Isothermal microcalorimetry (IMC) has been applied in a variety of biomineral studies, as detailed in Section 2.4.5, ranging from determining thermodynamics and reaction energies of microbial cultures associated with pyrite and chalcopyrite mineral particles (Rohwerder et al., 1998; Krok, 2016), to measuring activity of microorganisms that facilitate bioleaching of mine waste to determine and estimate their potential for ARD formation (Schippers et al., 1998; Schippers et al., 2000; Schippers et al., 2001; Kock and Schippers, 2006; Schwartz et al., 2006; Sand et al., 2007; Schippers et al., 2007). IMC has also been used to measure microbial activity during the leaching of copper in batch flasks and continuous stirred tank reactors (CSTR’s) (Hedrich et al., 2016; Krok, 2016; Hedrich et al., 2018).
Microcalorimetry has been shown to be a suitable technique for the detection and quantification of the activity of microorganisms that facilitate leaching, due to its ability to detect and measure low metabolic activities. It has also been shown to assist in understanding various impacts that microorganisms have on both bioleaching and ARD prediction.
In this chapter, the aim is to further develop the IMC method and use it to measure the metabolic activity of mineral associated microorganisms as a function of the surface area (m-2) of available mineral concentrate, with the aim of developing a method to investigate the
microbial-mineral association during the colonisation of mineral surface at the onset of heap bioleaching or during uncontrolled leaching in waste rock dumps. The work includes the measurement and assessment of chemical and bio-chemical pyrite reaction rates per available surface area. Emphasis is placed on the generation of a heat-flow curve that is not prematurely truncated due to substrate limitation.
4.2 Research approach
Metabolic activity was first determined in cells that were suspended in liquid media and thereafter, a method that allowed attachment and colonisation of the fixed area mineral surface was set up and the best configuration between saturated and unsaturated surfaces was determined for the surface area study. Post the selection of the best representative configuration, reproducibility studies were performed. Correct bead loading in ampoules was assessed and these short-term experiments were conducted in concert with investigations of oxidation rates and O2 availability or limitation. Column studies were also conducted for longer
4.2.1 Heat-flow measurements from suspended microbial cells
The maximum heat-flow of microbial cells, in the absence of mineral substrate, was determined. Mixed mesophilic iron and sulfur oxidising acidophiles were obtained from the stock reactor (Section 3.2) and cell counts were performed. Cells ranging from 1 × 105 to
1 × 108 cells ml-1, supplemented with 5 g L-1 Fe2+ in 0K media (making up a total volume of 2
ml) were transferred into the IMC ampoules. Cell free control ampoules contained 5 g L-1 Fe2+
in 0K media only. The ampoules were sealed and loaded into the IMC where microbial heat- flow was measured. After obtaining the maximum heat-flow, the IMC experimental run was stopped and the obtained maximum heat-flow, together with microbial cell numbers, was used to determine maximum heat-flow per cell. Iron consumed by the microbial cultures over the IMC experimental run was also measured using the spectrophotometric method detailed in Section 3.6.3.
4.2.2 Mineral colonisation
Glass beads were coated with the finely milled pyrite concentrate, as described in Sections 3.3.1 and 3.4, to provide a defined and reproducible surface area of well-liberated pyrite on which to study colonisation. These were colonised with the mixed mesophilic culture (Section 3.1) for use in IMC method development experiments. The beads were colonised in 250 ml Erlenmeyer flasks containing a working volume of 100 ml 0K basal salts medium (pH 1.6) with 0.5 g L-1 Fe2+, 100 mineral-coated glass beads and a total cell inoculum of1 × 108
cells ml-1. A control flask was not inoculated. The flasks were incubated at 30 °C for 72 hours
to allow sufficient time for microbial colonisation of the mineral surface. The flasks were gently agitated at 100 rpm, to ensure adequate mixing and distribution of the media, whilst preventing coated minerals from detaching off the surface of the beads. Microbial growth and activity were assessed at 24-hour intervals over the course of the 72-hour colonisation period. Wet chemistry assessment was through planktonic microbial cell counts (Section 3.6.4), measurement of the redox potential (Section 3.6.2) and soluble iron concentrations (Section 3.6.3) at 24-hour intervals. After the 72-hour colonisation period, the colonised beads were loaded into ampoules and the maximum heat-flow was measured.
4.2.3 Isothermal microcalorimetry experiments
The microbial activity of the colonised mineral surfaces was assessed using IMC described in Section 3.9. The IMC method was scrutinised with respect to the configuration of the ampoule contents including the solid and liquid phases, availability of reactants and intra-experiment repeatability.
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4.2.3.1 IMC ampoule liquid contents
In order to determine the most representative system for heat generation measurements, the degree of saturation and liquid composition in the ampoule were varied to observe the effect of the liquid phase conditions in IMC ampoules on the measured heat-flow from the colonising microbes on the pyrite coated beads (10).
