JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 92, No. 4,354359. 2001

Leaching of Mn, Co, and Ni from Manganese Nodules Using an Anaerobic Bioleaching Method

EUN YOUNG LEE,’ SEUNG-RIM NOH,’ KYUNG-SUK CHO,‘* AND HEE WOOK RW2x3

National Subsurface Environmental Research Laboratory, Ewha Womans University, 11-I Daehyun-dong, Seodaemun-gu, Seoul 120-750,’ Department of Chemical and Environmental Engineering,

Soong Sil University, I-l Sangdo-dong, Dongiak-gu, Seoul 1515-743,~ and Research Institute of Biological and Environmental Technology, Biosanit Co.,

600-16 Shinsa-dong, Kangnam-gu, Seoul 135-120,’ Korea

Received 16 April 2001iAccepted 25 July 2001

An anaerobic bioleaching of a manganese nodule by anaerobic Mn-reducing bacteria was eval- uated for the leaching of metals, Mn, Co, and Ni. Insoluble Mn4+ in the nodule could be reduced to soluble Mn*+ by dissimilatory Mn-reducing bacteria that use a carbon source and Mn4’ as an elec- tron donor and acceptor, respectively. As a result of the Mn reduction, Co and Ni could be leached from the loosed Mn matrix. Leaching experiments were carried out to optimize various process parameters, such as inoculation, pH, temperature, mineral salts, and particle size of the nodule used. The leaching efficiencies of Mn, Co, and Ni increased from 18, 7, and 10% to 77, 70, and 75%, respectively by the inoculation of the Mn-reducing enrichment culture broth. Metals could be effkiently recovered from the nodule in the ranges of pH from 5.0 to 6.5 and temperature from 30 to 45’C by anaerobic bioleaching. External addition of mineral salts was not necessary for Mn, Co, and Ni leaching from the nodule. The optimum ratio of nodule to glucose was 0.1 (w/w). To obtain a leaching effkiency above 70%, the particle size of the nodules must be less than 0.6 mm.

[Key words: metal resource, manganese nodules, manganese, cobalt, nickel, anaerobic bioleaching, Mn-reducing bacteria]

Metals such as Mn, Co, and Ni have been utilized widely in various industries. Mn is industrially important because it serves as a desulfurizing, deoxidizing, and alloying element in the manufacture of steel and cast iron (1). Co is used pri- marily in the manufacture of special steels, such as magnet steel, stellite steel for metal cutting, temperature-resistant alloys, carbide-type alloys, and corrosion-resistant steels (1). Ni is used in the manufacture of stainless steel and alloy steels. It is also utilized primarily as a catalyst in ceramics and magnets, and as salts and electrolytes for the metal- finishing application of electroplating (2). The metal re- sources are being exhausted globally as the demand of industrially important metals increases. Therefore, the de- velopment of new technology is very important to recover metals from low-grade ores and nodules that cannot be processed economically by conventional methods. In partic- ular, Mn nodules on ocean floors are potentially important natural sources of metals such as Mn, Cu, Co, and Ni (1). Mn in these nodules is mainly in an insoluble, tetravalent state (3). The Mn concentration by weight in the nodules may be as high as 25% or higher, and Co and Ni are present at variable concentrations, which can be as high as 2% for Ni and Cu, and 1% for Co (4).

The conventional methods for recovery of metals such as

* Corresponding author. e-mail: kscho@mm.ewha.ac.kr phone: t82-2-3217-2393 fax: +82-2-3277-3275

Mn, Co, and Ni from low-grade ores or Mn nodules have involved physicochemical processes. However, in recent years, increasing environmental awareness and the need for a cost-effective process have led to the consideration of biohydrometallurgical approaches. Bioleaching is a technol- ogy in which metal ions are extracted from low-grade ores and nodules by the direct or indirect actions of microor- ganisms. The advantages of the bioleaching process include the absence of noxious off-gases or toxic effluent, simplicity of plant operation and maintenance, economic and simple process requiring low-capital and low-operating costs, and applicability to various metals (5-9).

