7476
Ind. Eng. Chem. Res. 2008, 47, 7476–7482
Study of the First Isolated Fungus Capable of Heavy Crude Oil Biodesulfurization
Sarah Torkamani, Jalal Shayegan,* Soheila Yaghmaei, and Iran Alemzadeh
Chemical and Petroleum Engineering Department, Sharif Uni V ersity of Technology, Tehran, Iran
To meet stringent emission standards stipulated by regulatory agencies, the oil industry is required to bring
down the sulfur content in fuels. Oil supplies 38% of the worldwide energy, and as the light oil is limited and
meanwhile the energy demand is increasing, it is a must to use heavy crude oil and therefore desulfurize it
to meet environmental standards. As it is not feasible to desulfurize all the sulfur containing compounds of
heavy crude oil by the existing methods (such as hydro-desulfurization) we have focused on biodesulfurization
of heavy crude oil. We have isolated the first native fungus which has been identified as Stachybotrys sp. and
is able to remove sulfur and nitrogen from heavy crude oil selectively at 30 ° C. This fungus (labeled as WS4
with BBRC-9052 code) is able to desulfurize 76% and 64.8% of the sulfur content of heavy crude oil of
Soroush oil field and Kuhemond oil field (with the initial sulfur contents of 5 wt % and 7.6 wt %, respecticely)
in 72 and 144 h, respectively. We have studied the impacts of several parameters on heavy crude oil
biodesulfurization efficiency of WS4 fungus strain such as initial pH of the medium, water - oil ratio, and
number of spores in the suspension used for inoculation. A comparison between the heavy crude oil
biodesulfurization capability of WS4 fungus and the thermophilic BBRC-9016 bacterium, which is active at
45 ° C, has been conducted. The role of medium formulation on isolating a suitable desulfurizing microorganism
has been presented. This fungus strain has been isolated as a part of the heavy crude oil biodesulfurization
project initiated by Petroleum Engineering Development Company (PEDEC), a subsidiary of National Iranian
Oil Company.
Introduction
The benzothiophene (BTH)-specific pathway desulfurizes
BTH to 2-(2 -hydroxyphenyl)ethan 1-al (HPEal) and probably
The concentration of gases such as SO 2 , CH 4 , CO, CO 2 , and
inorganic sulfite. This pathway was identified in Gordonia sp.
NO X in the atmosphere has increased due to the primarily
strain 213E (NCIMB 40816).
14
cumulative effect of fossil feedstocks combustion. SO x is known
as air pollution and causes acid rains. 1,2
Heavy crude oils have
Some of the isolated microorganisms capable of sulfur
high heteroatom levels (such as N, S, O, Ni, V, and Fe). This
removal are listed in Table 1. They all have been isolated by
high concentration of heteroatoms makes their removal gen-
using DBT as the sulfur source during the isolation. The
erally much more difficult than from lighter crude oils. Hence,
substrates that they can desulfurize are also shown in Table 1.
most traditional refining practices have been found inadequate.
3
According to the table above, no work has been published
Today petroleum is treated by hydrodesulfurization (HDS)
on heavy crude oil biodesulfurization. Therefore, there is still
which is a catalytic process that converts organic sulfur to
a need to explore other microorganisms that may efficiently
hydrogen sulfide gas by reacting crude oil fractions with
biodesulfurize the heavy crude oil. According to published
hydrogen at pressures between 150 and 3000 lb/in.
2
and
reports, no biodesulfurizing fungus has ever been reported.
temperatures between 290 and 455 ° C, depending upon the feed
In this work, we have isolated the first fungus strain capable
level of desulfurization required and the catalyst used.
4 - 6
of efficient heavy crude oil biodesulfurization. Heavy crude oil
Although HDS can remove various types of sulfur compounds,
of the Soroush oil field (located in Persian Gulf, some 82 km
some types of heterocyclic sulfur compounds existing in
southwest of Kharg Island) and heavy crude oil of Kuhemond
petroleum cannot be removed.
