Journal of Soil Conraminarion. 3(3):27 1-283 (1 994) Assessment of the Potential for In Situ Bioremediation of Cyanide and Nitrate Contamination at a 1 Heap Leach Mine in Central New Mexico Carleton S. White and James T. Markwiese Department of Biology, University of New Mexico, Albuquerque, NM 87131 ABSTRACT: The potential for in siru bioremediation of cyanide (CN) and nitrate (NO,) contamination within the extracted ore (residue pile) and downgradient groundwater at a CN heap leach mine in New Mexico was assessed through the following steps: (1) identification of the relative abundance of CN-degrading microorganisms in the contaminated residue pile. (2) identification of amendments to enhance aerobic CN degradation and assimilatory NOj reduction and determination of the optimal carbon-to-nitrogen concentration for degradation. (3) assessment of optimum amendment's influence on biodegradation of CN and NO, by experiments with large-scale columns filled with residue pile material, and (4) evaluation of the potential for other adverse environmental effects, specifically acid rock drainage, due to application of the amendment. These investigations determined that application of a reduced carbon source (sucrose) significantly increased the rate of CN degradation and NO3 immobilization without increasing the probability of acid rock drainage., - I KEY WORDS: cyanide, nitrate. remediation. glucose amendment, available carbon, C/N ratio. ground water, gold mine. I. INTRODUCTION Beginning with placer mining in 1828, the Ortiz Mountains in central New Mexico (Figure 1) are one of the oldest gold-producing areas in the U.S. Active mining nearly ceased when extraction of gold was no longer profitable with the mining techniques of that time. However. a new "gold rush" started in the 1970s in the Ortiz Mountains and throughout the American West, partly due to improvements in the cyanide (CN) heap leach process to extract gold from low-grade ores (Grabowski et al., 1991). As described by Grabowski et al. (1991), a typical CN heap leach process utilizes the following steps. The gold-bearing ore is extracted, crushed to a nominal size, and piled on a constructed impervious pad. An alkaline cyanide-laden solution is sprayed on the pile. The CN solution is buffered at a ph of about 11 to keep 1058-8337/94/$.50 O 1994 by AEHS
FIGURE 1. Location of the Ortiz Mine in north-central New Mexico. CN from forming hydrogen cyanide (HCN), which can be lost through volatilization. CN in solution complexes with gold (and other metals) and cames the metals from the ore. The CN solution is captured by the impervious pad and recirculated through the pile to further extract CN-complexed metals. The gold is then recovered from the CN-complexed solution by adsorption processes on activated charcoal. After leaching, the crushed ore is either left on the pad or removed to another area for disposal..4t the Ortiz Mine, the CN-leached ore was rinsed and then transported to and dumped in an unlined arroyo (which is prohibited by current regulations), creating a residue pile that covers about 12 ha and ranges in depth from 10 to 30 m (Figure 2). Because of its toxicity to higher animals, CN is a strictly regulated compound in the U.S. In spite of current regulations, release of CN to the environment has occurred at several heap leach mining sites, including the Ortiz Mine. Analysis of the residue pile in 1987 by the mine operators found total CN to range from 1.5 to 23 mag, while nitrate-n (NO 3 -N) ranged from 1.5 to 62 mg/kg (Gold Fields Mining Corporation, unpublished data). Analyses of water samples from monitoring wells placed downgradient from the residue pile contained as much as 0.7 mg~l total CN and 102 mg/l (NO 3 -N). Apparently, residual CN and NO, drained from
FIGURE 2. Contour map of the pile constructed from residue ore (residue pile) at the Ortiz Mine. Contour intervals are 10 ft. the pile, resulting in a contamination plume that extends about 375 m downgradient from the residue pile. Remediation activities decreased the size of the contamination plume by pumping the groundwater, but the residue pile remains a potential source of CN and NO, to the local groundwater supply. In 1990, the mine operators initiated studies to determine remediation options for the residue pile. Because the residue pile was made from highly porous crushed ore, evaluation of the potential to bioremediate CN and NO, focused on aerobic microbial processes. In a review of the pathways and types of microorganisms involved in CN degradation. Wyatt and Palmer (1991) identified a number of different intermediates or end products of CN degradation, depending on the
