Cyanide Biodegradation

Cyanide Biodegradation

Management of mine drainage and mineral processing waste systems is a serious problem that could have major impacts on the U. S. mineral industry. Federal legislation (established by the Superfund Amendments and Reauthorization Act of 1986) provides that preferred remedial actions are those treatments that permanently and significantly reduce the volume, toxicity, or mobility of the hazardous substance. However, in many cases, treatment technology to meet stringent discharge requirements for toxic anions and heavy metals does not exist. The Bureau of Mines has a long history of research on minerals waste management and related environmental problems and policies. The Bureau now has a unique opportunity to develop or adapt mineral processing technologies to remediate site-specific contaminated mining and milling wastes and then apply these technologies to sites with similar problems.

A major remediation problem involves cyanide wastes generated by the heap leaching of low-grade precious metal ores. The use of heap leaching for treating low grade ores is expanding rapidly, particularly in the gold industry. Effective, low-cost remediation techniques, both for contaminated solutions and leached residues (spent heaps), will be needed during the closure phase of these operations. Initial Bureau research concentrated on using effective, relatively simple, chemical oxidation methods, such as the addition of sodium hypochlorite (NaOCl) or hydrogen peroxide (H2O2) , to remediate cyanide in contaminated solutions. Because of the high reagent costs associated with these chemical techniques, the Bureau subsequently turned to biological cyanide oxidation as a possible alternative treatment.

The fact that certain microorganisms, such as fungi and bacteria, can metabolize cyanide is well-known. Certain microbes use cyanide, thiocyanate, and cyanamide in their cellular metabolism tb synthesize amino acids. These compounds can also be used as nitrogen and carbon sources. For example, Bacillus megaterium converts potassium cyanide (KCN) to asparagine, asparatic acid, and carbon dioxide; whereas, Pseudomonas paucimobilis mudlock oxidizes free and complexed cyanide to carbonate and ammonia.

Several biological processes have been studied for cyanide decomposition from waste waters including trickling filters, activated sludge, fluidized bed reactors, and rotating biological contactors (RBCs). In general, these processes have been operated at a neutral or slightly basic pH and cyanide concentrations less than 200 ppm. For example, a full-scale biological treatment facility (RBC process) is currently being used to treat cyanide wastewaters at Homestake Mining Co., Lead, SD; the wastewater, a mixture of mine water and tailings impoundment water, generally contains <6.5 ppm total cyanide at a pH of 7 to 8.5. The bacteria used in this operation were isolated from process waters and gradually acclimated to increased cyanide and thiocyanate concentrations to produce a bacterial strain (Pseudomonas paucimobilis mudlock) with increased capacity to degrade cyanide; these bacteria oxidize free and complexed cyanide to carbonate and ammonia.

The Bureau conducted research to determine if biological oxidation could be used to treat a tailings pond water containing 280 ppm CN at pH 10.5. Cyanide-degrading bacteria, identified as Pseudomonas pseudoalcaligenes. were isolated from the tailings pond water; these bacteria, which were subsequently studied in batch and column tests, oxidized >90 pct of the cyanide in the pond water. The results of this research generated considerable interest among heap leach operations looking for possible low-cost closure technology. As a result, extensive testing was conducted using solution and ore samples from several gold heap leach operations.

Description of Cyanide-Contaminated Solutions and Residues

Cyanide-contaminated solutions from several precious metals processing operations were treated. These solutions included tailings pond water from an operating silver mine (S-1), leak collection system water from a dump leach operation (S- 2), barren solution from two heap leach operations (S-3 and S-5), and pregnant solution from two heap leach operations (S-4 and S-6). Concentrations of the primary contaminants in these solutions are presented in table 1.


Ore from a dump leach operation was used in exploratory tests to determine if biological oxidation could be used to destroy the residual cyanide in spent heaps. The ore sample was crushed and sized to -4 +10 mesh, leached with barren solution from the dump leach operation, and then treated with cyanide-oxidizing bacteria.

