MICROBIOLOGICAL PROBLEMS IN STRIP MINE AREAS:RELATIONSHIP TO THE METABOLISM OF

THIOBACILLUS FERROOXIDANS1' 2

DONALD G. LUNDGREN

Biology Department, Syracuse University, Syracuse, N. Y. 13210

ABSTRACTLUNDGREN, DONALD G. Microbiolog-

ical problems in strip mine areas: Rela-tionship to the metabolism of Thiobacillusferrooxidans. Ohio J. Sci. 75(6): 280,1975.

The role of the iron oxidizing bacteriain contributing to acid mine drainage isconsidered relative to the metabolism ofthe organism. To study acid productionan in vitro system was developed usingisolated cell envelopes from Thiobacillusferrooxidans, which retained the abilityto oxidize Fe2+. An oxygen electrode wasused to follow oxygen depletion as Fe2+

was oxidized. The kinetics of Fe2+ oxi-dization was analyzed over a temperaturerange of 15° to 60° C and temperatureeffects were plotted as Arrhenius plotsand Km's and Vmax's were determined atdifferent temperatures.

Activation energies of 4.5 kcal/molefor cytochrome oxidase and 11.4 kcal/mole for cytochrome c reductase werenoted for the two enzymes involved inFe2+ oxidation. The relative bindingaffinity of Fe2+ (Km) was relatively stableto temperature changes but reactionrates (Vmax) did increase as expected.

The power of the microbe is a con-tinuously misunderstood phenomenon insanitary biology. Because microbes areinconspicuous, they are often overlookedas agents of environmental problems. Asa general microbiologist, I have alwaysaccepted a doctrine advanced by micro-bial scholars—never underestimate thepower of the microbe. Also, I havelearned that within the field of microbialecology the concept of chemoautotrophyis extremely vital to one's understanding

iManuscript received January 10, 1975.2Presented as part of a symposium, Biological

Implications of Strip Mining, held at BattelleMemorial Institute, Columbus, Ohio, onNovember 15, 1974.

of how the microbe contributes to themovements of the earth and the variouselemental cycles of nature. This con-cept should not be strange to this audi-ence, for within this concept lies anunderstanding of the biological problemswe are discussing during the symposium.

Chemoautotrophic bacteria are thosethat obtain their energy necessary forgrowth and carbon assimilation from theoxidation of reduced sulfur, and ironcompounds, iron metals, ammonium ion,nitrite ion and hydrogen. The bulk oftheir carbon for existence comes fromCO2 assimilation.

Of immediate attention and interest tothis group are the iron and sulfur oxidiz-ing bacteria which are very much con-cerned with acid mine drainage, themining of copper and uranium and thedisplacements of large buildings. Timedoes not permit discussion of the lattertwo problems, but they are becomingmore and more important as man isfaced with energy depletion and con-struction needs.

To focus upon our immediate problemof acid mine drainage and the bacteriumThiobacillus ferrooxidans, a fundamentalreference to Singer and Stumm (1970)is important. These authors relate thefollowing conditions:

(+O2)(1) FeS2 >Fe2++S—compounds

(2)bacterium

>Fe2++O2catalyst

Fe3++FeS2-

The equations are sequential reactionsinitiated by (1) the oxidation or dissocia-tion of pyrite in which Fe2+ is releasedand (2) a cyclic process where ferrousiron is oxidized to ferric via oxygen in

No. 6 IRON OXIDATION 281

conjunction with a biological catalyst.The rate-limiting reaction is catalyzedby an acidophilic bacterium. The opti-mum pH of the reaction is around 2.5 to3.5; Fe2+ is continuously regenerated bythe reduction of Fe3+ by pyrite.

The environment contains many otherexamples of microbial-mineral interac-tions. Consider the following (Silvermanand Ehrlich, 1974):

"All living materials cause small scaletransformations of trace elements neces-sary for enzyme activation and function.Microorganisms can by either direct or in-direct action transform relatively largeamounts of mineral matter sufficiently toinfluence its geological distribution. Theydo this via certain enzymatic processesinvolving:a. Redox reactions ((igurc 1).

nitrogen sources thereby freeing complexinorganic ions. These can precipitate aswater insoluble hydroxides or salts at theappropriate pH and Eh.Non-enzymatic reactions can also influencethe transformations of mineral matter.a. Interactions with metabolic products.Products as H2S, H2CO3, HNO3, H2SO4react nonbiologically with dissolved in-organic matter.b. Absorption of mineral matter to cellsurfaces.Sheath bacteria carry out this function."