Most of the biotechnical processes for leaching of metals have been developed using aerobic microorganisms (6-S). However, the highly oxidized metal compounds such as MnO, and Fe,O, can be solubilized by reduction processes (10). Mn and Fe from MnO, and Fe,O, can be recovered by the direct or indirect actions of heterotrophic microorga- nisms that thrive under microaerobic or anaerobic condi- tions (1 l-l 5). In the former case, the microorganisms are capable of utilizing MnO, as a final acceptor of electrons in the respiratory chain of their metabolism, instead of oxygen (10). That is, anaerobic heterotrophs such as iron-reducing bacteria, manganese-reducing bacteria, and sulfur-reducing bacteria donate electrons, which are produced by the oxida- tion of organic substrates to Fe,O,, MgO, MnO,, SeO,, and V,O,, and leach the reduced metal ions into the medium (16,

354

VOL. 92,200l ANAEROBIC BIOLEACHING OF PRECIOUS METALS FROM Mn NODULES 355

17). In the second case, the reduction process is associated with the formation of reducing compounds, which are prod- ucts of their metabolism (13-15).

Since Mn in the nodules is mainly present as an insoluble MnO, (Mn4’) (3), Mn can be enzymatically reduced to a sol- uble divalent Mn ion (MrP) during the bacterial respiratory process. It appears that Co and Ni can be easily solubilized when Mn matrices, mostly MnO,, are removed from the nodules.

Several researchers (10, 12, 14, 18-22) have extensively investigated the leaching of Mn by aerobic and anaerobic microorganisms. The first experimental runs carried out in the selection and isolation of suitable microorganisms for the bioreduction of MnO, were performed using a TS me- dium (23, 24). The optimum leaching conditions were ob- tained with a mixed culture of microorganisms rather than with pure cultures (15,24).

The anaerobic bioleaching technology of metals has not been commercialized yet, because it has to be adapted ac- cording to each type of metals. Moreover, there is a demand for a less expensive and more environmentally friendly anaerobic bioleaching process. Selecting more efficient bac- terial cultures, optimizing operating parameters such as in- oculum, incubation temperature, initial pH, mineral salt, carbon source and particle size, and/or performing leaching in columns or reactors should be carried out. Therefore, in this study, we characterized and optimized the bioleaching of Mn and other precious metals such as Co and Ni from manganese nodules using an anaerobic bioleaching method.

MATERIALS AND METHODS

Mu nodule samples The Mn nodule sample, collected from the Clarion-Cliperton Fracture Zone in the Central and Western Pacific Ocean, was purchased from the Korean Institute of Geo- science and Mineral Resources. The composition of the nodule is shown in Table 1, and the concentrations of Mn, Co, and Ni of the nodule are 263.2, 1.5, and 12.2 mg.g-nodule-‘, respectively. The nodule sample used in this experiments, except in that on the effect of particle size, was pulverized using a bowl mill after being dried at room temperature. The average particle size was 0.6 mm in di- ameter.

Enrichment culture of Mn-reducing microorganisms Generally, the Mn-reducing organisms have Fe-reducing abilities, because Fe(II1) and Mn(IV) reductases share some common prop- erties, and the redox states of Fe and Mn have very close relation- ship. Therefore, the mixed culture of Mn-reducing microorganisms was obtained by the acclimation of Fe-reducing microorganisms in the nodule (25). Three kg of the nodule, 10 I of mineral salt me- dium, and 100 ml of the culture broth enriched with Fe-reducing microorganisms (25) were placed in a 20-Z reactor. The composi- tion of the mineral salt medium is as follows (g.r’): NaHCO,, 2.5; NH&I, 1.5; KH,PO,, 0.6; KCl, 0.1; citric acid, 4; MgSO,, 1. Ten g. k’ glucose was supplied to the medium as the carbon source, and the pH of the medium was adjusted to 6.3-6.5 with 25% NH,OH. Gases produced through fermentation during incubation were col-

lected with a balloon installed on the lid of the reactor. Enrichment of the microorganisms to qualitatively evaluate the more active strains was performed: cultures were incubated at 37’C and exam- ined periodically for Mn reduction, which was indicated by release of a gas and color change of the nodule. The color of the nodule changed from black to white because leached Mn*+ reacted with CO, and produced a white precipitate, in the form of MnCO,. When the color of the nodule changed to white, the culture broth was transferred to a fresh nodule medium to a final concentration of 10% (v/v). The enrichment culture broth of Mn-reducing micro- organisms which was obtained after five times of serial transfer, as kept in serum bottles and transferred into a fresh medium every 5 d, utilizing 10 ml of inoculum per 100 ml of the mineral salt medium supplemented with 10 g. b’ glucose and 1 g. f’ nodule.

Metal leaching experiments All experiments were carried out in 500-ml serum bottles. The bottles were charged with 1 g’ P Mn nodule, 400 ml of the mineral salt medium, 10 g.f’ glucose, and 10 ml of the enrichment culture broth of Mn-reducing micro- organisms (4.5 mg-protein). The pH ofthe medium was adjusted to 6.3-6.5 with 25% NH,OH. The bottles were sealed tightly with butyl rubber stoppers and incubated at 37°C. Gases formed during incubation were collected with a 50-ml syringe, which was in- stalled on each bottle.

