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BITE 8632 14 July 2011 Highlights

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" NMR relaxation measurements (T2) can provide iron oxidation activity of bacterial cells. " The iron oxidation ability of Acidithiobacillus ferrooxidans was investigated. " T2 shortening was found with increasing time as the bacteria oxidize Fe2+ to Fe3+ ions. " Bacteria concentration increased 80 times and high iron oxidation ability was found in 9K. " The measurement has the advantage of in situ, nonintrusive, and fast.

BITE 8632 14 July 2011

Bioresource Technology xxx (2011) xxxxxx 1

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Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

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An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement

S.N. Tan a, I. Burgar b, M. Chen a,
a b

CSIRO Process Science and Engineering, Clayton South VIC3169, Australia CSIRO Materials Science and Engineering, Clayton South VIC3169, Australia

a r t i c l e

i n f o

a b s t r a c t
NMR relaxation measurements can provide a simple means for understanding biological activity of cells in solution with known composition. It has the advantage that it is an in situ, non-intrusive technique, and the acquisition is fast. The iron oxidation ability of Acidithiobacillus ferrooxidans was investigated using NMR relaxation measurements. The transversal relaxation is characterized by a time constant, T2, which is sensitive to the chemical environment. Fe3+ ion has more signicant T2 shortening than Fe2+ ion. In the presence of A. ferrooxidans in solutions containing Fe2+ ion, T2 shortening was found with increasing time as the bacteria oxidize Fe2+ to Fe3+ ions. In the optimal growth medium, the bacteria concentration increased 80 times and high iron oxidation rate was found. In 10 mM K2SO4 medium, however, bacteria concentration remained almost unchanged and the iron oxidation rate was signicantly lower. 2011 Elsevier Ltd. All rights reserved.
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Article history: Received 2 May 2011 Received in revised form 24 June 2011 Accepted 26 June 2011 Available online xxxx Keywords: NMR Relaxation T2 Biooxidation Acidithiobacillus ferrooxidans Bioleaching

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1. Introduction Microorganisms are present in various forms in daily life, such as in food and beverages (Shah, 2000), waste recycling and dental health. Microorganisms also play an important role in industrial processes. In mineral processing, for example, bacteria are used as collectors in biootation and for promoting mineral oxidation to enhance extraction of metals, such as gold and copper, in the bioleaching process (Rohwerder et al., 2003; Watling, 2006; Cordoba et al., 2008). Microorganisms have specic functions in these processes. For instance, in probiotic food, bacteria can improve the balance of microora in the gut by producing a variety of substances to suppress pathogens and actively enhance health (Shah, 2000; Fuller, 1989). In bioleaching, bacteria such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans have the ability to oxidize iron and sulfur and thus promote metal dissolution (Rawlings, 2002, 2005). The investigation of the role of microbes in these processes can be grouped into three levels of complexity. First is the global level of the dynamic microbial community structure and its inuence on the environment (Zeng et al., 2010; Kowalchuk et al., 1999; Peters et al., 2000), followed by the understanding of the bioactivity and response of the cells to the environment, and the most fundamental level of cell chemistry and cell signaling processes (Pesci et al.,
Corresponding author.
E-mail address: Miao.Chen@csiro.au (M. Chen). 0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.06.088

1999; Blake and Shute, 1994). Among these, the understanding of the biological activity of bacteria and how it is inuenced by its environment, particularly their natural environment, is not straightforward. NMR relaxation method has been used to detect the activities of microbes for microbial food contamination application (Pascall et al., 2006; Krockel et al., 2001). This paper illustrates the use of NMR relaxation time to understand biooxidation activity of A. ferrooxidans cells. A. ferrooxidans is a naturally occurring bacterium in acid mine drainage (Watling, 2006) and it is the most commonly studied microorganisms for bioleaching applications. A. ferrooxidans has the ability to oxidize Fe2+ ions:

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2Fe2 2H 0:5O2 ! 2Fe3 H2 O

74
75 76 77

The oxidation product, Fe3+ ions, can solubilize metal suldes and hence enable the extraction of precious metals, such as copper and gold (Rawlings, 2002).

