Author Institution: Department of Bacteriology, University of Toledo, Toledo, Ohi

Joseph, J. Mehsen

The Ohio Journal of Science

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MICROBIOLOGICAL STUDY OF ACID MINE WATERS:PRELIMINARY REPORTJ. MEHSEN JOSEPHDepartment of Bacteriology, University of Toledo, Toledo, OhioWaters issuing from bituminous mines may be either acid or alkaline. Theacid mine waters are objectionable; their disposal or abatement is highly desirable.Many agencies are concerned in the reduction of this important industrial wastefor several reasons: pollution with acid wastes renders streams extremely acidic,pH 2.0 to pH 4.0, which results in the annihilation of the majority of commonlyencountered micro- and macroorganisms in water; high concentrations of ferric,ferrous, and sulfate compounds are hazardous to children using such streams asbathing areas; the aesthetic quality of these streams is in a deplorable conditiondue to the deposition of ferric hydroxide in the channels of the streams.FORMATION OF ACID MINE WATERThe oxidation of sulfur and sulfur-bearing compounds in coal seams resultsin the formation of large quantities of acid compounds available for solution inwater. It is assumed that ferric disulflde is oxidized to soluble ferrous sulfateand sulfuric acid:2FeS2 + 702 + 2H2O > 2FeSO4. (1)When these acid wastes enter streams they are exposed to atmospheric conditionsand atmospheric oxidation of ferrous sulfate to ferric sulfate and water occurs:4FeSO4 + 2H2SO4 + O2 > 2Fe2(SO4)_3 + 2H2O. (2)The subsequent hydrolysis of ferric sulfate yields ferric hydroxide:Fe2(SO4)3 + 6H2O -> 2Fe(OH)3 + 3H2SO4. # _ (3)Ferric hydroxide in conjunction with hydrated iron oxides is precipitated in thestream's course to impart a rusty color.Carpenter and Herndon (1933) and Hodges (1938) have reported analyses ofthis type of drainage which vary widely from one stream to another. The analysisof a stream gave the following results; pH 2.0; total acidity, 47,800 ppm; sulfates,41,700 ppm; total iron, 12,270 ppm. The author has reported the followingvariations for streams having a pH range from 2.0 to 2.9: total acidity, 196-106,000ppm; total residues, 7,700-40,000 ppm; calcium, 145-300 ppm; magnesium,20-55 ppm; sulfate, 100-18,300 ppm; ferric iron, 30-3,900 ppm; and ferrous iron,20-1,200 ppm.Numerous attempts have been made to abate or modify acid drainage issuinginto many streams. Sealing of abandoned mines has been instituted to reduce theoxygen concentration with the hope that less oxidation of sulfur compounds willensue, resulting in a smaller amount of available acid compounds for solution inwater. Chemical treatment to modify or rid water of objectionable compoundshas been studied. Since such treatment is expensive and results in the accumula-tion of large amounts of sludge, it is not feasible.Colmer and Henkle (1947) have approached the problem from a micro-biologicalpoint of view. They maintain that microorganisms are in part responsible forthe formation of large quantities of sulfuric acid and ferric hydroxide. Therefore,studies were undertaken to determine the role of microorganisms in the formationof acid mine waters. They were able to isolate two significant organisms; one wasan unidentified bacterium involved in the oxidation of ferrous sulfate to ferricsulfate, and the other was Thiobacillus thiooxidans which is capable of oxidizingsulfur compounds to sulfuric acid. It is suggested that the study of the relationshipTHE OHIO JOURNAL OF SCIENCE 53(2): 123, March, 1953.124 J. MEHSEN JOSEPH Vol. LIIIof microorganisms to the formation of sulfuric acid might provide a more effectivemethod for the abatement of objectionable acid wastes.Since conditions of extreme acidity and hyperconcentration of iron compoundsprovide a profound adverse environment for the survival of a great majority ofmicro- and macroorganisms indigenous to streams, the author has instituted theinvestigation of the diversity of genera and species tolerating such conditions.In an attempt to review the literature pertaining to the microbiological assayof acid mine waters it becomes apparent that such information is entirely lacking.A few theses and articles of many kinds were