Khaled F. Mossallam
Azerbaijan State Oil Academy, AZ1010, Baku, Azerbaijan
Phenols in our
environment come from various sources. For example, many are found
in the waste waters of industries such as petroleum refineries, glue and resin
manufacturers, coal processing, pulp and paper mills, and from the leaching of
municipal landfills. Phenol is toxic to fish at a level of 0.05 mg/l, therefore the
removing of phenols from waste water is therefore of great importance. Produced
water is the water pumped from aquifers associated with petroleum oil production, gas
production or coal bed methane production. Produced water from petroleum oil
reservoirs was produced in huge amounts and it contained several pollutants, such as
phenol and petroleum oil droplets. New methods were required to reduce the amounts
of toxic pollutants to permissible limits before rejecting this produced water to the
environment. The produced water contained the following 4.5 mg/L phenol, 300 mg/l
petroleum oil, Cl
62 mg/L and pH 6.0. The effect of addition of petroleum oil on the
strong depressing effect on the activity of the horseradish peroxidase at concentrations
up to 1 gm/L of petroleum oil. The effects of treating the produced water with enzyme
peroxidase extracted from horseradish plant, peroxide (H
have been studied. The horseradish peroxidase–hydrogen peroxide system decreased
the amount of phenol present in produced water by 80%. The most effective
concentrations for treating produced water were peroxidase at 2U/mL and 0.5mM
hydrogen peroxide (H
) at neutral pH.
Keywords: horseradish peroxidase, produced water, enzymatic treatment, phenol,
Produced water is the water pumped from aquifers associated with petroleum oil
production, gas production or coal bed methane production. The physical and chemical
properties of produced water vary considerably depending on the geographic location
of the field, the geological formation with which the produced water has been in
contact for years and the type of hydrocarbon product being produced. Produced water
properties and volume can vary throughout the lifetime of a reservoir. The major
aromatic hydrocarbons (phenol) (Veil et al., 2004).
Discharging produced water in marine environment without previous treatment has
which are deleterious to aquatic life. The main contributors to acute toxicity of
produced water have been found to be the aromatic and phenol fractions of the
dissolved hydrocarbons. Phenol is toxic to fish at levels of 0.0045-0.05 mg/L,
therefore the detoxification of phenols from produced water is therefore of great
importance (CCME, 2002).
Most of the methods used for transformation of wastes and pollutants and treating
the application of reagents and reaction conditions to transform and treat target
species. Conventional processes have proven to be efficient in the detoxification of
phenolic compounds. However, these processes have certain disadvantages and
limitations. The high cost and disposal of contaminated media are the disadvantages of
solvent extraction and activated carbon adsorption.
Biological processes make use of the natural metabolism of cells to
remove oil and aromatics from produced water.
The metabolic processes occur as a
result of a sequence of reactions conducted inside the cell that are catalyzed by
proteins called enzymes. An important advantage of biological systems is that they can
be used to carry out processes for which no efficient chemical transformations have
been devised. In addition, biological processes can often be conducted without the
harsh conditions that are necessary during chemical transformations.
Due to the large
water hold-up volume and bacterial culture contact time, these systems are very large
and heavy, and therefore only suitable offshore for low volume application. Also there
are operational and bacterial inhibition problems. The method is best suited to onshore
installations where space and volume are not limitations (IAOGP, 2002).
The following potential advantages of an enzyme-based treatment over conventional
compounds; action on, or in the presence of, many substances which are toxic to
microbes; operation at both high and low concentrations of contaminants; operation
over wide temperature, pH and salinity ranges; no shock loading effects; no delays
associated with acclimatization of biomass; reduction in sludge volume (no biomass
generation) and better defined system with simpler process control.
Produced water re-injection into a disposal well, or preferably the same reservoir from
regions, particularly onshore, but the method has some disadvantages. Re-injection can
be very energy intensive due to high pump pressure requirements, and thereby cause
increased greenhouse gas emissions (IAOGP, 2002).
peroxidase (HRP) and H
effluents. Treatment with horseradish peroxidase and hydrogen peroxide precipitates
97-99% of the phenol in a wide range of pH and phenol concentrations.
