Vol. 48, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1984, p. 48-550099-2240/84/070048-08$02.00/0Copyright © 1984, American Society for Microbiology

Growth Kinetics of Thiobacillus ferrooxidans Isolated from ArsenicMine Drainage

JOAN FORSHAUG BRADDOCK,* HUAN V. LUONG, AND EDWARD J. BROWNInstitute of Water ResourceslEngineering Experiment Station, University of Alaska, Fairbanks, Alaska 99701

Received 16 January 1984/Accepted 2 April 1984

Thiobacillus ferrooxidans is found in many Alaskan and Canadian drainages contaminated by metalsdissolved from placer and lode gold mines. We have examined the iron-limited growth and iron oxidationkinetics of a T. ferrooxidans isolate, AKI, by using batch and continuous cultures. Strain AKI is an arsenic-tolerant isolate obtained from placer gold mine drainage containing large amounts of dissolved arsenic. Thesteady-state growth kinetics are described with equations modified for threshold ferrous iron concentrations.The maximal specific growth rate (limax) for isolate AK1 at 22.5°C was 0.070 h-1, and the ferrous ironconcentration at which the half-maximal growth rate occurred (K,) was 0.78 mM. Cell yields varied inverselywith growth rate. The iron oxidation kinetics of this organism were dependent on biomass. We found noevidence of ferric inhibition offerrous iron oxidation for ferrous iron concentrations between 9.0 and 23.3 mM.A supplement to the ferrous medium of 2.67 mM sodium arsenite did not result in an increased steady-statebiomass, nor did it appear to affect the steady-state growth kinetics observed in continuous cultures.

The bacterium ThiobacillusJerrooxidans is responsible formetal leaching and acid production in water draining fromsulfide deposits. T. ferrooxidan.s can catalyze the dissolutionof metals either by directly attacking sulfide ores or byoxidizing reduced iron leading to indirect dissolution via theferric ion (3). Sulfides and metals (such as arsenic) arecommonly associated with lode gold deposits and are some-times associated with placer deposits (46). Recently, T.ferrooxidans has been isolated from streams in Alaska andnorthern Canada that are affected by mining.Brown et al. (6) found large numbers of T. Jerrooxidans in

drainage from 90% of the placer mines that they sampled.Concentrations of dissolved arsenic >10 parts per billion (10,uig/liter) were found in 30% of those same streams. T.ferrooxidans and arsenic were also present in streams affect-ed by lode mining. In addition, T. Jerrooxidans will leachsubstantial amounts of arsenic from placer gold mine materi-al (H. V. Luong, J. F. Braddock, and E. J. Brown, Geomi-crobiol. J., in press). These results suggest that T. Jerrooxi-dans may be directly involved in the leaching of arsenic andother metals associated with active and abandoned mines tosubarctic streams and groundwaters. However, the mecha-nisms by which these organisms are involved in the cyclingof metals such as arsenic are not fully understood.Many investigators have studied the microbiologically

mediated dissolution kinetics of various sulfide minerals (10,23, 26, 32, 42, 44). Although the growth kinetics of variousthiobacilli when grown on reduced sulfur compounds havebeen described (2, 18, 25, 31), the iron-limited growthkinetics of T. ferrooxidans are not well understood. Thepartial kinetic descriptions that are found in the literature(21, 27-29, 33, 43) show inconsistent results. This may bedue to the difficulties of growing this organism, particularlyin continuous culture, and to taxonomic uncertainty aboutisolated strains of acidophilic iron oxidizers (13).

In this study, we used batch and continuous cultures todescribe the iron-limited growth and iron oxidation kineticsof a T. ferrooxidans isolate (AKI) obtained from a streamnear Fairbanks, Alaska. This stream is affected by placermining and contains large amounts of dissolved arsenic. The

* Corresponding author.

growth and iron oxidation kinetics of isolate AK1 appear todeviate from traditional kinetic models. There was no appar-ent effect on the growth or iron oxidation kinetics of thisisolate when the initial ferrous iron concentration was in-creased from 9.0 to 23.3 mM. Additionally, the presence ofreduced arsenic in the feed ferrous iron did not affect thekinetics of this arsenic-tolerant isolate.

MATERIALS AND METHODSMicroorganisms. T. ferrooxidans AK1 was originally iso-

lated from Eva Creek, located near Fairbanks, Alaska, andis the same isolate as strain TF133 described by Martin et al.(34). Eva Creek is heavily affected by placer gold mining andis known to contain large amounts of dissolved arsenic (6).Growth medium. The growth medium was modified from

that described by Tuovinen and Kelly (45) and containedMgSO4 7H20 (0.4 g liter-1), (NH4)2SO4 (0.4 g * liter-1),KH,PO4 (0.1 g liter- i), and 10 N H2S04 to bring themedium to pH 1.85 to 1.95. Reduced iron as a sole energysource was supplied as FeSO4 7H,O, and concentrationsvaried in different experiments from 5 to 30 g * liter-1 forbatch cultures and 2.5 to 6.5 g liter-1 for continuouscultures. To eliminate iron precipitate formation, the compo-nents of the medium were autoclaved in two separatesolutions and were aseptically mixed after the solutionscooled to room temperature. Solution A contained theFeSO4 and about 2.5 ml of 10 N H2SO4 per liter of finalmedium volume. Solution B contained the remaining saltsdiluted in water. The water used throughout these experi-ments was American Society for Testing Standards Type Iquality.

