World J Microbiol Biotechnol (2015) 31:535–537 DOI 10.1007/s11274-015-1805-6
SHORT COMMUNICATION
Formate supplementation can increase nickel recovery by Halothiobacillus halophilus Mikhail Vainshtein • Tatiana Abashina Alexander Bykov • Alyona Repina • Elena Kaparullina
•
Received: 10 February 2014 / Accepted: 16 January 2015 / Published online: 23 January 2015 Springer Science+Business Media Dordrecht 2015
Abstract Acidophilic thiobacilli are traditional biotechnological agents for metal recovery from sulfide ores. Major industrial strains belong to autotrophic bacteria which are used without any organic supplements to stimulate the process. The autotrophic strain Acidithiobacillus ferrooxidans ATCC 21834 is known to use formate as a source of energy under special laboratory conditions. We showed the presence of formate dehydrogenase in the type strain of another autotrophic species Halothiobacillus halophilus representing another genus of thiobacilli. This finding prompted studies of bioleaching stimulation by formate. Canadian sulfide nickel ore was chosen for model investigation as leached substrate and the moderate acidophilic strain H. halophilus DSM 6132 was used as the leaching agent. In bench-scale bioleaching experiments, inoculation of the ore with H. halophilus supplemented with 0.3 % formate increased the recovery of nickel 70-fold as compared with formate-free inoculation (1008.0 vs. 13.8 mg Ni/L per 34 days). Bacteria H. halophilus belong to moderate acidophilic microorganisms; thus, the results were obtained with initial pH 7.4 and final pH 5.4. The mechanism of formate stimulation is under discussion. Keywords Bioleaching Thiobacilli Halothiobacillus halophilus Formate Nickel
M. Vainshtein (&) T. Abashina A. Bykov A. Repina E. Kaparullina Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Prospekt Nauki, Pushchino, Moscow Region 142290, Russia e-mail: [email protected] M. Vainshtein Pushchino State Institute of Natural Sciences, 3 Prospekt Nauki, Pushchino, Moscow Region 142290, Russia
Highlights Formate dehydrogenase activity with PMS was shown in Halothiobacillus halophilus. Formate supplementation affected the growth of H. halophilus. Supplementation of 0.3 % formate corresponded to a maximum growth of H. halophilus. The supplementation increased the recovery of nickel from ore 70-fold. Industrial recovery of metals from sulfide ores and dumps by bioleaching is a widespread biotechnological process. The most known industrial bioleaching bacteria are thiobacilli, which oxidize sulfide to sulfuric acid. They belong to the genera Acidithiobacillus, Halothiobacillus, and Thiobacillus, which had been earlier described within the single genus Thiobacillus (Robertson and Kuenen 2006). Some species of this group are heterotrophic, i.e., they need organic substrates, and have been known at least since the 1960s (Hallberg and Lindstro¨m 1994; London 1963; London and Rittenberg 1966). Metal recovery by bioleaching has an emphasis on classical acidophilic autotrophic bacteria (Walting 2006) because they do not need organic supplements. It has been shown that the autotrophic species A. ferrooxidans strain ATCC 21834 uses the organic compound, formate, as a source of energy (Pronk et al. 1991, 1992). This mechanism was suggested because the investigated bacteria (1) were stimulated by formate, (2) contained phenazine methosulfate (PMS) formate dehydrogenase to degrade this substrate, and (3) contained no enzymes responsible for direct formate assimilation. Stimulation of thiobacilli by a simple organic substrate could be of interest for industrial biotechnology. The type strain H. halophilus DSM 6132 was chosen as an object of study to show
123
536
formate utilization in a representative of other thiobacilli genera. This article is the first report on bench-scale nickel bioleaching by moderate acidophilic H. halophilus DSM 6132 stimulated by formate. A study of moderate acidophiles could be especially interesting as most investigated bioleaching processes are limited by strong acidic conditions (pH 1–2). The strong acidic conditions are favorable for metal extraction but imply high subsequent expenses to remediate the site. The strain H. halophilus was grown in medium DSM 518, pH was adjusted to 7.2. This medium was also used for experiments on bioleaching, the strain was not pregrown with formate. In