Download Comparison of degradation of benzene, toluene

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Ukpaka / Current Science Perspectives 2(4) (2016) 105-115
Current Science Perspectives 2(4) (2016) 105-115
iscientic.org.
Comparison of degradation of benzene, toluene and phenol in both fresh and salt
water media
C. P. Ukpaka
Department of Chemical/Petrochemical Engineering Rivers State University of Science and Technology Nkpolu PMB 5080,
Port Harcourt, Nigeria
*Corresponding author’s E-mail: [email protected]
A R T I C L E
I N F O
Article type:
Research article
Article history:
Received December 2015
Accepted June 2016
October 2016 Issue
Keywords:
Comparison
Degradation
Benzene
Toluene
Phenol
Fresh water
Salt water
A
B
S T
R
A
C
T
Research work was conducted to demonstrate the relationship between some
aromatic hydrocarbon in fresh and salt water media. The comparison of the
degradation rate of toluene in fresh and salt water media revealed a good match
in the substrate degradation per unit time. The rate of degradation of benzene,
toluene and phenol decreases with increase in period of exposure. The reaction
mechanisms were inhibited by various factors such as salinity, P + , temperature
etc. The medium of contamination play an activity role in effective remediation of
contaminated environment. The nature of the functional parameters of
environment may cause inhibition in the process in which the substance found in
the area may activate the programme. Degradation of Benzene, Toluene and
Phenol is faster in fresh water them salt water medium; this can be attributed to
the variation in the physiochemical parameters of the media considered during
the investigation.
© 2016 International Scientific Organization: All rights reserved.
Capsule Summary: The rate of degradation of benzene toluene and phenol was monitored and predicted in fresh and salt
water media in Niger Delta of Nigeria. The characteristics of the aquatic environment was used in examining the degradation
of the component investigated and results obtained reveals the significance of the physiochemical parameters (functional
parameters) on the system.
Cite This Article As: C. P. Ukpaka. Comparison of degradation of benzene, toluene and phenol in both fresh and salt water
media. Current Science Perspectives 2(4) (2016) 105-115
INTRODUCTION
The publication of Spies et al., (1996) cited in Okoh, (2006),
claims that Marine oil spills; especially large-scale spill
accidents can be hazardous to its coastal environment. They
cited the case of the oil spill of the North Slope crude oil into
the Prince William Sound, from Exxon Valdez, in 1989. This
case is still being referred to today, because of the death of
thousands of Sea bird and other marine mammals that the
caused pollution.
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105
In a more recent report, Knightes and Peters, (2006)
carried out a study on Polycyclic Aromatic Hydrocarbons
(PAHs), and described them as complex mixtures occur as
distinct organic phases, often called ‘non-aqueous-phase
liquids’. PAHs are called Colliod-associated solutes in
groundwater, soils and sediments. As for their hazardous
effects of the contaminants in the environment today,
knightes and Peters said that there is pressing need to
develop and improve on previous biochemical methods of
treating these pollutants (PAHs), in a controlled and effective
manner. Polycyclic Aromatic Hydrocarbons are so dangerous,
as environmental contaminants, that over 16 PAHs have been
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named as ‘priority contaminants’, by the US Environmental
Protection Agency (EPA). They are said to be extremely
carcinogenic. They are carcinogenic in that, they can induce
cancer or cancerous growth in humans.
Bioremediation is the process of employing the
biodegradative potentials of micro-organisms or their
attributes (Caplan, 1993, cited in Okoh, 2006). This definition
could not have been coined better!
Bioremediation is an effective technology in that it
also treats the physical and chemical aspects of the
contaminants alongside the biochemical aspect. Simply put,
bioremediation encourages volume reduction and
detoxification.
According to Zhu et al., (2001), a major setback in
bioremediation is the lack of guidelines regarding which and
how to use the technology. This problem would be solved as
the technology advances. At the moment, a lot of research is
being conducted on bioremediation.
Bio-augmentation is a potential strategy for Oil
bioremediation since the 1970s Zhu et al., (2001). Their
rationale behind the augmentation of microbial population is
that indigenous microbial population may not be able to
degrade a wide range of potential substrates, present in
complex mixtures such as petroleum. It is for this reason that
oil-degrading micro-organisms are added to the indigenous
microbial population. This practice may come in handy when
conducting experiments in this project.
Biostimulation involves the addition of rate-limiting
nutrients to speed up the biodegradation process. In most
shoreline ecosystems that have been heavily contaminated
with petroleum hydrocarbons, nutrients are likely the
limiting factors in the oil biodegradation. He stated that
laboratory studies have shown that addition of growth
limiting nutrients, namely Nitrogen and Phosphorous, have
enhanced the rate of oil biodegradation Venosa, (1998) cited
in Zhu et al., (2001).
Okoh, (2006) stated that is important to expose the
