Understanding a Core Pilin of the Type IVa Pili of Acidithiobacillus thiooxidans, PilV.

Pilins are protein subunits of pili. The pilins of type IV pili (T4P) in pathogenic bacteria are well characterized, but anything is known about the T4P proteins in acidophilic chemolithoautotrophic microorganisms such as the genus Acidithiobacillus. The interest in T4P of A. thiooxidans is because of their possible role in cell recruitment and bacterial aggregation on the surface of minerals during biooxidation of sulfide minerals. In this study we present a successful ad hoc methodology for the heterologous expression and purification of extracellular proteins such as the minor pilin PilV of the T4P of A. thiooxidans, a pilin exposed to extreme conditions of acidity and high oxidation-reduction potentials, and that interact with metal sulfides in an environment rich in dissolved minerals. Once obtained, the model structure of A. thiooxidans PilV revealed the core basic architecture of T4P pilins. Because of the acidophilic condition, we carried out in silico characterization of the protonation status of acidic and basic residues of PilV in order to calculate the ionization state at specific pH values and evaluated their pH stability. Further biophysical characterization was done using UV-visible and fluorescence spectroscopy and the results showed that PilV remains soluble and stable even after exposure to significant changes of pH. PilV has a unique amino acid composition that exhibits acid stability, with significant biotechnology implications such as biooxidation of sulfide minerals. The biophysics profiles of PilV open new paradigms about resilient proteins and stimulate the study of other pilins from extremophiles.

Bioleaching of sewage sludge for copper extraction using Acidithiobacillus thiooxidans: Optimization and ecological risk assessment.

In this study, Acidithiobacillus thiooxidans was used for the bioleaching of copper (Cu) from sewage sludge. In order to find optimization conditions, three factors including solid-to-liquid ratio (S/L) (0.01-0.2 %(w/v)), initial element sulfur (S0) (1-10 g/L), and initial pH (1-3) have been investigated. Based on response surface methodology (RSM) determined a significant reduced quadratic model with a p-value of 0.0022 (<0.05 significant level). The maximum Cu recovery was 85.3% in the optimum condition of S/L = 0.16% (w/v), S0 = 8.2 g/L, and pH = 1.4. Furthermore, a kinetic study based on a shrinking core model was performed and the result showed that chemical reaction was rate limiting in the extraction. Toxicity Characteristic Leaching Procedure (TCLP) results after bioleaching showed the bioleaching process detoxified sludge and the bioleached sludge residue was well within the regulatory limits for disposal. The germination seed with adding bioleached and unbioleached sludge to the soil was determined. Various parameters such as Germination Index (GI), Tolerance Index (TI), Vigor Index (VI), and stem length showed that the sewage sludge indices significantly increased than the sample soil with unbioleached sludge.

Disorder and amino acid composition in proteins: their potential role in the adaptation of extracellular pilins to the acidic media, where Acidithiobacillus thiooxidans grows.

There are few biophysical studies or structural characterizations of the type IV pilin system of extremophile bacteria, such as the acidophilic Acidithiobacillus thiooxidans. We set out to analyze their pili-comprising proteins, pilins, because these extracellular proteins are in constant interaction with protons of the acidic medium in which At. thiooxidans grows. We used the web server Operon Mapper to analyze and identify the cluster codified by the minor pilin of At. thiooxidans. In addition, we carried an in-silico characterization of such pilins using the VL-XT algorithm of PONDR® server. Our results showed that structural disorder prevails more in pilins of At. thiooxidans than in non-acidophilic bacteria. Further computational characterization showed that the pilins of At. thiooxidans are significantly enriched in hydroxy (serine and threonine) and amide (glutamine and asparagine) residues, and significantly reduced in charged residues (aspartic acid, glutamic acid, arginine and lysine). Similar results were obtained when comparing pilins from other Acidithiobacillus and other acidophilic bacteria from another genus versus neutrophilic bacteria, suggesting that these properties are intrinsic to pilins from acidic environments, most likely by maintaining solubility and stability in harsh conditions. These results give guidelines for the application of extracellular proteins of acidophiles in protein engineering.

Molecular Identification and Acid Stress Response of an Acidithiobacillus thiooxidans Strain Isolated from Rio Tinto (Spain).

