Elucidation of Iron(III) Bioleaching Properties of Gram-Positive Bacteria

Microbial-based iron reduction is an emerging technology used as an alternative to conventional chemical-based iron reduction. The iron reduction in kaolin refinement is vital for enhancing its commercial value. Extensive studies on microbial-based iron reduction mainly focus on Gram-negative bacteria, whereas little is understood about Gram-positive bacteria’s mechanism and potential application. This study aims to investigate the iron-reducing mechanism of two Gram-positive bacterial isolates, Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus). By varying the growth environment of bacteria and monitoring the biochemical changes during the process of iron reduction, the results show that Gram-positive bacterial iron reduction performance depends on the medium composition, differing from Gram-negative bacteria-based reduction processes. Nitrogen-rich medium facilitates the microbial basification of the medium, where the alkaline conditions impact the microbial iron reduction process by altering the gene expression involved in intracellular pH homeostasis and microbial growth. This discovery will contribute to the mineral refining processes and promote the development of microbial-based bioprocesses for ore purification, while also laying the foundation for investigating other Gram-positive bacterial iron-reducing ability.


■ INTRODUCTION
Kaolin is a silicate clay mineral exhibiting excellent physical and chemical properties and used primarily in papermaking, refractory materials, rubber, ceramics, plastic, coatings, and other industrial fields. 1,2 However, the iron contaminants in unprocessed kaolin ores reduce the whiteness of kaolin, limiting its industrial and pharmaceutical applications. 1,3 As an alternative to traditional physical-and chemical-based iron leaching processes, microbial-based processes are deemed advantageous due to their lower operational cost and environmentally sustainable approach.
Various studies found that some microorganisms extract iron oxides (the predominant form of iron-based contaminants) in kaolin clay by producing organic acids. Aspergillus niger can facilitate iron contaminant removal in kaolin ore, achieving industrial grade purity applicable for papermaking, coatings and fillers. 4 This microbial-based leaching process outperforms conventional bleaching methods by increasing the kaolin ore whiteness by over 50%, while generating lesser chemical waste. 5 However, the use of A. niger is limited as it generates an acidic environment during the leaching processes that can impact the environment. Other iron oxide extraction approaches use other iron-respiring bacteria such as Gramnegative Shewanella that reduce ferric(III)oxide to ferrous ions through electron transfer during microbial growth. 6−9 Currently, limited Gram-positive bacteria are found to possess the iron leaching capabilities, impeding the understanding of the iron-leaching mechanism in these bacteria.
Therefore, this study aims to investigate the ferric(III)oxide leaching mechanism of two Gram-positive bacteria under different growth conditions, and the different biochemical responses that occur throughout the bioleaching processes. liter); 10× diluted lysogeny broth, 0.1LB (1 g of tryptone, 0.5 g of yeast extract, and 1 g of sodium chloride per liter); glucose medium (15 g of glucose, 10 g of sodium chloride per liter); starch LB medium (1 g of starch, 0.5 g of glucose, 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride per liter); starch 0.1LB medium (0.1 g of starch, 0.05 g of glucose, 1 g of tryptone, 0.5 g of yeast extract, and 1 g of sodium chloride per liter); starch medium (1 g of starch, 0.5 g of glucose per liter). All media were prepared with double distilled water and sterilized at 121°C, 20 min. All microbial seeds were cultured overnight in LB medium at 37°C.
Microbial Culturing Conditions. Microbes were cultured in LB, 0.1LB, glucose, starch LB, starch 0.1LB, and starch medium with ferric(III)oxide or kaolin ore. Each sample had an initial medium volume of 50 mL, in which 0.3 g of ferric(III)oxide or 1 g of kaolin powder was added and inoculated with the respective microbes to a final optical density of 0.02 (λ = 600 nm). Each culture was in triplicate on a shaker at 160 rpm and room temperature. Samples were taken every 24 h for 14 consecutive days at a rate of 4% of the total volume. The samples were then subjected to biochemical tests. Each experiment included a group of the same condition but without microbes as the negative control.