limiting factors of biodegradation of petroleum hydrocarbons
in both Fresh and Sea water (Salt water). These limiting
factors should definitely affect the kinetic modeling of the
biodegradation of petroleum hydrocarbons, in subsequent
chapters. The limiting factors are: petroleum hydrocarbon
composition (PHC), physical state of the petroleum
hydrocarbons, mineral nutrients availability, salinity, pH,
temperature, oxygen, water and micro-organisms.
Enumerations and subsequent explanation of the roles
played by these mentioned factors, while not lengthy in
content, used as few words as possible in order to simplify
the factors.
The highpoint of Bossert and Bartha’s work, (1984)
cited in Okoh, (2006) is the approximation of the
temperature ranges for biodegradability of petroleum to be:
20-30o in fresh environment, and 15oC – 20oC , in water (sea
water)
environments.
Temperature
affects
the
biodegradability of petroleum hydrocarbons in two ways: the
properties of spilled petroleum hydrocarbons, and the
activity or population of micro-organisms. It was also stated
in their work that the biodegradability of petroleum
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106
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hydrocarbons decreases more viscous, the micro-organisms
become less Okoh, (2006).
In terms of the physical state of the petroleum hydrocarbons,
Morrrison and Boyd, (1974), noted that oxidation increases
the biodegradability of petroleum hydrocarbon by increasing
its availability. This enhances microbial activities.
Oxygen’s role in biodegrability of petroleum
hydrocarbons is very important one, depending on the type
of micro-organism. The Microbial utilization of hydrocarbons
require an exogenous “electron sink”. This electron sink,
according to them in the absence of molecular oxygen,
further biodegradation of partially oxygenated intermediates
may be supported by nitrate and Sulphate reduction. What
they didn’t explain clearly was the concept of the “electronsink” Reardon etal., 2003; Prasad, 2000; Okoh, 2002;
Egberongbe et al., 2006; Keyzig, 199).
Oxygen is most important to this project because a
lot of micro-organisms are aerobic, especially bacteria, while
some are facultative by nature. That is, they depend on
oxygen from aeration in order to survive in their substrates.
Oxygen is crucial for rapid bioremediation. Dibble and
Bartha, (1976) cited in Okoh, (2006).
Nutrients affect the biodegradability of petroleum
hydrocarbons. Inadequate mineral nutrients especially
Nitrogen and Phosphorus, often limits the growth of
hycarbon utilizing microbes in water. This case can be
especially found in fresh water plants for nutrients. On the
other hand, the nutrients level could increase as a result of
the deposal of industrial waters, which may and may not
contain nutrients Zhu, et al; (2001).
Nitrogen is at a low level in Sea (salt) water. This
publication contrasts the findings of Okoh in 2006. It is also
evident that the case of ‘bends’ which occurs when a sea
diver swims directly upwards especially from a very deep sea
level, is as result of the Nitrogen struggling to burst through
the skin pores of the Sea Diver who is a now suffering from
the ‘bends’ condition (Ukpaka, 2013, 2013a; 2013b; Ukpaka,
2015, 2015a; 2015b; 2015c; 2015d; Belcher et al., 1970).
The increase of salinity leads to a sharp drop in the growth
rate of micro-organism. This is only temporary as the culture
recovers and adapts to its harsh environment. The effect,
however, is difficult to quantify. It is transient, and applies to
mixed culture Richardson and Peacok (1991); Ukpaka (2014;
2014a; 2014b; 2014c; 2014d)
Inhibition can be illustrated through simple reaction
equation, where a substance ‘B’, when introduced to a
reactant ‘A’, causes the slowdown, pauses, or completely
stops further enzyme-substrate reaction of A to R. Levenspiel
(1999), as shown in equation (1a) below:
A+I
R
(1)
Equation (1a): Single reversible reaction.
The aim and objectives of the research work is to determine
the rate of degradation of each component investigated such
as benzene, toluene, and phenol as well as the significance of
the
water
medium
environment
in
enhancing
biodegradation. Another important aim and objective is to
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Fig. 1: Comparison of Degradation Rate of Toluene in Both Media
determine the specific rate of substrate, maximum specific
rate of substrate and determination of the equilibrium
constant.
The substance ‘I’ is known as an inhibitor. Inhibitor reacts
with enzyme ‘E’ to make the inert Y.
The Equation (1) illustrates competitive inhibition:
MATERIALS AND METHODS
Inhibition can be illustrated through simple reaction
equation, where a substance ‘B’, when introduced to a
reactant ‘A’, causes the slowdown, pauses, or completely
stops further enzyme-substrate reaction of A to R. Levenspiel,
(1999), as shown in equation (1a) below:
A+I
R
(1)
(1)
Equation (1a): Single reversible reaction.
Equation (1): Cooperate Inhibition reaction
Enzyme Inhibitors can be described as molecules that reduce
or abolish enzyme activity. There are several types of
inhibitation namely: Competitive, Non-competitive and
Mixed Inhibition.
Competitive Inhibition occurs when a substrate ‘A’
and a substrate ‘I’ competes for the same site on the enzyme
‘E’. The competition usually results in either the slowing
down or stops the reaction completely. Levenspiel (1999).
The following equation rates are derived from the reaction
equations above (equation 1):
TR 
K 3CEOC A
K 3CEOC A