Sample Analysis

Determination of total and weak acid dissociable cyanide (WAD) involved reflux distillation of acidified samples. The hydrogen cyanide (HCN) gas formed in the distillation was purged into an absorption solution containing sodium hydroxide (NaOH). Titrimetric or selective ion electrode methods were used for determining the free alkali cyanide. Cations such as copper, iron, and zinc were analyzed by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Atomic absorption spectrophotometry (AA) was used to analyze for arsenic, selenium, and silver. Low levels of arsenic were determined using a graphite furnace AA technique. Total selenium and selenite were determined using a hydride generation AA procedure. Solutions were analyzed for sulfate and nitrate using ion chromatography and for phosphate using a colorimetric procedure.

Experimental Procedures and Results

Initial biological research was conducted using tailings pond water (S-1) from an active silver mine. Bacteria that would degrade cyanide were isolated from the tailings pond water; using Roche identification tubes, the bacteria were identified as a Pseudomonas species. Subsequently, a fatty-acid based technique was used to identify the bacterial strain as Pseudomonas pseudoalcaligenes. These bacteria are not the same strain of bacteria as that being used in the Home stake process (Pseudomonas paucimobilis mudlock): however, they belong to the same family. Pseudomonas pseudoalcaligenes apparently oxidizes cyanide by the same mechanism as Pseudomonas paucimobilis mudlock as shown in the following equation (9):


Subsequently, several cyanide process solutions were treated using Pseudomonas pseudoalcaligenes to determine if these bacteria would oxidize the cyanide in solutions other than the original tailings pond water. Tests were conducted in single-pass, trickling column reactors packed with quartz chips; test results indicated that the bacteria oxidized cyanide in all the solutions tested. The research attracted the attention of gold heap leach operations looking for possible low-cost closure technology. As a result, extensive research was conducted to determine if biological cyanide oxidation was a viable means of remediating cyanide during the decommissioning of heap leach operations.

Laboratory Research to Establish Process Feasibility

Initial tests were conducted in a single-pass, trickling reactor consisting of three glass columns (2.54-cm ID by 61-cm high) packed with 0.635-cm quartz chips leaving a 10-cm headspace; the columns were connected in series. The columns were seeded with a mixture of 50 pct PGY broth (full-strength PGY is 5 g/L peptone, 2.5 g/L glycerol, and 0.5 g/L yeast) and tailings pond water; this mixture was inoculated with cyanide-degrading bacteria (Pseudomonas sp. isolated from tailings pond water). To build up biomass, the initial feed to the reactor consisted of tailings pond water with 10 pct PGY and a small inoculum of Pseudomonas sp. The retention time in the system was generally 4.5 h.

Treatment of Tailings Pond Water

To study the effectiveness of the system, a long-term test was conducted in which tailings pond water (S-1) was fed downflow through the columns at 0.12 mL/min (4.5 h retention time) for 253 days. Feed and effluent samples were analyzed periodically for cyanide as well as copper, iron, selenium, and zinc. Cyanide concentrations for feed and effluent solutions at various time intervals are presented in figure 1. The feed concentration varied from 82 to 281 ppm CN because of periodic dilution with varying amounts of PGY solution. Effluent concentrations (from column 3) ranged from 9 to 41 ppm CN. The average cyanide removal through the system for the 253-day test was about 87 pct.

heap-rinsing cyanide removal

The feed and effluent solutions were also analyzed for ammonia since ammonia is a product of cyanide degradation (equation 1); the ammonia concentration in the water increased from a feed concentration of 56 ppm to an effluent concentration of 136 ppm which indicates that the bacteria are oxidizing the cyanide. In addition, the nitrite concentration decreased from 29.5 to 21.3 ppm; the nitrate concentration increased from 135 to 172 ppm indicating that the bacteria in the system may be oxidizing the ammonia to nitrite and then to nitrate as seen in the Homestake process.

