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Sulfur, iron and solid phase transformations during the biological oxidation of pyritic mine spoil
SodBid. Biochrm.Vol.23.No.2.p~. 101-107.1991 PrintedinGreat Britain.All rig& lcymd
Copyrights
0038-0717/91 s3.OO+o.w 1991PergamoaPre~pk
SULFUR, IRON AND SOLID PHASE TRANSFORMATIONS DURING THE BIOLOGICAL OXIDATION OF PYRITIC MINE SPOIL J. R.
PICHTEL*
and W. A. DICK
Department of Agronomy, Ohio State University/Ohio Agricultural Research and Development Center. Wooster, OH 44691. U.S.A. (Accepted 25 September 1990)
Summary-Spoil material composed of pyrite, coal and rock was ground and inoculated with spoil microflora and monitored for pH. S@,-. reduced S compounds, total soluble Fe and Fe(H). A ‘I-day lag in production of both SG- and total soluble Fe was observed followed by a marked increase in concentration. Concentrations of w-. total soluble Fe and Fe(U) after 28 days were co 81. 91, and 59 mmol kg-’ spoil, respectively. Reduced S anions including thiosulfate. trithionate and tetrathionatc were detected in the spoil at all samplings during a 28day incubation. Final trithionate concentrations were greatest (1.07 mmol kg-’ spoil). Scanning electron microscope observations of the pyrite nvealcd a gradual pitting and erosion of particle faces after prolonged incubation. Certain faces appeared to have been preferentially weathered.
INTRODUClION
Surface mining of certain coal seams in the eastern portion of the United States results in the exposure of large quantities of pyrite (Fe&) which is readily oxidized in the atmosphere (Caruccio. 1968). Oxidation of pyrite is a complex process involving several types of oxidation-reduction reactions, hydrolysis and complex ion formation, solubility controls and kinetic effects (Nordstrom, 1982; Pichtel and Dick, 1991). The overall reaction is commonly written as: FeS, + (15/4)0, + (7/2)H,O -. Fe(OH), + 2H2S0, (1) The H,SO, produced can create hostile environments (Blevins et al.. 1970; Lundberg er 01.. 1977; Pons et al., 1982) by inhibiting the establishment and growth of plant cover, thereby resulting in soil erosion and acidification of adjacent soils and waters. Autotrophic bacteria of the genus Thiobacilhts have been studied extensively regarding their contribution to mine spoil acidification. Several species have been isolated from acid mine wastes: T. ferrooxiduns, which oxidizes pyrite, Fe(H) and S, T. thiooxidans which oxidizes pyrite and S. and T. acidophilus which oxidizes S (Unz and Lundgren, 1961; McGoran et d, 1969; Guay and Silver, 1975). These thiobacilli generate energy via the oxidation of reduced S and Fe compounds for the reductive assimilation of CO, and for growth. During S oxidation, various intermediate S compounds are formed (Kelly, 1982) and the environmental significance and fate of these compounds are not clearly understood. In soil or aquatic environments, S and Fe oxidizing microorganisms may occur together, their oxidation *Present address: Department of Natural Raourccs. Ball State University, Muncie, IN 47306. U.S.A.
reactions proceeding simultaneously. Concordant with biological oxidation, however, is abiotic S and Fe oxidation and, if conditions are appropriate, microbially-catalyzed SO:- reduction. The occurrence and quantities of various S compounds in mine spoils, therefore, represent a composite of many different reactions. A further complication to the study of the S, Fe and solid phase reactions of pyritic spoils is that the spoils may be dramatically altered by both biological and chemical mechanisms after only a brief exposure to O2 and water. A better understanding of such reactions at the time of initial exposure is needed. Our objectives were: (i) to assess pH change and production of SOi- and soluble Fe in fresh, inoculated spoils, (ii) to assay for the presence of certain reduced S oxyanions in the oxidizing spoils; and (iii) to observe pyrite surface morphology with progress of biological weathering. MATERIALS AND METHODS
Spoil material
To ensure the ready availability of unweathered spoil, a synthetic spoil composed of equal weights of pyrite, bituminous coal and a rock mixture (shale, sandstone and mudstone) was prepared. The coal and rock fractions were collected from the floor of an active surface mine in southeastern Ohio, sealed in plastic bags, placed into ice chests, and returned to the laboratory for storage at - 12°C. Pure, unweathered pyrite was not available from the mine floor and museum grade euhedral specimens were used. All components were crushed with a hammer, ground with a mortar and pestle, and sieved (< 150 pm). To remove free Fe and S salts present, the spoil was washed with 5 vol of N,-degassed, deionized water, lyophilized in a Labconco freeze dryer, then stored in plastic containers at - 12°C.