CM  C A  NC EOCM CM 1  NC EO C A
(2)
Equation (2): Competitive Inhibition equation.
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K 2  K3
, Michealis' cons tan t , mol / m3
K1
N 
And
Ch t    1  Pt  e8r t t

Where,
CM 
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K4
K5
In non-competitive Inhibition, a Substrate ‘A’ attacks the
Enzyme ‘E’ on one site, while, Inhibitor ‘I’ attacks the enzyme
on another site. ‘I’ would still be an inhibitor because, its
action of attacking the enzyme on a different site results in
the slowdown or stopping of the reaction. This form of
Inhibition is illustrated below. Substrate ‘A’ attacks the
enzyme to form ‘intermediate ‘X’. Intermediate ‘X’ is
completed into product ‘R’. On a different site but, on the
same enzyme, ‘I’ attacks the enzyme, either stopping it, or
slowing it down, as seen in equation (3) below.
(5)
Where, Ch(t)
=
time-varying
hopane-normalised
concentration, P = polar fraction of the Oil, r = ratio of the
average residual nitrogen concentration to Oil loading, ε =
assumed multiplicative error term, and α, δ, γ, ε = fitting
parameters determined from the multiple regression
analysis.
They also claimed that the equation (5) model
matched the experiment results, when parameters were
chosen to fit the data. The Model has a limitation though, and
that is because; the data set used was limited to only a small
area in the field.
Venos, et al. (1996) cited in Zhu, et al. (2001)
developed a model from field data first-order biodegradation
rate constants for Alkanes and several PAHs presents in Light
Crude oil. This model is shown below in equation (6):
e  kt
 A  A
  
 A   A 0
(6)
Where,
 A
 
 A
time-varying hopane-normalised concentration of an analyte,
 A
  
 A 0
Equation (3) is a non-competitive Inhibition reaction.
From Equation (3) above, equation (4) is derived:
TR 

time-varying hopane-normalised concentration of an analyte
K 3C EO C A
C M  C A  NC BO C M  LC AC B
K3
CE C
1  LC BO  0 A
 1  NC BO  
  C A
C M 
1

LC
BO 

First-order biodegradation rate constant for an analyte and is
was stated that actual first-order biodegradation rates are
not constant, but are a function of the residual nutrient
concentration. Equation (7) shows this relationship between
nutrient concentration and 1st –order biodegradation rates:
 N 

K obs  K max 
 Kn  N 
(4)
Biodegradation Kinetics
Oil biodegradation rates are difficult to prevent due to the
complexity of the environment Zhu et al. (2001). According
to Zhu, the Kinetic model development by the Exxon Valdez
monitoring computer programme is based on field studies
conducted by researchers from Exxon. This model is depicted
below in equation 5:
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108
(7)
Where, Kobs =
Observed
first-oder
hydrocarbon
biodegradation rates. Kmax
=Maximum
first-order
hydrocarbon biodegradation rates. Kn = Half-saturation
concentration for a specific nutrient (MnL-3), N = Interstitial
pore water residual nutrient conc. (MnL-3).
According to Reardon et al. (2002), the rate of
biodegradation of Phenol is shown below in equation (8) we
have
dS X

dt Yx / s
(8)
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Fig. 2: Comparison of the degradation rates of benzene in fresh and salt water

