APPLIED MICROBIOLOGY, May 1968, p. 695-702 Vol. 16, No. 5Copyright © 1968 American Society for Microbiology Printed in U.S.A.

Bacteriology of Manganese NodulesIII. Reduction of MnO2 by Two Strains of Nodule Bacterial

R. B. TRIMBLE AND H. L. EHRLICH

Department ofBiology, Rensselaer Polytechnic Institute, Troy, New York 12181

Received for publication 22 January 1968

MnO2 reduction by aerobic growing cultures of Bacillus 29 and coccus 32, isolatedfrom ferromanganese nodules, was assessed for 7 days. A 1-day lag was observedbefore the onset of MnO2 reduction by either culture. Addition of HgC12 to a finalconcentration of about 103 M caused a rapid cessation of MnO2 reduction by thegrowing cultures. Neither culture reduced MnO2 when grown under continuedanaerobiosis from the start of an experiment. However, if conditions were madeanaerobic after MnO2 reduction was initiated, reduction continued at a rate onlyslightly lower than that under aerobic conditions. Resting-cell cultures reducedMnO2 equally well aerobically and anaerobically, provided that ferricyanide waspresent to serve as electron carrier. These findings showed that oxygen is neededfor culture adaptation to MnO2 reduction, and that oxygen does not interferewith microbial MnO2 reduction itself. Both cultures caused sharp drops in the pH ofthe medium during MnO2 reduction: with coccus 32, during the entire incubationtime; with Bacillus 29, for the first 3 days. The Eh of the medium fluctuatedwith either culture and never fell below 469 mv with Bacillus 29 and below 394 mvwith coccus 32. The rates of glucose consumption and Mn2+ release by Bacillus29 and coccus 32 were fairly constant, but the rates of lactate and pyruvate pro-duction were not. Although acid production undoubtedly helped in the reductionof pyrolusite (MnO2) by the bacteria, it did not appear to be important in thereduction of manganese oxide in ferromanganese nodules, as shown by the re-sults with a nodule enrichment.

With suitable electron donors, several differentsoil microorganisms and bakers' yeast reducemanganese oxides to Mn"I (9, 10, 12, 14). Toobserve MnO2 reduction by such microorganisms,Mann and Quastel (10) and Hochster and Quastel(9) employed an electron carrier and an oxygen-free atmosphere of N2 and CO2 in their reactionsystem. On the other hand, Perkins and Novielli(12) and Vavra and Frederick (14) did not usean electron carrier nor did they exclude 02 fromtheir system. According to generally acceptedviews (1), microbial MnO2 reduction should befavored by an absence of 02, but neither Perkinsand Novielli nor Vavra and Frederick commentedon this in the discussion of their work.Reduction of manganese oxides in bacterial

enrichments of marine ferromanganese noduleswas first reported by Ehrlich (3). In later investi-gations, he demonstrated MnO2 reduction by purecultures of bacteria isolated from ferromanganese

1 Part of a dissertation submitted by R. B. Trimbleto the faculty of Rensselaer Polytechnic Institute inpartial fulfillment of the requirements for the M.S.degree in the Department of Biology.

nodules (5; H. L. Ehrlich, Bacteriol. Proc., p.42-43, 1964). Ehrlich's work showed that growingcultures of Bacillus 29 and coccus 32, as well asother microorganisms, reduced MnO2 to Mn"aerobically with glucose as electron donor. Underresting-cell conditions, he found that cultures notpreviously grown in the presence of MnO2required the addition of an electron carrier (ferri-cyanide) in order to reduce MnO2 with glucose assubstrate during an initial 3-hr period of incuba-tion. Ehrlich's experiments indicated that bothorganisms possessed an enzyme system whichreduced ferri- to ferrocyanide with electronsderived from the substrate, and another systemwhich reduced MnO2 to Mn" with electronsderived from ferrocyanide. However, the organ-isms lacked one or more enzyme componentsnecessary to convey electrons from the ferri-cyanide reducing system to the ferrocyanideoxidizing system. His experiments also indicatedthat growing cultures developed this missingcomponent after a period of contact with MnO2.Ehrlich called such cultures "adapted cultures."Whether this adaptation involved a mutation and

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selection phenomenon, an allosteric rearrangementof an existing component, or an induction was notdetermined.

Further details of bacterial MnO2 reduction bygrowing and resting-cell preparations of Baciilus29 and coccus 32 are given in this paper.