Wagner et al. (2001) used the same enzymatic technique (horseradish peroxidase and
) for the treatment of a petroleum refinery wastewater which contains high
amount of phenols. As a result of the treatment with enzyme HRP, the phenol content
of a refinery was reduced below the discharge limit. Phenols were transformed to less
biodegradable compounds and approximately 95% of toxicity was removed.
In this research, the same technique (horseradish peroxidase HRP and H
assessing the treatment of produced water in order to decrease the phenol content in
produced water before its discharge to the marine environment has been used. The
produced water contains several pollutants, a high amount of total hardness of
250mg/L, a high amount of oil and grease, a high chemical oxygen demand (COD),
and contains 4.5mg/l phenol. However, the effects of petroleum oil content on the
activity of HRP and enzymatic treatment are not clear in the previous literature.
The aim of this study was to study the effect of petroleum oil content on the enzyme
) for removing the phenol from the produced water have been determined.
2. MATERIALS AND METHODS
2.1 Materials and Equipments
HRP enzyme (EC 18.104.22.168) was extracted from horseradish in our laboratory. All the
) and 4-aminoantipyrine were used for analysis of phenol and
activity of horseradish peroxidase and are of analytical grade and were purchased from
Photoelectrocolorimeter (FEK-II) (wavelength 300 to 700 nm) was used for the
volume of 5 mL were used. Measurements of pH were made using a pH probe.
Centrifuge with 6000 max revolution per minutes (rpm) and with a capacity 200 mL
was used. Magnetic stirrer with coated magnetic bar was used for mixing reactants
with the produced water.
The horseradish peroxidase enzyme concentration and activity was determined by
reacted with the enzyme solution under controlled pH
and temperature conditions. The pH was
maintained constant with the addition of a
buffer solution of pH 7.4. The reaction resulted in the formation of a non-precipitating
coefficient of 7311 M
formation (Wright, 1995).
The concentration of total phenols was measured using a colorimetric method.
ferricyanide at a pH of 10 to form a stable reddish-brown colored antipyrine dye. The
amount of color produced is a function of the concentration of phenolic material
(Yiseon, 1998). Grease and oil, suspended solids (by filtration method) and COD were
all determined according to the procedures described in Standard Methods (1975).
Horseradish peroxidase enzyme was extracted in the laboratory from horseradish roots
water was 50 gram minced horseradish root to 500 milliliter of tap water. The
horseradish roots were purchased from market, washed and cut to very small pieces.
The minced horseradish-water mixture was vigorously mixed for 3 hours using an
electric mixer at high speed. The resulting solution was filtered and the supernatant
was centrifuged at 4000 revs./min. The supernatant liquid was the crude enzyme and
was preserved at -4°C. Every day the activity of HRP was analyzed before using the
enzyme in the research. The activity of the enzyme in each gram of horseradish root
contains 70 units. The activity of the enzyme is defined in units where one unit of
activity (U) is defined as the number of micromoles of hydrogen peroxide which are
consumed in one minute at pH 7.4 and 25°C.
Batch experiments were conducted at room temperature (25°C). The batch reactors
predetermined doses of each of HRP enzyme, hydrogen peroxide (H
polyethylene glycol (PEG) have been added and the mixing time of the produced
water with the reactants was kept at 4 hours. Polyethylene glycol was added to
minimize enzyme inactivation through interaction with the oil and grease present in
the produced water samples or through phenol oxidation reaction products during the
enzymatic treatment of produced water sample. PEG has been reported to exert a
strong protective effect and is a nontoxic compound. After treatment, the resulting
solution was centrifuged for 30 minutes at 6000 revs./min. The supernatant was then
analyzed for phenol level.
3.1 Effect of Petroleum Oil Addition on HRP Activity
The effect of adding petroleum oil on the activity of HRP was tested and is represented
were vigorously agitated with 0.0, 0.25, 0.75, 1.5 g/L petroleum oil for one hour. A
dose of HRP equivalent to 2 U/mL was added to each of the four sets and agitation
occurred further for another one hour.
