Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 74:1000±1004 (1999)

Short communication
Sulfur specificity in the bench-scale biological
desulfurization of crude oil by Rhodococcus
IGTS8†
Eric N Kaufman,1* Abhijeet P Borole,1 Robert Shong,2 Jerry L Sides2 and
Cliff Juengst3
1
Bioprocessing Research and Development Center, Chemical Technology Division, Oak Ridge National Laboratory, Oak Ridge,
Tennessee 37831-6226, USA
2
E&P Technology Department, Texaco, 3901 Briarpark, Houston, TX 77042, USA
3
UNOCAL Corporation, 14141 Southwest Freeway, Sugar Land, TX 77478, USA

Abstract: Biological removal of organic sulfur from petroleum feedstocks may offer an attractive
alternative to conventional thermochemical treatment due to the mild operating conditions and
greater reaction speci®city afforded by the nature of biocatalysis. Previous investigations have either
reported the desulfurization of model sulfur compounds in organic solvents or gross desulfurization of
crude oil without data on which sulfur species were being removed. This study reports initial sulfur
speciation data for thiophenic sulfur compounds present in crude oil which may be used as a guide both
as to which species are treated by the biocatalyst investigated as well as to where biocatalyst
development is needed to improve the extent of biological desulfurization when applied to whole
crudes. Biodesulfurization of two different crude oils in the 22±31 ° API speci®c gravity range with total
sulfur contents between 1 and 2% is demonstrated in 1-dm3 batch stirred reactors using wild type
Rhodococcus sp IGTS8. While analysis of the crudes before and after biodesulfurization did not reveal
a decrease in total sulfur, GC±MS did reveal signi®cant (43±99%) desulfurization of dibenzothiophenes
(DBT) and substituted DBTs. Fractionation of the whole crude, followed by analysis using gas
chromatography±sulfur chemiluminescence detection (GC±SCD) of the aromatic fraction of the Van
Texas crude oil, demonstrated a reduction of sulfur in this fraction from 3.8% to 3.2%. This research
indicates that IGTS8 may be capable of biodesulfurization of re®ned products such as gasoline and
diesel whose predominant sulfur species are dibenzothiophenes. Further biocatalyst development
would be needed for effective treatment of the spectrum of sulfur-bearing compounds present in whole
crudes.

Keywords: oil desulfurization; Rhodococcus; IGTS8; dibenzothiophene; biodesulfurization; sulfur speciation

INTRODUCTION model sulfur compounds such as dibenzothiophene,

Biological re®ning of fossil fuel feedstocks may offer an
attractive alternative to conventional thermochemical
treatment due to the mild operating conditions and
greater reaction speci®city afforded by the nature of
biocatalysis. Efforts in microbial screening and
development have identi®ed microorganisms capable
of model compound desulfurization (see for
example1±10), as well as petroleum desulfuriza-
tion,11,12 denitri®cation,12 demetalization,12 crack-
ing12,13 and dewaxing. Further investigation using
and manipulation of enzymatic pathways responsible
for these reactions6,14±17 has led to processes which are
approaching commercial application, particularly in
biological desulfurization of diesel whose primary
sulfur compound is dibenzothiophene (DBT).18,19
Dibenzothiophenes and substituted dibenzothio-
phenes are some of the most recalcitrant organic sulfur
compounds relative to hydrodesulfurization of crude
oil and re®ned transportation fuels.18 DBT has been
used as a sulfur model compound for over a decade of

* Correspondence to: Eric N Kaufman, Bioprocessing Research and Development Center, Chemical Technology Division, Oak Ridge
National Laboratory, Oak Ridge, Tennessee 37831-6226, USA
E-mail: ekn@ornl.gov
†
This article is a US Government work and is in the public domain in the United States
Contract/grant sponsor: Office of Oil and Gas Processing, US Department of Energy; contract/grant number: DE-AC05-96OR22464
Contract/grant sponsor: Lockheed Martin Energy Research Corp
(Received 16 February 1999; revised version received 2 June 1999; accepted 3 June 1999)