Where, S = Substrate concentration, T= time, = specific
growth rate,YX/S=
Biomass yield, andX= Biomass
concentration.
Author attributed the emergence of this model to the
non-volatile nature of phenol. As for the model for Toluene
and Benzene (being volatile hydrocarbons), the equation was
modified. This, according to them is because Toluene is
present in both gas and liquid phases in the bioreactor. Since
the microbial growth rate depends on the liquid phase
substrate concentration only, and biomass yield depends on
the change in total mass of substrate, the masses of Toluene
(liquid and gas) are shown in equations (9) and (10) below:
M TOT  M L  M G
  H  VG 
M TOT  M L 1  
   M L
  RT  VL 
(9)

dSL  S L X

dt
YX /S
Where,
(10)
109
(11)
SL = Liquid-phase
Their first setoff experience involved the use of P. Putida F1
microorganism to biodegrade benzene, toluene and phenol,
alongside their binary and tertiary mixtures. Their results
proved that their single-substrate experiment’s growth
kinetics fit the Monod model (toluene and benzene,
respectively). Equation (12) below show the Monod model:

Where, ML= mass of Hydrocarbon in liquid phase, MG = mass
of Hydrocarbon in Gas phase, MTOT = mass of Hydrocarbon in
the entire system, H = Henry’s constant, R = Gas constant. T =
temperature, VG = Gas-pahse volume,VI = Liquid - Phase
Volume,VG = Gas-phase volume, and VL = Liquid- Phase
volume.
From the equations above, during their experiments,
they discovered that the temperature and volume of liquid
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remained essentially constant. Therefore, the rate of
substrate consumption was written below in equation (11)
below, as:
m SL
K S S L
(12)

Bearing in mind that
Were
1 dX
X dt
max  max imum specific growth rate,
KS = Monod half-saturation constant.
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However, they chose to adopt the Monod model over
Andrew’s because the differences between the model fits
were small, also, Andrew’s model did not improve the
prediction of mixture experiments. They also adopted a
model for Cell growth on homologous substrate mixtures. In
this model, the specific growth rate is the sum of the specific
growth rate of each substrate. Equation (14) depicts this
model:

 max 1 S1
K S ,1  S1
μ
=

μ0 = specific growth rate at temperature T0,
R = gas constant,
3.2  103
Ea = activation energy =
KJ
Kmol
Richardson and Peacock (1991).
Effect of salinity on microbial growth rate
 max 2 S 2
K S ,2  S 2
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Increase in salinity leads to sharp drop in growth rate of
microbes. This is only temporary, as the culture recovers
after adaptation. The effect is however, difficult to quantify. It
is transient, and applies to mixed culture.
(14)
μ1 + μ2 + ……… μi
it should be noted though that this model is strictly a no
interaction model. This study is laudable. The never-say-die
attitude of the researchers to the various setbacks and
challenges they were in gigantic contributions to field of
Bioremediation. This piece will be directly relevant to this
project, as their findings would be improved on, and
innovated.
The model below was developed by Park and Marchaland,
(2005) to explain the effect of salinity on the maximum
specific growth rate of the biomass:
Effect of pH on microbial growth rate
Where, IS = Salinity inhibition constant (h-1), K1 = Substrate
inhibition constant (mg I-1).
Micro-organisms have an optimum pH for growth. This is
usually close to neutrality (7.0), the effect of hostile
environments, caused by unusually on pH specific growth
rate is similar to the product inhibition. That is, it is
considered as a modification of μm. Note that a number of
species of micro-organisms can actually cope and even grow
in spite of their harsh environments.
The equation (15) below shows the modified version of μm:
m
m  pH  
1
H   K
H 
K

a2

a1
Where,
Ka1 and Ka2 are constants.
[H+] = Hydrogen ion concentration.
The specific growth rate is not necessarily at maximum at the
optimum Ph.
The formula below is used to calculate the effect of
temperature on the microbial growth rate:
K S  S  S 2 / K1 
(17)
I S % NaCl 
0.01  % NaCL
(18)
Where, I*S = Constant depending on culture.
The PARK and MARCHLAND’S model was modified, with
simpler parameters introduced to show the relationship
between the maximum specific growth rate and salinity.
A basis of 1% salinity level was used. A specific
growth rate of 0.01 (h-1), and the estimated density of salt
water of 1.027 g/cm3 were also used as a basis. However, this
is a Hypothesis that would be tested in the experimental
chapter of this project: Salinity is directly proportional to the
density of sea water, but inversely proportional to the
maximum specific growth rate, as shown below in equation
(19).
S    1 /  m
(19)
S = salinity level (% g/cm3),
 = density of sea water.
S  Da  / m
(20)
 constant for the salinity inhibition of
Where, D
maximum specific growth rate.
(16)
It affects the Monod kinetic parameters Ks and μm.
Fixing the values
m T   Specific growth rate at temperature T ,
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IS 
 m  1s S
Where,
Effect of temperature on microbial growth rate

