Enhanced Dissolution of Oil

Shale by Bioleaching with

Thiobacilli
W. Craig Meyer and T. F. Yen
Appl. Environ. Microbiol. 1976, 32(4):610.

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1976,

Copyright C) 1976 American Society for Microbiology

p.

Vol. 32, No. 4

Printed in U.S.A.

610-616

Enhanced Dissolution of Oil Shale by Bioleaching with

Thiobacilli
W. CRAIG MEYER AND T. F. YEN*
University of Southern California, University Park, Los Angeles, California 90007

Received for publication 10 May 1976

Organic materials suitable in abundance and

composition for processing into fuel are located in a variety of rocks known as oil shales.
However, the mineral matrix entrapping these
fuel precursors greatly decreases their exposure, thus impeding the breakdown and extraction processes. Existing shale oil production
methods have only been able to recover a fraction of these avialable fuel precursors due to
their usage of ineffective mechanical or thermal breakdown methods.
Laboratory experiments have established
that sulfuric acid generated by the sulfur-oxidizing capabilities of Thiobacillus sp. can be used
to effect more efficient matrix destruction than
by either thermal or mechanical means. When
used in combination with the sulfate-reducing
bacterium Desulfovibrio sp., the sulfuric acid in
the spent sulfate leaching medium is recycled
back to sulfur to form a cyclic process (7).
The oil shales of the Green River formation
are unique since their mineral matrix is largely
composed of carbonate minerals capable of
being dissolved by biogenic acids through a
process termed bioleaching. The organic-rich
Green River oil shale contains approximately
40% acid-soluble minerals [CaCO:j and
CaMg(CO1)j2, which are almost entirely removed by exposure to the sulfuric acid medium
produced by thiobacilli. The purpose of this investigation was to determine the effects of bioleaching on the mineral matrix of the oil shales
of the Green River formation (western United
States) in order to develop a more efficient and
economic shale oil production process.

MATERIALS AND METHODS

Shale samples. Samples from the organic-rich
Mahogany Ledge of the Green River formation were
composed of fine-grained, indurated, calcareous, lacustrine, sedimentary rocks ranging in color from
tan to black depending on organic content. The obviously varved layering represents paired seasonal
laminae composed of relatively large carbonate
crystals deposited during the summer months and
finer-grained detrital clays and organics deposited
during the winter.
X-ray analysis of disordered powder mounts was
used to determine whole-rock mineralogy. Separate
preparations were made for the clay analysis by
using methods devised by Jackson (6). The indurated nature of the shale inhibited disaggregation,
but digestion in warm sodium-acetate solution released sufficient carbonate-free clay for analysis.
Replicate clay mounts were desiccated, treated with
glycol and then heated to 350 and 550C to promote
shifts in diagnostic peaks necessary for identification of individual clay species (2).
Bioleaching cultures. Samples of Thiobacillus
thiooxidans ATCC 8050 and Thiobacillus concretivorus ATCC 19703 were obtained from the American
Type Culture Collection. Stock cultures were maintained in Waksman medium (7) and distilled water
(1 liter). The medium was autoclaved and the surface was layered with elemental sulfur and adjusted
to pH 3.5 with concentrated H:,PO4.
Samples of Desulfovibrio vulgaris, Hildenborough strain, NCIB 8303, were obtained from J. M.
Akagi at the University of Kansas, Lawrence, Kan.
This strain was cultured on Postgate medium (7)
and distilled water (1 liter). Sterilization was effected by autoclaving plus membrane filtration, and
the medium was adjusted to a final pH of 7.2.
Cultures of T. thiooxidans were grown in contact

610

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Oil shale was subjected to bioleaching by cultures of thiobacilli. From X-ray,

electron microprobe, and thin-section petrographic analysis, the shale matrix
was found to contain tightly bonded carbonate minerals. When subjected to the
bioproduced acids, these carbonate minerals were removed successively from the
shale matrix. This process created pits and cavities which were gradually
enlarged as indicated by scanning electron micrographs of samples subjected to
leaching for varying lengths of time. At the end of 14 days, essentially all
available carbonates had been depleted from the solid matrix. The effected
increase in porosity and permeability of the oil shale then enhanced the exposure of fuel precursors, thus facilitating their production and conversion.

