Energy Sources , 23:305 —325, 2001

Copyright © 2001 Taylor & Francis

0090-8312 / 01 $12.00 1 .00

In-Vessel Bioremediation of Oil-Contaminated Peat

A. E. GHALY
J. B. PYKE
Biological Engineering Department
Dalhousie University, P.O. Box 1000, Halifax,
Nova Scotia, Canada B3J 2X4

Petroleum pollution of the ocean arises from activities undertaken to meet energy require-
ments such as extraction, transport, and general uses of petroleum products. Marine trans-
port of oil has greatly increased over the past few decades and oil spills affect the living
and non-living resources of the surrounding areas. In this study, the effectiveness of peat
in the remediation of oil contaminated water was tested and the in-vessel bioremediation
of the oil contaminated peat was evaluated.
The results indicated that kiln dried peat is an excellent absorbent for extracting oil
from water. It has the ability to remove 99.998% of the oil from contaminated water hav-
ing an oil slick of 1.3 cm in depth. Mixing the peat with the contaminated water allowed
for the coagulation of the peat with oil, and thus increased the oil removal efficiency, but
also increased the moisture content of the peat.
Evidence of three microbial populations (psychrophilic, mesophilic and ther-
mophilic) in the bioreactor demonstrated the potential of the in-vessel bioremediation
process to achieve a much higher oil degradation efficiency at a lower cost compared to
biopiling and lasd farming. The worming of the material in the bioreactor and the
increased moisture content were signs of the conversion of complex organic carbon by
these microbes into energy, CO2 and H2O. Higher temperatures caused by mesophilic and
thermophilic activities resulted in a higher conversion rate. The moisture content, pH and
the aeration rate were within the optimum range for the bioremediation process. The poul-
try manure provided all the required nutrients for microbial growth, but nitrogen appeared
to be the limiting factor. The bioremediation process achieved 56.7% reduction in oil con-
tent in about twelve days. Addition of a nitrogen source would have resulted in a much
higher reduction percentage. The original humic mixture was reduced to a denser, but
porous material that looked like a garden soil with a dark brown color and no oil odor.

Keywords absorption, air, contamination, in-vessel bioremediation, micronutrient,

moisture content, oil, peat, petroleum hydrocarbon, pH, temperature, water.

Petroleum pollution of the ocean arises from activities undertaken to meet energy require-
ments such as extraction, transport, and general uses of petroleum products. Oil accounted for
nearly 32% of the world’s total energy requirements; the greatest percentage of any energy

Received 27 April 1998; accepted 20 November 2000.

Address correspondence to A. E. Ghaly, Biological Engineering Department, Dalhousie Uni-
versity, P. O. Box 1000, Halifax, Nova Scotia, Canada, B3J 2X4.

305
306 A. E. Ghaly and J. B. Pyke

source. Marine transport of oil has greatly increased over the past few decades. Over 1.5 bil-
lion tons of crude oil and oil products are transported each year by more than 7000 tankers and
as a result the estimated input of oil into the marine environment is 3.2 million tons per year
(Doerffer, 1992). An oil spill affects the living and non-living resources of the area. Affected
living resources include: waterfowl, fisheries, marine food web, and marine mammals. These
biological resources have further reaching implications on humans through eating contami-
nated seafood, decreased fishery resources, altered marine ecosystems, and changed wildlife
habitats. Non-living resources include: the land, water, and air contaminated by oil. Exposure
to low concentrations of petroleum hydrocarbons can cause headaches, skin irritation, itchy
eyes and burning sensations in internal organs. As the concentration of the petroleum contam-
ination increases, the severity of the adverse health effects increases. Prolonged exposure to
high concentrations of petroleum hydrocarbons can cause liver and kidney diseases, bone
marrow damage, and increases a person’s probability of getting cancer (Ghaly et al., 1998).
Therefore, the remediation of oil contaminated water is of paramount importance.
Peat is partially fossilized plant matter (mostly hemicellulose, cellulose, and lignin)
which is formed in poorly oxygenated wetlands where the rate of accumulation of plant mat-
ter is greater than that of decomposition (Couillard, 1994; Viaraghavan & Rana, 1991). It is a
highly porous material with a porosity of approximately 95% and a specific area of 200 cm 2/g.
The polar functional groups of lignin in peat (which include alcohols, aldehydes, ketones,
acids, phenolic hydroxides, and ethers) are all involved in the formation of chemicals bonds.
Because of its polar and porous characteristics, peat has been reported to be an effective agent
in the remediation of oil-contaminated water and as an absorbent of contaminants and impu-
rities in wastewater (Couillard, 1994). Not only is peat capable of absorbing contaminants and
impurities, but it is less costly and its absorption capacity is greater than that of other common
absorbents such as volcanic grain glass, chemically treated glass wool and foamed plastic
(Ghaly et al., 1998 ). However, the oil-contaminated peat must be then disposed of in an envi-
ronmentally safe manner or bioremediated.
Bioremediation refers to the microbial transformation of hazardous chemicals (such as
hydrocarbons ) to less toxic and environmentally acceptable compounds (such as CO 2 and
H2 O). With hydrocarbon bioremediation, indigenous bacteria (Table 1) utilize the hydrocar-
bons as the substrate for energy and carbon for growth. However, the success of the bioreme-
diation process depends on the types of microorganisms involved, substrate utilization rate,
and the rate of oxygen transfer (King et al., 1992). The two most important bioremediation
technologies currently available are land farming and biopiling. In land farming, hydrocarbon
contaminated medium is spread over the top of a farm land in a thin layer and is then plowed
into the soil (Autry & Ellis, 1992). In this form of bioremediation, only the top one meter of
soil is actively involved in the process and fluctuation of environmental factors such as mois-
ture and temperature affects the rates of bioremediation. Furthermore, the process is quite
slow (20% reduction in hydrocarbon concentration in 6-7 months) and there is an increased
environmental concern over the loss of volatile organics to the atmosphere or leachating of
improperly biodegraded hydrocarbons to ground water. On the other hand, biopiling involves
the placement of hydrocarbon contaminated medium into containment cells supplied with air,
water and essential nutrients (Baker & Herson, 1994a). Although the technique is environ-
mentally sound, it is costly and maintaining a homogeneous biopile is difficult.
In this study, in-vessel bioremediation of hydrocarbon contaminated peat is evaluated. As
in composting, the process is designed for optimum conditions for microbial activities and
can, therefore, provide faster degradation rates and a much shorter operating time than land
farming and biopiling.
 In-Vessel Bioremediation 307