In some continuous-culture experiments, 2.67 mM re-duced arsenic (supplied as NaAsO2) was also incorporatedinto the culture medium. The arsenite was dissolved in water(pH 2) and was then aseptically filtered into the preparedmedium.Batch culture. For batch culture experiments, 5 to 10 ml of

exponentially growing cells was inoculated into 500-ml Er-lenmeyer flasks containing iron medium. The flasks wereplaced on a rotary shaker and were maintained at 22.5°C forthe duration of each experiment. Microbiological and chemi-cal parameters were measured periodically in small samples

48

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

GROWTH KINETICS OF T. FERROOXIDANS 49

SAMPLING

~~~~PORTMAGNETIC

STIRRER

PERISTALTIC WASTEPUMP

FIG. 1. Two-phase continuous-culture system used for cultiva-tion of T. ferrooxidans.

aseptically removed from each flask. Triplicate flasks wereused so that a mean and standard error could be calculatedfor the measured quantities at each sampling time.

Continuous culture. A two-phase continuous-culture appa-ratus (Fig. 1) was designed to provide the optimum condi-tions for iron oxidation by T. ferrooxidans. Culture mediumstored in a 20-liter glass carboy was pumped by a peristalticpump directly into a specially designed reactor vessel. Thereactor was adapted from a 1-liter boiling flask by adding an

effluent tube, atn air-sparging tube, and a sampling port. Theeffluent port was placed to maintain the volume of liquid inthe flasks at 500 to 600 ml. The exact volume of liquid wasmeasured for each flask. Compressed air to supply oxygenand carbon dioxide was added via the air port at a rate of 500to 600 ml * min- . The sampling port was sealed with asilicone stopper through which a small piece of glass tubingwas inserted. A small valve connected the glass tubing to a

sterile syringe. We could rapidly and efficiently removesterile samples with this system.A magnetic stirring device continually agitated the con-

tents of the reactor flask. The flasks were supported slightlyabove the stirrer to avoid any effect on the culture vesselfrom the heat generated by the motor of the stirrer. Siliconestoppers sealed the reactor vessel and feed carboy. Sterilecotton filters inserted in the feed carboy stopper and reactorvessel stopper maintained ambient pressure throughout thesystem. Glass tubing and a minimal amount of siliconetubing connected the feed carboy to the reactor vessel.The continuous cultures were maintained at 22 to 23°C in a

temperature-controlled room. At the start of an experiment,5 to 10 ml of exponentially growing cells was added to 200 to300 ml of fresh culture medium. The peristaltic pump was

then started, the vessel was filled, and continuous cultiva-tion was begun. Generally, a slow flow was used for severaldays until organism growth was observed, at which time thepump was adjusted to the desired flow rate. The flow rate foreach culture was measured several times to assure continu-ous and constant delivery at a given flow rate by each pump.The dilution rate (D) was calculated as the flow rate dividedby the volume of the culture vessel. The value of D wasassumed to be equivalent to the specific growth rate (,u) ofthe organism at steady state. After at least three residencetimes (R, where R = D-' or 7-1) had passed at a given flowrate, a steady state was assumed, and samples of the reactorvessel contents were removed for microbiological and chem-ical analysis.

Enumeration. Numbers of cells in laboratory cultureswere determined by epifluorescent direct-count microscopyby a method adapted from that described by Hobbie et al.(22). Measured aliquots (0.1 to 0.5 ml) of flask or reactor

vessel contents were placed in small test tubes containingfiltered water. Two drops of filtered 0.1% (in water) acridineorange solution were then added to each tube to stain thecells. The stained-cell suspension in each tube was thenfiltered with a 3-ml syringe and a Swinnex filter apparatus(Millipore Corp.) onto an irgalan black-stained (22) filter(0.2-,um pore size, 25-mm diameter; Nuclepore Corp.). Fil-tered water (three 2-ml portions) was used to rinse each tubeand the filter apparatus. The damp Nuclepore filter was thenplaced, with a drop of immersion oil, under a cover slip on amicroscope slide and was held at 4°C in the dark for 30 minto 1 h, after which 15 to 20 microscope fields were countedper slide by using a Zeiss Standard microscope with anepifluorescence light source. Duplicate or triplicate filterswere prepared and counted for each sample.

Ferrous iron. Ferrous iron concentrations in solution(filtered through a Millipore type HA filter [0.22-Rm poresize; 25-mm diameter]) and total ferrous iron concentrations(nonfiltered) were determined by the ortho-phenanthrolinespectrophotometric method of the American Public HealthAssociation (1). Ferrous iron concentrations were monitoredin continuous-culture feed reservoir medium to assure chem-ical stability for the duration of an experiment. Ferrous ironwas stable in the acid media for at least 6 months.

Arsenic. Arsenic concentrations were designated as ar-senic in solution (filtered through a Millipore type HA filter[0.22-iim pore size; 25-mm diameter]) or as total arsenic(including arsenic in solution and associated with bacterialcells).