Acidithiobacillus thiooxidans is of paramount importance in the development of biomining technologies. Being widely recognized as an extreme acidophile, extensive research has been dedicated to understanding its significant role in the extraction of several ores in recent years. However, there still exist significant molecular uncertainties surrounding this species. This study focuses on developing a taxonomic assignment method based on the sequencing of the 16S-5S rRNA cluster, along with a qPCR-based technology enabling precise growth determination. Additionally, an approach to understanding its response to acid stress is explored through RT-PCR and MALDI-TOF analysis. Our findings indicate that when subjected to pH levels below 1, the cell inhibits central (carbon fixation and metabolism) and energy (sulfur metabolism) metabolism, as well as chaperone synthesis, suggesting a potential cellular collapse. Nevertheless, the secretion of ammonia is enhanced to raise the environmental pH, while fatty acid synthesis is upregulated to reinforce the cell membrane.

Bioleaching of the α-alumina layer of spent three-way catalysts as a pretreatment for the recovery of platinum group metals.

Acid bioleaching of Al by Acidithiobacillus thiooxidans has been explored as an environmentally friendly pretreatment to facilitate the extraction of platinum group metals from spent three-way catalysts (TWC). Biogenic sulfur obtained from desulfurization bioreactors improved the production of acid by A. thiooxidans compared to commercially available elemental sulfur. The lixiviation abilities of bacteria-free biogenic acid and biogenic acid with exponential or stationary phase bacteria were compared against a control batch produced by commercial H2SO4. The maximum Al leaching percentage (54.5%) was achieved using biogenic acids with stationary-phase bacteria at a TWC pulp density of 5% w/v whereas bacteria-free biogenic acid (23.4%), biogenic acid with exponential phase bacteria (21.7%) and commercial H2SO4 (24.7%) showed lower leaching abilities. The effect of different pulp densities of ground TWC (5, 30, and 60% w/v) on Al leaching and bacterial growth was determined. While greater Al leaching yields were obtained at lower TWC pulp density solutions (54.5% at 5% w/v and 2.5% at 60% w/v), higher pulp densities enhanced microbial growth (2.3 × 109 cells/mL at 5% w/v and 9.5 × 1010 cells/mL at 60% w/v). The dissolution of the metal from the solid into the liquid phase triggered the production of biological polymeric substances that were able to absorb traces of both Al (up to 24.80% at 5% w/v) and Pt (up to 0.40% at 60% w/v).

Improvement of gold bioleaching extraction from waste telecommunication printed circuit boards using biogenic thiosulfate by Acidithiobacillus thiooxidans.

Cyanide usage in gold processing techniques has become increasingly challenging due to its toxicity and environmental impact. It is possible to develop environmentally friendly technology using thiosulfate because of its nontoxic characteristics. Thiosulfate production requires high temperatures, resulting in high greenhouse gas emissions and energy consumption. The biogenesized thiosulfate is an unstable intermediate product of Acidithiobacillus thiooxidans sulfur oxidation pathway to sulfate. A novel eco-friendly method was presented in this study to treat spent printed circuit boards (STPCBs) using biogenesized thiosulfate (Bio-Thio) obtained from Acidithiobacillus thiooxidans cultured medium. To obtain a preferable concentration of thiosulfate among other metabolites by limiting thiosulfate oxidation, optimal concentrations of inhibitor (NaN3: 3.25 mg/L) and pH adjustments (pH= 6-7) were found to be effective. Selection of the optimal conditions has led to the highest bio-production of thiosulfate (500 mg/L). The impact of STPCBs content, ammonia, ethylenediaminetetraacetic acid (EDTA), and leaching time on Cu bio-dissolution and gold bio-extraction were investigated using enriched-thiosulfate spent medium. The suitable conditions were a pulp density of 5 g/L, an ammonia concentration of 1 M, and a leaching time of 36 h, which led to the highest selective extraction of gold (65 ± 0.78%).

A new technique to evaluate Acidithiobacillus thiooxidans growth during a bioleaching process based on DNA quantification.