Table 1
Known Hydrocarbon Degrading Microorganisms

Category Compound Micro-Organism

Straight Chain Alkanes Methane Pseudomonas methanica, Myobacterium
fortuitum, Myobacterium Smegmatis
Ethane Pseudomonas methanica
Propane Pseudobacterium sublteum
Butane Pseudomonas fluorescens, Actinomyces
candidus, Corynebacterium
Branched Alkanes Isobutane Corynebacterium, Brevibacterium ery-
throgenes
Cyclic Alkanes Cyclopropane Nocardia sp., Xanthobacter sp.
Alkenes Ethylene Candida lipolytica, Pseudomonas
oleovorans
Aromatics Phenol Pseudomonas putida, Acinetobacter cal-
coaticus, Vibrio sp., Spirillum sp., Bacil-
lus sp., Norcardia sp., Chromobacter
sp., Flavobacterium sp.
Benzene Pseudomonas sp., Acinetobacter sp.,
Nocardi sp.
Ethylbenzene Nocardia sp., Acinetobacter sp.
Toluene Pseudomonas sp., Achromobacter sp.,
Pseudomonas aeruginosa, Micrococcus,
Acinetobacter sp.
Xylenes Pseudomonas putida, Nocardia sp.
Napthalene Nocardia sp., Nostoc sp., Cunning-
hanella elegans, Coccochloris sp.
Anthracene, Pyrene Achromobacter sp., Beijerinckia sp.
Benzo(a)anthracene Cunninghanella elegans, Beijerinckia
sp.
Benzo(a)pyrene Beijerinckia sp., Candida lipolytica

Objectives
The main objectives of this study were: a) to test the efficiency of a local peat product as an
oil sorbent, and b) to evaluate the effectiveness of in-vessel bioremediation in reducing the
total petroleum hydrocarbons concentration in the contaminated peat.

Experimental Apparatus
The experimental set up is shown in Figure 1. The bioreactor used in the study was con-
structed of 6.4 mm thick stainless steel. The sides of the bioreactor measured 340 mm ´ 280
mm with a radius of 150 mm at the lower end. The top of the bioreactor (340 mm ´ 800 mm)
was held in place by 4 hinges placed on one side, which allowed for the closing and opening
of the top. Four locking clamps were provided on the other side to ensure adequate sealing and
308 A. E. Ghaly and J. B. Pyke

Figure 1. Experimental setup.