7,8
Biotechnological processes
oil field (located near the Persian Gulf, 80 km southeast of
such as biodesulfurization can be considered either an alternative
Boushehr City) have been used as the sulfur source during all
or a complementary to the conventional oil refining technologies.
the isolation steps of this fungus. Four different media and
Furthermore, biodesulfurization may improve the conventional
various soil samples being contaminated continuously by oil
refining energetic efficiency and reduce its environmental
for at least 2 years have been used to increase the probability
impact. Also biodesulfurization has been considered not only
9
of isolating a suitable biodesulfurizing microorganism. The
for liquid fuels but for coal and gas processing as well.
10
In
contaminated soil samples have been used as the source of
literature, the model compound used to study deep liquid fuel
microorganisms. This newly isolated fungus strain desulfurizes
biodesulfurization is dibenzothiophene (DBT),
11 - 13
where vari-
the heavy crude oil of Soroush oil field and Kuhemond oil field
ous bacteria and yeasts have been isolated based on using DBT
selectively and with a high efficiency.
as the sulfur source during the isolation process. Two kinds of
desulfurization specific pathways have been recognized:
The DBT-specific pathway (4S pathway) desulfurizes DBT
Materials and Methods
to inorganic sulfite and 2-hydroxybiphenyl (HBP). This pathway
was originally identified in Rhodococcus erythropolis strain
Chemicals. DBT was purchased from Aldrich Chemical
IGTS8 (ATCC 53968).
14
Company (U.S.A.) to designate whether the isolated fungus
desulfurizes the complex sulfur containing compounds selec-
* To whom correspondence should be addressed. E-mail: shayegan@
tively or not. All other chemicals were of analytical grade and
sharif.edu.
commercially available.
10.1021/ie800494p CCC: $40.75
2008 American Chemical Society
Published on Web 08/28/2008
Ind. Eng. Chem. Res., Vol. 47, No. 19, 2008
7477
Table 1. Biodesulfurizing Microorganisms
microorganism
substrate
Sphingomonas sp. strain AD109
15
DBT
Rhodococcus sp. strain IGTS8 (ATCC 53968)
16
DBT
Rhodococcus erythropolis Ac-1514D
17
DBT
Rhodococcus ruber Ac-1513D
17
DBT
Gordonia sp. F.5.25.8
18
DBT
Paenibacillus sp. strain A11-2
19
DBT
Bacillus subtilis WU-S2B
20
DBT, alkylated DBTs
Mycobacterium phlei WU-F1
20
DBT, alkylated DBTs
21
strains UM9 and UM3 ( Rhodococcus )
DBT, dibenzosulfone
22
Rhodococcus erythropolis N1-36
DBT, dibenzosulfone
4
Rhodococcus sp. strain ECRD-1
4,6-DEDBT, sulfuric and thiophenic sulfur compounds, diesel range
fuels, Oregon Basin crude oil
Gordona sp. strain 213E (NCIMB 40816)
23
BTH
Rhodococcus sp. strain T09
24
BTH
Sinorhizobium sp. KT55
25
BTH
Corynebacterium ZD-1
26
BTH
27
Rhodococcus sp. strain P32C1
DBT, model oil (DBT in hexadecane), diesel oil
Mycobacterium phlei WU-0103
4
DBT, model oil (DBT in hexadecane), diesel oil
21
Nocardia sp. CYKS2 (KCTC 0432 Br)
DBT, petroleum, coal, diesel oil
Rhodococcus sp. strain WU-K2R
28
NTH
Strains DBVPG 6662 and DBVPG 6739 of Rhodosporidium toruloides
DBT, BTH, a large variety of thiophenic compounds
29
Mycobacterium phlei GTIS10
30
DBT, BTH, a large variety of thiophenic compounds
Rhodococcus sp. KT462
27
BTH, DBT
Gordona sp. CYKS1
28
model oil
Rhodococcus erythropolis , R. erythropolis XP
17
BTH, diesel oil, crude oil
Sphingomonas subarctica T7b
33
light gas oil (LGO), 4,6-dibutyl DBT, 4,6-dipentyl DBT
Rhodococcus erythropolis 1awq
34
hydro-desulfurized diesel oil
Mycobacterium goodii X7B
35
gasoline, diesel oil
Rhodococcus erythropolis XP
36
fluid catalytic cracking (FCC), straight-run (SR) gasoline oils
Isolation and Cultivation of Heavy Crude Oil Biodesul-
sample. Cell cultivation was carried out at 30 ° C on a rotary
furizing Microorganisms. Four different media were used for
shaker operated at 150 rpm. Potato dextrose agar (PDA) was
isolation to increase the probability of isolating a suitable
used as the solid medium for fungi isolation from turbid cultures
biodesulfurizing microorganism. Various soil samples being
and nutrient agar (NA) was used as the solid medium for bacteria
contaminated continuously by oil for at least 2 years have been
isolation.
