Effect of temperature on bioleaching of iron impurities from kaolin by Aspergillus niger fungal

ABSTRACT The microorganisms used in this study are isolates and purified strains of Aspergillus niger pistachios and NCIM 548 A. niger. All experiments, including testing of the pH, iron, citric acid, oxalic acid and sucrose concentrations in kaolin samples, were performed at temperatures of 30°C and 25°C with three repetitions. After analyzing and disinfecting the samples with distilled water on the 28th day, X-ray fluorescence (XRF) was used to analyze kaolin, especially for iron content, as well as to conduct color measurements. The results of the kaolin powder XRF showed 52% iron removal from kaolin by A. niger fungal isolated from pistachio skin after 14 days and 47.7% iron removal after 28 days at a temperature of 25°C and 33.8% iron removal after 28 days at a temperature of 30°C. The results of white measurements of treated kaolin samples showed that kaolin powders treated with A. niger pistachio skin isolate at 25°C and 30°C and A. niger NCIM 548 with fungal cells at 25°C showed the highest L* of 75.41, 74.72 and 74.49, respectively, which were increased in comparison with the L* sample of untreated kaolin, which registered 71.94. Thus, the results showed that the A. niger fungal isolated from pistachio skin at 25°C and 30°C achieved better iron removal than the other samples and that a temperature of 25°C achieved better results than a temperature of 30°C.


Introduction
Kaolin is among the most important minerals, mainly in the mineral composition of aluminum silicate (Al 2 Si 2 O 5 (OH) 4 ) [1][2][3]. The mineral properties, morphology and physical and chemical properties of kaolin make it suitable for many industrial applications, such as production of ceramics, paper, paints, fillers, cosmetics and pharmaceuticals [3][4][5][6]. These applications are heavily influenced by the condition of the soil and its content of impurities, including iron. The presence of iron in kaolin decreases its brightness and refractoriness [1,4,7,8], and causes a significant reduction in its value [9]. Even 0.4% of oxide and iron hydroxides may cause red and yellow colors in the soil [10]. These iron oxide/hydroxides may be hematite (red), maghemite (reddish brown), goethite (brownish yellow), lepidocrocite (orange), ferrihydrate (brownish red), etc. Similarly, iron ores such as hematite may contain clays like kaolin as contamination that can cause problems in the operation of blast furnaces [10]. The first beneficial step in the production of commercially available raw materials is to effectively remove iron oxides from kaolin. The iron removal process can be conducted physically, chemically or in a combination of both [1,11]. Most industries have employed potent chemical reductants such as dithionite or hydrazine to remove iron impurities, but these chemicals are associated with of iron reduction in nature [5]. Chemical techniques for the removal of iron have economical, technological and environmental disadvantages. Sodium hydrosulfite, in particular, is an expensive and dangerous chemical requiring specific and costly storage and transport arrangements. Iron leaching with this chemical is also fairly complex, requiring careful monitoring of the pH, density of the kaolin slurry, oxygen level and amount of added sodium hydrosulfite, as the Fe (III) reduction reaction may be impaired by concurrent reactions [12]. Its use also produces large amounts of effluents containing high concentrations of dissolved sulfates that require chemical treatment, often in large ponds, before disposal.
In recent decades, biological methods have been used to remove iron impurities from kaolin [7,9,13,14]. Microbial leaching is more efficient compared to chemical routes because it is environmentally friendly: it neither uses nor releases dangerous chemicals, uses far less energy since it operates at room temperature, reduces initial and operational costs and maintains the crystal structure of clay to a large extent.
In a biological process, microorganisms reduce Fe (III) , even in its structural form, for purposes other than assimilation of iron [3,15,16]. This occurs under anaerobic conditions in which soluble Fe (II) is produced by bacteria and removed from the kaolin [7,9,17,18]. These microorganisms can utilize hydrogen or organic compounds such as sugars, amino acids and even monoaromatic compounds and long-chain fatty acids as electron donors and oxidize them to produce carbon dioxide with Fe (III) serving as the sole electron acceptor [3,7,16,19]. The reactivity of Fe (III) as an electron acceptor is related to its degree of crystallinity.
Most studies on microbiological leaching have been carried out using microorganisms that produce acids such as bacillus sp [9,20] and Aspergillus niger [9,13,21,22], which are resistant to heavy metals. Organic acids produced by these microorganisms dissolve metals. Organic acids produced by A. niger fungal, in particular, cause whitening of kaolin from 56.5% to 80% within 40 h [23] because this fungus has the highest coefficient of oxalic acid production [3,21,22,24]. The process involves two steps: the first is culturing of the fungal and the second is kaolin acid leaching. The temperature and pH of the environment must be controlled. Maximum oxalic acid production occurs at a pH of 6-7. But when the pH is lower, citric acid predominates [21,23]. The use of oxalic acid produced by the fungal to dissolve iron impurities from kaolin has been the subject of many studies. The important factors for iron removal from kaolin in microbiological tests are the pH, strain type, temperature, amount of kaolin and time allotted to add kaolin to the microbe culture. The experimental results show that adding oxalic acid directly rather than biologically at a pH of 3 and heating the culture at up to 84°C for more than 5 h removes 44 wt% iron from raw kaolin [25].
Cameselle et al. [26] investigated the effects of the temperature, agitation rate, pH, citric acid and oxalic acid concentrations on iron dissolution. The dissolved iron concentration increased with increases in the temperature at a set pH. They also showed that the pH has a lower effect at low temperatures and that the oxalic acid concentration has greater importance. Iron dissolution increases with increases in the oxalic acid concentration.
Cameselle et al. [26] worked with only one fungi strain, but the present study used two different strains (NCIM 548 A. niger with and without fungal cells and A. niger fungal isolated from pistachio skin). The results were then compared with those for the initial sample.
The purpose of this study was to investigate the effects of temperature on the sucrose, citric acid, oxalic acid and pH concentrations in iron bioleaching by A. niger fungal. X-ray fluorescence (XRF) analyses were also performed at different temperatures at each stage to check the iron removal.

