JOURNAL OF BACTERIOLOGY, May 1968, p. 1495-1503Copyright © 1968 American Society for Microbiology

Activity of Microorganisms in Acid Mine Water

I. Influence of Acid Water on Aerobic Heterotrophs of a Normal Stream

JON H. TUTTLE, C. I. RANDLES, AND P. R. DUGAN

Faculty of Microbial and Cellular Biology, The Ohio State University, Columbus, Ohio 43210

Received for publication 21 February 1968

Comparison of microbial content of acid-contaminated and nonacid-contami-nated streams from the same geographical area indicated that nonacid streamscontained relatively low numbers of acid-tolerant heterotrophic microorganisms.The acid-tolerant aerobes survived when acid entered the stream and actuallyincreased in number to about 2 X 103 per ml until the pH approached 3.0. Theorganisms then represented the heterotrophic aerobic microflora of the streamscomprised of a mixture of mine drainage and nonacid water. A stream which was

entirely acid drainage did not have a similar microflora. Most gram-positive aerobicand anaerobic bacteria died out very rapidly in acidic water, and they compriseda very small percentage of the microbial population of the streams examined.Iron- and sulfur-oxidizing autotrophic bacteria were present wherever mine waterentered a stream system. The sulfur-oxidizing bacteria predominated over ironoxidizers. Ecological data from the field were verified by laboratory experimentsdesigned to simulate stream conditions.

High concentrations of iron, sulfate, and hydro-gen ions in streams located in coal-mining regionsare attributed to oxidation of pyritic mineralsassociated with coal. The oxidation of reducediron and sulfur compounds, particularly pyriteand marcasite, was associated with metabolicactivities of certain related chemosynthetic auto-trophic bacteria (3).The autotrophic bacteria in the Thiobacillus-

Ferrobacillus group are responsible for enzymaticoxidation of ferrous ions and reduced sulfur com-pounds with concomitant production of ferric,sulfate, and hydrogen ions. Aspects of the energymetabolism of iron-oxidizing autotrophs havebeen reported by Dugan and Lundgren (4). Therole of reduced sulfur as an energy source forautotropic bacteria has been reviewed by Peck(8). Silverman and Ehrlich have surveyed andreported microbial interactions with minerals(11).

Sulfuric acid and ferric ions thus producedhave a deleterious influence on the heterotrophicbiota of streams that receive the mine drainage.Ecological reports (2, 6, 7, 10) have indicatedthat H2SO4 could cause the killing of the normalmicroflora of affected waters and that aciduricspecies, notably fungi, appeared to thrive. Ehrlich(5) reported the isolation of large numbers ofRhodotorula from acidic copper mine effluentsand described an ecological relationship betweenprotozoa and Thiobacillus species which he cul-tured from the water.

This paper describes an investigation of themicrobial ecology of acid mine water, in additionto a study of the influences of acid mine water onthe microflora of a nonacid-polluted stream.During the investigation we have considered totalnumbers of organisms without species differ-entiation based on the thesis that it is totalphysiological activity which contributes to thesuccession of events that constitute an ecosystemand that microbial activity is a primary consid-eration in re-establishing the biota of a "healthy"stream.We used two separate investigative approaches.

The first approach was a field survey, which was

conducted for the purpose of correlating bacterialtypes and numbers with chemical components inthe stream. The second approach was to developan artificial stream system in the laboratory. Thelaboratory system would approximate streamconditions and allow a measure of control overvariables that cannot be controlled in the field.Variables such as temperature, stream flow,aeration, and organic content of water all in-fluence the number of microorganisms per unitvolume of water and must be controlled beforeobservations in the natural habitat can be inter-preted.