ENHANCED DISSOLUTION OF OIL SHALE

VOL. 32, 1976

with oil shale for 10 days to establish the feasibility

of using bacterial growth as part of an in situ oil
shale production method. At the end of the experiment, oxidation was terminated by autoclaving the
entire assembly. A portion of the culture was then
inoculated with a lactate growth substrate and D.
vulgaris and sparged with nitrogen to establish anaerobiosis. Growth was demonstrated by the development of a deep gray color indicative of FeS formation (3). At day 20, the flasks were reaerated and
reinoculated with T. thiooxidans.
For batchwise (flask bioleaching) and continuous
operations (column bioleaching), 14 liters of viable
Thiobacillus in Waksman medium (pH = 1.7) were
percolated through 50 g of crushed oil shale (-2 mm)
at the rate of 1 liter per day. To visualize the effects
of bioleaching, scanning electron micrographs were

IC

611

made of shale samples leached for varying periods of

time. Samples were cut into square wafers (approximately 2 by 2 by 0.5 cm) and polished with no. 600
grit before treatment. Unleached samples of polished and fractured shale (Fig. la and lb) were
photographed as controls to determine if any material was being removed by crystal plucking during
polishing. To further quantify the carbonate removed by bioleaching, whole-rock weight percentages of organic carbon, carbonate ion, and mineral
carbonate were determined on duplicate samples
(270-mesh) using the Leco gasometric analyzer (8).

RESULTS
Whole rock mineralogy. X-ray analysis of
disordered powder mounts from the organicrich Mahogany Ledge of the Green River for-

50__

FIG. 1. Morphology of shale matrix. (a) Control sample of fractured, unleached shale. Note absence of
solution pits. (b) Control sample ofpolished, unleached shale. Note absence of solution pits. (c) Thin section of
Green River oil shale showing dark organic material entrapped in granular mineral matrix (x 250).

APPL. ENVIRON. MICROBIOL.

MEYER AND YEN

612

formed.
Electron microprobe analysis of the samples
(Table 1) revealed that, with the exception of
calcium and magnesium, the distribution and
concentration of elements chosen for analysis
coincided with the mineralogy as determined
by X-ray diffraction. Calcium concentration
(10.8%), when compared to magnesium (2.6%),
yielded a ratio (Ca/Mg = 4.2) which was much
higher than would be expected if the carbonate
were predominately dolomite (Ca/Mg = 1). UnTABLE 1. Microprobe analysis of Green River oil
shalea
Element

Mean wt %

Maximum
wt %

Minimum wt
%

Ca
Mg
Al
Si
Fe

10.8
2.6
1.6
6.8
1.0

21.6
5.1
4.8
16.8
0.4

0.9
1.0
0.3
2.7
2.4

Based on 10 analyses for each element.

less some undiscovered source of calcium was

present, this suggests that calcite may be more
common in Green River oil shale than expected.
In general, the clays and associated organics
of the Mahogany Ledge samples were localized
in small aggregates (-20 ,utm) which formed diffuse dark-brown or narrow opaque strings
within the dark winter laminae. Light colored
bands were composed entirely of matrix crystals and a few relatively coarse, possible detrital, grains. The clays and visible organics owed
their intimate association to simultaneous deposition or linkage through physical and chemical processes (1).
The texture of the studied samples suggested
two events: (i) the deposition of varved lake
sediments and (ii) penecontemporaneous or secondary mineral precipitation. This explains the
superimposition of a granular mineral matrix
over the entire rock, filling the interstices and
decreasing permeability to effectively zero.
Therefore, the first step in liberating absorbed
or mechanically entrapped kerogens would be
to disrupt the crystalline matrix.
Effect of bioleaching. The unleached samples of polished and fractured shale used as
controls to determine if any material was being
removed by crystal plucking during the polishing showed no evidence of mechanical pitting,
verifying that pits observed in treated samples
were due to chemical action (Fig. 1). Several
shale samples were recovered, dried, and
weighed after a fixed number of days of bioleaching by Thiobacillus sp. and then by Desulfovibrio sp. The feasibility of coupling the reductive process of Desulfovibrio sp. between
two oxidative processes of Thiobacillus sp. in
contact with oil shale was confirmed by the
disappearance of the FeS gray color (produced
during Desulfovibrio sp. growth) upon aeration
and reinoculation of the flasks with T. thiooxidans. Thus, T. thiooxidans could use the FeS
produced by Desulfovibrio sp. to generate the
sulfuric acid used in bioleaching.
For batchwise (flask bioleaching) and continuous operations (column bioleaching), the pH of
the column effluent was monitored and found to
be buffered at pH 4.0 by the carbonates solubilized from the shale samples. Carbonate dissolution was also indicated by vigorous evolution
of C02, which ceased after 5 days. The rate of
weight loss (representing dissolved carbonate)
was maximized during the initial contact period and asymptotically decreased with time
(Fig. 2). After 14 days, the weight loss leveled
off, indicating the removal of available carbonate by this time. This was true for both T.
thiooxidans and T. concretivorus.

