Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

Bioremediation of Crude Oil-Contaminated Soil

in the Presence of Nickel, Zinc and Cadmium

Heavy Metals Using Bacterial and Fungal

Consortia-Bioaugmentation Strategy

1

Samuel Enahoro Agarry *, Ganiyu Kayode Latinwo , Ebenezer Olujimi Dada , Chiedu

Ngozi Owabor²

¹Biochemical and Bioenvironmental Engineering Laboratory, Department of Chemical Engineering, Ladoke Akintola University

of Technology, Ogbomoso, Nigeria

2

Department of Chemical Engineering, Federal University of Petroleum Resources, Effurun, Delta State, Nigeria

Received: 10/01/2019

Accepted: 27/02/2019

Published: 01/06/2019

Abstract

The study evaluated the effectiveness of indigenous bacterial consortia (Pseudomonas aeruginosa, Bacillus subtilis and

Micrococcus letus) and fungal consortia (Aspergillus niger, Aspergillus carmari and Penicillium notatum) as well as their

combination (bacterial-fungal consortia) as bioaugmentation agents in the soil bioremediation of petroleum hydrocarbons in the

absence and presence of nickel, zinc and cadmium heavy metals. Bioremediation was carried out in 10% w/w crude oil-

contaminated soil microcosms for 35 days in the absence and presence of nickel, zinc, and cadmium bioaugmented with or

without bacterial, fungal and bacterial-fungal consortia, respectively. In the heavy metal-free soil microcosms, 72.5%, 64% and

90.7% total petroleum hydrocarbon (TPH) biodegradation were attained with bacterial, fungal and bacterial-fungal consortia,

respectively, while 45% TPH biodegradation was achieved in the non-bioaugmented soil microcosm. In the heavy metal-soil

microcosms: nickel, zinc, cadmium and mixed form (nickel + zinc + cadmium), 79.2%, 81.4%, 75.3% and 68.2% TPH

biodegradation was correspondingly obtained with bacterial consortia; 69.4%, 66.4%, 68.2%, and 60.6% with fungal consortia;

while 99%, 98.5%, 95.7%, and 100% was respectively attained with bacterial-fungal consortia. The kinetics of TPH

biodegradation were adequately described by the first-order kinetics and half-life times were estimated. Soil microcosm

bioaugmented with bacterial-fungal consortia displayed the highest biodegradation rate constant with the lowest half-life times in

the absence and presence of heavy metals. Therefore, the results suggest that microbial consortia (bacterial and fungal) could be

very effective for soil bioremediation of crude oil in the presence of heavy metals.

Keywords: Bacteria; Bioaugmentation; Bioremediation; Crude oil; Fungi; Heavy metals

1

four thousand incidents of crude oil spills have been

estimated to have occurred in the Niger Delta region of

Nigeria, amounting to several million barrels of crude oil

1

Introduction

Nigeria is the largest oil producer in Africa with a

maximum oil production capacity of 2.5 million barrel per

day and the sixth largest oil producing country in the world

[

3]. About 90% of the contaminated sites in the USA are

soils that are contaminated with petroleum hydrocarbons

4].

[

1]. All over the world, oil is transported through pipelines,

[

vessels/ships, road, and rail, and as a result poses serious

danger to the environment in case of spills. In 2005, almost

every day, about nine incidents of oil pollution were

reported around the world [2]. Since 1960 till date, over

Crude oil consists of complex mixture of aliphatic and

aromatic hydrocarbons, asphaltene and resins which are

considered to be environmental pollutants [5]. In addition,

trace amount of heavy metals have been recognized to exist

in crude oils [3, 6]. The metals commonly considered as

pollutants includes aluminium, arsenic, cadmium,

chromium, copper, mercury, nickel, lead, iron, zinc and

some radionuclides [7]. These hydrocarbons and heavy

metals have been found to be toxic, mutagenic and

carcinogenic [8]. However, some of these metals such as

Corresponding author: Samuel Enahoro Agarry,

Biochemical

and

Bioenvironmental

Engineering

Laboratory, Department of Chemical Engineering, Ladoke

Akintola University of Technology, Ogbomoso, Nigeria. E-

mail: sam_agarry@yahoo.com.

179

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

copper, chromium, nickel, zinc and iron play an integral

role as micronutrients in the life metabolic processes of

microbes and can as well at higher concentrations become

toxic and inhibit various cellular or biochemical processes

by forming unspecific complex compounds within the

microbial cell [9]. While several other metals like

cadmium, lead, silver and mercury are not essential, do not

have any biological role and are potentially toxic to

microorganisms [9].

The toxicity of these hydrocarbons and metals

necessitated the need to develop methods or technologies

that are environmentally-friendly to remediate oil spills

contamination of the environment (soil or water). One of

such technology is bioremediation due to its efficiency and

cost-effectiveness as compared to physicochemical

technologies that are expensive [10]. Microbial species that

utilizes hydrocarbons as a source of carbon and energy are

referred to as hydrocarbonoclastic microorganisms. They

are the major agents of bioremediation and thus petroleum

hydrocarbons degradation due to their associated metabolic

abilities [2]. Reviews of literature on the petroleum

hydrocarbons degradation have confirmed that several

microorganisms majorly bacteria and fungi are capable of

utilizing petroleum hydrocarbons as the sole source of

carbon and energy [2, 11, 12].

The desire for environmentally-friendly and sustainable

technology towards contaminated environment has made

the evolving bioremediation technology a standard practice

for the treatment and restoration of contaminated

environment. Over three decades, considerable studies have

been carried out in both the laboratory and field in the area

of bioremediation with different modifications such as

nutrient supplementation (biostimulation) [13] and

microbial inoculation (bioaugmentation) for the

remediation of diverse range of contaminants [14 – 16]. In

all of these studies which did not involve heavy metals

interference, bioaugmentation strategy has been found to be

successful.

Bioaugmentation is normally recommended for

contaminated sites where the autochthonous microbial

populations is insufficient for contaminants degradation

and/or those in which the indigenous microorganisms do

not possess the necessary catabolic pathways for

contaminants metabolism [17, 18]. However, for

environment such as soil and water co- contaminated with

both metals and organic compounds, they are considered to

be crucially difficult to treat due to the mixed nature of the

pollutants [19]. Thus a co-contaminated environment

represent a serious problem in the bioremediation processes

[24] or the use of phytoremediation [25, 26].

There are certain number of metal-resistant

microorganisms that can detoxify metals, such as cadmium

[27, 28]. These metal-resistant and detoxifying

microorganisms possesses the capability to exhibit some

metal resistance mechanisms that allows them to function

in a metal co-contaminated environments and these include:

intracellular and extracellular sequestration; extracellular

precipitation; redox transformation; membrane efflux

system; exclusion by permeability barrier; and enzymatic

detoxification [9]. Despite the bioremediation successes

that have so far been recorded using bioaugmentation

strategies, very few studies involving bioaugmentation

strategy with the use of either single strain/pure culture or

mixed culture of two microbial strains for the

bioremediation of petroleum hydrocarbons such as

polycyclic aromatic hydrocarbons (PAHs) and heavy

metals co-contamination have been carried out [20, 29 –

32]. From these studies, varying forms and degrees of

organic compound degradation have been reported. For

instance, Alisi et al. [20] reported that complete

degradation of hydrocarbon n-C12–20 and total

disappearance of phenanthrene in diesel oil as well as 75%

diesel oil reduction was achieved by bacterial

bioaugmentation in the presence of heavy metals. Owabor

et al. [29] also reported that the degradation of naphthalene

in soil by inoculated microbial consortium made up of

Bacillus and Aspergillus niger species was gradually

inhibited as the concentration of each of the heavy metals

(Pb, Hg, Ni and Cr) increased from 40 to 200 mg/L.

Bioaugumentation strategies involving the use of

a

consortium consisting of Acremonium sp. and Bacillus

subtilis demonstrated high degradation efficiency in soil

that is heavily contaminated with crude oil [33], while the

presence of heavy metals have been revealed to impact on

the fungal-bacterial synergism in polycyclic aromatic

hydrocarbons (PAHs) degradation [31].

In spite of these information that revealed that soil

bioremediation in the presence of heavy metals can be

achieved with bioaugmentation strategy, the strategy is still

faced with a number of challenges with regard to the

presence of heavy metals, which often result in the toxicity

and inhibition of microbial growth and limit degradation

ability. There is still paucity of information on the influence

of heavy metals as single or mixture form on

bioaugmentation strategy using mixed bacterial consortia,

fungal consortia and bacterial-fungal consortia in the

bioremediation of petroleum hydrocarbons in crude oil

contaminated soil.