Figure 1 is a schematic diagram of the streamsystem that was studied in the field. This systemwas divided into five sampling stations. Samplesite B was highly acid water resulting from

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TUTTLE, RANDLES, AND DUGAN

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Wade Pile 4 B'II

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FJG. 1. Schematic diagram ofthe stream system. The system is divided into five sampling stations. A representsa waste pyritic coal slag pile. B is a highly acid stream emanating by drainage from A. C is acid water which isdiluted by water from other streams. D is a nonacid stream in the same geographical area. E is referred to as amixed stream resulting from C and D.

autotrophic bacterial activity present in a largepile of low-grade coal and pyritic mineralslocated at position A. This mineral waste heap isoften referred to as a "gob" pile. The porousmineral deposit is easily permeated with air andrain water which made it suitable for growth ofiron- and sulfur-oxidizing bacteria. Water flowingfrom B became somewhat diluted downstream atpoint C because it mixed with additional acid andnonacid drainage. An essentially neutral stream(pH 6.2 to 8.2) in the same geographical regionis shown at D. Stream D flows into the dilutedacid stream C and becomes stream E. E is re-ferred to as a mixed system.

MATERIAL AND MErHODS

Samples. Water samples were taken in the field insterile 8-oz (237 ml) bottles and held in a Styrofoamcabinet until they reached the laboratory and werethen refrigerated at 8 C. All water samples were platedon bacteriological culture media within 24 hr afterthey were taken from the stream.

Chemical determination. Total dissolved iron wasmeasured colorimetrically by the phenanthrolinemethod according to the procedure described for aHach Field Kit (Hach Chemical Co., Des Moines,Iowa). Sulfate was determined turbidimetrically withBaSO4 precipitate as described by the Hach procedure,andpH was determined with a Beckman pH meter.

Media and growth conditions. Samples (1 ml) ofsuitable dilutions were pour-plated on Tryptone Glu-cose Extract (TGE) Agar (Difco), which was supple-

mented with 0.5 g of yeast extract per liter (TGYE) andon Sabouraud Dextrose (SD) Agar (Difco). The cul-tures were incubated at 25 zt 2 C in the air for 3 days.

Anaerobic microorganisms, other than Desulfovi-brio, were determined by adding 1.0-ml portions of asingle-tube dilution series to 9.0 ml of ThioglycollateMedium (Difco). The reciprocal of the highest dilutionthat showed growth after 7 days at 25 2 C, as deter-mined by appearance ofturbidity in the anaerobic zoneof the tubes, was taken as the number of organisms.

Sulfate-reducing bacteria were enumerated with astandard three tube most probable number (MPN)method (1), according to the tube culture techniquedescribed by Postgate (9), with Desulfovibrio desulfuri-cans medium no. 3. Positive tubes were black after in-cubation at 25 ± 2 C for 6 to 21 days.

Iron-oxidizing chemoautotrophic bacteria werecounted by a five tube MPN technique in the saltsmedium of Silverman and Lundgren (12). Positivetubes were determined after 15 days at 25 :1: 2 C bythe presence of a dark red-brown precipitate. Uninocu-lated control tubes and those in which growth did notoccur contained a light yellow-brown precipitate atthe end of the incubation period due to autooxidationof ferrous iron.

Sulfur-oxidizing chemoautotrophic bacteria wereenumerated in the same manner as were the iron oxi-dizers. Elemental sulfur (0.1 g per 10 ml) replaced theFeSO,&7HOH in the culture medium, and positivetubes were taken as those in which sufficient acid wasproduced to cause five drops of a 1% thymol bluesolution to turn red (red pH 1.2 to yellow 2.8). Con-trol tubes and negative growth tubes gave a yellow in-dicator reaction.

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MICROORGANISMS IN ACID MINE WATER

Artificial stream system. The apparatus shown inFig. 2 was assembled to allow for mixing of acid-con-taminated stream water and neutral stream waterusing controlled flow rates and mixing conditions inan attempt to simulate field conditions. Glass bottles(5 gal) were fitted with 3.0 mm outer diameter glasstubing. A peristaltic pump was calibrated to deliver areproducible flow of water from the reservoir bottlethat contained acidic mine water taken from the fieldat location C (Fig. 1) into the receiving bottle whichwas filled to 10-liter capacity with neutral streamwater taken from the field at D. The receiving bottlewas aerated through a glass-frit dispersion tube tomaintain aerobic conditions and to facilitate mixing.The pumping rate was standardized at 180 ml/hr. Thereceiving bottle was filled after 3 days of elapsed time.The system was maintained at ambient temperature(25 47 2 C).A duplicate control apparatus was used simul-

taneously in which neutral water from station D (Fig.1) was pumped into neutral water. This control ac-counted for changes in microbial populations in thereceiving bottle which were not attributable to theaddition of acidic water.