cultivation of H. halophilus, as the optical density (OD) was measured (at 600 nm), the maximum OD increase was observed at a formate concentration of 0.3 %; higher concentrations (0.5–0.6 %) inhibited the growth. For instance, on the 4th day of cultivation, the measured OD reached 2.0 with 0.3 % formate while it was 0.12 in a blank and only 0.08 at 0.6 % formate. The mechanism of formate use by bacterial cells discussed by Pronk with coauthors (1991) seems to be open for discussion; anyway, we should point out that the biochemical decline of 0.3 % formate inside the cell results in a more than 10-fold increase of inorganic carbon, which is the growth substrate for autotrophic bacteria. Our analyses of the H. halophilus culture showed that these bacteria contained formate dehydrogenase during the growth with formate. To analyze the enzyme activity, bacterial cells were collected at the beginning of the stationary growth stage, precipitated (centrifugation at 6,000 g for 15 min), then washed with buffer solution and re-suspended. Further, the cells were disrupted by a Qsonica S-4000 touch-screen sonicator (Misonix, USA) in tubes placed in ice. The disruption procedure was repeated six times, 30 s each, at 1-min intervals. Unbroken cells were separated by centrifugation at 14,000g for 40 min. The resultant supernatant was used as a target extract to analyze the enzyme activity. Formate dehydrogenase was estimated by the reduction of 2,6dichlorophenolindophenol (DCPIP) (Johnson and Quayle 1964). The reaction mixture contained (lM): phosphate buffer (pH 7.0), 50.0; DCPIP, 0.075; PMS, 0.5; formate as substrate, 50.0; cell extract. NAD?-dependent formate dehydrogenase was estimated by the reduction of NAD? (Johnson and Quayle 1964). The reaction mixture contained (lM): tris–HCl buffer (pH 7.5), 50.0; NAD, 0.25; formate as substrate, 50; cell extract. The reactions were started by addition of formate. The bacteria were found to have a formate dehydrogenase activity with PMS and no activity with NAD?. In cell-free extracts of H. halophilus pre-grown in batch culture, the average activity of formate dehydrogenase was 10–18 nm/min • mg protein.
123
World J Microbiol Biotechnol (2015) 31:535–537
Efficiency of formate supplementation on the bioleaching of ore by H. halophilus was evaluated by the amount of nickel recovery, while changes in pH and sulfate concentrations served as confirming indicators. Specimens of sulfide nickel ore were obtained from Mining and Mineral Sciences Laboratories (CANMET, Ottawa, Canada) within the framework of ISTC Project 3624 ‘‘Bioleaching of nickel ores with moderate acidophiles’’. According to the Canadian data, the ore was of the following composition (%): Ni, 0.8; Cu, 0.2; Fe, 13.9; Mg, 2.7; pyrrhotite Fe(1-x)S, 33.0; pentlandite (Fe, Ni)9S8, 3.0; chalcopyrite CuFeS2, 0.7; hornblende Ca2(Mg)O–2(Fe)2–4(Al, Fe)(Si7Al)O22(OH)2, 13.0, plagioclase (Na, Ca)AlSi3O8, 22.0; hypersthene (Mg, Fe)SiO3, 5.0. Dry ore samples (40 g each) were placed into 750-ml flasks with liquid medium DSM 518 (200 ml), i.e., the solid and liquid phases were at a ratio of 1:5. All experiments were done in triplicate under aerobic conditions at 28 C on a shaker (180 rpm). Variants with and without formate were compared; the initial formate concentration was 0.3 %. The resultant nickel and sulfate concentrations in solution were analyzed by ion chromatography (Compact IC, columns 6.1010.300 Metrosep: 3 9 150 mm). Initial pH values and their changes were analyzed using an Ecotest-120 pH meter. The initial formate concentrations were 0.0 % in blank versions and 0.3 % in the experiments. The study showed that formate supplementation stimulated the bioleaching activity of the autotrophic thiobacilli H. halophilus: the recovery of nickel into solution increased up to 1008.0 mg Ni/L per 34 days with formate versus 13.8 mg Ni/L without formate (Table 1). Both the decrease of pH and increase of sulfate concentration confirmed the increase of bacterial leaching activity: it was more noticeable in the experiments with formate than in blanks. As far as nickel bioleaching is accompanied with pH decrease and sulfate production, we may suggest that H. halophilus realized the same mechanism of metal leaching as other thiobacilli, namely: oxidation of sulphur compounds related to acidification. We should also mention that