Total arsenic (micrograms per liter range) was analyzed bythe graphite furnace atomic absorption method described byWilson and Hawkins (46) as modified with a nickel matrix(19) or (milligrams per liter range) by the flame atomicabsorption method with an Electrodeless Discharge Lamp at193.7 nm as described for the Perkin-Elmer model 4000atomic absorption spectrophotometer (Publication 0303-0152, 1982, The Perkin-Elmer Corp., Norwalk, Conn.).

Ionic species of arsenic were determined in culture fil-trates by treating 2.0 ml of sample with 0.2 ml of a modifiedMurphy and Riley (36) molybdate reagent, containing 4 ml ofammonium molybdate (30 g - liter-'), 5 ml of 10 N H2SO4, 2ml of potassium antimony tartarate (1.36 g liter-'), and0.108 g of ascorbic acid. The samples were incubated for ca.6 h, at which time the arsenate fraction was extracted with

TABLE 1. Continuous-culture parameters

Symbol Parameter Units

SR Influent limiting nutrient con- mMcentration

S Ambient limiting nutrient con- mMcentration (external limitingnutrient concentration)

St Threshold mMYcarbon Yield mg of C *mMYcell Yield cells * mM-'

Specific growth rate h-'Gross iron oxidation rate mM * h-'

I1max Maximum growth rate h-I'Vmax Maximum iron oxidation rate mM - h-'K,^ Growth half-saturation constant mMKFe Iron oxidation half-saturation mM

constant

1'carbon Net iron oxidation rate mmol clmg of C h1'cell Cellular iron oxidation rate fmol cell' h'as Steady-state affinity liters mg of C` h`

VOL. 48, 1984

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

50 BRADDOCK, LUONG, AND BROWN

1.4

0.02 - 0.20.15 '

120 X0.oX, +

1-. N-

.c *n0 0 E+ a o

_u _

40 o oE *o o

9.02

0at

7.0

6.0

5.0

Time, hr

FIG. 2. Ferrous iron in solution (A), log1l number of cells (0),and carbon yield and cell yield (O) as functions of time for batch-grown cells.

isoamyl alcohol. The aqueous (arsenite-containing) phasewas analyzed, as described above, by atomic absorptionspectrophotometry (4). Arsenate ion concentrations werecalculated as the differences between total arsenic andaqueous phase concentrations.Carbon. Organic carbon was measured by the wet-chemi-

cal method described for the Technicon Auto Analyzer II(industrial method no. 451-76W/A, 1978, Technicon Instru-ments Corp., Inc., Tarrytown, N.Y.). This method wasselected for its sensitivity (0.4 to 20.0 mg of C * liter-') andsmall sample volume requirement (2 ml).

Kinetic measurements. A list of continuous-culture param-eters is found in Table 1. The relationships among theseparameters were used to describe the growth and ironoxidation kinetics of T. ferrooxidans in continuous culture.Growth rates (pL) were plotted against residual ferrous ironconcentrations with a modified Monod model (8). The modi-fied Monod relationship can be expressed as

I- = .max(S - St)[K + (S - St)' (1)

and can be linearized by the relationship

(S - St)([u)-. = Kp,(PmaxJ' + (S - St)(LmaxFW (2)

(see Table 1 for definition of symbols). This linearization wasselected rather than the more commonly used double-recip-rocal plot since it is less biased by deviations at low substrateconcentrations (11) and, for these experiments, yields amore accurate estimate of the maximum growth rate (P.max).Alternatively, P-max was estimated with equations analogousto thqse from the internal stores model (17, 40).e iron oxidation rate (v) was analyzed as a function of theresidual ferrous iron concentration, specific growth rate, andcarbon yield (Ycarbon). The relationship between v and theexternal ferrous iron concentration (S) was linearized asdescribed for the Monod model. Regression analysis wasused to estimate best-fit lines for all linear relationships.Curvilinear relationships were drawn by hand to indicategeneral trends in the data. The significance of the slope oflinear relationships at the 95% confidence level was deter-mined with F tests.

RESULTSBatch culture. The results of a batch culture experiment in

which ferrous iron in solution, organic carbon, and numbers

0.06r

0.051-

0.041-

0.03 1;Z/4 000

0.02-

0.01 F

0.0 0.5 1.0 1.5 2.0 2.5

External ferrous iron, mM

FIG. 3. Steady-state growth rate as a function of external ferrousiron concentration for T. ferrooxidans. Symbols represent influentferrous iron concentrations (mM) as follows: K, 9.0 to 9.5; *, 9.0 +1.3 to 2.7 mM As3"; A, 17.9; 0, 22.4 to 23.3.

of cells were monitored over time are shown in Fig. 2. Theinitial ferrous iron concentration in the culture medium was108 mM in this experiment. These data can be used topredict an apparent maximum specific growth rate, a maxi-mum iron oxidation rate (Vmax), and growth yields for thisisolate.The plot of logl0 numbers of cells over time can be used to

estimate the apparent P-max. The apparent P-max was calculat-ed to be 0.089 h- l for strain AK1 in this experiment and in aduplicate experiment (data not shown). Generation time (tg),the time required for the population to double during expo-nential growth, was about 7.7 h. The relationship betweenferrous iron in solution and time was used similarly toestimate an apparent maximum iron oxidation rate of 3.34mmol liter-' h-' for this isolate.The organic-carbon and cell number data were used to

calculate growth yields, Ycarbon and Ycell, for strain AK1.The Ycarbon ranged from 0.12 to 0.20 mg of carbon per mmolof Fe2+ oxidized, whereas the Ycell demonstrated an increase

40

30

20E 20

cn

cn10

0

0

0

K,, = O. 78mAltno =W 0. 070hr -/

0.0 0.5 1.0

S-St, mM

1.5 2.0

FIG. 4. Linearization of the Monod growth curve (thresholdcorrected) for T. ferrooxidans. The equation for the line at the 95%confidence level is y = (14.30 + 2.83)x + 11.12 ± 2.78 with r = 0.87.Symbols are the same as those in Fig. 3.