102
J. R.
RCHTEL
Fresh spoil samples were brought into solution by sodium carbonate fusion (Dick et uf., 1985). Aliquots were analyzed for Al. Ca. Mg, Fe and Mn by atomic absorption spectrophotometry using a Varian Model AA-475 spectrophotometer; for Ba. Cd, Cu. Ni, Pb, Sr and Zn by atomic emission spectrophotometry with a Jarrel-Ash Model 975 inductively-coupled plasma-atomic emission spectrophotometer; and for P by the calorimetric method of Murphy and Riley (1962) using ascorbic acid as a reducing agent. Total S content was determined in a separate aliquot using the methylene blue method of Johnson and Nishita (1952). Pyritic-S was measured by titrimetric determination of pyritic iron and sulfate-S was measured by precipitation with BaClr to form BaSO, (ASTM, 1976). X-ray diffraction analyses were conducted on the clay-sized fraction of the rock mixture sample and on a finely ground ( c 53 pm) pyrite sample. Sample preparation was as described by Kinter and Diamond (1956). Kittrick and Hope (1963), Rich and Barnhisel (1977) and Whittig (1965). A Philips XRG-3100 generator with a Cu and a DMS41 control panel was used for the X-ray studies. Surface morphology changes of the pyrite component of the spoil, with progress of bactcrially-mcdiatcd weathering. was monitored by scanning electron microscopy. I g pyrite (< I50 pm) was added to a 250 ml Erlcnmcycr flask containing 100 ml dcionizcd water and inoculated with IO ml of T. J2rrooxiduns strain TFP-I [containing co IOpg ml-’ bacterial protein (Lowry et al.. 1951)], cultured as dcscribcd by Norris and Kelly (1982). Flasks were shaken (140 rev min-‘) on a rotary shaker at room tcmpcraturc. Scanning clcctron micrographs wcrc taken of fresh (non-inoculated) pyrite, and after 28 and 70 days of cxposurc. The solids were removed by centrifugation (12.000~) and freeze-dried. Samples were mounted on an AI stub with double-stick tape and coated with a 20 nm-thick gold-palladium alloy in a vacuum evaporator. Electron micrographs were taken with an International Scientific Instruments Model ISIelectron microscope. Spoil incubations
Fresh spoil (I g), deionized water (100 ml), and inoculum were added to 250 ml Erlenmeyer flasks. The flasks were plugged with polyethylene and kept at room temperature on a rotary shaker (140 rev min-‘). The inoculum for all exposures of the fresh mine spoil was 10 mg of weathered spoil (< ISOpm) collected from the surface of an abandoned coal mine site located in Southeastern Ohio. Initial pH was 6.0. All work was conducted in triplicate. Periodically. three replicate flasks were removed from the shaker and the suspensions were filtered through Whatman No. 42 filter paper. The pH of each sample was recorded and IO ml aliquots were frozen at -12°C. All samples were analyzed for various chemical properties at the same time after thawing in an N, atmosphere. intermediates (thiosulfate, Sulfur oxidation trithionate and tetrathionate) were determined by the calorimetric procedure of Kelly et al. (1969). Na-thiosulfate (Fisher Scientific), K-tetrathionate (Kodak), and K-trithionate, prepared by the
and W. A. DICK method of Stamm and Goehring (1942). were used as standards. Sulfide was determined as HrS by the methylene blue procedure (Johnson and Nishita. 1952). Sulfite and sulfate were estimated by ion chromatography using a Dionex Model I4 ion chromatograph. Samples, preserved with 0. I ml cont. HNO, IOml-’ aliquot, were assayed for total soluble Fe by atomic absorption spectrophotometry. Fe(B) was measured using a calorimetric method which is accurate in the presence of large amounts of Fe(III) (Tamura er al.. 1974) and Fe(III) was calculated as the difference between total and Fe(H). Deionized water degassed with N: was used for dilutions. RESULTSAND
DlSCUBSlON
The synthetic spoil contained 103 and 179 g kg-’ total S and Fe, respectively (Table I). Pyritic- and sulfate-s comprised 25.2 and 0.13%. respectively, of the total S fraction. The pyrite fraction of the spoil was essentially pure. as X-ray diffractograms revealed only a trace of contamination. possibly by quartz. Minerals in the spoil, as determined by X-ray analysis, included kaolinite. clay mica, vermiculite. chlorite, quartz and gocthite. Secondary minerals characteristic of a weathered mining environment. such as jarositc and ferric sulfates and hydroxides. were not observed due to the unwcathcred state of the spoil. Fresh. unwcathercd pyrite appeared as irregular nonporous grains with smooth surfaces [Fig. l(a)]. After 28 days of weathering by T. ferrooxiduns. surface erosion and fissures bccamc evident due to acid etching and oxidation and solubilizttion by ferric iron (Nordstrom, l9g2) [Fig. l(b), (c)l. Copious
quantities of bacterial cells were attached to particle surfaces. After 70 days particles were severely eroded or pitted and most surfaces had become highly irregular [Fig. l(d), (e)]. Surface erosion such as that shown in Fig. l(b) and I(c) may be partially due to particle abrasion during shaking of the suspensions. However, the deep pitting and fissures observed after 70 days [Fig. l(d) and l(e)] are presumed to be the result of the activity of T. ferrooxiduns. Table I. Chemical analysis of the synthetic spoil Element
Concentration B kg-’
FC s AI Ca Mg al
179 103 32.4 2.50 2.00 I .70 mg kg-’
Mll Pb Ba P CU Sr Cd Ni
527 527 267 265 257 xl 18 13
J. R. PKHTEL and W. A. DICK
Fig. 1, (d-c) Fig. I. !~GIII ning electron micrographs of pyrite (C 150 pm) (a) prior to weathering; (b), (c) after 28 d ay!3 of weathering: (d). (e) after 70 days of weathering.
fnspection of electron micrographs of pyrite weathered for 70 days suggested that particle faces possessed differential weathering tendencies. Certain faces had been considerably transformed while others appeared afmost unaffected. Suscept~biiity to weathering could have been due to the exposure of broken chemical bonds on a particle fact. Sulfate production in the spoil suspension was initially slow (Fig. 2) but began to increase after 7 days and a sharp increase was observed after 14 days. After 28 days ca 7.8% of the total S was oxidized to Soi- and by 38 days Saconcentrations accounted for 35.6% of the total S. Although 25.2% of the original S was in the pyritic form. thiobacilli and other spoil microorganisms could have oxidized various inorganic S compounds (e.g. thiosulfate and polythionatcs) and organic sulfides of biogenic origin in addition to pyritic S (Kelly, 1982).