H. halophilus belongs to the group of moderate acidophilic bacteria active in the pH limits from 7 to 5 which are favorable for ecological safety and curtailment of remediation expenses in nickel bioleaching. The increasing activity in bioleaching could be explained by an increasing yield of bacteria, especially when a supplemented source of inorganic carbon was provided via declined formate. We suggest that the ability to use formate can be widespread in various representatives of thiobacilli, A. ferrooxidans and H. halophilus species including. With respect to biotechnology, it means that recovery of metals from sulfide ores, at least nickel recovery, by industrially significant autotrophic acidophilic bacteria can be increased by formate supplementation. The presented
World J Microbiol Biotechnol (2015) 31:535–537 Table 1 Effects of formate supplementation on nickel recovery and bioleaching of sulfide nickel ore inoculated with H. halophilus
Concentrations of extracted nickel are given as absolute values (mg Ni/L) and percentages of initial content in ore
Time, days
537
H. halophilus DSM 6132; formate, 0.3 % pH
2?
SO4 , mg/L
Ni
mg/L
H. halophilus DSM 6132; formate, 0.0 % pH
%
Ni2? mg/L
SO42-, mg/L %
0
7.4
0.00
0.00
156 ± 1
7.4
0.00
0.00
8
6.0
1.61 ± 0.01
0.10
3,035 ± 20
6.0
0.06 ± 0.01
0.00
229 ± 2
14
5.8
3.1 ± 0.4
0.19
3,271 ± 25
6.2
0.86 ± 0.07
0.05
2,235 ± 14
20
5.4
572 ± 2
35.75
3,747 ± 26
6.0
1.8 ± 0.2
0.11
3,046 ± 23
29.2 ± 0.7
34
5.4
1,008 ± 8
63.00
5,809 ± 39
5.8
13.8 ± 0.1
0.86
3,443 ± 27
43
5.4
1,116 ± 8
69.75
7,557 ± 52
5.6
35.4 ± 0.5
2.21
3,532 ± 22
model experiments showed that addition of formate into the leaching medium affected the activity of the bacterial population and greatly increased its biotechnological yield. Acknowledgments The work was partially supported by ISTC Project 3624 ‘‘Bioleaching of nickel ores with moderate acidophiles’’ and by the Russian Federal Program, Agreement 14.604.21.0048 ‘‘Developing effective bioleaching technology of valuable metals from solid waste’’. Conflicts of interest of interests.
2-
The authors declare that there are no conflicts
References Hallberg KB, Lindstro¨m EB (1994) Characterization of Thiobacillus caldus sp. nov., a moderately thermophilic acidophile. Microbiology 140:3451–3456. doi:10.1099/13500872-140-12-3451
Johnson PA, Quayle JR (1964) Microbial growth on C1-compounds. Oxidation of methanol, formaldehyde and formate of methanolgrown Pseudomonas AM1. Biochem J 93:281–290 London J (1963) Thiobacillus intermedius nov. sp. A novel type of facultative autotroph. Arch Mikrobiol 46:329–337 London J, Rittenberg SC (1966) Effects of organic matter on the growth of Thiobacillus intermedius. J Bacteriol 91(3):1062–1068 Pronk JT, Meijer WM, Hazeu W, van Dijken JP, Bos P, Kuenen JG (1991) Growth of Thiobacillus ferrooxidans on formic acid. Appl Environ Microbiol 57(7):2057–2062 Pronk JT, van Dijken JP, Bos P, Kuenen JG (1992) High yield method of growing Thiobacillus ferrooxidans on formate. Australian Patent AU 646559. Applicant - Technische Universiteit Delft. Date of accepted 24.02.1994 Robertson LA, Kuenen JG (2006) The genus Thiobacillus. The Prokaryotes, 3rd edn. Springer, Singapore, pp 812–827 Walting HR (2006) The bioleaching of sulfide minerals with emphasis on copper sulfides. Hydrometallurgy 84:81–108
123
SHORT COMMUNICATION
Formate supplementation can increase nickel recovery by Halothiobacillus halophilus Mikhail Vainshtein • Tatiana Abashina Alexander Bykov • Alyona Repina • Elena Kaparullina
•
Received: 10 February 2014 / Accepted: 16 January 2015 / Published online: 23 January 2015 Springer Science+Business Media Dordrecht 2015
Abstract Acidophilic thiobacilli are traditional biotechnological agents for metal recovery from sulfide ores. Major industrial strains belong to autotrophic bacteria which are used without any organic supplements to stimulate the process. The autotrophic strain Acidithiobacillus ferrooxidans ATCC 21834 is known to use formate as a source of energy under special laboratory conditions. We showed the presence of formate dehydrogenase in the type strain of another autotrophic species Halothiobacillus halophilus representing another genus of thiobacilli. This finding prompted studies of bioleaching stimulation by formate. Canadian sulfide nickel ore was chosen for model investigation as leached substrate and the moderate acidophilic strain H. halophilus DSM 6132 was used as the leaching agent. In bench-scale bioleaching experiments, inoculation of the ore with H. halophilus supplemented with 0.3 % formate increased the recovery of nickel 70-fold as compared with formate-free inoculation (1008.0 vs. 13.8 mg Ni/L per 34 days). Bacteria H. halophilus belong to moderate acidophilic microorganisms; thus, the results were obtained with initial pH 7.4 and final pH 5.4. The mechanism of formate stimulation is under discussion. Keywords Bioleaching Thiobacilli Halothiobacillus halophilus Formate Nickel
