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.