MATERIALS AND METHODS

Media. Stock cultures were carried on Difco StockCulture Agar made up in distilled water. Inocula ofdesired cultures were prepared on slants in Rouxbottles, each containing 100 ml of seawater-nutrientagar consisting of (in g per liter of natural seawater):peptone, 5; beef extract, 3; agar, 15.

Bacterial cultures. Two strains of bacteria, a spore-forming Bacillus, designated strain 29, and an un-identified coccus, called coccus 32, were used in pureculture work. The Bacillus and coccus had been pre-viously isolated from ferromanganese nodules ob-tained from the Atlantic Ocean (3). Stock cultureswere maintained at 25 -4 1 C. Frequent checks forpurity were made by microscopic observation andplating.

Preparation of inoculum. Bacillus 29 or coccus 32was grown up for 24 hr on Roux bottle slants of sea-water-nutrient agar. Cells were harvested and washedthree times by centrifugation in undiluted or 10-folddiluted seawater. The washing procedure was carriedout aseptically, when the cells were to be used as theinoculum in growth experiments. The final cell titerof each suspension was adjusted to between 108 and109 cells per ml. A l-ml amount of final suspensionwas used for inoculation of reaction vessels.

Manganese dioxide. Reagent-grade MnO2 (J. T.Baker Chemical Co., Phillipsburg, N.J.) was used.The MnO2 was washed 10 times in 200-ml volumes ofdistilled water in order to remove fine, colloidalparticles of the oxide from larger ones. It was thendried under a heat lamp and was used in experimentsimmediately thereafter.

Anaerobic incubation. Anaerobic conditiions weremaintained in a large vacuum desiccator containing50 ml of distilled water on the bottom for humiditycontrol. The desiccator was evacuated to a resdualpressure of less than 60 mm of Hg with an aspiratorpump. Dry, purified nitrogen gas (Union CarbideCorp., Linde Div., New York, N.Y.) was then intro-duced to a pressure of 760 mm of Hg. This procedurewas repeated two more times, leaving a residualpressure of less than 0.15 mm Hg of 02 (13).

Experimenital procedure. Growing-cell experiments(long-term) were performed by autoclaving 1-g por-tions of MnO2 under 10 ml of distilled water in anappropriate number of 125-ml Erlenmeyer flasks at15 psi for 30 min. After sterilization, the water wasaseptically removed by pipette and was replaced with18 ml of sterile, natural seawater. To this were added1 ml of a sterile, distilled water solution of 1.0% pep-tone and 1 ml of another sterile, distilled water solu-tion of either 2, 10, or 20%C glucose. A 1-ml amount ofaseptically washed cell suspension was added to experi-mental flasks, while an additional 1 ml of sterileseawater was added to control flasks. In some experi-

ments, additional controls of inoculated flasks withthe above nutrient solution, but without MnO2, wereincluded. Each experimental condition, includingcontrols, was set up in duplicate and sampled induplicate at appropriate times. Incubation was at25 4 1 C.

Chemical and physical changes in the medium ofgrowing cultures were determined in separate pairs ofexperimental and control flasks, at desired times, byassaying for dissolved manganese, residual glucose,and lactic and pyruvic acids. Cell populations in thesupematant fluid were estimated turbidimetrically ina Spectronic-20 colorimeter (Bausch & Lomb, Roch-ester, N.Y.). Determinations of pH and Eh weremade with a model 7 or model 10 pH meter (CorningGlass, Corning, N.Y.). For Eh determinations, aplatinum electrode against a calomel reference elec-trode was used. Readings of observed potentials wereconverted to standard potentials (Eh) by adding avalue of +244 mv, the Eh of the calomel electrode at25 C.

Resting-cell experiments (short-term) were carriedout for 3 hr in 50-ml Erlenmeyer flasks. Each flaskreceived 0.1 g of washed MnO2, which was overlaidwith 4.9 ml of 10-fold diluted seawater, 1 ml of 0.25M NaHCO3 buffer (pH 7.0) containing 1% glucose,0.1 ml of 0.01 M potassium ferricyanide, and 1 ml ofwashed cells suspended in 10-fold diluted seawater.In control flasks, 1 ml of 10-fold diluted seawater re-placed the cell suspension. Flasks to which ferri-cyanide reagent was not added received 0.1 ml of10% seawater. Each experimental condition andcontrol was set up in duplicate and incubated at roomtemperature for 3 hr. Immediately thereafter, a 1.5-ml sample was removed from each flask, centrifugedat 3,500 X g, and assayed for dissolved manganese.The residual solution in each flask was then acidifiedwith 0.05 ml of 10 N H2SO4 and incubated at roomtemperature for 30 min. A 3-mi sample of solutionwas then removed from each flask, centrifuged at3,500 X g, and assayed for manganese.To determine if pyruvate would cause a dismutation