APPL. ENVIRON. MICROBIOL.

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

GROWTH KINETICS OF T. FERROOXIDANS 51

1.2

1.0I..

I-

-

7

62E

0-

ac0._.

a0ax

cp

0.00 0.01 0.02 0.03 0.04 0.05

0.6

Q4

0.06

A

a

c

0.5 1.0 1.5 2.0 2.5

Growth rate, hr-1

FIG. 5. The steady-state yield of cellular carbon as a function ofgrowth rate for T. ferrooxidans. Symbols are the same as those inFig. 3.

over time from 0.25 109 to 1.20 109 cells per mmol of Fe2+oxidized. In a duplicate experiment (data not shown), a

similar final Ycell value of 1.3 109 cells per mmol of Fe'2oxidized was calculated. These Ycarbon and Ycell valuessuggest that the amount of carbon per cell decreased over

time in batch-grown cells. At the stationary phase of growth(near the end of the experiment), there was ca. 0.12 pg ofcarbon per cell.

Continuous culture. (i) Growth kinetics. The Monod rela-tionship between growth rate and external ferrous ironconcentration for isolate AKi is shown in Fig. 3. Thisrelationship describes the growth of this isolate at severaldifferent influent ferrous iron concentrations (SR) and in thepresence and absence of reduced arsenic. An apparentthreshold ferrous iron concentration (St; x intercept) ofabout 0.25 mM was estimated from the results in Fig. 3. Thisextrapolated threshold value represents ferrous iron unavail-able to the organism for growth (8).The apparent threshold value was applied to a lineariza-

External ferrous Iron, mM

FIG. 7. Steady-state gross ferrous iron oxidation rate as a func-tion of external ferrous iron concentration at the three differentinfluent ferrous iron concentrations (A, B, C) as defined by thesymbols in Fig. 3.

tion of the Monod (35) growth curve (Fig. 4). The apparentP-max was 0.070 h- l, and the threshold-corrected growth half-saturation constant (K, ) was 0.78 mM (Fig. 4).

Ycarbon, expressed as carbon produced per iron oxidized as

a function of growth rate, is shown in Fig. 5. The steady-state Ycarbon decreased with the increasing dilution rate forthis isolate. This decrease is apparently related to a decreasein carbon per cell as a function of growth rate (Fig. 6) for thisisolate. The carbon-to-cell ratio was not constant whenstrain AK1 was grown in batch cultures (Fig. 2).

(ii) Iron oxidation kinetics. The Michaelis-Menten relation-ship between the gross (total) iron oxidation rate and theexternal ferrous iron concentration at three different influentferrous iron concentrations is shown in Fig. 7. The apparentthreshold ferrous iron concentration for gross iron oxidationat three SR values was ca. 0.25 mM, the same value

5

40.5 r

0.4 FzU

u

C.)

C

0.0

U.a

I-)

0.3 F

b-

U 21

D

0.2 V0

0.1 F

ao

c

B

A

0

0.5 1.0 1.5 2.0

0.0 L-

0.00 0.01 0.02 0.03 0.04 0.05

Growth rate, hr-1FIG. 6. The steady-state carbon/cell ratio as a function of growth

rate. Symbols are the same as those in Fig. 3.

S-St, mM

FIG. 8. Threshold-corrected linearization of Fig. 7. The equa-tions for the lines at the 95% confidence level are as follows: A, y =

(0.45 + 0.39)x + 0.91 0.48 with r = 0.85; B, y = (0.57 + 0.78)x +

1.06 + 1.00 with r = 0.47; and C, y = (2.44 + 0.56)x + 0.87 + 0.39

with r = 0.91. Symbols are the same as those in Fig. 3.

. 0.5x0e

*~04.- OA"0E

u 0.3

la 0.2

0.1

VOL. 48, 1984

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

52 BRADDOCK, LUONG, AND BROWN

- 0.57 D>

£

E 0.4 - O/E

00

'R0.2 0z ,|

0 %~~~~~~

0~~~~

0..

0.0 0.5 1.0 1.5 2.0 2.5

External ferrous iron, mM

FIG. 9. Net iron oxidation rate as a function of external ferrousiron concentration. The equation for the line at the 95% confidencelevel is y = (0.22 ± 0.04)x - 0.05 ± 0.04 with r = 0.91. Symbols are

the same as those in Fig. 3.

estimated for growth kinetics. This indicates that iron oxida-tion and growth kinetics are tightly coupled. Threshold-modified linearization of the gross iron oxidation rate versus

the external ferrous irori concentration is shown in Fig. 8.This figure can be used to predict a vmmax and a half-saturationconstant (KFe) for different values of SR. The apparent vmaxpredicted from the reciprocal slope of the best-fit lines whenSR = 9.0 mM was 0.41 mmol * liter-' h- l. The Vmax for SR =

22.4 to 23.3 mM was 2.22 mmol * liter-' h-. The KFe isestimated from vmax times the y intercept. Thus, KFe was

0.36 and 2.02 mM for SR = 9.0 and 22.4 to 23.3, respectively.Insufficient data were available at low dilution rates to yielda significant linear relationship when SR = 17.9 mM, so no

attempt was made to estimate I'max or KFe for this value ofSR .The Michaelis-Menten relationship between net iron oxi-

dation rate (icarhon) and external ferrous iron concentrationis shown in Fig. 9. Since saturation was not apparent, we

assumed that the relationship between 1'carhon and S was

linear (first order). A theoretical maximum net iron oxidationrate must exist; therefore, the rates we observed for AKimust be significantly below the maximum. The x intercept(threshold) concentration of the linear relationship (Fig. 9) is0.24 mM. The slope of the line (Fig. 9) is mathematicallyanalogous to steady-state affinity (as) as defined by Button(9). The a, for isolate AKI is ca. 0.22 liters * mg of C'and is a good parameter to use for comparative measures ofspecific (subsaturating) iron oxidation rate at low iron con-

centrations.(iii) Arsenic. Reduced arsenic (1.33 or 2.67 mM) was added