Ferrous Ion and pH Measurement. Samples taken daily were quick-frozen with liquid nitrogen and stored at −20°C and finally measured together. The reaction equation of ferrous ion with 1,10-phenanthroline is as follows: Ferrous ions can form stable orange complexes with 1,10phenanthroline. 10 The samples were mixed with 1,10phenanthroline, ammonium acetate, and sodium fluoride in a ratio of 1:2:2:5 and incubated in the dark under room temperature for 1 h to measure its absorption at 492 nm wavelength through a Tecan microplate reader (Mannedorf, Canton of Zurich, Switzerland). The pH was measured directly by a Mettler-Toledo pH meter (Zurich, Canton of Zurich, Switzerland).
Microbial Growth Rate Measurement. Microbial growth rates were measured daily under sterile conditions. The taken samples were diluted with sterile water by 10−20 orders of magnitude according to microbial growth and spread on antibiotic-free LB agar plates. The plates were incubated at 37°C overnight. The number of colony forming units was recorded according to the number of microbes and dilution ratio.
Determination of Total Ferric(III)oxide Content in Kaolin Clay. Kaolin was treated with 37% (w/v) hydrochloric acid at room temperature for 12 h to convert iron to an ionic state. The resulting solution was mixed with hydroxylamine hydrochloride in a ratio of 1:1 to reduce iron ions, and the concentration of ferrous ions was subsequently determined using 1,10-phenanthroline.
RT-PCR Analysis. The levels of gene expression under different conditions were determined by real-time PCR system (RT-PCR). Sample RNA extraction, reverse transcription into cDNA, and RT-PCR were performed using Promega kits (Madison, Wisconsin, USA) according to the manufacturer's instructions. The quantity and quality of RNA were evaluated by Metash B-500 spectrophotometer (Shanghai, China). The mRNA levels of related genes were determined by Thermo Fisher Scientific real-time PCR system (Waltham, Massachusetts, USA) using BRYT Green dye. The primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RT-PCR was performed in 20 μL reaction mixture containing 10 μL of GoTaq qPCR Master Mix (contains BRYT Green dye, carboxy-X-rhodamine, GoTaq hot start enzyme, MgCl 2 , deoxy-ribonucleoside triphosphate, patented reaction buffer), 0.4 μL of forward primer (10 μM), 0.4 μL of reverse primer (10 μM), 0.2 μL of carboxy-X-rhodamine (1×), 7 μL of nuclease-free water, 2 μL of template cDNA. Thermal cycling includes the following conditions: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s and 60°C for 1 min.
Siderophore Detection. Detection of siderophore in the culture medium was performed by chrome azurol S (CAS) assay. 11 The CAS solution was prepared as follows: Solution No. 1: 0.0027 g of FeCl 3 ·6H 2 O was dissolved in 10 mL of HCl of 10 mM. Solution No. 2: 60.5 mg of CAS powder was dissolved in 50 mL of distilled water. Solution No. 3: 4.037 g of piperazine was added to 30 mL of distilled water, followed by 8 mL of concentrated HCl until pH was 5.6 to completely dissolved. Solution No. 4: 21.9 mg of HDTM dissolved in 50 mL of distilled water. 1.5 mL of solution No. 1 was added to 7.5 mL of solution No. 2, mixed with solution Nos. 3 and 4, and brought up to 100 mL using distilled water.
In the CAS assay method, the samples and CAS solution were mixed in equal volumes, and the absorbance of obtained mixture was measured at 630 nm wavelength through microplate reader. The reference blank contained equal volumes of CAS solution and uninoculated medium. The percentage of siderophore units was estimated using the following formula where A s was the absorbance of the mixture of samples and CAS solution and A r was the absorbance of the reference blank. Scanning Electron Microscopy. The morphology of microbes and kaolin was characterized by scanning electron microscopy. The microbes and kaolin were collected by centrifugation at 5000 rpm at room temperature, fixed with 2.5% glutaraldehyde for more than 4 h, and washed two times with 0.1 M phosphate-buffered saline (PBS). The samples were then dehydrated over a graded ethanol series (30%, 50%, 70%, 90%, and 100%) and dried at room temperature overnight. Finally, the samples were visualized on HITACHI Regulus 8100 scanning electron microscope (Tokyo, Japan).