between MnO2 and Mn2+, 18 ml of sterile seawaterwas added to six 125-ml Erlenmeyer flasks containingsterilized MnO2. Two experimental flasks received 1 mlof sterile 2 X 10-2 M MnSO4- H20 and 1 ml of 1.0 Msodium pyruvate; two control flasks received 1 ml ofsterile 2 X 10-2 M MnSO4- H20 and an additional 1ml of sterile seawater; and two other control flasksreceived an additional 2 ml of sterile seawater each.All flasks were incubated at room temperature for aspecified length of time. Samples of supernatant fluidwere then assayed for manganese concentration.

Analytical methods. Manganese in solution wasdetermined by a persulfate method described byEhrlich (3, 5). Since this method does not discriminateamong the various valence states of manganese,periodic tests with benzidine-acetate reagent wereused to detect Mn"'l and MnIV (6), for which thisreagent was specific under the conditions of theseexperiments. Relative sensitivities of the benzidinetest and the persulfate assay are of the same order ofmagnitude. However, the benzidine test does not lenditself to precise quantitative measurement.

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Residual glucose was measured by mixing 2-miportions of 50-fold diluted samples of culture fluidwith 4 ml of anthrone reagent in test tubes (15 X 150mm) and measuring color development at 540 myafter 15 min (11). The anthrone reagent consisted of0.2 g of anthrone in 100 ml of 95% HSO4.

Lactic acid was determined on 1.0-, 0.2-, or 0.1-mlsamples of culture fluid, depending on its concentra-tion, according to the colorimetric method of Barkerand Summerson (2). Since pyruvic acid interfereswith this assay, all measurements of lactic acid werecorrected for pyruvic acid present. Color develop-ment was measured at 560 m,u.

Pyruvic acid was measured on 1.0-, 0.2-, or 0.1-nmlsamples of culture fluid, depending on its concentra-tion, by the colorimetric method of Friedemann andHaugen (7). Color development was measured at 520m,u.

All color determinations were made with a Spec-tronic-20 colorimeter.

In the rate studies of manganese release shown inFig. 1, changes in manganese concentration in thesupernatant fluid were measured by repeated sam-pling of the same duplicate flasks. This required correc-tion of the measurements for volume changes and for

10

8

I_

x

I"I

4n8

6

4

0

FIG.Bacillusthe procdBacillus.HgCh. (HgC/2 tigrowingdays of iactivity i

manganese removal resulting from repeated samplino.Corrections were made according to the followingrelationship, which normalized all data on the basis ofan initial 21 -ml volume of supernatant fluid:

An21 - 0.4(n - 1] + 0.4(An-,)21

= corrected manganese concentration per ml

where A. = manganese concentration of the nthsample of supernatant fluid; A,-, = manganese con-centration of the (n - I)th sample of supernatantfluid; and it = the number of 0.4-ml samples of super-natant fluid removed from the reaction vessel. Thecorrections are necessary only when A, > A,-,.When A. = A.-,, the nth sample contains the samemanganese concentration on a normalized basis as the(n - 1)th sample because MnO2 was not reducedduring this time.

RESULTS

MnO2 reduction by growing cultures. The actionof Bacillus 29 and coccus 32 on MnO2 in a mediumcontaining an initial concentration of 0.95%glucose and 0.048% peptone is showninFig. 1,curves A and B. A fairly constant rise in the con-centration of soluble manganese was noted afterthe first day of incubation at 25 C. No manganesewas detected in the supernatant fluid of uninocu-lated flasks (Fig. 1, curve E). Inoculated flaskscontained dissolved manganese which reactedwith persulfate, but not with benzidine-acetatereagent, indicating that the valence state of thedissolved manganese was Mn".