The effect of this cyanide degradation system on other water contaminants is shown in table 2. The solution assays indicate that most of the iron and zinc are removed in this system. The copper and selenium, which are not removed, may be present as metal-selenium bonded complexes, known as selenocyanates; cyanide solutions are known to dissolve selenium from selenides with the subsequent formation of selenocyanates.

heap-rinsing biological treatment

Treatment of Heap Leach Process Solutions

The next phase of the research was initiated to show that the cyanide-oxidizing bacteria isolated from the tailings pond water (S-1) could be used to treat cyanide solutions from other sources. These process solutions included leak collection system water from a dump leach operation (S-2), barren solution from a heap leach operation (S-3), and pregnant solution from a heap leach operation (S-4). In addition to cyanide, these solutions contained other contaminants such as arsenic, copper, iron, selenium, and zinc (table 2). As with the tailings pond water, these solutions were pumped downflow through the reactor system at a flowrate of 0.12 mL/min (retention time of 4.5 h). In general, a 10-pct PGY addition was used.

The S-2 solution was effectively treated with over 90 pct cyanide removal. The solution treated least effectively was S-3, the solution with the high selenium concentration (30 ppm Se). The residual cyanide may be present as a metal selenocyanate complex since the selenium and copper effluent concentrations remained high.

Because of the high cyanide concentration (520 ppm) in S-4, this solution was diluted to 150 ppm initially to prevent shocking the system; the cyanide concentration was gradually increased in 25 to 50 ppm increments. This solution was effectively treated up to a cyanide concentration of about 280 ppm; above this concentration, the bacteria were not effective. Table 2 shows the feed and effluent concentrations for the primary contaminants in these process solutions. Since the objective of treating these solutions was to show that Pseudomonas pseudoalcaligenes could be used to destroy cyanide in solutions other than the original tailings pond water, only total cyanide concentrations were determined; the solutions were not analyzed for weak acid dissociable (WAD) cyanide.

Overall, column test data indicate that the cyanide-oxidizing bacteria isolated from a tailings pond water destroyed a high percentage of the cyanide in several heap leach process solutions; certain cyanide complexes, which appear to be copper and selenium complexes, were not destroyed.

Laboratory Research to Bacterially Decommission Heap Leach Operations

The trickling column reactor studies demonstrated the generic application of biological cyanide oxidation to gold heap leach process solutions. Research then turned to the possibility of using this technology during closure of heap leach operations. Currently, when a heap leach operation is decommissioned, the common practice is to rinse the spent heaps with water until the desired effluent cyanide concentration is achieved; in most states, the discharge standard is 0.2 ppm WAD cyanide. If fresh water is used for rinsing, an evaporation scheme must be developed to maintain the water balance. Regardless of whether fresh water rinsing or recycled rinsing is used, chemical oxidation is eventually needed to destroy cyanide in the rinse solutions.

As an alternative to the conventional rinsing procedure, the Bureau conducted research to determine if biological oxidation could be used to destroy cyanide in the rinse solution on a continuous basis. A conceptual approach would involve establishing a bioreactor in the heap leach circuit. For example, many operations use carbon adsorption columns for gold recovery; these columns could be used as bioreactors with the activated carbon serving as a growth surface for the bacteria. The cyanide in the process solution would be destroyed by biological oxidation; the solution would then be recycled through the system until the discharge cyanide concentration is achieved. Biological cyanide destruction in a spent heap or in a holding pond, such as the pregnant or barren pond at a heap leach site, was also investigated. Laboratory research was conducted in (1) trickling column reactors and upflow carbon columns to simulate carbon adsorption tanks, (2) flasks to simulate holding ponds, and (3) columns packed with cyanide -leached ore to simulate spent heaps.