[

20]. This is because metals cannot be biologically

Hence, this present study aimed to explore the

effectiveness of mixed indigenous bacterial consortia,

mixed indigenous fungal consortia and mixed bacterial-

fungal consortia as bioaugmentation agents in the soil

bioremediation of petroleum hydrocarbons in crude oil in

the absence and presence of heavy metals (nickel, zinc and

cadmium) in single or mixture form. The rates of petroleum

hydrocarbons biodegradation were determined from the

application of first-order kinetics and the biodegradation

half-life times were estimated.

degraded or modified like toxic organic compounds, but

their speciation and bioavailability may change with

varying environmental factors [21, 22]. Also, metals may

inhibit the hydrocarbon biodegradation through its

interaction with microbial enzymes that are directly

involved in the biodegradation or through its interaction

with microbial enzymes involved in general metabolism

[23]. Nevertheless, to overcome this difficulty is to either

employ the use of bioaugmentation with microorganisms

that has the ability to resist and detoxify metal as well as

the use of organic compound-degrading microorganisms

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

2

. Materials and Methods

isolated from the collected oil polluted soil samples using

the serial dilution and pour or spread plate method [28].

The soil samples were sieved and 10 g of it were added into

90 mL de-ionized water in a conical flask, vigorously

shaken and left overnight. For bacteria isolation, 0.1 mL

2.1 Chemicals and Reagents

All the chemicals and reagents are of analytical grade

such as: n-hexane, calcium chloride solution, zinc chloride,

cadmium sulphate, nickel nitrate, nutrient agar (NA), and

potato dextrose agar (PDA).

-1

aliquots of clear soil suspension and its dilutions (from 10

-3

–

10 ) were poured and spread on a NA plate containing

2

.2 Sample Collection

Soil samples were obtained from a farmland in Oleh

crude oil droplets, Nystatin (to prevent fungal growth) and

the plate was then incubated at 37°C for 24 hours; and the

grown bacterial colonies were isolated [35]. For fungi

isolation, 0.1 mL aliquots of the soil suspension and its

town (Latitude 5.4589° N and Longitude 6.2031° E) of

Delta State in the Niger-Delta region of Nigeria. It has no

pollution history and devoid of hydrocarbon contamination.

It was the top soil layer of the farmland not exceeding a

depth of 25 cm from the surface that was excavated with a

shovel, collected into a black polyethylene bag and brought

to the laboratory. Crude oil used for artificial pollution was

obtained from Warri Refinery and Petrochemical Company

located in Warri (5.5544° N, 5.7932° E) in the Niger-Delta

region of Nigeria. The microbial strains used in this study

were cultured from the soil sample obtained from a

previously oil contaminated soil in the Niger-Delta region

of Nigeria.

-1

-3

dilution (10 – 10 ) were introduced into sterile Petri

dishes containing PDA and droplets of crude oil and then

o

incubated at ambient temperature (28 ± 2 C) for 72 – 120

hours. Streptomycin antibiotic (10 mg/L) was added into

the PDA medium to inhibit any form of bacterial growth.

After 3-5 days of fungal growth, the spore bearing mycelia

were then carefully sectioned, teased out and stained on a

slide using lactophenol cotton blue stain and later observed

with a light microscope. The bacterial and fungal colonies

with different morphologies obtained after incubation were

sub-cultured and purified repeatedly by streaking on sterile

NA and PDA plates, respectively. Pure cultures of isolated

bacterial species were identified and characterized on the

basis of Bergey’s manual [36]. Standard conventional

methods that involved various cultural, morphological,

physiological and biochemical tests were performed

namely gram staining, catalase test, indole test, citrate

utilisation test, urease test, motility test, oxidase test,

coagulase test, glucose utilization, fructose utilization,

lactose utilization, lipase test [37]. The isolated bacterial

species were identified as Pseudomonas aeruginosa,

Bacillus subtilis, Staphylococcus aureus, Staphylococcus

epidermidis, Escherichia coli and Micrococcus letus.

Pseudomonas, Bacillus, Staphylococcus, E.coli and

Micrococcus species have been reported to have the

potential to biodegrade petroleum hydrocarbons [38, 39].

The fungal isolates were identified and characterized on the

basis of cultural, microscopic (septation of mycelium,

shape, form, diameter and texture of spore/conidia), and

macroscopic (pigmentation, shape, diameter, colony

appearance and texture) features [28, 40]. The cultural and

morphological features of the fungal isolates were then

compared with those described by Samson et al. [41] as

well as using the keys of Mackie and McCartney [42]. The

fungal isolates were identified as Aspergillus niger,

Aspergillus carmari, and Penicillium notatum. Aspergillus

and Penicillium species have been confirmed to have the

potential to biodegrade the petroleum hydrocarbons in

crude oil [43, 44].

2.3 Characterization of Soil

Physical, chemical and microbiological properties of the

top soil samples at the onset of the remediation were

characterized. The soil samples were sieved, pulverized and

air-dried. Then, the samples were analyzed for pH,

moisture content, total organic carbon, total nitrogen, and

available phosphorus using standard methods [34]. The soil

was also analyzed for total hydrocarbon degrading bacteria

(

THDB) and total hydrocarbon degrading fungi (THDF)

count using pour plate method [28]. The metals (calcium,

magnesium, nickel, zinc and cadmium) concentrations in

the soil samples were determined after wet digestion of 0.5

3 4

g of the soil n 10 mL of concentrated HNO and HClO

7:1 v/v) and the concentrations measured using atomic

(

absorption spectrometry (AAS) with a flame furnace

nebulizer (Perkin-Elmer). The physical and chemical

characteristics of the soil were as follows: pH (6.8 ± 0.3),

moisture content (10.2 ± 0.2%), total organic carbon (0.76

±

0.03%), total nitrogen (0.12

phosphorus (0.46 ± 0.24%), nickel (< 0.01 mg/kg), zinc (<

.01 mg/kg), cadmium (< 0.01 mg/kg), calcium

14.24meq/100g) and magnesium (9.85meq/100g). The

± 0.01%), available

0

(

microbiological properties of the soil were as follows:

THDB count (0.8 ± 0.01 ×10 cfu/g) and THDF count (0.2

±

hydrocarbon degrading microorganisms in the test soil

sample is very small and thus there is the need to bio-

augment the soil with more hydrocarbon degrading

microorganisms.

2

0.03 ×10 cfu/g). This revealed that the population of the

2.5 Heavy Metal Tolerance Index of Bacterial and Fungal

Isolates

2

.4 Source of Bacterial and Fungal Consortia

The bacteria and fungi species used for this study were

Isolated bacterial and fungal species were assessed

either as pure isolates or as mixed isolates (consortium) for

181

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

2

+

2+

their heavy metal tolerance or resistance to Cd , Zn and

2.7 Experimental Design and Bioremediation Protocol

The soil sample from the farmland was sieved with a 5

mm mesh sieve to remove large stones and debris.

Thereafter, the sieved soil samples were sun-dried by

spreading on a flat clean surface for one week and then

pulverized. A known weight of the soil sample (150 g) was

each introduced into sixteen separate clean dry plastic

containers (i.e. soil microcosm) and moistened to about

20% water holding capacity with sterilized de-ionized

water. Thereafter, 18 mL of crude oil corresponding to 15 g

(i.e. 10% w/w) was measured and introduced into each of

the soil in the sixteen plastic containers and the contents

were thoroughly stirred. The sixteen soil microcosms were

2+

Ni . Eight mm diameter circular extract or disks from 10

day old pure cultures of each bacterial isolates and fungal

isolates were inoculated into separate corresponding NA

and PDA Petri-dishes or plates supplemented with each of

the sterilized 31.13 mg/L Ni (NO ) , 20.84 mg/L ZnCl and

3 2 2

8.55 mg/L CdSO salts solution corresponding to 10 mg/L

each of Ni , Zn and Cd heavy metals, respectively. The

inoculated plates were incubated at ambient temperature

28±2°C) for at least 7 to 10 days to establish their growth.

The experiments were performed in triplicates with

bacterial isolates in NA plate and fungal isolates in PDA

plate without supplementation with heavy metals that

served as control. The radial growth was determined by

measurement of the culture spread from the center of the

colony or inoculated portion. The inoculated portion

diameter was subtracted from the diameter of growth [45].

The tolerance index (TI), which is an indication of the

microbes response to the stress of heavy metal was

calculated from the growth of microbial isolates exposed to

the heavy metals divided by the growth of the microbial

isolates in the absence of heavy metals [27]. The microbial

isolates heavy metal tolerance can be rated as follows [28]:

1

4

2+

(

labelled B-T

1

to B-T16

.

The 10% w/w artificial

contamination was adopted so as to achieve severe

contamination because beyond 3% w/w concentration, oil

has been reported to be increasingly deleterious to soil biota

and crop growth [13, 46]. Soil microcosm B-T

the control (i.e. natural bioattenuation). After the oil

contamination, 31.13 mg/L of nickel nitrate (Ni(NO ),

18.55 mg/L cadmium tetraoxosulphate (VI) (CdSO ), and

20.84 mg/L zinc chloride (ZnCl ) solutions each of which

2+

corresponded to 10 mg/L of Ni , Cd and Zn

1

served as

3 2

)

4

2

2+

0

.00–0.39 (very low tolerance), 0.40–0.59(low tolerance),

.60–0.79 (moderate tolerance), 0.80–0.99 (high tolerance)

concentrations, respectively, were added singly into the

contaminated soil microcosms while 70.52 mg/L of mixed

2+

heavy metal salts solution corresponding to 30 mg/L (Ni

2+ 2+

and 1.00- >1.00 (very high tolerance). The higher the TI

values, the higher the microbial species tolerance or

resistance to the heavy metal.