Subsequent variations in water-mixing procedurewere used. Neutral water was pumped into acid water,and, in this case, acid water was added to acid wateras a control. This control accounted for changes inmicrobial populations in acid water which were notattributable to dilution by neutral water or a pH rise.

Samples were taken at the time pumping com-menced (0 time) and at random intervals over a 3-dayperiod from all receiving bottles (i.e., two samplebottles plus two control bottles) during each experi-mental run.

Sulfate, iron, and hydrogen ion concentrations weredetermined on all samples by the techniques previouslydescribed.

Portions (1 ml or appropriate dilutions) of eachsample were pour plated on SD Agar and TGYEAgar. All plates were incubated at 28 2 C in air for3 days, and colonies were enumerated.

Fresh water was brought to the laboratory from thefield for each experiment. Therefore, control valuesdiffered in each experiment (Fig. 3 and 5, and Table1) because of changing conditions in the field.

Buffering capacity of neutral water in the mixedstream system is demonstrated in Fig. 4. A 100-mlsample of water obtained at site D (Fig. 1) was ti-trated with 0.02 N H2SO4 .

Figure 5 illustrates the changes in pH and dissolvediron and sulfate in mixed water when neutral waterwas added to 10 liters of acid water at the rate of 180ml/hr for a 3-day period.The survival of heterotrophic aerobic microorga-

nisms recovered on TGYE and SD Agar while eitherneutral water was added to 10 liters of acid water atthe rate of 180 ml/hr or acid water was added to neu-tral water at the same rate is shown in Fig. 6.

RESULTS

An initial survey of acidic streams over a sev-eral square-mile area indicated that bacterial andyeast populations at the various sites examinedwere similar with respect to types of colonies onthe plating media used.The values (Table 1) represent a compilation

of data taken at the locations shown in Fig. 1.The data represent an average of a minimum of

AirI

WaterFlow _-Vent

| %- SamplingDuct

Receiving Bottle

FIG. 2. Apparatus used as an artificial stream system to simulate the mixing ofacidic water with neutral water.

Reservoir Bottle

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%/ Millimol~~~~~~~~~~~~~~~eutr+dleWtoe0r M f oe

14 __N__

2g/-T-4| | < six vallles. Compar~~~~ieWater misofhteotO ___ __in_water as acidity Hcasedtomlocater

4 waterand.neutralstreamwytmater whows tihate wthe 0ctua

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- _ _ water andtneutralstrat wer w,entierate witrehse.-0 - in water as acidity increased from locations D to

C to B.Figure 3 presents data obtained from the arti-

500 ~~~~~~~~ficial stream system and shows that the actualpH of the mixture, when acid water (C) wasadded to neutral water (D), did not decrease as

__--__ - - ~rapidly as predicted by simple dilution (theo-retical curve). The theoretical curves plotted inFig. 3 and 5 show anticipated ion concentrations

300 - - - in the receiving bottles. All theoretical curveswere calculated from equation 1. The theoretical

00~t/I Iion concentration (equation 1) in receiving bottle200- - - - is equal to the sum of two determinations: (i)

average concentration in reservoir bottle fromT T-10 hr to T hr times liters of water pumped'°° - - - during 10 hr divided by total liters of water in

receiving bottle at T hr, plus (ii) theoretical con-centration in the receiving bottle calculated at

0 lo 20 30 40 50 60 70 0 the previous time interval (T-10 hr) times litersTime in roor

3. Changes in (A) pH, (B) dissolved iron, and water control; *, mixture resulting from addition ofdlfate in an artificial stream system when acidic acid water to neutral water; A, calculated theoreticalwas added to neutral water. Symbols: o, neutral curve; *, acid water control.

FIG(C) Slwater

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- - - - - of water in receiving bottle at T-10 hr divided bytotal liters of water in receiving bottle at T hr.