 E  1 1 

m T   0 exp a   

 R  T T0 


110
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0.01 
Ukpaka / Current Science Perspectives 2(4) (2016) 105-115
Da 1.027
0.01
Headquarters, Diobu. The water runs around Eagle Island, to
Iwofe –Siapem Company and up to the Aka Naval Base and
beyond. The water is used for bathing around the Eagle
Island, and fishing around the Aka (Naval base) region. The
samples were gotten from the following sites:
(21)
 
 Da  0.00009737 h 1
Site I: Siapem Company, which is an upstream operation
company. The water serves as a passage way for vessels and
local fishermen.
Substituting the constant above into equation (17) we have

 m Da S
K S  S  S 2 / K1 
Site II: The Siapem Company water front. This water is a
few kilometers from the Aka Naval base. It is used as a way
for boats to go to sea.
(22)
Henry’s Equation
Henry’s equation will be used to calculate the masses and
volumes of volatile hydrocarbons, which are present in both
gas and liquid phases in the bioreactor. Since the microbial
growth rate depends on the liquid-phase substrate
concentration only, and biomass yield depends.
Description of Sampling Site/Station (Fresh Water Source)
The location of sampling site is the new Calabar River, which
passes through Choba Community. It flows from Aluu
through to Ibefaway at Emohua Local Government area and
then to Kalabari area and lined up with the Niger River. The
river is used for bathing, drinking and washing. Samples
were obtained from the following station / site:
Site I: Aluu Community which is the upstream with little
contamination from the inhabitants through activities like
washing of cloths and plates and dumping of used water from
domestic works, bathing and dredging activities.
Site II: Spot close to the Wilbros Company just directly under
the bridge. This station is polluted with a lot of diesel oil
from the barges. Also some meters away are the local
dredges and standard dredgers, carrying out their dredging
activities.
Site III: is the Choba Community, which is located a few
kilometers from the Wilbros Company. Here the diesel-oil
also pollutes the water and a lot of feacal pollution (human
wastes) is seen here because the inhabitants defecate, bathe,
wash and dump refuse on this site. Other activities like
fishing and swimming are also carried out.
Sample collection
Sub-surface water sample was collected at three (3) different
locations or stations from the New Calabar River, in sterile
sample bottles. This was taken from the sub-surface water
and the sample collected was not up to the brim of the
sample bottles. The sample were immediately kept in an ice
bag and taken to the laboratory within two (2) hours of
collection. Samples were also collected for dissolved oxygen,
biochemical oxygen demand, salinity pH and conductivity.
Description of sampling site/ station (salt water)
The salt water samples were collected from the Eagle Island
waters. Eagle Island is situated behind AGIP Port Harcourt
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111
Site III: This is the Aka Naval base. It is more saline than the
two previous sites. The Nigerian Naval base runs her
activities on these waters.
Sample collection
Sub-surface water sample was collected at three (3) different
locations or sites from the Aka Naval Base, Saipem Company
water front, and the Eagle Island waters, respectively. This
was taken from sub surface water. The samples were not up
to the brim of the sample bottles. The samples were
immediately kept in ice bags. They were promptly taken to
the laboratory within two hours of collection. The Benzene,
Toluene and Phenol samples were collected in sterile
containers from Oil Test Group of Companies laboratory,
Trans-Amadi, Port Harcourt. Dissolved oxygen, Biochemical
oxygen demand, salinity pH and conductivity tests were
conducted on the water samples.
Temperature
Mercury in Glass thermometer was dipped into water for 3
minutes, and then the result gotten was read and recorded.
The unit for temperature is Celsius/ Centigrade.
Biochemical oxygen demand
Water samples collected in the same way as the DO were
incubated at 20oC for five days. At the end of the incubation
period, the samples were treated in the same manner as the
DO samples stated previously to determine the dissolved
oxygen. To ensure the presence of oxygen the BOD samples
were dissolved and diluted before incubation and the DO of
the dilution water determined. DO at day 5 was determined
