setup 5 1500 g of polluted soil + 150 ml of diesel + 50 g sodium nitrate + 20 ml
of water
42
2.0 g NaCl, 15 g of Agar powder, 100 ml of distilled water amended with 0.01 g to inhibit the growth of fungi. Then, it was
sterilized by autoclaving at 121 °C and 15 psi for 15 min. and allowed to cool to about 45 °C. The already prepared medium
was poured into Petri dishes and allowed to gel after which 0.1 ml of the inocula was spreaded onto the plates with spreading
rod under aseptic conditions. A sterile filter paper (Whatman No. 1) was impregnated with diesel oil and was aseptically placed
on the cover of the Petri- dishes and covered. The plates were incubated by inversion for 7 – 10 days at 28 ºC.
2.7 Statistical Analysis
Statistical analyses were carried out on the mean ± SD values of the data obtained from experimental studies using GraphPad
Prism Version 7.00. Two way ordinary analysis of variance (ANOVA) and Dunnet comparison test were used to test for
significance at 95 % confidence intervals (P < 0.05) among the various treatments during the 56 days experimental study.
3.0 RESULTS
3.1 Physicochemical properties of Unpolluted Soil
The result of the physicochemical properties of the soil sample before contamination is presented in Table 2. From the results,
the pH, conductivity, temperature, moisture content, nitrate, phosphate and total organic carbon were: 7.40, 500 µS/ cm, 27.10 oC, 20.01 %, 2.00 mg/ kg, 1.80 mg/ kg and 4.95 %, respectively.
Table 2: Physicochemical properties soil sample before contamination
Parameter Value
pH 7.40
Conductivity (µS/ cm) 500
Temperature (oC) 27.10
Moisture content (%) 20.01
Nitrate (NO3) (mg/ kg) 2.00
Phosphate (PO4) (mg/ kg) 1.80
Total organic carbon (TOC) (%) 4.95
3.2 Physicochemical and Microbiological Properties of Biostimulating Agents
The results of the physicochemical and microbiological properties of animal nutrients used for biostimulation study are
presented in Table 3. From the result, the pH, conductivity, temperature, moisture content, nitrate, phosphate, and total organic
carbon result for cow dung were: 7.50, 340 µS/ cm, 28.20 oC, 54.01 %, 4.00 mg/ kg, 2.20 mg/ kg, 6.95 % and 7.00, 280.00 µS/
cm, 29.60 oC, 50.54 %, 5.00 mg/ kg, 2.10 mg/ kg, 5.25 % for cow dung and poultry dropping, respectively. The total petroleum
hydrocarbon content, total hydrocarbon bacteria count and total culturable hydrocarbon utilizing bacteria count of cow dung
and poultry dropping were 5, 571.03 mg/kg, 9.05 LogCfu /g, 8.81 LogCfu /g and 5, 446.62 mg/kg, 8.92 LogCfu /g and 9.04
LogCfu /g respectively.
Table 3: Physicochemical and microbiological properties of animal nutrients used for biostimulation study
Parameters Cow dung Poultry waste
pH 7.5 7.0
Conductivity (µS/cm) 340 280
Temperature (oC) 28.2 29.0
Moisture content (%) 40.01 50.54
Nitrate (mg/kg) 4.0 5.0
Phosphate (mg/kg) 2.2 2.1
Total organic carbon (%) 6.95 5.25
Total petroleum hydrocarbon (mg/kg) 5, 571.03 5, 446.62
THBC (LogCfu /g) 9.05 8.92
TCHUB (LogCfu /g) 8.81 9.04
3.3 Base Line Features of Diesel Impacted Soil
The result of the baseline physicochemical and microbiological properties of the diesel impacted soil is presented in Table 4.
From the results, the pH, conductivity, temperature, moisture content, nitrate, phosphate and total organic carbon were: 7.40,
500 µS/ cm, 27.10 oC, 72.01 %, 2.00 mg/ kg, 1.80 mg/ kg and 4.95 %, respectively. The total petroleum hydrocarbon content
was 47, 619.05 mg/kg while the total heterotrophic bacterial count and total culturable hydrocarbon utilizing bacterial count
were 6.53 LogCFU/ g and 6.73 LogCFU/ g, respectively.