DiSCUSSIONThe data (Table 1) suggest that the hetero-

trophic microflora, which can be recovered froman acid stream on an acid-growth medium (SDAgar), probably represents transient organismswhich are not indigenous to the acid stream. Forexample, an average of 5,776/ml was found in arelatively nonacid stream. When this water wasmixed with acid water in approximately equal

4 volumes, the average recoverable heterotrophicpopulation dropped by 78%. The dilution can beapproximated by the change in sulfate and ironconcentration when stream C mixes with stream

3 - ___ ___ ___ - - D to form E. No significance was attached todifferences between values obtained on TGYEAgar as compared to SD Agar. The low numberof organisms recovered from locations B and C

0o - suggests that a small population of transientsi heterotrophs was tolerant to the acid environ-

0t ment but did not proliferate there.

a \ \ Anaerobic bacteria were present in neutral. 5 - _ _ waters, in small numbers (10 or fewer per ml) in

IL \ _"_diluted acid water (C, E), and absent in highlyc0 _ _ acid water (B); this suggests that they were not-b 4

la t\_acid tolerant. This may be caused by (i) the high_ _ _ Eh of acidic waters which results from high con-

centrations of oxidized compounds, (ii) a lowO2 X_\ concentration of organic material, and (iii) the

2 __ + s _ absence of those microorganisms that utilize___, \ ,=- __ oxygen while metabolizing organic materials. On

Mt:l - o two occasions, the anaerobic species isolatedo _ from diluted acid water included sulfate-reducing

bacteria. Sulfate-reducing organisms were either

c lacking or present in very small numbers in acidwater. This probably precludes biological sulfateremoval from these streams under natural condi-

400 tions; no evidence of the sulfate removal processwas observed.The predominating aerobic heterotrophic

oo_ _<_ ___ microorganisms in the acid water were yeasts and300 ___ ___ - molds with eight different gram-negative bacilli

recovered. The bacilli were generally nonmotile,oxidase-negative, aerobic or facultative species.The bacteria formed white- or cream-colored

0

200 ~<; ~ colonies on the media employed. One aerobicoxidase-positive bacterium was also isolated. Theorganisms were not thoroughly characterized but

e' FIG. 5. Changes in (A) pH, (B) dissolved iron, andz - - (C) sulfate in cn artificial stream system when neutral

water was added to acidic water. Symbols: o, neutralwater control; , mixture resulting from addition ofneutral water to acid water; A, calculated theoreticalcurve; *, acid water control.

30 40 50 60 70 eo

Time in Hours

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TUTTLE, RANDLES, AND DUGAN

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Time In Hours

so

FIG. 6. Enumeration ofheterotrophic aerobic micro-organisms in an artificial stream system as determinedon (A) Tryptone Glucose Yeast Extract Agar and (B)Sabouraud Dextrose Agar. Symbols: o, in mixtureresulting from acid water added to neutral water; *, inmixture resulting from neutral water added to acidwater; ,, neutral water control; *, acid water control.

are tentatively identified as Achromobacter andPseudomonas. No gram-positive bacteria wereever isolated from the acid streams with thetechniques employed. The organisms that were

examined seemed to be transients, entering froma nonacid environment. We must, therefore,presume that the gram-negative bacteria have agreater permeability barrier to hydrogen ions inthe environment which gives the gram-negativessurvival advantage. The permeability barriermay be related to the higher lipid content of thegram-negative cell envelope as compared to thatof the cell wall of gram-positive bacteria.Comparison of the iron- and sulfur-oxidizing

chemosynthetic autotrophic bacteria indicatedthat the sulfur oxidizers were nearly alwayspresent in greater numbers by a factor of 10 thanwere the iron-oxidizing bacteria. Since the ironoxidizers also oxidize sulfur, the counts of sulfur-oxidizing bacteria may represent both types. Thedifference can also be attributed to the increasedenergy available in reduced sulfur compoundsover that of ferrous iron. The concentration ofsulfate was always greater than 10 times that ofiron in the same water. The difference betweensulfate and iron concentrations has usually beenattributed to removal of iron by precipitation asferric hydroxides. However, the observed highsulfate concentration may support the viewsexpressed by Silverman and Ehrlich (11), in thatoxidation of Fe++ will result in formation ofS04-2 from S-2.