as in dissolved oxygen above and the BODs calculated using
the following (A__ B) X Df. Where A is initial DO of dilution
water, Bin DO after 5days incubation and DF in dilution
water.
Conductivity
Measurement for pH, salinity, and conductivity were done
using Horiba water Clucker (model U-10) after calibrating
the instrument with the standard Horiba solution. The units
of measurement, for salinity and conductivity are in
percentage (%) and μs/cm respectively.
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Table 1: Test Results from Samples Sites (Fresh and Salt Water)
Locations
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BOD (mg/L)
Salinity %
EC
Fresh
Salt
fresh
Salt
Fresh
salt
Fresh
Salt
Fresh
Salt
Fresh
Salt
Location 1
Location 2
Location 3
19
19
19
17
17
15
6.1
6.3
6.2
7.2
7.3
7.3
4.5
4.1
4.1
5.9
6.3
6.4
18.3
18.3
18.3
19.6
19.5
19.4
0
0
0
1
1.1
1.4
17
15
15
19
19.6
19.9
Location 1
Location 2
Location 3
Location 1
Location 2
Location 3
Total
20
20
20
19.5
19.5
20
176.2
20
20
17
15
15
16
152.5
8.2
7.9
8.9
7.7
7.5
6
63.7
8.5
7.9
8.9
8.8
8.9
8.8
73.6
6.9
7.3
8.9
6.8
6.z5
5.9
51.4
WEEK II
6.9
7.9
7.9
7.8
7.7
7.8
64.6
12.2
9.1
12.2
15.4
10.1
15.3
129.2
19.5
19.3
19.5
19.3
19.4
19.6
175.1
0
0
0
0
0
0
0
1.1
1.1
1.4
1
1
1.6
10.7
21
16
15
18
14
16
147
22.8
19.6
19.5
19.3
19
119.2
177.9
19.6
16.9
21.2
8.2
5.7
7.2
14.4
19.5
0
1.19
16.3
19.8
0.3
1.9
0.5
0.7
0.6
0.8
1.7
0.1
0
0.21
0.6
1.1
Experiment to determine total heterotrophic bacteria
Media: The media used for isolation of the organisms are:
nutrient agar, manifold salt agar, and petroleum hydrocarbon
agar. The solution for the nutrient agar contained 28g of
powdered nutrient agar and 1 litre of distilled water Manitol
contained NaCl (0.55mg) and 1 litre of distilled water, 0.5g of
NH4Cl, 5g of Na2HPO4, 0.5g of KHPO4, 5ml of Toluene. The
media were filtered with a filter paper of hole-size 20μm.
Chemicals: NaCl powdered nutrient agar (Biology
Laboratory, RSUST, Port Harcourt), Toluene (Oil test Group of
Companies, Port Harcourt), distilled water (Biology
Laboratory, RSUST, Port Harcourt), glycerol (Biology
Laboratory, RSUST, Port Harcourt). All chemicals used for
media preparation were reagent grade.
of the test organism. This is in order to identify the total
heterotrophic bacteria that are present in the colony.
Experiment to determine the degradation rate of aromatic
hydrocarbon content
Aim: The aim of this experiment is to determine the rate of
biodegradation of aromatics hydrocarbons, using aromatic
hydrocarbon-utilizing bacteria. The bacteria isolated are
Pseudomonas Putida (from pseudomonas sp).
Micro-organism used: Pseudomonas Putida was gotten
from the isolation of total petroleum hydrocarbon utilizing
bacteria from samples of fresh water. The isolation and
enumeration of the micro-organisms was carried out in the
Biology laboratory, Port Harcourt.
Apparatus: Hot oven, Conical flask, Test tubes, Cotton wool,
Aluminum tool, Thermometer, Petri dishes, Filter paper
(20μm hole size), Pipette (sterile), Glass spreader (sterile),
Bijor storage bottle, Microscope, Wire loop and Wooden
spatula.
Media: The media used was an aqueous nutrient agar. It
contained 1 litre of distilled water, in order to form a
solution. It also contained about 0.5g of NH4Cl, 5g of Na2HPO4,
5.0g/I of KH2PO4. The Benzene, Toluene and phenol were
added.
Experimental Procedure: All glass waves were sterilized in
a hot air oven for 1 hour at 160oC and left to cool. Culture was
extracted and re-grown in another Petri dish. Plate count
method was used to count the number of colonies of bacteria
formed. The diluents were then prepared using normal
saline.
Chemicals: Benzene (Oil Test Group of Companies, Port
Harcourt, HPLC Grade), Toulene (Oil Test Group of
Companies, Port Harcourt, HPLC grade), Phenol (Oil Test
Group of Companies, Port Harcourt, H2S04 acid (Biology
Laboratory, RSUST, Port Harcourt),(CH3CN), (Biology
Laboratory, RSUST, Port Harcourt over 99.5% purity),
chlorofoam and p-xylene (Baxter GC Grade, Institute of
Pollution Studies, RSUST, Port Harcourt), De-ionized water
(RSUST Biology laboratory).
Analytical Methods: One ml of the original was transferred
in triplicate into test tubes containing 9ml of normal saline.
The sample was then inoculated. The sample was isolated,
using Gram’s stain method to prepare the micro-organism for
observation under the microscope. The following tests were
conducted: Biochemical test, Oxides test, Voges-Pros Kaner