The potential of Acidithiobacillus (Thiobacillus) genus members, namely Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, for bioleaching purposes is known. Specifically, previous studies have shown the potential of A. thiooxidans strain DSM 26636 used in bioleaching processes to remove metals in high-metal-content matrices. All Acidithiobacillus growth-monitoring techniques available to date, including sulfate production, commonly used, present disadvantages. Thus, the current work shows a technique based on DNA quantification to evaluate the growth of A. thiooxidans DSM 26636, which is useful even in the presence of a high-metal-content residue. This proposed methodology may represent a functional complementary tool to evaluate Acidithiobacillus growth to develop biometallurgical applications.

Co-inoculation with beneficial microorganisms enhances tannery sludge bioleaching with Acidithiobacillus thiooxidans.

Bioleaching of tannery sludge is an efficient and environmentally friendly way for chromium (Cr) removal, which supports the sustainable development of the leather industry. Acidithiobacillus thiooxidans has been reported effective in Cr bioleaching of tannery sludge. However, little is known about whether the presence of other benefiting species could further improve the Cr leaching efficiency of A. thiooxidans. Here, we studied the enhancing roles of four species namely Acidiphilium cryptum, Sulfobacillus acidophilus, Alicyclobacillus cycloheptanicus, and Rhodotorula mucilaginosa in chromium bioleaching of tannery sludge with A. thiooxidans by batch bioleaching experiments. We found that each of the four species facilitated the quick dominance of A. thiooxidans in the bioleaching process and significantly improved the bioleaching performance including bioleaching rate and efficiency. The bioleaching efficiency of Cr in the tannery sludge could reach 100% on the sixth day by co-inoculating A. thiooxidans and four auxiliary species. The achievements shed a light on the role of the community-level interactions on bioleaching and may also serve as guidance for managing bioleaching consortiums for better outcomes.

Optimization of zinc bioleaching from paint sludge using Acidithiobacillus thiooxidans based on response surface methodology.

The reduction of zinc metal in the paint sludge, a hazardous waste, was investigated using Acidithiobacillus thiooxidans by a two-stage bioleaching process. This process was performed using the response surface methodology (RSM) method based on the central composite design (CCD). Four variables, a temperature range of 32-34.5-37 °C, rotation speed of shaker 120-150-180 rpm, pH of 4.2-3.2-2.2, and particle sizes of 1-2-3 mm, were used to optimize the experiments. The results showed that with a constant pulp density of 10 g/L at 32 °C, shaker speed of 120 rpm, a particle size of 1 mm and pH of 4.2, the highest removal predicted by the software (Design Expert version 11) was 22.89%. Repeating the experiments confirmed a decrease in zinc to the nearest predicted point. According to the ANOVA result, the rotation speed of the shaker has the greatest effect on the bioleaching process, followed by the two variables of the rotation speed of shaker and pH together affects. After the bioleaching process, energy dispersive X-ray (EDX) and mapping analysis showed quantitative changes in the chemical composition of the paint sludge, and morphological changes of texture were confirmed by scanning electron microscopy (SEM).

Quorum Sensing Signaling Molecules Positively Regulate c-di-GMP Effector PelD Encoding Gene and PEL Exopolysaccharide Biosynthesis in Extremophile Bacterium Acidithiobacillus thiooxidans.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Spent sulfuric acid plant catalyst: valuable resource of vanadium or risky residue? Process comparison for environmental implications.

The enormous amount of spent catalysts generated worldwide may pose a risk to the environment because of their high load of metals, including vanadium. The latter may be mobilized and released to the environment if managed improperly. Moreover, the catalysts could be considered as secondary resources rather than waste. This study aimed at the efficient extraction of vanadium from spent desulfurization catalyst (SDC) from a sulfuric acid production plant. The raw SDC and the post-extraction residues were characterized in terms of their chemical and phase composition. The metal mobility from the materials was examined with both single-step and multi-step extractions. The environmental risk assessment was performed using sequential extraction. The study revealed that both tested methods (citric acid leaching and bioleaching with Acidithiobacillus thiooxidans) enable the extraction of nearly 96% of V from SDC with a simultaneous reduction of metal mobility. However, the bacterial treatment was found more suitable. The leached residue was mostly (> 90%) composed of SiO2, which makes it a potential candidate for application in construction (e.g., concrete mixtures) after additional examinations. The study highlights the need to develop a metal extraction process for SDC in a way that metal-free residue could be a final product.