We made the assumption that theorganism which acts as a catalyst in thefield is going to perform the same way inthe laboratory. Consequently, labora-tory studies were devoted to the cultural,physiological and biochemical investiga-tion of this bacterium.

This work is focused upon the mechan-

Oxidation ofmineral matter

Organiccarbon

Reduction ofmineral matter

cN

TJX

O

a>

Oxidation oforganic carbon

Anabolicmachinery

of cell

Anabolicmachinery

of cell

Anabolicmachinery

of cell

Chemosyntheticautotroph

Photosyntheticautotroph

Heterotroph

FIGURE 1. Schematic drawing (Silverman and Ehrlich, 1964), showing oxidation and reduction ofmineral matter.

b. Digestion of metal complexes. Someinorganic ions in aqueous solution, partic-ularly ions of iron, copper, maganese, zinc,calcium and magnesium from chelates orcomplexes with certain organic chemicals.Complexing agents generally stabilize in-organic ions in solution. Many bacteriacan use the complexing agent as carbon or

ism of action of the biologically catalyzedoxidation of ferrous iron.

Figure 2 shows an electron micrographof the catalytic organism T. ferrooxidans,isolated initially from a Pennsylvanianacid stream. It is in the inner laver

282 DONALD G. LUNDGREN Vol. 75

FIGURE 2. Electron micrograph of T.ferrooxidans showing the layered envelope components. OL,outer layer; ML, middle layer; CM, cytoplasmic membrane. 24, 300X.

No. 6 IRON OXIDATION 283

SIMPLIFIED MODEL OF THE CELL ENVELOPE

OF GRAM NEGATIVE BACTERIA-

THIOBACILLUS

FIGURE 3. Model of a typical cell envelope of a gram-negative bacterium, T. ferrooxidans. Ironoxidation occurs in the cytoplasmic membrane of the bacterium.

(CM) where ferrous iron oxidation occurs.Further, within this narrow zone a micro-environment exists which separates anacid milieu (pH 2.5) from the cell cytosol(pH about 6.0).

Figure 3 shows a typical model of theenvelope of T. ferrooxidans, based onwhat is known of the cell envelope ofgram-negative bacteria. Costerton et al.(1974) offer an excellent review of an in-depth appraisal of the cell envelope ofgram-negative bacteria. The left sideof the figure outlines those reactionswhich this paper will deal with, andmore< .specifically, that portion of thefigure which describes the loss of theelectron from Fe2+ oxidation. Diagram-atically this can be described as:

Energy in the form of ATP is generatedthrough the electron transport system.Noteworthy is the observation from themodel that the electron transport systemresides in the cytoplasmic membrane ofthe cell, and that it is one of the simplestelectron transport systems known tobacteria. Lundgren et at. (1974) havepublished an extensive review of the ionoxidizing bacteria and their electrontransport systems.

The electron transport system of T.ferrooxidans was investigated with cleancell envelope preparations. Figure 4 isan electron micrograph of cell envelopesprepared from intact cells. On the basisof cell protein, the envelopes containabout 40% of the iron oxidation activity

or as

Fe+2/ \

Cyt £ + £ i(reduced)

Cyt c_ + £x

(oxidized)

Cyt a(oxidized)

Cyt a_(reduced)

284 DONALD G. LUNDGREN Vol. 75

FIGURE 4. Electron micrograph of a cell envelope preparation isolated as described in the text.The envelope contains the "iron oxidase" enzymes. The three layers of the envelopeare the same as those shown in the insert of figure 2.

of intact cells. Bodo and Lundgren(1974) have described the isolation aswell as the application of the cell en-velopes to the study of iron oxidation.