78

MS Fe

H !M

0:5H2 Sn Fe

n ! 2 2
3+

0:5H2 Sn Fe3 ! 0:125S8 Fe2 H 0:125S8 1:5O2 H2 O ! SO2 2H 4

2+

80
81 82 83 84 85

The Fe ions produced in reaction 2 may be oxidized to Fe ions (reaction 1) and sulfur may be oxidized to sulfate by A. ferroxidans cells (Rawlings, 2005). Therefore, A. ferrooxidans cells act as biocatalysts and signicantly enhance the bioleaching rate of sulde minerals.

Please cite this article in press as: Tan, S.N., et al. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.06.088

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Common methods to quantify the iron oxidation rate of bacteria rely on chemical (titration and spectroscopy) and electrochemical (redox potential (Eh)) techniques. Chemical methods have the disadvantage that they are ex situ techniques and sample preparation steps such as removal, dilution, pH adjustment and reagents additions are required. The preparation can potentially cause contamination or shifting of chemical equilibria of the Fe oxidation state, and this method is not suitable when the early oxidation behavior is of interest (e.g. <30 min). Eh determination has the advantages of fast acquisition and being an in situ method. However, this method is limited to ideal solution (i.e. dilute solution). At high ion concentration, the true activities of the ions must be used and this complicates the data analysis. NMR relaxation time measurement also has the advantages of being an in situ and non-intrusive technique with fast acquisition. The data acquisition time take less than 1 min, the viability of the bacteria is not affected by the magnetic eld and the measured samples (<2 ml) can be returned to the solution. Furthermore, it has the added benets of being suitable for a wide range of ion concentrations, with minimal possibility of cross contamination. The NMR relaxation method is based on the response of proton nuclei to a magnetic eld. When a magnetic eld is applied, proton nuclei build up a magnetic moment parallel to the external magnetic eld. A radio frequency pulse perpendicular to the magnetic eld ips the magnetization of the proton nuclei in the direction perpendicular to the external magnetic eld. After the pulse, the magnetization of the proton nuclei gradually restores to equilibrium and returns to the direction parallel to the external magnetic eld. The relaxation time of water in a homogeneous eld is about 12 s, but this can be signicantly shortened in the presence of a non homogeneous eld, such as due to magnetisable species (Fe, Co, Ni, etc.) or a change in the mobility of protons in hydration shells around ions or other solutes. The proton relaxation time is therefore sensitive to the chemical environment, such as the concentration of Fe2+ and Fe3+ ions in solution. Thus NMR relaxation technique is a potential tool to quantify iron oxidation rate of bacteria since the chemical composition of the solution changes when the bacteria oxidize Fe2+ ions in solution to Fe3+ ions. The objective of this work is to illustrate the use of NMR relaxation time as a tool to understand bacterial processes. The iron oxidizing ability of A. ferrooxidans cells will be investigated. This work consists of three parts: (a) the interaction between protons and Fe2+ and Fe3+ ions, (b) the interaction between protons and bacterial cells, and (c) the interaction between protons, bacterial cells and Fe2+/Fe3+ ions.