Iron and sulfur oxidizers appeared to have apositive correlation with acid concentration. Thepresence of these bacteria in the neutral streamprobably indicates contamination by acid effluentsfrom an unobserved source. The data also suggestthat the autotrophic bacteria are tolerant toaverage pH values up to 6.6. Generally, sulfuroxidizers have a wide pH range.

Values of the various parameters measured inthe field had a wide variance, which can be at-tributed to daily fluctuations in uncontrolledenvironmental variables. For example, rainfallcan cause a sudden increase in cell numbers in astream as a result of runoff from surroundingland. The increase in cell numbers would befollowed by a sudden decrease in numbers be-cause of dilution. This is particularly true of theautotrophic bacteria, which appear to proliferatein the "gob" pile (Fig. IA). The organisms, aswell as their metabolic by-products, acid, sulfate,and iron ions, are flushed out of the waste pileduring a heavy rain causing a sudden surge inconcentration in the stream. If the rainfall is pro-longed, the reservoir in the waste pile is depleted,and the concentrations of acid, sulfate, and ironions are diluted markedly in the stream.A laboratory investigation was conducted for

the purpose of controlling environmental vari-ables to assess more accurately the field observa-tions.

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TUTTLE, RANDLES, AND DUGAN

The actual iron concentration in the receivingbottle did not increase as rapidly as predicted(Fig. 3). Sulfate concentration increased morerapidly than expected, to a maximum of about 65,.g/ml greater than theoretical. An explanation ofthis phenomenon may be that (i) neutral wateracts as a buffer in the mixed water system, (ii)iron precipitates from solution, and (iii) sulfateis formed in the mixed water probably as theresult of oxidation of reduced sulfur compoundscontributed by the neutral water. Figure 4 dem-onstrates a buffering effect by neutral waterover the entire pH range of the titration. How-ever, buffering capacity probably varied withenvironmental conditions in the field. The secondpossibility mentioned above is not unexpectedbecause iron was observed to precipitate asyellow-orange material on the bottom of thereceiving bottle. As indicated in Fig. 3B, ironalso precipitated in the acid water control(reservoir). This factor influenced the actual ironconcentration in the mixture because, as timeprogressed, less iron per hr was added to thereceiving bottle. The calculations involved in thedetermination of the theoretical curve (see equa-tion 1) allow for this factor. The difference be-tween the actual and theoretical iron concentra-tions can then be attributed to precipitation ofiron in the receiving bottle. The unexpected in-crease in the sulfate concentration in the mixture(Fig. 3C) is attributable to changes in the mixtureand not in the acid water contained in the reser-voir bottle. The sulfate concentration in theneutral water control remained constant through-out the experiment. The discrepancy betweenactual and calculated curves may be due to bio-logical, as well as chemical and physical factors.Production of sulfate via microbial oxidation ofreduced sulfur compounds in the neutral wateris a possibility. Sulfate may also be released fromprecipitated ferric iron resulting from variationin pH.When neutral water (D) was added to acid

water (C), the actual pH in the mixture increasedmore rapidly than the theoretical pH (Fig. 5).This is consistent with the observed bufferingcapacity of the neutral water. The flat portion ofthe actual pH curve between 32 and 45 hr prob-ably indicates oxidative production of hydrogenion, which compensates for an anticipated rise inpH caused by dilution by neutral water.The iron concentration (Fig. SB) in the mixed

water (receiving bottle) decreased more rapidlythan predicted by dilution of the acid water withneutral water. Virtually all of the iron in the-mix-ture precipitated as the pH increased. This isprobably due to oxidation of ferrous to ferriciron and the decreased solubility of ferric iron

with increasing pH. The iron concentration ofthe neutral water control remained constantthroughout the experiment. Precipitation oc-curred rapidly during the first 8 hr in the acidwater control and at a slower rate after 8 hr.

After 15 hr, the actual sulfate concentration inthe mixture (Fig. 5C) was greater than could beaccounted for on the basis of dilution. No addi-tional sulfate was produced in the acid wateralone, and the data obtained in this mixed systemare in accordance with the data obtained whenacid water was added to neutral water (Fig. 3C).