 In-Vessel Bioremediation 309

easy locking of the top cover when the bioreactor was in operation. A rubber gasket lining was
used to prevent air leakage from the bioreactor during operation. The top cover and end walls
of the bioreactor were insulated with 25.4 mm thick styrofoam layer. The sides of the biore-
actor were insulated by means of a 10 mm thick air space between double walls and a 25.4
mm thick styrofoam layer placed on the outer wall of the reactor. There were three holes at
the bottom of the reactor which were connected to a manifold by 6.4 mm diameter tygon tub-
ing and used for aeration. The top cover also had three 60 mm holes which were used as sam-
pling ports. These were covered during operation with rubber stoppers.
Inside the reactor, a 6.4 mm diameter solid stainless steel shaft was mounted on two bear-
ings. There were 5 stainless steel collars on the shaft in which five bolts of 101.6 mm long,
6.4 mm diameter each were mounted. The shaft was rotated by a permanent magnet variable
speed (0-250 rpm), 3/4 hp electric motor (Model No. 2X846, D.C., Dayton Electric Mfg. Co.,
Chicago, Illinois) and was coupled directly to a gearbox (Model No. 4Z29B, Dayton Electric
Mfg. Co., Chicago, Illinois) of a 30:1 gear reduction ratio. The speed of the motor was con-
trolled by a speed controller (Model No. 60648, Dayton Electric Mfg. Co., Chicago, Illinois).
The air was supplied to the bioreactor by a 3/4 hp compressor (Model No. M01 FC 75-
7.5, Sanborn, Markam, Ontario) with the airflow regulated at 2.5 L/min. The supply air was
passed through a 4.38 L plexiglass canister (35.0 cm ´ 15.0 cm in. diameter) filled with hydro-
scopic silica gel (Type 3-8 mesh-ACS Grade, Fisher Scientific, Fair Lawn, New Jersey) which
allowed the air to completely dry. The dried air was then passed through a flowmeter (Model
No. N103-5G, Cole Parmer, Chicago, Illinois) before entering the bioreactor. The condensate
from the saturated exhaust air was collected in a 0.63 L plexiglass water trap. The outlet air
was then dried in another 4.38 L plexiglass canister (35.0 cm ´ 15.0 cm in diameter) filled
with hydroscopic silica gel (Type 3-8 mesh-ACS Grade, Fisher Scientific, Fair Lawn, New
Jersey). Another flowmeter (Model No. N103-5 ST, Cole Parmer, Chicago, Illinois) was used
to measure the flow rate of the outlet air before it was exhausted through tygon tubing into the
laboratory fume hood. Two rubber septums were located on the air inlet and outlet lines to
provide access for gas sampling.
Temperature measurements were taken using ten thermocouples (Model No. LM 35CZ,
National Semiconductor, Chicago, Illinois). The thermocouples and their location are shown
in Figure 1. The thermocouples T1-T3 were used to measure the temperature of the center of
composting mass. The thermocouples T4-T6 were located on the front wall of the reactor,
while thermocouples T7-T9 were located on the back wall of the reactor. The thermocouple
T10 was used to measure the ambient air temperature. All thermocouples were linked to a da-
talogger (Model No. 1000, Omega Tempscan Engineering, Stamford, Connecticut) which was
connected to a microcomputer (Model 486, Comax, Halifax, Nova Scotia) and a printer
(Model No. PR 9102, Raven Mfg., Tokyo, Japan) for the storage and printing of data files.

Methodology
Peat Collection
A 9 kg peat sample was collected for the study. The peat was of CANSORB Organic Oil
Absorbent type, manufactured by AVP CANSORB, Berwick, Nova Scotia, Canada. The sam-
ple was obtained from the manufacturer in a sealed plastic bag. It was stored in the Biotech-
nology Laboratory of the Biological Engineering Department at Dalhousie University in
Halifax, Nova Scotia, Canada until needed. Some characteristics of the peat used in this study
are presented in Table 2.
310 A. E. Ghaly and J. B. Pyke

Table 2
Some Characteristics of the Peat Used in the Study

Item Value
Absorption ratio 8.00
Moisture content (%) 7.10
Bulk density (Kg/m 3 ) 151.00
Organic carbon
Cellulose (%) 25.40
Hemicellulose (%) 72.20
Lignin (%) 2.40
Ultimate Analysis (%)
C 49.56
H 5.09
O 42.25
N 0.62
S 0.22
Cl 0.03
Proximate Analysis (%)
Volatiles 87.75
Fixed carbon 10.02
Ash 2.23

Oil Spill Experiment

A motor oil (Quaker State, 10 W 30) was used in this study. Some characteristics of the oil are
presented in Table 3. Initially, an oil spill on the water surface was simulated to test the abil-
ity of the peat to bind with an oil slick on the water surface. Fifteen liters of water were placed
into a large holding container (57.5 cm in length ´ 33.0 cm in width ´ 41 cm in height). The
oil was “spilled” onto the surface of the water until a thick layer (1.3 cm) of oil covered the
surface of the water. The amount of oil used was 2400 ml. Peat was applied onto the water sur-
face to absorb the oil. Agitation of the oil and peat allowed for coagulation to occur. The appli-
cation of peat was halted once there were no signs of an oil slick on the water surface and the
amount of peat used was determined. The peat/oil mixture was skimmed off of the surface of
the water with a strainer and placed into another holding container (46 cm in length ´ 40 cm
in width ´ 41 cm in height). After all of the peat/oil mixture had been removed, the remain-
ing water was filtered through a coarse filter paper (No. 41, 15.0 cm in diameter, Whatman
International Ltd., Maidstone, England) placed on a filtration system (Figure 2) to remove any
peat remaining in the water. The filtration system consisted of a large capacity, 2-piece PVC
Buchner filtration funnel (Model No. 4280-1500, Nalge, Sybron International, New York,
New York) placed on a 2 L Ehrlenmeyer vacuum flask (No. 5340-2L, Pyrex, New York, New
York) connected to a vacuum pump (Model No. 5-High Purity, Edwards Pumps, Sussex, Eng-
land). The filter paper (with the captured peat) was placed into the peat/oil mixture to mini-
mize procedural error. A sample was taken from the filtrate (water) for total petroleum
hydrocarbon analysis.
 In-Vessel Bioremediation 311

Table 3
Some Characteristics of Motor Oil (10 W 30) * Used in the Study

Item Value
Viscosity at 40°C(Centistokes ) 119.00
Bulk density (Kg/m 3 ) 892.00
Ultimate Analysis (%)
C 85.40
H 13.00
O 0.00
N 0.13
S 0.32
Cl 0.05
Proximate Analysis (%)
Volatiles 97.70
Fixed Carbon 1.20
Ash 1.10

*Approved by the American Petroleum Institute for Use in Turbo Engines.

