Biological reduction of iron to the elemental state from ochre deposits of Skelton Beck in Northeast England

Ochre, consequence of acid mine drainage, is iron oxides-rich soil pigments that can be found in the water drainage from historic base metal and coal mines. The anaerobic strains of Geobacter sulfurreducens and Shewanella denitrificans were used for the microbial reduction of iron from samples of ochre collected from Skelton Beck (Saltburn Orange River, NZ 66738 21588) in Northeast England. The aim of the research was to determine the ability of the two anaerobic bacteria to reduce the iron present in ochre and to determine the rate of the reduction process. The physico-chemical changes in the ochre sample after the microbial reduction process were observed by the production of zero-valent iron which was later confirmed by the detection of elemental Fe in XRD spectrum. The XRF results revealed that 69.16% and 84.82% of iron oxide can be reduced using G. sulfurreducens and S. denitrificans respectively after 8 days of incubation. These results could provide the basis for the development of a biohydrometallurgical process for the production of elemental iron from ochre sediments.


INTRODUCTION 29
Water seepage from old ironstone mine workings into Skelton Beck is caused by rising 30 ground-water levels from the old Longacre Pit in Skelton, which is choked with iron ochre. 31 The iron deposits have coloured the beck red and threaten wildlife. Government agencies 32 worldwide often face challenges towards remediation of water contaminated by historical 33 mining activities (Mayes et al. 2009). 34 Acid Mine Drainage (AMD) is one of the most challenging environmental problems 35 faced today due to historic mine activities. Looking at the mining history it has been stated 36 that a total of about 1,150 million tons of heavy metals have been mined since the Stone Age 37 (Sheoran and Sheoran, 2006;Simpson et al. 2014). The typical characteristic of AMD is the 38 significantly low pH and high concentration of heavy metals (Engleman and McDiffett, 39 1996). This problem results from the microbial oxidation of iron pyrite in the presence of 40 water and air generating acidic leachates containing toxic metal ions and sulphates (Mohan 41 and Chander, 2006). 42 The oxidation of iron disulfides (pyrite) occurs in the beginning, this oxidation reaction 43 may release ferrous iron, sulfate and H + in the aqueous solution (Reaction 1). Ferrous iron is 44 simultaneously oxidized to the ferric form (Reaction 2), which is hydrolysed and ferric 45 hydroxide is generated. 46 47 2FeS 2 (Pyrite) + 7O 2 + 2H 2 O 2Fe 2+ (ferrous iron) + 4SO 4 2 (Sulphate) + 4H + (acid) (1) 48 4Fe 2+ + O 2 + 4H + 4Fe 3+ + 2H 2 O (2) 50 51 Komnitsas and Pooley (1990) reported acceleration of pyrite oxidation by bacterially 52 generated ferric iron. Other microorganisms like Thiobacillus ferrooxidans derive energy 53 from the oxidation of ferrous to ferric iron (Cheng et al, 2009 Thus it can be observed that eventually SO 4 2− concentration is increased (Reaction 3) and 58 the pH is decreased due to the iron disulphide dissolution. Although the reactions causing 59 oxidation of pyrite and formation of AMD occur in an abiotic environment, lithotrophic 60 microbes can be helpful to accelerate this process (Komnitsas and Pooley, 1990;Mayo et al., 61 2000;Cheng et al, 2009). 62 One of the consequences of acid mine drainage is the formation of ochre. It has been 63 reported that the term "ochre" is used for materials having iron or iron-rich ore minerals in 64 the range of 3 to 30% which are found in the water drainage from coal and other mines 65 (Popelka-Filcoff, 2008). The main chromophores responsible for the red and yellow pigments 66 are hematite (α-Fe 2 O 3 ) and goethite (FeO(OH) respectively (Gill et al., 2007). At high Fe(III) 67 and sulphate concentrations and low pH jarosite, capable of adsorbing several other heavy 68 metals present in AMD, is formed (Komnitsas et al., 1995). 69 The objective of the study is to develop a sustainable procedure to extract elemental iron 70 from the naturally prevailing and hazardous ochre. The water in Skelton Beck is regarded as 71 harmful to human and aquatic lives and had been a matter of concern since 1866, the 72 its natural environment, which will make the procedure more promising and reliable. 75

PREPARATION OF OCHRE SAMPLES 78
The ochre sample was collected from Skelton Beck river bed, receiving acid mine discharge. 79 Prior to the experiment, the ochre sample was pre-dried in an oven at 40 o C for 12 h. The 80 dried sample was slowly grinded to obtain fine powder (<10 μm), which was then sieved 81 manually with the help of sieve with apertures of 500 μm and stored in refrigerator for further 82 use. The Scanning electron microscope image of the fresh ochre is presented in Figure 1. 83 Some agglomeration as a result of drying was noticed. 84