collected. The culture was ltered through Whatman lter paper (No. 1) to remove any precipitates such as jarosite. The bacteria in the ltrate were collected on a 0.2 lm Whatman regenerated cellulose syringe lter (Uniow), washed with pH 2 sulfuric acid solution to remove iron, metabolites and salts from the culture medium, and then redispersed in 10 mM K2SO4 or 9K at pH 1.8. Bacterial concentration was determined with the optical density method (Varian Cary 500 UV Vis spectrophotometer). To study the interaction between proton and Fe2+ and/or Fe3+ ions, the following samples were prepared in 9K or 102 M K2SO4 medium. (a) 05.4 103 M Fe2+ or Fe3+ ions (b) Varying ratio of Fe2+ and Fe3+ ions with a total iron concentration of 5.4 103 M (c) Varying ratio of Fe2+ and Fe3+ ions with a total iron concentration of 0.16 M To investigate the interaction between protons and bacteria, samples containing 0, 5.4 103 M or 0.16 M Fe3+ ions in 9K or 10 mM K2SO4 medium in the absence and presence of bacteria (5.1 108 cell/ml) were prepared. In order to investigate the oxidizing ability of A. ferrooxidans cells, samples containing 1 106 cell/ml of A. ferrooxidans cells in 9K and 10 mM K2SO4 medium solutions with 5.4 103 M or 0.16 M Fe2+ ions were prepared. Abiotic controls were also studied. The proton relaxation time and bacterial concentration were determined over a period of time up to 5 days. Bacterial concentration was monitored using the Thoma counting cell method. The loss of cells due to cell attachment to glass and biolm formation was not considered. 2.2. NMR relaxation time A 20 MHz Minispec spectrometer was used to determine the transversal relaxation time, T2, at a eld strength of 0.5 T. The Carr-Purcell-Meiboom-Gill (CPMG) method Palmer et al., 1992 was used. The experiment consists of a 90 pulse followed by a series of 180 pulses. For samples containing no or 5.4 103 M Fe ions, the duration spacing between the rst 90and 180 pulse was 1000 ms and the duration between the subsequent 180 pulses was 2000 ms. For solutions containing 0.16 M Fe ions, the duration spacings were 100 and 200 ms, respectively. The magnitude of every second echo was measured to construct the characteristic CPMG decay curve. A total of 16 scans were performed for each measuring point in the decay curve. Prior to measurements, eld adjustment was carried out to ensure that the free induction decay signal is smooth. Single exponential tting with a baseline correction was performed on the rst 10 and 200 ms of the signal for samples containing 0.16 M and 5.4 103 M iron, respectively. Repeated measurements provide an accuracy of T2 within 5%. 3. Results and discussion 3.1. Proton and iron interactions The relaxation rate, 1/T2, is a probability of proton relaxation, measuring the effectiveness of relaxation mechanisms. The presence of Fe3+ ions results in more signicant T2 shortening than Fe2+ ions (Fig. 1). Thus Fe3+ ions are more effective in enhancing the proton relaxation than Fe2+ ions in both media. To compare the enhancement of proton relaxation by iron ions in both media, the enhancement, R, which is the slope of the data in Fig. 1, was determined (Table 1).

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2. Methods 2.1. Solution preparation The Fe and Fe solutions used in this work were prepared using analytical grade FeSO47H2O and Fe2(SO4)37H2O, in either 10 mM K2SO4 or 9K (Silverman and Lundgren, 1959). The 9K medium is the optimal growth medium for A. ferrooxidans and is composed of 3 g/l (NH4)2SO4, 0.5 g/l MgSO47H2O, 0.5 g/l K2HPO4 and 0.1 g/l KCl. UV treated deionized water was used in all solution preparation. The pH of all solutions was adjusted to 1.8 using aqueous H2SO4, unless otherwise stated. All glassware was rinsed with concentrated sulfuric acid. No attempt was undertaken to sterilize the glassware and media. 100 ml 9K and 4.45 g of FeSO47H2O were added to a 250 ml Erlenmeyer ask. The medium was inoculated with 10 ml of A. ferrooxidans (DSM14882) culture at a cell concentration of 5 108 cell/ml. The culture was incubated in an orbital shaker at 160 rpm, 30 C for 3 days. Bacteria in the stationary phase were
2+ 3+

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Please cite this article in press as: Tan, S.N., et al. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.06.088

BITE 8632 14 July 2011

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(a)

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30 20 10 0 0 2 4 6

Fe concentration, mM

(b)

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1/T2, s

20 15 10 5 0 0 2 4 6

Fe concentration, mM
Fig. 1. 1/T2 of Fe2+() and Fe3+(d) ion in (a) 10 mM K2SO4 and (b) 9K medium as a function of iron concentration.