Further manganese release stopped very soonafter the addition of 0.2 ml of 10-1 M HgCI2(final concentration about 10-3 M) to growing,MnO2-reducing cultures of either Bacillus 29 orcoccus 32. Curves C and D (Fig. 1) show theresults obtained with coccus 32 when HgCl, wasadded after 3 and 6 days, respectively. When thecells of coccus 32 were poisoned with HgCl2 atthe time of inoculation, no soluble manganeseappeared throughout the 7-day period of incuba-tion. Similar results were obtained with Bacillus29.

A=, E_ Effect of anaerobiosis on growing and resting-cellcultures. When growing cultures of Bacillus 29 or

0 2 3 4 5 6 7coccus 32 were made anaerobic immediately

daYS after inoculation and were then kept anaerobic

1. MnO2 reduction by growing cultures of for 5 to 7 days, no manganese was found in the29 and coccus 32, and the effect ofHgC12 upon supernatant fluid. However, if growing culturesess. Curves A and B represent the activities of of either strain of bacteria were made anaerobic

29 anid coccus 32, respectively, in the absence of after the cells had begun to reduce MnO2, as

_urves C and D represent the effect of addingo a final concentration of about 10-3 to a evidenced by finding Mn" in the supernatantMnO2-reducing culture ofcoccus 32 at 3 and 6 fluid of the reaction vessels, the reduction con-ncubation, respectively. Curve E represents the tinued anaerobically at a rate only slightly lowerin an uninoculated control. than the aerobic rate during a 5-day period. At the

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TABLE 1. Aerobic and anaerobic MnO2 reduction byresting cells of Bacillus 29 and coccus 32

Mn (mumoles) per mlof supernatant fluida

Culture Condition

Aerobic Anaerobic

29 Systemb- cells + (a)e 12.8 12.0System + cells + (a) 12.8 12.0System - cells + (b)c 12.8 12.0System + cells + (b) 89.6 91.6

32 System - cells + (a) c 1.6 1.6System + cells + (a) 1.6 1.6System - cells + (b)c 1.6 1.6System + cells + (b) 12.9 16.0

aAfter acidification at the end of 3 hr. Acidi-fication accomplished by addition of 0.05 ml of 10N H2SO4 to each reaction vessel.

b System consisted of 0.1 g of MnO2; 4.9 ml of10% seawater; 1.0 ml of 0.25 M NaHCO3 (pH 7.0)containing 1% glucose; and 1.0 ml of inoculum or10% seawater.

c (a) 0.1 ml of 10% seawater; (b) 0.1 ml of 0.01M KYFe(CN)6.

end of these experiments, the pH was slightlyhigher in the flasks in which MnO2 was beingreduced anaerobically than in similar flasksincubated aerobically.

Ehrlich (5) has mentioned that preliminarywork with resting cells of Bacillus 29, which hadnot been adapted to MnO2 reduction, showedMn" release from MnO2 anaerobically in evacu-ated Thunberg tubes, provided that an electroncarrier, potassium ferricyanide, was present in thereaction mixture. The preliminary findings wereconfirmed in experiments in which 50-ml Erlen-meyer flask cultures were incubated in a vacuumdesiccator under a nitrogen atmosphere.Additional experiments with coccus 32, notpreviously tested under anaerobic conditions,yielded similar results. Typical findings for eachorganism are summarized in Table 1. After acidi-fication, some manganese appeared even in thesupernatant fluid of uninoculated flasks. Thismanganese was not removed from the super-natant fluid by centrifugation for 15 min at 4,000x g and was only detectable by the persulfatemethod, indicating that it was Mn". Whetherthis manganese was initially present in the MnO2substrate as an impurity or whether it resultedfrom chemical reduction during the experiment isnot known. Before acidification, no manganesewas recovered in the supernatant fluids from theexperiment with coccus 32, nor, with one excep-tion, in the supernatant fluids from the experimentwith Bacillus 29. The unacidified supernatant

fluids in flasks with the complete reaction mixtureand Bacillus 29 contained 13.6 m,umoles of Mnper ml after aerobic incubation and 7.2 mumolesof Mn per ml after anaerobic incubation. Aswill be discussed later, the recovery of less Mn"tfrom the anaerobic than from the aerobic re-action of Bacillus 29 prior to acidification wasprobably due to the formation of less acid by thecells in the supernatant fluid under anaerobic con-ditions.