+ Zn + Cd ) concentration was added as shown in Table

1. The soil microcosms were left for 14 days to allow for

aging or equilibration. After day 14, mixed bacterial

(Pseudomonas aeruginosa, Bacillus subtilis and

Micrococcus letus) and mixed fungal (Aspergillus niger,

Aspergillus carmari, and Penicillium notatum) inoculums

were inoculated into the contaminated soil microcosms as

also shown in Table 1 and thoroughly mixed together with

2

.6 Preparation of Bacterial and Fungal Inoculum

The bacterial and fungal inoculums were prepared

according to the method of Ma et al. [15]. To prepare and

generate the bacterial inoculum required for

bioaugmentation, the bacterial isolates were separately

o

grown in a NA broth at 30 C and shaken in an orbital

a

stirring rod. The inoculated contaminated soil

shaker at 120 rpm for 24 h. Thereafter, the cultures were

centrifuged at 10,000 rpm for 10 min. Then, the cell pellets

obtained were washed twice with phosphate buffer (0.1 M,

pH 7.0), and again re-suspended in a fresh phosphate buffer

and the resulted suspensions were mixed together in equal

proportions and used as mixed bacterial culture (bacterial

consortia) for the study. Similarly, to prepare fungal

inoculums for bioaugmentation, the fungal isolates were

microcosms were covered with aluminum foil and then

incubated for 35 days. At intervals of 3 days, small volume

(30 mL) of sterilized de-ionized water was added to the

contaminated soil microcosms and stirred together so as to

maintain the moisture content. At intervals of 7 days, soil

sample was taken to carry out analyses for residual TPH,

microbial (bacterial and fungal) count and pH, respectively.

o

grown separately in a PDA liquid medium at 30 C and

2.8 pH Measurement

shaken in an orbital shaker at 100 rpm for 72 h. Then, the

cultures were centrifuged and the fungal mycelia obtained

were washed twice with phosphate buffer, and thereafter re-

suspended in a fresh phosphate buffer and the resultant

suspensions were mixed in equal proportions and used as

mixed fungal culture (fungal consortia). The volume of the

liquid medium was 30 mL with 10 cfu/ml of either

bacteria or fungi to be added to each of the contaminated

soil microcosms.

The pH of the soil samples was measured in the course

of the bioremediation studies according to the following

procedure: 10 g of the soil sample was accurately weighed

and added into 10 mL of de-ionized water in a conical

flask. The mixture was allowed to stand for 15 min after

which the flask was placed in an orbital shaker (SSL1-

model) and agitated at 150 rpm for 30 min. At the end of

the agitation, the mixture was allowed to stand for 10 min

and the pH value was read on an already calibrated pH

meter (JENWAY 3020-model).

6

182

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

Table 1: Soil microcosm artificially contaminated with crude oil and heavy metal and bioaugmented with microbial species

Soil Microcosm Code

Description

B-T

1

2

3

4

5

6

7

8

9

(Control)

Soil + Oil (Natural Bioattenuation)

Soil + Oil + Bacterial Consortium

Soil + Oil + Bacterial Consortium + Nickel

Soil + Oil + Bacterial Consortium + Zinc

Soil + Oil + Bacterial Consortium + Cadmium

Soil + Oil + Bacterial Consortium + Nickel + Zinc + Cadmium

Soil + Oil + Fungal Consortium

Soil + Oil + Fungal Consortium + Nickel

Soil + Oil + Fungal Consortium + Zinc

B-T10

B-T11

B-T12

B-T13

B-T14

B-T15

B-T16

Soil + Oil + Fungal Consortium + Cadmium

Soil + Oil + Fungal Consortium + Nickel + Zinc + Cadmium

Soil + Oil + Bacterial- Fungal Consortium

Soil + Oil + Bacterial-Fungal Consortium + Nickel

Soil + Oil + Bacterial-Fungal Consortium + Zinc

Soil + Oil + Bacterial-Fungal Consortium + Cadmium

Soil + Oil + Bacterial-Fungal Consortium + Ni + Zn + Cd

TPH TPH

o

f

100

2

.9 Determination of Total Petroleum Hydrocarbon

The total petroleum hydrocarbon (TPH) content of the

%TPH 

(1)

TPH_o

artificially contaminated soil samples was determined

according to the following procedure: 5 g of the soil sample

was weighed into a plastic bottle and 25 mL of n-hexane

was added to the soil sample and the mixture shaken on an

orbital shaker (SSL1-model) at 250 rpm for 20 min and

then allowed to stand. Filtration of the mixture was done

and the hexane solvent in the filtrate was allowed to

evaporate at room temperature in a fume hood. The

concentration of extracted residual TPH in the filtrate was

then determined or measured at an absorbance of

wavelength 400 nm using the UV-VIS Spectrophotometer

where TPH_oand TPH _fare the initial and residual TPH

concentrations, respectively.

2.11 Determination of TPH Biodegradation Rate and

Half-life Time

The rate of TPH biodegradation was determined from

the application of first-order kinetic model equation as

given in Eq. (2) [13, 47]:



o

kt

TPH  TPH e

(2)

t

(

JENWAY 6715-model).

where TPH

O

and TPH

t

are the initial TPH and residual TPH

2

.10 Determination of Total Hydrocarbon-Degrading

t

concentration (mg/kg) at time in soil (mg/kg), k is the

−1

Bacterial and Fungal Count

Quantification of the total hydrocarbon-degrading

bacteria (THDB) and total hydrocarbon-degrading fungi

biodegradation rate constant (day ), and t is time (day).

The biodegradation half-life time (t1/2) was calculated from

the biodegradation rate constant (k) using Eq. (3) [13, 47]:

(

THDF) present in the soil samples was determined by the

pour plate count method [28]. Soil samples (10 g) was

transferred into sterilized Erlenmeyer conical flasks

containing 90 mL of sterile 0.9% (m/v) NaCl solution and

then shaken in an orbital shaker at 150 rpm for 15 min.

Samples (1mL) were subjected to a serial 10-fold dilution

procedure and cultivated in a NA medium for THDB at 30

ln2

(3)

k

3

. Results and Discussion

3

.1 Heavy Metal Tolerance Index of Bacterial and Fungal

Isolates

o

C for 48 h and in a PDA medium for THDF at 30 C for 72

h, respectively. The number of colony forming units (cfu)

was counted in each sample and expressed as colony-

forming units per gram of dry soil (cfu/g dry soil). All

microbiological counts and experiments were carried out in

triplicate. The TPH concentration was expressed as mg of

petroleum hydrocarbons per kg of dry soil [13]. Eq. (1) was

employed to calculate the percent TPH biodegradation [13]:

In ascertaining the tolerance of the bacterial and fungal

2+ 2+ 2+

isolates to heavy metals (Ni , Zn and Cd ) of 10 mg/L

concentration, the tolerance index (TI) was estimated and

the values are presented in Table 2. The tolerance rating of

Pseudomonas aeruginosa and Bacillus subtilis (being

bacterial isolates) as well as Aspergillus niger, Aspergillus

carmari and Penicillium notatum (fungal isolates) to 10

2+

mg/L each of Ni , Zn and Cd were observed to be high,

with TI ranging between 0.80 and 0.99. Similar

observations have been reported for Aspergillus sp and

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Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

Penicillium sp as well as Pseudomonas aeruginosa and

Bacillus subtilis using higher concentration of Cd [48, 49],

Ni [49] and Zn [49], respectively. In addition, high

tolerance of Pseudomonas sp., Bacillus sp, Aspergillus

sp.,and Penicillium sp. for heavy metals such as Zn, Ni and

Cd has been reported [50, 51]. Meanwhile, the tolerance

rating of Pseudomonas aeruginosa and Bacillus subtilis

amount [53]. On the other hand, Smrithi and Usha [54]

have reported the metal tolerance by bacteria isolates from

tannery effluent in the decreasing order of Ni > Zn > Cd

while Amalesh et al. [55] stated that the decreasing order

for metal tolerance is Cd > Ni.

On the other hand, the tolerance ratings of the bacterial

consortia (Pseudomonas aeruginosa, Bacillus subtilis and

Micrococcus letus), fungal consortia (Aspergillus niger,

Aspergillus carmari and Penicillium notatum) and

bacterial-fungal consortia (Pseudomonas aeruginosa,

Bacillus subtilis, Micrococcus letus, Aspergillus niger,

Aspergillus carmari and Penicillium notatum) to 10 mg/L

(

being bacterial isolates) as well as Aspergillus niger,

Aspergillus carmari and Penicillium notatum (fungal

isolates) to 30 mg/L of the combined or mixed heavy

2+

metals (Ni + Zn + Cd ) were observed to be moderately

low, with TI ranging between 0.10 and 0.62.