Determination of Microbial Biofilm Formation. Glass surfaces with microbial biofilm growth were stained with crystal violet solution (1 g/L) for 30 min followed by vigorous washing with distilled water twice. The stained surface was dried at 60°C for 10 h and then suspended in 10 mL of absolute ethanol. The resulting solution was measured at an absorption wavelength of 595 nm.

■ RESULTS AND DISCUSSION
Based on an earlier study, we identified two Gram-positive bacteria that reduce iron differently compared to Gramnegative bacteria. 12 We further found that the ferric(III)oxide reduction ability of the Gram-positive bacteria B. cereus and S. aureus are nutrient-dependent. Nitrogen-rich environments elicit microbial ferric reduction by ammonia biosynthesis that raises the media pH. In this study, we further evaluate the ferric(III)oxide reduction ability and its impact on other microbial behavior (pH and microbial growth rate) by altering the carbon sources complemented with a nitrogen-rich medium. Generally, ferric(III)oxide salts exist in the αferric(III)oxide crystalline form, whereas in kaolin clay, ferric contaminants exist in the form of crystalline ferric(III)oxide and amorphous ferric(III) oxide. Therefore, the iron-reducing ability of B. cereus and S. aureus was tested on ferric(III) oxide salt (Fe 2 O 3 ) and kaolin. We altered the nitrogen source in the culturing media using either 0×, 0.1×, or 1× lysogeny broth (LB) as a culturing base, supplemented with carbon sources of starch (0.1% w/v) or glucose (0.05% w/v).
Ferric Reduction Properties of B. cereus. We found that B. cereus cultured with starch showed a slightly weaker degree of alkalization (Figure 1, Figure S1). B. cereus cultured in    days of incubation (Figure 2A,B,E,F, Figure S2). Interestingly, the use of 0.1× LB showed different pH profiles when cultured with either Fe 2 O 3 or kaolin, showing basification and acidification, respectively, affecting the ferric reducing ability of S. aureus ( Figure 2C,D). S. aureus reduces ferric contaminants in kaolin when cultured in both LB-starch and 0.1LB-starch and achieved the maximum reduction within 2 days of culture ( Figure 2B,D). In contrast, S. aureus cultured in 0.1LB-starch medium showed 2-fold lower Fe 2 O 3 reduction ( Figure 2C). Similar to B. cereus, starch and glucose cultures rapidly acidify to about pH 4, inhibiting S. aureus growth and thus no ferric reduction ( Figure 2E,F, Figure S2E,F). We found that S. aureus cultured in LB-starch, 0.1LB-starch, and glucosestarch removed 29.7%, 32.5%, and 5.9% of ferric(III)oxide contaminants, respectively ( Figure 2G−I).
Transcriptomics Analysis of B. cereus Bioleaching Mechanism. In order to better follow the cellular processes involved in microbial-mediated bioleaching, we investigated the iron reduction-related genes of B. cereus and S. aureus under different media conditions ( Figure 3). The B. cereus's asb gene cluster is responsible for the biosynthesis of the catecholate siderophore, petrobactin, which plays an essential role in intracellular ferric iron transport. These siderophores can be used to reduce oxidative stress while increasing iron acquisition and virulence. 13−15 We observed that B. cereus cultured in starch-supplemented nitrogen-rich media (0.1× and 1× LB) showed an up-regulation of asb gene cluster ( Figure 3A), consistent with the increased siderophores and ferrous ion concentration resulting from the bioleaching process of both Fe 2 O 3 and kaolin ores. This phenomenon is evident in B. cereus cultured under 0.1LB-starch-Fe 2 O 3 conditions, where an approximately 10-fold increase in the asb gene cluster expression was observed during the highest concentration of the leached ferrous ion. The siderophore content in these conditions increased rapidly and stabilized at approximately 60% at the beginning of the culture, almost reaching the maximum value of all culture conditions ( Figure  4B). Similarly, cultures in the absence of starch (0.1LB-Fe 2 O 3 ) also increased the asb gene cluster expression during the highest ferrous ion concentration, while the siderophore content reached approximately 60% (Figures S1, S3, and S8). Interestingly, the addition of any carbon-based nutrients (glucose or starch) triggers the acidification of the surrounding medium ( Figure 1E,F, S1E,F).