# 1999 Society of Chemical Industry. J Chem Technol Biotechnol 0268±2575/99/$17.50 1000


Figure 1. Biochemical pathway showing the sequence of DBT oxidation by
IGTS8.
research. DBT desulfurization by the `4S' pathway as
performed by Rhodococcus IGTS820,21 conserves hy-
drocarbon value as opposed to desulfurization via
mineralization (see Fig 1). In this mechanism, sulfur is
oxidized and removed from the petroleum as sulfate
which partitions into the biocatalyst-containing aque-
ous phase. IGTS8 and related biocatalysts have been
shown to be capable of desulfurization of DBT,
substituted DBTs, and other sulfur species in model
hydrocarbon systems.3,4,22±24 There have been
published reports of diesel24 and whole crude oil
desulfurization.10±12,19,25 However, most reports of
crude biodesulfurization investigate only the total
sulfur content of the crude before and after biodesul-
furization, and do not reveal sulfur speciation data
which may guide further biocatalyst development. The
one study in which known sulfur species or `markers'
are used as internal standards utilized a proprietary
biocatalyst which is not available for further research
and publication.19 The present study utilizes the
widely available wild type Rhodococcus IGTS8 and
reports initial data revealing that while this biocatalyst
is capable of desulfurizing DBT related compounds, it
does little to reduce the total sulfur content of the
crude oil.
EXPERIMENTAL PROCEDURE
Crude oil samples, `Sand Flat' and `Van Texas', were
obtained via Texaco and UNOCAL respectively. Both
crudes have an API speci®c gravity of 22±31 °, with the
Sand Flat being 2.0% (w/w) and the Van Texas 0.96%
(w/w) sulfur. In both of these oils, volatiles were not
present due to elevated temperature production
(99 °C) of the wells. Experiments were performed
J Chem Technol Biotechnol 74:1000±1004 (1999)
Biodesulfurization of crude oil
in batch stirred reactors utilizing 50 g of frozen
Rhodococcus sp wild type strain IGTS8 (ATCC
53968) cell paste which were brought up to 750 cm3
with 0.156 mol dmÿ3 (pH 7.5) potassium phosphate
buffer. Cells were supplied as a frozen paste and had a
cell dry weight of 0.28 g gÿ1 of original frozen material.
These cells exhibited a speci®c activity typically
between 1 and 5 mg DBT oxidized dry gÿ1 of bio-
catalyst hÿ1 when DBT served as the sole substrate in a
buffer/hexadecane system.26 The cells were suspended
in the phosphate buffer prior to addition to the reactor.
The reactor vessel used was a 1-dm3 VirTis Omni-
Culture fermenter (model 178657, Gardiner, NY),
utilizing a six-bladed Rushton-type impeller with two
baf¯es. The reactor was kept at 30 °C, agitated at 800
rpm, and aerated with room air at a rate of 0.2
standard dm3 minÿ1. A water condenser was used on
each reactor to capture volatiles which were expected
to be minimal or non-existent considering the fact that
the operating temperature was much less than that of
the oil reservoir. In each experiment, 250 cm3 of crude
oil was treated with 750 m3 of the aqueous phase.
Samples (30 cm3 from the top of the organic phase)
taken during the course of biological treatment were
collected after ceasing the agitation and aeration for
5 min to allow the aqueous and organic phases to
separate. These samples and oil remaining in the
reactor at the end of the run were centrifuged at 6000
rpm in a Beckman Model TJ-6 centrifuge to dewater
the oil. Samples were then boiled in a closed container
for 30 min to halt biological activity.
Two sets of experiments were conducted. The ®rst
was conducted with a treatment time of 48 h on both
oils investigated. A control was run during this
experiment which contained no biocatalyst. Surfactant
(0.2 cm3 of Petrolite MU2000) was added to abiotic
control experiments to mimic the surfactant released
by the microorganisms. Without a surfactant present
(either released by the biocatalyst or supplied as
MU2000), buffer salts stabilized the asphaltene
groups in the oil and resulted in a thick paste that
could not be moved by the reactor impellers. Total
sulfur analysis was conducted using a Leco SC432
(Englewood, CO) on samples collected at the begin-
ning of the run and after 48 h of treatment. In addition,
individual sulfur species were analyzed by gas chroma-
tography±mass spectrometry (GC±MS). Where un-
ambiguous mass/charge data are available, GC±MS
analysis allows quanti®cation of individual sulfur
species in the whole crude. DBT and eight methylated
derivatives were quanti®ed by GC±MS. The analysis
was performed using an HP5890 GC equipped with
electronic pressure control. A 30 m  0.25 mm ID
DB-5 column with 0.25 mm ®lm thickness was used.
Helium was used as a carrier gas at 1 cm3 minÿ1. The
temperature program included a holding time of 2 min
at 40 °C, followed by a ramp at 20 °C minÿ1 to 150 °C,
followed by a second ramp at 3 °C minÿ1 to 310 °C and
a ®nal holding time of 13 min. A MicroMass 70SE
mass spectrometer was coupled to the GC with a
1001
EN Kaufman et al

direct interface. The MS was operated at a resolving Table 1. GC–MS analysis of DBT and C1 and C2 substituted DBTs in Sand