43
Table 4: Base line physicochemical and microbiological properties of diesel impacted soil
3.4 Changes in the Physical Properties of Organic and Inorganic Nutrients Treatments
The result of the changes in pH of diesel polluted soil amended with organic and inorganic nutrients during 56 days incubation
is shown in Figure 1. From the result, all the five treatments of the polluted garden soil were slightly acidity (4.50) to slightly
alkaline (7.80) from day 0 hour to 56th days of the study. In the control (without nutrients), the pH for the 0 hour to 56th were
slightly alkaline (7.27 - 7.97). The result of the changes in conductivity of diesel polluted soil amended with organic and
inorganic nutrients during 56 days incubation is shown in Figure 2. The result showed that conductivity decreased from 23.80
µS/ cm to 0.51 µS/ cm in all the five treatments of the polluted garden soil. In addition, control experimental set-up showed a
slight decrease in conductivity from 22.70 µS/ cm – 0.73 μS/ cm. The result of the changes in temperature of diesel polluted
soil amended with organic and inorganic nutrients during 56 days incubation is shown in Figure 3. From the result, all the five
treatments of the polluted garden soil and the control had mesophilic temperature range 26.70 – 31.70 and 27.40 – 29.00,
respectively. The result of the changes in moisture content of diesel polluted soil amended with organic and inorganic nutrients
during 56 days incubation is shown in Figure 4. The result revealed that the day 1 had the highest moisture content value of
89.05 % on the control set up while the day 14 had the least moisture content value of 37.95 % on the NH4Cl set up.
Fig. 1. Changes in pH of diesel polluted soil amended with organic and inorganic nutrients during 56 days incubation
012345678
control cow dung poultrywaste
NH4CL NaNO3
day 1 7.9 7.2 7.8 6.2 5.7
day14 7.3 7.02 6.9 5.2 6.5
day 28 7.27 7.5 6.5 5.6 6.2
day 42 7.8 7.6 7.7 5.5 6.8
day 56 7.97 7.42 6.6 4.9 5.4
pH
day 1day14day 28day 42day 56
Parameter Value
pH 7.40
Conductivity (µS/ cm) 500
Temperature (oC) 27.10
Moisture content (%) 72.01
Nitrate (NO3) (mg/ kg) 2.00
Phosphate (PO4) (mg/ kg) 1.80
Total organic carbon (% TOC) 4.95
Total petroleum hydrocarbon (mg/ kg) 47, 619.05
THBC (LogCfu/ g) 6.53
TCHUB (LogCfu/ g) 6.73
44
Fig. 2. Changes in conductivity of diesel polluted soil amended with organic and inorganic nutrients during 56 days incubation
1 da y
1 4 da y
2 8 da y
4 2 da y
5 6 da y
0
1 0
2 0
3 0
4 0
S tim u la tin g p e rio d
Te
mp
era
ture
(C
)
c o n t r o l
c o w d u n g
p o u lt ry w a s te
N H 4C l
N a N O 3
1 d
ay
14
da
y
28
da
y
42
da
y
56
da
y
0
2 0
4 0
6 0
8 0
1 0 0
S tim u la tin g p e rio d
Mo
istu
re c
on
ten
t (%
)
c o n tro l
c o w d u n g
p o u ltry w a s te
NH 4 C l
N aNO 3
0
5
10
15
20
25
30
control cow dung poultrywaste
NH4CL NaNO3
day 1 22.7 23.8 28.1 2.95 10.98
day14 2.41 4.97 4.7 5.02 6.88
day 28 2.97 3.1 3.33 1.49 1.29
day 42 0.37 0.47 0.11 0.39 0.34
day 56 0.73 0.51 0.79 0.48 0.25
Co
nd
uct
ivit
y (µ
S/ c
m)
day 1
day14
day 28
day 42
day 56
Fig. 3. Changes in temperature of diesel polluted soil amended with organic and inorganic nutrients during 56 days
incubation
Fig. 4. Changes in moisture content of diesel polluted soil amended with organic and inorganic nutrients during 56
days incubation
45
3.5 Changes in the Chemical Properties of Organic and Inorganic Nutrients Treatments
3.5.1 Changes in concentration of nitrate levels
The result of the changes in the concentration of nitrate level of diesel polluted soil amended with organic and inorganic
nutrients during 56 days incubation is shown in Figure 5. The result showed that the day 1 had the highest value of nitrate level
(7.00 mg/ kg) on the cow dung treated set up while day 56 had the least value of nitrate level (2.00 mg/ kg) on the NaNO3
treated set up during the 56th days of biostimulation study.