In the artificial stream system, the numbers ofmicroorganisms in the neutral water controlsfluctuated with time, although they were gen-erally present at 106/ml or greater. Comparisonof the neutral control curve shown in Fig. 6A(approximately 106/ml) to that in Fig. 6B (ap-proximately 104/ml) suggests that less than 10%of the organisms in neutral water were tolerant tothe acidity ofSD Agar (pH 5.5). This observationwas verified by comparing the sharp drop innumbers of organisms over the first 20 hr whenacid was added to neutral water to the change inpH (Fig. 3A). A plot of the total counts oforganisms versus time appears quite similar tothe pH versus time plot.The numbers of organisms per ml of acid water

control increased during the first 20 hr (Fig. 6B),which shows that the acid water (pH 2.5) wasable to support growth of organisms up to a levelof about 2 X 103/ml. Addition of neutral waterto acid water resulted in organism counts stabi-lizing at about 5 X 104/ml (Fig. 6A), whereasthe counts were approximately 103 when acidwater was added to neutral water. This again in-dicates that the acid water contained an acid-tolerant microflora which was maintained whenneutral water was added and that relatively fewacid-tolerant microbes entered the system withthe neutral water. Acid water added to neutralwater resulted in a lower stabilization value)which is related to the relative number of acid-tolerant microbes present.

ACKNOWLEDGMENTSThis work was supported by allotment grant no.

14-01-0001-805,980 from the Office of Water ResourceResearch, U.S. Department of Interior.We are grateful for the assistance of Carol

Macmillan and for the use of facilities at the WaterResources Center of The Ohio State University.

LITERATURE CITED1. AMERICAN PUBLIC HEALTH ASSoCIATION. 1960.

Standard methods for examination of water andwaste water, 11th ed. American Public HealthAssociation, Inc., New York.

2. CAMPBELL, R. S., 0. T. LIND, G. L. HARP, W. T.

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GEILING, AND J. E. LETrER. 1965. Water pollu-tion studies in acid mine lakes: changes in waterquality and community structure associatedwith aging. Proc. Symp. Acid Mine DrainageRes., Mellon Inst., Pittsburgh, Pa., p. 188-198.

3. COLMER, A. R., AND M. E. HINKLE. 1947. Therole of microorganisms in acid mine drainage.Science 106:253-256.

4. DUGAN, P. R., AND D. G. LUNDGREN. 1965. En-ergy supply for the chemoautotroph Ferro-bacillus ferrooxidans. J. Bacteriol. 89:825-834.

5. EHRLICH, H. L. 1963. Microorganisms in aciddrainage from a copper mine. J. Bacteriol.86:350-352.

6. JOSEPH, J. M. 1953. Microbiological study of acidmine waters: preliminary report. Ohio J. Sci.53:123-137.

7. JOSEPH, J. M., AND D. E. SHAY. 1952. Viability ofEscherichia coli in acid mine waters. Am. J.Public Health 42:795-800.

8. PECK, H. D., JR. 1962. Symposium on metabolismof inorganic compounds. V. Comparativemetabolism of inorganic sulfur compounds inmicroorganisms. Bacteriol. Rev. 26:67-94.

9. POSTGATE, J. R. 1963. Versatile medium for theenumeration of sulfate-reducing bacteria. Appl.Microbiol. 11:265-267.

10. RILEY, C. V. 1965. Limnology of acid mine waterimpoundments. Proc. Symp. Acid Mine Drain-age Res., Mellon Inst., Pittsburgh, Pa., p. 175-187.

11. SILVERMAN, M. P., AND H. L. EHRLICH. 1964.Microbial formation and degredation of min-erals. Advan. Appl. Microbiol. 6:153-206.

12. SILVERMAN, M. P., AND D. G. LUNDGREN. 1959.Studies on the chemoautotrophic iron bac-terium Ferrobacillus ferrooxidans. I. An im-proved medium and a harvesting procedure forsecuring high cell yields. J. Bacteriol. 77:642-647.

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