Environmentally Benign Bio-Leaching Extraction of Rare Earth Elements from Non-Conventional Resources

The bio-leaching approaches are environmentally benign and economically viable. We investigated the bacterial Acidothiobacillus ferrooxidans (Af) and the fungal Aspergillus niger (An) for bio-leaching of REEs from a phosphate ore, and examined the factors governing the leaching processes. Based on the shake flask leaching studies, an increase in surface area with the increase in pulp density level (1-15 wt.%) was found to lead to an increase in the amounts of REEs leached. In contrast, with an increase in the surface area by reducing the sizes of particles, from 75 μm to 32 μm, led to a decrease in the amounts of REEs leached. The maximum bioleached percentages of REEs from concentrate phosphate and tailings were (81% & 48%) by At. ferrooxidans and (65% & 38%) by A. niger, respectively using particle size of 75 μm and at a pulp density of 1 (g/50 mL).


Introduction
Rare Earth Elements (REEs) are essential for many important applications, such as in electronics, energy generation and defense. The common resources for economic extraction of REEs are typically carbonates such as bastnaesite and phosphate as monazite, which are scarcely available on earth crust [1]. REEs are abundantly available in many clay and phosphate ores where REEs substitute Ca 2 + or Si 4 + of the mineral lattices. The conventional acid leaching of REEs from oxides are economically unfeasible for the clay and phosphate ores because of significantly low. REE contents in these ores [2][3][4]. In some cases, REEs could be extracted from these ores via chemical leaching routes as they constitute reasonable amounts of REEs (0.1-1% REEs). A major challenge faced in general in chemical leaching processes is the loss of REEs in the phosphate/gypsum precipitates/crystals, which are overcome in a step-by-step manner by sequentially removing impurities such as fluorine and calcium, including steps that carried out at elevated temperatures [5]. On the other hand, bio-leaching approaches are environmentally benign and economical alternatives. Acidophilic bacteria has been are used for bio-leaching of Au/Cu/Ni from low grade ores industrial scale. Studies on bioleaching of REEs from red mud (Al 2 O 3 ) using Penicillium tricolor and Aspergillus niger (An), and, of bio-leaching (lab-scale) of phosphate rocks using Acidithiobacillus ferrooxidans (Af) have been reported [6,7]. We conducted bacterial (Af) bio-leaching of REEs from a phosphate ore in shake flasks and in columns, and we examined the key factors that govern leaching kinetics.

Florida phosphate samples
FPT (Florida phosphate) samples including phosphate concentrate and tailings (with particle sizes about 197 µm and 147 µm), respectively were obtained from Florida University (USA). Phosphate concentrate (~80% fluorapatite) contained REEs about ~800 ppm and tailings (~80% clays) contained REEs about ~304 ppm, respectively. These samples were ground for different particle sizes by Fisher mortar grinder, model 155, U.S.A Table 1. 30 °C on a rotary shaker (150 rpm/min). Experiments were carried out for duration of 24 h. The resulting solutions were analyzed using a spectrophotometer to determine the percentage of REEs leached by the respective fungal strain.
Bioleaching of REEs by At. ferrooxidans and A. niger at different particle size and pulp density To determine optimal particle size range where dissolution will be the highest, bioleaching tests were carried out with particles of 32, 53, 75 and 105 μm size for different pulp densities of 0.1, 0.5, 1, and 1.5 g/50 mL. The suspension was maintained at constant temperature of 30 °C and air sparged with simultaneous adjustment of solution pH with diluted sulphuric acid in the case of leaching with bacteria. Solution agitation was done at 150 rpm/min stirring speed. The inoculum volume fraction was 1% for bacteria and fungi.

Analytical method
For the determination of the REEs content in the bioleaching samples, the samples were filter through a Whatman No. 1 filter paper. Filtration of the samples allowed the separation of the mineral particulates with bacterial and fungal cells from the leaching media. The filtrate was further centrifuged at 7000 rpm for more than 10 minutes to remove micro-particulates. The filtrated was treated with 1-2 mL of ArsenazoI (obtained from Sigma Aldrich), where the addition of the amount of the chelating dye was standardized; 2-3 mL/L. The content of REEs was determined spectrophotometrically using the colorimetric technique (a Shimadzu model UV-260-UV visible spectrophotometer), where a characteristic peak at ~520 nm in the spectra allowed determination of REEs content in the liquor [9].

Growth behavior of At. ferrooxidans and A. niger with and without FPT tailing samples
The data in Figure 1 and Figure 2 show that the growth rate of At. ferrooxidans and A. niger. As the density of the culture increases, the rate of division decreases until the bacteria reaches a concentration at which they no longer divide but are viable. Estimation of optical density carried out using spectrophotometer allowed us to determine the bacterial and fungal growth. The exponential phase of bacterial growth started at 30 h after the lag phase and continued until 50 h when it reached the stationary phase. During the exponential phase, each microorganism is dividing at constant intervals. Thus, the population doubles as shown in Figure 1. Regarding the pH, initially increased due to the consumption of H + by the bacteria and thereafter decreased due to the abiotic hydrolysis of ferric sulfate. The bacterial catalyzed oxidation of ferrous to ferric ion can be expressed as follows [6]:

At ferropxidans
The abiotic hydrolysis of ferric sulphate follows this reaction: Collection (ATCC). At. ferrooxidans was grown in 9K medium 1% FPT tailing samples were treated with At. ferrooxidans and A. niger in 9K and Modified Czapek's-Dox broth medium, respectively. All flasks were incubated at 30 °C using a Remi rotary shaker at 150 rpm/min for biomass measuring.
Volatile solids (VS) were determined as a measure of biomass production. VS was used rather than dry weight because monazite sand becomes entrapped in the biomass during bioleaching. By measuring VS, the organic portion of the total dry weight could be measured. Samples comprising the entire contents of a bioleaching flask were filtered on glass microfiber filters, dried overnight at 105 C, cooled to room temperature, and weighed. Samples were then ashed at 550 C for four hours, cooled to room temperature, and weighed again. The difference between the dried weight and the ashed weight was determined as VS.