2+

Pseudomonas aeruginosa showed a relatively higher TI

each of Ni , Zn and Cd were observed to be very high,

with TI ranging between 1.81 and 2.38. These very high TI

values showed that microbial consortia can tolerate the

presence of heavy metals more than the individual

component in the consortium. In addition, the TI for the

2+

for Ni , Zn and Cd and this was followed by Bacillus

subtilis, Micrococcus letus, Escherichia coli,

Staphylococcus aureus and Staphylococcus epidermidis,

respectively. Aspergillus niger showed a relatively higher

2+

TI for Zn and Cd and then followed by Aspergillus

carmari and Penicillium notatum, respectively.

combined mixture of heavy metals (Ni + Zn + Cd )

was found to be 0.83, 0.80 and 1.66 for bacterial, fungal

and bacterial-fungal consortia, respectively. These

relatively high TI values indicate that microbial consortia

can tolerate the presence of combined or mixed heavy

metals more than their separate components in the

consortium.

Furthermore, the heavy metal tolerance by the six bacteria

species (Pseudomonas aeruginosa, Bacillus subtilis,

Micrococcus luteus, Escherichia coli, Staphylococcus

aureus and Staphylococcus epidermidis) is in this

decreasing order of Zn > Ni > Cd while the metal tolerance

by the three fungal species (Aspergillus niger, Aspergillus

carmari and Penicillium notatum) is in this decreasing

order of Zn > Cd > Ni. A similar observation has been

reported by Oaikhena et al. [52] for metal tolerance by five

bacteria species (Pseudomonas aeruginosa, Staphylococcus

aureus, Escherichia coli, Proteus vulgaris and Klebsiella

pneumoniae). The higher tolerance levels for zinc and

nickel could be attributed to their classification as

micronutrients that are needed by the bacteria in trace

3.2 Bacterial and Fungal Consortia Degradation of

Petroleum Hydrocarbons

Figure 1 shows the level of TPH biodegradation in non-

bioaugmented soil microcosm (B-T

bio-augmented alone with bacterial consortium (microcosm

B-T ), fungal consortium (microcosm B-T ) and bacterial-

fungal consortium (B-T12), respectively.

1

) and in soil microcosm

2

7

Table 2: Tolerance index of bacterial and fungal isolates in growth media supplemented with 10 ppm of heavy metal

Microbial Isolates

Tolerance Index

Cd²⁺

0.88

0.80

0.60

0.55

0.25

0.20

0.85

0.82

0.80

1.86

1.84

2.25

Ni²⁺

0.90

0.88

0.77

0.75

0.45

0.80

1.88

1.81

2.34

Zn

2+

Ni²⁺+ Zn²⁺+ Cd²⁺

Pseudomonas aeruginosa

Bacillus subtilis

Micrococcus luteus

Escherichia coli

Staphylococcus aureus

Staphylococcus epidermidis

Aspergillus niger

Aspergillus carmari

Penicillium notatum

Bacterial consortium

Fungal consortium

0.92

0.85

0.80

0.70

0.56

0.54

0.99

0.94

0.90

1.90

1.87

2.38

0.62

0.58

0.44

0.30

0.15

0.10

0.61

0.60

0.56

0.83

0.80

1.66

Bacterial-fungal consortium

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100000

80000

60000

40000

20000

0

7

14

21

28

35

Remediation Time (Days)

Control (Natural Bioattenuation)

Fungal Consortium-Metal Free

Bacterial Consortium-Metal Free

Bacterial-Fungal Consortium-Metal Free

Figure 1: TPH biodegradation profile by bacterial consortium, fungal consortium and bacterial-fungal consortium in the absence of heavy metals

Nickel, Zinc and Cadmium). Bars indicate the average of triplicate samples while the error bars show the standard deviation.

(

As shown in Figure 1, within the first 21 days of

remediation in the soil microcosms B-T , B-T and B-T12, a

in the different consortium biodegradation efficiency. The

higher percent TPH biodegradation observed in bacterial-

fungal consortium might be ascribed to the synergistic

metabolic activities by the consortium of three bacteria and

three fungi isolates with larger number of different types of

enzymes which might have enhanced the degradation

process than separate bacterial and fungal consortiums with

fewer types of enzymes. These results thus suggest that the

consortium which consists of both bacterial and fungal

TPH degraders may lead to very rapid and more complete

degradation as compared to separate consortium of bacteria

or consortium of fungi and it emphasized the role of fungal

species with degradation capability in the synergistic TPH

degradation. Also, the bacterial consortium yielded an

increase of 11% compared to the fungal consortium.

2

7

rapid decrease in TPH amounting to over 50% degradation

was attained. The degradation efficiency of these consortia

increased with time and at the end of day 35 remediation

period, 100, 000 mg/kg of TPH concentration was reduced

to 27,500 ± 1750, 36,000 ± 1900 and 9,300 ± 1100 mg/kg

TPH corresponding to 72.5%, 64% and 90.7%

biodegradation or reduction in soil bio-augmented with

2

7

bacterial consortium (B-T ), fungal consortium (B-T ) and

bacterial-fungal consortium (B-T12), respectively.

Meanwhile, in the non-bioaugmented soil microcosm (B-

), the TPH concentration of 100,000 mg/kg was reduced

T

1

to 55,000 mg/kg amounting to 45% TPH biodegradation at

the end of day 35 remediation period. The bacterial

consortium, fungal consortium and bacterial-fungal

consortium correspondingly resulted in an increase of 61%,

3.3 Influence of Heavy Metals on Bacterial and Fungal

Consortia Degradation of TPH

42.2% and 101.5% TPH biodegradation in relation to the

2+

percent TPH biodegradation obtained in the non-

bioaugmented soil microcosm (natural bioattenuation) or

control. The increased degradation by the consortium in the

bio-augmented soil microcosms as compared to the non-

bio-augmented microcosm may be attributed to greater

population of TPH degraders. Similarly, the bacterial-

fungal consortium resulted in an increase of 25% and

The influence of the presence of heavy metals (Ni ,

Zn²⁺and Cd ) as individual metals and as combined or

mixture form on bacterial consortium, fungal consortium

and bacterial-fungal consortium degradation of TPH was

evaluated. Figure 2(A) shows the level of TPH

biodegradation in soil microcosm bio-augmented alone

2+

with bacterial consortium (B-T ), bacterial consortium +

2

+

2+

41.7% TPH biodegradation with respect to the percent

3 4

Ni (B-T ), bacterial consortium + Zn (B-T ), bacterial

2+ 2+

consortium + Cd (B-T

degradation attained by the bacterial consortium and fungal

consortium, respectively. These increases in TPH

biodegradation represent a significant difference (p < 0.05)

5

), and bacterial consortium + Ni

6

), respectively.

2+

+ Zn + Cd (B-T

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(A) Bacterial Consortium

(B) Bacterial Consortium

100000

16

14

12

10

8

6

4

2

0

5

0000

0

7

14

21

28

35

Remediation Time (Days)

Bacterial Consortium-Metal Free

Bacteria Consortium + Nickel

Bacterial Consortium + Zinc

Bacterial Consortium + Cadmium

Bacterial Consortium + Nickel + Zinc + Cadmium

Ni

Zn

Cd Ni + Zn +

Cd

Heavy Metals

Figure 2: (A) TPH biodegradation profile by bacterial consortia in the absence and presence of heavy metals (Nickel, Zinc and Cadmium). (B)

Amount of heavy metal (Nickel, Zinc and Cadmium) bioavailable to bacterial consortia. Bars indicate the average of triplicate samples while the

error bars show the standard deviation.

A rapid decrease in TPH amounting to over 50%

reduction was attained within the first 21 days of the

remediation in all the soil microcosms augmented with

bacteria and heavy metals. At the end of day 35

remediation period, 100,000 mg/kg of TPH concentration

was reduced to 27,500 ± 1750, 20,800 ± 1500, 18,600 ±

augmented with bacterial consortium, the highest

percentage of TPH biodegradation or reduction was

achieved in soil microcosm that contained the presence of

2+

Zn (81.4%). This is relatively and closely followed by

2+

that of bacterial consortium + Ni (79.2%), bacterial

2+

consortium + Cd (75.3%), bacterial consortium alone

2+

(72.5%), and bacterial consortium + Ni + Zn + Cd

2+

1450, 24,700 ± 1300 and 31,800 ± 2000 mg/kg TPH

corresponding to 72.5%, 79.2%, 81.4%, 75.3% and 68.2%

degradation or reduction in soil bio-augmented with

bacterial consortium-metal free, bacterial consortium +

(68.2%), respectively. Thus there are significant differences

(p < 0.05) in the percent TPH degradation influenced by the

2+

individual and combined mixture of Zn , Ni and Cd

2+

Ni , bacterial consortium + Zn , bacterial consortium +

heavy metals. This observation means that the degree of

bioavailability of the heavy metals to the bacterial

consortium differs as shown in Figure 2(B). Figure 2(B)