The pH of the inoculated spoil suspension decreased from 6.0 to 3.7 after 28 days of exposure (Fig. 3). It declined further to pH 3.0 after 38 days and to 1.9 after 150 days. This decline would probably have been more rapid had the solution been replaced periodically with fresh deionized H,O because T. ferrooxidans is known to excrete organic compounds (Schnaitman and Lundgren, 1965) which slows autotrophic growth and metabolism. A negligible decline in pH occurred when sodium azidc (IO-‘M) was added to the spoil suspensions. This indicates that pyrite oxidation, at lcast initially, was due to biological activity rather than physicochemical processes. Soluble sulfide or sulfite were not detected at any of the sampling times, which included times as short as 2 h. They were either not produced during exposure or were transformed rapidly to other S species. Both sulfide and sulfite are unstable in
105
Chemical transformations during pyrite oxidation 100
;,,
,
I
I
I
I
._ i
75
-
I 3 cn al ' 4 a
50-
'-j ul 1
25-
z
o1
I
I
I
I
0124
I
I
I
I
7
14
21
28
Time
of Incubation
(days)
Fig. 2. Changes in sulfate-s concentrations of inoculated synthetic spoil suspensions during 28 days of exposure. LSD,,,, values are for comparison of concentrations in the presence or absence of the microbial inhibitor, NaN, (IO-‘M).
acidic solutions, being spont;~ncously oxidized in the prcscncc of oxygen (Roy and Trudingcr, 1970; Goldhabcr. 1980). Thiosulf’atc. trithionatc and tctrathionntc wcrc dctcctcd throughout the B-day cxposurc (Fig. 3). After an initial lag. maximum concentrations of thiosulfatc (0.97 mmol kg-’ spoil) and tctrrrthionatc (0.26 mmol kg-’ spoil) were obtained on day 7. Both ions
dcclincd in concentration beyond day 7. while trithionatc incrcascd sharply after day 14. It is possiblc that thiosulfatc or tctrathionatc may have been precursors to trithionatc during spoil oxidation as their diminution is coincident with the incrcasc in trithionatc content (Kelly and Syrctt. 1966; Landcsman CI ol., 1966; Sinha and Walden. 1966; Kelly. 1982). Howcvcr. in contrast, trithionate may
-l
7
6
5
PH
0124
7
Time
21
14
of
Incubation
28
(days)
Fig. 3. Changes in ptl and production of the sulfur oxyanion intermediates thiosulfate. trithionate tctrathionatc in inoculated spoil suspension during 28 days of incubation.
and
106
J. R. Ptcnrr.t and W. A. DICK
100
0
0124
7
14
Time
of Incubation
21
(clays)
Fig 4. Iron solubilization in inoculated spoil suspensionsduring 28 days of exposure. LSD,,, values are of total Fe concentrations in the presence or absence of the microbial inhibitor, NaN, (IO ’ M).
for comnarison
considcrcd a precursor to thiosulfatc and tctrathionatc. Trithionatc accumulation in the spoil mixture would then be a result of inhibition of the transformation process of trithionatc to thiosulfatc and tctrathionatc. Also, during the abiotic dccomposition of acidified thiosulfatc. trithionatc was the primary thionatc produced (Pollard PI crl.. lY64). The dcclinc in thiosulfate concentration may be partly attributed to its instability in acidic solutions (Pollard er ul.. 1964). The pH trend, superimposed upon the trend for the sulfur oxyanions (Fig. 3). reveals the presence of increasing suspension acidity with time. Thiosulfate may also be oxidized by iron(II1) hydroxide on the surface of a pyrite particle (Miller, 1980) according to the following equation: bc
2S,Oj-
+ 2Fe(OH),
+ S,Ob+ 2Fe(OH)2
+ 20H
-
(2)
The interaction of soluble reduced S compounds, such as thiosulfate, with metal oxides in spoils may be of practical significance due to their mobilization of potentially toxic metals, such as Fe, Al. Mn. Trithionate and tetrathionatc are stable in acid conditions (Roy and Trudinger, 1970; Goldhaber. 1980). Trends of Fe solubilization in the spoil suspensions (Fig. 4) paralleled those of SOi- production shown in Fig. 2. Fe(II) production remained below dctcction lcvcls until day 7. then increased steadily. Beyond day 28, the Fe( III) form dominated. attaining 182.6 mmol compared to 87.7mmol Fc(ll) kg spoil-’ on day 38 (data not shown). This result agrees with the data of Vuorincn et al. (1983. 1985). who leached both pyrite and uranium arc with T. /‘errooxidans. After several leachings and at a pH cu 2.0. Fe(lII) was always many times higher than that of Fe(ll). In our work. IO-’ M sodium azide was a highly effective inhibitor of Fe
solubilization as pH remained bctwccn 5.5 and 6.2 and iron was not detected in the azide-trcatcd flasks during cxposurc. Biological iron oxidation is a cytochrome-mediated process and azide ion is known to be a polcnl cytochrome inhibitor (Duncan cf ~1.. 1967). The total amount of Fe in solution, i.e. Fe(lI) plus Fe(Ill), was greater than the quantity of SO;--S produced (compare Figs 2 and 4). Different quantities of Fe and S0i-S may have precipitated as oxides or other insoluble compounds such as the mineral hydronium jarosite (HzFe,(SO,),(OH),) (Duncan and Bruyncsteyn. 1971). The incongruity is also due to the presence of non-sulfate S species such as thiosulfate and various thionates in solution. Our results indicate that S and Fe in freshly exposed pyritic spoil is rapidly oxidized by microorganisms resulting in acid production and a sharp decline in pH to a value of I.9 after I50 days. The oxyanions thiosulfate. trithionate, and tetrathionate are produced during the oxidation process. During oxidation T. firrooxidms stems to attack certain faces of the pyrite particles preferentially. Ackno,\,/rclR~~vn/s-We thank Dr 0. H. Tuovinen for supplying T. fcrroo.riduns strain TFP-I and Dr J. M. Bigham for assisting in the mineralogical analyses. Salaries and research support wcrL: provided by State and Federal funds appropriated IO the Agricultural Research and Dcvelopment Ccmcr. The Ohio State University.