Figure 2. Filtration System.

Bioremediation Experiment
The peat/oil mixture weighed 6.41 Kg. An amount of chicken manure (1.28 Kg) was added
to the peat/oil mixture as a source of nitrogen and micronutrients. Some characteristics of the
poultry manure are presented in Table 4. The mixture was then inoculated with a liquid solu-
tion (2 L or 2 Kg) of aerobic microbes obtained from a petroleum aeration tank. Another 1.73
312 A. E. Ghaly and J. B. Pyke

kg of dry peat (uncontaminated ) and 2.25 Kg of water were also added to the mixture to bring
the final moisture content to about 60%. The entire mixture (peat/oil/manure/water/inoculum )
was first mixed thoroughly and then placed into the bioreactor. Table 5 shows the various con-
stituents of the final mixture. A representative sample was then collected from the mixture for
total petroleum hydrocarbon and moisture content analyses.
The bioreactor was started and the mixing speed was maintained at 5 rpm whereas the air
flow rate was maintained at 240 L/h (3 v/v/h). The reactor temperature in the vessel was
recorded at half-hour intervals for the duration of the experiment. The moisture content and
total petroleum hydrocarbon were measured at the beginning of the experiment and every sec-
ond day throughout the experiment. Samples were taken at 2 day intervals for the total petro-
leum hydrocarbons and moisture content analyses. At the end of 15 days, the bioreactor was
turned off and the contents removed. A sample of the final mixture was collected for total
petroleum hydrocarbons and moisture content analyses. After the experiment was terminated
the compost (peat/oil mixture) was sealed and kept for further biodegradation studies.

Table 4
Some Characteristics of the Poultry Manure Used in the Study

Item Value
pH 6.90
Moisture content (%) 75.00
Bulk density (Kg/m 3 ) 966.00
Ultimate Analysis (%)
C 42.00
H 4.78
O 38.15
N 6.72
S 0.45
Cl 0.65
Proximate Analysis (%)
Volatiles 82.37
Fixed Carbon 17.37
Ash 7.25
Micronutrients (mg/Kg)
K 2260.00
P 3310.00
Fe 1450.00
Al 560.00
An 310.00
Mn 250.00
B 87.00
Cu 54.00
Ni 19.00
Co 4.00
Cd 1.00
Mg 1.00
 In-Vessel Bioremediation 313

Table 5
The Constituents of the Mixture Used in the Bioreactor

Item Value
Initial Oil-Peat mixture (Kg) 6.41
— Added Peat (Kg) 3.29
— Spilled Oil (Kg) 2.14
— Absorbed Water (Kg) 1.02
Added chicken manure (Kg) 1.28
Added inoculum (2L or 2 L) 2.00
Added dry peat (Kg) 1.73
Added water to bring moisture content to 60-70 % (Kg) 2.25

Total weight of mixture (Kg) 13.711

Oil-Peat mixture moisture content = 48.63 % w/w

Final mixture moisture content = 60.51 % w/w
Final mixture oil concentration = 15.61 % w/w
Final mixture C:N ratio = 59:1

Analyses
Proximate Analysis
The proximate analysis was performed to determine the weight fractions of volatiles, ash and
fixed carbon in the uncontaminated peat, manure and oil samples. The ASTM Standard
Method for Proximate Analysis of Coal and Coke (D-3172-73 through D-3174-82 and D-
3175-82) was used (ASTM, 1986). Because peat and manure are highly volatile fuels, the
modified version for sparkling fuels was used. Thus, the values of volatiles and ash were
determined on dry matter basis. The fixed carbon was then obtained by subtracting from 100
the sum of volatile matter and ash contents.

Ultimate Analysis
Ultimate analysis was performed to determine the elemental composition of the uncon-
taminated peat, manure, and oil samples. The weight fractions of carbon, hydrogen, nitro-
gen, sulfur, chlorine, and ash were determined, and the weight fraction of oxygen in the peat
and oil samples were calculated by the difference. Carbon, hydrogen, and nitrogen weight
fractions were determined using a Perkin-Elmer LECO CHN Elemental Analyzer (Model
No. 240, International Equipment Company, Needham Heights, Massachusetts). Sulfur was
determined using the LECO induction furnace (LECO S-Analyzer, International Equip-
ment Company, Needham Heights, Massachusetts). Chlorine was determined by following
the Mercuric Nitrate Method given in Standard Methods for the examination of Water and
Wastewater (APHA, 1985). The ASTM Standard Test Method for Ash in Wood (D-1102-
84) was followed to determine the ash percentage in the peat sample (ASTM, 1986).
Because of the lack of a simple, direct method for determining oxygen in biomass fuels, it
is usually estimated by subtracting the sum of carbon, hydrogen, nitrogen, sulfur, chlorine,
and ash from 100.
314 A. E. Ghaly and J. B. Pyke