Identification of a gene encoding a novel thiosulfate:quinone oxidoreductase in marine Acidithiobacillus sp. strain SH.

Sulfur-oxidizing bacteria that are halophilic and acidophilic have gained interest because of their potential use in bioleaching operations in salt-containing environments. Acidithiobacillus sp. strain SH, which was previously identified as Acidithiobacillus thiooxidans based on its 16S rRNA gene sequence, is a chemolithoautotrophic marine bacterium exhibiting sodium chloride-stimulated thiosulfate-oxidizing activities. A novel thiosulfate:quinone oxidoreductase from strain SH (SH-TQO) has been purified from its solubilized membrane fraction. The gene for SH-TQO was determined from the draft genome sequence of the strain SH. Amino acid sequences of peptides generated by the in-gel trypsin digestion of SH-TQO were found in a protein encoded by locus tag B1757_09800 of the genome of the strain SH. The gene encoded 444 amino acids with a signal peptide of 29 amino acids and was annotated to encode a porin. The gene was located in a unique genomic region, not found in A. thiooxidans strains, suggesting that the strain SH acquired this region through a horizontal gene transfer. A protein-protein basic local alignment search revealed that sulfur-oxidizing bacteria, such as Acidithiobacillus species have proteins homologous to SH-TQO, though the degree of homologies was relatively low. The protein, DoxXA, which is homologous to TQO from Acidianus amvibalens, was also found in the genomic region.

Effect of biogenic jarosite on the bio-immobilization of toxic elements from sulfide tailings.

The discharge of toxic elements from tailings soils in the aquatic environments occurs chiefly in the presence of indigenous bacteria. The biotic components may interact in the opposite direction, leading to the formation of a passivation layer, which can inhibit the solubility of the elements. In this work, the influence of jarosite on the bio-immobilization of toxic elements was studied by native bacteria. In batch experiments, the bio-immobilization of heavy metals by an inhibitory layer was examined in the different aquatic media using pure cultures of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. A variety of analyses also investigated the mechanisms of metals bio-immobilization. Among different tests, the highest metal solubility yielded 99% Mn, 91% Cr, 95% Fe, and 78% Cu using A. ferrooxidans in 9KFe medium after ten days. After 22 days, these percentages decreased down to 30% Mn and about 20% Cr, Fe, and Cu, likely due to metal immobilization by biogenic jarosite. The formation of jarosite was confirmed by an electron probe micro-analyzer (EPMA), X-ray diffraction (XRD), and scanning electron microscope (SEM). The mechanisms of metal bio-immobilization by biogenic jarosite from tailings soil confirmed three main steps: 1) the dissolution of metal sulfides in the presence of Acidithiobacillus bacteria; 2) the nucleation of jarosite on the surface of sulfide minerals; 3) the co-precipitation of dissolved elements with jarosite during the bio-immobilization process, demonstrated by a structural study for jarosite. Covering the surface of soils by the jarosite provided a stable compound in the acidic environment of mine-waste.

Comparison of three different bioleaching systems for Li recovery from lepidolite.

Three different biological systems, the consortium of autotrophic bacteria Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans, heterotrophic fungus Aspergillus niger and heterotrophic yeast Rhodotorula mucilaginosa, were investigated for lithium extraction from lepidolite. The bacterial consortium was the most effective, 11 mg l-1 of Li was dissolved in the absence of nutrients within 336 days. Fungal and yeast bioleaching was faster (40 days), however, with lower extraction efficiency. Bioaccumulation represented a main process of Li extraction by R. mucilaginosa and A. niger, with 92 and 77% of total extracted Li accumulated in the biomass, respectively. The X-ray diffraction analysis for bioleaching residue indicated changes caused by microorganisms, however, with differences between bacterial leaching and bioleaching by fungi or yeasts. The final bioleaching yields for bacterial consortium, A. niger and R. mucilaginosa were 8.8%, 0.2% and 1.1%, respectively. Two-step bioleaching using heterotrophic organisms followed by autotrophic bioleaching could lead to the increase of the process kinetics and efficiency. Bioaccumulation of Li offers strong advantage in Li extraction from solution.

