The Effect of Nutrient Supplementation on Growth and
Leaching Performance of Bioleaching Bacteria
R.P. van Hillea, L.V. Bromfieldb, S.S. Bothac, G. Jonesd, A.W. van
Zyle and S.T.L. Harrisonf
Centre for Bioprocess Engineering Research, Department of Chemical Engineering,
University of Cape Town, Private Bag, Rondebosch 7701, South Africa
Email: arob.vanhille@uct.ac.za, fsue.harrison@uct.ac.za
Keywords: Acidithiobacillus ferrooxidans, nitrogen metabolism, iron oxidation rate,
biomass yield
Abstract. Heap bioleaching operations are often faced with extended and unpredictable lag periods
after inoculation, prior to the establishment of a stable oxidising environment, during which the heap is
fully colonised or the inoculum overcomes the sub-optimal conditions resulting from acid
agglomeration. Supplementation of laboratory scale (4kg ore) leach columns with soluble nitrogen,
particularly as yeast extract, significantly reduced the lag time and promoted bacterial growth, resulting
in a 50-95% increase in copper recovery post-inoculation. The effect of yeast extract addition to
Acidithiobacillus ferrooxidans in controlled oxidation tests was investigated. Initial exposure of a
stock culture to yeast extract resulted in a transient, dose dependent inhibition at concentrations of 0.5
g.l-1 and below. At 1.25 g.l-1 inhibition was complete over the time scale of the experiment. The
inhibition phase was characterised by observable changes in cell morphology and ultrastructure.
Despite the initial inhibition, the biomass yield at the end of the experiments was equivalent, or higher,
in the presence of yeast extract. Cultures were adapted to growth on yeast extract as the sole nitrogen
source and adapted cultures showed the highest rates of iron oxidation and cell growth, in the presence
of 0.5 and 1 g.l-1 of yeast extract.
Introduction
Heap bioleaching has proven to be a viable technology for the economic recovery of base metals,
particularly copper, from low grade deposits. Mineral sulphides may be oxidised through the action of
ferric ions and acid. Acidophilic iron and sulphur oxidising micro-organisms are responsible for the
regeneration of these reactive elements. Therefore, their active presence is required for the sustainable
operation of heap bioleaching processes. From an operational perspective it is desirable to obtain
complete colonisation of the heap in as short a time as possible and commercial operations may be
plagued by extended or unpredictable lag periods. A number of strategies such as forced inoculation
and inoculation during agglomeration have been developed in an attempt to optimise colonisation. The
addition of the inoculum during agglomeration facilitates an even distribution of organisms throughout
the heap, but exposes the organisms to potentially unfavourable conditions of pH, ionic strength and
dissolved ions. In either the presence or absence of forced inoculation, it is desirable to accelerate
microbial growth during the initial phase to ensure full colonisation of the heap.
Nitrogen is the second most important element, after carbon, in the synthesis of new cell mass so the
provision of adequate nitrogen is important where high cell growth rates are desired. In tank leaching
operations nitrogen is typically provided in the form of inexpensive fertiliser grade ammonium
sulphate, but this may not be feasible or economically viable in extensive heap leach operations [1]. A
number of important bioleaching organisms, including At. ferrooxidans and L. ferrooxidans are capable
of fixing atmospheric nitrogen, although the nitrogenase enzyme responsible is inhibited by oxygen. In
addition, the energy requirement is extensive, with 537 kJ.mol-1 required to break the first bond alone.
The oxidation of one mole of ferrous iron yields only 33.9 kJ. However, once a heap is sufficiently
colonised it may be beneficial for the organisms to have to oxidise significant amounts of iron to fix
nitrogen, particularly as carbon dioxide may becoming limiting.
Organic supplements such as yeast extract or peptone are included in the laboratory growth media for a
number of important bioleaching organisms, particularly the thermophilic archae. The Acidithiobacilli
have been described as obligate chemolithoautotrophs and organic compounds have been reported to be
toxic [2]. However, conflicting reports have suggested that the addition of yeast extract enhances the
yield of At. caldus and At. ferrooxidans is capable of growth on glucose and formate [3].