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mation was used to determine whole-rock mineralogy. Quartz (SiO2) and dolomite
(CaMG(CO3)2) were the predominant mineral
species. A limited amount of calcite (CaCO3)
was also present, probably representing residual primary carbonates not yet subjected to
diagenesis. Thin sections of the shale showed
distinct banding of dark components enclosed
in mono- and polymineralogical crystal aggregates, which superimposed a granular texture
over the entire rock (Fig. lc). This granular
matrix was found to be dolomite and calcite.
Other minor detrital or authigenic minerals
present in this rock included plagioclase and
orthoclase feldspar, analcite, pyrite, montmorillonite, and illite.
Montmorillonite was the dominant clay mineral. This species was in a poorly crystallized
phase and often not detectable by X-ray unless
present in excess of 10% of the sample (2). The
presence of a montmorillonite peak in the
whole-rock pattern suggested, therefore, that it
might be present in significant quantities. If
the quartz and carbonate were removed from
the shale, the expansive forces of swelling
montmorillonite would be useful in disaggregating the residual fraction. Garrels and Mackenzie (5) showed that, with time, most clay
species will alter to montmorillonite, illite, or
chlorite, suggesting that the illite detected in
our analysis may represent primary clay, degraded mica, or potassium-enriched clays of
other species. It was not feasible to determine
quantitatively the clay content without data on
the whole-rock distribution of elemental oxides,
and, as yet, these analyses have not been per-