Moisture Content
Moisture content tests were performed on the as-received peat (uncontaminated peat) and
manure samples by the oven drying method, according to the ASTM D 3173-73 procedure
(ASTM, 1983 ). Samples weighing approximately 4 to 5 g each were placed in large alu-
minum dishes. The dishes containing the samples were placed in an air forced drying oven
(Isotemp Oven Model No. 655F, Fisher Scientific, Toronto, Ontario) at 105°C for at least 24
hours. The moisture content of the samples was then calculated on wet basis as follows:

WW 2 DW
MCwet 5 3 100 (1)
WW

where:
MC wet is the moisture content on wet basis (%)
WW is the wet weight of the sample (kg)
DW is the dry weight of the sample (kg)
Moisture content tests were also performed on the oil-contaminated peat samples using
a combination of the oven drying procedure ASTM D3173-17 (ASTM, 1983) and a modified
solvent extraction procedure. Contaminated peat (oil, water, and peat mixtures) samples
weighing approximately 10 g each were placed in a 100 ml round bottomed centrifuge test
tubes. Fifty ml of a chromatographic grade solvent (hexane) was added to each of the tubes
containing the contaminated peat mixture. The solvent and contaminated peat were then
mixed for 10 minutes using a vortex mixer (Model No. M16715, Thermolyne, Dubuque,
Iowa). The vortex mixing allowed for sufficient liquid/solid contact and thus complete oil
recovery from the contaminated peat sample by the hexane. The mixtures were then cen-
trifuged at 2000 rpm for 40 minutes. The supernatants (hexane/oil/water emulsion) were care-
fully decanted and quantitatively transferred into 100 ml round bottomed centrifuge test tubes.
The centrifuged plugs of peat were placed in large aluminum dishes and the moisture content
of the peat was then calculated on wet basis according to the ASTM D3173-173 procedure
(ASTM, 1983 ). The supernatant (hexane/oil/water emulsion) samples were then centrifuged
at 2000 rpm for 20 minutes to break down the emulsion into layers. The samples were then
held at –4°C for 12 hours. The top layer, which consisted of the hexane solvent containing oil,
remained in a liquid state and was decanted and kept for further gas chromatographic analy-
sis. The bottom layer, which consisted of frozen water, was then weighed using a Mettler Bal-
ance (Model AE200, Mettler Instrumente AG, Griefensee, Zurich). The moisture content of
the contaminated peat samples were then calculated.

Oil Content
The oil content in the contaminated peat and filtrate water was determined using a gas chro-
matograph (Model No. 5890-SII, Hewlett Packard, Atlanta, Georgia). The gas chromatograph
was calibrated by injecting 1.0 µL of the hexane extracted oil onto a 25 m ´ 0.2 mm, 0.33 µm
film thickness, 5% diphenyl siloxane megabore capillary column. A split ratio of 5:1 was
employed using the split mode of the injection port. The injection port was set at 180°C and
the flame ionization detector was set at 250°C. The oven containing the column was first
maintained at 40°C for 4 minutes and then increased at a rate of 10°C/min until a final tem-
perature of 350°C was reached. This final temperature was held for 5 minutes. The carrier gas,
helium, was held at a flow rate of 1.2 ml/min.
 In-Vessel Bioremediation 315

Results and Discussions

Remediation of Oil-Contaminated Water
A real oil spill was simulated in this experiment using 2.4 L of motor oil which formed a 1.3
cm thick oil slick on the surface of 15 L of water as shown in Figure 3. Peat was then added
to the oil-water mixture as shown in Figure 4. When the mixture was agitated, the oil and peat
coagulated (the oil was absorbed by the peat) as shown in Figure 5. After the removal of the
peat-oil mixture, there was no sign of oil on the water surface as shown in Figure 6.
Table 6 shows the results of oil recovery from the contaminated water. The initial oil con-
centration in the water was 142 720 mg/L (16% v/v or 14.27 w/w) while the oil concentration
in the remediated water was only 3.1 mg/L (or 0.00031% w/w), thus resulting in an oil recov-
ery of 99.998%. Viaraghavan and Mathavan (1989) achieved 80% oil removal efficiency from
contaminated water percolating through horticultural peat at a flow rate of up to 2.88 L/h. This
is much lower than the removal efficiency of 99.998% achieved in this present study.
The results obtained from this study illustrate the effectiveness of the kiln-dried peat in
absorbing oil from contaminated water. However, the moisture content of the oil-peat mixture
was 48.63% showing an absorption ratio (peat:oil/water ) of approximately 1:1 which is much
lower than the absorption ratio of 8 reported by the manufacturer. This may suggest the use
of lesser amounts of peat per Kg of oil, or the ability of the peat to absorb more oil. In a study
by Ghaly et al. (1998) it was reported that the same peat absorbed up to 5.7 times its weight
in diesel fuel oil.
According to Martin (1991 ), several possible interactions take place between peat and
contaminants. These interactions include: a) cation exchange with H + found in the –COOH
phenolic hydroxyl and heterocyclic groups, b) the interaction of metallic cations to form
chelate complexes, and c) the formation of anion-cation bonds. These interactions are, how-
ever, dependent on the characteristics of the peat and the contaminant. Couillard (1994)
reported that heat treatment of peat frees some functional groups and increases the number of
available exchange sites. In addition, oleophilic waxes are brought to the surface and give the
peat hydrophobic properties.