test, Catalase test, Methyl Red test, Sugar Fermentation test
and Coagulase test. The tests were conducted on the colony
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Apparatus: GC Spectrophotometer, correlated to biomass
concentration, Filter paper (0.33μm), Gas chromatograph,
Thermometer, 2 ml screw cap vials, 25 μ! Gas-tight syringe
and10ml Test tubes
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Fig. 3: Comparison of Degradation Rate of Benzene in Both Media
Method used: Cell concentration was measured as optical
density of 600 mM (oD600) using GC spectrophotometer. It
was correlated to bio mass concentration. 0.22 μm filtered
samples were used as optical density blanks. Deionized water
was used as the OD blank for toluene (1.00 OD600=
1000mg/L). Benzene, Toluene and Phenol concentrations
were mentioned by gas chromatography. Aqueous samples
were extracted (0.75ml of sample to 0.75ml of chlorofoam
continuing 25mg/l p-xylene as an internal standard). The
chlorofoam layer was removed and analyzed using an HP
5890 II gas chromatography equipped with a mass selective
detector (HP 5971A). Samples were stored at 4oC in 2ml
screw cap vials with Teflon-lived rubber septa until analysis.
Chlorofoam was used to extract the Benzene, Toluene and
Phenol standard solutions. The gas phase concentrations for
the volatile hydrocarbons (Benzene and Toluene) were
determined through gas chromatography. Samples were
injected into the gas chromatography equipment using 25Μl
gas tight syringe. Aqueous intermediated were formed in the
biodegradation experiments.
RESULTS AND DISCUSSIONS
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113
The results obtained from the investigation are presented in
the paper, in terms of comparison of benzene, toluene and
phenol degradation in both fresh and salt water media.
Comparison of the degradation rates of toluene in fresh
and salt water
The degradation rate of Toluene was similar irrespective of
the water present (fresh or salt water). Salinity only inhibited
it slightly. This is depicted in figure (1)
Comparison of degradation rate of phenol in fresh and salt
water
The degradation pattern of Phenol in both fresh and salt
water followed the same trend at first. This was during the
lag period. However, the patterns deviated with phenol,
degrading better in fresh water than salt water. This is
illustrated in Figure 2.
Comparison of the degradation rates of benzene in fresh
and salt water
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The degradation rates of Benzene in both fresh and salt water
were roughly the same. Slight occasional deviation was only
briefly experienced. This illustrated in Figure 3.
After a rather interesting study of the
biodegradation of petroleum hydrocarbons, the following
recommendations have been made.
More work needs to be done on the role of salinity in
the degradation of petroleum hydrocarbons in water. Judging
from the Biodegradation of Phenol and Benzene, especially
phenol, in salt water, there is no doubt that they were
inhibited by their harsh saline environment.
The assumptions made on the kinetic model
equation for the degradation of petroleum hydrocarbons
should be proven and compared to the experimental data.
The success of this equation would make bioremediation
much easier for Engineers and other professionals in the field
of Biochemical engineering.
An improved version of petroleum-utilizing bacteria
should be genetically engineered. This will enable a broader
range of petroleum hydrocarbons to be biodegraded.
A ‘super-bacteria’ should be developed, which would
be used to completely degrade Phenol at a very fast rate. In a
situation where there is limited time to degrade Phenol, the
microbe could become useful.
CONCLUSIONS
The following conclusion was drawn from the investigation
such as:
1.
Physicochemical properties of the fresh and salt
water media influence the rate degradation that is to say that
the active site of microbe will be inhibited.
2.
The degradation of benzene is faster than toluene
whereas toluene is faster than phenol in both fresh and salt
water media.
3.
The reaction pathway is the same as observed in the
bioreactor set up
4.
The microorganism in non diluted on the reactor
was capable of degrading the substrate, thereby producing
product that are environmental friendly.
5.
The major inhibiting factors considered during the
investigation includes, Ph, temperature and salinity for both
salt and fresh water media.