Fig. 5. Changes in the concentration of nitrate level of diesel polluted soil amended with organic and inorganic nutrients during
56 days incubation.
3.5.2 Changes in concentration of phosphate levels
The result of the changes in the concentration of phosphate level of diesel polluted soil amended with organic and inorganic
nutrients during 56 days incubation is shown in Figure 6. The result showed that the day 1 and day 14 had the highest and least
values of nitrate levels of 4.80 mg/ kg and 1.90 mg/ kg on the cow dung treated set up during the 56 th days of biostimulation
study.
Fig. 6. Changes in the concentration of phosphate level of diesel polluted soil amended with organic and inorganic nutrients
during 56 days incubation
01234567
control cow dung poultrywaste
NH4CL NaNO3
day 1 6 7 6 2.6 2
day14 3.8 3.6 2 4.1 3.8
day 28 3.8 4.2 3.8 4.2 4.2
day 42 4 3.8 4.4 3.8 4
day 56 4.8 5.8 4.6 6.4 4.2
Nit
rate
(M
g/ k
g) day 1day14day 28day 42day 56
00.5
11.5
22.5
33.5
44.5
5
control cow dung poultrywaste
NH4CL NaNO3
day 1 4 4.8 4.6 4.4 4.2
day 14 2 1.9 2.2 2.3 2.2
day 28 2.3 2.3 2.1 2.2 2.3
day 42 2.3 2.3 2.1 2.2 2.2
day 56 2.8 2.6 2.7 3.1 2.9
Ph
osp
hat
e (M
g/ k
g)
day 1
day 14
day 28
day 42
day 56
46
3.5.3 Changes in concentration of total organic carbon levels
The result of the changes in the total organic carbon level of diesel polluted soil amended with organic and inorganic nutrients
during 56 days incubation is shown in Figure 7. The result showed that the day 1 had the highest value of total organic carbon
level (4.55 %) on the poultry waste treated set up while day 42 had the least value of total organic carbon level (0.36 %) on the
control untreated set up during the 56th days of biostimulation study.
1 da y
1 4 da y
2 8 da y
4 2 da y
5 6 da y
0
1
2
3
4
5
S tim u la tin g p e rio d
To
tal
org
an
ic c
arb
on
(%
) c o n t r o l
c o w d u n g
p o u lt ry w a s te
N H 4C l
N a N O 3
Fig. 7. Changes in the total organic carbon level of diesel polluted soil amended with organic and inorganic nutrients during 56
days incubation
3.5.4 Changes in concentration of total petroleum hydrocarbon levels
The result of the changes in the total petroleum hydrocarbon level of diesel polluted soil amended with organic and inorganic
nutrients during 56 days incubation is shown in Figure 8. In the cow dung and poultry waste amended option, it decreased from
12, 150.67 mg/ kg to 3, 685.96 mg/ kg and 12, 165.45 mg/ kg to 3, 541.08 mg/ kg while in the NH4Cl and NaNO3 amended
options, it decreased from 12150.67 mg/ kg - 3554.92 mg/ kg and 12165.45 mg/ kg - 3312.36 mg/ kg, respectively. In the
control experiment, the total petroleum hydrocarbon decreased at day 1 from 35, 473.76 mg/ kg to 20, 000 mg/ kg at day 56.