Bioremediation of Oil-Contaminated Peat

The oil-peat-manure mixture placed in the bioreactor was homogeneous. About 15% of the
peat particles ranged in length from 5 to 30 mm with a maximum width of 2.8 mm, 25%
ranged in length from 1 to 20 mm and had a width ranging from 1 to 2.5 mm and 60% were
dust (less than 1 mm in both length and width) The poultry manure consisted of mostly fine
materials except for a few gravel sized pellets of approximately 5 mm in diameter.
The first visual observation was made after 8 days from the start of the bioremediation
experiment. Upon opening of the bioreactor cover, a warm, moist air with a slightly oily
odor was detected. The composition of the mixture had undergone a significant change and
the volum e was reduced to approxim ately 75% of the original volum e. The original humic
porous mixture was reduced to a denser, spongy, and clay like material. The moisture con-
tent at this point (64.25% ) was higher than the initial value of 60.51% which may be a result
of the increased density of the material and the metabolic activity of the microorganisms.
The second visual observation was made at the end of the experiment (after 16 days). The
final material was similar to that which was inspected after 8 days from the start of the
experiment except for the moisture content which was slightly lower (62.00% ). The mix-
ture was porous and looked like a garden soil with a dark brown color and no oily odor.
316 A. E. Ghaly and J. B. Pyke

Figure 3. Oil slick formation on the surface of the water.

The growth of a pure microbial population under optimum batch condition undergoes
several stages as shown in Figure 7. These are: a) the lag phase, which represent the time
required for the cells to acclimatize themselves to the new environment and to produce the
enzymes required for the utilization of the substrate, b) the exponential growth phase, during
which the growth rate has a constant maximum value, c) the stationary phase, during which
the growth rate is zero, and d) the death phase, during which the cells die faster than the new
cells are produced. If the degradation process of the organic compound involves exothermic
 In-Vessel Bioremediation 317

Figure 4. Addition of peat to the surface of the oil slick.

reactions, heat will be released and a rise in the temperature of the media will be associated
with microbial growth as shown in Figure 7. However, several species of microbes are
involved in the bioremediation process and these can be classified on the basis of temperature
range in which they grow into three main groups as shown in Figure 8-a: a)psychrophilic (-
10-25°C), b) mesophilic (15-40°C) and c) thermophilic (30-80°C). A typical temperature
curve for a biological process in which these three groups of microbes are involved is shown
in Figure 8-b.
318 A. E. Ghaly and J. B. Pyke

Figure 5. Coagulation of the oil and peat with surface agitation.

The results obtained from the bioremediation experiment exhibited the effects of the
metabolic activities of these three groups of microbes on the temperature of the moisture in
the bioreactor. The worming of the material and the increased moisture content demonstrated
the conversion of complex organic carbon compounds (peat, manure, and oil) by the microbes
into energy, H2O and CO2 in a process called mineralization of carbon or cell respiration
(Baker & Herson, 1994). A relatively small part of this energy is needed for work done within
the microbial cells in synthesizing substances and structural cell parts (enzymes, protoplasms,
cell walls, etc.) whereas the rest appears as heat. Viewed in a simplified manner, cell respira-
 In-Vessel Bioremediation 319

Figure 6. Final water appearance after the removal of the oil-peat mixture.

tion is analogous to slow combustion. The combustion (i.e. cell respiration) is slow because
it takes place at relatively low temperatures; even in a cold environment of 3-5°C, respiration
continues as a result of the metabolic activities of the psychrophilic organisms.
The bioremediation temperature profile (Figure 9) clearly represents the three successive
microbial populations which were involved in the bioremediation of the oil contaminated
peat. The initial temperature of the oil-peat-manure mixture was 17°C which quickly rose to
26°C after 48 hours, owing to the metabolic activities of the psychrophilic bacteria. The tem-
perature remained constant for the next 24 hours indicating the shift in population from the
320 A. E. Ghaly and J. B. Pyke

Table 6
Oil Recovery from Contaminated Water Using Peat as the Absorbent

Item Value
Water used (ml) 15,000.0
Oil spill (ml) 2,400.0
Oil concentration in water before peat addition (mg/L) 142,720.0
Oil concentration in water after peat-oil mixture was removed (mg/L) 3.1

Oil recovery with peat (%) 99.998

Oil density = 0.892 g/L

Figure 7. Microbial growth and medium temperature for a pure culture under optimal batch
conditions.

psychrophilic group to the mesophilic group (i.e. the start of the stationary phase for the psy-
chrophilic population and/or the lag phase for the mesophilic population). Thereafter, the tem-
perature continued to rise on the fourth day reaching 34°C and then remained constant for 20
hours indicating the shift in population from the mesophilic group to the thermophilic group
(i.e. the start of the stationary phase for the mesophilic population and/or the lag phase for the
thermophilic population ). Thereafter, the temperature continued to rise reaching 46°C on the
sixth day then decreased gradually reaching 25°C by the eleventh day and finally remained
constant until the end of the experiment on the sixteenth day.
 In-Vessel Bioremediation 321

Figure 8. Specific growth and temperature of bioremediation microorganisms .