Figure 1 presents the data obtained for HRP activity relative to 0.0 g/L of petroleum
decreased to 40%, a further increase in the petroleum oil addition caused a further
decrease on the activity of HRP. At a dose of 1.5 g/L petroleum oil the HRP activity
was decreased down to 10%. From these results, it may be deduced that high doses
(0.75g/L -1.5 g/L) of petroleum oil have a strong depressing effect on HRP activity,
which is mainly attributed to the physical adsorption of petroleum oil on the active
sites of the enzyme peroxidase. The amount of petroleum oil in the produced water
sample was 300 mg/L which means that the presence of petroleum oil in the produced
water would cause a depressing effect on the Horseradish peroxidase treatment, which
can be tolerated by using high doses of the enzyme peroxidase.
Petroleum oil, gm/liter
Figure 1 The effect of petroleum oil addition on the activity of
horseradish peroxidase, at a HRP dose 2 U/ml
at constant petroleum oil content equals 0.75gm/L. A model of four vial glasses each
containing 20mL distilled water were vigorously agitated with 0.75 g/L petroleum oil
for one hour. A doses of HRP equivalent to 2.0, 4.0, 6.0, 8.0 U/mL was added to the
and after two hours. As the time of agitating petroleum oil with the enzyme HRP in the
distilled water increases the activity of HRP decreased due to increasing the chance for
adsorption of petroleum oil droplets on the active sites of the enzymes. Also, by
increasing the dose of HRP the activity of HRP increases.
Horseradish peroxidase undergoes a cyclic reaction when reacting with phenolic
Ei + H
Ei + PhOH ' Eii + PhO
Eii + PhOH "
E + PhO + H
The enzyme starts in its native form (E) and is oxidized by hydrogen peroxide (H
to form an active intermediate compound known as compound 1 (Ei). Compound 1
oxidizes one molecule of phenol (PhOH) to form a phenol free radical (PhO) and
become compound II (Eii). Compound II oxidizes a second phenol molecule to
produce another phenol free radical and complete the cycle by returning to its native
form E. The free radicals polymerize and form insoluble compounds which precipitate
The polymerization reaction is illustrated in equation (4):
PhO + PhO Polymer of aromatic products
3.3 Chemical Analysis of Produced Water
Table 1 summarizes the produced water characteristics as determined in the laboratory.
Petroleum oil and Grease
3.4 Effect of pH on the Treatment of Produced Water
These tests were conducted to study the effect of pH on the removal percentage of the
aromatic substance (phenol) from the produced water by using HRP enzyme and
hydrogen peroxide (H
50 mL, the HRP dose was 1.0 U/mL of the solution, the H
the PEG dose was 400 mg/L. The time of mixing the produced water sample with the
reactants was 4 hours. The pH of each sample was adjusted to be between pH 3.0 and
pH 11 using concentrated HCl or NaOH. The pH of the sample was readjusted before
stirring and after addition of PEG, H
and HRP doses. Stirring of the produced
water with the reactants (HRP, H
and PEG) has been carried out at medium speed
of approximately 200revs/min to allow for oxidation of phenol by using H
oxidation products, which were less toxic to the environment, the remaining phenol in
the treated sample was decreased and a measurement of the phenol by colorimetric
method was thereafter performed.
Figure 3 shows that the optimal percentage of removal of phenol from the reaction
of phenol. The removal percentage of phenol decreased at with increasing acidity and
decreasing alkaline conditions. This could have possibly been due to the effect of oil
and grease on the activity of HRP - a decrease in the suspended solids and oil and
grease content with the increase in pH has been noticed (some clarification of
ions may have
some effect on the oxidation reaction of phenol by using H
and HRP system. This
study has therefore demonstrated that HRP is mostly active at neutral and medium
alkaline conditions and is probably suitable for the treatment of phenol in the produced
water at medium alkaline conditions (7.0 – 9.0).