M. Vainshtein (&) T. Abashina A. Bykov A. Repina E. Kaparullina Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 5 Prospekt Nauki, Pushchino, Moscow Region 142290, Russia e-mail: [email protected] M. Vainshtein Pushchino State Institute of Natural Sciences, 3 Prospekt Nauki, Pushchino, Moscow Region 142290, Russia
Highlights Formate dehydrogenase activity with PMS was shown in Halothiobacillus halophilus. Formate supplementation affected the growth of H. halophilus. Supplementation of 0.3 % formate corresponded to a maximum growth of H. halophilus. The supplementation increased the recovery of nickel from ore 70-fold. Industrial recovery of metals from sulfide ores and dumps by bioleaching is a widespread biotechnological process. The most known industrial bioleaching bacteria are thiobacilli, which oxidize sulfide to sulfuric acid. They belong to the genera Acidithiobacillus, Halothiobacillus, and Thiobacillus, which had been earlier described within the single genus Thiobacillus (Robertson and Kuenen 2006). Some species of this group are heterotrophic, i.e., they need organic substrates, and have been known at least since the 1960s (Hallberg and Lindstro¨m 1994; London 1963; London and Rittenberg 1966). Metal recovery by bioleaching has an emphasis on classical acidophilic autotrophic bacteria (Walting 2006) because they do not need organic supplements. It has been shown that the autotrophic species A. ferrooxidans strain ATCC 21834 uses the organic compound, formate, as a source of energy (Pronk et al. 1991, 1992). This mechanism was suggested because the investigated bacteria (1) were stimulated by formate, (2) contained phenazine methosulfate (PMS) formate dehydrogenase to degrade this substrate, and (3) contained no enzymes responsible for direct formate assimilation. Stimulation of thiobacilli by a simple organic substrate could be of interest for industrial biotechnology. The type strain H. halophilus DSM 6132 was chosen as an object of study to show
123
536
formate utilization in a representative of other thiobacilli genera. This article is the first report on bench-scale nickel bioleaching by moderate acidophilic H. halophilus DSM 6132 stimulated by formate. A study of moderate acidophiles could be especially interesting as most investigated bioleaching processes are limited by strong acidic conditions (pH 1–2). The strong acidic conditions are favorable for metal extraction but imply high subsequent expenses to remediate the site. The strain H. halophilus was grown in medium DSM 518, pH was adjusted to 7.2. This medium was also used for experiments on bioleaching, the strain was not pregrown with formate. In cultivation of H. halophilus, as the optical density (OD) was measured (at 600 nm), the maximum OD increase was observed at a formate concentration of 0.3 %; higher concentrations (0.5–0.6 %) inhibited the growth. For instance, on the 4th day of cultivation, the measured OD reached 2.0 with 0.3 % formate while it was 0.12 in a blank and only 0.08 at 0.6 % formate. The mechanism of formate use by bacterial cells discussed by Pronk with coauthors (1991) seems to be open for discussion; anyway, we should point out that the biochemical decline of 0.3 % formate inside the cell results in a more than 10-fold increase of inorganic carbon, which is the growth substrate for autotrophic bacteria. Our analyses of the H. halophilus culture showed that these bacteria contained formate dehydrogenase during the growth with formate. To analyze the enzyme activity, bacterial cells were collected at the beginning of the stationary growth stage, precipitated (centrifugation at 6,000 g for 15 min), then washed with buffer solution and re-suspended. Further, the cells were disrupted by a Qsonica S-4000 touch-screen sonicator (Misonix, USA) in tubes placed in ice. The disruption procedure was repeated six times, 30 s each, at 1-min intervals. Unbroken cells were separated by centrifugation at 14,000g for 40 min. The resultant supernatant was used as a target extract to analyze the enzyme activity. Formate dehydrogenase was estimated by the reduction of 2,6dichlorophenolindophenol (DCPIP) (Johnson and Quayle 1964). The reaction mixture contained (lM): phosphate buffer (pH 7.0), 50.0; DCPIP, 0.075; PMS, 0.5; formate as substrate, 50.0; cell extract. NAD?