collected from evaporation ponds of Tehran refinery and
Analytical Methods. Sulfur content of heavy crude oil was
Kuhemond oil field. The mentioned contaminated soil samples
determined by a Horiba (Japan) sulfur-in-oil analyzer SLFA-
contain useful microorganisms. By using these different media
2800 using the X-ray fluorescence technique. In 1967 X-ray
and different soil samples, we have isolated various types of
fluorescence was approved as an ASTM standard method for
microorganisms and compared their sulfur removal efficiency
the determination of sulfur.
38
ASTM D5291 Standard Test
to choose the best one. It was observed that one of the isolated
Method has been used to determine the total carbon, nitrogen,
fungus strains (labeled WS4) was the best biodesulfurizing
and hydrogen content of Soroush heavy crude oil. The analyzer
isolated microorganism. As most of the isolated microorganisms
is Foss Heraeus CHN-O-RAPID (Germany). Research Institute
capable of sulfur removal from DBT and its derivatives are
of Petroleum Industry (RIPI) has conducted the two described
bacteria of Rhodococcus sp. (this can be seen in Table 1), we
analyses. Iranian Organization of Science and Technology
have applied four media which were proper for Rhodococcus
(IROST) and Tarbiat Modares University are using the mor-
growth. These media formulations have been chosen from
phological interpretation and biochemical and molecular tech-
Handbook of Microbiological Media
37
and been modified for
niques to identify the isolated fungus strain.
becoming proper for biodesulfurizing microorganisms. The
Heavy Crude Oil. Heavy crude oil of Soroush oil field and
modifications have been conducted by omitting their various
Kuhemond oil field have been used as the sole sulfur source
sulfur sources such as sulfates, yeast extract, and so forth.
during the isolation process. Heavy crude oil of Soroush oil
The formulations of the mentioned media are as follows:
field has an API gravity of 15.5 and sulfur content of 5 wt %.
Medium no. 1 contains 5 g of glucose, 0.4 g of KH 2 PO 4 ,
Heavy crude oil of Kuhemond oil field is known as one of the
1.6 g of K 2 HPO 4 , 1.5 g of NH 4 Cl, 0.17 g of MgCl 2 · 6H 2 O,
heaviest Iranian oils with the API gravity of 14 and sulfur
0.09 g of CaCl 2 · 2H 2 O, and 50 mg of Na 2 WO 4 in 1 L of distilled
content of 7.6 wt %. These heavy crude oils have been kindly
water.
provided by Petroleum Engineering Development Company
Medium no. 2 contains 2 g of NH 4 NO 3 , 2 g of KH 2 PO 4 , 3 g
(PEDEC).
of Na 2 HPO 4 , 0.2 g of NaCl, 0.02 g of CaCl 2 , and 10 mL of
Selective Sulfur Removal. It is important that the biodes-
n -hexadecane in 1 L of distilled water.