2

+

2+

Cd , and bacterial consortium + Ni + Zn + Cd ,

respectively. The results revealed that the individual

2+

presence of Ni , Zn and Cd exerted a significant

positive influence on the TPH bacterial consortium

degradation by increasing the TPH percent degradation by

reveals that Zn was more bioavailable to the consortium

2

+

2+

and followed by Ni and Cd , respectively. Therefore, the

2+

potential bioavailability of Ni , Zn and Cd in soil

strongly decreased after bioaugmentation with bacterial

consortium after 35 days’ incubation. However,

9.2%, 12.3% and 3.9%, respectively, relative to the TPH

degradation by the bacterial consortium that is heavy metal-

free. The relatively enhanced TPH biodegradation or

reduction may be due to the relative bioavailability of the

individual metals to the bacterial consortium (Figure 3(A))

which tend to stimulate growth as well as to the high metal

TI of the bacterial consortium (Table 2). While the

2+

bioavailable mixed heavy metals (Ni + Zn + Cd ) in

soil microcosm only decreased slightly after 35 days’

incubation. Thavamani et al. [30] reported that there was

complete phenanthrene biodegradation in soil solution by

bacterial consortium (Alcaligenes sp., Pseudomonas sp.,

Pandoraea sp. and Paenibacillus sp) in the presence of 5

mg/L of cadmium. Nonetheless, according to Sandrin and

Hoffman [57] making comparisons between heavy metals

2+

combined equal concentration mixture of Ni , Zn and

2+

Cd (30 mg/L) exerted a considerable negative impact on

the TPH bacterial consortium degradation by significantly

(

p < 0.05) suppressing or decreasing the TPH bacterial

concentrations

that

inhibit

organic

compounds

degradation by 5.9%. This indicated that the presence of

mixed heavy metals (or multi-heavy metals) acts

synergistically to impose a greater inhibitory response to

the growth of the bacterial consortium thereby imposing a

relatively partial inhibition to the degradation than that

imposed by single or individual heavy metals. A similar

observation has been reported [56]. The different

bioaugmentation treatments ranking with respect to

biodegradation as reported by different researchers is

difficult. This is because more often than not, significant

variation is usually observed in the inhibitory range for

heavy metals due to different conditions of experiments

used such as time of exposure, pH etc. [58]. Figure 3(A)

shows the TPH biodegradation profile in soil microcosms

bio-augmented with fungal consortium-metal free, fungal

2+

consortium + Ni , fungal consortium + Zn , fungal

2

+

2+

increasing TPH biodegradation level was B-T

6

< B-T

2

< B-

consortium + Cd , and fungal consortium + Ni + Zn +

2+

Cd , respectively.

T

5

< B-T < B-T . That is, in all the soil microcosms bio-

3

4

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(A)

(B)

100000

2

5

0

2

50000

15

1

0

5

0

7

14

21

28

35

Ni

Zn

Cd

Ni + Zn +

Cd

Remediation Time (Days)

Fungal Consortium-Metal Free

Fungal Consortium + Nickel

Fungal Consortium + Zinc

Heavy Metals

Fungal Consortium + Cadmium

Figure 3: (A) TPH biodegradation profile by fungal consortia in the absence and presence of heavy metals (Nickel, Zinc and Cadmium). (B)

Amount of heavy metal (Nickel, Zinc and Cadmium) bioavailable to fungal consortia. Bars indicate the average of triplicate samples while the

error bars show the standard deviation.

Similarly, within the first 21 days of the remediation

period the decrease observed in TPH resulted to about 50%

reduction in all the soil microcosms augmented with fungal

consortium and heavy metals. At day-35 remediation

period, the TPH concentration of 100,000 mg/kg was

reduced to 36,000 ± 1900, 30,600 ± 1700, 34,200 ± 1600,

and 3.8%, respectively, relative to the percent TPH

biodegradation attained by the fungal consortium-metal

free. This enhancement could also be due to the relative

bioavailability of the individual metals to the fungal

consortium as shown in Figure 3(B) as well as the

concentration tolerance of the individual heavy metals by

2+

the consortium (Table 2). Figure 3(B) reveals that Ni was

2+

31,800 ± 1500 and 39,400 ± 1850 mg/kg TPH which

corresponded to 64%, 69.4%, 66.4%, 68.2%, and 60.6%

more bioavailable to the consortium and followed by Cd

2+

TPH biodegradation in soil augmented with fungal

and Ni , respectively. Meanwhile, the combined mixture

2+

consortium, fungal consortium + Ni , fungal consortium +

of Ni + Zn + Cd of 30 mg/L concentration exerted a

considerable negative impact on the TPH fungal

2

+

2+

Zn , fungal consortium + Cd , and fungal consortium +

2+

Ni + Zn + Cd , respectively. Similarly, in all the soil

microcosms bio-augmented with fungal consortium, the

consortium degradation by significantly (p < 0.05)

decreasing the TPH fungal consortium degradation by

5.3%. This observation also revealed that the presence of

mixed heavy metals with relatively higher concentration

acts synergistically to impose a greater inhibitory response

to the growth of the fungal consortium hence imposing a

partial inhibition to the TPH degradation than that imposed

by single heavy metals. A similar observation has been

reported (Anahid et al., 2011).

2+

microcosm containing Ni had the highest percentage of

TPH reduction (69.4%) and was relatively followed by that

2+

of fungal consortium + Cd (68.2%), fungal consortium +

2+

Zn (66.4%), fungal consortium-metal free (64%) and

2+

2+ 2+

fungal consortium + Ni

+ Zn + Cd (60.6%),

respectively. Hence, the bioaugmentation treatments

ranking with respect to increasing TPH biodegradation

level is B-T11 < B-T

significant differences (p < 0.05) in the percent TPH

7

< B-T

9

< B-T10 < B-T

8

. Thus there are

The TPH biodegradation profile in soil microcosms

bio-augmented with bacterial-fungal consortium (metal-

2+

free), bacterial-fungal consortium + Zn , bacterial-fungal

2+ 2+

biodegradation influenced by the individual and combined

2+

mixture of Ni , Zn and Cd heavy metals. This

consortium + Ni , bacterial-fungal consortium + Cd , and

2+ 2+ 2+

2+

observation indicated that Ni , Zn and Cd exerted a

relatively positive influence and thus enhanced the TPH

biodegradation by the fungal consortium by 8.4%, 6.6%

bacterial-fungal consortium

+

Ni

+

Zn

+ Cd ,

respectively, is shown in Figure 4(A).

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2019, Volume 7, Issue 1, Pages: 179-195

(A)

(B)

100000

30

25

20

15

10

5

0

7

14

21

28

35

Remediation Time (Days)

Bacterial-Fungal Consortium-Metal Free

Bacterial-Fungal Consortium + Nickel

Bacterial-Fungal Consortium + Zinc

Bacterial-Fungal Consortium + Cadmium

Ni

Zn

Cd

Ni + Zn +

Cd

Heavy Metals

Bacterial-Fungal Consortium + Nickel + Zinc + Cadmium

Figure 4: (A) TPH biodegradation profile by bacterial-fungal consortium in the absence and presence of heavy metals (Nickel, Zinc and

Cadmium). (B) Amount of heavy metal (Nickel, Zinc and Cadmium) bioavailable to bacterial-fungal consortium. Bars indicate the average of

triplicate samples while the error bars show the standard deviation.

Similarly,

a

fast decrease in TPH concentration

and Acremonium sp.) [59]. In addition, Shen et al. [60]

have reported that 10 mg/L concentration of cadmium

inhibited the biodegradation of phenanthrene by indigenous

microorganisms in soil microcosms where the soil pH is

8.18 while on the other hand, 1 mg/L of cadmium inhibited

resulting to over 70% reduction was achieved within day 21

of the remediation period in all the soil microcosms bio-

augmented with bacterial-fungal consortium and heavy

metals. At the end of day 35, the TPH concentration

(

1

100,000 mg/kg) significantly reduced to 9,300 ± 1100,

,000 ± 850, 1,500 ± 900, 4,300 ± 1000 and 0 mg/kg TPH

phenanthrene

microorganisms in soil of pH 7.6 [61].

biodegradation

by

indigenous

corresponding to 90.7%, 99%, 98.5%, 95.7% and 100%

degradation in soil bio-augmented with bacterial-fungal

consortium (metal free), bacterial-fungal consortium +

The ranking of the different bacterial-fungal consortium

bioaugmentation treatments with respect to TPH reduction

or biodegradation was B-T12 < B-T15 < B-T14 < B-T13 < B-

2+

Ni , bacteria-fungal consortium + Zn , bacterial-fungal

T16. The ranking revealed that in all the soil microcosms

2

+

2+

consortium + Cd , and bacterial-fungal consortium + Ni

+

bio-augmented with bacterial-fungal consortium, the

highest percent TPH biodegradation was attained in soil

microcosm containing the presence of the mixed heavy

2+ 2+ 2+ 2+

Zn + Cd , respectively. The mixture of Ni , Zn and

2+

Cd of 30 mg/L concentration elicited a complete TPH

biodegradation by the bacterial-fungal consortium. This

indicated that the bacterial-fungal consortium acted

synergistically to pose strong resistance to the synergistic

toxicity and inhibitory effect imposed by the presence of

the mixed heavy metals. In addition, the results also

2

+

2+

metals (Ni + Zn + Cd ) (i.e. 100%). This is closely

2+

followed by bacterial-fungal consortium + Ni (99%),

bacterial-fungal consortium + Zn (98.5%), bacterial-

fungal consortium + Cd (95.7%) and bacterial-fungal

consortium-metal free (90.7%), respectively. Furthermore,

in comparison with bacterial and fungal consortia, the

bacterial-fungal consortium in the absence and presence of

2+

2

+

2+

revealed that the single presence of Ni , Zn and Cd and

their mixture exerted a significant positive effect on the

TPH biodegradation by bacterial-fungal consortium by

increasing the percent TPH degradation by 9.2%, 8.6%,

heavy metals achieved

a

higher percent TPH

biodegradation than that due to bacterial and fungal

consortia, respectively. This is respectively followed by

that due to bacterial consortium and fungal consortium in

the absence and presence of heavy metals. This observation

suggest that both bacteria and fungi species play very

significant role in TPH degradation or removal, and that the

presence of both types of microorganisms may result in

synergy of degradation activity during bioaugumentation

[15]. Nevertheless, the bacterial consortium, fungal

consortium and bacterial-fungal consortium have

demonstrated their ability to degrade TPH with high

degradation efficiency even in the presence of heavy metals

in relation to the non-bioaugmented soil microcosm

(control or natural bioattenuation).