power of 5000 and used selected ion monitoring Flat crude oil

(SIM). Dibenzothiophene was quanti®ed with IGTS8, 48 h, Abiotic control,

response factors derived from a DBT standard. The IGTS8, 0 h, ppm 48 h, ppm
concentrations of the methylated DBT derivatives Sulfur species ppm (% change) (% change)
were determined by employing d10-phenanthrene as
DBT 273.4 65.1 (ÿ76%) 266.5 (ÿ3%)
an internal standard. Unfractionated Sand Flat oil was
C1-DBT #1 181.5 21.3 (ÿ88%) 206.7 (‡14%)
also analyzed by gas chromatography±sulfur chemi-
C1-DBT #2 102.7 37.8 (ÿ63%) 123.3 (‡20%)
luminescence detection (GC±SCD) before and after C1-DBT #3 106.0 31.8 (ÿ70%) 118.9 (‡12%)
treatment as described below. C2-DBT #1 6.4 2.6 (ÿ59%) 6.3 (ÿ2%)
The second experiment was conducted only with C2-DBT #2 17.1 6.7 (ÿ61%) 21.3 (‡25%)
Van Texas oil with a treatment time of 6 days. A C2-DBT #3 7.3 4.1 (ÿ44%) 9.1 (‡25%)
GC±SCD method was used to determine the sulfur C2-DBT #4 24.2 7.2 (ÿ70%) 31.1 (‡29%)
content of the aromatic fraction of the oil. In C2-DBT #5 34.0 16.1 (ÿ53%) 42.2 (‡24%)
conventional gas chromatographic analysis of whole C2-DBT #6 10.7 6.1 (ÿ43%) 13.2 (‡23%)
oil, only a portion of the sample elutes from the C2-DBT #7 10.6 5.3 (ÿ50%) 11.9 (‡12%)
column. To allow facilitated observation of sulfur in
the treated oil, whole oil samples were fractionated
according to ASTM method D2007. An extended Tables 1 and 2). The control reactor for Sand Flat oil
ASTM D2887 procedure was used for chromato- which did not contain biocatalyst did not reveal such a
graphic separation of the aromatic fraction of the decrease in these sulfur species. The slight increase in
crude oil. The extended ASTM D2887 procedure is a sulfur species reported in the control reactor was not
gas chromatographic simulated distillation method to corroborated by GC±SCD analysis (Fig 2), and may
obtain comparable data to the ASTM D2892 physical be taken as an indication of sample variability in
distillation for crude oil. An AC (Analytical Controls correcting for the water content of the oil. The rate of
Inc, 3448 Progress Drive, Bensalem, PA 19020) conversion of the unsubstituted and C1 and C2
software on Hewlett-Packard Chemstation was used substituted DBT species measured (as listed in Tables
to control an HP 5890 GC and produce the simulated 1 and 2) after a period of 48 h, was 10.18 mg total
distillation reports. Sulfur analysis was performed by DBTs gÿ1 dry cell mass for the Sand Flat crude oil and
modifying the ASTM D2887 procedure by adding a 9.46 mg total DBTs gÿ1 dry cell mass for the Van
Sievers Chemiluminesence sulfur speci®c detector Texas crude. This is an order of magnitude less than
after the ¯ame ionization detector. The dual detectors the 96.0 mg DBT gÿ1 dry cell mass, measured for pure
produce a chromatogram for the hydrocarbon simu- DBT in model organic systems, also in a 48 h period.26
lated distillation and a chromatogram for the sulfur The reason for this apparent decreased rate of
distribution in the sample. The GC method employed oxidation may be due to the fact that total DBT
a 10 m  0.53 mm AC Sim Dist methyl silicone column substrate is underestimated by measuring only un-
with a 0.9 mm ®lm thickness. Helium was used as a substituted and C1 and C2 substituted DBT species
carrier gas. The GC oven program began at 40 °C with and that the intrinsic rate of oxidation may be
a ramp of 15 ° minÿ1 to 380 °C followed by a holding inhibited by components of the crude oil. The variable
time of 4.5 min. The injector port was temperature rate at which DBT and other individual sulfur
programmed to begin at 125 °C and increase to 380 °C compounds were oxidized between the two different
at 40 ° minÿ1. The ASTM D2887 calls for carbon oils is consistent with observations that properties of
disul®de as the diluent for the oil samples but, in this the oil, inhibitory compounds, and the need to adapt
procedure, tri¯uoro-trichloroethane was used since to various sulfur substrates all play roles in determin-
sulfur determination was one of the analytical objec-
tives. In the case of fractionated oil analysis, total
Table 2. GC–MS analysis of DBT and C1 and C2 substituted DBTs in Van
sulfur was calculated from GC±SCD data by integrat- Texas crude oil
ing the chromatograms and comparing their areas to
those of known standards resulting in an accuracy of Sulfur species IGTS8, 0 h, ppm IGTS8, 48 h, ppm (% change)
0.01% S. This integration could not be performed on
DBT 158.6 3.1 (ÿ98%)
whole oil GC±SCD data since the baseline failed to C1-DBT #1 153.1 0.8 (ÿ99%)
return to zero and the resulting area was indetermi- C1-DBT #2 72.0 3.3 (ÿ95%)
nant. C1-DBT #3 54.2 1.5 (ÿ97%)
C2-DBT #1 3.7 0.3 (ÿ92%)
C2-DBT #2 23.9 0.7 (ÿ97%)
RESULTS AND DISCUSSION C2-DBT #3 9.7 0.7 (ÿ93%)
GC±MS analysis revealed a 40±90% reduction in C2-DBT #4 25.4 0.4 (ÿ98%)
unsubstituted, C1, and C2 substituted DBTs in Sand C2-DBT #5 28.8 2.6 (ÿ91%)
Flat and decreases of >90% for these compounds in C2-DBT #6 8.2 1.1 (ÿ87%)
C2-DBT #7 6.7 0.7 (ÿ90%)
Van Texas oil after 48 h of biological treatment (see