examined, but data pertinent to theauthor's work are not accessible.These studies were first begun to determine the viability of Escherichia coli,the organism used as the index of fecal pollution, in acid mine streams, but werelater merged into a study of all viable microbes. The task at hand was to isolate,cultivate, observe, and attempt identification of the various kinds of organismscapable of tolerating conditions existing in acid waters and to observe the director indirect effects of acid waters upon the existing microorganisms. A preliminarystudy was also made for the special groups, i.e., colon-typhoid, sulfur bacteria,iron bacteria, actinomycetes, and fungi.MATERIALS AND METHODSMicroflora. Samples were collected from acid streams in northern West Virginiaand Pennsylvania in previously sterilized (autoclaved) sampling bottles by sub-merging the mouths of the bottles against the current of the stream (most asepticalmethod), and filled to capacity to eliminate any air space. The presence ofsufficient oxygen would result in the oxidation of ferric sulfate to ferric hydroxideand sulfuric acid. Analyses of all samples were made as soon as possible aftercollection.The above water samples were plated on various agars suspected of supportingthe growth.of a few or a single organism. Various types of broth combinationswere inoculated with aliquots of mine water. Streaks were made for these brothtubes onto agar in an attempt to procure pure cultures of the growing organisms.Pure cultures were numbered and transferred to fresh media at intervals in orderto maintain identity and viability. The preparation of suitable media proved tobe the most difficult task;In order to isolate special group organisms, elective cultural methods wereemployed. All waters were inoculated into the synthetic medium of Waksmanand Starkey (1922) for the cultivation of sulfur bacteria, artifical mine watermedium for the cultivation of iron bacteria, peptone malt agar for fungi, andpotato glycerol agar for actinomyces.Diatom study. In collecting samples of diatoms, pyrex resistant glass wasused in order to reduce the deteriorating effects induced by the absorption ofalkali from ordinary glass. On each trip to the mine streams definitely markedspots were examined continually so as to thoroughly study the algae present.Subsequently, various other areas were picked and thoroughly studied. After agreat number of samples were collected from numerous areas, no cross-sectionaldata of the diatom flora were obtained. Precise notes were taken as to the timeof sampling, the place and the exact spot so that in case further study is necessitatedone may return to the exact location for sampling. All of the above data wereplaced on each sampling bottle, and if worthy of preservation, it was recordedin a record book. Samples were taken by the removal of surface mud and scrapingfrom rocks and twigs on the stream bed.Of great importance is the immediate examination of samples, since algae tendto deteriorate rapidly. If samples were to be stored for any length of time, specialmethods of preservation were employed, namely, fixation with King's fluid, picro-sulfuric acid, or osmic acid. One of the best methods for the study of livingNo. 2 ACID MINE WATERS 125diatoms is by the hanging drop mount preparation. Motility and similar functionsperformed by delicate structures are very difficult to preserve, thus, immediateobservation is recommended and almost indispensable.The internal structure of diatoms was not considered in their identification.However, the ornamental sculpturing of their cell wall, their shape, and theirsize were utilized to accurately determine genus and species names.The method employed for elucidating cell wall sculpturing was the siliceouscorrosion method. This procedure may be outlined briefly. Treat the materialcontaining diatoms with concentrated HCl until effervescence ceases. Allowmaterial to remain in HCl for two days to remove any debris adhering to thediatoms, and then filter through coarse muslin. Centrifuge the filtrate and washthe remaining precipitate several times with water. The washed precipitate istreated with concentrated H2SO4 and a small crystal of K^C^Oy to char or dissolveany