5

.5% and 10.3%, respectively, relative to the percent TPH

degradation attained by the bacterial-fungal consortium

metal free). The observed relative enhancement of TPH

(

biodegradation may be due to high relative bioavailability

of the individual metals and their mixture as presented in

Figure 4(B) as well as the relative high combined or mixed

metal concentration tolerance of the bacterial-fungal

2+

consortium (Table 2). It has been reported that Ni

supplementation at a concentration of 5 mM (294 mg/L)

suppressed significantly (p≤ 0.01) fluorene, phenanthrene

and anthracene removal from broth media, while it

increased fluoranthene degradation by 4.8 % relative to the

control using fungus-bacterial consortium (Bacillus subtilis

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(B) Bacterial Growth

(A) Microbial Growth in the

20

Absence of Metals

10

0

10

20

30

40

0

10

20

30

40

Time (Days)

Bacteria Count in Control

Fungi Count in Control

Bacterial Consortium (Metal Free)

Bacteria Consortium + Nickel

Bacterial Consortium + Zinc

Bacterial Consortium (Metal Free)

Fungal Consortium (Metal Free)

Bacterial Consortium + Cadmium

(D)Bacteria-Fungi Growth

(C) Fungal Growth

1

5

30

20

10

0

1

0

5

0

10

20

30

40

0

10

20

30

40

Time (Days)

Fungal Consortium (Metal Free)

Fungal Consortium + Nickel

Fungal Consortium + Zinc

Fungal Consortium + Cadmium

Fungal Consortium + Nickel + Zinc + Cadmium

Bacteria + Fungi (Metal Free)

Bacteria + Fungi + Nickel

Bacteria + Fungi + Zinc

Bacteria + Fungi + Cadmium

Bacteria + Fungi + Nickel + Zinc + Cadmium

Figure 5: (A) Growth profile of bacterial consortium, fungal consortium and bacterial-fungal consortium in the absence of heavy metals (Nickel,

Zinc and Cadmium). (B) Growth profile of bacterial consortium in the absence and presence of heavy metals (Nickel, Zinc and Cadmium). (C)

Growth profile of fungal consortium in the absence and presence of heavy metals (Nickel, Zinc and Cadmium). (D) Growth profile of bacterial-

fungal consortium in the absence and presence of heavy metals (Nickel, Zinc and Cadmium).

g⁻¹which corresponded to a percentage increase of 167%,

81.4% and 423%, respectively. On the other hand, the

bacterial count and the fungal count in the non-augmented

soil microcosm correspondingly increased from 8.1 to 8.9 ×

3

.4 Microbial Growth Profile

Figure 5 shows the profiles of microbial growth in the

non-augmented soil microcosm and soil microcosms

augmented with bacterial and fungal consortia in the

2+

4

−1

4

−1

presence and absence of Ni , Zn and Cd heavy metals.

The profiles of bacterial growth in the non-augmented soil

microcosm and soil microcosms augmented with bacterial

consortium-metal free, fungal consortium-metal free and

bacterial-fungal consortium-metal free as shown in Fig.

10 cfu-g and from 2.8 to 3.5 × 10 cfu-g corresponding to

a percentage increase of 10 and 25%, respectively. This low

percentage increase in both bacterial and fungal growth

showed that the non-augmented soil microcosm had more

of non-hydrocarbon utilizing microbes than the

hydrocarbon utilizing bacteria and fungi when compared to

the soil microcosm bio-augmented with bacterial and

fungal consortia that are hydrocarbon utilizers. This

accounted for the low TPH biodegradation achieved in the

non-augmented soil microcosm (natural bioattenuation).

Figure 5(B) shows the profiles of bacterial growth in

the soil microcosms augmented with consortia of bacteria,

5(A) indicates that the bacterial counts generally increased

from day 0 to day 21 in each of the augmented and non-

augmented soil microcosms. For soil microcosm bio-

augmented with bacterial consortium-metal free, fungal

consortium-metal free and bacterial-fungal consortium-

metal free, the bacterial counts increased from 4.8 to 12.8 ×

6 −1 6 −1 6

0 cfu-g , 4.3 to 7.8 × 10 cfu-g and 3.2 to 16.9 × 10 cfu-

1

189

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

Ni , Zn , Cd and the mixture of Ni , Zn and Cd²⁺,

respectively. As shown in Figure 5, it is seen that the

bacterial counts generally increased from day 0 to day 21 in

each of the augmented soil microcosms. For soil

microcosm bio-augmented with bacterial consortium-metal

2

+

2+

microcosms’ bio-augmented with fungal consortium + Cd

2+

+

Zn

9

(B-T10), fungal consortium (B-T ), fungal

2+

consortium-metal free (B-T ) and fungal consortium + Ni

+ Zn + Cd (B-T11), respectively. This observation

7

2+

suggests that the 10 mg/L concentration of each Ni , Zn

2+

and Cd enhanced the growth of the fungal population

2+ 2+ 2+

free, bacterial consortium + Ni , bacterial consortium +

2

+

2+

Zn , bacterial consortium

+ Cd , and bacterial

while the mixed heavy metals (Ni + Zn + Cd ) of 30

mg/L total concentration suppressed the growth. The

enhanced growth of the fungal consortium may be due to

the synergistic interaction of the fungal species in the

consortium to positively tolerate 10 mg/L concentration

2

+

2+

consortium+ Ni + Zn + Cd , the bacterial counts

6

−1

increased from 4.8 to 12.8 × 10 cfu-g , 3.2 to 16.9 ×

6 −1 6 −1 6 −1

0 cfu-g , 2.2 to 11.5 × 10 cfu-g , 2.1 to 9.8 × 10 cfu-g ,

6 −1

1

and 4.2 to 10.6 × 10 cfu-g which corresponded to a

percentage increase of 167%, 428%, 423%, 367% and

2

+

2+

each of Ni , Zn and Cd respectively. While the fungal

consortium growth suppression due to the presence of

1

52%, respectively. This results revealed that soil

microcosm bio-augmented with bacterial consortia in the

presence of Zn heavy metal exhibited the highest bacterial

growth. This is closely followed by soil microcosms’ bio-

augmented with bacterial consortium + Ni , bacterial

consortium + Cd , bacterial consortium-metal free and

bacterial consortium + Ni + Zn + Cd , respectively.

This observation suggests that the 10 mg/L concentration of

the single heavy metals (Ni , Zn and Cd ) used in this

2+

combined mixture of heavy metals (Ni + Zn + Cd )

may probably be due to the concentration (30 mg/L) being

too high to act synergistically to impose a greater inhibitory

response to the growth of the fungal consortium.

2+

Figure 5(D) shows the growth profiles of bacteria and

fungi in the soil microcosms augmented with bacterial-

fungal consortium-metal free, bacterial-fungal consortium

2+

2+ 2+

+ Ni , bacterial-fungal consortium + Zn , bacterial-fungal

2+ 2+

study had enhancement effect on the growth of the bacteria

population with Zn

consortium + Cd and bacterial-fungal consortium + Ni

+

2+

displaying the most growth

Zn + Cd , respectively. Generally, it is seen that the

bacterial-fungal counts generally increased from day 0 to

day 21 in each of the augmented soil microcosms. The

enhancement while the combined mixture effect of the

three heavy metals suppressed the growth of the bacteria

population. The results also suggest that the combined

mixture of heavy metals acts in synergy to impose more

toxic effect to bacterial growth than the single heavy metal.