The enhancement of Fe2+ ions is similar in both medium but the enhancement of Fe3+ ions in 10 mM K2SO4 is 81% higher than in 9K (Table 1). The enhancement ratio, dened as the ratio of enhancement of Fe3+ to Fe2+ ions, in 10 mM K2SO4 was 21.5. This is similar to the enhancement ratio of 25.5 reported by Prasad et al. measured at a higher eld strength of 1.5 T (Prasad et al., 1991). The lower enhancement found for Fe3+ ion in 9K media may be linked to the presence of additional ions, such as sulfate, phosphate and chloride, in 9K medium. These ions may be associated with Fe3+ ions and therefore fewer protons are available to interact with Fe3+ ions and consequently, the enhancement is lower. The relaxation rate of samples containing mixtures of Fe2+ and 3+ Fe ions at a total iron concentration of 5.4 103 M and 0.16 M, respectively, was determined. The relaxation rate of the solution increases with increasing fraction of Fe3+ ions in both media (Fig. 2). This trend is in agreement with the above discussion showing that Fe3+ ions are more hydrated and therefore more effective in enhancing proton relaxation time than Fe2+ ions. For solutions with 5.4 103 M total iron concentration, the slope of the plot of relaxation rate versus fraction of Fe3+ ions is 2.7 times higher when prepared in 10 mM K2SO4 than in 9K (Table 2). The smaller slope in 9K may be linked to the presence of counter ions in the medium that interacts with Fe3+ ions and reducing the number of protons interacting with the Fe3+ ions, again consistent with the preceding discussion. At a total iron concentration of 0.16 M, the slope of the plot of relaxation rate versus fraction of Fe3+ ions in both 10 mM K2SO4 and 9K medium are similar (Table 2). This may be linked to the fact that the number of protons interacting with iron is

-1

1/T2, s

-1

(a)
Table 1 Enhancement and enhancement ratio of Fe2+ and Fe3+ in 10 mM K2SO4 and 9K.

70 60 50

1/T2, s

Enhancement, R R2 < 0.99 Fe2+ in 10 mM K2SO4 Fe3+in 10 mM K2SO4 Fe2+ in 9K Fe3+ in 9K 0.47 10.10 0.47 5.57

Enhancement ratio RFe3 =RFe2 21.5

-1

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11.9

10 0

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75

100

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The enhancement of proton relaxation due to the presence of paramagnetic ions is well documented (Eisinger et al., 1962). The large difference in the enhancement of Fe2+ and Fe3+ ions may be linked to the differences in their hydration. Experimental and theoretical studies indicate that the rst hydration shell thickness is 2.102.28 and 1.962.05 for Fe2+ and Fe3+ ions, respectively (Moin et al., 2010), each ion being coordinated with six water molecules (Moin et al., 2010). The Fe3+ ion has a more tightly bound rst hydration shell than Fe2+ ion. The second hydration shell of Fe3+ ion is also stronger than for Fe2+ ion, consisting of an average of 15 and 13 water molecules, respectively, by Monte Carlo simulation (Degreve and Quintale, 1996) and 13.6 and 13 water molecules, respectively, by molecular dynamics (Moin et al., 2010). Molecular dynamic simulations also suggest that both ions inuence the water structure beyond the second hydration shell (Moin et al., 2010). The more signicant T2 shortening by Fe3+ ions compared to Fe2+ ions is probably linked to the larger number of protons interacting with Fe3+ ion as it has a compact hydration core and a more highly hydrated second hydration shell.