MATERIALS AND METHODSOrganism and culture procedures. T. ferro-

oxidans was grown on the standard 9K mediumin 16-liter Nalgene carboys under forced aera-tion. The 9K medium was adjusted to pH 3.2prior to inoculation, with H2SO4 and one ml ofa 15% (w/v) cell suspension used as an in-oculum. Carboys were aerated and main-tained in a 30°C incubator.

Cells were harvested in a Sharpies centrifugeafter determining the absence of Fe2+ iron inthe culture medium by adding, 2,2' dipyridylto a sample of the culture and noting theabsence of a red color. Fe+2 was completelyoxidized by 60-70 hr. The collected cell pastewas suspended in 40-80 ml of acidified (pH 3.0with H2SO4) glass distilled water, and the ac-companying ferric iron precipitates removed bylow speed centrifugation (750 rpm) in a refrige-rated RC2-B Sorvall centrifuge equipped witha SS-34 rotor. The cell suspension was de-canted into clean 50-ml centrifuge tubes andcentrifuged at 17,500 rpm for 15 min. Thesupernatant was discarded, and the cell pelletsuspended and centrifuged at 17,500 rpm. Thewashing step was repeated three to four timesuntil no iron (Fe3+ or Fe2+) was detected in thesupernatant. The final cell suspension wasmade up to a 20% (w/v) stock with acid water.

Preparation of crude envelopes. Fifteen ml ofthe 20% suspension of T. ferrooxidans were di-

luted to approximately 40 ml with distilledwater, and centrifuged at 17,500 rpm for 15 min.as described. The cell pellet was suspended in20 ml, 0.01 M PO43- buffer (pH 7.0) and storedat 4°C overnight. The cell suspension wasdiluted to 40 ml with distilled water and cen-trifuged at 17,500 rpm for 15 min. The super-natant was discarded and the pellet suspendedin 40 ml of glass distilled water and centrifugedas described earlier. The supernatant wasdiscarded and the cell pellet suspended to afinal volume of 20 ml. The cell suspension waspassed 3 times through an Aminco Powerdriven French Pressure Cell Press at a gagepressure of 20,000, and the lysate collected.Whole cells were removed from the lysate bycentrifuging at 7,500 rpm for 10 min; the super-natant was passed through a 0.45jum Milliporefilter and 1 mM MgSO4 added to the filtrate.The filtrate was centrifuged at 40,000 rpm for1 hr in an International B-60 preparative ultra-centrifuge equipped with an A-211 rotor. Theresulting pellet was suspended in 0.01 M PO4

3~(pH 7.0) buffer containing 1 mM MgSO4 to aprotein concentration of 20 to 25 mg protein/ml.This preparation was designated the cell en-velope fraction.

Assays. Iron oxidation by both intact cellsand cell envelopes of T. ferrooxidans was as-sayed by measuring oxygen uptake with a Clarkoxygen electrode. The components added toa water cooled, 2 ml volume reaction vesselcontaining the electrode were: either 0.1 ml ofa 1% (w/v) suspension of whole cells or 0.1 mlof the cell envelope preparation (20-25 mgprotein/ml); 1.8 ml of 0.02 M B-alanine-SO4

2~buffer (pH 3.5); 0.1 ml of 0.2 M FeSO4-7H2O in

No. 6 IRON OXIDATION 285

pH 2.5, H2SO4. The iron initiated the reactionand was delivered with a microliter syringe.All assays were normally conducted at 28°Cand were agitated by means of a magneticstirring bar. Auto-oxidation of FeSO4 waschecked during each set of assays by omittingwhole cells or envelopes and adding 0.1 ml ofthe buffer in its place. Results were alwaysexpressed as averages of at least these separateexperiments. Different cell batches were al-ways used to check results.

For studying temperature effect upon ironoxidation, a temperature range of 15 to (iO°Cwas used. Constant temperatures wereachieved by using a circulating water bath.Temperature effects on iron oxidation wereplotted as Arrhenius plots. Km's for theaffinity of iron and the Vmax of iron oxidationwere also determined for cell envelopes exposedto varying temperatures.

RESULTSThe linearity of O2 uptake with in-

creasing concentrations of cell envelopesextended over an envelope protein con-centration range of 2-10 mg of protein.The pH activity curve for the cell en-velopes showed an optimum at 2.5.