This paper investigates the effect of ammonium sulphate and yeast extract supplementation of bioleach
columns and the effect of supplementation and nitrogen deprivation on iron oxidation by At.
ferrooxidans under controlled conditions in shake flasks.
Materials and methods
Microbial cultures. Stock cultures of Acidithiobacillus ferrooxidans (DSM 584), Acidithiobacillus
caldus (DSM 8584) and Leptospirillum ferriphilum (ATCC 49881) were maintained at 30°C, 45°C and
37°C on their respective defined media (DSM 71, 150a and 882). Stock cultures of At. ferrooxidans
depleted of nitrogen and adapted to growth on yeast extract (0.5 g.l -1) were obtained by repeated sub
culturing using appropriate media.
Mineral ore. Bioleaching columns were packed with a low-grade copper sulphide ore containing
0.69% copper, predominantly as chalcopyrite, chalcocite and covellite, and 4% pyrite by mass. Ore
was acid agglomerated by mixing 1 kg or ore with 70 ml ddH 2O and 4.3 ml 98% H2SO4.
Leach columns. Heap leaches were simulated in PVC columns packed with 4 kg of acid agglomerated
ore. Temperature was maintained at 32 ± 0.2°C and air blown in at the column base at a rate of 200 ±
10 ml.min-1. Feed solution (1.3 g Fe2+.l-1, Fe3+ 0.7 g.l-1, pH 1.2) was pumped into the column from the
top through a distributor plate at a rate of 40 ml.h-1. Columns were drip-inoculated with a mixed
mesophilic culture containing equal proportions of At. ferrooxidans, At. caldus and L. ferriphilum at a
cell loading of 1011 cells per ton. Nitrogen supplementation was achieved by adding ammonium
sulphate (60 mg.l-1, 12.7 mg N.l-1), yeast extract (31.2 mg.l-1, ± 3 mg N.l-1) or both to the feed solution.
Effluent samples were collected daily and analysed for volume, pH, redox potential, ferrous and total
iron, copper and planktonic cell number.
Shake flasks. The effect of various supplementation regimes on the iron oxidation performance of At.
ferrooxidans was determined using shake flasks at 30°C. A 300 ml volume of sterilised basal salt
solution (0.5 g.l-1 MgSO4, 0.5 g.l-1 KH2PO4, 0.1 g.l-1 CaCl2, pH 1.8) was inoculated with At.
ferrooxidans to an initial cell concentration of 1.6 x 10 5 cells.ml-1. Sterilised ferrous sulphate solution
was added to ensure an initial Fe2+ concentration of approximately 2.5 g.l-1 and where appropriate
nitrogen or carbon supplements were added to the desired concentration. At predetermined intervals a
3 ml sample was removed aseptically and analysed for pH, redox potential, ferrous iron (1-10
phenanthroline method) and cell concentration (direct cell counting). Data were used to calculate total
and specific iron oxidation rates and microbial yields. Culture purity was confirmed using restriction
digest analysis of the 16S rRNA gene [4] and real-time PCR with Acidithiobacillus specific primers.
Results and discussion
Effect of nutrient addition on bioleach column performance. Nitrogen addition significantly
reduced the lag phase prior to the establishment of a stable redox potential in excess of 600 mV. In the
absence of any nitrogen addition, the establishment of the stable oxidising environment occurred over a
period of 21 days during which the redox increased slowly from 450 to 600 mV. Ammonium sulphate
supplementation reduced the period of establishing this regime to 12 days, while yeast extract addition
reduced it further to eight days. Associated with the reduced duration of the phase of establishing this
regime was an increase of up to six fold in the planktonic cell numbers detected in the leach solution
eluted. The increase in cell concentration was more rapid and of a greater magnitude where yeast
extract was added. While the increase in cell number may result from the presence of heterotrophic
organisms on the ore, unsterilised prior to agglomeration, the increase in ferrous oxidation rate and
copper extraction suggested an enhanced microbial population associated with bioleaching. Copper
recovery post-inoculation approached two fold the control value in the case of yeast extract
supplementation (Figure 1).