Fe , %

3+

(b)

1500 1250 1000

1/T2, s

-1

750 500 250 0

25

50

75

100

Fe , %
Fig. 2. 1/T2 of Fe and Fe ion in 9K () and 10 mM K2SO4(4) at total iron concentration of (a) 5.4 103 M and (b) 0.16 M.
2+ 3+

3+

Please cite this article in press as: Tan, S.N., et al. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.06.088

BITE 8632 14 July 2011

4 Table 2 Enhancement of iron solution in 10 mM K2SO4 and 9K. Total Fe concentration, M 5.4 103 0.16 Medium 10 mM K2SO4 9K 10 mM K2SO4 9K Slope* 0.64 0.24 12.8 10.7 S.N. Tan et al. / Bioresource Technology xxx (2011) xxxxxx

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Slope of the plot of relaxation rate versus fraction of Fe3+ ions present in solution (Fig. 2).

Table 3 T2 of medium in the presence and absence of Acidithiobacillus ferrooxidans cells (5.1 108 cell/ml). Medium T2, ms No bacteria 10 mM K2SO4 10 mM K2SO4 + 5.4 103 M Fe3+ 10 mM K2SO4 + 0.16 M Fe3+ 9K 9K + 5.4 103 M Fe3+ 9K + 0.16 M Fe3+ 1818 22 17.6 0.05 0.833 0.004 1770 15 33.5 0.1 0.841 0.005 With bacteria 1821 25 16.9 0.1 0.839 0.006 1654 30 32.3 0.1 0.840 0.004

presence and absence bacteria (Table 3). The presence of bacteria does not signicantly inuence the relaxation time. The bacteria concentration was 5.1 108 cell/ml. An A. ferrooxidans cell has an average dimension of 1.2 0.5 0.2 lm3 (Gehrke et al., 1998) and occupies an approximate volume of 0.12 lm3. Therefore, the volume fraction of the bacteria in the solution is approximately 0.006%. The relaxation time of protons interacting with bacteria is expected to shorten due to their decreased mobility. Since the majority of the protons in the solution interact with water and Fe3+ ions, the presence of the small volume fraction of bacteria is not likely to signicantly inuence the relaxation time. At low Fe3+ ion concentrations, a slightly lower T2 was found in the presence of bacteria in both media, but at high Fe3+ ion concentration, T2 was unaffected by the presence of bacteria. The decrease in T2 arising from the bacteriaproton interaction is too small to measure at high Fe3+ ion concentration as signicantly more protons interact with Fe3+ ions. 3.3. Proton, bacteria and iron interaction Fig. 3a shows the relaxation rate as a function of time in 10 mM K2SO4 and 9K solutions containing 5.4 103 M Fe2+ ions in the presence and absence of bacteria. In the absence of bacteria, the relaxation rate remained constant with time. In the presence of bacteria, an enhancement in the relaxation rate with time was found. The effect is more pronounced in 10 mM K2SO4 medium. The enhancement in relaxation rate with time is due to the oxidation ability of A. ferrooxidans as Fe2+ was oxidized to Fe3+ ions (reaction 1). The Fe3+ ion concentration, determined from Fig. 2a is shown in Fig. 4a. In the absence of bacteria, no or insignicant amounts of Fe3+ ions were present in both media, indicating

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csignicantly greater than other ions present in the medium. The iron concentration is at least 10 times larger than the concentration of other electrolyte species. Therefore, the presence of small amounts of other ions does not inuence the proton relaxation time of solutions with high total iron concentration. 3.2. Proton and bacteria interaction The relaxation time of solutions containing 0, 5.4 103 M and 0.16 M Fe3+ in 10 mM K2SO4 or 9K were determined in the

261 262 263

(a)
-1

70 60

(a)

6 5 4

Fe , mM

50

3 2 1 0 -1 0 10 20 30 40 50

1/T2, s

30 20 10 0 0 10 20 30 40 50

3+

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time, h

time, h

(b)

800 700 600

(b)

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500 400 300 200 100 0 0 25 50 75 100 125 150

Fe , mM

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3+

25

50

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150

time, h
Fig. 3. 1/T2 as a function of time in 9K (diamonds) and 10 mM K2SO4 (triangles) containing (a) 5.4 103 M and (b) 0.16 M Fe2+ ions in the presence (,N) and absence (e,4) of Acidithiobacillus ferrooxidans.

time, h
Fig. 4. The calculated Fe3+ ion concentration as a function of time in 9K and 10 mM K2SO4 medium containing (a) 5.4 103 M and (b) 0.16 M Fe2+ ions in the presence (,N) and absence (e,4) of Acidithiobacillus ferrooxidans.