ulfurizing microorganism does not use the carbon of the sulfur
Medium no. 3 contains 20 g of sucrose, 0.8 g of K 2 HPO 4 ,
containing compounds. In other words, the isolated fungus strain
0.2 g of KH 2 PO 4 , 0.001 g of FeCl 3 , and 0.001 g of Na 2 -
should have the ability to use the sulfur of oil without using its
MoO 4 · 2H 2 O in 1 L of distilled water.
carbon. This is called selective sulfur removal. This character-
Medium no. 4 contains 5 g of glucose, 5 g of CaCO 3 , 1 g of
istic is checked by conducting one of the three tests explained
K 2 HPO 4 , and 1 g of NaCl in 1 L of distilled water.
below:
Cultivation was carried out in 500 mL flasks containing 100
(a) The carbon source must be omitted from the medium,
mL of medium, 5 mL of heavy crude oil, and 0.5 g of soil
and instead DBT must be added to it to be both the sulfur and
7478
Ind. Eng. Chem. Res., Vol. 47, No. 19, 2008
Table 2. Isolated Microorganisms by Applying Heavy Crude Oil of Kuhemond Oil Field as the Sulfur Source
sulfur removal efficiency
code of
applied medium for
applied source
from heavy crude oil of
a
type
microorganism
isolation
for isolation
Kuhemond oil field (%)
bacteria
b-MK2-2
medium no. 1
soil samples collected from
43.4
b-AK1-2
medium no. 3
evaporation ponds of
51.3
b-MK3
medium no. 1
Tehran refinery
40.7
b-AK4
medium no. 3
soil sample collected from
20
b-RK4
medium no. 2
Kuhemond oil field
27.6
b-MK4
medium no. 1
43.4
fungi
WK2
medium no. 4
soil sample collected from
40.8
WK4
medium no. 4
Kuhemond oil field
26.3
a
Each code represents one strain.
the carbon source for the isolated fungus. If the fungus strain
flasks (with 500 mL volume) containing 100 mL of medium
does not grow, it means that it does not use the carbon of DBT
and 10 mL of heavy crude oil.
(which is a model compound). Therefore, it desulfurizes
complex sulfur containing compounds selectively.
Experimental Conditions
(b) The carbon source must be omitted from the medium,
and instead heavy crude oil must be added to it to be both the
Heavy Crude Oil Biodesulfurization Conditions. WS4
sulfur and the carbon source for the isolated fungus. If the fungus
fungus strain was grown aerobically in 500 mL flasks while 10
mL of suspension of 1.3 × 10 7 spores/mL in serum (which
strain does not grow, it means that the fungus strain does not
contains 9 g NaCl in 1 L of distilled water) was inoculated into
use the carbon of heavy crude oil and, therefore, is able to
100 mL of medium containg 10 mL of heavy crude oil and
desulfurize it selectively.
then incubated on a rotary shaker at 30 ° C at 150 rpm for 6
(c) The carbon, hydrogen, and nitrogen contents of the heavy
days. Shaking makes it possible that the fungus in the medium
oil have to be measured before and after the biodesulfurization
be more in contact with the oil and even be attached to the oil.
process to make sure that the carbon of oil has not been used
The remaining sulfur content of heavy crude oil and pH of the
by the fungus strain and the fungus strain removes the sulfur
medium were analyzed and examined every day. Heavy crude
from oil selectively.
oil was mechanically separated from the medium and has been
We have performed all the three tests explained above to
sent to Research Institute of Petroleum Industry (RIPI) for
make sure that the biodesulfurizing process has been done
determination of total sulfur content.
selectively.
Impact of Initial pH of the Medium on Fungus Biodesul-
furization Efficiency. pH of the medium is equivalent to 7.64.