Chemical and physical changes in the culturemedium during bacterial MnO2 reduction. Studiesof chemical, physical, and population changes inthe presence and absence of MnO2 reduction byBacillus 29 at an initial glucose concentration of0.48% and by coccus 32 at an initial glucoseconcentration of 0.60% are summarized in Table2. Bacillus 29 and coccus 32 consumed moreglucose with MnO2 than without MnO2. Therates of glucose consumption and Mn" releaseby Bacillus 29 and coccus 32 were relativelyconstant for most of the observation period.Lactate production by Bacillus 29 reached a peakon the fourth day of incubation and thereafterleveled off. The total lactate produced by Bacillus29 with MnO2 was greater than without MnO2.Pyruvate production by Bacillus 29 increasedcontinually with MnO2 but leveled off after thefourth day without MnO2. Lactate productionby coccus 32 was negligible with or without MnO2.Pyruvate production by coccus 32 increasedprogressively with or without MnO2, but the totalyield was greater with MnO2 than without MnO2.It is also seen in Table 2 that Bacillus 29 in thepresence of MnO2 caused a sharp pH drop to 5.4in 3 days followed by a slow rise. This pH patternwas even more apparent in an experiment with aninitial glucose concentration of 0.95%. Withcoccus 32, the pH tended to drop continuallyduring the entire experimental period. With bothBacillus 29 and coccus 32, pH dropped more inthe absence of MnO2 than in its presence.Observed changes in Eh, fluctuating between 469and 617 mv with MnO2 and 474 and 594 mvwithout MnO2 in the case of Bacillus 29, andbetween 394 and 519 mv with MnO2 and 419 and549 mv without MnO2 in the case of coccus32, provided further evidence that MnO2 reduc-tion by bacteria is not dependent on strong re-ducing conditions.

Qualitatively similar results were obtainedwith Bacillus 29 at initial glucose concentrationsof 0.95 and 0.095%, and with coccus 32 at 0.48%.At an initial concentration of 0.095%, Bacillus 29exhausted the glucose in 3 days, requiring theaddition of fresh glucose to a final concentrationof 0.095% for continued activity for the nexttwo 3-day periods.

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TABLE 2. Chemical and physical changes during growth of Bacillus 29 and coccus 32 in the presence andabsence ofMnOea

Man-Glucose consumed ganese Pyruvate Lcaepoue

Cul- | Time \(mpumoles/ ld produced L tp;rmodes/MI) E

h (MV) pH Cells/mi (X 107)Cul-Time ~~(mptmoles (mjumoles/ml) (poe/lture (days) /ml)

+MnO2 -MnOs +MnO2 +MnO2 -MnO2 +MnO2 -MnO2 +MnO2 -MnO2 +MnOs -MnOs +MnO2 -MnO2

29 0 0 0 0 0 0 0 0 469 474 7.3 7.2 1.6 1.61 1,720 655 25 43 32 461 449 528 482 6.6 6.7 3.8 4.03 3,060 1,239 240 190 153 1,635 1,612 502 512 5.4 5.6 4.5 4.84 3,686 2,342 420 438 330 2,430 1,955 573 514 5.7 5.5 4.5 5.07 6,120 3,635 1,060 705 243 2,080 1,387 615 594 5.7 5.5 4.6 5.28 8,750 5,286 1,260 879 330 2,040 1,705 617 589 5.5 4.9 4.8 5.0

11 11,893 7,281 1,830 936 225 2,430 1,761 602 589 5.8 5.1 5.0 5.0

32 0 0 0 0 0 0 0 0 426 426 7.7 7.7 2.5 2.51 480 0 0 50 52 0 0 394 419 7.0 6.9 4.6 5.82 1,440 1,440 0 84 75 9 5 519 490 6.8 6.5 6.2 7.03 130 138 79 491 496 6.6 6.4 6.2 7.07 3,000 2,650 490 1,025 575 174 68 484 495 5.7 4.7 6.0 4.89 4,900 3,125 780 1,650 980 250 124 514 549 5.4 4.5 5.8 4.211 5,500 3,600 1,020 2,625 1,120 250 123 475 509 5.6 4.3 5.4 4.8

a Composition of culture medium is described in text.

