Applied and Environmental Microbiology

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

ThiobacilliShale by Bioleaching with Enhanced Dissolution of Oil

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1976, p. 610-616Copyright C) 1976 American Society for Microbiology

Vol. 32, No. 4Printed in U.S.A.

Enhanced Dissolution of Oil Shale by Bioleaching withThiobacilli

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

Received for publication 10 May 1976

Oil shale was subjected to bioleaching by cultures of thiobacilli. From X-ray,electron microprobe, and thin-section petrographic analysis, the shale matrixwas found to contain tightly bonded carbonate minerals. When subjected to thebioproduced acids, these carbonate minerals were removed successively from theshale matrix. This process created pits and cavities which were graduallyenlarged as indicated by scanning electron micrographs of samples subjected toleaching for varying lengths of time. At the end of 14 days, essentially allavailable carbonates had been depleted from the solid matrix. The effectedincrease in porosity and permeability of the oil shale then enhanced the expo-sure of fuel precursors, thus facilitating their production and conversion.

Organic materials suitable in abundance andcomposition for processing into fuel are lo-cated in a variety of rocks known as oil shales.However, the mineral matrix entrapping thesefuel precursors greatly decreases their expo-sure, thus impeding the breakdown and extrac-tion processes. Existing shale oil productionmethods have only been able to recover a frac-tion of these avialable fuel precursors due totheir usage of ineffective mechanical or ther-mal breakdown methods.Laboratory experiments have established

that sulfuric acid generated by the sulfur-oxidiz-ing capabilities of Thiobacillus sp. can be usedto effect more efficient matrix destruction thanby either thermal or mechanical means. Whenused in combination with the sulfate-reducingbacterium Desulfovibrio sp., the sulfuric acid inthe spent sulfate leaching medium is recycledback to sulfur to form a cyclic process (7).The oil shales of the Green River formation

are unique since their mineral matrix is largelycomposed of carbonate minerals capable ofbeing dissolved by biogenic acids through aprocess termed bioleaching. The organic-richGreen River oil shale contains approximately40% acid-soluble minerals [CaCO:j andCaMg(CO1)j2, which are almost entirely re-moved by exposure to the sulfuric acid mediumproduced by thiobacilli. The purpose of this in-vestigation was to determine the effects of bio-leaching on the mineral matrix of the oil shalesof the Green River formation (western UnitedStates) in order to develop a more efficient andeconomic shale oil production process.

MATERIALS AND METHODS

Shale samples. Samples from the organic-richMahogany Ledge of the Green River formation werecomposed of fine-grained, indurated, calcareous, la-custrine, sedimentary rocks ranging in color fromtan to black depending on organic content. The ob-viously varved layering represents paired seasonallaminae composed of relatively large carbonatecrystals deposited during the summer months andfiner-grained detrital clays and organics depositedduring the winter.

X-ray analysis of disordered powder mounts wasused to determine whole-rock mineralogy. Separatepreparations were made for the clay analysis byusing methods devised by Jackson (6). The indur-ated nature of the shale inhibited disaggregation,but digestion in warm sodium-acetate solution re-leased sufficient carbonate-free clay for analysis.Replicate clay mounts were desiccated, treated withglycol and then heated to 350 and 550C to promoteshifts in diagnostic peaks necessary for identifica-tion of individual clay species (2).

Bioleaching cultures. Samples of Thiobacillusthiooxidans ATCC 8050 and Thiobacillus concretivo-rus ATCC 19703 were obtained from the AmericanType Culture Collection. Stock cultures were main-tained in Waksman medium (7) and distilled water(1 liter). The medium was autoclaved and the sur-face was layered with elemental sulfur and adjustedto pH 3.5 with concentrated H:,PO4.

Samples of Desulfovibrio vulgaris, Hildenbor-ough 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 ef-fected by autoclaving plus membrane filtration, andthe medium was adjusted to a final pH of 7.2.