VOL. 32, 1976

ENHANCED DISSOLUTION OF OIL SHALE

613

Shale bioleached for 2 days (Fig. 4a) had a

pitted, spongy-appearing surface caused by the
solubilization of carbonate minerals. Bioleaching for 1 week (Fig. 4b) resulted in no increase
in the density of pits per unit area, but an
increase in average pit size was apparent. Two
weeks of exposure to bioleaching medium (Fig.
4c) further increased pit size but did not result
ty
~~~~~T.
CONCRETIVOROUS
in the formation of additional pits.
As a quantitative indication of the volume of
carbonate removed by solution, the cross-sectional dimensions of solution pits were meaO
2.1
5.0
h.0t.5
12.5
15.0
sured on photomicrographs of shale leached for
(DAYS)
varying lengths of time. The number of pits on
FIG. 2. Percentage of weight loss of Green River each sample varied from 29 to 33, independent
oil shale as a function of time (in days). A column is of time, suggesting that solubilization began
used for leaching by passing 14 liters of culture me- simultaneously at all soluble sites immediately
dium through at the rate of 1 literlday. The bioleach- after exposure to the leaching medium. Avering solution is derived from Thiobacillus thiooxidans

,0

and Thiobacillus concretivorus.

To determine the effect of bioleaching on

mineralogy, the X-ray pattern of raw shale was
compared with that of an identical sample that
had undergone a 38.4% weight loss during bioleaching. The qualitative mineral composition
of both samples proved identical. However, the
peak intensities of the carbonate minerals
were strongly decreased, suggesting that they
had been dissolved and partially removed (Fig.
3). Peak size was not linearly related to the
amount of mineral present, and, without addition of matching internal standards, a quantitative estimate of carbonate removal was not
possible.
Using the Leco gasometric analyzer on duplicate samples revealed that by weight raw shale
contained about 33% mineral carbonate and
10% carbon as organic compounds (Table 2).
Assuming an average hydrocarbon chain of C16
in the Green River material, the added hydrogen would bring the total weight of organic
constituents to approximately 11%.
To quantify how effectively carbonate was
removed by bioleaching, samples of crushed
(16-mesh) bioleached shale were analyzed for
residual carbonate (Table 2). The sample used
for this experiment had lost 36.5% of its weight
during leaching, and it was found to have approximately 2.3% residual mineral carbonate.

1)
0

28
26 32
31
30
29
-2 e
o2E
FIG. 3. Selective ranges of X-ray diffraction patterns of the raw shale (solid line) and bioleached oil
shale (dotted line). The removal of carbonate minerals such as dolomite and calcite is practically complete as indicated in the right-hand section. Quartz
peak to the left serves as an internal reference.

TABLE 2. Mean weight-percent of carbon compounds in whole-rock and bioleached samples of Green River oil
shale
Shale

Carbonate % Carbonate %
ion
carbon
20.09
4.02
1.35
0.27

Raw shale (whole rock)

Bioleached shale (residual carbonate)
a
Organic carbon = total carbon - carbonate carbon.
b Total carbon for this sample has not been determined.

Mineral %
carbonate
33.47

2.31

Total % carbon

%
Organice
carbon

13.69
_b

9.67
_b

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TIME

APPL. ENVIRON. MICROBIOL.

MEYER AND YEN

614

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4e

uw'm I-

VOL. 32, 1976

ENHANCED DISSOLUTION OF OIL SHALE

reservoir that could be filled with water and

inoculated with acid-forming bacteria. Mineral
salts leached from the carbonate-rich shale
would provide an aqueous solution within the
shale formation similar to Waksman medium
to which addition of sulfur or the internal sulfur of the shale (1 to 2% contained in pyrite)
would permit the bacteria to generate the acid
necessary to dissolve the mineral matrix and
expose the kerogen for extraction.
In this work it seemed appropriate to utilize
autotrophic microorganisms for acid production
since their energy is secured from the oxidation
of inorganic compounds and their carbon source
from atmospheric carbon dioxide, bicarbonates, or carbonates liberated during the bioleaching process. The three choices ofdivergent
autotrophic groups which could have been utilized were: (i) the nitrifying bacteria, (ii) the
sulfur oxidizers, and (iii) the iron oxidizers. The
nitrifying bacteria were not used because their
process could not be converted readily into a
cyclic process.
It is known that under certain situations the
naturally occurring sulfur cycle might be operated to advantage. Dilute sulfuric acid (ca. 0.1
N) produced biologically from thiobacilli acts
with bioderived lipids as surface active agents.
The dolomite and calcite in the shale matrix is
removed through this acid leaching. The dissolved sulfate salts and suspended sulfate
slurry is processed anaerobically using Desulfovibrio. This reduces sulfate to sulfide which is
readily oxidized by air to recycle the sulfur
needed by the thiobacillus culture. The energy
and carbon sources for the sulfate reducers are
supplied through the organics from shale process wastewater. The yeast and vitamins required for the Postgate medium are readily obtained from the lysed remains of dead thiobacillus cells. The feasibility of this continuous
cyclic process has been demonstrated in our
laboratory. Furthermore, iron oxidizers such as
Thiobacillus ferrooxidans or Ferrobacillus ferrooxidans are compatible with this sulfur-oxidizing system.