Test for possible reaction between MnO2 andmetabolic products. An experiment was performedin order to determine if pyruvate might complexMnIII and thus promote the reaction, MnO2 +Mn2+ + 4H+ = 2Mn3+ + 4H20, which wouldresult in solution of MnO2. As described inMaterials and Methods, the setup consisted oftwo experimental flasks containing MnO2 and aseawater solution of MnSO4 and sodium pyru-vate, two control flasks containing MnO2 and aseawater solution of MnSO4 but without sodiumpyruvate, and two more control flasks containingMnO2 and seawater without eitherMnSO4 or sodi-um pyruvate. No change in soluble manganeseconcentration was measured with persulfate overan 8-day period in any of the six flasks. All su-pernatant fluids gave a negative test with benzi-dine acetate reagent. The pH of the reaction mix-ture was 6.8 during this time. To determine if thedismutation between MnO2 and Mn2+ would haveoccurred at a lower pH, 0.4 ml of 0.1 N H2SO4was added and the pH in the flasks was loweredto about 4. Still, no change in soluble manganeseconcentration was measured with persulfateduring the next 4 days. The supernatant fluids alsoremained unreactive with benzidine-acetate rea-gent. It is concluded that pyruvate cannot pro-mote a dismutation between MnO2 and Mn2+ bycomplexing MnIII.

Chemical and physical changes in a noduleenrichment undergoing bacterial oxide reduction.A 17.6-g portion of an Atlantic ferromanganesenodule, supplied in 1961 by the Woods Hole

Oceanographic Institution, Woods Hole, Mass.,was aseptically crushed, placed in a sterile 3-literFernbach flask, and overlaid with 1 liter ofsterile seawater containing 0.5% glucose and0.05% peptone. The glucose and peptone weresterilized separately. A 5-ml sample was asep-tically removed from the enrichment on successivedays, examined for the presence of microorgan-isms growing from the nodular material, andassayed for pH, Eh, dissolved manganese, andresidual glucose. The results of the assays areshown in Fig. 2. Unlike the experiments withpure cultures that reduced MnO2, the initial pHin this enrichment did not drop when the man-ganese in the nodule substance began to bereduced by the outgrowing bacteria. Glucose wasprogressively consumed as manganese was solu-bilized over the 16 days of the experiment. Wetmounts of crushed nodular material revealed fourdistinct morphological forms of organisms: motilerods, nonmotile cocci, curved rods, and largespherical cells. Of a total of 2,640 mg ofmanganese (as Mn°) in the nodule substanceinitially present in the flask, 248 mg were solu-bilized in 16 days. This represents a solubilizationof about 10% of the manganese in the nodule.

DISCUSSION

The rapid cessation of Mn" release after HgC12poisoning of the bacteria suggested an enzymaticrole of the cells in MnO2 reduction (Fig. 1).A much more gradual slowing down in Mn"

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25 t

20 4-

0

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0

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FIG. 2. Chemical and physical chlanges in an enirichmenit culture made with a portion ofan Atlantic ferromanga-nese uiodule in tlhe presence ofglucose and peptonie. Compositioni of the enirichment is described in text.

release would have been expected if metabolic endproducts, whose reactivity was unaffected byHgCl,, had been responsible for MnO2 reductioninstead of enzymatic interaction by the bacteria.

Since the cells used in these experiments werenot in contact with MnO2 while they were beingcultured in the preparation of the inoculum, andsince it has been previously shown that such cellscannot reduce MnO2 in the first 3 hr of contactwithout added ferricyanide (5), the 1-day lag inFig. 1 is partly attributable to a missing com-ponent in the electron transport system for MnO2reduction at the time of inoculation, which theculture synthesized during the lag period. As willbe explained later, the lag may also be partlyattributable to an initially insufficient quantity ofacid for dissolving Mn(OH)2, the probable directproduct of MnO2 reduction.The observation that displacement of oxygen

by nitrogen at the beginning of a growth experi-ment with unadapted Bacillus 29 or coccus 32prevented Mn" release from MnO2 indicated that02 is required for adaptation of the culture toMnO2 utilization. This conclusion was furthersupported by noting that displacement of air bynitrogen, after MnO2 reduction was initiated by

the culture, did not significantly reduce the rateof MnO2 reduction. The observation that anaero-biosis did not stimulate MnO2 reduction by thesenow adapted cells paralleled the observation thatMnO2 reduction by unadapted cells, utilizing ferri-cyanide as electron carrier, was not stimulatedor inhibited by anaerobiosis (Table 1). With addedferricyanide, the unadapted organisms must havepossessed a complete, oxygen-insensitive systemfor electron transport from electron donor toMnO2. The observed lack of sensitivity of bac-terial MnO2 reduction to 02 and, indeed, theabsence of any significant increase in the rate ofMnO2 reduction under anaerobic conditions arecontrary to the generally held notion that suchreduction should proceed preferentially underanaerobic conditions (1). The results indicatedthat adapted cultures use MnO2 in preference to02 as the terminal electron acceptor, and that insuch cells MnO2 and 02 do not compete witheach other. Trimble (M.S Thesis, RensselaerPolytechnic Institute, Troy, N.Y., 1967) hasnoted that unadapted, nongrowing cells ofBacillus 29, with ferricyanide as electron carrier,use significantly less 02 in the presence of MnO2than in its absence.