to the ferrous iron medium in several continuous-cultureexperiments. The results indicated that reduced arsenic was

not oxidized in the presence of isolate AK1 growing at fourdifferent growth rates ranging from 0.007 to 0.034 h-l.Virtually none of the reduced arsenic added was recoveredin the oxidized form.

DISCUSSIONGrowth kinetics. We have used batch and dilute (low

biomass) continuous cultures to describe the growth kinetics

of an arsenic-tolerant subarctic isolate of T. ferrooxidans.The growth and iron oxidation kinetics of this isolate deviatefrom traditional kinetic models.The conventional Monod model which was originally

derived from Michaelis-Menten enzyme kinetics is useful fordescribing the carbon-limited growth of heterotrophs whenthe growth-limiting substrate is incorporated as cellularmaterial. When T. ferrooxidans is grown on reduced iron,the energy-limiting substrate is not incorporated as cellularmaterial. The Monod model depends on a constant yieldwhich links specific growth rate to substrate removal. TheMonod model has also been modified to accommodate thepossibility that yield may increase with increasing growthrate (i.e., maintenance energy; see reference 39). The modelis less readily adaptable to the possibility that yields maydecrease with increasing growth rate.

Decreasing yields with increasing growth rate have com-monly been observed for a variety of microorganisms grownin chemostats under the limitation of inorganic ions such asphosphate, magnesium, silicate, ammonium, and nitrate andof vitamins such as B12 (5, 12, 30, 40). The internal-storeskinetic model originally proposed by Droop (17) and modi-fied by Rhee (40) has been used to describe growth rates as afunction of internal rather than external concentrations oflimiting substrates. It is essential in this kinetic model thatyields vary inversely with growth rate since concentrationsof intracellular substrate increase with increasing growthrate (38, 40). The application of either the Monod or theinternal-stores model to the observed growth of dilute, iron-limited cultures of isolate AK1 is limited. Despite theserecognized limitations, either model can be used to mathe-matically describe some growth and iron oxidation functionsfor this isolate.The Monod model was used to estimate iron-limited

growth parameters for strain AKi with respect to externalferrous iron concentrations. The values we found for PLmaxand K. were somewhat lower than those reported for otherisolates of T. ferrooxidans. Values reported for other isolatescultured in chemostats include the following: a Pmax of 0.14h-V and a K of 37 mM (43); a Pmax of 0.161 h-l and a K of3.8 mM (33); and a -max of 1.25 to 1.78 h-1 and a K of0.7 to2.4 mM (27, 28). These apparent discrepancies are probablya result of growing the organism under different physiologi-cal regimes (i.e., pH and temperature) and of variations inthe microorganisms classified as T. ferrooxidans. Severalinvestigators recently demonstrated that variations of taxo-nomic importance may occur among different isolates of T.ferrooxidans. Variations have been found in DNA basecomposition studies (20, 34), physiological studies (41), andmorphological studies (13). Growth and iron-oxidation kinet-ics of these iron-oxidizing bacteria may be yet another wayof describing a functional classification for these microorga-nisms.Our results also show apparent threshold ferrous iron

concentrations of about 0.25 mM for both iron-limitedgrowth and iron oxidation. The threshold ferrous iron con-centration observed here represents a concentration of sub-strate unavailable to strain AKi for ehergy utilization but isnot analogous to maintenance energy since the same thresh-old exists for both growth rate and iron oxidation rate (i.e.,maintenance energy is observed as a positive threshold onlyfor growth in conventional kinetic models). Thresholds havenot been reported for other T. ferrooxidans strains growingon reduced iron. However, most other studies have usedmuch higher iron concentrations, resulting in much morebiomass in the continuous-culture vessel. Eccleston and

APPL. ENVIRON. MICROBIOL.

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

GROWTH KINETICS OF T. FERROOXIDANS 53

Kelly (18) did observe a maintenance energy coefficient for aT. Jerrooxidans strain grown in either tetrathionate- orthiosulfate-limited chemostat cultures. However, the lowestdilution rate used in these studies was 0.02 h'. The appar-ent threshold of 0.25 mM for both growth and iron oxidationindicates that growth and iron oxidation are tightly coupledin isolate AKI. The threshold value may be an indication ofinhibition of ferrous oxidation at high ratios of ferric iron toferrous iron. However, increasing ferric iron/ferrous ironratios do not change specific (biomass-adjusted) ferrousoxidation rates (7).The yield of organic carbon for strain AK1 was relatively

constant in batch cultures, ranging from 0.12 to 0.20 mg ofcarbon per mmol of iron oxidized. However, steady-state(continuous-culture) data indicate a decreasing curvilineardependence of Ycarbon on the specific growth rate. particular-ly below the point where p. = 0.03 h- 1 (Fig. 5). If Y,[rbon databelow this point had not been available, the yield might havebeen assumed to be constant with increasing dilution rate.Yce,i (number of cells mmol of iron oxidized-') also de-creased as a function of p. (data not shown). The exponentialfunction (Fig. 5) can be approximated by the linearization InY = -22.19p. - 0.89 (r = -0.89) which describes theobserved relationship between Y.r,,tn and p.The steady-state carbon/cell ratio also appears to decrease