Bacterial Strain. We planned to compare the sulfur removal
We have set its pH to 6.5 by adding HCl and 9, 10, and 12 by
ability of our isolated biodesulfurizing microorganism with that
adding NaOH and then monitoring the fungus growth and the
of a bacterium, which has been isolated by using DBT as the
medium pH for 6 days. We analyzed the remaining sulfur
sulfur source. The thermophilic BBRC-9016 bacterium strain
39
content of heavy crude oil of Soroush oil field after 6 days to
that has been isolated in Sharif University of Technology in
determine the impact of initial pH of the medium on fungus
2006 belongs to a microbial collection of Biochemical and
biodesulfurization efficiency.
Bioenvironmental Research Center of Sharif University of
Impact of Water - Oil Ratio on Fungus Biodesul-
Technology. We chose this bacterium and measured its sulfur
furization Efficiency. The volume ratio of heavy crude oil of
removal efficiency from heavy crude oil of Soroush oil field
Soroush oil field to medium was set to be 1/20, 1/10, 1/5, 2/5,
and heavy crude oil of Kuhemond oil field by BBRC-9016
and 3/5. Sulfur content of heavy crude oil of Soroush oil field
strain. This bacterium is active at 45 ° C in a medium containing
was analyzed after 6 days and pH was examined every day.
4 g of K 2 HPO 4 , 1 g of NH 4 Cl, 0.5 g of KH 2 PO 4 , 0.2 g of
Impact of Number of Spores per mL of Suspension
MgCl 2 · 6H 2 O, 0.02 g of CaCl 2 , 0.01 g of NaCl, 10 mL of metal
Used for Inoculation on Fungus Biodesulfurization Effi-
solution, and 1 L of distilled water. The metal solution contains
ciency. Various suspensions of spores in serum were prepared:
0.1 g of Na 2 MoO 4 , 0.5 g of FeCl 2 · 4H 2 O, 0.5 g of ZnCl 2 , 0.5 g
containing 1.3 × 10 7 , 1.6 × 10 7 , 1.5 × 10 6 , and 6.8 × 10 5
of MnCl 2 · 4H 2 O, 0.05 g of CuCl 2 , 0.05 mg of Na 2 WO 4 , and 1
spores/mL. Sulfur content of heavy crude oil of Soroush oil
L of water. The maximum specific DBT desulfurization rate
field and Kuhemond oil field was analyzed after 6 days, and
for BBRC-9016 was calculated and is equivalent to 0.04 mmol
pH was examined every day.
DCW h
- 1
DBT g
- 1
which occurs at logarithmic growth phase
while following the 4S pathway.
Results and Discussion
Bacterial Cells Preparation for Heavy Oil experiments.
Effects of Medium Formulation and Sulfur Source on
We have added the isolated bacterium by a sterile loop from
Microorganism Isolation. Four media and two different heavy
its slant to 100 mL of nutrient broth. This preculture was shaken
crude oils were applied to isolate a proper microorganism
on a rotary shaker at 150 rpm and temperature of 60 ° C. After
capable of heavy crude oil biodesulfurization. Various bacteria
24 h it was observed that the bacterium has grown in nutrient
and fungi have been isolated which are listed in Tables 2
broth because its optical density was determined and was
and 3.
equivalent to 0.9. We added a small amount of this preculture
The isolated microorganisms (listed in Tables 2 and 3)
to 100 mL of fresh nutrient broth to set its optical density equal
use the sulfur content of heavy crude oil while they are growing
to 0.2. Finally we have added 10 mL of this subculture to the
in the medium and, therefore, remove the sulfur from oil. The
Ind. Eng. Chem. Res., Vol. 47, No. 19, 2008
7479
Table 3. Isolated Microorganisms by Applying Heavy Crude Oil of Soroush Oil Field as the Sulfur Source
sulfur removal efficiency
code of
applied medium for
applied source
from heavy crude oil of
a
type
microorganism
isolation
for isolation
Soroush oil field (%)
bacteria
b-MS1-1
medium no. 1
soil samples collected from
76
b-MS1-2
medium no. 1
evaporation ponds of
52
b-AS2
medium no. 3
Tehran refinery
72
b-AS3-1-a
medium no. 3
75
b-AS3-1-b
medium no. 3
74
b-RS2
medium no. 2
40
fungi
WS4
medium no. 4
soil sample collected from
76
Kuhemond oil field
a
Each code represents one strain.
