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Growth and respiration kinetics of Thiobacillus ferrooxidans limited by CO2

and O2

Article  in  Biologia · January 1984

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 B 10 LOG I A (BRATISLAVA) 39, 4, 429-434, 1984

GROWTH AND RESPIRATION KINETICS OF THIOBACILLUS

FERROOXIDANS LIMITED BY C02 AND 02

Martin Man d I

Veterinary Research. Institute, 62132 Brno, Czechoslovakia

Mandl M., Growth and res.piration kinetics of Thiobacillus ferrOOxidans

limited by C02 and 02. Bl016g1a (Bratislava) 39, 429-434, 1984.
Thiobacillus ferrooxtdans was batch cuUlva'ted i;n mineral medium 9R.
After eXp<lnentlal growth phase (doubling :time 6.4 h 1 limitation by Cal
induced the linear growth phase but the metabol1c efficiency of Fe 2 + re­
mained. unchanged: about 4 % was used for NAD reduction, the rest for
ATP generation in both phases. Michaelis-Menten c<mstant for 03 was
(0.63 :10 0.22) ,umol.l- 1 ; non-growing cells oxidiZing Fe2+ had O>llly a low
oxygen demand. A 15 min oxygen-limitation (incubation in a closed cell)
caused lowering of the respiration activity. which reached normal values
altelr 20 min aeratioill.

Thiobacillus ferrOxidans is an attractive bacterium for use in biohydro­

metallurgy. It obtains energy by reaction (1): Fe 2+ electrons enter the elec­
tron transport chain via cytochrome c (Z a v ar z in, 1972; S i I v e ,r, 19?8).
4 FeS0 4 + 2 H2S0 4 + 02 = 2 Fe2(S04h + 2 H2 0 (1)
The reversal of electron "transpoTt from cytochrome c to NAD yields NADH
for CO 2 reduction in the Calvin cycle.
ATP
Fe2 + + H+ + NAD = Fe 3 + + NADH (2)

Insufficient CO 2 supply limits cell growth and insufficient 02 supply restricts

energy metabolism. This may reduce :metal extraction rate during bacterial

'"

Symbols:
C dissolved oxygen concentration In culture medium
Co oxygen concentration at time t=O
Cs saturation concentration of dissolved oxygen
kLa volumetric oxygen transfer coefficient
Km Michaelis-Menten constant for 02
Ks saturation constant for Fe 2 +
Ks saturation constatnt for COl
Q oxygen uptake rate
S concentration of Fe 2 +
S concentration of C02 in culture medium
t time

429
leaching of sulphide ores (T 0 r m a, 1977; B r i e r ley. 1978). K e I I y and
Jon e s (1978 J described factors affecting metabolism and Fe2+ oxidation in
suspensions and batch cultures and demonstrated Fe 2 + oxidation by non-gro­
wing ,cells in the absence of CO 2 which indicated growth-uncoupled oxidation
of Fe 2 +.
This study demonstrates that the metabolic efficiency of Fe 2 + is unaffected
by growth limitation with CO 2, It also describes the effect of oxygen-limitation
on oxygen uptake by cells.

Material and methods

Microorganism, medium and culture conditions. The microorganism used was Thio­
bacillus ferroxidans isolated from mine waters of the North Bohemian brown coal mi­
ning area by Ing. E. Beranova. Ten per cent active culture was inoculated into growth
medium 9K Silverman-Lundgren [S i I ve r man and L un d g r e n, 1959] adJusted
to pH 2 and was grown on a, rotary shakeiI' at 28 oC in 500 ml ,culture flasks C<Jntainiil1g
100 ml medium.
Determination of Fe2+. Fe2+ was determined mangaJlomet'ricalJy (Tom! l! e 'k, 1958).
Bacterial growth. Bacterial growth was measured turbidimetrically after acidifica­
tioo with H 2 S04 (resuatant H2S04 con.cenrra1ion in the culture: about 0.5 mol.1- 1 ).
The: medium was deC<J!c'I'ized by binding Fe3+ with H3P04 to give a ,colo'lll"lesscomp!ex.
Bacterial turbidity was measured, at 4,20 run in a Perktn-Elmer Coleman, 124 D spe'ctro­
ph 01 ometw.
Measurement ot oxygen uptake rate. Oxygen uptake rate during baciteri:al growth
was measured with a Z 151 polarographic 02-electrooe (EJectrofaCot TB 31] in a closed
system (thermostated 25 m! flask), Q = -dC/dt. The effe,ct .of oxygen-limitation was
studied in an open system using the dynamic method where saturation of the medium
with atmospheric oxygen occurred concurrently with oxygen uptake by the bacteria:
dc/dt = kLa (C s - C) - Q (3}