with increasing dilution rate for isolate AKI (Fig. 6). Weobserved, when counting AK1 cells by epifluorescencemicroscopy, that cell size markedly increased at low dilutionrates. Other investigators have reported similar observations(D. W. Tempest and 0. M. Neijssel, Abstr. Third Int. Symp.Microbial Ecol., 1983, 112, p. 8). Our data indicate that atlow growth rates strain AK1 is most efficient at utilizingreduced iron. The highest Ycarbo)n we observed was 0.50 mgof C per mmol of iron oxidized at p = 0.007 h- l. This yield isclose to the theoretical maximum growth yield of 0.53 mg ofC per mmol of iron for T. ferlooxidans growth at pH 2 (24).The steady-state growth kinetics of strain AKI apparently

do not depend on the initial ferrous iron concentration (Fig.3) over the range of SR values used in these experiments.Additionally, the growth kinetics of this isolate are notaffected by the addition of reduced arsenic to the ferrousiron-containing feed.

Iron oxidation kinetics. The gross ferrous iron oxidationrate at steady state depends on the influent ferrous ironconcentration (see Fig. 7 and 8 for strain AK1). The imjx andthe KFe both appear to increase with increasing SR. Thisincrease in maximum iron oxidation rates with increasing SRwas also observed by Guay et al. (21). Guay et al. examinedthe steady-state iron oxidation kinetics of a T. jerrooxidansstrain at influent ferrous iron concentrations of 89 to 161mM. Their strain did not reach its maximum capacity foriron oxidation even at an SR of 161 mM.The observed dependence of 1'niax on influent substrate

concentration is predicted from Michaelis-Menten kineticsapplied to substrate oxidation in a chemostat. The Michaelis-Menten parameter, i'mj,x. is a relative rather than a specificvalue. The apparent maximum rate of the enzyme is attainedwhen all of the enzyme sites are saturated with substrate.Therefore, if more enzyme is present at a given substrateconcentration, the apparent V'm.tx is greater. When the influ-ent ferrous iron concentration is increased, a larger concen-tration of organic carbon (as a function of ambient substrateconcentration or of growth rate) is produced by strain AKI.Assuming that cellular carbon represents about 50% of thedry weight of the AK1 cell under iron-limited, steady-stateconditions, an increase in cell dry weight (biomass) should

result in an increased concentration of ferro-oxidase per unitvolume. Therefore, the total activity of the enzyme is greaterat increasing influent ferrous iron concentrations as long asthe cells remain iron limited.The gross iron oxidation rate can be converted to a

specific rate by defining iron oxidation per unit of carbon('carhon) or per numbers of cells (OceIl). These specific iron

oxidation rates are analogous to the specific growth rateconstant described in the Monod growth model. The l carbonas a function of the S-S, is independent of the SR for strainAKI (Fig. 9). Therefore, enzyme concentration, not ferricinhibition of ferrous-iron oxidation, appears to be responsi-ble for the results observed in Fig. 7 and 8, which may alsobe used as evidence that ferrous iron remained the limitingfactor in these experiments. Additionally, reduced arsenicadded to the feed does not affect the net iron oxidation rateof this organism.

Expressed mathematically, 1'carhon p :Ycarbo 1. When11 Ycarbj l is plotted against Y.,trh- ' by analogy to theinternal-stores growth model, the relationship described bythe equation pL = 0.070 - 0.179Y(r = 0.933) predicts a p-maxof 0.070 h-', the same value predicted by the Monodequation.The steady-state 1'carhon as a function of S (Fig. 9) for strain

AKI appears to be a linear relationship. Since a theoreticalmaximum net iron oxidation rate must exist, the functionmust asymptotically approach a maximal value. I'his indi-cates that the observed rates are significantly lower than themaximum rate obtainable by strain AK1. The maximum ratewould presumably occur when much higher ferrous ironconcentrations are added to iron-starved batch cultures (30).

Arsenic. T. fe'rooxidans oxidizes many reduced metalsincluding cuprous copper (37), elemental selenium (44), anduranous sulfate (14-16). Furthermore, T. fjrrooxidans maybe able to obtain energy from some of these inorganicoxidations (15, 16).

Strain AK1 was isolated from placer gold mine drainagecontaining dissolved arsenic. Speciation of the dissolvedarsenic in this stream indicated that arsenate species andother species (including arsenite) were present in aboutequal amounts (6). Arsenate was not the only species presentdespite the fact that the stream was saturated with oxygen.

In laboratory cultures, strain AK1 was tolerant to at least900 mg of arsenite or arsenate liter-' when the organismwas grown in a medium containing ferrous iron (J. M.Forshaug, M.S. thesis, University of Alaska, Fairbanks,1983).For these reasons, we hypothesized that T. fe,,ooxidans

isolate AKI can oxidize reduced arsenic and derive energyfrom such an oxidation. However, the results of steady-stateexperiments to test this hypothesis were inconclusive. Whenarsenite was added to the continuous-culture medium con-taining ferrous iron, we observed no oxidation of the arseniteion. The presence of either 1.33 or 2.67 mM arsenite in themedium did not yield an increase in biomass, nor did it haveany observable effect on growth-kinetic or iron oxidationkinetic parameters being measured. If arsenite can be usedfor energy production by isolate AK1, an observable in-crease in biomass would be expected.Summary. The importance of the iron-oxidizing thiobacilli