A similar observation of growth suppression for Bacillus

sp. CPB4 in the presence of combined mixture of lead (Pb),

copper (Cu), zinc (Zn) and cadmium (Cd) has been

reported [62]. The suppression or inhibition of the bacterial

6

bacterial-fungal counts increased from 7.6 to 17.2 × 10 cfu-

−

1

6

−1

6

−1

g , 4.3 to 19.4 × 10 cfu-g , 5.5 to 17.8 × 10 cfu-g , 5.0 to

6

−1

6

−1

15.8 × 10 cfu-g , and 6.7 to 30.7.3 × 10 cfu-g , and

which corresponded to a percentage increase of 126%,

351%, 224%, 216% and 358%, for soil microcosm bio-

augmented with bacterial-fungal consortium-metal free,

2+

Ni , bacterial-fungal

2+

bacterial-fungal consortium

+

2+

consortium + Zn , bacterial-fungal consortium + Cd , and

2+ 2+ 2+

growth exhibited by the combined mixture of Zn , Ni

2

+

and Cd may be due to the fact that the bioavailable mixed

heavy metals of 30 mg/L concentration might probably be

high for the bacterial consortium to exert a synergistic toxic

effect. It has been documented in the literature that all

heavy metals exhibit toxicity to living microbial cells at

certain concentration [35, 63]. This could have been

responsible for the lower TPH biodegradation observed in

the soil microcosm containing combined mixture of the

heavy metals (Ni + Zn + Cd) bio-augmented with bacterial

consortium.

bacterial-fungal consortium

+

Ni

+

Zn

+ Cd ,

respectively. This results indicated that bio-augmented soil

microcosm (B-T16) containing bacterial-fungal consortium

+ Ni + Zn + Cd²⁺displayed the highest bacterial-fungal

2

+

2+

growth. This is closely followed by soil microcosms’ bio-

2+

augmented with bacterial-fungal consortium + Ni (B-T13),

2+

bacterial-fungal consortium + Zn (B-T14), bacterial-fungal

2+

consortium + Cd (B-T15) and bacterial-fungal consortium-

metal free (B-T12), respectively. This observation suggests

2+

that the 10 mg/L concentration of each Ni , Zn and Cd

Figure 5(C) shows the growth profiles of fungi in the

soil microcosms augmented with fungal consortium-metal

free, fungal consortium + Ni , fungal consortium + Zn ,

as well as the 30 mg/L of the mixed heavy metals enhanced

the growth of the bacterial-fungal population. The

enhanced growth of the bacterial-fungal consortium may be

due to the synergistic interaction of the bacterial-fungal

species in the consortium to positively tolerate 10 mg/L

2+

2

+

2+

fungal consortium + Cd and fungal consortium + Ni +

2+

Zn + Cd , respectively. Generally, it is seen that the

fungal counts generally increased from day 0 to day 21 in

each of the augmented soil microcosms. The fungal counts

2+

concentration each of Ni , Zn and Cd and the 30 mg/L

of the mixed heavy metals, respectively.

6

−1

6

increased from 4.3 to 7.8 × 10 cfu-g , 1.9 to 6.4 × 10 cfu-

−1

6

−1

6

−1

g , 4.4 to 9.8 × 10 cfu-g , 3.9 to 10.7 × 10 cfu-g , and

3.5 pH profile during TPH biodegradation

6

−1

3

.8 to 6.5 × 10 cfu-g , and which corresponded to a

percentage increase of 81.4%, 237%, 123%, 174% and

1.1%, for soil microcosm bio-augmented with fungal

The un-impacted soil pH before crude oil

contamination was 5.1. However, after crude oil

contamination, the soil pH reduced to 4.5. This observed

reduction in soil pH as a result of crude oil contamination is

in agreement with the findings of Osuji and Nwoye [64].

The reduction in soil pH implies increased soil acidity

which is a problem for agricultural soils.

7

2+

consortium-metal free, fungal consortium + Ni , fungal

2+

consortium + Zn , fungal consortium + Cd and fungal

2+

consortium + Ni + Zn + Cd , respectively. This results

indicated that bio-augmented soil microcosm (B-T

8

)

2+

containing fungal consortium + Ni displayed the highest

fungal growth. This is relatively followed by soil

190

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

(A) Bioaugmentation without Metals

(B) Bacterial Consortium

1

0

5

0

1

0

5

0

10

20

30

40

0

10

20

30

40

Remediation Time (Days)

Bacterial Consortium (Metal Free)

Bacterial Consortium + Nickel

Bacterial Consortium + Zinc

Control (Natural Bioattenuation)

Bacterial Consortium (Metal Free)

Fungal Consortium (Metal Free)

Bacterial-Fungal Consortium (Metal Free)

Bacterial Consortium + Cadmium

Bacterial Consortium + Nickel + Zinc + Cadmium

(

D)Bacterial-Fungal Consortium

(

C)Fungal Consortium

1

0

5

0

10

0

10

20

30

40

0

10

20

30

40

Remediation Time (Days)

Bacterial-Fungal Consortium (Metal Free)

Bacterial-Fungal Consortium + Nickel

Bacterial-Fungal Consortium + Zinc

Bacterial-Fungal Consortium + Cadmium

Bacterial-Fungal Consortium + Nickel + Zinc + Cadmium

Fungal Consortium (Metal Free)

Fungal Consortium + Nickel

Fungal Consortium + Zinc

Fungal Consortium + Cadmium

Fungal Consortium + Nickel + Zinc + Cadmium

Figure 6. (A) Soil pH profile in the course of TPH biodegradation by bacterial consortium, fungal consortium and bacterial-fungal consortium in

the absence of heavy metals (Nickel, Zinc and Cadmium). (B) Soil pH profile in the course of TPH biodegradation by bacterial consortium in the

absence and presence of heavy metals (Nickel, Zinc and Cadmium). (C) Soil pH profile in the course of TPH biodegradation by fungal

consortium in the absence and presence of heavy metals (Nickel, Zinc and Cadmium). (D) Soil pH profile in the course of TPH biodegradation

by bacterial-fungal consortium in the absence and presence of heavy metals (Nickel, Zinc and Cadmium).

The resulting increased acidity could be due to the fact

that hydrocarbons contain many free cations causing them

to have properties of a weak acid. Figure 6 shows the soil

pH profile in the course of TPH biodegradation in the

absence and presence of heavy metals and microbial

consortium. The results in Figure 6 as depicted for

microbial consortium in the absence of heavy metals

pH values (6.6 – 7.8) obtained for the various

bioaugmented and non-bioaugmented soil microcosms

were within the range for optimum microbial activities.

3.6 Kinetics and half-life of TPH biodegradation

First-order kinetics model equation (Eq. 2) fitted to the

biodegradation data was used to determine the rate of TPH

biodegradation (i.e. biodegradation rate constants (k)) in the

various bioaugmentation treatments. The results are

presented in Table 3. The results in Table 3 as revealed by

(

Figure 6(A)), bacterial consortium in the presence of

heavy metals (Figure 6(B)), fungal consortium in the

presence of heavy metals (Figure 6(C)) and bacterial-fungal

consortium in the presence of heavy metals (Figure 6(D))

revealed that the pH of the crude oil contaminated soil

microcosms increased in the course of the TPH

biodegradation. The observed changes in the soil pH values

of the various soil microcosms were due to the removal or

degradation of the crude oil contaminant. The range of soil

2

high correlation coefficient (R ) indicated that the TPH

biodegradation data fitted well to the first-order kinetic

model. The half-life time (t1/2) of TPH biodegradation in

the absence and presence of heavy metals was calculated

using Eq. (3), and the results are given in Table 3.

191

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

Table 3: Biodegradation rate constants and correlation coefficients obtained from the fitting of first-order kinetic model to TPH

biodegradation data and the calculated half-life time

-1

2

Soil Treatment

B-T (Control)

Bacterial Consortium (B-T

Bacterial Consortium + Nickel (B-T

Bacterial Consortium + Zinc (B-T

Bacterial Consortium + Cadmium (B-T

Bacterial Consortium + Nickel + Zinc + Cadmium (B-T

Fungal Consortium (B-T

Fungal Consortium + Nickel (B-T

Fungal Consortium + Zinc (B-T

Fungal Consortium + Cadmium (B-T10

Fungal Consortium + Nickel + Zinc + Cadmium (B-T11

Bacterial- Fungal Consortium (B-T12

Bacterial-Fungal Consortium + Nickel (B-T13

Bacterial-Fungal Consortium + Zinc (B-T14

Bacterial-Fungal Consortium + Cadmium (B-T15

Bacterial-Fungal Consortium + Ni + Zn + Cd (B-T16

k

(day )

t₁_/₂(days)

R

1

0.010

0.043

0.049

0.051

0.046

0.040

0.035

0.040

0.037

0.039

0.032

0.060

0.129

0.118

0.084

0.158

69.3

16.1

14.1

13.6

15.1

17.3

19.8

17.3

18.7

17.8

21.7

11.6

5.4

0.9553

0.9952

0.9981

0.9994

0.9974

0.9942

0.9930

0.9933

0.9956

0.9946

0.9894

0.9966

0.9977

0.9949

0.9972

0.9992

2

)

3

)

4

)

5

)

6

)

7

)

8

)

9

)

5.9

8.3

4.4

)

-1

It is important to note that a higher biodegradation rate

constant indicates a faster or higher rate of biodegradation

and subsequently a lower half-life time. It could be seen

from Table 3 that for soil microcosms augmented with

+ Cd²⁺had a higher k (0.158 day ) and lower t1/2 (4.4 days)

than that augmented with bacterial-fungal consortium +

Ni (k = 0.129 day and t1/2 = 5.4 days), bacterial-fungal

2+

-1

2+

-1

consortium + Zn (k = 0.118 day and t1/2 = 5.9 days),

2

+

-1

microorganisms in the absence of heavy metals (B-T

2

, B-T

7

bacterial-fungal consortium + Cd (k = 0.084 day and t1/2

= 8.3 days) and bacterial-fungal consortium (metal free) (k

and B-T12) and non-augmented soil microcosm, the soil

microcosm augmented with bacterial-fungal consortium

-1

= 0.060 day and t1/2 = 11.6 days), respectively.