-dependent formate dehydrogenase was estimated by the reduction of NAD? (Johnson and Quayle 1964). The reaction mixture contained (lM): tris–HCl buffer (pH 7.5), 50.0; NAD, 0.25; formate as substrate, 50; cell extract. The reactions were started by addition of formate. The bacteria were found to have a formate dehydrogenase activity with PMS and no activity with NAD?. In cell-free extracts of H. halophilus pre-grown in batch culture, the average activity of formate dehydrogenase was 10–18 nm/min • mg protein.
123
World J Microbiol Biotechnol (2015) 31:535–537
Efficiency of formate supplementation on the bioleaching of ore by H. halophilus was evaluated by the amount of nickel recovery, while changes in pH and sulfate concentrations served as confirming indicators. Specimens of sulfide nickel ore were obtained from Mining and Mineral Sciences Laboratories (CANMET, Ottawa, Canada) within the framework of ISTC Project 3624 ‘‘Bioleaching of nickel ores with moderate acidophiles’’. According to the Canadian data, the ore was of the following composition (%): Ni, 0.8; Cu, 0.2; Fe, 13.9; Mg, 2.7; pyrrhotite Fe(1-x)S, 33.0; pentlandite (Fe, Ni)9S8, 3.0; chalcopyrite CuFeS2, 0.7; hornblende Ca2(Mg)O–2(Fe)2–4(Al, Fe)(Si7Al)O22(OH)2, 13.0, plagioclase (Na, Ca)AlSi3O8, 22.0; hypersthene (Mg, Fe)SiO3, 5.0. Dry ore samples (40 g each) were placed into 750-ml flasks with liquid medium DSM 518 (200 ml), i.e., the solid and liquid phases were at a ratio of 1:5. All experiments were done in triplicate under aerobic conditions at 28 C on a shaker (180 rpm). Variants with and without formate were compared; the initial formate concentration was 0.3 %. The resultant nickel and sulfate concentrations in solution were analyzed by ion chromatography (Compact IC, columns 6.1010.300 Metrosep: 3 9 150 mm). Initial pH values and their changes were analyzed using an Ecotest-120 pH meter. The initial formate concentrations were 0.0 % in blank versions and 0.3 % in the experiments. The study showed that formate supplementation stimulated the bioleaching activity of the autotrophic thiobacilli H. halophilus: the recovery of nickel into solution increased up to 1008.0 mg Ni/L per 34 days with formate versus 13.8 mg Ni/L without formate (Table 1). Both the decrease of pH and increase of sulfate concentration confirmed the increase of bacterial leaching activity: it was more noticeable in the experiments with formate than in blanks. As far as nickel bioleaching is accompanied with pH decrease and sulfate production, we may suggest that H. halophilus realized the same mechanism of metal leaching as other thiobacilli, namely: oxidation of sulphur compounds related to acidification. We should also mention that H. halophilus belongs to the group of moderate acidophilic bacteria active in the pH limits from 7 to 5 which are favorable for ecological safety and curtailment of remediation expenses in nickel bioleaching. The increasing activity in bioleaching could be explained by an increasing yield of bacteria, especially when a supplemented source of inorganic carbon was provided via declined formate. We suggest that the ability to use formate can be widespread in various representatives of thiobacilli, A. ferrooxidans and H. halophilus species including. With respect to biotechnology, it means that recovery of metals from sulfide ores, at least nickel recovery, by industrially significant autotrophic acidophilic bacteria can be increased by formate supplementation. The presented
World J Microbiol Biotechnol (2015) 31:535–537 Table 1 Effects of formate supplementation on nickel recovery and bioleaching of sulfide nickel ore inoculated with H. halophilus
Concentrations of extracted nickel are given as absolute values (mg Ni/L) and percentages of initial content in ore
Time, days
537
H. halophilus DSM 6132; formate, 0.3 % pH
2?