Kaolin materials
Samples of kaolin soil for testing were provided by the International Powder Technology Company. The mine from which the kaolin samples were taken was located in Iranʼs Yazd province. After preparation of the kaolin samples, XRF spectroscopy was used to determine its mineral contents. The results are presented in Table 1. Based on these analyses, the kaolin soil samples contained concentrations of 5.26% Fe 2 O 3 and 3.68% Fe. The XRF analyses were carried out at the Razi Metallurgy Research Center in an environment with a temperature of 25°C and a humidity of 30%. The test reference standard is ASTM E1621-13. The microorganisms used in this study were isolates and purified strains of A. niger pistachios and NCIM548 A. niger.

Growth of microorganisms
Newly grown microorganism strains were used to improve their leaching operation performance. For this purpose, microorganisms that had been stored in a refrigerator were first transferred to a fresh culture medium under a hood in the presence of a flame. The fungal strains were then placed in an incubator (IKA with KS 4000i) for 7 days at 30°C in preparation for transfer to a leaching culture medium. Before transfer of the microorganisms to leaching test flasks, the strains were first transferred to a flask containing a sterile solution of 0.1% Tween 80 and 0.9% NaCl in order to disperse the cell spores and enable easier, more accurate counting under a microscope. The concentration of cells in the solution was adjusted to 107 spores/cm 3 by counting under a microscope. Appropriate volumes of this solution were entered into flasks containing the culture medium (final volume: 100 ml) to obtain the desired concentration under hooded, sterile conditions.

Culture medium for microbial leaching and performance of bioleaching
The fungal environment is presented in Table 2. Inoculation of the microorganisms into 500-ml flasks containing 100 ml of culture medium and 5 g of kaolin sample was conducted. All the flasks were then placed on a rotary shaker and shaken at a speed of 160 rpm at two temperatures, 30°C and 25°C.