The B. cereus's sigB and napA regulate intracellular and extracellular homeostasis. sigB was upregulated in B. cereus in the presence of kaolin ores or Fe 2 O 3 regardless of the culturing condition resultant from the microbial iron metabolism. B. cereus grown in nitrogen-rich and carbon-rich media produces various byproducts that alter the surrounding pH, leading to stress response factor expression of sigB and Na + /H + antiporter napA. These two stress response factors regulate cellular ATP and Na + /H + homeostasis. 16,17 Alternatively, Bacilli can metabolize nitrogen-rich nutrients to produce ammonia that basifies the surrounding medium. 18−20 Conversely, simple sugars such as glucose are metabolized through the glycolytic pathway to produce pyruvate and soluble carbon dioxide that acidifies the surrounding medium. 21,22 Complex polysaccharides such as starch are first hydrolyzed to produce dextrin, later converted to monosaccharides used in bacterial metabolism. 22 The slow process of hydrolyzing these polysaccharides influences the gradual acidification of the surrounding media, as observed in the earlier described results (Figure 1A−D).
napA imports H + into the cellular cytoplasm to maintain intracellular homeostasis, providing conducive conditions for ferric ion reduction. Basic pH levels upregulate napA, where cultures in 0.1LB-starch-Fe 2 O 3 measured a 100-fold increase in napA expression, showing the best ferric bioleaching compared to the other culturing conditions ( Figure 1C). Similarly, cultures of 0.1LB-Fe 2 O 3 showed upregulated napA levels during the peak of the iron reduction process (Figures S1C  and S3A). The acidic environment generated by cultures in sugar-based media down-regulates napA expression by 100fold. These results are indicative of the role of napA in regulating the homeostatic balance of pH in the environment.
Transcriptomics Analysis of S. aureus Bioleaching Mechanism. We similarly investigated the various S. aureus genes involved in iron reduction, including the iron-regulated surface determinant (Isd) system (ferric transport and internalization), SbnI (siderophore biosynthesis), and DacA (pH homeostasis). We observed minimal changes in IsdG expression from S. aureus cultured in LB-starch media despite showing no impairment to the bacterial growth. In contrast, lowered nitrogen-based nutrition was found to upregulate IsdG approximately 100-fold in 0.1LB-starch cultures while inhibiting growth, affecting ferric bioleaching in kaolin clay cultures ( Figure 3B). Similarly, the S. aureus cultures in 0.1LB exhibited a good ferric reduction in Fe 2 O 3 while upregulating IsdG ( Figure S3B). We further observed SbnI up-regulation by 100fold in 0.1LB-starch-kaolin and 0.1LB-Fe 2 O 3 . SbnI regulates the ferric binding siderophore staphyloferrin biosynthesis, facilitating ferric internalization. 23,24 The ferric-bound stapyloferrin is then reduced by iron uptake oxidoreductase (IruO), a flavin adenine dinucleotide (FAD)-containing nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase. 25,26 Therefore, corresponding to the up-regulation of SbnI, it can be seen that the siderophores content reached a high stable level of approximately 50% in 0.1LB-starch ( Figure  4E). About 50% of siderophores in the nitrogen-rich medium were also maintained in 0.1LB ( Figure S8E).