The oxidation of ferrous iron and con-comitant uptake of oxygen involves atwo component electron transport sys-tem-cytochrome c reductase and cyto-chrome oxidase. The present under-standing of the characteristics of theseenzymes is summarized in table 1. Thedata comes mostly from the research ofBlaylock and Nason (1963) and Din etal., "(1967).

Using metabolic inhibitors and the en-

TABLE 1Characteristics of Iron Cytochrome C Reductase

and Cytochrome Oxidase.

EnzymeIron Cytochrome c Reductase(partially purified)1. Closely associated with membranes; dif-

ficult to solubilize.2. Composed of two components: protein and

RNA. High stability to heat, proteolyticenzymes and RNAase.

3. High salt cone, causes an inactivation bydissociation of the two components.

4. Purified enzyme contains one single protein,MW 30,000; holoenzyme, MW = 110,000.

5. One atom of Fe++ associated with the pro-tein portion.

(>. Km of 1 x 10-;i M for Fe++; 9.5 x IO-5 M forCyt c.

7. No sulfhydryl group in enzyme.8. No flavin compound in enzyme action.9. pH optimum, 5.7-7.0.

Cytochrome Oxidase(crude preparation)1. pH optimum 4.3-4.72. Km for reduced Cyt c, 4.6 x 10"e M3. heat sensitive4. inhibited bv KCN, NaN3, Na,S

velope preparation, and measuring oxy-gen uptake involving the intact electrontransport chain, other information hasbeen obtained. Table 2 shows that ironoxidation was inhibited by cyanideand azide, both inhibitors of cytochromeoxidase, and also by silver nitrate anduranyl ions; these ions can affect electron

TABLE 2

Effect of Inhibitors on Iron Oxidation by Cell Envelopes.

Compound

NaN3NaCNAgNO,Uranyl acetateAntimycin A4-Hydroxy-2-n-heptylquinoline N-oxideSodium AmytalAtabrineIodoacetamideQuinacrine**Sodium diethyldithiocarbamateBathocuproine

Final concentration(M)

1 x 10~4

1 x 10-4

1 X 10-31 X 10-32 x 10"4

1 X 10-31 X 10-31 X 10-31 X 10-35 x 10"4

1 X 10-31 X ID"3

Inhibition%

1001008555

00000

00

* Cuvette contained: inhibitor in appropriate solvent; .02 M /3-alanine-SO4"2 buffer, pH 3.5; .2 M FeSO4 in acid water; 0.1 ml of envelopes (0.2 mgprotein) .

**12% stimulation.

286 DONALD G. LUNDGREN Vol. 75

transport functions by reacting with bio-logical membranes. The other inhibitorshad little affect upon oxidation.

The envelope preparation represents afunctional electron transport systemwhere the electron from iron is movedalong the chain to its ultimate acceptoroxygen. In the past, in vitro demon-strations of the "iron oxidase" have beendifficult, and this report presents a clear-cut methodology involving low pH'swhere spontaneous iron oxidation isminimal. Figure 4 shows the envelopepreparation.

Because of the technical problems as-sociated with the oxidation catalyzed bythese enzymes, our laboratory has at-tempted to study the "iron oxidase"system by using the intact electrontransport chain located in cell envelopes.One approach has been to use tem-perature studies of iron oxidation withassays done over the range of 10°C to70°C using both intact cells and cellenvelopes. The data were then plottedas Arrhenius plots, and as Lineweaver-Burk plots to assess energy changes andkinetics properties of these enzymaticreactions.

Figure 5 shows the effect of tempera-ture upon iron oxidation of both whole

range was seen. An Arrhenius plot ofthese data are shown in figure 6 for cell

10 20 30 40 50 60 70

TEMP C

FIGURE 5. Temperature effects upon iron oxi-dation using cell envelopes and in-tact cells.

cells and cell envelopes. Oxidation ratesincreased with temperature until 60°Cfor cell envelopes, and then decreasedrapidly. A sharper drop-off occurredwith envelopes than with intact cellswhere a broader maximum temperature

FIGURE 6. Temperature data for cell envelopesshown in Figure 5 plotted as anArrhenius Plot. E = activation- en-ergy or Arrhenius constant.

envelopes. A definitive break occurredin the curve at 35°C. The break repre-sents a transition of the two curves andthe slope of the lines are equal to anArrhenius constant (or activation energy)for each of the enzyme reactions com-prising the electron transport chain of"iron oxidase". Calculated values for

-6.9-

-7.0-

ARRHENIUS PLOT(Whole Cells)

E =12 8 Kcol/mole

3.0 31 3.2 3 3(55°C) (35°C)

- L x i o + 3

T'K

3.4 3.5 3.6

FIGURE 7. Temperature data for whole cellsshown in Figure 5 plotted as anArrhenius plot.