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The immediate product of MnO2 reduction atnear neutral pH appears to be a relativelyinsoluble form of Mn". It may be Mn(OH)2,which would be brought into solution by acidifi-cation. The lactic and pyruvic acids produced byBacillus 29 and the pyruvic acid produced bycoccus 32 undoubtedly accounted for readysolubilization of Mn" in growing cultures butnot in resting cultures. Unlike growing cultures,resting cultures, acting on MnO2 in a mediumbuffered with NaHCO3, could not have producedenough free acid to release much or any Mn"Iformed. Consequently, acidification with H2S04was required to bring the Mn",, which the restingcultures had formed, into solution.The quantitative observations on chemical and

physical changes in the culture medium duringMnO2 reduction by Bacillus 29 and coccus 32in air can be summarized as follows. Out of thetotal large population in the inoculum introducedinto a reaction vessel, only a portion of the cells,whose exact number depends on available sur-face on the MnO2, came into direct contact withMnO2 and reduced it. A period of adaptation wasnecessary before the cells on the MnO2 reduced it,providing that the inoculum had not been grownon MnO2 and that ferricyanide was not added tothe reaction mixture. During reduction of theMnO2 with glucose, the Bacillus 29 cells on theMnO2 produced lactic and pyruvic acid, and thecoccus 32 cells mainly pyruvic acid. The cells re-maining free in the liquid medium also producedthe respective acids, but by reducing 02 with glu-cose. The total acid thus formed caused a markeddrop in pH. When the pH ofthe supernatant fluidhad fallen below 6.0, the cells in the supernatantfluid slowed down or stopped growing and pro-duced little acid. However, the cells on the MnO2continued to grow and reduce MnO2 according tothe reaction, MnO2 + 2(H) = Mn(OH)2, buttheydid not produce acid rapidly enough for bringingall Mn(OH)2 formed into solution. Acid pre-viously formed by the cells in the supernatantfluid was consumed in dissolving Mn(OH)2 and,as a consequence, the pH in the supernatant fluidrose until it reached a level at which cells not incontact with MnO2 metabolized again. The cellpopulation in the supernatant fluid increasedslowly as cells on MnO2 multiplied and pushed atleast half of the progeny of each cell generationinto the medium because of the limited surfaceavailable on the MnO2. Depending on the pre-vailing pH, the cells that had been pushed intothe liquid medium may or may not have beenactive in oxidizing glucose to lactic and pyruvicacids. Under anaerobic conditions, cells in thesupernatant fluid were inactive, but cells on

MnO2 metabolized, if they were adapted to MnO2reduction or if ferricyanide was present.The interactions during MnO2 reduction

described above assign a proper function tometabolically formed acids. In general, acids canaffect dissolution of MnO2 in only two ways: (i)by reducing MnO2 chemically to Mn", as con-centrated HCl does, or (ii) by promoting thereaction MnO2 + Mn2+ + 4H+ = 2Mn3+ + 2H20through complexing of Mn3+, which prevents itsreoxidation to tetravalent manganese. The Mn3+complex would be soluble. In this study, wefound that pyruvic acid, an a-keto acid, is unableto complex Mn3+ at either neutral or acid pH. Ithas previously been shown that pyrophosphoricacid causes an accumulation of Mn"' by complexformation under similar experimental conditions(4).Although in pure culture experiments with