Cultures of T. thiooxidans were grown in contact610

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ENHANCED DISSOLUTION OF OIL SHALE 611

with oil shale for 10 days to establish the feasibilityof using bacterial growth as part of an in situ oilshale production method. At the end of the experi-ment, oxidation was terminated by autoclaving theentire assembly. A portion of the culture was theninoculated with a lactate growth substrate and D.vulgaris and sparged with nitrogen to establish an-aerobiosis. Growth was demonstrated by the devel-opment of a deep gray color indicative of FeS forma-tion (3). At day 20, the flasks were reaerated andreinoculated with T. thiooxidans.

For batchwise (flask bioleaching) and continuousoperations (column bioleaching), 14 liters of viableThiobacillus in Waksman medium (pH = 1.7) werepercolated through 50 g of crushed oil shale (-2 mm)at the rate of 1 liter per day. To visualize the effectsof bioleaching, scanning electron micrographs were

made of shale samples leached for varying periods oftime. Samples were cut into square wafers (approxi-mately 2 by 2 by 0.5 cm) and polished with no. 600grit before treatment. Unleached samples of pol-ished and fractured shale (Fig. la and lb) werephotographed as controls to determine if any mate-rial was being removed by crystal plucking duringpolishing. To further quantify the carbonate re-moved by bioleaching, whole-rock weight percent-ages of organic carbon, carbonate ion, and mineralcarbonate were determined on duplicate samples(270-mesh) using the Leco gasometric analyzer (8).

RESULTSWhole rock mineralogy. X-ray analysis of

disordered powder mounts from the organic-rich Mahogany Ledge of the Green River for-

IC 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 ofsolution pits. (c) Thin section ofGreen River oil shale showing dark organic material entrapped in granular mineral matrix (x 250).

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mation was used to determine whole-rock min-eralogy. Quartz (SiO2) and dolomite(CaMG(CO3)2) were the predominant mineralspecies. A limited amount of calcite (CaCO3)was also present, probably representing resid-ual primary carbonates not yet subjected todiagenesis. Thin sections of the shale showeddistinct banding of dark components enclosedin mono- and polymineralogical crystal aggre-gates, which superimposed a granular textureover the entire rock (Fig. lc). This granularmatrix was found to be dolomite and calcite.Other minor detrital or authigenic mineralspresent in this rock included plagioclase andorthoclase feldspar, analcite, pyrite, montmo-rillonite, and illite.

Montmorillonite was the dominant clay min-eral. This species was in a poorly crystallizedphase and often not detectable by X-ray unlesspresent in excess of 10% of the sample (2). Thepresence of a montmorillonite peak in thewhole-rock pattern suggested, therefore, that itmight be present in significant quantities. Ifthe quartz and carbonate were removed fromthe shale, the expansive forces of swellingmontmorillonite would be useful in disaggre-gating the residual fraction. Garrels and Mac-kenzie (5) showed that, with time, most clayspecies will alter to montmorillonite, illite, orchlorite, suggesting that the illite detected inour analysis may represent primary clay, de-graded mica, or potassium-enriched clays ofother species. It was not feasible to determinequantitatively the clay content without data onthe whole-rock distribution ofelemental oxides,and, as yet, these analyses have not been per-formed.

Electron microprobe analysis of the samples(Table 1) revealed that, with the exception ofcalcium and magnesium, the distribution andconcentration of elements chosen for analysiscoincided with the mineralogy as determinedby X-ray diffraction. Calcium concentration(10.8%), when compared to magnesium (2.6%),yielded a ratio (Ca/Mg = 4.2) which was muchhigher than would be expected if the carbonatewere predominately dolomite (Ca/Mg = 1). Un-

TABLE 1. Microprobe analysis of Green River oilshalea

Element Mean wt % Maximum Minimum wtwt % %

Ca 10.8 21.6 0.9Mg 2.6 5.1 1.0Al 1.6 4.8 0.3Si 6.8 16.8 2.7Fe 1.0 0.4 2.4

a Based on 10 analyses for each element.

less some undiscovered source of calcium waspresent, this suggests that calcite may be morecommon in Green River oil shale than ex-pected.