The effect of the inoculation of the enrichment culture broth of Mn-reducing microorganisms on the leaching efficiencies of Mn, Co, and Ni from the nodule was evaluated by comparing the leach- ing capacities with and without inoculation of the enriched culture broth.

The leaching characteristics of the metals from the nodule with time were evaluated. Six of the above bottles were cultured under the same condition, and the supematant was sampled from the bot- tle at 8 h intervals. The pH, oxidation-reduction potential (ORP), and the concentrations of glucose and organic acids such as acetic acid, propionic acid, butyric acid and valeric acid were measured.

The effects of temperature, pH, particle size, and mineral salts on the leaching efftciencies of the metals were investigated. The ranges of temperature and pH were from 20 to 45°C and from 5.0 to 7.0, respectively. The particle sizes were classified such as follows: cO.15, 0.15-0.3, 0.3-0.6, 0.6-1.18, 1.18-2.0, 2.0-2.8, 2.8-4.0mm. The effect of the addition of mineral salts on the leaching efficiency was compared between using the mineral salt medium and using tap water. The effect of carbon source on the leaching efficiency was investigated. The carbon sources used were 10 g. r’ glucose, sucrose, and maltose. The bottles used in this experiments were incubated at 37°C. All experiments were duplicated, and the leaching efftciency was calculated as the mean value of the duplicates.

Analytical methods The enriched culture broth sampled from each bottle was separated into the supematant and precipitate by centrifugation at 3000 rpm for 5 min. The concentrations of Mn, Co and Ni in the supematant were analyzed using an atomic-ab- sorption spectrometer (AAlOO, Perkin Elmer, USA) after filtration with a 0.45 pm filter paper. The precipitates were washed twice with distilled water and dried at 37°C. 1% H,SO, was added into the dried precipitates to dissolve MnCO,, which was formed from the reduced Mn (Mn*‘) with CO, (20). Then, the Mn concentration in the dissolved solution was measured using an atomic-absorption spectrometer as described above. The glucose concentration was measured using a glucose analyzer (YSI 2357, USA). The concen-

Composition Mn

TABLE 1. Composition of manganese nodule

co Ni CU Fe A’@, CaO W MgO N%O Concentration

(mg g-nodule&) 263.2 1.5 12.2 4.7 67.1 54.6 21.3 13.8 30.6 30.4

356 LEE ET AL. .I. BIOSCI. BIOENG.,

trations of acetic acid, propionic acid, butyric acid, and valeric acid were measured using a gas chromatograph (HP 5890 II plus, USA) equipped with a flame ionization detector and a Supelco Wax col- umn (Supelco, USA). The temperatures of the oven, injector, and detector were 150,210, and 25O”C, respectively.

the concentrations of glucose and organic acids during Mn, Co, and Ni leaching from the nodule with Mn-reducing enrichment culture. Because organic acids such as acetate,

RESULTS AND DISCUSSION

Effect of inoculation of Mu-reducing enrichment cul- ture Mn is soluble at low reduction potentials and precipi- tates in oxidizing environments (26). Insoluble Mn4’ can be reduced to soluble Mn2+ by Mn-reducing bacteria, and then leached from the Mn nodule. Since expensive metals such as Co and Ni are contained in MnO, in Mn nodules, these metals can be easily leached from the nodules if MnO, is decomposed. When the Mn nodule was treated anaerobi- cally for 48 h without an inoculum, the leaching effkiencies of Mn, Co, and Ni were 18, 7, and lo%, respectively (Fig. 1). The leaching efficiencies of Mn*+, Co and Ni increased respectively to 80, 75, and 79% after 48 h by the inoculation of Mn-reducing enrichment culture. This result indicates that the dissimilatory Mn-reducing microorganisms that use metal ions as electron acceptors and organic compounds as electron donors can leach the metals from the manganese nodule. When glucose is supplemented, the overall reac- tions involved in the Mn reduction by Mn-reducing bacteria can be expressed as follows (20):

(b)

12Mn02+C,H,,0,+24Hf a 12Mn2’+6C0,+18H20

(1)

Mn2++C02+H,0 3 MnC0,+2H’ (2)

It was considered that Ni and Co leached from the nodule because the MnO, matrix as removed. Figure 2 shows the scanning electron micrograph of the Mn nodule before and after the bioleaching treatment. As shown in Fig. 2, the slit on the surface of the nodule after the bioleaching treatment enlarged and deepened due to the removal of the Mn matrix from the nodule.