322 A. E. Ghaly and J. B. Pyke

Figure 9. Temperatures and Water and Oil Contents recorded during the bioremediation
experiment.

According to Walworth and Reynolds (1995), Cacciatore and MacNeil (1995) and Skinner
(1991), the success of bioremediation (or contaminant degradation) depends on the type of
microbes involved and the transport of the substrate and oxygen from the medium to the bacte-
ria. Furthermore, the biodegradability rate of a given hydrocarbon depends upon the C:N ratio
(26:1 to 35:1), moisture content (40-80% with an optimum of 60%), pH (6.5 to 8.5), aeration
rates (0.5 to 3 V/V/h) and availability of trace amounts of micronutrients such as cobalt, mag-
nesium, manganese, copper, and molybdenum. In this experiment, the moisture content
(60.51% ), pH (6.9), and aeration rate (3 V/V/h) were at their optimum values. In addition, the
poultry manure provided all of the required micronutrients for microbial growth and metabolism
as shown in Table 4. However, the nitrogen appeared to be the growth limiting substrate as the
C:N ratio was only 59:1. The sudden decline in the temperature after the sixth day was a sign of
nitrogen deficiency since there was sufficient carbon source and moisture. The lack of any acer-
tainable ammonia smell during the performed visual observation on the eighth day confirmed
the use of nitrogen source (which was provided in the poultry manure) by the microbes. Also,
the biodegradation of the hydrocarbons was halted after the eighth day (Figure 9) as was appar-
ent from the levelling off of the total hydrocarbon concentration in the mixture.
The analyses performed on the oil-peat mixture before the start and after the completion of
the bioremediation experiment are presented in Table 7. The amount of oil in the initial mixture
was 2.14 kg which was reduced to 0.94 kg at the end of the experiment resulting in a bioreme-
 In-Vessel Bioremediation 323

Table 7
Biodegradation efficiency

Item Value
Initial oil concnetration in the initial mixture (% w/w) 15.61
Amount of oil in the initial mixture (Kg) 02.14
Oil concentration in the final mixture (%w/w) 09.20
Amount of oil in the final mixture (Kg) 00.94

Bioremediation efficiency (%) 56.07

Table 8
Various hydrocarbons found in the motor oil

Boiling Point (°C) Weight Fraction (%) Hydrocarbons

200–300 02.0 Hexadecane (C 16 H 34 )
Heptadecane (C 17H 36 )
300–400 08.0 Octadecane (C 18H 38 )
Nonane (C19H 40 )
Eicosane (C20H 42 )
400–500 10.0 Heneicosane (C 21 H 44 )
Docosane (C 22H 46 )
Tricosane (C 23 H 48 )
Tetracosane (C 24H 50 )
500–1000 80.0 Pentacosane (C 25 H 52 )
Hexacosane (C 26 H 54 )
Heptacosane (C 27 H 56 )
Octacosane (C 28 H 58 )
Nonacosane (C29H 60 )
Triacosane (C 30H 62 )
Dotriacosane(C31H 64 )

* according to the ASTM Method D 5134 (Modified ) Distillation.

diation efficiency of 56.07%. This is much higher than the 20-25% achieved by CAPP (1994)
in 4 months and is comparable to that of 50-70% achieved by Jack et al. (1994) in 6 months.
The rate of biodegradation of hydrocarbons is inversely proportional to the compounds
molecular weight (Matthews, 1995). Also, hydrocarbons with rings or many branches are slower
to biodegrade (Environmental Protection Agency (EPA), 1993). The motor oil used in this study
contains several hydrocarbons with higher densities as shown in Table 8. The bioremediation
efficiency of only 56.07% could be partly due to the lack of a nitrogen source and partly due to
the oil containing mostly heavy molecular weight hydrocarbons (C12H12 - C31H 64 ).
However, the results obtained in this study illustrate the potential of peat utilization in the
remediation of oil contaminated water and the bioremediation of oil. Peat seems to play the
role of an active agent for biological degradation, thereby enhancing the biodegradative
processes of hydrocarbons. The final weight of the material was 10.25 Kg (Table 9). The
reduction of 3.46 Kg was due to: a) the breakdown of oil (1.20 Kg) to CO2 and H 2O owing to
324 A. E. Ghaly and J. B. Pyke

Table 9
Mixture Weight Reduction

Item Value
Initial weight of mixture (Kg) 13.71
Final weight of mixture (Kg) 10.25
Weight reduction 03.46
— Reduction due to oil degradation (Kg) 01.07
— Reduction due to respiration and evaporation (Kg) 02.39

the bioremediation process and b) the respiration of the organic material (peat and poultry
manure) and the evaporation of some water from the mixture, both amounted to 2.26 Kg.