REFERENCES ASTM (1976) SrondurJ Test Merhodr for Total Sul’ir in [he Analysis of Cool und Coke. American Sociely of Testing Materials, Annual Book of Standards. D3 177-75. pp. 380-386. U.S. Government Printing Office, Washington.
Chemical
transformations
Blevins R. L.. Bailey H. H. and Ballard G. E. (1970) The effect of acid mine water on floodplain soils in the western Kentucky coalfields. Soil Science 110, 191-196. Caruccio F. T. (1968) An evaluation of factors affecting acid mine drainage production and the ground water interactions in selected areas of western Pennsylvania. In Second Symposium on Coal Mine Drainage Research, pp. 107-152. Bituminous Coal Research Inc., Monroevillc. Dick W. A., Page J. R. and Jewclle K. E. (1985) Direct analysis of soil suspensions by inductively-coupled plasma-atomic emission spectrometry for determination of total metals. Soil Science 139, 21 I-218. Duncan D. W. and Bruynestcyn A. (1971) Enhancing bacterial activity in an uranium mine. Canadian Mining and Merallurgical Bulletin 64, 32-36. Duncan D. W.. Landcsman J. and Walden C. C. (1976) Role of Thiobacillus firooxidans in the oxidation of sulfide minerals. Canadian Journal of Microbiology 13, 397-403. Goldhaber M. 9. (1980) Experimental Study of Pyrite Oxidation af pH 69and WC’. United States Department of the Interior. Geological Survey. Open-file report 80-1272. U.S. Government Printina Office. Washinaton. Guay R. and Silver M. (1975) Thiob&illus ocidophilu; sp. nov: isolation and some physiological characteristics. Canudiun Journal of Microbiology 21. 281-288. Johnson C. M. and Nishita H. (1952) Microestimation of sulfur in plant materials, soils. and irrigation waters. Anal.vricul-Chemistry 24. 736742. _ Kellv D. P. (1982) , Biochcmistrv of the chcmolithotroohic oxidation of inorganic sulfur. Philosophicul Transaciions of rhe Royal Sockry of London 2%. 490-528. Kelly D. P. and Syrctt P. J. (1966) (“S) Thiosulphatc oxidation by Thiohucillus strain C. Biochcnricul Journul YU. 537-54s. Kelly D. P.. Chambers L. A. and Trudingcr P. A. (1969) Cyanolysis and spcctrophotomctric estimation of trithionatc in mixture with thiosulfatc and tctrathionatc. Anulyricul Chcmisrry 41. 89X 90 I. Kintcr E. B. and Diamond S. (1956) A new method for preparation and trrdtmcnt of oriented-aggregate spccimrns and soil clays for X-ray dilfraction analysis. Soil Scirncr 81, I I I-120. Kittrick J. A. and Hope E. W. (1963) A procedure for the particle sizr xparntion of soils for X-ray diflraction analysis. Soil Scirncr 96, 319-325. Lnndrsman J.. Duncan D. W. and Walden C. C. (1966) Oxidation of inorganic sulfur compounds by washed cell suspensions of Thiobucillus ferrvo.riduns. Cunudiun Journul of Microbiology 12, 957-964. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin-phenol reagent. Journul of Biologicul Chemisrry 193. 265-275. Lundberg P. E.. Bennrt 0. L. and Math& E. L. (1977) Tolerance of bermudagrass selections to acidity. I. EtTccts of lime on plant growth and mine spoil material. Agronomy Journul 69, 9 13-9 16. McGoran C. J. M., Duncan D. W. end Walden C. C. (1969) Growth of Thiobucillus firroosiduns on various substrates. Cunadiun Journul-of Microbiology 15. 135-l 38. Millrr S. D. (1980) Sulfur and hvdronen ion bulfcrina in pyritic strip‘mine’spoil. In Biogebchckry of Ancien,&d Modern Girirunntenrs (P. A. Trudingcr. M. R. Walter and B. J. Ralph. Eds). pp. 537-543. Springer. Berlin.
during Murphy
pyrite oxidatioo
107
J. and Riley J. P. (1962) A modified single solution
method for the determination of phosphorus waters. Analytica Chimica Acra 2l. 21-26.