No. 6 IRON OXIDATION 287

cell envelopes were 4.5 kcal/mole forcytochrome oxidase and 22.8 kcal/molefor cytochrome c reductase. Results ofsimilar experiments with whole cells gavevalues of 3.0 and 12.8 kcal respectivelyfor the two enzymes (figure 7). Therelationship also was found at pH 2.5,with activation energies of 4.5 and 11.4kcal determined for cytochrome oxidaseand cytochrome c reductase respectively.At pH 4.5, no break in the linearity ofthe curve at 35°C was noted; however,at this pH oxidation rates were muchslower and reproducibility was poor.

Figure 8 shows a series of Km plots forapparent iron affinities to sites on en-

KmxlO"3

6.0-

5.0-

4.0-

3.0-

2.0-

1.0-

Km vs. TEMP

pH 3.5

6.0-1

5.0-

4.0-

3.0-

2.0-

1.0-

10 20 30 40 50 °C

pH 2.5

10 20

6.0-

5.0

4.0-

3.0-

2.0-

1.0-

30 40

pH 4.5

50 °C

10 20 30 40 50 °C

FIGURE 8. Km values (M) plotted against tem-perature for three different pH's.The amount of Fe2+ added to thecuvette ranged from 7.5 to 40 ^mol.

velopes at three pH's. In three separateexperiments iron binding values fell in afairly reproducible fashion with an in-crease in temperature at pH 3.5 whereasat the other two pH's changes in Kmwere less over the temperature rangeexamined. Generally these values arecomparable to those of intact cells. Forthe moment there appears to be limitedinformation that can be collected fromsuch experiments. It is apparent, how-ever, that the "iron oxidase" system re-mains intact for a limited period of timeat higher temperatures and there isrelatively little change in affinity. Themaximum reaction rates (Vmax) did in-crease with temperature as expected.

The work presented represents anothercomponent of information relative tobiological iron oxidation. By chippingaway at this process through research,the full story will be told. At that time,the design of preventative measuresaimed at interferring with iron oxidationin nature will have more meaning.

Acknowledgments. Recognition of the contri-bution of experimental work reported here goesto Dr. Carl Bodo and George Barvinchak bothof Syracuse University.

LITERATURE CITEDBlaylock, B. A. and A. Nason. 1963. Electron

transport system of the chemoautotrophFerrobacillus ferrooxidans. 1. Cytochromec-containing iron oxidase. J. Biol. Chem. 238:3453-3463.

Bodo, C. and D. G. Lundgren. 1974. Ironoxidation by cell envelopes of Thiobacillusferrooxidans. Can. J. Microbiol. 20: 1647-1652.

Costerton, J. W., J. M. Ingrams and R. J.Cheng. 1974. Structure and function of thecell envelope of gram-negative bacteria.Bacteriol. Rev. 38: 87-110.

Din, G. A., I. Suzuki and H. Lees. 1967.Ferrous iron oxidation by Ferrobacillus fer-rooxidans. Purification and properties ofFe++-cytochrome c reductase. Can. T. Bio-chem. 45: 1523-1456.

Lundgren, D. G., J. R. Vestal, and F. R.Tabita. 1974. The Iron-Oxidizing Bacteria.In Microbial Iron Metabolism, Ed. J. B.Neilands. Chapter 18. Academic Press,N.Y.

Silverman, M. P. and H. L. Ehrlich. 1964.Microbial formation and degradation ofminerals. Adv. Appl. Microbiol. 00-153-206.

Singer, P. C. and W. Stumm. 1970. AcidicMine Drainage: The rate limiting step.Science 167: 1121-1124.