Figure 3 The effect of pH on the removal percentage of phenol
Mixing of the enzyme peroxidase with hydrogen peroxide and the phenol (at
Figure 4 demonstrates the effect of stirring time of peroxidase on the remained phenol.
peroxide with phenol for four hours.
The optimum HRP dose was determined as 0.3 mM H
at 400 mg/L. These tests were conducted at the optimum pH value 7.0 determined in
the previous set of experiments, and for time of mixing 4 hours. Horseradish
Peroxidase was added in predetermined amounts in order to determine the effect of
HRP dose on the phenol content in the produced water sample after enzymatic
Figure 5 shows that with an increase in the HRP dose, the removal of phenol increases
the solution started to be constant at this dose.
The initial phenol concentration was 4.5 mg/L and at a 0.3 mM H
phenol concentration of treated produced water was 1.1 mg/L. A high dose of HRP of
2.0 U/mL was required and this could have been necessary due to the inactivation
effect of HRP by the high amount of suspended solids, and oil and grease in the
Figure 5 The effect of horseradish dose on the removal of phenol
These experiments were conducted to determine an optimum hydrogen peroxide dose
Figure 6 shows that as the H
down to an optimum value 0.9 mg/L. The high dose of H
enzymatic oxidation of other dissolved contaminants present in the produced water
and due to the enzymatic oxidation of phenol. From these results, it may be stated that
the optimum doses and conditions for the enzymatic treatment of produced water were
at pH 7.0, an HRP dose of 2 U/mL, an H
dose 0.5 mM and a PEG concentration of
400 mg/L which a collectively achieved a final phenol concentration 0.9 mg/L
corresponding to a phenol removal of phenol 80%. The final phenol concentration in
the treated samples of 0.9 mg/L was still however still more than the required phenol
concentration 0.0045-0.05 mg/L. This high value of the residual phenol may be set on
account of the presence of high concentration of oil and grease in the produced water
sample at a value of 300 mg/L, which necessitates a previous pre-treatment by air
flotation to reduce the amount of oil and grease to the minimum concentration of less
than 20 mg/L before the enzymatic treatment.
This study has assessed the feasibility of treating produced water by the enzymatic
found to have a strong depressing effect on the activity of horseradish peroxidase
enzymatic activity, due to the physical adsorption of petroleum oil particles on the
active sites of the enzyme. The most effective concentrations for treating produced
water were a horseradish peroxidase dose of 2 U/mL, 0.5 mM hydrogen peroxide and
a PEG concentration of 400 mg/L, at neutral pH. The enzymatic treatment of produced
water gives a final phenol concentration 0.9 mg/L at a removal of 80%.
This research was supported by the Azerbaijan State Oil Academy. The authors are
Academy) and Professor Siyavus Qarayev (President of Azerbaijan State Oil
Academy) for their encouragement, help and institutional support during the research.
International Association of Oil & Gas Producers (IAOGP), January 2002. Aromatics
Klibanov, A.M., Alberti, B.N., Monis, E.D., Felshin, L.M., 1980. Enzymatic removal
of toxic phenols and anilines from wastewaters. J. App.
phenols from coal-conversion waste waters. Science, 221, 259-261.
Nicell, J.A., Al-Kassim, L., Bewtra, J.K., Taylor, K.E., 1993. Treatment of waste
waters by enzyme catalyzed polymerization and precipitation. Biodeterioration
, 7, 1-8.
Standard Methods for the Examination of Water and Wastewater. 1975. 14
working water quality guidelines for phenols. Summary Report.
Veil, J.A. et al., (2004). A white paper describing produced water from production of
crude oil, natural gas, and coal bed methane. Report prepared for: U. S. Department
of National Energy Technology Laboratory Under contract W-31-109-ENG-38.
Wagner, M., Nicell, J.A., 2001. Peroxidase-catalyzed removal of phenols from
petroleum refinery wastewater. Water Science & Technology, 43, 253-260.
Wright, H., 1995. Characterization of soybean peroxidase for the treatment of phenolic
waste water, Master of Engineering Thesis, McGill University, Montreal, Canada.
Yiseon Han, (1998). Arthromyces ramosus peroxidase catalyzed phenol removal,