1002 J Chem Technol Biotechnol 74:1000±1004 (1999)


Figure 2. GC–SCD analysis of whole Sand Flat
oil before and after biological treatment. Inset:
Abiotic control reveals overlapping GC–SCD
chromatograms before and after treatment,
indicating no change in sulfur compounds.
ing the ultimate rate of sulfur oxidation.27 Despite the
signi®cant removal of sulfur from DBT species,
negligible decrease in the total sulfur of the whole
crudes was evidenced when the unfractionated oil was
analyzed for total sulfur using the Leco SC-432. It
should be noted that the individual DBT sulfur species
which were measured by GC±MS accounted for less
than 1% of the total sulfur in each oil. Thus, it is not
surprising that these dramatic decreases in individual
components do not translate into changes in the total
sulfur. These results are further illustrated in the
GC±SCD chromatograms of whole Sand Flat oil
shown in Fig 2, where peaks associated with unsub-
stituted as well as substituted DBT are removed in the
biologically treated sample without signi®cantly alter-
ing the total area under the sulfur response curve. The
abiotic control revealed no change in sulfur species as
seen by GC±SCD analysis.
Figure 3. GC–SCD analysis of the aromatic
fraction of Van Texas oil before and after biological
treatment.
J Chem Technol Biotechnol 74:1000±1004 (1999)
Biodesulfurization of crude oil
The GC±SCD analysis of the aromatic portion of
Van Texas oil allowed quanti®cation of sulfur removal
within this fraction with unambiguous chromatogram
baseline accounting. The aromatic fraction of the Van
Texas oil accounted for 22% of the oil's original
volume. As shown in Fig 3, the total sulfur content of
this aromatic fraction was reduced from 3.8 to 3.2% in
6 days of treatment with IGTS8. The dramatic
decreases in peak areas associated with unsubstituted
and C1 and C2 substituted DBT species con®rms the
decreases evidenced in the GC±MS analysis shown in
Table 1. Note that GC analysis does not report
information for species with boiling points exceeding
565 °C since these compounds do not elute from the
column. In the chromatograms shown in Fig 3, these
high boiling point compounds accounted for 16±18%
of the hydrocarbon present.
These initial results are the ®rst that report sulfur
1003
EN Kaufman et al
speciation data in crude oil that has been treated with a
publicly available biocatalyst. While it appears that this
biocatalyst is capable of desulfurizing DBT and
substituted DBTs which are the majority of sulfur
species present in FCC gasoline and diesel and that
only improvements in the rate of desulfurization are
needed for the commercialization of these processes, a
great deal of research is needed for oil biodesulfuriza-
tion to be realized. The sulfur speci®c oxidation of
DBT by Rhodococcus resulted from over 15 years of
research using DBT as the model organic sulfur
compound in coal and oil. Detailed sulfur speciation
studies and biocatalyst development is needed to
achieve desulfurization of the broad spectrum of
organic sulfur species present in crude oil and to
realize the promises of petroleum biodesulfurization.
ACKNOWLEDGEMENTS
IGTS8 was the kind gift of Energy Biosystems
Corporation, The Woodlands, TX. The authors wish
to acknowledge the contributions of Dr Zbigniew Wilk
and APTI Geosciences, Houston, TX in conducting
the GC±MS analysis. The technical assistance of
James B Harkins is gratefully acknowledged.
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J Chem Technol Biotechnol 74:1000±1004 (1999)