MnO2 acid was important for Mn" release intothe supernatant fluid, it appeared less importantin the enrichment culture with manganese nodulesubstance (Fig. 2). Mn" was readily releasedinto the medium despite the fact that the pHduring the course of manganese oxide reductionnever dropped markedly below the initial pH.Whether the greater ease of MnII release atneutral pH was attributable to the different typesof oxides involved, or whether a difference inmicroorganisms accounted for it, remains to bedetermined. In the open sea, a sharp drop in pHto below 5.0, even in a microenvironment, seemsunlikely because an open system is involved. Insuch a system, any acid formed metabolically ona nodule surface during manganese oxide reduc-tion may be rapidly neutralized and would diffuseor be swept away by circulating waters. Releaseof Mn2+ from the reduction process may here bepromoted by the complexing of Mn2+ with sulfate,bicarbonate, or organic compounds, such asamino acids. Sulfate and bicarbonate complexeshave been described by Hem (8).The chemistry of MnO2 reduction in the experi-

ments described in this paper may be summarizedby the following reaction:

glucose bacteria) ne- + nH+ + end products (1)

n/2MnO2 + ne +

nH+ -- adapted cells I

or unadapted cells + Fe(CN)63-

n/2Mn(OH)2

(2)

n/2Mn(OH)2 + nH+ -* n/2Mn2+ + nH2O (3)

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TRIMBLE AND EHRLICH

n/2MnO2 + ne- +

2nH+ adapted cellsor unadapted cells + Fe(CN)63 (2) + (3)

n/2Mn2+ + nH20

Reaction 2 may actually proceed in two steps,involving an initial reduction of MnO2 toMnOOH, followed by a reduction of MnOOHto Mn(OH)2; firm evidence for such a sequence ofreactions has not been found. The protons inreaction 3 are derived from acid end productsby bacteria on MnO2 and by those not on MnO2.When insufficient amounts of such acids areavailable, some or all solubilization of Mn(OH)2would have to depend on complexing by sulfate,bicarbonate, or other agents of Mn2+ formed bydissociation of Mn(OH)2.

ACKNOWLEDGMENrS

This investigation was supported by contract 591(22), work unit number NR 103-665, between theOffice of Naval Research, Department of the Navy,and Rensselaer Polytechnic Institute. I

LITERATURE C1TED

1. Alexander, M. 1961. Introduction to soil micro-biology. John Wiley and Sons, Inc., New York.

2. Barker, R. S., and W. H. Summerson. 1941. Thecolorimetric determination of lactic acid in bio-logical material. J. Biol. Chem. 138:535-554.

3. Ehrlich, H. L. 1963. Bacteriology of manganesenodules. I. Bacterial action on manganese innodule enrichments. Appl. Microbiol. 11:15-19.

4. Ehrlich, H. L. 1964. Microbial transformations ofminerals, p. 43-60. In H. Heukelekian and N. C.

Dondero [ed.], Principles and applications inaquatic microbiology. John Wiley and Sons,Inc., New York.

5. Ehrlich, H. L. 1966. Reactions with manganeseby bacteria from marine ferromanganesenodules. Develop. Ind. Microbiol. 7:43-60.

6. Feigl, F. 1946. Qualitative analysis by spot tests.Inorganic and organic applications, 3rd ed.,p. 133-135. Elsevier Publishing Co., Inc., NewYork.

7. Friedemann, T. E., and G. E. Haugen. 1943.Pyruvic acid. II. The determination of ketoacids in blood and urine. J. Biol. Chem. 147:415-442.

8. Hem, J. D. 1963. Chemical equilibria and rates ofmanganese oxidation. Geological Survey Water-Supply Paper 1667-A. U.S. Govt. PrintingOffice, Washington, D.C.

9. Hochster, R. M., and J. H. Quastel. 1952. Man-ganese dioxide as terinal hydrogen acceptorin the study of respiratory systems. Arch. Bio-chem. Biophys. 36:132-146.

10. Mann, P. J. G., and J. H. Quastel. 1946. Man-ganese metabolism in soils. Nature 158:154-156.

11. Neish, A. C. 1952. Analytical methods for bac-terial fermentations. Report no. 46-8-3 (sec.rev.), Natl. Res. Council, Canada.

12. Perkins, E. C., and F. Novielli. 1962. Bacterialleaching of manganese ores. U.S. Bureau ofMines, Report of Investigations 6102. U.S.Govt. Printing Office, Washington, D.C.

13. Umbreit, W. W., R. H. Burris, and J. F. Stauffer.1957. Manometric techniques. Burgess Publish-ing Co., Minneapolis.

14. Vavra, J. P., and L. R. Frederick. 1952. The effectof sulfur oxidation on the availability ofmanganese. Soil Sci. Soc. Am. Proc. 16:141-144.

702 APPL. MICROBIOL.

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