-1

had a higher k (0.060 day ) and lower t1/2 (11.6 days) than

-

1

that augmented with bacterial consortium (k= 0.043 day

4

Conclusions

and t1/2= 16.1 days) and fungal consortium (k= 0.035 day^-¹

From this study, it can be concluded that the bacteria

and t1/2= 19.8 days), respectively. While the non-augmented

species (Pseudomonas aeruginosa, Bacillus subtilis and

Micrococcus letus) and fungi species (Aspergillus niger,

Aspergillus carmari and Penicillium notatum) used as

consortia in this study showed high tolerance and resistance

to nickel, zinc and cadmium heavy metals in soil. The

addition of hydrocarbon utilizing and metal-resistant

microorganisms in the form of consortia either as bacterial

consortia (Pseudomonas aeruginosa, Bacillus subtilis and

Micrococcus letus), fungal consortia (Aspergillus niger,

Aspergillus carmari and Penicillium notatum) or bacterial-

fungal consortia (Pseudomonas aeruginosa, Bacillus

subtilis, Micrococcus letus, Aspergillus niger, Aspergillus

carmari and Penicillium notatum) provided an enhanced

TPH biodegradation (>60%) in the presence and absence of

single or individual heavy metals (nickel, zinc and

cadmium) with the bacterial-fungal consortia providing the

highest enhancement. Complete TPH biodegradation

-

1

soil microcosm had the least k value of 0.010 day and the

highest t1/2 value of 69.3 days. The value obtained for the

non-bioaugmented soil microcosm is close to the k value of

-1

0

.015 day obtained for the natural bioattenuation of soil

microcosm contaminated with lubricating motor oil [13].

For soil microcosms augmented with bacterial

consortium in the presence of heavy metals, the soil

2+

augmented with bacterial consortium + Zn had a

-

1

relatively higher k (0.051 day ) and lower t1/2 (13.6 days)

2+

than the one augmented with bacterial consortium + Ni (k

-1

0.049 day and t1/2 = 14.1 days), bacterial consortium +

2+ -1

=

Cd (k = 0.046 day and t1/2 = 15.1 days), bacterial

-1

consortium (metal free) (k = 0.043 day and t1/2= 16.1

days) and bacterial consortium + Ni + Zn²⁺+ Cd²⁺(k=

2+

-1

0

.040 day and t1/2= 17.3 days), respectively. Furthermore,

for soil microcosms augmented with fungal consortium in

the presence of heavy metals, the soil augmented with

(

100%) and enhanced microbial growth in soil can be

2+

fungal consortium + Ni had a relatively higher k (0.040

achieved in the presence of mixed heavy metals(Nickel +

Zinc + Cadmium) using bacterial-fungal consortia while a

reduction in TPH biodegradation and decreased microbial

growth is attained with the use of bacterial or fungal

consortia. These features of a bacterial-fungal consortium

such as mixed-heavy metal resistance and high TPH

removal ability make it a very attractive candidate for TPH

biodegradation in co-contaminated soil environment. The

rate of TPH biodegradation in the presence and absence of

heavy metals and inoculated microbial consortia can be

described by biodegradation rate constant obtained from the

-

1

day ) and lower t1/2 (17.3 days) than the one augmented

with fungal consortium + Cd²⁺

1

(

= 0.039 day and t1/2

-1

=

k

7.8 days), fungal consortium + Zn₍_k₌₀_.₀₃₇_d_a_y-1 and

2+

t

1/2 = 18.7 days), fungal consortium (metal free) (k = 0.035

-

1

2+

day and t1/2 = 19.8 days) and fungal consortium + Ni +

Zn + Cd (k = 0.032 day and t1/2 = 21.7 days),

respectively.

2+

-1

Finally, for soil microcosms augmented with bacterial-

fungal consortium in the presence of heavy metals, the soil

augmented with bacterial-fungal consortium + Ni²+ Zn

+

2+

192

Journal of Environmental Treatment Techniques

2019, Volume 7, Issue 1, Pages: 179-195

4

.

Das, N., and Chandran, P. (2011). Microbial

degradation of petroleum hydrocarbon contaminants:

An overview. Research Int. 2011, Article ID 941810,

application of first order kinetics. The rate constant (

k

)

-

1

-1

ranges between 0.035 day and 0.060 day for soil

microcosm bioaugmented with bacterial, fungal and

bacterial-fungal consortia, respectively in the absence of

13 pages. Doi:10.4061/2011/941810.

-1

5. Yasin, G., Bhanger, M. I., Ansari, T. M. et al. (2013).

Quality and chemistry of crude oils. J. Pet. Technol.

Altern. Fuels 4, 53–63.

heavy metals and 0.010 day for non-bioaugmented soil

microcosm (natural bioattenuation). A half-life time (t1/2) of

69.3 days was attained for TPH biodegradation in non-

6.

Ogbo, E. M., and Okhuoya, J. A. (2011).

Bioavailability of some heavy metals in crude oil

contaminated soils remediated with Pleurotus tuber-

regium Fr. Singer. Asian J. Biological Sci. 4, 53-61

bioaugmented soil microcosm. This t1/2 was reduced to

between 16.1, 19.8 and 11.6 days in soil microcosm

bioaugmented with bacterial consortia, fungal consortia and

bacterial-fungal consortia, respectively. Similarly, the rate

-

1

-1

7. Shtangeeva, I. (2006). Phytoremediation of trace

element contaminated soil with cereal crops: role of

fertilizers and bacteria on bioavailability. In: Trace

Elements in the Environment. Biogeochemistry,

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K.S. Sajwan and R. Naidu (Eds.). CRC Press, Taylor

and Francis Group, Boca Raton, pp: 549-581.

constant (k) ranges between 0.032 day and 0.158 day for

soil microcosm bioaugmented with bacterial, fungal and

bacterial-fungal consortia, respectively in the presence of

single and mixed heavy metals Therefore, the inoculation

of bacterial consortia, fungal consortia and bacterial-fungal

consortia that possesses heavy metal resistance and

petroleum hydrocarbons degradation ability is a promising

bioaugmentation strategy to enhance in-situ and ex-situ soil

bioremediation of soil contaminated with both petroleum

hydrocarbons and heavy metals.

8.

Samanta, S. K., Singh, O. V., and Jain, R. K. (2002).

Polycyclic aromatic hydrocarbons: environmental

pollution and bioremediation. Trends Biotechnol. 20,

243–248.

9

.

Said, W. A., and D. A. Lewis. (1991). Quantitative

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degradation of organic chemicals. Appl. Environ.

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Aknowledgment

The authors wish to thank the staff of Thermosteel

Laboratories, Warri, Delta State of Nigeria for providing

the facilities used for the soil physical, chemical and

microbial analyses.

1

0. Ojo, O. A. (2006). Petroleum hydrocarbon utilization

by native bacterial population from a wastewater canal

in Southwest Nigeria. Afr. J. Biotechnol. 5(4), 333–337.

1. Ameen, F., Moslem, M., Hadi, S., et al. (2016).

Biodegradation of diesel fuel hydrocarbons by

mangrove fungi from Red Sea coast of Saudi Arabia.

Saudi. J. Biol. Sci. 23(2), 211–218.

Ethical issue

Authors are aware of, and have complied with the best

practice in publication ethics specifically with regard to

authorship, dual submission, and manipulation of figures,

competing interests and compliance with policies on

research ethics. Authors have adhered to publication

requirements that this submitted work is original and has

not been published elsewhere in any form of language.

1

2. Zhang, J. H., Quan-Hong, X., Hui, G., et al. (2016).

Degradation of crude oil by fungal enzyme preparations

from Aspergillus spp. for potential use in enhanced oil

recovery. J. Chem. Technol. Biotechnol. 91, 865–875.

3. Agarry, S. E., Aremu, M. O., and Aworanti, O. A.

(2013). Kinetic modelling and half-life study on

bioremediation of soil co-contaminated with lubricating

motor oil and lead using different bioremediation

strategies. Soil and Sediment Contam.- An Int. J. 22(7),

Competing interests

The authors wish to declare that there is no conflict of

interest in this research work.

8

00 – 816.

Authors’ contribution

1

4. Iordache, M., and Borza, I. (2012). The bioremediation

potential of earthworms (Oligochaeta: Lumbricidae) in

a soil polluted with heavy metals. J. Food Agric.

Environ. 10(2), 1183–1186.

5. Ma, X-K., Ding. N., and Peterson, E. C. (2015).

Bioaugmentation of soil contaminated with high-level

crude oil through inoculation with mixed cultures

including Acremonium sp. Biodegradation 26 (3), 259-

All the authors of this study have completely

contributed to the data collection, data analyses and

manuscript writing.

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