In Configuration 1, the microbially-colonised pyrite-coated beads were placed in the ampoule and saturated with 0K media (pH 1.6) supplemented with 0.5 g L-1 of Fe2+. This configuration
was equivalent to the set-up for planktonic cells used in Section 4.2.1. The use of Fe2+ solution
was informed by the frequent use of a low ferrous iron concentration during the start of shake flask or column experiments to initiate microbial activity; hence, the media was supplemented with 0.5 g L-1 Fe2+.
Configuration 2 was set-up in the same manner as Configuration 1, except that the saturating liquid phase used was cell free leachate (obtained from colonisation studies; Section 4.2.2). Through this selection, this configuration resembles a continuation of the experimental physicochemical conditions in the colonisation studies.
In Configuration 3, unsaturated colonised pyrite-coated beads were used to more closely resemble the unsaturated environment typical heap bioleaching or waste mine dump conditions. This configuration was adapted from Krok et al. (2013).
4.2.3.2 IMC reproducibility
To assess the IMC method reproducibility, four biological repeats were loaded into the ampoules and these were run against chemical repeats (non-colonised controls). In this section, the heat-flow data produced over time was integrated, thereby generating a sigmoidal heat data curve, which shows the cumulative heat produced over time.
4.2.3.3 Pyrite oxidation rate quantification
Increasing volumetric microbially facilitated pyrite oxidation rates are expected as a function of increasing surface loadings. However, when these rates are normalised, similar specific rates are expected per unit surface area from samples treated the same way i.e. experiments conducted for the same period using the equivalent microbial culture. For pyrite oxidation rate measurements, microbially colonised mineral-coated beads were prepared in 100 ml Erlenmeyer flasks with a total working volume of 40 ml and loaded with 40 mineral coated beads; following inoculation, these were incubated at 30 °C for 24 hours. Abiotic controls were prepared in the same manner, without the inoculation. All other conditions remained the same as in Section 4.2.2. Pyrite oxidation rates (chemical and biochemical) were studied through assessing and measuring various loadings of mineral coated glass beads. This was done to correlate normalised maximum heat-flow (m-2) across the different surface loadings. The
number of mineral-coated beads (using pyrite concentrate) that were loaded into the IMC
ampoules, and their associated surface area, were 1 (1.13 × 10-4 m2 total surface area),
2 (2.26 × 10-4 m2), 3 (3.39 × 10-4 m2) and 4 (4.52 × 10-4 m2) beads.
The maximum heat generated in these systems was recorded and oxidation was determined according to Schippers and Bosecker (2005) and Kock and Schippers (2006). Complete oxidation of FeS2 to Fe3+ and sulfate produces a reaction energy ΔfH0 of −1546 kJ mol−1. Using
this value, together with molecular mass of FeS2 (0.12 kg mol−1), the measured maximum
heat-flow a(μW) and the sample weight w(g), the pyrite oxidation rate r can be calculated using the following equation:
𝑟 ( 𝜇𝑔 𝑘𝑔 𝑠 ) = 1 ∆𝑓𝐻°(𝑚𝑜𝑙𝑘𝐽) × 𝐹𝑒𝑆2( 𝑘𝑔 𝑚𝑜𝑙) × 𝑎(𝜇𝑊) × 1 𝑤(𝑔) Equation 4.1 𝑟 ( 𝜇𝑔 𝑘𝑔 𝑠 ) = 1 −1546(𝑘𝐽 𝑚𝑜𝑙) × 0.12 (𝑚𝑜𝑙𝑘𝑔) × 𝑎(𝜇𝑊) ×𝑤(𝑔)1 Equation 4.2
The equation was adapted to present the reaction rate as a function of surface area (A; m2)
and not mass (g) as follows:
𝑟 ( 𝜇𝑔 𝑚2 𝑠 ) = 1 −1546(𝑘𝐽 𝑚𝑜𝑙) × 0.12 (𝑘𝑔 𝑚𝑜𝑙) × 𝑎(𝜇𝑊) × 1 𝐴(𝑚2) Equation 4.3
4.2.3.4 IMC ampoule gaseous reagent limitation
For the experiments on gaseous reagent limitation in the IMC ampoules, microbially colonised mineral-coated beads were prepared in 100 ml Erlenmeyer flasks with a total working volume of 40 ml, loaded with 40 mineral-coated beads and incubated at 30 °C for 24 hours. All other conditions remained the same as in Section 4.2.2. Limitation in microbial activity not due to pyrite content was observed from initial heat-flow curves. As the ampoules were sealed, it was postulated that either O2 or CO2 may be the limiting factor to microbial activity and thus the
heat generated. Studies were performed to validate the effect of O2 and CO2 availability on
microbial activity within the sealed vial. Two colonised mineral-coated beads were transferred into ampoules, whereafter the air was displaced from the system by introducing either N2 or
CO2 at a rate of 0.1 or 0.5 L min-1 respectively for 30 seconds. The available headspace in the
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microcalorimeter and the heat-flow output was monitored until the maximum activity was reached.