best isolated microorganism by using the heavy crude oil of
Kuhemond oil field as the sulfur source is the b-AK1-2
bacterium which removes sulfur from the Kuhemond heavy
crude oil with an efficiency of 51.3% in 6 days. The best isolated
microorganisms by using the heavy crude oil of Soroush oil
field as the sulfur source are the b-MS1-1 bacterium and WS4
fungus strain both removing sulfur from the Soroush heavy
crude oil with a 76% sulfur removal efficiency. According to
the sulfur removal analysis, the b-MS1-1 bacterium requires 6
days to remove sulfur from the Soroush heavy crude oil with a
76% sulfur removal efficiency while it takes the WS4 fungus
strain only 3 days to achieve this efficiency. Another important
point is the WS4 fungus strain and b-MS1-1 bacterium efficiency
Figure 1. Sulfur removal from heavy crude oil of Soroush oil field by WS4
in removing sulfur from Kuhemond heavy crude oil, which is
fungus strain and the resulting pH change.
64.8% and 42.1%, respectively (it takes both of them 6 days to
achieve this efficiency).
Hence, the WS4 fungus strain is considered a more proper
biodesulfurizing microorganism in comparison to b-MS1-1
bacterium and b-AK1-2 bacterium. Although the WS4 fungus
strain has been isolated by Soroush heavy crude oil as its sulfur
source, it shows a higher sulfur removal efficiency from
Kuhemond heavy crude oil in comparison to that of b-AK1-2
bacterium which had been isolated by Kuhemond heavy crude
oil. Therefore the kind of sulfur source during the isolation has
a great role in isolating the suitable biodesulfurizing microor-
ganism.
It has been observed that three fungi strains have been isolated
which are labeled as WS4 fungus, WK2 fungus, and WK4
Figure 2. Sulfur removal from heavy crude oil of Kuhemond oil field by
WS4 fungus strain and the resulting pH change.
fungus. As can bee seen in Tables 1 and 2, the applied medium
during their isolation had been medium no. 4. Therefore, it can
be concluded that medium no. 4 has a proper formulation for
fungi growth and, hence, for isolating biodesulfurizing fungi.
Iranian Organization of Science and Technology (IROST)
and Tarbiat Modares University are performing the identification
of WS4 fungus strain, and by now, it has been determined that
its genius is Stachybotrys . It is not possible to measure the
biomass of WS4 fungus strain, as part of the biomass is attached
to heavy crude oil and inseparable. According to observations,
the WS4 fungus strain grows pretty well by using the sulfur
content of heavy crude oil.
Heavy Crude Oil Biodesulfurization. As can be observed
in Figure 1, WS4 fungus strain decreases the sulfur content of
Figure 3. Impact of initial pH of the medium on sulfur removal efficiency
heavy crude oil of Soroush oil field from 5 wt % to 1.2 wt %
from heavy crude oil of Soroush oil field by WS4 fungus strain.
(in other words, its sulfur removal efficiency is equivalent to
fungus strain desulfurizes the heavy crude oil of Soroush oil
76%) in 72 h while the pH of the medium increases from 7.64
field faster and with a higher efficiency in comparison to that
to 8.19.
of Kuhemond oil field. It is because the sulfur content of heavy
As can be observed in Figure 2, WS4 fungus strain decreases
crude oil of Soroush oil field is much less than that of Kuhemond
the sulfur content of heavy crude oil of Kuhemond oil field
oil field. The other reason might be that heavy crude oil of the
from 7.6 wt % to 2.7 wt % (in other words its removes 64.8%
Kuhemond oil field is heavier than that of Soroush oil field.
of the Kuhemond heavy crude oil) in 144 h while the pH of the
Hence, Kuhemond heavy crude oil might have more sulfur
medium increases from 7.64 to 8.09. As is observed the WS4
containing compounds with a complex structure, which are not
7480
Ind. Eng. Chem. Res., Vol. 47, No. 19, 2008
Figure 8. Impact of number of spores per milliliter of suspension used for
inoculation on fungus biodesulfurization efficiency.
