Biological Cyanide Degradation

Cyanide heap leaching is the predominant technology used in processing low-grade gold ores. During closure of a heap leach operation, residual cyanide must be removed from the process and waste solutions, as well as the heap. Biological cyanide oxidation is a proven, economical technology for destroying cyanide in process and waste waters and spent heaps. The use of biological cyanide degradation eliminates the need for toxic or corrosive chemical oxidizers and has been implemented at full scale at treatment costs usually <$0.50/1,000 gal. The Applied Biosciences biological cyanide destruction (BCND) process has been demonstrated to effectively treat cyanide concentrations up to 350 mg/L. Treated process solutions and wastewaters also contained other contaminants such as arsenic, copper, iron, silver, selenium, mercury, nitrate and zinc, much of which was removed in the cyanide degradation process. Under optimal conditions, microorganisms can rapidly oxidize free cyanide in some process solutions from over 250 mg/L down to 0.1 mg/L in 4 to 5 hr. Cyanide is degraded to ammonia and carbon dioxide, with 50% of the cyanide carbon liberated as CO2. In general, carbon tank based bioreactors degrade cyanide at significantly higher rates than process or wastewater pond configured treatments, which usually degrade cyanide more rapidly than in heap treatments. Investigation of cell-free enzyme preparations as an alternative to live microbial cyanide degradation shows promise. Advantages of enzymes over live microbes for cyanide degradation include: (1) the ability to tolerate and degrade higher cyanide concentrations (>1,000 mg/L), (2) nutrients to support live microbial cells are not required, (3) the effects of other toxic contaminants, such as metals, found in mining waters are eliminated and (4) the potential for greatly increased kinetics. Introduction Management of mineral processing and associated waste systems is a concern that has a major impact on the U.S. minerals industry. High on the list of concerns with mineral processing and associated waste systems, even though most precious metal processing industries are zero discharge facilities, are cyanide wastes. Although cyanide occurs naturally at low levels in plants and some microorganisms, public attention has been drawn to the use of cyanides in industrial processes because cyanide is a potent inhibitor of cellular metabolism (1,2). This has particularly been the case with mining and mineral processing industries and the use of heap leaching for treating lowgrade ores. Heap leaching is a particularly effective and low-cost process and is relatively environmentally benign as long as waste streams and leached residues (spent heaps) remain zero discharge. However, during the closure phase of these operations, effective, low-cost, environmentally sound remediation techniques, both for contaminated solutions and spent heaps are needed. A number of processes are available for treatment of cyanide containing wastes ranging from sulfur dioxide-air, hydrogen peroxide, biological processes, carbon adsorption, evaporation, alkaline chlorination, ferrous sulfide, zinc sulfate, ozonation, electrochemical and ultraviolet treatments (3,4). However, because of treatment functionality, capital costs, operating costs, treatment volume/time, reliability, environmental concerns, technical feasibility issues and industry and public acceptance none is considered a good alternative for all sites (5). It is a well-known fact that certain microorganisms, such as fungi and bacteria, can metabolize cyanide. Certain microbes use cyanides to synthesize amino acids, as carbon and nitrogen sources and others oxidize free and metal-complexed cyanides to carbonate and ammonia (6,7). Biological process alternatives are usually examined because of the following chemical process chacteristics. Chemical Processes • Can be costly • Often are only partially effective • Can replace cyanide with another undesirable pollutant • Are generally only suitable for treatment of aqueous-based waste forms Several biological processes have been studied and some successfully implemented to remediate cyanide containing spent process waters and wastewaters including trickling filters (8), activated sludge treatments (9), fluidized bed reactors (10) and rotating biological contactors (11). In general, these processes have been operated at a neutral or slightly basic pH’s and at cyanide concentrations less than 200 mg/L. The biological cyanide degradation (BCND) process, developed by Applied Biosciences, has a number of advantages over chemical treatments including: • The process degrades all cyanide – ionic cyanide, WAD cyanide, ferrocyanides, thiocyanates and other metal cyanide complexes • Different microbes are available that have optimum cyanide degradation at temperatures from ~4° C to >30° C • Can be configured to degrade cyanide at the contamination source, the spent heap, by actually turning the heap into a part of the bioreactor • The process has been demonstrated effective with cyanide concentrations >350 mg/L and at solution pH’s from 7.5 to >11.5 • The process can be configured to use existing mine equipment to hold capital investment costs to a minimum • Typical nutrient costs for the BCND