Cyanide Destruction in Process Solutions

A heap leach barren solution (S-5) and a heap leach pregnant solution (S-6), which were low in selenium, were treated in trickling column reactors (the same systems in which solutions S-1, S-2, S-3, and S-4 were treated) with the objective of showing that bio-oxidation can be a viable alternative to chemical treatment for remediating cyanide in process solutions during heap leach closure. The target effluent cyanide concentration for these tests was 0.2 ppm WAD cyanide which is the discharge standard for WAD cyanide set by most state regulatory agencies. The feed and effluent concentrations for the primary contaminants in these solutions are shown in table 3; for both of these solutions, an effluent WAD cyanide concentration of 0.1 ppm was achieved with a 4.5 h retention time. The columns were inoculated with Pseudomonas pseudoalcaligenes and 5- to 10-pct PGY was used as a nutrient.


The heap leach pregnant solution (S-6) was subsequently treated in upflow carbon columns (2.54-cm ID glass columns) to determine if activated carbon, such as that used for gold recovery in many heap leach operations, can be used as a growth surface for Pseudomonas pseudoalcalipenes. At a 4.5 h retention time, the effectiveness of the carbon system compared favorably to the trickling column systems with WAD cyanide decreasing from 55 ppm to 0.1 ppm in both cases. Subsequently, tests were conducted at retention times as low as 13 min to simulate the retention time in the carbon adsorption columns of an actual heap leach operation; effluent WAD cyanide concentrations of 3 to 5 ppm were achieved. In these preliminary test series, the nutrient was 5- to 10-pct PGY.

Although PGY is an effective nutrient for Pseudomonas pseudoalcalipenes the cost would be prohibitive for large-scale work such as a field test involving the treatment of several hundred gallons of solution per minute. Low-cost nutrient sources, such as phosphate and phosphate plus dextrose, showed promise as alternative nutrient sources. For example, when treating the S-6 water in a trickling column system (retention time of 4.5 h), an effluent WAD cyanide concentration of <1 ppm was achieved using 26 ppm phosphate (as phosphoric acid) plus 500 to 1000 ppm dextrose.

Overall, the column test data show that biological treatment can be used to decrease the cyanide concentrations in certain gold heap leach solutions to acceptable discharge levels. In addition, the use of carbon adsorption columns as bioreactors for cyanide remediation shows promise as a heap leach decommissioning technique.

Cyanide Oxidation in Ponds

A series of flask tests was conducted to simulate biological oxidation of cyanide in a pond. The water used in these tests was the tailings pond water from which the cyanide-oxidizing bacteria were isolated. The objective of the tests was to determine the effect of variables such as nutrient addition, bacteria addition, and aeration on cyanide oxidation. The contents of the flasks were as follows: (1) tailings pond water (TPW), (2) tailings pond water plus 10 pct PGY, (3) tailings pond water plus bacteria (Cl-I), and (4) tailings pond water plus bacteria and 10 pct PGY. The water was not sterilized; thus, bacteria were present in all the samples. An inoculum of Pseudomonas pseudoalcaligenes was added to two of the flasks. The flasks were placed on the bench top and sampled periodically for cyanide over 81 days. In addition, four duplicate flasks were placed on a shaker to allow some aeration of the water.

Figure 2 shows cyanide concentrations for the quiescent tests. The most effective cyanide oxidation occurred when bacteria and nutrient were added to the water; the cyanide decreased from 240 ppm down to 57 ppm. Adding nutrient to feed the bacteria already present in the water decreased the cyanide concentration from 227 to 83 ppm. Shaker test data are shown in figure 3. Improvement in cyanide oxidation was observed only in the bacteria and nutrient added flask; the final cyanide concentration was 23 ppm as compared to 57 ppm in the quiescent test. No improvement was seen in the other flasks. Although some cyanide loss was observed in the control flasks (those flasks containing only tailings pond water), the primary mechanism involved in the destruction of cyanide in these tests appears to be biological oxidation. These preliminary data

heap-rinsing cyanide flask tests

heap-rinsing shaker flask tests

indicate that cyanide destruction in a pond may be possible provided nutrient is added to the pond; aeration of the pond would probably improve the effectiveness of the bacteria.