In general, the clays and associated organicsof the Mahogany Ledge samples were localizedin small aggregates (-20 ,utm) which formed dif-fuse dark-brown or narrow opaque stringswithin the dark winter laminae. Light coloredbands were composed entirely of matrix crys-tals and a few relatively coarse, possible detri-tal, grains. The clays and visible organics owedtheir intimate association to simultaneous dep-osition or linkage through physical and chemi-cal processes (1).The texture of the studied samples suggested

two events: (i) the deposition of varved lakesediments and (ii) penecontemporaneous or sec-ondary mineral precipitation. This explains thesuperimposition of a granular mineral matrixover the entire rock, filling the interstices anddecreasing permeability to effectively zero.Therefore, the first step in liberating absorbedor mechanically entrapped kerogens would beto disrupt the crystalline matrix.

Effect of bioleaching. The unleached sam-ples of polished and fractured shale used ascontrols to determine if any material was beingremoved by crystal plucking during the polish-ing showed no evidence of mechanical pitting,verifying that pits observed in treated sampleswere due to chemical action (Fig. 1). Severalshale samples were recovered, dried, andweighed after a fixed number of days of bio-leaching by Thiobacillus sp. and then by Desul-fovibrio sp. The feasibility of coupling the re-ductive process of Desulfovibrio sp. betweentwo oxidative processes of Thiobacillus sp. incontact with oil shale was confirmed by thedisappearance of the FeS gray color (producedduring Desulfovibrio sp. growth) upon aerationand reinoculation of the flasks with T. thiooxi-dans. Thus, T. thiooxidans could use the FeSproduced by Desulfovibrio sp. to generate thesulfuric acid used in bioleaching.For batchwise (flask bioleaching) and contin-

uous operations (column bioleaching), the pH ofthe column effluent was monitored and found tobe buffered at pH 4.0 by the carbonates solubi-lized from the shale samples. Carbonate disso-lution was also indicated by vigorous evolutionof C02, which ceased after 5 days. The rate ofweight loss (representing dissolved carbonate)was maximized during the initial contact pe-riod and asymptotically decreased with time(Fig. 2). After 14 days, the weight loss leveledoff, indicating the removal of available carbon-ate by this time. This was true for both T.thiooxidans and T. concretivorus.

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O,0 t

ty ~~~~~T.CONCRETIVOROUS

O 2.1 5.0 t.5 h.0- 12.5 15.0TIME (DAYS)

FIG. 2. Percentage of weight loss of Green Riveroil shale as a function oftime (in days). A column isused for leaching by passing 14 liters of culture me-dium through at the rate of1 literlday. The bioleach-ing solution is derived from Thiobacillus thiooxidansand Thiobacillus concretivorus.

To determine the effect of bioleaching onmineralogy, the X-ray pattern ofraw shale wascompared with that of an identical sample thathad undergone a 38.4% weight loss during bio-leaching. The qualitative mineral compositionof both samples proved identical. However, thepeak intensities of the carbonate mineralswere strongly decreased, suggesting that theyhad been dissolved and partially removed (Fig.3). Peak size was not linearly related to theamount of mineral present, and, without addi-tion of matching internal standards, a quanti-tative estimate of carbonate removal was notpossible.Using the Leco gasometric analyzer on dupli-

cate samples revealed that by weight raw shalecontained about 33% mineral carbonate and10% carbon as organic compounds (Table 2).Assuming an average hydrocarbon chain of C16in the Green River material, the added hydro-gen would bring the total weight of organicconstituents to approximately 11%.To quantify how effectively carbonate was

removed by bioleaching, samples of crushed(16-mesh) bioleached shale were analyzed forresidual carbonate (Table 2). The sample usedfor this experiment had lost 36.5% of its weightduring leaching, and it was found to have ap-proximately 2.3% residual mineral carbonate.