Figure 4. pH change of the medium for various cases having different initial
pH values.
Figure 9. pH change during Soroush heavy crude oil biodesulfurization
while having various numbers of spores per milliliter of suspension used
for inoculation.
Figure 5. Impact of oil - water ratio on WS4 fungus biodesulfurization
efficiency.
Figure 10. Heavy crude oil of Soroush oil field biodesulfurization efficiency
of WS4 fungus strain and BBRC-9016 bacterium.
Figure 6. pH change for various oil - water ratios.
Figure 11. Heavy crude oil of Kuhemod oil field biodesulfurization
efficiency of WS4 fungus strain and BBRC-9016 bacterium.
Figure 7. Impact of number of spores per milliliter of suspension used for
inoculation on fungus biodesulfurization efficiency.
lacks a nitrogen source, this fungus uses the nitrogen of heavy
crude oil for its growth. The nitrogen removal is being done
easily biodesulfurized. It was observed, by increasing the
selectively (which means that the WS4 fungus strain does not
aeration volume, that the heavy oil has became more dispersed
use the carbon of Soroush heavy crude oil). According to
which might be the result of emulsifier production by the fungus.
published reports, the sulfur removed selectively would appear
Selective Sulfur Removal. It was observed that the WS4
in the form of sulfite in the medium while the nitrogen removed
fungus strain is able to remove sulfur from heavy crude oil and
selectively would appear in the form of ammonia in the medium,
so the biodesulfurizing microorganism can use them easily.
40,41
DBT selectively. According to CHN test results, the initial
nitrogen content of Soroush heavy crude oil is equal to 1 wt %,
Hence, selective nitrogen removal would lead to increase of
and WS4 fungus strain removes 50% of it in 6 days without
pH of the medium while the selective sulfur removal would
using the carbon of oil. As the medium of WS4 fungus strain
lead to decrease of the pH of the medium. When both selective
Ind. Eng. Chem. Res., Vol. 47, No. 19, 2008
7481
sulfur and nitrogen removal happens, the final pH depends on
As can be seen in Figures 10 and 11, WS4 fungus strain has
the amount of the produced sulfite and ammonia.
higher sulfur removal efficiency in comparison to BBRC-9016
Impact of Initial pH of the Medium on Fungus Biodesul-
bacterium. The heavy crude oil of Soroush oil field and
furization Efficiency. Sulfur removal efficiency of heavy crude
Kuhemnod oil field have been used to perform this comparison.
oil of Soroush oil field by WS4 fungus strain decreases by
The higher sulfur removal efficiency of WS4 fungus strain
increasing the initial pH of the medium. It is shown in Figure
indicates that the type of sulfur source during the isolation
3. The pH change of the medium will lead to a final pH of
process is of great importance. The sulfur source during the
nearly 8 after 6 days no matter what the initial pH is. Only
isolation of WS4 fungous and BBRC-9016 bacterium has been
when the initial pH is too high, WS4 fungus strain cannot be
Soroush heavy crude oil and DBT, respectively. There is various
active and the resulting pH change is negligible which can be
sulfur containing compounds in heavy crude oil. The ability of
observed in Figure 4.
a microorganism to desulfurize DBT does not ensure that the
As is shown in Figure 4, the WS4 fungus strain is able to
microorganisms is able to desulfurize other types of sulfur
grow and biodesulfurize the heavy oil in a wide range of pH
containing compounds too.
values, and the best pH range is from 6.5 to 8.2. The pH suitable
for the growth of a fungus is correlated with the pH of the soil
Conclusion
from which the fungus is isolated.