Recovery of phosphorus from municipal wastewater treatment sludge through bioleaching using Acidithiobacillus thiooxidans.

Conventional wastewater treatment plants remove phosphorus, which is captured in sewage sludge. Increasing attention is paid to suitable process pathways that allow recovery and recycling of phosphorus. One of the processes under investigation is acid leaching and recovery of phosphorus, but this requires considerable chemical additives, which could be avoided by stimulating acidification via microbiological processes. This study investigated phosphorus leaching from sewage sludge by biogenic sulfuric acid, using Acidithiobacillus thiooxidans. Sulfur supplementation and solid to liquid ratio were varied to examine how these factors affected phosphorus leaching yield. Chemical leaching by sulfuric acid from sewage sludge and thermally-treated sludge was conducted to compare with bioleaching from sewage sludge. Sewage sludge samples were collected from wastewater treatment plants in Ghent, Belgium, and Delft, The Netherlands. Both bioleaching and chemical leaching were conducted at laboratory scale using shake flask technique, and highest phosphorus leaching yield and time was determined using one-way ANOVA statistical tests. Biogenic sulfuric acid produced by A. thiooxidans extracted phosphorus from both sludge samples. The highest phosphorus leaching yield observed was 48 ± 0% for 17 days from Ghent samples and 57 ± 4% for 27 days from Delft samples with 5.0% (w/v) sulfur supplementation and 1.0% (w/v) solid to liquid ratio. Chemical leaching took shorter than bioleaching, but the leaching yield was lower, i.e. 41 ± 1% for 4 h from Ghent samples, 44 ± 1% for 1 h from Delft samples, 48 ± 1% for 1 h from thermally-treated Ghent samples and 51 ± 2% for 4 h from thermally-treated Delft samples. During phosphorus bioleaching, pH increase was observed during the early stage which hampered the activity of A. thiooxidans and therefore increased phosphorus leaching time. This study suggests that creating conditions for A. thiooxidans to overcome acid neutralizing capacity of sewage sludge is needed to extract phosphorus effectively.

Effect of microbial nutrients supply on coal bio-desulfurization.

Research on coal desulfurization is very important for economic, social, and environmentally sustainable development. In this study, three batches of shake flask experiments were conducted for coal bio-desulfurization using Acidithiobacillus ferrooxidans to explore the relationship between microbial nutrients (iron-free M9 K medium) supply and coal bio-desulfurization efficiency. The results showed that the removal rates of pyritic sulfur and total sulfur from coal effectively increased following reintroduction of coal into the filtrate from previous batch. The removal rates of pyritic sulfur and total sulfur were 55.6% and 10.0%, 77.1% and 16.1%, and 86.5% and 28.2%, respectively, in the three batch experiments without iron-free M9 K medium addition. In contrast, the removal rates of pyritic sulfur and total sulfur reached 87.5% and 28.2%, 89.1% and 31.6%, and 92.0% and 29.1%, respectively, in the three batch experiments with 6.7% iron-free M9 K medium addition. However, addition of excessive iron-free M9 K medium was detrimental to coal bio-desulfurization because of the synthesis of jarosite (MFe3(SO4)2(OH)6, M = K+, NH4+) and gypsum (CaSO4·2H2O), which further declined the pyritic sulfur bio-oxidation efficiency and total sulfur removal efficiency.

Minimization of metal sulphides bioleaching from mine wastes into the aquatic environment.

The continuous presence of toxic elements in the aquatic environments around mine tailings occurs due to bioleaching or chemical extraction promoted by the mining operations. Biogenic passivation treatment of tailings dams can be a new environment-friendly technique to inhibit the solubility of heavy metals. In spite of current bioleaching researches, we tried to minimize the mobility of the trace elements in the laboratory scale through the formation of a passivation layer in the presence of a mixed culture of Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. The X-ray diffraction (XRD) and scanning electron microscope (SEM) represented the jarosite generation as an inhibitory layer on the mineral surfaces of the tested materials. More detailed observations on electron probe micro-analyzer (EPMA) showed the co-precipitation of metals with the passivation layer. Thereby, the passivation layer demonstrates potential in elements immobilization which, in turn, can be optimized in the natural systems. Our working hypothesis was to exploit and optimize the formation of the passivation layer to maximize the immobilization of heavy metals (e.g., Cu, Cr). The optimization process of bioleaching experiments using indigenous bacteria caused a reduced solubility for Cu (from around 20% to 4.5%) and Cr (from around 30% to 10.6%) and the formation of 6.5 gr passivation layer. The analyses finally represented the high efficiency of the passivation technique to minimize metals bioleaching in comparison to earlier studies.