process are <$0.50/1,000 gallons • The process can be configured to treat nitrates and remove metals, such as selenium, arsenic, mercury, etc. The bacteria used in the BCND process have been isolated from long-time cyanide contaminated sites and are matched with site bacteria to provide maximum cyanide degradation in environments with high cyanide and/or metal concentrations. It should be remembered that when dealing with diverse mining solutions, both chemical and biological processes require treatability tests to ensure economical operation that meets site discharge or closure requirements. This paper presents an overview of the biotreatability studies required for a successful full-scale implementation of any bioprocess, including a cyanide degradation process. It also includes BCND case studies, data comparing microbial and enzymatic cyanide degradation. A number of microorganisms are known to possess various enzymes capable of converting cyanide into compounds that may serve as carbon and nitrogen substrates. Such enzymes include formamide hydro-lyase, L-3-cyanoalanine synthase, thiosulfate sulfurtransferase, and oxygenases (2,12). Adams, et.al has also reported on enzymatic cyanide degradation by Alcalagenes and Pseudomonas sp. (13,14). Enzymatic cyanide degradation is being investigated with the dual purpose of isolating new microorganisms capable of cyanide degradation and the potential of providing low cost alternatives to cyanide degradation at extreme cyanide concentrations. Low cost treatments could possibly be provided through economical semi-purified enzyme preparations. Enzymatic cyanide destruction offers potential tolerance to much higher cyanide concentrations and greatly increased degradation kinetics. Materials and Methods Biotreatability Testing For the case studies presented, biotreatability testing was adjusted for specific site differences and pollutant/environmental characteristics to help ensure full-scale process success. As with most treatability studies, the tests conducted are largely dependent on site-specific factors. Testing covered site specific geochemical, microbiological and hydrological parameters and included large laboratory reactor testing that provided engineering information, microbiological performance data and costing criteria for operation of the full-scale treatment system. Full-Scale Case Studies Case Study 1. Following a biotreatability study to examine the indigenous microbial population and to establish the most cost-effective nutrient formulation, the effectiveness of the BCND process was studied in laboratory bioreactors under simulated site environmental conditions. After the biotreatability study determined that the BCND process was capable of degrading cyanide to discharge criteria under site conditions using acceptable retention times and costs, a full-scale process was implemented using the carbon tanks as bioreactors. Case Study 2. Biotreatability testing determined that the indigenous microbial population was not degrading cyanide. Bioaugmentation using BCND process microbes was used to degrade cyanide in waters and spent heaps. Laboratory tests compared the BCND process with fresh water rinsing, FeSO4 and H2O2 treatments in 6” x 6’ plastic pipe columns. At full scale, carbon tanks were used to generate an optimized microbial inoculum needed to degrade cyanide in the barren pond waters and spent heaps. Microbes and Culture Techniques Trypticase Soy Broth (TSB) media adjusted to pH 9.7 was used to grow cultures of various Pseudomonas sp. Stock microbial cultures at ~2 x 10 cells per milliliter were diluted 1:10 in TSB at pH 9.5 containing 200 mg/L cyanide and grown overnight on a shaker at room temperature (~25° C). For samples tested as mixtures, microbial cultures were grown individually and then combined for testing. Since growing cells readily deplete cyanide at higher cell densities, additional cyanide was added at various intervals during cell growth to bring the culture media back to 200 mg/L. Cell Concentration and Enzyme Extraction Prior to harvesting Pseudomonas sp. cells for enzyme extraction, spectrophotometric measurements of the cell densities verified that the microbial population had reached the desired cell density. One liter of TSB containing the desired Pseudomonas sp. was centrifuged and washed in 50 mM Hepes buffer solution. This process yielded between 6 and 10 ml of packed cells for extraction and testing. The cell pellet was resuspended in Hepes buffer solution at a 2:1 ratio and extracted using various methods, including using a bead-beater chamber filled 2/3 full with glass beads and encased in ice. Cells were homogenized for 30 second intervals six times, with 60 second intervals between each homogenization cycle. The cell debris was centrifuged and the supernatant removed and subjected to ammonium sulfate cuts. Twice washed extract protein content was determined using a Bovine Serum Albumin (BSA) standard curve with accepted R values greater than 0.99. Typical enzyme extract dilutions, used for determination of protein content assays, were ten-fold, twentyfold, and fifty-fold. Cyanide Degradation Assays Free cyanide for all tests and standards used to ensure equipment accuracy was obtained from dilutions of freshly prepared stock solutions of KCN