Shake flask experiments with At. ferrooxidans. The bioleach column experiments consistently
demonstrated improved performance when supplemented with nitrogen, particularly organic nitrogen
in the form of yeast extract. To validate this observation a series of experiments were performed under
controlled conditions using a pure culture of At. ferrooxidans. Batch flasks were inoculated with low
cell concentrations to ensure at least a 100 fold increase in cell number over the duration of the
experiment, such that physiological effects could be observed clearly. Cultures that had not been
exposed to organic nitrogen (yeast extract or peptone) previously were initially inhibited, showing
reduced rates of iron oxidation and cell growth. The inhibition was dose dependent, with complete
inhibition observed at 1.25 g.l-1 yeast extract (Figure 2). Despite the transient inhibition at lower yeast
extract concentrations, complete iron oxidation was achieved and, significantly, the final cell
concentrations were higher in these flasks, indicating a greater biomass yield.
In addition,
morphological changes were observed, particularly cell elongation and the formation of chains up to
five cells long. Molecular analyses confirmed the presence of a monoculture of At. ferrooxidans. This
is consistent with previous observation of At. ferrooxidans grown in the presence of sewage sludge [5].
The culture was adapted to yeast extract (0.5 g.l-1) as the sole nitrogen source by repeated sub-culturing
and the adapted culture showed the highest rate of iron oxidation (Figure 2) and cell growth (Table 1).
Removal of all soluble nitrogen did not have a significant effect of iron oxidation or cell growth rate
when the flasks were inoculated with cells grown in complete media. However, on sustained nitrogen
depletion the oxidation and growth rates were significantly reduced (Table 1), suggesting the cells
maintain an endogenous nitrogen store sufficient for a small number of cell divisions which is
subsequently depleted.
10
9
8
Cumlative Cu 2+ (g)
7
6
5
4
3
2
1
0
0
10
20
30
40
50
60
Time post inoculation (days)
No supplements
YE
Ammonium sulphate
YE + ammonium sulphate
Figure 1: Cumulative copper leached from columns,
supplemented as labelled, post inoculation with mixed
mesophilic culture. Excludes copper released during 6
day acid water (pH 1.8) wash.
Figure 2: Ferrous iron oxidation by At. ferrooxidans
supplemented with various nitrogen sources. Adapted
culture had been repeatedly sub-cultured in standard
media with 0.5g/l yeast extract as sole N source. Initial
inoculum 1.6 x 105 cells.ml-1 in all cases.
Table 1: Summary of kinetic parameters obtained during batch oxidation tests
µ max
Yield
Fe2+ oxidation rate
(h-1)
(cells.µmol Fe2+ ox-1)
(mmol.l-1.h-1)a
Stock
Full
0.109
489
0.85
Stock
N depleted
0.109
330
0.79
Nitrogen depleted
N depleted
0.042
106
0.33
Stock
YE 0.25 g.l-1
0.107
414
0.38
Stock
YE 0.5 g.l-1
0.081
510
0.17
YE adaptedb
YE 0.5 g.l-1
0.111
419
0.62
c
-1
YE adapted
YE 0.5 g.l
0.147
492
1.50
YE adaptedc
YE 1.0 g.l-1
0.140
517
1.27
a
data taken from T = 24-48h, while uninhibited cultures were in mid-log phase
b
-1
culture adapted to YE (0.5 g.l ) as sole nitrogen source for 2 weeks
c
culture adapted to YE for 4 weeks
Culture
Medium
Specific Fe2+ oxidation rate
(mmol.l-1.h-1.cell-1 x 10-8)a
8.98
7.93
12.0
7.51
4.62
7.26
9.24
8.14
The kinetic data are summarised in Table 1. The µ max values for uninhibited cultures are within the
range considered accurate (0.047-0.23 h-1) [6]. The effects of adaptation and nitrogen depletion are
clearly evident. The iron oxidation rate data has been selected to represent the mid-log period in
uninhibited cultures. Transient inhibition of the stock culture exposed to yeast extract occurred during
this period, accounting for the low iron oxidation rates (Figure 2). From 48 hours onward the overall
and specific iron oxidation rates were consistent with uninhibited cultures. Despite the significantly
reduced growth rates observed in the nitrogen depleted cultures they demonstrated the highest specific
oxidation rates, possibly as a response to the energetic demands of nitrogen fixation. In a colonised
heap this suggests that high iron oxidation could be maintained even in regions of the heap where cell
growth was retarded by nitrogen and carbon dioxide limitation.