Fig. 8. Changes in the total petroleum hydrocarbon level of diesel polluted soil amended with organic and inorganic nutrients
during 56 days incubation
0
5000
10000
15000
20000
25000
30000
35000
40000
control cow dung poultrywaste
NH4CL NANO3
day 1 35473.76 12150.67 12165.45 12150.67 12165.45
day 14 31605.32 5537.09 5564.48 5542.24 5549.39
day 28 27736.88 5470.46 5455.54 5425.94 5527.92
day 42 23868.44 5482.46 5479.45 5500.55 5482.46
day 56 20000 3685.96 3541.08 3554.92 3312.36
To
tal
pet
role
um
hyd
roca
rbo
n (
mg/
kg)
day 1
day 14
day 28
day 42
day 56
47
3.6 Changes in the Microbiological Properties of Organic and Inorganic Nutrients Treatments
3.6.1 Changes in total heterotrophic bacteria count
The result the changes in total heterotrophic bacterial count (THBC) of diesel polluted soil amended with organic and inorganic
nutrients during 56 days incubation is shown in Figure 9. From the result, the cow dung and poultry wastes amended option
increased from 8.72 Logcfu/ g and 8.48 Logcfu/ g at day 1 to 8.83 Logcfu/ g and 8.90 Logcfu/ g at the 56th day. The total
heterotrophic bacterial count in the control experiment ranged between 8.53 logcfu/ g to 8.91 logcfu/ g. In both cases the growth
of the heterotrophic bacterial organisms was lowest at day 1 and highest on the 56th day of study.
Fig. 9. Changes in total heterotrophic bacterial count (THBC) of diesel polluted soil amended with organic and inorganic
nutrients during 56 days incubation
3.6.2 Changes in total culturable hydrocarbon utilizing bacterial count (TCHUB)
The result the changes in in total culturable hydrocarbon utilizing bacteria count (TCHUB)) of diesel polluted soil amended
with organic and inorganic nutrients during 56 days incubation is shown in Figure 10. From the result, the cow dung and poultry
wastes amended option increased from 8.54 Logcfu/ g and 8.16 Logcfu/ g at day 1 to 8.85 Logcfu/ g and 8.86 Logcfu/ g at the
56th day. The in total culturable hydrocarbon utilizing bacteria count in the control experiment ranged between 8.93 logcfu/ g
to 8.87 logcfu/ g. Also, in both cases the growth of the hydrocarbon utilizing bacterial organisms was lowest at day 1 and
highest on the 56th day of study.
8.1
8.2
8.3
8.4
8.5
8.6
8.7
8.8
8.9
9
9.1
control cow dung poultrywaste
NH4CL NaNO3
day 1 8.52 8.72 8.48 8.83 8.9
day14 8.62 8.74 8.65 8.85 8.89
day 28 8.89 8.7 8.82 9.03 8.99
day 42 8.9 8.84 8.86 8.94 9.08
day 56 8.91 8.83 8.9 8.98 9.09
To
tal
het
ero
tro
phic
bac
teri
a co
unt
(lo
gcf
u/
g)
day 1
day14
day 28
day 42
day 56
48
Fig. 10. Changes in in total culturable hydrocarbon utilizing bacteria count (TCHUB)) of diesel polluted soil amended with
organic and inorganic nutrients during 56 days incubation
4. Discussion
Over the times, petroleum release has affected the physico - chemical and biological characteristics of soil, with resultant little
food productivity by decreasing the nutrients accessibility to the soil through improved soil and petroleum fraction toxicity.
Organic and inorganic fertilizers have been found to be resourceful in that they contribute essential nutrients which are needful
for effective remediation of contaminated soil.