6.
In this investigation lag phase and other phase was
also experienced in the bioreactor set up.
REFERENCES
Belcher, R., Nutten, A.J., Macdonald, A.M.G., 1970. Quantitative
inorganic analysis. 3rd edn. London: butterworths 47.
Egberongbe, F.O.A., Nwilo, P.C., Badejo, O.T., 2006. Oil Spill
disaster monitoring along Nigerian coastline, disaster
preparedness and management: shaping the change,
Munich, Germany 67-84.
Knightes, C.D., Peters, C.A., 2006.
Multisubstrate
biodegradation kinetics for binary and complex mixtures
of polycyclic aromatic hydrocarbons. Environmental
Toxicology and Chemistry, 25(7), 1746-1756.
www.bosaljournals/csp/
114
iscientic.org.
Keyzig, E., 1999. Advanced engineering mathematics. 8th edn.
New York: John Wiley and Sons, Inc. 1-21.
Levenspiel, O., 1999. Chemical reaction engineering 3rd edn.
New York: John Wiley and Sons, Inc. 318-329.
Morrison, R.T., Boyd, R.N., 1974. Organic Chemistry. 3rd edn.
Boston: Allyn and Bacon, Inc. 318-329.
Okoh, A.I., 2002. Assessment of the potentials of some
bacterial isolates for application in the bioremediation of
petroleum hydrocarbon polluted soil. Ph.D thesis.
Obafemi Awolowo University, Ile-Ife, Nigeria 45-78.
Okoh, A.I., 2006. Biodegradation alternative in the cleanup of
petroleum hydrocarbon pollutants. Biotechnology &
Molecular Biology, Review 1(2), 38-50.
Park, C., Marchland, E.A., (2006). Modeling salinity inhibition
effects during biodegradation of perchlorate. Journal of
Applied Microbiology 101, 222-233.
Prasad, R., 2000. Petroleum Refining Technology. Nai Sarak
Delhi: Khanna Publishers 346-349.
Reardon, K.F., Mosteller, D.C., Rogers, J., DuTeau, N.M., Kim, KH., 2002. Biodegradation kinetics of aromatic
hydrocarbon mixtures by pure and mixed bacterial
cultures. Environmental Health Perspectives, 119(6),
1005-1010.
Richardson, J.F., Peacock, D.G., 1991. Biochemical reactors
and process control. Coulson and Richardson Chemical
Engineering 3rd edn. Linecare House: ButterworthHeinemann 3.
Ukpaka, C. P., 2014.
Investigating the characteristics of
salinity on hydrocarbon
degradation in salt water.
International Journal of Novel Research in Engineering &
Pharmaceutical Sciences 1(05), 15-24.
Ukpaka, C. P., 2014a. Modelling the rate of diffusion of
petroleum hydrocarbon obtained in Niger Delta area of
Nigeria in a pond system, International Journal of Novel
Research in Engineering & Pharmaceutical Sciences,
1(05), 25-34.
Ukpaka, C. P., 2014b. Investigation into the competitive
inhibition of hydrocarbon degradation in pond system
for wet season upon the influence of momentum transfer,
International Journal of Novel Research in Engineering &
Pharmaceutical Sciences, 1(05), 35-44.
Ukpaka, C. P., 2014c. Evaluation of pneumatic proportional
control to predict and monitor constant area variable
pressure drop using different fluids in orifice plate,
International Journal of Novel Research in Engineering &
Pharmaceutical Sciences, vol.1, no. 05, pp. 8 – 14.
Ukpaka, C. P., 2015.
Investigation into the effect of
momentum transfer on de-oxygenation of wastewater
treatment in pond system for wet season. International
Journal of Novel Research in Engineering &
Pharmaceutical Sciences 2(04), 85-106.
Ukpaka, C. P., 2015a. Development of mathematical model to
control the distillate and reflux ratio of a distillation
column using ramp input application of response.
International Journal of Novel Research in Engineering &
Pharmaceutical Sciences 2(04), 1-23.
Ukpaka, C. P., 2015b. Evaluation of microbiological corrosion
of carbon steel in salt water environment of Niger Delta
region. Physical Chemistry Pakistan 17(1), 21 – 26.
[email protected]
ISSN: 2410-8790
Ukpaka / Current Science Perspectives 2(4) (2016) 105-115
iscientic.org.
Ukpaka, C. P., 2015c. Application of algorithm of Runge
Kutta method in monitoring and predicting alphatic
hydrocarbon degradation, International Journal of Novel
Research in Engineering & Pharmaceutical Sciences, vol.
2(03), 29-48.
Ukpaka, C. P., 2015d. Development of mathematical model
to control the distillate and reflux ratio of a distillation
column using ramp input application of response.
International Journal of Novel Research in Engineering &
Pharmaceutical Sciences 2(04), 1-23.
Zhu, X., Venosa, A.D., Suidan, M.T., Lee, K., 2001. Guidelines
for the Bioremediation of Marine Shorelines and Fresh
Water Wetlands. U.S. Environmental Protection Agency,
Contract 68-c7-0057, 1-163.
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