Just as in B. cereus cultures, S. aureus cultures can alter the surrounding pH by using different nutrients. DacA, a membrane-bound diadenylate cyclase, is upregulated in S. aureus under an acidic environment to produce cyclic di-AMP that triggers nitrogen metabolism to restore normal cell function and ferric iron transport. In sugar-based media (starch and glucose), DacA was upregulated by 100-fold attributed to the acidification of the media (Figures 3B and S3B). The addition of starch to 0.1LB also showed acidification of the media while upregulating DacA, which is attributed to the S. aureus's ability to rapidly break down starch to glucose. Generally, S. aureus upregulates Isd proteins via ferric uptake repressor (Fur) inactivation in the presence of iron. 27 The down-regulation of Fur is often observed in S. aureus cultured under an iron-reducing environment. Intriguingly, we observed that Fur up-regulation in the presence of starch, regardless of the state of the microbial growth or the leached iron profile, except for S. aureus grown in a glucose solution, where no readout was observed due to poor microbial viability ( Figure  S3B).

Microbial Localization on Kaolin Ores.
Earlier studies also showed that microbial localization on mineral ores plays a crucial role in facilitating iron reduction, giving rise to the fundamental question of the ability of these two microbes to localize on the surface of the kaolin clay to facilitate iron internalization and reduction. 28 We discovered that the different culturing conditions also affect microbial localization and biofilm formation on the clay surface ( Figure S9). The accumulated biofilm produced by B. cereus cultured in LB and glucose medium showed 2-fold higher compared to 0.1LB medium ( Figure 5A). Contrariwise, B. cereus and S. aureus produced the least biofilm under 0.1LB and starch 0.1LB compared to other conditions ( Figure 5), attributed to the excessive microbial growth in nutrient-rich medium such as LB medium. In the presence of a simple carbon source, the acidification of the surrounding environment coupled with the lack of growth essential nutrients promote biofilm formation. Intriguingly, the addition of complex sugars such as starch inhibited the biofilm formation but increased its iron-reducing ability. These observations were consistent with our scanning electron microscopy visualized images of microbial localization on the mineral surface ( Figure 6). We observed the biofilm formation of both microbes cultured with kaolin ore for 24 h at room temperature under low (OD 600 nm : <0.5) and high (OD 600 nm : >1.5) cellular density in nutrient sufficient medium. Low cellular density of B. cereus showed scattered microbial microcolonies on the kaolin ores, increasing the microbial microcolonies and kaolin ore contact area ( Figure 6D). With higher B. cereus density, we observed extensive biofilm formation that completely covers the surface of the kaolin ores ( Figure 6E). Similarly, the low cellular density S. aureus inoculum showed dispersed microcolonies formation on the kaolin ore ( Figure 6G), while higher starting S. aureus density also showed extensive biofilm formation on the ore surface ( Figure 6H). Various literature reports have illustrated the role of biofilm-forming communities in facilitating the iron acquisition and iron reduction. Various Gram-negative and Gram-positive bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni, and Staphylococcus aureus cause iron corrosion by readily forming biofilm on the iron surfaces. The rate of iron corrosion was much slower in planktonic microbial cultures due to ineffective microbial iron metabolism. 29−31 Based on our current experimental results, a controlled level of biofilm mass facilitates better iron reduction, allowing microbial localization on the clay surface for effective iron reduction. Excessive biofilm mass would hinder the contact between microbes and clay, thus reducing the iron-reducing ability. Therefore, moderate biofilm formation through regulating the media composition can further enhance the microbial iron metabolism on kaolin ores.

■ CONCLUSIONS
In conclusion, we investigated the iron reduction mechanism of Gram-positive bacteria under different culture conditions. We discovered that Gram-positive bacteria B. cereus and S. aureus use nitrogen sources to increase the medium's pH, facilitating the iron reduction process by regulating intracellular pH homeostasis and bacterial growth. Meanwhile, the level of biofilm formation was also found to affect iron reduction. This work established the connection of the various genes in regulating the microbial bioleaching process. It helps understand the microbial iron leaching process that enables the screening of other Gram-positive bacteria exhibiting good ironreduction ability in the future. This discovery sets the stage for further development of engineered microbes that can more effectively and cleanly remove iron contaminants from samples.
Nine figures and one table encompassing the ferric(III)oxide reduction of the different Gram-positive bacteria in different media, the qPCR results and analysis of the microbial expressed genes, the microbial siderophore production levels, and the biofilm growth, along with the corresponding images documenting these changes (PDF)