Glucose addition (0.5 g.l-1) had no effect of iron oxidation, growth rate or biomass yield, suggesting
heterotrophic growth was not significant (data not shown). However, Chen and Suzuki [7] have
demonstrated the oxidation of endogenous substrates by At. ferrooxidans, with a respiratory quotient
(CO2 produced/O2 consumed) of 1.0 which implies a carbohydrate electron donor. Ferric iron acted as
the electron acceptor. It is possible that components of the yeast extract were able to enter the cell and
act as substrates for oxidation, once the downhill reaction (NADH oxidation) pathway had been
induced. An alternative explanation could be the presence of NADH in the yeast extract which is
imported into the cell, reducing the energy required to reduce NAD + prior to carbon fixation. This
would account for the increased yields observed in the presence of yeast extract. Similarly, the
transport of amino acids into the cell would reduce the energy cost of protein synthesis, but it is
unlikely that this alone could account for the effects observed.
Conclusions
The addition of soluble nitrogen, as ammonium sulphate and/or yeast extract, significantly reduced the
time required to establish a stable bio-oxidation environment and increased cell growth and copper
leaching in bioleaching columns inoculated with a mixed culture of mesophilic bioleaching bacteria.
Under controlled conditions At. ferrooxidans demonstrated a period of adaptation to yeast extract
supplementation after which both growth and iron oxidation rates increased as did biomass yield. In
heap leach operations short term supplementation immediately post-inoculation could reduce the
duration and unpredictability of the initial colonisation phase.
Acknowledgements
The authors gratefully acknowledge the Department of Science and Technology and the National
Research Foundation of South Africa for funding received through the South African Research Chair
Initiative.
References
[1] D.E. Rawlings, in: Biomining, edited by D.E. Rawlings and D.B. Johnson, Springer-Verlag, Berlin
Heidelberg (2007). p. 186.
[2] J.H. Tuttle, P.R. Dugan. Inhibition of growth, iron, and sulfur oxidation in Thiobacillus
ferrooxidans by simple organic compounds. Canadian Journal of Microbiology Vol. 22 (1976). p. 719730.
[3] O.I. Rzhepishevska. Physiology and Genetics of Acidithiobacillus species: Applications for
Biomining. Thesis, Umeå University, Umeå (2008).
[4] N.J. Coram-Uliana, R.P. van Hille, W.J. Kohr, S.T.L. Harrison. Development of a method to assay
the microbial population in heap bioleaching operations. Hydrometallurgy Vol 83 (2006). p. 237-244.
[5] R. Matlakowska, A. Sklodowska. Isolation and evaluation of indigenous iron- and sulphuroxidising bacteria for heavy metal removal from sewage sludge. Proceedings of the 15 th IBS, Nereus
Group, Athens (2003). p. 265-276.
[6] S. Molchanov, Y. Gendal, I. Ioslvich, O. Lahav. Improved experimental and computational
methodology for determining the kinetic equation and the extant kinetic constants of (FeII) oxidation
by Acidithiobacillus ferrooxidans. Applied and Environmental Microbiology Vol. 73 (2007). p. 17421752.
[7] Y. Chen, I Suzuki. Electron transport pathways for the oxidation of endogenous substrate(s) in
Acidithiobacillus ferrooxidans. Canadian Journal of Microbiology Vol 52 (2006). p. 317-327.