Conclusions
The kiln dried peat is an excellent absorbent for extracting oil from water. It has the ability to
remove 99.998% of the oil from contaminated water having an oil slick of 1.3 cm in depth.
Mixing the peat with the contaminated water allowed for the coagulation of the peat with oil,
and thus increased the oil removal efficiency, but also increased the moisture content of the
peat. The final absorption ratio was 1:1 (oil/water:peat ) as compared to the 7:1 ratio given by
the manufacturer. The use of a surfactant to break the oil slick may result in a higher absorp-
tion ratio and thus reduce the amount of peat used.
Evidence of three microbial populations (psychrophilic, mesophilic, and thermophilic) in
the bioreactor demonstrated the potential for the refinement of the in-vessel bioremediation
process to achieve a much higher oil degradation efficiency at a lower cost compared to biopil-
ing and land farming. The worming of the material in the bioreactor and the increased mois-
ture content were signs of the conversion of complex organic carbon by these microbes into
energy, CO 2 and H2O. Higher temperatures caused by mesophilic and thermophilic activities
resulted in a higher conversion rate.
The moisture content, pH and the aeration rate were within the optimum range for the
bioremediation process, and the poultry manure provided all the required micronutrients for
microbial growth. However, the sudden decrease in the temperature indicated that the nitro-
gen was the limiting nutrient in the bioremediation process. The C:N ratio should be main-
tained in a range of 25:1 to 35:1 by the continuous addition of ammonium nitrogen. The
bioremediation process achieved 56.07% reduction in oil content in about twelve days. Addi-
tion of a nitrogen source would have resulted in a much higher reduction percentage.
The volume of the material was reduced to approximately 75% of its original value and
the moisture content increased slightly (from 60.51 to 62.00% ). The original humic mixture
was reduced to a denser (but porous) material that looked like a garden soil with a dark brown
color and no oily odor.

References
APHA, 1995. Standard Methods for the Examination of Water and Wastewater. American
Public Health Association, Washington, D.C., U.S.A.
ASTM, 1983. Annual of ASTM Standard Methods American Society for Testing and Mate-
rials. Philadelphia, Pennsylvania, U.S.A.
 In-Vessel Bioremediation 325

ASTM, 1986. Annual of ASTM Standard Methods American Society for Testing and Mate-
rials. Philadelphia, Pennsylvania, U.S.A.
Autry, A. R. and G. M. Ellis. 1992. Bioremediation: An Effective Remedial Alternative for
Petroleum Hydrocarbon-Contaminated Soil. Environmental Progress , 11(4):318–323.
Baker, K. H. and D. S. Herson. 1994a. Bioremediation of Surface and Subsurface Soils. In:
Bioremediation. McGraw-Hill, New York.
Baker, K. H. and D. S. Herson. 1994b. Bioengineering of Soils and Ground Waters. In: Biore-
mediation. McGraw-Hill, New York.
Cacciatore, D. A. and M. A. McNeil. 1995. Principles of Soil Bioremediation, BioCycle
2(10):61–64.
Couillard, D. 1994. Use of Peat in Wastewater Treatment. Water Resources. 28(60):1261–1274.
Doerffer, J. W. 1992. Oil Spill in the Marine Environment. Pergamon Press, New York.
Environmental Protection Agency (United States). 1993. An Overview of Underground Stor-
age Tanks Remediation Options. USEPA Press, Washington, D.C.
CAPP, 1994. Bioremediation of Petroleum Hydrocarbons. BioReactor Project Newsletter (1),
Canadian Association of Petroleum Producers, Calgary, Alberta.
Ghaly, R. A., J. B. Pyke, A. E. Ghaly and V. I. Urgusal. 1998. Remediation of Diesel Conta-
minated Soil Using Peat, Energy Sources 21(9):785–800.
Jack T. R., M. M. Francis and L. G. Stehmeier. 1994. Disposal of Slop Oil and Sludges by
Biodegradation. Research in Microbiology , 145(1):49–52.
King, R. B., G. M. Long and J. K. Sheldon. 1992. Practical Environmental Bioremediation.
Lewis Publishers, Florida.
Martin, A. M. 1991. Biological Degradation of Wastes. Elsevier Applied Science Publishers,
London, U. K.
Matthews, J. E. 1995. Handbook of Bioremediation. Lewis Publishers, Florida.
Skinner, J. H. 1991. Bioremediation Research Issues. In: Environmental Science Research,
41(1): Environmental Biotechnology for Waste Treatment. Plenum Press: London, Eng-
land.
Viraraghavan, T. and G. N. Mathavan. 1989. Use of Peat in Wastewater Treatment. Sypo-
sium’87 Wetlands Peatlands, American Society of Agricultural Engineers, St. Joseph,
MI, 1(4):223–232.
Viraraghavan, T. and S. M. Rana. 1991. Use of Adsorption Models for the Design of Peat-
Based Onsite Systems. Proceedings of the Sixth National Symposium on Individual and
Small Community Sewage Systems: On-site Wastewater Treatment, American Society of
Agricultural Engineers, St. Joseph, MI, p. 165–172.
Walworth, J. L. and C. M. Reynolds. 1995. Bioremediation of a Petroleum-Contaminated
Cryic Soil: Effects of Phosphorous, Nitrogen, and Temperature. Journal of Soil Contam-
ination, 4(3):299–310.