organic material present. Repeat the H2SO4 -bichromate treatment severaltimes. When the diatoms are scrupulously cleaned, they are infiltrated withCanada balsam and mounted on microscope slides. The slides thus prepared areused for taxonomic study.Microfauna. Isolation and identification of the organisms comprising themicrofauna were based entirely upon the microscopic study of cellular morphology.Various water samples, atrocious surface deposits, and surface soils were collectedand examined. Cultivation of this group was not attempted, since microscopicobservation proved sufficient for identification. Seasonal examinations were madein order to account for any possible variation of the microfauna with the season.RESULTS AND DISCUSSIONMicrofloraBacteria. By the exploitation of every method for isolation, either original orduplicated, 40 species of bacteria were obtained in pure cultures. Of the 40species, 14 were gram positive, spore-forming rods belonging to the genus Bacillus,2 were gram-negative rods belonging to the genera Escherichia and Aerobacter, and20 were gram-positive cocci belonging to the genera Micrococcus and Sarcina.The remaining species were members of the genera Thiobacillus, Crenothrix, andMicrosporium.Colmer and Henkle (1947) have reported frequent isolations of Thiobacillusthiooxidans from acid mine streams which provide confirmative evidence. Ellison(1919) reported the isolation of Microsporium desulphuricans and Vibrio desulph-uricans from surface mud in acid mine water.Table 1 illustrates very clearly the presence of bacteria in a paucity of numbersin acid mine streams, and table 3 shows that the members are equally dividedbetween the genera Bacillus and Micrococcus.Fungi. It is interesting to note that fungi are not as well represented as onewould anticipate in view of the existing acid conditions conducive to their growth.Organisms of the genera Aspergillus, Helminthosporium, Penicillium, Trichoderma,Cladosporiutn, Alternaria, and Trichothecium were isolated and identified. Therelative quantitative plate counts for fungi and bacteria may be seen in table 1.Actinomyces. Members of the genus Actinomyces were not isolated directlyfrom acid mine streams, but the examination of soils moistened with mine waterseepage showed their presence.Diatoms. Thorough investigation of the abominable brownish and greenish-brown deposits on the stream beds revealed the presence of myriads of diatomshaving an almost ubiquitous distribution. Navicula viridis appeared to be theonly existing form, but by very meticulous and effectual methods many othergenera and species were identified. The multitude of diatoms in every acidmine stream examined has led the author to suggest that these organisms may be126 J. MEHSEN JOSEPH Vol, LIIIused as an index of acidity. A total of 8 genera and 18 species of the class Bacil-larieae have been characterized, and are recorded in table 2. It is beyond doubtthat diatoms are the most abundant forms of life to be found in acid mine streams.TABLE 1Quantitative plate counts for acid mine streams and soils along their banks.Stream pHBacterial Countper mlFungal Countper mlActinomyces Countper gm of soilABCDEFGHIJ2.02.42.52.83.03.153.33.94.04.04,80018,00039,00024,000180,000150,00083,000470,000560,000340,00090,00060,000120,000118,000160,000190,000180,000110,000590,000640,0003,6006,40013,0007,00013,00021,0007,50016,00026,00018.000TABLE 2Diatoms isolated from acid mine streams which have been identified.1.2.3.4.5.6.Navicula viridisNavicula lanceolataNavicula oblongaNavicula spp.Navicula spp.Navicula spp.7.8.9.10.11.12.Surirella biseriata 13.Surirella saxonica 14.Denticula spp. 15.Nitzschia sigmoidea 16.Homoeocladia vermicularis 17.Homoeocladia spp. 18.Cocconeis placentulaCocconeis pediculusCocconeis spp.Synedra ulnaMeridion circulareRhoiosphenia curvataTABLE 3Microorganisms isolated from acid mine waters.BACTERIA:FUNGI:ACTINOMYCES:DIATOMS:PROTOZOA:TROCHELMINTHES:Bacillus subtilisBacillus spp.B. lentusB. cereusB. mycoidesMicrococcus spp.M. pyrogenes var. albusAspergillus sp.Trichoderma sp.Helminthosporium sp.M. pyogenes var. aureusM. variansM. desulphuricansEscherichia coliAerobacter aerogenesThiobacillus thioparusT. thiooxidansCrenothrix polysporaAlternaria sp.Penicillium sp.Trichothecium sp.Cladosporium sp.Several unidentified species(see table 2)Amoeba proteusEuglena viridisRotifersE. mutabilisParamecium caudatumMicrofaunaAnimal life fares no better in these acid streams, since only the phyla Protozoaand Trochelminthes were identified.Protozoa were represented by Euglena viridis and Euglena mutabilis of theNo. 