In this study, the interactive effects of organic and inorganic nutrients in the remediation of diesel contaminated soil was
evaluated and the result in Table 2 revealed that the soil is neutral to slightly alkaline in pH, moderate in conductivity, low in
nitrate, phosphate and total organic carbon contents, high in moisture content with moderate/mesophilic temperature. The result
in Table 3 revealed that both nutrients are neutral to alkaline in pH, moderate to high in conductivities, high in moisture contents
with moderate/mesophilic temperatures, low in nitrates, phosphates and total organic carbon contents, high number of total
hydrocarbon bacteria counts and total culturable hydrocarbon utilizing bacteria counts for both cow dung and poultry dropping,
respectively. The presence important limiting nutrients such as nitrate and phosphates which are pertinent growth dynamic for
microbial growth have implicated in cow and poultry wastes by Ezekoye et al 2017 and Orji et al. 2012a. They reported that
the addition of these nutrients is a significant aspect in accomplishing efficient biodegradation of hydrocarbons. The baseline
result in Table 4 also showed slightly alkaline in pH, high in conductivities, low in moisture contents with moderate/mesophilic
temperatures, low in nitrates, phosphates and total organic carbon contents, high quantities of total petroleum hydrocarbon
contents and is in line with the report of Ezekoye et al. 2017. There were lower but significant counts of total heterotrophic
bacteria and total culturable hydrocarbon utilizing bacteria with higher counts of total culturable hydrocarbon utilizing bacteria
than total heterotrophic bacteria revealing the likely toxicity of the diesel on the native soil heterotrophic bacteria as well as
diesel pollution history of study area. The baseline data results showed that the hydrocarbon utilizing microorganisms in the
polluted soil is reasonably satisfactory for bioremediation studies and corroborates with previous studies (Ebuehi et al., 2005;
Orji et al. 2012a, Ezekoye et al. 2017).
The result in Figure 1 showed that both nutrients had varying slightly acidic to alkaline pH observations which is favourable
for the growth most organisms observed in this study. This pH variation could be due to metabolites secreted at diverse phases
of the bioremediation study and significant differences (P < 0.05) were detected among the control and all the treatment set up
and is in-line with previous reports by Romanus et al. (2015) and Agarry and Jimoda (2013). The result in Figure 2 revealed
that the observed significant (P < 0.05) decreased in conductivity of both treatment and control set ups could be due to
absorption of nutrients ions and salts by the high microbial populations recorded and is similar to previous studies by Abu and
Akomah (2008) and Orji et al (2012a). Zhu et al. (2001) reported that in-situ and ex-situ bioremediation conductivity tests
7.6
7.8
8
8.2
8.4
8.6
8.8
9
9.2
control cow dung poultrywaste
NH4CL NaNO3
day 1 8.93 8.54 8.16 8.6 8.7
day14 8.68 8.63 8.67 8.81 8.99
day 28 8.72 8.69 8.78 8.81 9
day 42 8.86 8.83 8.84 8.9 8.99
day 56 8.87 8.85 8.86 8.93 9.01
Tota
l cu
ltu
rab
le h
ydro
carc
bo
n u
tiliz
ing
bac
teri
a (l
ogc
fu/
g)
day 1
day14
day 28
day 42
day 56
49
results are as surrogate values for salinity and total dissolved solid but it is frequently used than the latter’s due to easiness of
dimension. The result in Figure 3 showed that the temperature ranges favour the growth and survival of mesophilic
microorganisms as no significant (P > 0.05) moderate temperature were observed in all the treatments and control. John and
Okpokwasili (2012) reported that temperature is among the key environmental factor that affect microbiological activity. The
result in Figure 4 revealed that there were fluctuations in the moisture content values of all the four treatments and control with
no significant differences (P > 0.05) detected among their means. The moisture contents values were higher in the polluted and
nutrient treated soil than the unpolluted soil and biostimlating organic and inorganic nutrients. This differences could be
attributed to the weekly addition of sterile water to the experimental set ups. Ezekoye et al. (2015) reported that biodegradation
is lively as long as the soil moisture content was in the range between 20.00 % and 80.00 % of the maximum water holding
capacity.