For a large-scale operation, utilization of bioleaching in conjunction with an in situ production method would eliminate the need for mining, crushing, transportation, and elimination
of waste material. If in situ production does not

FIG. 4. Scanning electron micrographs showing visual effects of bioleaching. (a) Development of solution
pits in polished samples leached for 2 days. (b) Development of solution pits in polished samples leached for 1
week. (c) Development of solution pits in polished samples leached for 2 weeks. (d and e) Paired microphotographs (stereopair) from which pit depth is calculated. AB and A'B' connect the same points on both
photographs. Calculation is based on D = PI2M sin (0/2) where P is parallax (difference in distance between
the same two points on a stereopair), M is magnification, 0 is the difference in the tilt at which the photographs
are taken and D is the depth of the pit. In this case the calculated number is 2.2 ,um.

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age pit size (calculated as cross-sectional area

using the two extreme diameters for each pit)
did vary directly with time, increasing from a
minimum of 24.8 ,um2 after 2 days to a maximum of 54.3 ,tm2 after 2 weeks.
Paired scanning electron micrographs of the
same area (Fig. 4d and e) were used as a stereopair to indicate depth of field. The solution pit
chosen for measurement was selected at random from a shale sample bioleached for 2
weeks. Modification of an equation used for
aerial photograph interpretation made it possible to calculate the pit depth as being 2.2 ,um
between the stereopair in Fig. 4.
The irregular bottom of this pit is a typical
effect of solution. The shelf-like false bottom
and the small penetration of the true bottom
indicated that continued solution would result
in further deepening. This fact, coupled with
observed lateral enlargement with time, should
result in vertical and lateral interconnection of
soluble sites and an increase in porosity and
permeability. This would prove to be an important mechanism to facilitate exposure of fresh
surface to the leaching medium and form conduits for the migration of liberated hydrocarbons.
DISCUSSION
Kerogens in the Green River shale were
trapped in an inorganic mineral matrix composed primarily of quartz and carbonate minerals. Liberation of hydrocarbons was directly
related to the degree of matrix destruction exposing kerogen for extraction. The removal of
oil shale mineral matrix by bioleaching began
immediately upon exposure to the leaching medium, developing porosity and permeability
otherwise nonexistent in untreated shale. As
the leaching process continued, more rock surface was brought into contact with the leaching
medium, leading to increased matrix solution
and exposure of organic fuel precursors and
enlargement of pathways for the migration of
liberated hydrocarbons.
Application of bioleaching techniques (3, 7)
should provide many advantages over presently used retorting technology. With explosives or compressed air, it is possible to fracture
the shale while still in the ground (P. J. Closman, U.S. Patent 3,565,171, 1971), providing a

615

616

APPL. ENVIRON. MICROBIOL.

MEYER AND YEN

prove practical, pretreatment of shale by bioleaching would remove about 40% of the mineral matrix, reducing shipping costs by a similar amount. In addition, the remaining shale
would be enriched in organic material, making
recovery more economical since laboratory experiments indicate that shale previously releasing 25 gallons (ca. 95 liters) of oil per ton of rock
by retorting alone would provide 35 to 40 gallons (ca. 133 to 152 liters) of oil if pretreated by
bioleaching (19).

LITERATURE CITED
1. Bradley, W. H. 1970. Green River oil shale - concept of

identification. Geol. Soc. Am. Spec. Pub. 126:1.

3. Findley, J., M. D. Appleman, and T. F. Yen. 1974.
4.

5.
6.
7.

8.

9.

Degradation of oil shale by sulfur-oxidizing bacteria.

Appl. Microbiol. 28:460-464.
Findley, J., M. D. Apleman, and T. F. Yen. 1976. Microbial degradation of oil shale, p. 175-182. In Science
and technology of oil shale. Ann Arbor Science Publishers, Ann Arbor, Mich.
Garrels, R. M., and F. T. Mackenzie. 1971. Evolution of
sedimentary rocks. W. W. Norton, Inc., New York.
Jackson, M. L. 1958. Soil chemnical analysis-advanced
course. Prentice-Hall, Englewood Cliffs, N.J.
Kim, K. E., H. A. Higa, and T. F. Yen. 1976. Sulfur
recovery by Desulfovibrio in a bioleached method of
oil shale production, p. 157-162. In Science and technology of oil shale. Ann Arbor Science Publishers,
Ann Arbor, Mich.
Kolpack, R. L., and A. S. Bell. 1968. Gasometric determination of carbon in sediments by hydroxide absorption. J. Sediment. Petrol. 38:617-620.
Moussavi, M., and T. F. Yen. 1975. Environmental
considerations of bioleaching as a conditioning step in
retorting of oil shale, p. 143-146. In Energy and the
environment. Inst. Environ. Sci. 1975 Proc., 21st
Annu. Meet., vol. 1.

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ACKNOWLEDGMENTS
Thanks are extended to G. U. Dinneen for supplying the
shale samples used in this study, Kathleen Kim for her aid
in establishing and maintaining bacterial cultures, Jack
Worrell for taking the scanning electron micrographs used
in this report, and Donna Jue for technical assistance. This
work was supported by National Science Foundation grant
GI-35683, AER-74-23797, and A.G.A. BR-18-12.

origin extended. Geol. Soc. Ann. Bull. 81:985-1000.

2. Carroll, D. 1970. Clay minerals: a guide to their X-ray