Shale bioleached for 2 days (Fig. 4a) had apitted, spongy-appearing surface caused by thesolubilization of carbonate minerals. Bioleach-ing for 1 week (Fig. 4b) resulted in no increasein the density of pits per unit area, but anincrease in average pit size was apparent. Twoweeks of exposure to bioleaching medium (Fig.4c) further increased pit size but did not resultin the formation of additional pits.As a quantitative indication of the volume of

carbonate removed by solution, the cross-sec-tional dimensions of solution pits were mea-sured on photomicrographs of shale leached forvarying lengths of time. The number of pits oneach sample varied from 29 to 33, independentof time, suggesting that solubilization begansimultaneously at all soluble sites immediatelyafter exposure to the leaching medium. Aver-

1)0

7 26 32 31 30 29 28-2e o2E

FIG. 3. Selective ranges of X-ray diffraction pat-terns of the raw shale (solid line) and bioleached oilshale (dotted line). The removal of carbonate min-erals such as dolomite and calcite is practically com-plete as indicated in the right-hand section. Quartzpeak to the left serves as an internal reference.

TABLE 2. Mean weight-percent ofcarbon compounds in whole-rock and bioleached samples ofGreen River oilshale

Shale Carbonate % Carbonate % Mineral % Total % car- Organice %carbon ion carbonate bon carbon

Raw shale (whole rock) 4.02 20.09 33.47 13.69 9.67Bioleached shale (residual carbonate) 0.27 1.35 2.31 _ b _ b

a Organic carbon = total carbon - carbonate carbon.b Total carbon for this sample has not been determined.

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age pit size (calculated as cross-sectional areausing the two extreme diameters for each pit)did vary directly with time, increasing from aminimum of 24.8 ,um2 after 2 days to a maxi-mum of 54.3 ,tm2 after 2 weeks.

Paired scanning electron micrographs of thesame area (Fig. 4d and e) were used as a stereo-pair to indicate depth of field. The solution pitchosen for measurement was selected at ran-dom from a shale sample bioleached for 2weeks. Modification of an equation used foraerial photograph interpretation made it possi-ble to calculate the pit depth as being 2.2 ,umbetween the stereopair in Fig. 4.The irregular bottom of this pit is a typical

effect of solution. The shelf-like false bottomand the small penetration of the true bottomindicated that continued solution would resultin further deepening. This fact, coupled withobserved lateral enlargement with time, shouldresult in vertical and lateral interconnection ofsoluble sites and an increase in porosity andpermeability. This would prove to be an impor-tant mechanism to facilitate exposure of freshsurface to the leaching medium and form con-duits for the migration of liberated hydrocar-bons.

DISCUSSIONKerogens in the Green River shale were

trapped in an inorganic mineral matrix com-posed primarily of quartz and carbonate min-erals. Liberation of hydrocarbons was directlyrelated to the degree of matrix destruction ex-posing kerogen for extraction. The removal ofoil shale mineral matrix by bioleaching beganimmediately upon exposure to the leaching me-dium, developing porosity and permeabilityotherwise nonexistent in untreated shale. Asthe leaching process continued, more rock sur-face was brought into contact with the leachingmedium, leading to increased matrix solutionand exposure of organic fuel precursors andenlargement of pathways for the migration ofliberated hydrocarbons.

Application of bioleaching techniques (3, 7)should provide many advantages over pres-ently used retorting technology. With explo-sives or compressed air, it is possible to fracturethe shale while still in the ground (P. J. Clos-man, U.S. Patent 3,565,171, 1971), providing a

reservoir that could be filled with water andinoculated with acid-forming bacteria. Mineralsalts leached from the carbonate-rich shalewould provide an aqueous solution within theshale formation similar to Waksman mediumto which addition of sulfur or the internal sul-fur of the shale (1 to 2% contained in pyrite)would permit the bacteria to generate the acidnecessary to dissolve the mineral matrix andexpose the kerogen for extraction.

In this work it seemed appropriate to utilizeautotrophic microorganisms for acid productionsince their energy is secured from the oxidationof inorganic compounds and their carbon sourcefrom atmospheric carbon dioxide, bicarbon-ates, or carbonates liberated during the bio-leaching process. The three choices ofdivergentautotrophic groups which could have been uti-lized were: (i) the nitrifying bacteria, (ii) thesulfur oxidizers, and (iii) the iron oxidizers. Thenitrifying bacteria were not used because theirprocess could not be converted readily into acyclic process.

