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SME Annual MeetingFeb. 19 - 22, 2012, Seattle, WA

1 Copyright © 2012 by SME

Preprint 12-034

EVALUATION OF CYANIDE PRODUCING MICROORGANISMS TO RECOVER GOLD FROM LOW-GRADE ORE

D. Shin, Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of KoreaJ. Jeong, Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of Korea

B.D. Pandey, Nat. Metallurgical Lab. - Council of Scientific & Industrial Research (CSIR), Jamshedpur, IndiaJ. Lee,

,Korea Inst. of Geoscience and Mineral Resources, Daejeon, Republic of Korea

INTRODUCTION

The gold leaching using cyanide from ore or waste has beenalmost universally in use [1]. Recently, biological gold bioleaching usingcyanide producing bacteria, e.g. Chromobacterium violaceum ,Pseudomonas fluorescens , or P. aeruginosa , has been investigated asenvironmental friendly and cost effective method. Among them, C.violaceum has been reported as effective cyanide producing bacteriumon gold leaching [2, 3]. C. violaceum produces cyanide using glycineas a direct precursor and subsequently detoxify the cyanide by β-cyanoalanine synthase [4]. However, until today only 0.026 – 16.9 mg/Lof cyanide production in YP medium has been reported in theliteratures by C. violaceum [4-6]. Comparing with adding cyanidechemical, biogenic cyanide concentration is lower than the cyanideconcentration added in traditional cyanidation. Since cyanide alsoforms complexes with a number of other elements such as iron, nickel,zinc, silver, copper, and platinum, it is very important for gold recoveryto investigate the enhancement of biological cyanide production andset optimal experimental condition.

Most of early studies on gold bioleaching by C. violaceum havebeen performed to investigate gold leaching mechanism using goldpowder, and recently various resources such as ores, electronic scrap,and printed circuit boards of waste electronics [7]. Few studies havedealt with gold bioleaching from low-grade ore. In this research thefeasibility of gold bioleaching by C. violaceum from low-grade orescontaining gold was evaluated and fundamental study was performedfor further scale-up study. The aim of this study was to improve thepotential of gold bioleaching by C. violaceum from gold-containing ore;in particular, enhancement of biological cyanide production.

MATERIALS AND METHODS

Bacterial strains and culture conditionsChromobacterium violaceum DSM 30191

T and Acidithiobacillus

ferrooxidans DSM 14882T were purchased from DSMZ (Braunschweig,

Germany) culture collection. Cyanide-producing bacterium, C.violaceum was grown in yeast extract/peptone medium (YP medium;peptone 10 g, yeast extract 5 g, distilled water 1 L) at 30°C. Forcyanide production assay, 200 mL of YP medium were added in 250mL Erlenmeyer flasks and adjusted pH 6.7, 8, 9, 9.5, 10, and 11 with 1M NaOH. The glycine concentration in YP medium was changed in therange of 0.5, 1, 2, 5, 10, and 20 g/L. Five-milliliter of pregrown C.violaceum in YP medium was inoculated. The flasks were incubatedfive days on a shaking incubator (220 rpm) at 30°C. Ten milliliters ofsamples were taken every day to analyze bacterial density and cyanideconcentration. Bacterial density was determined spectrophotometricallyby monitoring optical density at 600 nm.

For pretreatment, A. ferrooxidans was maintained in the 9Kmedium [8] which was filter-sterilized and adjusted to pH 1.8 with 0.1 NH2SO4. The bacterial culture was cultivated on a shaking incubator with220 rpm, at 30°C.

Ore characterizationThe gold and silver content of ore samples was analyzed by fire

assay and copper was analyzed by inductively coupled plasmaemission spectrometry (ICP-ES). The mineralogy of the ores was

analyzed by X-Ray Diffraction. The analytical data were shown in Table1. Some of the samples for the experimental tests were prepared bygrinding and sieving to


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pH to 11 when glycine was added.4) The ores was incubated with A. ferrooxidans for 7 days

before incubating with C. violaceum. The rest experimentalprocedure was the same with 1).