in natural
Nordstrom D. K. (1982) Aqueous pyrite oxidation and the subsequent formation of secondary iron minerals. In Acid SuIjarc Wcarhering (J. A. Kittcrick. D. S. Fanning and L. R. Hossner, Eds), pp. 37-56. Soil Scimcc Society of American. Madison. Norris P. R. and Kelly D. P. (1982) The use of mixed microbial cultures in metal recovery. In Microbial Inreracrions and Communiries (A. T. Bull and J. H. Slater, Eds). pp. 443-474. Academic Press, New York. Pichtel J. R. and Dick W. A. (1991) Influence of biological inhibitors on the oxidation of pyritic mine spoil. Soil Biology & Biochemistry 23, 109-I 16. Pollard F. H.. Nickless G. and Glover R. 9. (1964) Chromatographic studies on sulphur compounds. Part IV. The decomposition of acidified thiosulphate and polythionate solutions. Journal of Chromatography 15. 518-532. Pons L. J.. Van Breeman N. and Dricssen P. M. (1982) Physiology of coastal sediments and development of potential soil acidity. In Acid Sulfate Weurhering (J. A. Kittrick, D. S. Fanning and L. R. Hossner, Eds). pp. I-18. Soil Science Society of America. Madison. Rich C. 1. and Bamhisel R. 1. (1977) Preparation of clay samples for X-ray diffraction analysis. In Minerals in Soil EnrQironmmfs (J. 9. Dixon and S. B. Weed. Eds). pp. 797-808. Soil Science Society of America, Madison. Roy A. 9. and Trudinger P. A. (1970) Oxidation of inorganic sulphur compounds by microorganisms and plants. In The Biochrmktry of Inorganic Compounds of Sulfur. Cambridge University Press, Cambridge Schnaitman C. and Lundgren D. G. (1965) Organic compounds in the spent medium of F~,rrohocillusfrrro~~.~idun.~. Cunudiun Journul of Microbiology Il. 23-27. Sinha D. 9. and Walden C. C. (1966) Formation of polythionatcs and their interrelationships during oxidation of thiosulfatc by Thiobucillus ferrooxiduns. Cunudiun Journul of Microbiology 12. IO4I - 1054. Stamm H. and Goehring M. (1942) Zur kenntnis dcr polythionsaurcn und ihrer bildung. Zeirschri/ fur Anorgun&he und Allgemeine Chemie 250, 226-228. Tamura H.. Goto K., Yotruyunagi T. and Nagayamu M. (1974) Spcctrophotometric determination of iron with I,IO-phenanthroline in the prcscncr of large amounts of iron(lll). Taiunru 21. 314-318. Unz R. F. and Lundgren D. G. (1961) A comparative nutritional study of three chemoautotrophic bacteria: Ferrobucillus frrrooxiduns. Thiobucillus ferrooxiduns. and Thiobucillus thiooxiduns. Soil Science 92. 302-3 13. Vuorinen A., Hiltunen P. and Tuovinen 0. H. (1985) Speciation of ferrous and ferric iron associated with the indirect bacterial leaching of uranium ore materials. Journal of Fermenrurion Technology 63. 337-342. Vuorinen A.. Hiltunen P., Hsu J. C. and Tuovinen 0. H. (1983) Solubilization and speciation of iron during pyrite oxidation by Thiobucillus ferrooxiduns. Geonricrobiolog) Journal 3, 95-l 19. Whittig L. D. (1965) X-ray diffraction techniques for mineral identification and mineralogical composition. In hferhods of Soil Anul.vsis. Purr I (C. A. Black. Ed.), pp. 681-687. American Society of Agronomy. Madison.
Copyrights
0038-0717/91 s3.OO+o.w 1991PergamoaPre~pk
SULFUR, IRON AND SOLID PHASE TRANSFORMATIONS DURING THE BIOLOGICAL OXIDATION OF PYRITIC MINE SPOIL J. R.
PICHTEL*
and W. A. DICK
Department of Agronomy, Ohio State University/Ohio Agricultural Research and Development Center. Wooster, OH 44691. U.S.A. (Accepted 25 September 1990)
Summary-Spoil material composed of pyrite, coal and rock was ground and inoculated with spoil microflora and monitored for pH. S@,-. reduced S compounds, total soluble Fe and Fe(H). A ‘I-day lag in production of both SG- and total soluble Fe was observed followed by a marked increase in concentration. Concentrations of w-. total soluble Fe and Fe(U) after 28 days were co 81. 91, and 59 mmol kg-’ spoil, respectively. Reduced S anions including thiosulfate. trithionate and tetrathionatc were detected in the spoil at all samplings during a 28day incubation. Final trithionate concentrations were greatest (1.07 mmol kg-’ spoil). Scanning electron microscope observations of the pyrite nvealcd a gradual pitting and erosion of particle faces after prolonged incubation. Certain faces appeared to have been preferentially weathered.
INTRODUClION
Surface mining of certain coal seams in the eastern portion of the United States results in the exposure of large quantities of pyrite (Fe&) which is readily oxidized in the atmosphere (Caruccio. 1968). Oxidation of pyrite is a complex process involving several types of oxidation-reduction reactions, hydrolysis and complex ion formation, solubility controls and kinetic effects (Nordstrom, 1982; Pichtel and Dick, 1991). The overall reaction is commonly written as: FeS, + (15/4)0, + (7/2)H,O -. Fe(OH), + 2H2S0, (1) The H,SO, produced can create hostile environments (Blevins et al.. 1970; Lundberg er 01.. 1977; Pons et al., 1982) by inhibiting the establishment and growth of plant cover, thereby resulting in soil erosion and acidification of adjacent soils and waters. Autotrophic bacteria of the genus Thiobacilhts have been studied extensively regarding their contribution to mine spoil acidification. Several species have been isolated from acid mine wastes: T. ferrooxiduns, which oxidizes pyrite, Fe(H) and S, T. thiooxidans which oxidizes pyrite and S. and T. acidophilus which oxidizes S (Unz and Lundgren, 1961; McGoran et d, 1969; Guay and Silver, 1975). These thiobacilli generate energy via the oxidation of reduced S and Fe compounds for the reductive assimilation of CO, and for growth. During S oxidation, various intermediate S compounds are formed (Kelly, 1982) and the environmental significance and fate of these compounds are not clearly understood. In soil or aquatic environments, S and Fe oxidizing microorganisms may occur together, their oxidation *Present address: Department of Natural Raourccs. Ball State University, Muncie, IN 47306. U.S.A.
reactions proceeding simultaneously. Concordant with biological oxidation, however, is abiotic S and Fe oxidation and, if conditions are appropriate, microbially-catalyzed SO:- reduction. The occurrence and quantities of various S compounds in mine spoils, therefore, represent a composite of many different reactions. A further complication to the study of the S, Fe and solid phase reactions of pyritic spoils is that the spoils may be dramatically altered by both biological and chemical mechanisms after only a brief exposure to O2 and water. A better understanding of such reactions at the time of initial exposure is needed. Our objectives were: (i) to assess pH change and production of SOi- and soluble Fe in fresh, inoculated spoils, (ii) to assay for the presence of certain reduced S oxyanions in the oxidizing spoils; and (iii) to observe pyrite surface morphology with progress of biological weathering. MATERIALS AND METHODS
Spoil material
To ensure the ready availability of unweathered spoil, a synthetic spoil composed of equal weights of pyrite, bituminous coal and a rock mixture (shale, sandstone and mudstone) was prepared. The coal and rock fractions were collected from the floor of an active surface mine in southeastern Ohio, sealed in plastic bags, placed into ice chests, and returned to the laboratory for storage at - 12°C. Pure, unweathered pyrite was not available from the mine floor and museum grade euhedral specimens were used. All components were crushed with a hammer, ground with a mortar and pestle, and sieved (< 150 pm). To remove free Fe and S salts present, the spoil was washed with 5 vol of N,-degassed, deionized water, lyophilized in a Labconco freeze dryer, then stored in plastic containers at - 12°C.