It is known that under certain situations thenaturally occurring sulfur cycle might be oper-ated to advantage. Dilute sulfuric acid (ca. 0.1N) produced biologically from thiobacilli actswith bioderived lipids as surface active agents.The dolomite and calcite in the shale matrix isremoved through this acid leaching. The dis-solved sulfate salts and suspended sulfateslurry is processed anaerobically using Desul-fovibrio. This reduces sulfate to sulfide which isreadily oxidized by air to recycle the sulfurneeded by the thiobacillus culture. The energyand carbon sources for the sulfate reducers aresupplied through the organics from shale proc-ess wastewater. The yeast and vitamins re-quired for the Postgate medium are readily ob-tained from the lysed remains of dead thioba-cillus cells. The feasibility of this continuouscyclic process has been demonstrated in ourlaboratory. Furthermore, iron oxidizers such asThiobacillus ferrooxidans or Ferrobacillus fer-rooxidans are compatible with this sulfur-oxi-dizing system.For a large-scale operation, utilization of bio-

leaching in conjunction with an in situ produc-tion method would eliminate the need for min-ing, crushing, transportation, and eliminationof waste material. If in situ production does not

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

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prove practical, pretreatment of shale by bio-leaching would remove about 40% of the min-eral matrix, reducing shipping costs by a simi-lar amount. In addition, the remaining shalewould be enriched in organic material, makingrecovery more economical since laboratory ex-periments indicate that shale previously releas-ing 25 gallons (ca. 95 liters) of oil per ton of rockby retorting alone would provide 35 to 40 gal-lons (ca. 133 to 152 liters) of oil if pretreated bybioleaching (19).

ACKNOWLEDGMENTSThanks are extended to G. U. Dinneen for supplying the

shale samples used in this study, Kathleen Kim for her aidin establishing and maintaining bacterial cultures, JackWorrell for taking the scanning electron micrographs usedin this report, and Donna Jue for technical assistance. Thiswork was supported by National Science Foundation grantGI-35683, AER-74-23797, and A.G.A. BR-18-12.

LITERATURE CITED1. Bradley, W. H. 1970. Green River oil shale - concept of


origin extended. Geol. Soc. Ann. Bull. 81:985-1000.2. Carroll, D. 1970. Clay minerals: a guide to their X-ray

identification. Geol. Soc. Am. Spec. Pub. 126:1.3. Findley, J., M. D. Appleman, and T. F. Yen. 1974.

Degradation of oil shale by sulfur-oxidizing bacteria.Appl. Microbiol. 28:460-464.

4. Findley, J., M. D. Apleman, and T. F. Yen. 1976. Micro-bial degradation of oil shale, p. 175-182. In Scienceand technology of oil shale. Ann Arbor Science Pub-lishers, Ann Arbor, Mich.

5. Garrels, R. M., and F. T. Mackenzie. 1971. Evolution ofsedimentary rocks. W. W. Norton, Inc., New York.

6. Jackson, M. L. 1958. Soil chemnical analysis-advancedcourse. Prentice-Hall, Englewood Cliffs, N.J.

7. Kim, K. E., H. A. Higa, and T. F. Yen. 1976. Sulfurrecovery by Desulfovibrio in a bioleached method ofoil shale production, p. 157-162. In Science and tech-nology of oil shale. Ann Arbor Science Publishers,Ann Arbor, Mich.

8. Kolpack, R. L., and A. S. Bell. 1968. Gasometric deter-mination of carbon in sediments by hydroxide absorp-tion. J. Sediment. Petrol. 38:617-620.

9. Moussavi, M., and T. F. Yen. 1975. Environmentalconsiderations ofbioleaching as a conditioning step inretorting of oil shale, p. 143-146. In Energy and theenvironment. Inst. Environ. Sci. 1975 Proc., 21stAnnu. Meet., vol. 1.

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