SO4 , mg/L
Ni
mg/L
H. halophilus DSM 6132; formate, 0.0 % pH
%
Ni2? mg/L
SO42-, mg/L %
0
7.4
0.00
0.00
156 ± 1
7.4
0.00
0.00
8
6.0
1.61 ± 0.01
0.10
3,035 ± 20
6.0
0.06 ± 0.01
0.00
229 ± 2
14
5.8
3.1 ± 0.4
0.19
3,271 ± 25
6.2
0.86 ± 0.07
0.05
2,235 ± 14
20
5.4
572 ± 2
35.75
3,747 ± 26
6.0
1.8 ± 0.2
0.11
3,046 ± 23
29.2 ± 0.7
34
5.4
1,008 ± 8
63.00
5,809 ± 39
5.8
13.8 ± 0.1
0.86
3,443 ± 27
43
5.4
1,116 ± 8
69.75
7,557 ± 52
5.6
35.4 ± 0.5
2.21
3,532 ± 22
model experiments showed that addition of formate into the leaching medium affected the activity of the bacterial population and greatly increased its biotechnological yield. Acknowledgments The work was partially supported by ISTC Project 3624 ‘‘Bioleaching of nickel ores with moderate acidophiles’’ and by the Russian Federal Program, Agreement 14.604.21.0048 ‘‘Developing effective bioleaching technology of valuable metals from solid waste’’. Conflicts of interest of interests.
2-
The authors declare that there are no conflicts
References Hallberg KB, Lindstro¨m EB (1994) Characterization of Thiobacillus caldus sp. nov., a moderately thermophilic acidophile. Microbiology 140:3451–3456. doi:10.1099/13500872-140-12-3451
Johnson PA, Quayle JR (1964) Microbial growth on C1-compounds. Oxidation of methanol, formaldehyde and formate of methanolgrown Pseudomonas AM1. Biochem J 93:281–290 London J (1963) Thiobacillus intermedius nov. sp. A novel type of facultative autotroph. Arch Mikrobiol 46:329–337 London J, Rittenberg SC (1966) Effects of organic matter on the growth of Thiobacillus intermedius. J Bacteriol 91(3):1062–1068 Pronk JT, Meijer WM, Hazeu W, van Dijken JP, Bos P, Kuenen JG (1991) Growth of Thiobacillus ferrooxidans on formic acid. Appl Environ Microbiol 57(7):2057–2062 Pronk JT, van Dijken JP, Bos P, Kuenen JG (1992) High yield method of growing Thiobacillus ferrooxidans on formate. Australian Patent AU 646559. Applicant - Technische Universiteit Delft. Date of accepted 24.02.1994 Robertson LA, Kuenen JG (2006) The genus Thiobacillus. The Prokaryotes, 3rd edn. Springer, Singapore, pp 812–827 Walting HR (2006) The bioleaching of sulfide minerals with emphasis on copper sulfides. Hydrometallurgy 84:81–108
123