102
J. R.
RCHTEL
Fresh spoil samples were brought into solution by sodium carbonate fusion (Dick et uf., 1985). Aliquots were analyzed for Al. Ca. Mg, Fe and Mn by atomic absorption spectrophotometry using a Varian Model AA-475 spectrophotometer; for Ba. Cd, Cu. Ni, Pb, Sr and Zn by atomic emission spectrophotometry with a Jarrel-Ash Model 975 inductively-coupled plasma-atomic emission spectrophotometer; and for P by the calorimetric method of Murphy and Riley (1962) using ascorbic acid as a reducing agent. Total S content was determined in a separate aliquot using the methylene blue method of Johnson and Nishita (1952). Pyritic-S was measured by titrimetric determination of pyritic iron and sulfate-S was measured by precipitation with BaClr to form BaSO, (ASTM, 1976). X-ray diffraction analyses were conducted on the clay-sized fraction of the rock mixture sample and on a finely ground ( c 53 pm) pyrite sample. Sample preparation was as described by Kinter and Diamond (1956). Kittrick and Hope (1963), Rich and Barnhisel (1977) and Whittig (1965). A Philips XRG-3100 generator with a Cu and a DMS41 control panel was used for the X-ray studies. Surface morphology changes of the pyrite component of the spoil, with progress of bactcrially-mcdiatcd weathering. was monitored by scanning electron microscopy. I g pyrite (< I50 pm) was added to a 250 ml Erlcnmcycr flask containing 100 ml dcionizcd water and inoculated with IO ml of T. J2rrooxiduns strain TFP-I [containing co IOpg ml-’ bacterial protein (Lowry et al.. 1951)], cultured as dcscribcd by Norris and Kelly (1982). Flasks were shaken (140 rev min-‘) on a rotary shaker at room tcmpcraturc. Scanning clcctron micrographs wcrc taken of fresh (non-inoculated) pyrite, and after 28 and 70 days of cxposurc. The solids were removed by centrifugation (12.000~) and freeze-dried. Samples were mounted on an AI stub with double-stick tape and coated with a 20 nm-thick gold-palladium alloy in a vacuum evaporator. Electron micrographs were taken with an International Scientific Instruments Model ISIelectron microscope. Spoil incubations
Fresh spoil (I g), deionized water (100 ml), and inoculum were added to 250 ml Erlenmeyer flasks. The flasks were plugged with polyethylene and kept at room temperature on a rotary shaker (140 rev min-‘). The inoculum for all exposures of the fresh mine spoil was 10 mg of weathered spoil (< ISOpm) collected from the surface of an abandoned coal mine site located in Southeastern Ohio. Initial pH was 6.0. All work was conducted in triplicate. Periodically. three replicate flasks were removed from the shaker and the suspensions were filtered through Whatman No. 42 filter paper. The pH of each sample was recorded and IO ml aliquots were frozen at -12°C. All samples were analyzed for various chemical properties at the same time after thawing in an N, atmosphere. intermediates (thiosulfate, Sulfur oxidation trithionate and tetrathionate) were determined by the calorimetric procedure of Kelly et al. (1969). Na-thiosulfate (Fisher Scientific), K-tetrathionate (Kodak), and K-trithionate, prepared by the
and W. A. DICK method of Stamm and Goehring (1942). were used as standards. Sulfide was determined as HrS by the methylene blue procedure (Johnson and Nishita. 1952). Sulfite and sulfate were estimated by ion chromatography using a Dionex Model I4 ion chromatograph. Samples, preserved with 0. I ml cont. HNO, IOml-’ aliquot, were assayed for total soluble Fe by atomic absorption spectrophotometry. Fe(B) was measured using a calorimetric method which is accurate in the presence of large amounts of Fe(III) (Tamura er al.. 1974) and Fe(III) was calculated as the difference between total and Fe(H). Deionized water degassed with N: was used for dilutions. RESULTSAND
DlSCUBSlON
The synthetic spoil contained 103 and 179 g kg-’ total S and Fe, respectively (Table I). Pyritic- and sulfate-s comprised 25.2 and 0.13%. respectively, of the total S fraction. The pyrite fraction of the spoil was essentially pure. as X-ray diffractograms revealed only a trace of contamination. possibly by quartz. Minerals in the spoil, as determined by X-ray analysis, included kaolinite. clay mica, vermiculite. chlorite, quartz and gocthite. Secondary minerals characteristic of a weathered mining environment. such as jarositc and ferric sulfates and hydroxides. were not observed due to the unwcathcred state of the spoil. Fresh. unwcathercd pyrite appeared as irregular nonporous grains with smooth surfaces [Fig. l(a)]. After 28 days of weathering by T. ferrooxiduns. surface erosion and fissures bccamc evident due to acid etching and oxidation and solubilizttion by ferric iron (Nordstrom, l9g2) [Fig. l(b), (c)l. Copious
quantities of bacterial cells were attached to particle surfaces. After 70 days particles were severely eroded or pitted and most surfaces had become highly irregular [Fig. l(d), (e)]. Surface erosion such as that shown in Fig. l(b) and I(c) may be partially due to particle abrasion during shaking of the suspensions. However, the deep pitting and fissures observed after 70 days [Fig. l(d) and l(e)] are presumed to be the result of the activity of T. ferrooxiduns. Table I. Chemical analysis of the synthetic spoil Element
Concentration B kg-’
FC s AI Ca Mg al
179 103 32.4 2.50 2.00 I .70 mg kg-’
Mll Pb Ba P CU Sr Cd Ni
527 527 267 265 257 xl 18 13
J. R. PKHTEL and W. A. DICK
Fig. 1, (d-c) Fig. I. !~GIII ning electron micrographs of pyrite (C 150 pm) (a) prior to weathering; (b), (c) after 28 d ay!3 of weathering: (d). (e) after 70 days of weathering.