Prorvided 1:hat the .record of oxygencon:centl'atiJ:rl a'S a func1:ion of time is reasOlIliablr

accurate, it is enough to .compute the dC/dt value by numerical derivation of the
experimell!ta~ l'elat:olIl C = f(t) from fiVle paints. Less dispe'l'Sion of the Q values, ho­
wever, is achieved by delrivatio[l from three pmnts (H a I a m a, 1979). The Cs and
kLa values were determined after additiOn of NaN3 to the respiring bact'eria. Then
Q = 0 and the solution to equation (3) is
In(C s - C) = In(C s - Co) - kLa . t (4)
Limitation ot respiratiOn by oxygen. Bacteria in the closed meaS!U.Ting ceiJl COlIliSU'"
med pra.ctically all the o~ygen. They weTa then left undm- .oxygen-Um~ted 'condiJtions
for 15 minutes. Afterwards the, flask was opened and Cihanges in the respil'atiOO- rate
of b~cteria were computed from equation [3) under conditions of
kLa = (4.88: O.02) h-l

X biomass concentration
Ys / o ratio of Fe 2 +' consumption to 02 consumption during bacterial oxidation of
Fe 2 + . Ys/o = Yx / o . y ;/~ = dS/dC
Yx/o growth yield on oxygen
Yx/s growth yield on Fe2 +
1.1. specific growth rate
jJm maximum specific growth rate

430
 The Michaelis-Menten constant for oxygen was determined in the bacterial suspens­
ion as the oxygen concentra'tion that reduced maximum oxygen uptake rate by one­
-half.
Where desirable, results v~eTe expressed in 'terms of 95 % confidence' interval.

Results and discussion

The ,ClQurse of cultivation is shown in ~g. 1. The exponential growth phase

with a doubling time of 6.4 h was followed by a linear phase of both growth
and substrate consumption and by a constant oxygen uptake rate. Acid medium
and poor aeration [kLa ctrca 16 h -1 J create conditions for growth ldmitation
by C02. This conclusion is supported by kinetic analysis.
In the light of the Monad kinetics, which is not at variance with the experi­
mental data. the linear phase indicates limitation of gaseous substrate. Since
there was no limitation by oxygen. the concentration of which never fell be­
low 0.16 mmol.l- 1, limitation by CO 2 is inferred.
If we disregard oxygen consumption unrelated to bacterial growth and
assume a twofold limitation (by CO, and Fe2+ J, then the following equation
holds for oxygen uptake rate:

1 dX 1 1 S
Q=---.-- = - - .•uX- • fLm X • [5 ]
Yx/ o dt Yx / o Yx / o

If CO 2 concentration approaches Ks ' the growth is limited and specific growth

rate fl decreases in consequence of S/(Ks+S] proportionally to the growth of

d
ci
----.
20 ~2°1 .; 0.6
'c '7_100L
E
":" . 15
-. lBof
-0
0.4
~ 10 ~60 Q
'-­ tf
a '--40
0.2
5
20

o o~~--~----~----~---
20 40 60
t [h]
Fig. 1. Fe2 -r oXlda'tlon and oxygen uptake rate (QJ during the growth of Thioba­
emus lerroOxidans (O.D.)

431
biomass X. Q remains constant till the Fe2+ concentration decreases to Ks
(7.04 mmolJ -1) and, in consequence, limitation by energy substrate takes
place.
A further reduction of aeration made CO 2-limitation more pronounced by
shortening the exponenti.al phase and slowing down the rate of Fe2+ oxida­
tion in the linear phase.
Correlation between the optical density and Fe2+ and 02 consumption indi­
cates that the change of the yield coefficients was negligible (within measu­
ring error). Based on the rates of bacterial Fe2+ oxidation and respir,ation, it
is possible to assess the Fe2+ proportion reducing NAD and the Fe 2 + propor­
tion reducing oxygen tn general1:e ATP. The ratio of Fe2+ consumption to O2
consumption during bacterial growth (Ys/o = dS/dC J ex,ceeds the stoichio­
metric ratio of Fe2+ to 02 in equatlon (lJ (4 Fe 2 +/O z ) by that proportion of
Fe 2 + consumption that reduces NAD on the reversal of the electron transport
chain ,and does not reduce oxygen (Equation 2). Provided that the entire Fe z+
substrate served fOlr 02 reduction (non-growing culture J, then Ys/o = 4.
During bacterial growth

- dS/dt = Y s/0 • Q (61

FTom equation (6) it is possible to compute Y s/0 at each time point of cultiva­
tion. In the linear phase the dS/dt value is constant; in the exponential phase
it was computed by numeri-cal derivation of the relation S = f (t].
The loog doubling time is related to the small Fe 2 + proportion serving fOT
grDwth. Better growth conditions
were obtained by K ell y et al.
..,.---' (1977) in continuous cuHivaUon .
• 00
.£; 5,0
It might have been expected
that cell growth conditions v-.,rould
- ..
.~
E