Analytical methodsTotal cyanide concentration was analyzed by ion-selective

electrode (ISE) for cyanide (Orion ionplus combination cyanideelectrode; 9606BNWP, Thermo Scientific, USA) and titration againststandard AgNO3 solution. Gold, copper, silver content in leached liquorwas analyzed by atomic absorption analysis (AAnalyst 400, Perkin-Elmer, USA).

RESULTS AND DISCUSSIONS

Cyanide productionCyanide formation by C. violaceum was observed within five days

of incubation time with various pH (Figure 1 (a) and (b)) and glycineconcentrations (Figure 2 (a) and (b)). Characteristically, the peak valueof cyanide concentration was observed at two days of incubation andthe concentration decreased with time after that possibly due tocyanide-utilizing enzyme activity. The bacterial growth reached late-exponential phase at one day and stationary phase after two days ofincubation. C. violaceum starts to produce cyanide from lateexponential phase and the cyanide-utilizing enzyme is induced afterthe start of stationary phase [4]. The highest cyanide production (i.e.,20 mg/L) was observed at pH 9 with glycine 5 g/L even though thebacterial growth was higher at pH less than 9. It is because cyanideexists primarily as CN

- rather than as HCN at pH above values 9.31.

Therefore, pH 9 and 5 g/L of glycine was used throughout this study.

(a)

Incubation time (day)

0 1 2 3 4 5

B a c t e r i a l g r o w t h ( O D 6 0 0

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

pH 6.7

pH 8

pH 9

pH 9.5

(b)


0 1 2 3 4 5

C y a n i d e c o n c e n t r a t i o n ( m g / L )

0

10

20

30

40

pH 6.7

pH 8

pH 9

pH 9.5

Figure 1. (a) Cyanide production and (b) bacterial density of C.violaceum with various pH in YP medium containing glycine 5 g/L.

(a)


0 1 2 3 4 5

B a c t e r i a l g r o w

t h ( O D 6 0 0

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

glycine 0.5 g/L

glycine 1 g/L

glycine 2 g/L

glycine 5 g/L

glycine 10 g/L

glycine 20 g/L

(b)


0 1 2 3 4 5

C y a n i d e c o n c e n t r a t i o n ( m g / L )

0

10

20

30

40

glycine 0.5 g/L

glycine 1 g/L

glycine 2 g/L

glycine 5 g/Lglycine 10 g/L

glycine 20 g/L

Figure 2. (a) Cyanide production and (b) bacterial density of Cviolaceum with various glycine concentrations in YP medium with pH 9

Shake flask cultureTo optimize sampling time and pulp density, the bioleaching o

gold, copper, and silver was determined in YP medium with glycine 5g/L at pH 9 using H ore during five days and with 20, 50, 100 g/L opulp density. The results showed that copper was leached firstly andgold was started to be leached after 2 days (Figure 3) due to thedifferences between their standard potential (E

0

Au

3+

/Au : 1.52 V and

E 0

Cu

2+

/Cu : 0.34 V). The H ore was added at the same time with Cviolaceum inoculation.

During the experimental period, leached metals wereaccumulated continuously. From 20 to 100 g/L of pulp density was used(Figure 4) since the gold concentration was too low to analyze in lessthan 10 g/L of pulp density. The H ore was added after cultivating Cviolaceum overnight. With 20 g/L of pulp density, the maximum goldbioleaching yield was observed (i.e., 22.48±9.39%) comparing withhigher pulp density. Throughout this study, 20 g/L of pulp density andsampling at five days of incubation had been used. During theexperimental period, the pH was kept in the range of pH 8

The bioleaching yield of gold of the all ores was analyzed in batchcultures and then for enhancing bioleaching efficiency variousenhancement factors were applied as follows: 1) pre-incubation, 2grinding, 3) pH adjustment, 4) pretreatment. In the case of batchculture (i.e., the ores were added at the same time with C. violaceuminoculation), the results showed very low bioleaching efficiency thathe bioleaching efficiencies of gold were 0%, 42.19±65.36%, and8.68±4.35% for R, S, and H, respectively (Figure 5 (a)). In the goldleaching using cyanide, copper presents difficulties due to the lack oselectivity of cyanide for gold over copper. For copper and silver


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copper was leached from R, S, and H at 12.06±3.06%, 1.46±0.34%,and 18.70±1.85%, respectively, and silver was leached from R and Hat 21.72±2.92% and 4.87±0.55%, respectively. Since copper may formcomplex with cyanide than gold, the biogenic cyanide would beconsumed before reacting with gold.