FIG. 2. Scanning electron micrograph of the Mn nodule before (a) and after (b) bioleaching treatment.

Characterization of bioleaching of Mn, Co, and Ni Figures 3(a) and (b) show the time profiles of pH, ORP, and

FIG 1. Effect of the inoculation of enrichment culture broth of Mn- reducing microorganisms on the metal leaching effkiencies from the Mn nodule after 48 h of incubation. A, Without inoculation; B, with in- oculation.

- Propionate

& Valerate

-600

3.5 -c

,.6 -2

-5 b 8

-.4 g

2

-.3 ;rr c

-.2 j$

5 -.I ‘2

- 0.0 g 0 IO 20 30 40 50 60

Time (h)

FIG. 3. Time profiles of pH, ORP, and concentrations of organics during metal leaching from the Mn nodule.

VOL. 92,200l ANAEROBIC BIOLEACHING OF PRECIOUS METALS FROM Mn NODULES 357

butyrate, valerate, and propionate were accumulated during the fermentation reaction of glucose, the pH of the medium decreased from 6.5 to 4.8 after 40-h incubation. During the incubation, the initial ORP 300 mV decreased to -480 mV after 30 h, indicating a more favorable reduction environ- ment. Approximately 10 g. t-l glucose was exhausted after 48 h, and 0.73 g.t-’ acetic acid, 3.11 g’t’ butyric acid, 0.43 g. t’ propionic acid and 0.05 g’t’ valeric acid were accu- mulated in the medium (Fig. 3b). The metal extraction was clearly detected after 40 h of incubation when the concen- tration of glucose decreased, and the ORP dropped drasti- cally to -480 mV (Fig. 4). The concentrations of Mn, Co, and Ni in the nodule are 263.2, 1.5, and 12.2 mg.g-‘, respec- tively, as shown in Table 1. The concentrations of Mn, Co, and Ni that leached in the medium were respectively 208.0, 1.1, and 9.6 mg . g-l and the maximum leaching efftciencies were 80,75, and 79%.

The leaching efficiencies of Mn, Co, and Ni have been reported as follows: 90% leaching of Mn from low-grade ores required 90 d (27); 95-100% of Mn by heterotrophic mixed culture of microorganisms from manganiferous min- erals was obtained at 48 h (21); 50% leaching of Co and 60% leaching of Ni by Aspergillus sp. and Penicillium sp. from laterite required almost 50 d (28); 96% of Mn, 89% of Co and 87% of Ni by Thiobacillus ferrooxidians from cobalt-rich ferromanganese crusts required 7 d (29). In these studies, aerobic leaching processes have been performed. In this study, it required only 2 d to obtain the maximum metal leaching efficiencies from the nodule by anaerobic Mn-reducing microorganisms. The results of the present study and those of other studies could not be compared directly because the type, composition, and size of ores are different. However, the anaerobic bioleaching process using

0' I I I I 1-o 0 IO 20 30 40 50 60

Time (h) 6.0

PH

6.5

FIG 4. Time profiles of Mn (a), Co (b), and Ni (c) leaching from FIG. 6. the Mn nodule.

Effect of pH on the metal leaching efficiencies after 48 h of incubation.

anaerobic reducing microorganisms could effectively achieve metal leaching from the high-oxidation-state metals such as Mn oxide, because highly oxidized metal compounds can be solubilized by reduction processes (10).

Effects of temperature, pH, type of mineral salt, and particle size on the bioleaching The leaching efficiency depends largely on the activity of the microorganisms and on the chemical and mineral composition of nodules to be leached. The maximum yields of metal extraction can be achieved only when the leaching conditions correspond to the optimum growth conditions of the bacteria (6).

The effect of temperature ranging from 20 to 45°C on leaching efficiency after 48 h is shown in Fig. 5. The opti- mum leaching efficiencies ranged from 30 to 45°C. The leaching efficiencies were very low at temperatures below 20°C. Recently, at higher temperatures (50-8O”C), thermo- philic bacteria are reported to be used for metal leaching (30). However, the optimum incubation temperature rang- ing from 30 to 37°C is suitable for metal leaching from the nodule considering the increasing cost with increase in tem- perature.

Figure 6 shows the effect of the initial pH of the medium on the extraction of Mn, Co, and Ni by bioleaching after 48 h. The bioleaching reaction was active below weak-acid

Temperature (“C)

FIG. 5. Effect of temperature on the metal leaching efficiencies after 48 h of incubation.