fnspection of electron micrographs of pyrite weathered for 70 days suggested that particle faces possessed differential weathering tendencies. Certain faces had been considerably transformed while others appeared afmost unaffected. Suscept~biiity to weathering could have been due to the exposure of broken chemical bonds on a particle fact. Sulfate production in the spoil suspension was initially slow (Fig. 2) but began to increase after 7 days and a sharp increase was observed after 14 days. After 28 days ca 7.8% of the total S was oxidized to Soi- and by 38 days Saconcentrations accounted for 35.6% of the total S. Although 25.2% of the original S was in the pyritic form. thiobacilli and other spoil microorganisms could have oxidized various inorganic S compounds (e.g. thiosulfate and polythionatcs) and organic sulfides of biogenic origin in addition to pyritic S (Kelly, 1982).
The pH of the inoculated spoil suspension decreased from 6.0 to 3.7 after 28 days of exposure (Fig. 3). It declined further to pH 3.0 after 38 days and to 1.9 after 150 days. This decline would probably have been more rapid had the solution been replaced periodically with fresh deionized H,O because T. ferrooxidans is known to excrete organic compounds (Schnaitman and Lundgren, 1965) which slows autotrophic growth and metabolism. A negligible decline in pH occurred when sodium azidc (IO-‘M) was added to the spoil suspensions. This indicates that pyrite oxidation, at lcast initially, was due to biological activity rather than physicochemical processes. Soluble sulfide or sulfite were not detected at any of the sampling times, which included times as short as 2 h. They were either not produced during exposure or were transformed rapidly to other S species. Both sulfide and sulfite are unstable in
105
Chemical transformations during pyrite oxidation 100
;,,
,
I
I
I
I
._ i
75
-
I 3 cn al ' 4 a
50-
'-j ul 1
25-
z
o1
I
I
I
I
0124
I
I
I
I
7
14
21
28
Time
of Incubation
(days)
Fig. 2. Changes in sulfate-s concentrations of inoculated synthetic spoil suspensions during 28 days of exposure. LSD,,,, values are for comparison of concentrations in the presence or absence of the microbial inhibitor, NaN, (IO-‘M).
acidic solutions, being spont;~ncously oxidized in the prcscncc of oxygen (Roy and Trudingcr, 1970; Goldhabcr. 1980). Thiosulf’atc. trithionatc and tctrathionntc wcrc dctcctcd throughout the B-day cxposurc (Fig. 3). After an initial lag. maximum concentrations of thiosulfatc (0.97 mmol kg-’ spoil) and tctrrrthionatc (0.26 mmol kg-’ spoil) were obtained on day 7. Both ions
dcclincd in concentration beyond day 7. while trithionatc incrcascd sharply after day 14. It is possiblc that thiosulfatc or tctrathionatc may have been precursors to trithionatc during spoil oxidation as their diminution is coincident with the incrcasc in trithionatc content (Kelly and Syrctt. 1966; Landcsman CI ol., 1966; Sinha and Walden. 1966; Kelly. 1982). Howcvcr. in contrast, trithionate may
-l
7
6
5
PH
0124
7
Time
21
14
of
Incubation
28
(days)
Fig. 3. Changes in ptl and production of the sulfur oxyanion intermediates thiosulfate. trithionate tctrathionatc in inoculated spoil suspension during 28 days of incubation.
and
106
J. R. Ptcnrr.t and W. A. DICK
100
0
0124
7
14
Time
of Incubation
21
(clays)
Fig 4. Iron solubilization in inoculated spoil suspensionsduring 28 days of exposure. LSD,,, values are of total Fe concentrations in the presence or absence of the microbial inhibitor, NaN, (IO ’ M).
