39

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: ubabright4real@yahoo.com; 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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