in catalyzing biogeochemical cycles has only recently beenrecognized. With the depletion of high-grade ores. methodsof bioleaching for the recovery of valuable metals are

becoming increasingly important. At some time in the future.genetic manipulation may be used to produce highly efficientleaching microorganisms (3). The iron-limited growth kinet-

VOL. 48, 1984

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

54 BRADDOCK, LUONG, AND BROWN

ics have not been well described for T. jerooxidans. Pub-

lished kinetics are inconsistent. These inconsistencies prob-ably result from apparent strain-to-strain differences andfrom the difficulties in culturing this organism, especially in

iron-limited continuous cultures. A complete kinetic modelmust describe both cell growth and substrate utilization withsome function that relates the specific growth rate to thesubstrate utilization rate. The yield constant links the equa-

tions of the Monod model. Alternatively, the Monod modelcan be modified to accommodate an increasing yield withincreasing growth rate when a maintenance coefficient isobserved. Other kinetic models have been developed (17. 38.40) to describe the growth of microorganisms as a function ofinternal rather than external concentrations of a limitingnutrient.We have described the growth kinetics, ferrous iron

oxidation kinetics, and arsenite oxidation potential of an

arsenic-tolerant T. fjerrovidans isolate. AK1. The growth ofthis organism is described in both transient (batch) cultureand steady-state (continuous) culture. We have observedthat the growth and iron oxidation kinetics of this autotro-phic isolate deviate from Monod kinetics. The kinetics ofthis isolate show some similarities to kinetic models derivedto describe substrate-limited growth from internal ratherthan external substrate concentrations. These observationsindicate that additional studies are needed, particularly atlow dilution rates, to accurately model the growth andsubstrate utilization of T. jerrooxidans and possibly otherautotrophic microorganisms.We have also described a method for the successful

continuous cultivation of T. Jfrrooxiidans in dilute, iron-

limited continuous cultures. This continuous-culture methodcan be used to predict the growth and iron oxidation kineticsof other strains of T. jerrooxidins and to investigate theability of T. jerrooxiclans to oxidize and produce energy fromreduced metals other than iron. Such studies are importantfor understanding the biogeochemical cycling of many met-als and for applying these microorganisms to biohydrometal-lurgy.

ACKNOWLEDGMENTS

Support for this study was provided in part by grants B-024-ALASand A-070-ALAS from the Office of Water Policy. U.S. Departmentof the Interior. and by grant MIN-1-81 from the Alaska Council on

Science and Technology.

LITERATURE CITED

1. American Public Health Association. 1975. Standard methods forthe examination of water and wastewater, 14th ed.. p. 207-213.American Public Health Association. Inc.. Washington. D.C.

2. Bounds, H. C., and A. R. Colmer. 1972. Comparisoni of thekinetics of thiosulfate oxidation by three iron-sulfur oxidizers.Can. J. Microbiol. 18:735-740.

3. Brierley, C. L. 1982. Microbiological mining. Sci. Am. 247:44-53.

4. Brown, E. J., and D. K. Button. 1979. A simple method ofarsenic speciation. Bull. Environ. Contam. Toxicol. 21:37-42.

5. Brown, E. J., and D. K. Button. 1979. Phosphate-limited growthkinetics of a green alga. J. Phycol. 15:305-311.

6. Brown, E. J., H. V. Luong, and J. M. Forshaug. 1982. Theoccurrence of 7Thioba(c(illius f'erloo.xitlaoins and arsenic in subarcticstreams affected by gold mine drainage. Arctic 35:417-421.

7. Brown, E. J., H. V. Luong, and j. M. Forshaug. 1983. Geomi-crobiology of arsenic associated with gold deposits in Alaska. p.

571-580. In G. Rossi and A. E. Torma (ed.). Recent progress inbiohydr-ometallurgy. Associlzione Mineraria Sarda. Iglesias.Itally.

8. Button, D. K. 19)78. O)n the theory of control of microbiall growth

kinetics by limiting nutrient concentrations. Deep-Sea Res.25:1163-1177.

9. Button, D. K. 1983. Differences between the kinetics of nutrientuptake by microorganisms. growth and enzyme kinetics. TrendsBiochem. Sci. 8:121-124.

10. Chang, Y. C., and A. S. Myerson. 1982. Growth models of thecontinuous bacterial leaching of iron pyrite by Thiobacilliusferroo.voidans. Biotechnol. Bioeng. 24:889-902.

11. Cornish-Bowden, A. 1979. Fundamentals of enzyme kinetics, p.15-33. Butterworth. Woburn. Mass.

12. Cunningham, A., and P. Maas. 1982. The growth dynamics ofunicellular algae. p. 167-188. In M. J. Bazin (ed.). Microbialpopulation dynamics. vol. 1. CRC Press. Inc.. Boca Raton. Fla.

13. DiSpirito, A. A., M. Silver, L. Voss, and 0. H. Tuovinen. 1982.Flagella and pili of iron-oxidizing thiobacilli isolated from auranium mine in northern Ontario, Canada. AppI. Environ.Microbiol. 43:1196-1200.

14. DiSpirito, A. A., and 0. H. Tuovinen. 1981. Oxygen uptakecoupled to uranous sulfate oxidation by Thiobacillhs ferrooxi-(lans and T. acidophilus. Geomicrobiol. J. 2:275-291.

15. DiSpirito, A. A., and 0. H. Tuovinen. 1982. Uranous ionoxidation and carbon dioxide fixation by Thiobacillius ferrooxi-dlans. Arch. Microbiol. 133:28-32.