Methods of analysis
In this study, an A. niger strain was used to remove iron from kaolin. It was also suggested that, in order to increase the removal rate of iron, a gross kaolin sample be added to the culture medium at the beginning of the experiment [22,27]. Sampling was carried out on days 2, 7, 14, 21 and 28. After removal of a small volume (10 ml) of liquid from the flasks and separation of the liquid phase from the solid phase by centrifuge, the pH, iron, citric acid, oxalic acid and sugar concentrations in the samples were measured to obtain the results presented below. For observation of changes in the mineralogy after bioleaching, X-ray diffraction (XRD) measurements were obtained (Philips X'pert PRO XRD system) from 5°to 80°2θ CuKα, at 0.02°θ per step. Since the composition changes were very low after the bioleaching process, no effect was observed on the intensity of the XRD peaks. For this reason, XRF analyses were used to specify the percentages of compositions. After analysis and disinfection of the kaolin samples with distilled water on the 28th day of analysis, XRF (ARL 8410 instrument, tube node: Rh, 60 kv) was performed to measure the contents of the kaolin, especially the iron content, as well as color measurements. All experiments were performed at temperatures of 30°C and 25°C with three repetitions. The O-Phenanthroline method was used to measure the iron concentration [28]. Finally, a PYE UNICAM spectrophotometer device (spectrophotometer with T80+ UV/ VIS spectrometer) was used at 510 nm to record the absorption. The results were compared with standard solutions to calculate the dissolved iron content, and the amount of dissolved iron was calculated. The Marrier and Boulet method [29] was used to measure the citric acid content in the medium. This method is based on the reaction reported by Ferret and Hermann, in which pyridine and pyridine acetic anhydride react with citric acid. Conducting this reaction, with the temperature controlled at 32°C ± 2°C for half an hour, produced a yellow solution. The absorption was measured at a wavelength of 425 nm using a spectrophotometer and compared to a control sample. Finally, the citric acid concentration was calculated and compared with that of the standard samples [29]. Oxalic acid measurement was performed using the method of Manganometry [28], and the Nelson and Somogyi method was used to measure the sugar content [30,31]. In this method, four solutions with the specifications given in reference [30,31] were combined. The absorbance was then recorded using a UNICAM PYE spectrophotometer at a wavelength of 500 nm and compared to that of the standard solutions.

Analyses of color properties
The kaolin powders were sent to the Color Science Technology Research Institute after 28 days of treatment with fungal strain. They were placed in a furnace set to 1000°C for 1 h if the microbial biomass mixed with the kaolin powder was burned at this temperature. The color coordinates were measured according to the ASTM E308, D2244 standard. The laboratory environment had a controlled moisture content of 29% and a temperature of 25°C. The samples were placed on a quartz glass powder and measured with an uncertainty of 95% with a spectrophotometer (XRite SP-64) using Color I Control software.

Results and discussion
3.1. Results for NCIM 548A. niger fungal without fungal cells In order to evaluate the functions of the metabolites produced by fungal cells in the culture medium, NCIM 548 A. niger fungal cells were initially isolated from the culture medium in sterile condition after 7 days. Sterilized kaolin powder (5 g) was then added to the culture medium without fungal cells and samples were taken on specific days. The final results of kaolin bioleaching are given in Table 3. The table shows that, following a reduction in the temperature from 30°C to 25°C on day 7, the amounts of citric acid and oxalic acid increased and pH decreased as a result. After day 14, in addition to the increase in acidity, the iron concentration also increased. After day 7, the concentration of iron increased and the pH decreased, which corresponds to the results obtained by Hosseini et al. [22] and Cameselle et al. [26]. It can therefore be concluded that A. niger NCIM 548 fungal reacts better at a temperature of 25°C than at 30°C and is able to produce more organic acids, as a result of which its rate of iron removal is also higher. The XRF results for kaolin samples after 28 days at 30°C and 25°C are shown in Table 4. The results of XRF analysis of kaolin samples at 25°C (  XRF samples in this study that the samples performed better at 30°C, which contradicts the results for iron, organic acids and pH measurements reported in Ref. [26].
The results for NCIM 548 A. niger fungal without fungal cells showed that iron dissolution increased with increases in the leaching time, which is consistent with the results of Ref. [26].