for comnarison
considcrcd a precursor to thiosulfatc and tctrathionatc. Trithionatc accumulation in the spoil mixture would then be a result of inhibition of the transformation process of trithionatc to thiosulfatc and tctrathionatc. Also, during the abiotic dccomposition of acidified thiosulfatc. trithionatc was the primary thionatc produced (Pollard PI crl.. lY64). The dcclinc in thiosulfate concentration may be partly attributed to its instability in acidic solutions (Pollard er ul.. 1964). The pH trend, superimposed upon the trend for the sulfur oxyanions (Fig. 3). reveals the presence of increasing suspension acidity with time. Thiosulfate may also be oxidized by iron(II1) hydroxide on the surface of a pyrite particle (Miller, 1980) according to the following equation: bc
2S,Oj-
+ 2Fe(OH),
+ S,Ob+ 2Fe(OH)2
+ 20H
-
(2)
The interaction of soluble reduced S compounds, such as thiosulfate, with metal oxides in spoils may be of practical significance due to their mobilization of potentially toxic metals, such as Fe, Al. Mn. Trithionate and tetrathionatc are stable in acid conditions (Roy and Trudinger, 1970; Goldhaber. 1980). Trends of Fe solubilization in the spoil suspensions (Fig. 4) paralleled those of SOi- production shown in Fig. 2. Fe(II) production remained below dctcction lcvcls until day 7. then increased steadily. Beyond day 28, the Fe( III) form dominated. attaining 182.6 mmol compared to 87.7mmol Fc(ll) kg spoil-’ on day 38 (data not shown). This result agrees with the data of Vuorincn et al. (1983. 1985). who leached both pyrite and uranium arc with T. /‘errooxidans. After several leachings and at a pH cu 2.0. Fe(lII) was always many times higher than that of Fe(ll). In our work. IO-’ M sodium azide was a highly effective inhibitor of Fe
solubilization as pH remained bctwccn 5.5 and 6.2 and iron was not detected in the azide-trcatcd flasks during cxposurc. Biological iron oxidation is a cytochrome-mediated process and azide ion is known to be a polcnl cytochrome inhibitor (Duncan cf ~1.. 1967). The total amount of Fe in solution, i.e. Fe(lI) plus Fe(Ill), was greater than the quantity of SO;--S produced (compare Figs 2 and 4). Different quantities of Fe and S0i-S may have precipitated as oxides or other insoluble compounds such as the mineral hydronium jarosite (HzFe,(SO,),(OH),) (Duncan and Bruyncsteyn. 1971). The incongruity is also due to the presence of non-sulfate S species such as thiosulfate and various thionates in solution. Our results indicate that S and Fe in freshly exposed pyritic spoil is rapidly oxidized by microorganisms resulting in acid production and a sharp decline in pH to a value of I.9 after I50 days. The oxyanions thiosulfate. trithionate, and tetrathionate are produced during the oxidation process. During oxidation T. firrooxidms stems to attack certain faces of the pyrite particles preferentially. Ackno,\,/rclR~~vn/s-We thank Dr 0. H. Tuovinen for supplying T. fcrroo.riduns strain TFP-I and Dr J. M. Bigham for assisting in the mineralogical analyses. Salaries and research support wcrL: provided by State and Federal funds appropriated IO the Agricultural Research and Dcvelopment Ccmcr. The Ohio State University.
REFERENCES ASTM (1976) SrondurJ Test Merhodr for Total Sul’ir in [he Analysis of Cool und Coke. American Sociely of Testing Materials, Annual Book of Standards. D3 177-75. pp. 380-386. U.S. Government Printing Office, Washington.
Chemical
transformations
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during Murphy
pyrite oxidatioo
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method for the determination of phosphorus waters. Analytica Chimica Acra 2l. 21-26.
in natural
Nordstrom D. K. (1982) Aqueous pyrite oxidation and the subsequent formation of secondary iron minerals. In Acid SuIjarc Wcarhering (J. A. Kittcrick. D. S. Fanning and L. R. Hossner, Eds), pp. 37-56. Soil Scimcc Society of American. Madison. Norris P. R. and Kelly D. P. (1982) The use of mixed microbial cultures in metal recovery. In Microbial Inreracrions and Communiries (A. T. Bull and J. H. Slater, Eds). pp. 443-474. Academic Press, New York. Pichtel J. R. and Dick W. A. (1991) Influence of biological inhibitors on the oxidation of pyritic mine spoil. Soil Biology & Biochemistry 23, 109-I 16. Pollard F. H.. Nickless G. and Glover R. 9. (1964) Chromatographic studies on sulphur compounds. Part IV. The decomposition of acidified thiosulphate and polythionate solutions. Journal of Chromatography 15. 518-532. Pons L. J.. Van Breeman N. and Dricssen P. M. (1982) Physiology of coastal sediments and development of potential soil acidity. In Acid Sulfate Weurhering (J. A. Kittrick, D. S. Fanning and L. R. Hossner, Eds). pp. I-18. Soil Science Society of America. Madison. Rich C. 1. and Bamhisel R. 1. (1977) Preparation of clay samples for X-ray diffraction analysis. In Minerals in Soil EnrQironmmfs (J. 9. Dixon and S. B. Weed. Eds). pp. 797-808. Soil Science Society of America, Madison. Roy A. 9. and Trudinger P. A. (1970) Oxidation of inorganic sulphur compounds by microorganisms and plants. In The Biochrmktry of Inorganic Compounds of Sulfur. Cambridge University Press, Cambridge Schnaitman C. and Lundgren D. G. (1965) Organic compounds in the spent medium of F~,rrohocillusfrrro~~.~idun.~. Cunudiun Journul of Microbiology Il. 23-27. Sinha D. 9. and Walden C. C. (1966) Formation of polythionatcs and their interrelationships during oxidation of thiosulfatc by Thiobucillus ferrooxiduns. Cunudiun Journul of Microbiology 12. IO4I - 1054. Stamm H. and Goehring M. (1942) Zur kenntnis dcr polythionsaurcn und ihrer bildung. Zeirschri/ fur Anorgun&he und Allgemeine Chemie 250, 226-228. Tamura H.. Goto K., Yotruyunagi T. and Nagayamu M. (1974) Spcctrophotometric determination of iron with I,IO-phenanthroline in the prcscncr of large amounts of iron(lll). Taiunru 21. 314-318. Unz R. F. and Lundgren D. G. (1961) A comparative nutritional study of three chemoautotrophic bacteria: Ferrobucillus frrrooxiduns. Thiobucillus ferrooxiduns. and Thiobucillus thiooxiduns. Soil Science 92. 302-3 13. Vuorinen A., Hiltunen P. and Tuovinen 0. H. (1985) Speciation of ferrous and ferric iron associated with the indirect bacterial leaching of uranium ore materials. Journal of Fermenrurion Technology 63. 337-342. Vuorinen A.. Hiltunen P., Hsu J. C. and Tuovinen 0. H. (1983) Solubilization and speciation of iron during pyrite oxidation by Thiobucillus ferrooxiduns. Geonricrobiolog) Journal 3, 95-l 19. Whittig L. D. (1965) X-ray diffraction techniques for mineral identification and mineralogical composition. In hferhods of Soil Anul.vsis. Purr I (C. A. Black. Ed.), pp. 681-687. American Society of Agronomy. Madison.