2 ACID MINE WATERS 127class Mastigophora, by Paramecium of the class Ciliata, and by Amoeba of theclass Sarcodina. Euglena viridis is per se as abundant as the entire group ofdiatoms, for they are very acid-tolerant organisms. Trochelminthes were foundvery sparingly and only as rotifers. Lackey (1938) has reported the existenceof rotifers, ciliates, and flagellates in acid waters which provides a corollary tothese observations.In passing it might be mentioned that higher animal and plant forms than thosereported here are not to be found in acid mine waters, save a few plant forms.Thus, one can readily see that these waters constitute a very objectionable class.Even though conditions in acid mine streams appear highly adverse for the viabilityof microorganisms, Joseph (1951) has shown by means of longevity studies andactual isolation of Escherichia coli that the transmission of disease is still possible.SUMMARYThe most highly acid streams will support the growth of a few microscopicorganisms.Bacteria are represented by the genera Bacillus, Micrococcus, Sarcina,Escherichia, Aerobacter, Thiobacillus, Crenothrix, and Microsporium. The bacteriaare almost equally divided among the genera Bacillus and Micrococcus, but theirabundance relative to their numbers in alkaline waters indicates an intensivedetrimental effect of acid mine water. This is also borne out by the fact thatvariant or aberrant forms of the surviving organisms are frequently encountered.Escherichia coli is capable of surviving acid mine waters, therefore, its toleranceis indicative of the ability of the typhoid bacillus to also be tolerant and probablypresent.Fungi are relatively abundant and are represented by seven genera. Quanti-tative plate counts for fungi range from 60,000 to 640,000 per ml, but these countare much lower than one would anticipate. It is also interesting to note thatActinomyces were not isolated from acid mine waters, but were present in thesoil along the banks of the streams. Quantitative plate counts for Actinomycesrange from 3,600 to 26,000 per gram of soil.Diatoms are very abundant in acid mine streams, and are represented primarilyby Navicula viridis. The author suggests the use of this organism as an index ofacid conditions, since little skill is required for its recognition. A total of 8 generaand 18 species of diatoms have been isolated from various acid streams andidentified.There is a great reduction in the microfauna of acid mine waters, since theyare shy of every phylum, except for a few members of the phyla Protozoa andTrochelminthes. Protozoa are represented by Euglena viridis, Euglena mutabilis,Amoeba, and Paramecia. Euglena viridis is the most abundant organism, and itsassociation with acid waters indicates its use as an index for acidity. Trochelminthesare represented only by one member, rotifers.REFERENCESCarpenter, L. V., and L. K. Herndon. 1933. Sterilization of sewage by acid mine drainage.W. Va. Univ. Eng. Exp. Sta. Bull. No. 10.Colmer, A. R., and M. E. Henkle. 1947. Study of acid mine drainage from bituminous mines.W. Va. Univ. Agri. and Eng. Exp. Sta. Bull. No. 373.Ellison, D. 1919. Iron Bacteria. Frederick A. Stokes Co., New York.Hodges, W. W. 1938. Study of acid mine drainage. W. Va. Univ. Eng. Exp. Sta. Bull.No. 18.Joseph, J. M. Viability of E. coli in acid mine water: an indirect study of disease transmission.Jour. Amer. Pub. Health. To be published.Lackey, J. B. 1938. Flora and fauna of acid mine waters. Pub. Health Reports, 53: 1499-1507.Waksman, S. A., and R. L. Starkey. 1922. Cultivation of sulfur bacteria. Jour. Gen.Physiol., 5: 285.

Microbiological Study of Acid Mine Waters : Preliminary Report

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Microbiological Study of Acid Mine Waters : Preliminary Report

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