and all tests were performed on a flow injection cyanide analyzer (OI Analytical). Enzyme controls were denatured by heating equal amounts of the enzyme samples in an 85° C water bath for 15 minutes. All tests were done between pH 9.5 and 11. Results presented are examples of tests done within a variance of pH of 0.2. Cells were harvested in late log phase and yielded approximately 2 x 10 cells/mL. Cells were processed using a bead-beater, ammonium sulfate, and various detergents. The bead-beater, which uses glass beads to homogenize microbial cells, does not fractionate every cell; approximately 10 to 10 cells remain viable. However, the bead-beater tests yielded results that are more consistent from experiment to experiment than most other extraction methods. All media were run through the cyanide analyzer individually to insure no interference occurred. Controls of standardized cyanide concentrations, distilled water, denatured whole cells and enzyme preparations were included with each test series and demonstrated no significant cyanide degradation. Additionally, three known cyanide concentrations were run with each test to assure that the cyanide analyzer was functioning properly and that results were linear. Results and Discussion Biotreatability Tests Whether used as a primary treatment technology, a pretreatment or as a polishing step, successful applications of biological treatments involve site characterization, bio-assessment, biotreatability testing, and bioremediation monitoring. Often these important steps are over looked or considerably shortened because of initial assessment costs. These steps are necessary to answer the following frequently asked questions: Can bioremediation work for my cyanide problem? How fast will it work? What volumes and flow rates can be processed? How much will it cost? Site characterization, assisted through site maps, historical documentation, and sampling must determine, as best possible, the horizontal and vertical extent of the contamination. In most cases for mining cyanide biotreatment applications, this can be achieved by answering three basic questions: (1) What is the form and concentration of the contaminants of interest and any cocontaminants? (2) What contamination carrier, soils or liquids, must be treated and what characterization is required to define the soil chemistry and site hydrology? (3) Are the indigenous microbial populations capable of cyanide oxidation and what are their concentrations? Site bio-assessment can include the following: determination of the indigenous microorganisms; bio-availability of the contaminants; interferences caused by co-contaminants, ions, or required treatment conditions; site environmental conditions including pH, oxidation/reduction potential, temperature, dissolved oxygen, available carbon, nitrogen, and phosphate concentrations; and nutrient or amendment stability within the proposed remediation system. More detailed biotreatability studies are needed to identify factors and parameters involved in determination in situ versus ex situ or bioreactor treatment. For in situ treatment, a determination must be made as to whether site adjustments or amendments of nutrients, oxygen and/or microbes can result in transformation of cyanide under the environmental conditions present. This approach requires a more thorough site characterization and bio-assessment, and biotreatability testing often is more difficult. For ex situ application, the objective is to determine the optimum conditions for cyanide and other target compound transformations. This testing can use various types, sizes, and configurations of reactor systems where microbes, nutrients and oxygen concentrations can be controlled, monitored, and optimized more readily. Laboratory and Full-Scale Treatments Case Study 1. Following a biotreatability study to examine the indigenous microbial population for potential use in the development of a site optimized microbial mix and to establish the most cost-effective nutrient formulation, the effectiveness of the BCND process was studied in laboratory bioreactors under simulated site environmental conditions. Cyanide levels were reduced from ~70 mg/L to 0.01 mg/L in 34 days. After a laboratory biotreatability study determined that the BCND process was capable of degrading cyanide to discharge criteria under site conditions using acceptable retention times and costs, a full-scale process was implemented, Figure 1. The full-scale implementation used modified carbon adsorption tanks as bioreactors to remove ~35 mg/L cyanide from process and wastewaters totaling ~2.3 million gallons. Cyanide levels had dropped ~50% through active evaporation efforts. Treatment times were approximately 3 months to reduce cyanide levels to below 0.02 mg/L; the site treatment goal. Water was cycled through the carbon tanks at flow rates of up to 300 gal/min to determine optimal retention times; as bacterial cyanide oxidation is a function of retention time, nutrient concentration and microbial density. Nutrient and microbial inoculum costs for treating these waters were ~$0.39/1,000 gallons. Figure 1. Case Study 1 laboratory and field cyanide biodegradation results in process and wastewaters. Case Study 1 0 10 20 30 40 50 60 70 80 Days [C N ] m g/ L Laboratory (BCND)