3.2.
Results for NCIM 548A. niger fungal with fungal cells The results of the previous series of the experiments shown for different times reveal that the amount of organic acids produced does not increase due to the absence of fungal cells. It is therefore likely that this will affect the amount of iron removal. It was consequently decided to add spores of the fungal as well as kaolin to the culture medium this time, and to conduct fungal growth analyses simultaneously and compare the results with those of previous tests. The results of bioleaching and the XRF results for the kaolin samples after 28 days at 30°C and 25°C temperatures are shown in Tables 5 and 6. Table 5 shows that the iron and sucrose concentrations increased and the PH decreased with decreases in the temperature after day 7 and that the amount of oxalic acid also increased in addition to the concentration of iron and sucrose after day 14. The amounts of iron and oxalic acid in the leaching culture medium were relatively high at 25°C compared to a temperature of 30°C, but the production of citric acid at a temperature of 30°C was slightly higher than at 25°C. pH reduction at 25°C was higher, however, than at 30°C. The XRF results for the treated kaolin sample show a 41.6% decrease in Fe 2 O 3 and Fe content at 25°C, while other constituents also show a significant decrease compared to the standard. The XRF results for the kaolin sample show a 37.2% decrease in Fe 2 O 3 and Fe content at 30°C. The reduction Table 3. Results of the kaolin bioleaching by NCIM 548 A. niger after 28 days without fungal cells. percentages of other components were also higher at 25°C. The results of measurements of the amount of iron in the culture medium indicate that iron reduction at 25°C was approximately twice as high as at 30°C, but these results show little change in the XRF analysis. The results for iron reduction at lower temperatures also contradict the results reported by Cameselle et al. [26]. Cameselle et al. [26] showed that iron dissolution increased with increases in leaching time, but own results for NCIM 548 A. niger fungal with fungal cells showed that iron dissolution decreased after 21 days at 25°C and 30°C.

Results forA. niger fungal isolated from pistachio skin
Similar experiments were carried out for A. niger fungal isolated from pistachio skin and sampling was conducted at different time intervals. The results after 1 month are presented in Table 7 according to fungal cell. XRF analysis of the treated kaolin samples was conducted after 14 days at 25°C and after 28 days at the two temperatures of 30°C and 25°C for purposes of comparison (Tables 8 and 9). Pellets were prepared from kaolin powder treated with A. niger isolated from pistachio skin at 25°C after 28 days and from a standard kaolin sample and heated in a furnace (NUVE MF207 model) at 1000°C for 1 h. After removal of the pellets from the furnace and cooling, the changes in color were observed. Images of the treated and standard kaolin samples are shown in Figure 1. The results of the iron XFR at 25°C after 14 days showed a reduction of 52% in Fe 2 O 3 and Fe content and a reduction to 47.7% after 28 days. These results indicate that the time for the microbial culling with this fungus strain is 14 days, after which there is no significant reduction in the amount of iron in the environment. The results of iron measurements also confirm these results. The amount of sulfur in the treated kaolin sample after 14 days also shows a further decrease compared to other samples. The XRF results for a kaolin sample at 30°C showed a reduction of 33.8% in Fe 2 O 3 and Fe content. The results of these experiments also indicate that a temperature of 25°C achieves better performance than 30°C. The results for A. niger fungal isolated from pistachio skin also showed that iron dissolution decreased after 14 days at 25°C and after 21 days at 30°C with no increase with increases in leaching time, which contradicts the results reported by Cameselle et al. [26]. In addition, the image presented in Figure  1 shows a significant change in the color of the treated sample compared to the original kaolin sample.