---'
.. t
i
be' more favourable in the expo­
nential phase !thanin the CO 2-li­
0
E
mited phase, but the use of Fe z+
~
:t.
for growth waseql.1l8.11y low in
bath phases. In the exponential
O 2,5 phase Ys/o = 4.14+0.06, in the
lmea·r 'phase Ys/ o = 4.18: 0.07.
The d1fference between the two
phases was no~ signifi,cant
PJtO.05 J. Comparison of the stoi­
chiometric ration of Fe 2 + to O2
(equalling 4 ) with the mean va­
o~~--~--~--~--~
lue f,or Ys/o (4.16) shows that al­
510 15 20 most 4·' % o! Fe 2 + served for
t [min] growth and the rest of the sub­
Fig. 2. Changes' in the respiration rate st,rate served for energy genera­
after a 15 min limitation by oxygen tion. These conditions were inde­
Measured by the dynamic method (equa­ pendent of CO 2-lirnita tion.
tion 3). Limitation Of respiration by
Oxygen uptake rate before. 02-1imitation:
Q = (5.25: 0.12) ,umoU-l.mln- 1. oxygen. A 15 min oxygen-lim.:ta­

432
 tion produced only short-time changes in the respiration activity (Fig. 2]
which returned to. the original level after about 20 min aeration. The brief
02-limitation thus caused no marked impairment in energy metabolism.
Km = {0.63+0.22) .umol.l- 1• The Jimiting 0, concentration wa'S therefore very
low. Compared to CO 2-limitation, the conditions of 02-limitation are negligible.
In a suspension of 108 cells/ml at a kLa exceeding 4 h -1, respiration was not
limited. This implies that non-growing cells oxidiz!ng Fe 2+ have a low de­
mand for oxygen.

Acknowledgement. I wish to thank Doc. Ing. D. Harama, CSc., for his interest and
helpful suggestions and assistance in preparing the manuscript.

Translated by J. Sochorova

References

BRIERLEY, C. L., CRC Critical Reviews in Microbiol{lgy, 6(3) : 207-262, 1978.

HAI:AMA, D., in Sbornik referatu z celostatnlho seminare "Funkce mikroarganismu
v procesu zneciSteni vad", CSAV, 81-83, Bratislava, 1979.
KELLY, D. P., JONES, C. A., in Metallurgical Applications pf Bacterial Leaching and
Rela,ted Microbiological Phenomena, Murr, L. E., Torma, A. E., Brierley, J. A. eds.
19-44, Academic Press, New York, San Francisc::J~ London, 1978.

KELLY, D. P., ECCLESTON, M., JONES, C. A., in Conference Bacterial Leaching,

Schwartz, W. ed. 1-7, Verlag Chemie, Weinheim, New York, 1977.

SILVER, M., in Metallurgical Applications of Bacterial Lea·ching and Related Micro­

biological Phenomena, Murr, L. E., Torma, A. E., Brierley, J. A. eds. 3-17, Academic

Press, New York, San FranCiSCO, London, 1978.

SILVERMAN, M. P., LUNDGREN, D. C., J. Bacterinl., 77 : 642-647, 1959.

TOMicEK, 0., Kvantitatlvni analysa. SZN, 204-205, Praha, 1958.

TORMA, E. A., in Advances in Biochemical Engineering 6, Ghose, T. K., Fiechter, A.•

Blakebrough, N. eds. 2-37, Springer-Ve.rlag, Ber:lin, Heidelberg, New York, 1977.

ZAVARZIN, G. A: Litotrofnyje mikroorganizmy. Nauka, 204-211, Moskva, 1972.

KINETIKA ROSTU A RESPIRACE THIOBACILLUS FERROOXIDANS LIMITOVANA

COl A 02

Martin Man d 1

V jednorazove kultivacl (minercllni medium 9K) po exponencj,alnf fazi rustu (doba

zdvojeru 6,4 h) nastala lineami faze' v du.sIedku limil\:a·ce oxidem uhliciltYm. Vyuzi,telIllOSt
Fe2 + substratu pro ru.st se vsak nezmeniJa - v obou fazIch asi 4 %. 'fiedukovala NAD,
zbytek substratu slouzil tvorbe ATP. Michaelis.ova konstatnta pro 02 = (0,63 + 0,2e)
.umoU -1, nerostoud kultura oxidujid Fe2 + nema velke naroky na aera<:i. Limitace
respirace kyslikem (15 min. inkubace v uzavren~m syst~mu) vyvolala kratkodobe sni­
zen! respiral:ni aktivity, pu.vodnich hodnot se dosahlo po 20 min. aaraee.

Doslo: 25. 5. 1983

433

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