The result in Figure 5 revealed that the nitrate levels of cow dung and poultry waste amended polluted soil increased to 7.0 mg/
kg and 6.0 mg/ kg at day one and later decreased to 5.8 mg/kg and 4.6 mg/ kg on day 56 with the polluted soil baseline values
of 2.0 mg/ kg. The nitrate levels of NH4Cl and NaNO3 nutrients in the amended polluted soil slightly increased to 2.6 mg/ kg
and 2.0 mg/ kg at day one and later increased to 6.4 mg/ kg and 4.2 mg/ kg on day 56 with the polluted soil baseline values of
2.0 mg/ kg during the bioremediation study. The nitrate levels of the control set up increased and decreased from 2.0 mg/ kg -
6.0 mg/ kg and 6.0 mg/ kg to 4.8 mg/ kg. There was significant differences detected (P < 0.5) for the five experimental set ups
at 95 % interval. The result in Figure 6 showed that the concentration of phosphate increased to 4.8 mg/ kg in the cow dung
and 4.6 mg/ kg in the poultry waste amended set ups and later decreased to 2.6 mg/ kg and 2.7 mg/ kg; while the concentration
of phosphate increased to 4.8 mg/ kg in the NH4Cl and 4.2 mg /kg in the NaNO3 amended set ups and later decreased to 3.1
mg/ kg and 2.9 mg/ kg during the 56th days study with the polluted soil baseline values of 1.8 mg/ kg. The phosphate levels of
the control set up increased and decreased from 1.8 mg/ kg - 4.0 mg/ kg and 4.0 mg/ kg to 2.8 mg/ kg. There was significant
differences detected (P < 0.5) for the five experimental set ups at 95 % interval. The initial increased on day 1 and later
decreased in day 56 clearly showed that these two limiting nutrients (nitrate and phosphate) were absorbed by the bacterial
degraders in the experimental set ups for cellular metabolism during the bioremediation period further revealing that there is
positive association between nitrate and phosphate nutrient utilization and is similar to the observations in previous studies
(Zhu et al. 2001; Orji et al. 2012a; Ezekoye et al. 2015; 2017).
Also, the result in Figure 7 showed that the concentration of total organic carbon (TOC) slightly decreased to 4.49 % in the
cow dung and 4.55 % in the poultry waste amended set ups and further decreased to 3.24 % and 1.50 %; while the concentration
of total organic carbon (TOC) slightly decreased to 4.8 % in the NH4Cl and 4.2 % in the NaNO3 amended set ups and further
decreased to 2.40 % and 2.34 % during the 56th days study with the polluted soil baseline values of 4.95 %. The concentration
of total organic carbon (TOC) of the control set up decreased and further decreased from 4.95 % - 3.69 % and 3.69 % to 2.73
%. There was no significant differences detected (P < 0.05) for the five experimental set ups at 95 % interval. A non-significant
(P > 0.05) positive association was observed between changes in conductivity and total organic carbon (TOC) of the amended
diesel contaminated soil and corroborates with the finding of previous study (Orji et al. 2012a). The result in Figure 8 showed
that there were varying reductions in the amount of total petroleum hydrocarbon (TPH) in all the four treatments and their
control. There was significant decrease from 12,150.67 mg/ kg to 3,685.96 mg/ kg in the cow dung amended set up; 12,165.45
mg/ kg – 3,541.08 mg /kg in the poultry waste amended set up; decreased from 12,150.67 mg/ kg to 3,554.92 mg/ kg in the
NH4Cl amended set up and 12,165.45 mg/ kg – 3,312.36 mg/ kg in the NaNO3 amended set ups from day 1 to the 56th days
study with the polluted soil baseline values of 47,619.05 mg/ kg. The amount of total petroleum hydrocarbon (TPH) in the
control set up decreased from 35,473.76 mg/ kg – 20,000 mg/ kg. There was significant differences detected (P < 0.05) for the
five experimental set ups at 95 % interval. The highest reduction was in the order: NaNO3 > poultry waste > NH4Cl > cow
dung amended set ups in comparison to the unamended control set up. The significant high hydrocarbon losses or reductions
in all the four treatment set ups could be attributed to the addition of the organic and inorganic nutrients which act as necessary
nitrogen and phosphorus sources thereby increasing the number of hydrocarbon utilizers in the treated polluted soil and
enhanced the biodegradation of diesel oil. The low reduction or loss observed in the control set up could be attributed to natural
attenuation processes reflecting inactive remediation and significance of limiting nutrients which were absent in the non-treated
control set up. Similar results were published by Obasi et al. (2013) who observed highest significant loss of TPH in treatments
amended with Poultry manure and Cow dung (PM + CM) followed by Poultry manure (PM) treatment. In another similar
observation, Ezekoye et al. (2015) reported that NPK fertilizer treatment option showed more effectiveness in removing
THC/TPH from impacted medium, followed by Non-sterile Poultry Waste and Sterile Poultry Waste. Chikere et al. (2009),
observed and reported that the NPK 20:10:10 fertilizer option reduced TPH from 3, 666.0 mg/ kg to 89.68 mg/ kg for 57 th days
where as urea fertilizer option reduced TPH from 3, 666 mg/ kg to 162 mg/ kg for 57 th days. In the poultry droppings option,
the TPH was reduced from 3, 666.0 mg/ kg of soil to 135.01 mg/ kg of soil.