Results of XRD, reflection spectra and color coordinate analyses
To investigate any biogenic alteration of kaolin after bioreduction, mineralogical analyses were carried out using XRD (Figure 2). The mineral material exhibited the preferred orientation of the kaolin pattern with all peaks clearly resolved. Analysis of the XRD pattern revealed that the mineral was composed mainly of highly  crystalline kaolinite, which is an accessory mineral found in both biotreated and blanc kaolin materials. Similarly, no crystalline by-product was produced during bioreduction. The XRD patterns obtained following treatment with A. niger appear to be unaltered with similar peaks before and after the treatment. This may be due to that the mineral is of high crystallinity. Figure 3 of the reflection spectrum chart shows five samples produced at visible wavelengths from 400 to 700 nm in comparison with the control sample. As the wavelength rises from blue to red, the magnitude of the reflection increases. At wavelengths of 400-410 nm, the reflection has a steep slope at first and then a gradual slope. The powder treated with NCIM 548 A. niger fungal with fungal cell at a temperature of 30°C shows the highest reflection value and the powder treated with NCIM 548 A. niger fungal without fungal cells at 25°C shows the lowest reflection value. Table 10 shows a comparison between color indices. The closer L* is to 100, the whiter the sample color. Also, a* and b* show the red and yellow indices of the sample, respectively. As shown in the table, sample 6 has the highest L*, which is equivalent to 75.41, after which samples 5 and 4 show the highest values for L* of 74.72 and 74.49, respectively. L* of the original kaolin powder is 71.94. These three samples also show the smallest amount of a* and b* in comparison with other samples. Compared to the initial powder of kaolin (sample 7), a* shows decreases in samples 6 and 5 of 45% and 47% respectively, and a decrease of 35% in sample 4. Also, b* shows a 63% decrease in samples 6 and 5, moreover, and a 31.82% decrease in sample 4. It can be concluded, therefore, that kaolin powders treated with A. niger fungal isolated from pistachio skin at two temperatures of 25°C and 30°C as well as NCIM 548 A. niger fungal at 25°C show the highest degrees of whiteness compared to other samples. Pellets were prepared from the five treated samples and the original and placed in a furnace at a temperature of 1000°C for 1 h, as shown in Figure 4. As seen in the figure, samples 1, 2 and 5 show more color variations than the other samples and the control sample, which is consistent with the results for whitening and iron removal from the kaolin samples.

Conclusion
This study examines the effect of temperature on the removal of iron from kaolin by a biological method using A. niger strains isolated from pistachio skin and  A. niger NCIM 548 fungal. The kaolin samples contained 5.26% iron oxide, and after kaolin treatment, the best iron concentration of 1039 ppm was obtained by A. niger fungal isolated from pistachio skin at 25°C. The results for kaolin powder XRF showed 52% iron removal from kaolin after 14 days. But after treatment for 28 days with the same fungal, the iron concentration was 752.750 ppm, which XRF analysis showed to represent about 47.7% iron removal from the kaolin powder. The same fungal at 30°C showed iron content of 757.5 ppm after 28 days.
Kaolin powder treated with A. niger NCIM 548 with fungal cells was also tested for the amount of iron in a 735 ppm culture medium after 21 days, and XRF results showed 41.6% iron removal from kaolin powder after 28 days at 25°C and 37.2% removal at 30°C. The results of white measurements of treated kaolin samples showed that kaolin powder treated with A. niger with pistachio skin at 25°C and 30°C and A. niger NCIM 548 with fungal cells at 25°C contained the highest L*; the results showed 75.41, 74.72 and 74.49, respectively, which had increased   in comparison with the L* sample of the original kaolin, which was 71.94. Therefore, the results showed that the A. niger fungal isolated from pistachio skin at 25°C and 30°C achieved more efficient iron removal than the other fungal. Also, a temperature of 25°C showed better results than a temperature of 30°C. The results of the experiments therefore indicated that 14 days is suitable for removal of iron from kaolin powder by this fungal strain, after which period there is no increase in iron removal.