Please cite this article in press as: Tan, S.N., et al. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.06.088

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conditions to enhance biooxidation ability and thus contribute to the optimization of bioleaching process efciency. The measurement has the advantage of in situ, non-intrusive, and fast. Acknowledgement This work was supported by CSIRO CEO Science Leader program. References

bacteria, 10 cell/ml

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Fig. 5. Acidithiobacillus ferrooxidans concentration as a function of time in culture medium containing 0.16 M Fe2+ ions in 9K () and 10 mM K2SO4(N).

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insignicant degrees of Fe2+ ions oxidation. But in the presence of bacteria, the Fe3+ ion concentration increases with time in the rst 24 h, after which the Fe3+ ion concentration remained constant. Converting the results to concentration shows (Fig. 4a) that the evolution of Fe3+ ion concentration with time is independent of the medium. This suggests that the metabolic activity of the bacteria is the same in both media at low iron concentration. Fig. 3b shows the relaxation rate as a function of time of 0.16 M Fe2+ in 10 mM K2SO4 and 9K. The corresponding Fe3+ ion concentration, determined from Fig. 2b, is shown in Fig. 4b. In the absence of bacteria, the Fe3+ ion concentration increases very slightly with time. The slight increase in Fe3+ ion concentration is due to the abiotic oxidation of Fe2+ ion by dissolved oxygen in the solution. In the presence of bacteria, the Fe3+ ion concentration increases and then reaches a plateau with time, due to the iron oxidation ability of A. ferrooxidans. The Fe3+ ion concentration at the plateau is 67% higher in 9K medium than in 10 mM K2SO4. The bacteria concentration in 10 mM K2SO4 remained almost unchanged with time (Fig. 5), suggesting the Fe2+ is being oxidized by the inoculated A. ferrooxidans without signicant cell propagation. The insignicant cell propagation is likely linked to the fact that the 10 mM K2SO4 media does not contain the essential nutrients, such as phosphorus, for cell growth. The bacteria concentration increased by about 80 times in 9K medium after 75 h. 9K medium is known to be the optimal growth environment for A. ferrooxidans (Silverman and Lundgren, 1959). Therefore, A. ferrooxidans cells are able to oxidize iron and be capable of cell propagation in 9K medium. The NMR method provides a non-invasive means of monitoring iron oxidation rate in situ and correlating this with cell growth. 4. Conclusion NMR relaxation measurements can provide understanding of iron oxidation activity of bacterial cells in solution of known composition. When A. ferrooxidans is present in solutions containing Fe2+ ions, T2 reduction is found with increasing time as the bacteria oxidizes Fe2+ to Fe3+, since the Fe3+ ion is more effective in enhancing the proton relaxation than the Fe2+ ion. NMR relaxation measurements can be a useful tool to optimize the culturing