The populations of total heterotrophic and hydrocarbon utilizing bacteria are shown in Figures 9 and 10. The base line values
for the total heterotrophic bacterial count (THBC) and total culturable hydrocarbon utilizing bacteria count (TCHUBC) were
6.53 Logcfu/ g and 6.73 Logcfu/ g, respectively. In addition, during the study period, bacterial populations of the cow dung
(CD) and poultry waste (PW) amended options increased from 8.72 Logcfu/ g to 8.83 Logcfu/ g, 8.48 Logcfu/ g to 8.90 Logcfu/
g and 8.54 Logcfu/ g to 8.85 Logcfu/ g and 8.16 Logcfu/ g to 8.86 Logcfu/ g for THBC and TCHUBC from day 1 to day 56,
respectively. Also, bacterial populations of the NH4Cl and NaNO3 amended options increased from 8.83 Logcfu/ g to 8.98
Logcfu/ g, 8.90 Logcfu/ g to 9.09 Logcfu/ g and 8.60 Logcfu/ g to 8.93 Logcfu/ g and 8.70 Logcfu/ g to 9.01 Logcfu/ g for
50
THBC and TCHUBC from day 1 to day 56, respectively. The total heterotrophic bacterial count in the control experiment
ranged between 8.52 Logcfu/ g to 8.91 Logcfu/ g and 8.93 Logcfu/ g to 8.87 8.93 Logcfu/ g for THBC and TCHUBC from day
1 to day 56, respectively. In all the set ups, the growths of the heterotrophic and hydrocarbon bacterial organisms were lowest
at day 1 and highest on the day 56 except in the control where there was decreased in TCHUBC. There was significant
differences detected (p < 0.05) in the four experimental set ups in comparison to their controls. The hydrocarbon utilizing
bacterial organisms responded well to the nutrient amendments with the organic (cow dung and poultry wastes) and the
inorganic (NH4Cl and NaNO3) nutrients. The response of native hydrocarbon utilizing bacteria to the bioremediation treatments
was largely positive with significant higher counts obtained as study progressed. The bacterial species in this study
demonstrated capabilities to either degrade or utilize the diesel hydrocarbon fractions as carbon and energy sources and these
findings are supported by previous studies (Okolo et al., 2005; Obire et al. 2008; Orji et al. 2012a; 2012b; Ezekoye et al. 2015;
2017).
5. Conclusion and Recommendation
The result of this research study has revealed that cheap fertilizers/nutrients such as cow dung, poultry waste, NH4Cl and
NaNO3 are effective in the supply of limiting nutrients necessary for the growth of microorganisms and subsequent
enhancement of bioremediation of diesel impacted soil. It was observed that as the TPH of the diesel decreased during the 56th
days study period, the population of the hydrocarbon utilizing bacterial isolates increased significantly (P < 0.05) in all the
treated options with the highest reduction observed in the order: NaNO3 > poultry waste > NH4Cl > cow dung amended set
ups in comparison to the unamended control set up. This cost effective organic fertilizers can be harnessed into preserved
forms and be used for bioremediation.
Further research attention should be given for larger scale studies or pilot-scale studies on the use of cow dung, poultry waste,
NH4Cl and NaNO3 nutrients to bio- remediate diesel impacted garden soils is therefore recommended.
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How to cite this article Uba, B.O., Okoye, E.L., Ebodi-Henry, J.N., and Okoye, W.K. (2019). Organic and Inorganic Nutrients Mediated Enhanced Bioremediation
of Diesel Contaminated Soil. Tropical Journal of Applied Natural Sciences, 2(3): 39-51.
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