1 I Mm Ezm co

_ -Ni

358 LEE ET AL. J. BIOSCI. BIOENG.,

100 1 uMn

A B

FIG. 7. Effect of the addition of mineral salt medium on metal leaching efficiencies after 48 h of incubation. A, Mineral salt medium; B, tap water.

pH 6.5 and decreased drastically at neutral pH 7.0. That is, the bioleaching reaction was more active at weak-acid pH than at neutral pH. These results correspond with the de- cline in pH with time (Fig. 3a) and the leaching pattern of metals (Fig. 3b). The Mn-reducing microorganisms used in this experiment had active bioleaching ability below weak- acid pH 6.5.

Figure 7 shows the effect of mineral salt addition on the leaching efficiencies after 48 h. Regardless of the addition of mineral salts, the leaching efficiency did not differ much. The Mn-reducing microorganisms in the enrichment culture broth used in this study could grow without the addition of mineral salts; the mineral leached from the manganese nod- ule alone was able to support their growth. This indicates that this bioleaching process proceeds with only the addi- tion of a carbon source and without extra supplementation of mineral salts. The comparison of the experiment using tap water with that using the mineral salt medium in this study showed that bioleaching is an economical process.

As described above, because the bioleaching of Mn from the nodule involves a dissimilatory reduction reaction, the carbon source was supplied as an electron donor (16, 17,20, 26). Thus, the removal efficiencies were compared among the conditions where three kinds of sugars, glucose, sucrose, and maltose, were supplied. All sugars yielded the leaching capacities of more than 70% (data not shown). Table 2 shows the leaching efficiencies of Mn, Co, and Ni at differ- ent nodule/glucose ratios. There is no significant increase in leaching efficiency when the ratio of nodules to sugar was more than 0.1.

The effect of mineral particle size on metal leaching from

TABLE 2. Leaching efficiencies of Mn, Co, and Ni at various nodule/glucose ratios (w/w) after 48 h of incubation

Nodule/glucose _ Leached metal concentration (mg’g-nodule-‘) ratio (w/w) Mn co Ni

0.05 197.13 (75%) 1.32 (88%) 9.69 (79%) 0.1 210.56 (80%) 1.18 (79%) 9.10 (74%) 0.2 161.63 (61%)” 0.77 (51%) 6.53 (53%) 0.3 163.32 (62%)” 0.83 (55%) 5.72 (47%) 0.4 151.90 (58%y 0.62 (41%) 4.42 (36%)

a Leaching efficiency.

TABLE 3. Leaching efficiencies of Mn, Co, and Ni at various particle sizes of the nodules after 48 h of incubation

Patticle size Metal leaching efficiency (%)

(mm) Mn co Ni < 0.15 84 91 80 0.15-0.30 85 82 84 0.30-0.60 85 82 80 0.60-1.18 74 68 70 1.18-2.00 65 53 56 2.00-2.80 54 42 42 2.80-4.00 42 27 24

the manganese nodule was investigated. The leaching effl- ciencies increased with the decrease in the particle size (Table 3). When the particle size was 0.60-1.18 mm, the leaching efficiencies were more than 74, 68, and 70% for Mn, Co, and Ni, respectively. In contrast, when the particle size as 1.18-2.00 mm, the leaching efficiencies decreased to 65% for Mn, 53% for Co, and 56% for Ni. By decreasing the particle size, the surface area per unit mass of mineral is increased and, as such, improved mass transfer and en- hanced bioleaching rates are achieved (3 1).

In conclusion, the proposed anaerobic bioleaching is a promising method to recover precious metals, such as Mn, Co, and Ni, from the Mn nodule. The anaerobic bioleach- ing method has some advantages such as rapid reaction rate, low capital investment, excellent leaching efficiency of metals, and being an environmentally friendly process. The results of this study also provided fundamental information on the optimum pH, temperature, mineral salts, and particle size for large-scale operations. On the other hand, the mi- crobial information on the enrichment culture used in this study is very important. Some bacteria from the enrichment culture were isolated. When anaerobic metal leaching was investigated using these isolates, the leaching efficiency was not good. This result indicates that the characterization of the total microbial population is necessary. To obtain the information on the total microbial population, the microbial population of the enrichment culture is characterized by 16s rDNA analysis using DGGE (denaturing gradient gel elec- trophoresis). The microbial information obtained from this research will be published.

ACKNOWLEDGMENTS

The funding for this research was provided by the National Re- search Laboratory Program of the Korean Ministry of Science and Technology, and the Brain Korea 21 Project of the Korean Minis- try of Education.

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