Bioleaching experiments
The experiments were conducted with mesophilic acidophilic bacterial culture of At. ferrooxidans enriched in ferrous sulphate 9K medium. The culture was adapted to rare earth elements concentration of 1-10 ppm in 9K medium prior to experimentation. Bioleaching experiments were performed in a 500 mL Erlenmeyer flask with phosphate concentrate or tailings at 1% pulp density in 250 mL of 9K medium adjusted to pH 1.7 using 20% H 2 SO 4 under aseptic conditions. Experiments were carried out for duration of (2-3) days using a Remi rotary shaker at 150 rpm/min and 30 °C. In each flask 1 mL of start stationary phase bacterial culture (approximately 5 × 108 cells mL -1 ) was used as inoculum.
A. niger bioleaching experiments were carried out in 500 mL Erlenmeyer flasks with 250 mL where (3-5 days age) or one mL of fungal culture (approximately 1 × 107 spores mL -1 ) of Modified Czapek's-Dox liquid media and test samples (1%). Sterilization of the samples and media was done by autoclaving (121 °C). Each flask was inoculated with 2 mL of spore suspension of the respective ore-adapted fungal strain. All flasks were incubated at

Shake flask bacterial leaching of REEs from Florida phosphate concentrate and tailing samples
Based on bioleaching studies of REEs from FTP samples, by At. ferrooxidans and A. niger at different incubation periods, as shown in Figure 3 and Figure 4, it was found that the amount of REEs leached from phosphate concentrate and tailings by At. ferrooxidans increased with the time of incubation up to three days. The highest REEs leaching efficiencies were (70% & 45%) from phosphate concentrate and tailings, respectively at the mentioned conditions. After that, the amount of REEs leached was negligible. This decrease in leaching attributed to poor acid production capabilities of the bacteria after three The following reaction shows the formation of jarosite: The oxidation of ferrous ions by At. ferrooxidans is responsible for the large increase in the redox potential (Eh) of Fe 3+ /Fe 2+ .
The values of dry weight for At. ferrooxidans and A. niger in the presence of Florida phosphate tailing (FPT) samples, as the time of incubation increased. This result may be due to the attachment of bacterial/fungal cells to the surfaces of mineral samples. Also, it has to be noted that the toxicity of the leaching materials can affect the mechanism of biooxidation of sulphur and iron for At. ferrooxidans and thus reduce the metabolic products (citric acid, amino acids and  A. niger on bio-dissolution of organic acids in leaching media. The leaching process with A. niger was attributed to a range of different type of reagents produced during its growth, which are low-molecular weight organic acids (LMWOAs), comprising aliphatic compounds with 1 -3 carboxylic acid functional groups and methoxy, hydroxy, carboxylic acid substituted benzoic and cinnamic (aromatic) acids. Because they form stable complexes with metals, LMWOAs play a significant role in mineral transformation. Organic acids promote mineral dissolution by 1) Donating H + to protonpromoted dissolution processes; 2) Forming inner-sphere surface complexes that dislodge structural metals from the days. The leaching reactions are considered to be via indirect action of At. ferrooxidans on bio-oxidation of ferrous sulphate (Fe 2+ ) to form ferric sulphate (Fe 3+ ) in leaching media. The indirect action of At. ferrooxidans suggests that H 2 SO 4 and Fe 2 (SO 4 ) were produced during of bio-oxidation reaction. The key reactions governing the leaching process are given below.
In addition to, the highest REEs leaching efficiencies by A. niger were (42.5% & 22.5%) from phosphate concentrate and tailings, respectively at the mentioned conditions. The leaching reactions are considered to be via direct action of  Figure 7 and Figure 8). The maximum bio-dissolution of REEs from FPT were (81% & 48%) by At. ferrooxidans as mineral surface and 3) The formation of aqueous metalligand complexes that reduce the relative solution saturation with respect to minerals undergoing dissolution [4]. The key reactions governing the leaching process are given below.

Probable interaction pathways:
Citric acid is a tricarboxylic acid which contains three carboxylic moieties and one hydroxyle group (pka = 6.39) as the possible donor of a proton (H + ) at 30 °C.   The sizes of the particles determine the surface area, which in turn determined the amounts of REEs leached. Smaller surface area of the larger particle size fraction of 105 μm can be expected to result in a decrease in the number of active microbial attachment sites that could promote dissolution [10]. Lower recovery at finer particle size fractions might be attributed to possible cell damage and deactivation, which could be caused by finer particles arising from probable oxygen deficiency due to limiting air flow rate. Reduction of particle size below a critical level could increase the extent of the particle-particle collision and impose severe attrition which might disrupt the structure of the cells [11]. Optimal previously appeared in Figure 5 and Figure 6 where (65% & 38%) by A. niger for concentrate phosphate and tailings as found in Figure 7 and Figure 8, respectively a particle size of 75 μm and at a pulp density of 1 (g/50 mL). The results reveal that dissolution generally increased with the decrease in particle size down to 75 μm but decreased as particle size decreased further to < 53 μm. Also, results showed that by increasing the pulp density from 0.1 to 1 (g/50 mL), the leaching was increased for three days of incubation. The bioleaching behavior of particles of different particle size can be explained by considering the surface area of the mineral that is available for the microbes.