PRE-CULTURE OF THE TWO BACTERIAL STRAINS TO PREPARE BIOMASS FOR INOCULATION 86
Anaerobic bacterial strains used were Geobacter sulfurreducens (DSM 12127) and 87 Shewanella denitrificans (DSM 15014). The strains were selected based on their ability to 88 solubilise minerals (Table 1). Sterile nutrient broth (CM0001, Oxoid, UK) was used as 89 medium for G. sulfurreducens (GS) and S. denitrificans (SD). It was prepared with de-ionised 90 water for GS and filtered sea water for SD respectively. Abiotic and biotic controls were 91 maintained for both the cultures. 10 mL of inoculum (48 h growth) was inoculated in 90 mL 92 nutrient broth and complete anaerobic condition was maintained in Drechsler bottle by 93 replacing nitrogen gas, which was confirmed with the help of the risazurine anaerobic 94 indicator (BR0055, Oxoid, UK) and 5% air dried ochre was used in the experiment. Both of 95 the cultures were sub-cultured for 48 h. The bacterial growth was measured using UV visible 96 spectrophotometer (Camspec model: M550) at absorbance of 600 nm.
The sub-cultured microorganisms were inoculated in nutrient broth with ochre and were 100 incubated for 8 days in anaerobic condition. The culture was monitored at regular time 101 intervals (2 days) to assess the rate of iron synthesis from ochre by both the strains. For the 102 analysis of solid residues, the culture broth with ochre was transferred to centrifuge tubes. 103 The centrifugation was carried out at 10,000 rpm for 10 min at 4 o C. Absolute ethanol was 104 added to the pellet thus to sterilise any organisms left and was allowed to dry by placing in 105 the vacuum oven at 64 o C for 12 h. When the pellet was completely dried, it was homogenised 106 with the help of mortar and pestle until a fine powder was formed, which was then analysed 107 by X-Ray powder diffraction (XRD), X-Ray fluorescence (XRF) and Scanning electron 108 microscope (SEM). The X-ray powder diffraction was carried out for all the powdered form of the reduced 118 samples using Cu Kα target. For each sample, the scanning time was set for 50 minutes with 119 graphical axis range from minimum of 2θ = 20° to maximum of 70°. The peak intensity and 120 the peak position for all the samples after the X-ray scanning were obtained and thus used for 121 identifying the different forms of iron present in the sample.
The samples were prepared by mounting 10 mg of each reduced sample on aluminium 125 sample holder using double sided tape. These holders were coated with thin film of carbon. 126 Once inside the SEM, random vertical and lateral movements around the microscope stage 127 was carried out to select the appropriate image of the compound present. Then energy 128 dispersive X-ray analysis (EDX) was used to determine the chemical composition of the 129 selected area. The sample was studied by comparing four different spectrums of the area of 130 interest within the sample, to minimise error. 131

GROWTH STUDY 134
The bacterial growth was monitored using UV visible spectrophotometer at the absorbance of 135 600 nm. The optical densities of the inocula were 0.927 and 1.753 for GS and for SD 136 respectively. These measurements were used to set-up the experiments. The colour of the biotic control before incubation was yellow, identical to the ochre 157 colour, but as incubation time increased, its colour changed gradually to black ( Figure 5). 158 This showed that some indigenous microorganisms present in the ochre were able to bring 159 some biotic change in the ochre in the presence of nutrient broth. Acidophilic to 69% for GS and 65 to 84 for SD within the first week of incubation. As the colour of 210 sample inoculated with SD changed much faster than the sample inoculated with GS, it can be 211 said that SD is much more effective and efficient in the reduction process. 212

MINERALOGICAL ANALYSIS USING SCANNING ELECTRON MICROSCOPE (SEM) 214
The GS treated sample was analysed by EDX in SEM. Four different EDX spectra were 215 recorded and the results are presented in Table 4. The main elements were iron (35.4%), 216 oxygen (48.4%), carbon (13.8%), silicon (0.88%) and calcium (1.31%). Four different EDX 217 spectra were also recorded for the sample treated by SD (Table 5). In addition to Fe, O, Ca, Si 218 and Cl, the sample also contained Na, Mg and S. The presence of sodium could have been 219 due to the use of sea water in the preparation of nutrient broth for SD. However the 220 percentage by weight of sodium, Magnesium and sulfur is almost negligible as they were just Archaeol. Sci. 35, 752-762. doi:10.1016/j.jas.2007.05.018 298 299 Sheoran, A. S., and Sheoran, V. (2006. Heavy metal removal mechanism of acid mine 300 drainage in wetlands: A critical review. Miner. Eng. 19, 105-116. doi: 301 10.1016Eng. 19, 105-116. doi: 301 10. /j.mineng.2005