Acidithiobacillus thiooxidans and its potential application.

Acidithiobacillus thiooxidans (A. thiooxidans) is a widespread, mesophilic, obligately aerobic, extremely acidophilic, rod-shaped, and chemolithoautotrophic gram-negative gammaproteobacterium. It can obtain energy and electrons from the oxidation of reducible sulfur, and it can fix carbon dioxide and assimilate nitrate, nitrite, and ammonium to satisfy carbon and nitrogen requirement. This bacterium exists as different genomovars and its genome size range from 3.02 to 3.97 Mb. Here, we highlight the recent advances in the understanding of the general biological features of A. thiooxidans, as well as the genetic diversity and the sulfur oxidation pathway system. Additionally, the potential applications of A. thiooxidans were summarized including the recycling of metals from metal-bearing ores, electric wastes, and sludge, the improvement of alkali-salinity soils, and the removal of sulfur from sulfur-containing solids and gases.

Adaptive mechanism of Acidithiobacillus thiooxidans CCTCC M 2012104 under stress during bioleaching of low-grade chalcopyrite based on physiological and comparative transcriptomic analysis.

Acidithiobacillus thiooxidans (A. thiooxidans) is often used for sulfur-bearing ores bioleaching, but its adaptive mechanism to harsh environments remains unclear. Here, we explored the adaptive mechanism of A. thiooxidans in the process of low-grade chalcopyrite bioleaching based on the physiology and comparative transcriptome analysis. It was indicated that A. thiooxidans maintains intracellular pH homeostasis by regulating unsaturated fatty acids, especially cyclopropane fatty acids, intracellular ATP, amino acid metabolism, and antioxidant factors. Comparative transcriptome analysis indicated that the key genes involved in sulfur oxidation, sor and soxABXYZ, were significantly up-regulated, generating more energy to resist extreme environmental stress by more active sulfur metabolism. Confocal laser scanning microscope analysis found that down-regulation of flagellar-related genes was likely to promote the biofilm formation. System-level understanding of leaching microorganisms under extreme stress can contribute to the evolution of these extremophiles via genetic engineering modification work, which further improves bioleaching in future.

Column bioleaching of metals from refinery spent catalyst by Acidithiobacillus thiooxidans: Effect of operational modifications on metal extraction, metal precipitation, and bacterial attachment.

The feasibility of column bioleaching in the recovery of valuable metals (Ni, V, Mo, and Al) from an uncrushed petroleum refinery spent hydroprocessing catalyst using Acidithiobacillus thiooxidans has been reported. Different operational strategies such as submerged bioleaching in continuous mode, submerged bioleaching in resting period mode, free flow bioleaching in continuous mode, and free flow bioleaching in resting period mode were tested to find out the optimum bioleaching strategy for the recovery of metals from spent hydroprocessing catalyst. Among various operational modifications, submerged bioleaching in continuous mode was considered as the best strategy in which about 82.9% of Ni, 33.4% of Al, and 22.7% of Mo were leached after 315 h of column operation. The maximum yield of V (53.6%) in this column was achieved in 105 h, after which, a rapid decrease in its yield was observed, possibly due to its precipitation. The field emission scanning electron microscopy (FESEM) analysis revealed the presence of V in precipitates. The modified kinetic models showed that the leaching of Al, V and Mo followed the chemical control model, whereas the dissolution of Ni was controlled by diffusion control reaction. The bacterial attachment study with FESEM indicated that the metal toxicity was induced on bacterial cells attached to the sulfur particles. The results of the current study indicate that column bioleaching of spent hydroprocessing catalyst is effective in leaching of Ni and V, whereas leaching of Al and Mo require further treatments.