16. DiSpirito, A. A., and 0. H. Tuovinen. 1982. Kinetics of uranousand ferrous iron oxidation by Tlhiohbc('illius f'('OO-i-oxdn.s. Arch.Microbiol. 133:33-37.

17. Droop, M. R. 1968. Vitamin B,2 and marine ecology. IV. Thekinetics of uptake. growth and inhibition in Monochr.vsis lhtheri.J. Mar. Biol. Assoc. U.K. 48:689-733.

18. Eccleston, M., and D. P. Kelly. 1978. Oxidation kinetics andchemostat growth kinetics of Tliioba(cillius ferroo.idalns on te-trathionate and thiosulfate. J. Bacteriol. 134:718-727.

19. Ediger, R. D. 1975. Atomic absorption analysis with the graphitefurnatce using matrix modification. At. Absorpt. Newsl. 14:127-132.

20. Guav, R., M. Silver, and A. E. Torma. 1976. Base compositionof DNA isolaited from 7lhiobacillhs fierrooxidans grown ondifferent substrates. Rev. Can. Biol. 35:61-67.

21. Guay, R., M. Silver, and A. E. Torma. 1977. Ferrous ironoxidation and uranium extraction by Tlhioba(illius ferroo.idans.Biotechnol. Bioeng. 19:727-740.

2 . Hobbie, J. E., R. J. Daley, and S. Jasper. 1977. U se ofNuclepore filters for counting bacteria by fluorescence micros-copy. Appl. Environ. Microbiol. 33:1225-1228.

23. Hoffman, M. R., B. C. Faust, F. A. Panda, H. H. Koo, and H. M.Tsuchiya. 1981. Kinetics of the removal of iron pyrite from coalby microbial catalysis. Appl. Environ. Microbiol. 42:259-271.

24. Ingledew, W. J. 1982. Tliiob(acillis fe('lrloox.iitdans: the bioenerget-ics of an acidophilic chemolithotroph. Biochim. Biophys. Acta683:89-117.

25. Justin, P., and D. P. Kellv. 1978. Growth kinetics of 7liiobacillhsclenitrificains in anaerobic and aerobic chemostat culture. J.Gen. Microbiol. 107:49-58.

26. Karaivko, G. 1., S. 1. Kuznetsov, and A. I. Golonizik. 1977. Thebacterial leaching of metals from ores. Technicopy. Ltd.. Stone-house. U.K.

27. Kelly, D. P., M. Eccieston, and C. A. Jones. 1977. Evaluation ofcontinuous chemostat cultivation ofTIhiohbcillins fi ,.,.oovidaccnson ferrous iron or tetrathionate. p. 1-7. In W. Schwartz (ed.),Gesellschaft fur biotechnologische Forschung Conference-Bacterial Leaching. Verlag Chemie GmbH. Weinheim. FederalRepublic of Germany.

28. Kelly, D. P., and C. A. Jones. 1978. Factors affecting metabo-lism and ferrous iron oxidation in suspensions and batch cul-tures of 71hiobacillus fierrooxidans: relevance to ferric iron leachsolution regeneration. p. 19-43. In L. E. Murr. A. E. Torma,and J. E. Brierley (ed.). Metallurgical applications of bacterialleaching and related microbiological phenomena. AcademicPress, Inc.. New York.

29. Lacey, D. T., and F. Lawson. 1970. Kinetics of the liquid-phaseoxidation of acid ferrous sulfate by the bacterium Thiobczcillhsfcnrooxidans. Biotechnol. Bioeng. 12:29-50.

30. Lang, D. S., and E. Jl. Brown. 1981. Phosphorus-limited growth

Appi- ENVIRON. MICROBIOL.

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from

GROWTH KINETICS OF T. FERROOXIDANS 55

of a green alga and a blue-green alga. Appl. Environ. Microbiol.42: 1002-1009.

31. Leefeldt, R. H., and A. Matin. 1980. Growth and physiology ofThliobacillls notvel/ius under nutrient-limited mixotrophic condi-tions. J. Bacteriol. 142:645-650.

32. Lundgren, D. G., and M. Silver. 1980. Ore leaching by bacteria.Annu. Rev. Microbiol. 34:263-283.

33. MacDonald, D. G., and R. H. Clark. 1970. The oxidation ofaqueous ferrous sulphate by Thiobacillusferrooxidans. Can. J.Chem. Eng. 48:669-676.

34. Martin, P. A., P. R. Dugan, and 0. H. Tuovinen. 1981. PlasmidDNA in acidophilic, chemoautotrophic thiobacilli. Can. J. Mi-crobiol. 27:850-853.

35. Monod, J. 1942. Recherches sur la croissance des culturesbacteriennes. Hermann S.A., Paris.

36. Murphy, J., and J. P. Riley. 1962. A modified single solutionmethod for the determination of phosphate in natural waters.Anal. Chim. Acta 27:31-36.

37. Nielsen, A. M., and J. V. Beck. 1972. Chalcocite oxidation andcoupled carbon dioxide fixation by Thiobacillus ferrooxidans.Science 175:1124-1126.

38. Nyholm, N. 1976. A mathematical model for microbial growthunder limitation by conservative substrates. Biotechnol.Bioeng. 18:1043-1056.

39. Pirt, S. J. 1975. Principles of microbe and cell cultivation, p. 4-75. John Wiley & Sons, Inc., New York.

40. Rhee, G.-Y. 1980. Continuous culture in phytoplankton ecology.p. 151-203. In M. R. Droop and H. W. Jannasch (ed.), Ad-vances in aquatic microbiology, vol. 2. Academic Press. Inc.,New York.

41. Silver, M. 1978. Metabolic mechanisms of iron oxidizing thioba-cilli, p. 3-17. In L. E. Murr, A. E. Torma. and J. A. Brierley(ed.), Metallurgical applications of bacterial leaching and relatedmicrobiological phenomena. Academic Press, Inc., New York.

42. Silver, M., and A. E. Torma. 1974. Oxidation of metal sulfidesby Thiobacillus ferrooxidans grown on different substrates.Can. J. Microbiol. 20:141-147.

43. Tomizuka, N., M. Yagisawa, J. Someya, and Y. Takahara. 1976.Continuous leaching of uranium by Thiobacillus ferrooxidans.Agric. Biol. Chem. 40:1019-1025.

44. Torma, A. E., and F. Habashi. 1972. Oxidation of copper (11)selenide by Thiobacillus ferrooxidans. Can. J. Microbiol.18:1780-1781.

45. Tuovinen, 0. H., and D. P. Kelly. 1974. Use of microorganismsfor recovery of metals. Metall. Rev. 19:21-31.

46. Wilson, F. H., and D. B. Hawkins. 1978. Arsenic in streams.stream sediments, and ground water, Fairbanks area, Alaska.Environ. Geol. 2:195-202.

VOL. 48, 1984

on October 22, 2020 by guest

http://aem.asm

.org/D

ownloaded from