42
According to the fact that
the pH of the soil from which the WS4 fungus strain is isolated,
The following conclusions have been achieved:
equal to 7.4, it is reasonable that the best pH range for growth
(i) WS4 fungus strain is isolated fungus for the first time
and therefore biodesulfurization activity of WS4 fungus strain
which is capable of heavy crude oil biodesulfurization. Its
is from 6.5 to 8.2. According to Figure 4, while the initial pH
selective sulfur removal makes it proper for the industrial use.
of the medium is set to be 6.5 and 7.64, the pH increases which
(ii) WS4 fungus strain removes sulfur from crude oil,
indicates that not only the sulfur removal occurs but also the
therefore it can be considered for upstream sulfur removal.
nitrogen has been removed and the produced sulfite and
(ii) It is able to be active at neutral and higher pH because
ammonia has totally increased the pH. But when the initial pH
the pH of the soil from which it is been isolated is almost neutral.
is equal to 9, 10, and 12, we can observe that pH decreases
(iv) WS4 fungus strain is able to desulfurize the sulfur content
which indicates that nitrogen removal has not been done in large
of heavy crude oil of Soroush oil field and Heavy crude oil of
amounts and is a sign of its less gowth and less activity at very
Kuhemond oil field with efficiencies of 76% and 64.8%
high pH.
respectively in 3 and 6 days.
(v) WS4 fungus strain desulfurizes the Soroush oil faster and
Impact of Oil - Water Ratio on WS4 Fungus Biodesul-
with a higher efficiency in comparison to Kuhemond heavy oil
furization Efficiency. Sulfur removal efficiency of this newly
because this oil contains much less sulfur and is lighter (which
isolated fungus depends on the oil - water ratio as shown in
means that has less complex sulfur containing compounds) in
Figure 5. Sulfur removal efficiency decreases by increasing the
comparison to Kuhemond heavy crude oil.
oil - water ratio. In addition, the final pH is about 8, no matter
(vi) WS4 fungus strain has higher sulfur removal efficiency
what the oil - water ratio is (Figure 6).
from heavy crude oil in comparison to thermophilic BBRC-
Increasing the oil + water ratio would decrease the biodes-
9016 bacterium, because of the difference between the sulfur
ulfurizing efficiency as the fungus strain uses the sulfur content
sources being used during their isolation.
of heavy crude oil for growth and, therefore, requires a limited
(vii) The best oil - water ratios for WS4 fungus were found
amount of sulfur.
to be 1/10 and 1/20, and as there was not much difference
Impact of Number of Spores per Milliliter of Suspen-
between the sulfur removal efficiency at these two conditions,
sion used for Inoculation on Fungus Biodesulfurization
the 1/10 ratio is recommended for the industrial study.
Efficiency. If the suspension used for inoculation contains a
(viii) Number of spores per milliliter of suspension used for
greater number of spores per milliliter, the sulfur removal
inoculation and the initial pH of the medium have important
efficiency will increase. It must be noticed that the optimum
roles in the sulfur removal efficiency of the WS4 fungus strain.
number of spores per milliliter is equal to 1.3 × 10 7 . As the
(ix) The capability of this fungus to remove nitrogen
sulfur is used for the WS4 fungus strain growth, a small number
selectively from heavy crude oil makes it more suitable for
of spores requires less sulfur and a very large number of spores
industrial application.
would make them compete for gaining sulfur and other required
(x) Using various media during the isolation process increases
nutrients. This means that a very small or very large number of
the probability of isolating a suitable biodesulfurizing microor-
spores would lead to less sulfur removal efficiency. It is shown
ganism.
in Figures 7 and 8. The final pH is also about 8 for any number
of spores per milliliter which is shown in Figure 9.
Supporting Information Available: Additional details and
According to Figure 9 when the number of spores per
photographs. This information is available free of charge via
milliliter is equal to 1.3 × 10 7 the pH is higher in comparison
the Internet at http://pubs.acs.org.
to the case when the number of spores per milliliter is equal to
6.8 × 10 5 which indicates that the nitrogen removal efficiency
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