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Blake, R.C., Shute, E.A., 1994. Respiratory enzymes of Thiobacillus-ferrooxidans kinetic properties of an acid-stable iron-rusticyanin oxidoreductase. Biochemistry 33, 92209228. Cordoba, E.M., Munoz, J.A., Blazquez, M.L., Gonzalez, F., Ballester, A., 2008. Leaching of chalcopyrite with ferric ion. Part IV: the role of redox potential in the presence of mesophilic and thermophilic bacteria. Hydrometallurgy 93, 106 115. Degreve, L., Quintale, C., 1996. The interfacial structure around ferric and ferrous ions in aqueous solution: the nature of the second hydration shell. J. Electroanal. Chem. 409, 2531. Eisinger, J., Shulman, R.G., Szymansk, Bm., 1962. Transition metal binding in DNA solutions. J. Chem. Phys. 36, 1721. Fuller, R., 1989. Probiotics in man and animals. J. Appl. Bacteriol. 66, 365378. Gehrke, T., Telegdi, J., Thierry, D., Sand, W., 1998. Importance of extracellular polymeric substances from Thiobacillus ferrooxidans for bioleaching. Appl. Environ. Microbiol. 64, 27432747. Kowalchuk, G.A., Naoumenko, Z.S., Derikx, P.J.L., Felske, A., Stephen, J.R., Arkhipchenko, I.A., 1999. Molecular analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in compost and composted materials. Appl. Environ. Microbiol. 65, 396403. Krockel, L., Poser, R., Schwagele, F., 2001. Microbially induced changes of the relaxation times of intact eggs low resolution nuclear magnetic resonance measurements (NMR). Fleischwirtschaft 81, 113116. Moin, S.T., Hofer, T.S., Pribil, A.B., Randolf, B.R., Rode, B.M., 2010. A quantum mechanical charge eld molecular dynamics study of Fe(2+) and Fe(3+) ions in aqueous solutions. Inorg. Chem. 49, 51015106. Palmer, A.G., Skelton, N.J., Chazin, W.J., Wright, P.E., Rance, M., 1992. Suppression of the effects of cross-correlation between dipolar and anisotropic chemical-shift relaxation mechanisms in the measurement of spin relaxation rates. Mol. Phys. 75, 699711. Pascall, M.A., Ravishankar, S., Ghiron, K., Lee, B.T., Johannessen, J.N., 2006. Evaluation of magnetic resonance for detection of bacterial contamination in low-acid, shelf-stable packaged soymilk. J. Food Prot. 69, 16681674. Pesci, E.C., Milbank, J.B.J., Pearson, J.P., McKnight, S., Kende, A.S., Greenberg, E.P., Iglewski, B.H., 1999. Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96, 1122911234. Peters, S., Koschinsky, S., Schwieger, F., Tebbe, C.C., 2000. Succession of microbial communities during hot composting as detected by PCR-single-strandconformation polymorphism-based genetic proles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66, 930936. Prasad, P.V., Nalcioglu, O., Rabbani, B., 1991. Measurement of 3-dimensional radiation-dose distributions using MRI. Radiat. Res. 128, 113. Rawlings, D.E., 2002. Heavy metal mining using microbes. Annu. Rev. Microbiol. 56, 6591. Rawlings, D.E., 2005. Characteristics and adaptability of iron- and sulfur-oxidizing microorganisms used for the recovery of metals from minerals and their concentrates. Microbial Cell Factories 4. Rohwerder, T., Gehrke, T., Kinzler, K., Sand, W., 2003. Bioleaching review part A: progress in bioleaching: fundamentals and mechanisms of bacterial metal sulde oxidation. Appl. Microbiol. Biotechnol. 2003, 239248. Shah, N.P., 2000. Probiotic bacteria: selective enumeration and survival in dairy foods. J. Dairy Sci. 83, 894907. Silverman, M.P., Lundgren, D.G., 1959. Studies on the chemoautotrophic iron bacterium Ferrobacillus-ferrooxidans. 1. An improved medium and a harvesting procedure for securing high cell yields. J. Bacteriol. 77, 642647. Watling, H.R., 2006. The bioleaching of sulphide minerals with emphasis on copper sulphides a review. Hydrometallurgy 84, 81108. Zeng, W.M., Qiu, G.Z., Zhou, H.B., Peng, J.H., Chen, M.A., Tan, S.N., Chao, W.L., Liu, X.D., Zhang, Y.S., 2010. Community structure and dynamics of the free and attached microorganisms during moderately thermophilic bioleaching of chalcopyrite concentrate. Bioresour. Technol. 101, 70687075.

Please cite this article in press as: Tan, S.N., et al. An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement. Bioresour. Technol. (2011), doi:10.1016/j.biortech.2011.06.088