0 1 2 3 4 5

M e t a l l e a c h e d ( % )

0

5

10

15

20

Au

Cu

Ag

Figure 3. Gold, copper, and silver bioleaching yield (% of total gold,copper, and silver) by C. violaceum using H ore with 50 g/L of pulpdensity.

Pulp density (g/L)

20 50 100

M

e t a l l e a c h e d ( % )

0

5

10

15

20

25

30

35

Au

Cu

Ag

Figure 4. Gold, copper, and silver bioleaching yield (% of total gold,copper, and silver) by C. violaceum using H ore with 20, 50, and 100g/L of pulp density.

The methodology of pre-incubation was that C. violaceum wascultivated in YP medium without glycine overnight and the ore samplesand glycine was added into the pre-incubation culture. As shown inFigure 5 (b), the bioleaching efficiencies were enhanced to

4.36±41.67%, 131.55±73.93%, and 18.51±8.51% for R, S, and H,respectively, as the effect of pre-incubation. The experimental error andstandard deviation was very high especially in S ore possibly due topoor mixing and distribution of the ore samples. Since the bioleachingefficiency was much enhanced by introducing the methodology of pre-incubation, pre-incubation had been used throughout this research.

By grinding, the bioleaching efficiency was enhanced as shown inthe Figure 6. Using ground sample (< 180 µm), the bioleachingefficiencies were enhanced to 60.93±27.18% and 28.31±7.97% for Rand H, respectively. In the case of S, almost all of gold was alreadyleached out in pre-incubation so the leaching yield did not so increase.Most of cyanidable gold were leached out in ground and pre-incubationsample.

The most effective pH for gold cyanidation has been reported apH 11 [9]. When adding glycine, pH of the C. violaceum culture wasadjusted to 11 and the H was added and incubated 5 days (Figure 7).However, the gold bioleaching yield didn’t change very much.

(a)

R S H


0

50

100

150

200

250

Au

Cu

Ag

(b)

Rosia Montana Saramacca Hollister


0

50

100

150

200

250

Au

Cu

Ag

Figure 5. Gold, copper, and silver bioleaching yield (% of total goldcopper, and silver) by C. violaceum with 20 g/L of pulp density at pH 9(a), Bioleaching assay in batch culture; (b) Bioleaching assay in pre-incubation culture.

R S H


0

50

100

150

200 Au

Cu

Ag

Figure 6. Gold, copper, and silver bioleaching yield (% of total goldcopper, and silver) by C. violaceum with 20 g/L of pulp density at pH 9using ground ore (


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the gold leaching agent (i.e., cyanide) cannot reach them [10, 11]. Thebiooxidative pretreatment may be a useful tool to oxidize sulfide matrixof the ore and liberate gold particle from the ore. Therefore, except forS ore that most iron existed as ferric form, R and H ores wereincubated with A. ferrooxidans for 7 days and then filtrated, dried andincubated again with C. violaceum . However, the bioleaching efficiencywas not enhanced after biooxidation (data not shown), it may bebecause of short biooxidation time (i.e., 7 days). Overall, the

methodology of pre-incubation and grinding was the most effective toenhance the bioleaching yield of gold. Further study is underway thatcyanide capturing system has been operated to concentrate cyanideconcentration and remove biofilm formation on the surface of ore. Also,total cyanide produced by C. violaceum , the partitioned ratio of cyanidebetween air and aqueous phase, and available cyanide for bioleachingwill be analyzed.

pH (unadjusted) pH (adjust to 11)


0

20

40

60

80

100

Au

Cu

Ag

Figure 7. Gold, copper, and silver bioleaching yield (% of total gold,copper, and silver) by C. violaceum using H ore with 20 g/L of pulpdensity at pH 9 and 11 using ground ore (

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