Treatment of Cyanide-Leached Ore

Exploratory tests were conducted to investigate the feasibility of using cyanide-oxidizing bacteria to destroy the residual cyanide in spent ore during heap leach closure. Initially, a column test was conducted with leached ore from an active heap leach operation to determine if cyanide-oxidizing bacteria would survive with ore as the growth surface. A 7.62-cm ID column was packed with a 50.8-cm bed of leached ore weighing 3650 g. A solution containing about 1 ppm CN (the solution used was the runoff water from the same heap leach operation) was inoculated with Pseudomonas pseudoalcaligenes plus 5-pct yeast as nutrient and pumped downflow through the column. Cell counts indicated that the Pseudomonas pseudoalcaligenes bacteria attached to the leached ore. No conclusions could be drawn as to the effect of the bacteria on the residual cyanide in the ore. However, when solution containing about 200 ppm CN (barren solution from the heap leach operation) was pumped through the column, cyanide oxidation was observed indicating that the Pseudomonas pseudoalcaligenes bacteria survived with ore as the substrate.

Tests designed primarily to establish operating procedures were then conducted in 5.08-cm ID columns containing 50.8-cm beds of heap leach ore (1500 g of material), crushed and sized to -4 + 10 mesh. The ore was leached by pumping barren solution, containing 200 ppm CN, downflow through the columns at 0.12 mL/min for several days. Following the cyanide leach, the columns were allowed to drain and biological treatment was initiated to destroy the residual cyanide in the ore.

Initially, the effects of inoculation technique and aeration were investigated. The inoculation techniques used were one-pass and solution recycle. One-pass inoculation involved pumping one pore-volume of bacteria-inoculated solution through a column; whereas, with solution recycle, one pore-volume of inoculated solution was pumped through a column several times. Aeration of both the ore and the feed solution was investigated. Test results were disappointing in that the recycled, inoculated solutions contained >2 ppm CN after one month. These concentrations were much higher than anticipated because the bacteria did not survive the one month test period; viable bacteria were found only in the reservoirs of the one-pass columns. Because the bacteria did not survive, the effect of aeration, either of the column or the solution, was not determined. The problems encountered in this test series may have resulted from improper or insufficient nutrient addition to the solutions.

As a result, a test series was conducted to investigate the effect of various PGY and yeast concentrations on the bacteria and the subsequent effect on cyanide destruction; both one-pass and solution recycle inoculations were used. In these tests, inoculation was followed by a one week rest period; the columns were then rinsed with 1.5 pore volumes of fresh water. Test results indicate the most effective procedure involved either one-pass or solution recycle inoculation using PGY as the nutrient. For example, the final cyanide concentration in the rinse water of columns inoculated with a 50 pct PGY solution was <0.2 ppm total cyanide; whereas, the final cyanide concentrations in the rinse from water only tests (columns that were rinsed with a pore volume of water, followed by a one week rest period, followed by. a 1.5 pore volume rinse) were about 1 ppm. These data indicate that the bacteria are destroying a portion of the residual cyanide in the leached ore.


Biological oxidation using bacteria (Pseudomonas pseudoalcaligenes) indigenous to a tailings pond water was investigated; this treatment showed promise in trickling reactor studies with >85 pct cyanide removal over a 253 – day test period. Subsequent research showed that Pseudomonas pseudoalcallgenes oxidized cyanide in other precious metals process solutions as well; effluent WAD cyanide levels as low as 0.1 ppm were achieved. The generic application of biological cyanide oxidation for solution treatment led to research involving heap leach closure using this technology. A conceptual flowsheet was proposed in which the metals processing portion of the plant, possibly the carbon adsorption columns, or a collection pond is used as a bioreactor. The cyanide in the process solution is destroyed in the bioreactor and the treated water is used to rinse residual cyanide from the spent heaps. The water is recycled until the effluent WAD cyanide concentration meets discharge standards. In addition, exploratory research showed that Pseudomonas pseudoalcalipenes survived in a column with heap leach ore as the growth surface. Test data indicated that the bacteria destroyed a portion of the residual cyanide in the spent ore.