organisms and environmental conditions. The generalized aerobic degradation of CN is expressed as: CN + 0? + microorganisms + nutrients + CO, + NH, + microbial biomass A wide range of aerobic microorganisms, including actinomycetes, fungi, and bacteria, are known to degrade CN. Microorganisms are used successfully to degrade CN-laden effluent in an aerobic biotreatment facility operated by the Homestake Mining Company at their gold mine in Lead. SD (Mudder and Whitlock, 1984). Cyanide contains one atom of carbon (C) and one atom of nitrogen (N), which are joined by a triple bond. Biodegradation of CN can produce other potentially toxic compounds. The end product for N in the CN molecule is ammonium (NH,), which is readily converted to NO, by aerobic organisms in many environments. The State of New Mexico drinking water standard for NO,-N is set at 10 mg/l (New Mexico Water Quality Control Commission. 1992). If extensive anoxic zones existed within or below the residue pile, either oxidation of ammonium could not occur or denitrification processes could convert NO, to N20 or imocuous nitrogen gas. The major pathway for removal of NO3 from aerobic systems is by reduction to ammonia and subsequent assimilation into the organic-n pool as glutarnate or glutamine (assimilatory nitrate reduction; Cole, 1987). This process is widely utilized in water treatment facilities. Both assimilatory nitrate reduction and degradation of CN are favored by an increase in available organic carbon sources (Furuki et al., 1972; Trelawny et al., 1956; Ludzack and Schaffer, 1960). In general, sugars aid in the degradation of CN. Furuki et al. (1972) reported that degradation of CN was greatest when CN-degrading microorganisms were supplied glucose and fructose but was lower when supplied with galactose. Raef et a!. (1977) reported that CN actually combines with glucose to form more readily biodegradable compounds. Sucrose, a complex sugar made from glucose and fructose, is known to be an effective immobilizer of available N, including NO,, in field experiments (White et al., 1988). In addition to CN and NO,, the residue pile contains high amounts of iron pyrite Wols' gold). When exposed to the atmosphere, oxidation of these sulfide minerals produces sulfuric acids, which can lead to "acid rock drainage", a severe environmental problem. Thus, remediation of the CN and NO, contamination must be carefully evaluated to ensure that concurrent stimulation of acid rock drainage does not occur. This article summarizes the research steps used to identify the potential for bioremediation of the residue pile at the Ortiz Mine site by application of sucrose, an inexpensive and readily available form of carbon. We also evaluate the potential of creating acid conditions with a remediation procedure that uses water leaching or sucrose amendments. Early stages of this research are detailed in Markwiese and White (1991, 1992).
II. METHODS A. Field Sampling Samples were obtained from the residue pile in June 199 1. A hollow-stem auger was used to sample at approximate 3-m intervals to a depth of about 30 m or until native soil was encountered at a total of eight locations on the pile. A total of 74 samples were obtained, of which 27 were used in the experiments. In the laboratory, the entire sample was weighed. One quarter of the sample was reweighed and oven dried to determine total sample dry weight. The remainder of each soil sample was sieved (2-mm screen). Approximately one tenth of the <2-mm fraction was weighed and dried to determine moisture content, and the rest was placed in cold storage. B. Enumeration of CN-Degrading Organisms Microbiological procedures generally follow those in Wollurn (1982). Only the organisms associated with the <2-mm fraction were used in enumeration experiments; however, the results are expressed on the whole-sample basis (number per gram of residue pile material). CN-degrading organisms were plated on a mineral salts agar composed of the following in 0.8 1 of deionized (DI) water: 0.4 g KH,PO,, 0.6 g Na,HPO,, 0.2 g MgSO,, 0.01 g CaCl,, 0.02 g MnSO,, 0.015 g FeSO,, and 15 g Bacto Agar (Difco). Another solution containing 23 g of KOH and 3.256 g of KCN per liter was prepared. Both the KCN solution and the media were sterilized by autoclaving at 121 "C for 30 min. Immediately before pouring the plates, 200 ml of the KCN solution was mixed with 800 ml of media. The KCN solution was analyzed later to verify the CN concentration. Using the <2-mm fraction, a 10-g subsarnple of residue pile material was added to 95 ml of sterile DI water. These suspensions were serially diluted to reach a working range of colony forming units (CFUs). Spreads (0.1 ml) of serially diluted residue pile suspension were plated in triplicate. Plates were incubated at 25 C and the colonies counted after 6 to 13 d. Colonies grown on this agar have the capability to derive their C and N from the CN. Appropriate control plates, both without CN and spreads of serially diluted sterilized water (without residue pile material), had either no or few CFUs. C. Batch Cultures to Determine Optimum CN-Degrading Amendment The experimental design consisted of two levels of carbon amendment (using sucrose) coupled with or without phosphate (P) amendment and controls. Each treatment contained three replicate batch cultures. The basic medium for the batch cultures was the mineral salts solution used for the plates (without agar), in which
a total of six treatment solutions were prepared. The treatments included (I) sucrose and phosphorous amendment to give a final carbon:nitrogen:phosphorous (C:N:P) ratio of 20: 1:O. 1, (2) sucrose and phosphorous amendment to give a final C:N:P ratio of 10: 1 :O. 1, (3) sucrose amendment to give a C:N ratio of 20: 1 without P, (4) sucrose amendment to give a final C:N ratio of 10:l without P, (5) no amendment, and (6) sterilized (per methods above) no amendment as an abiotic control. All solutions were adjusted to a ph of about 11 with 1 N NaOH to reduce volatile loss of HCN, which occurs at lower ph, because HCN volatilization could not be distinguished from the degradation of CN. Analyses of the <2-mm fraction of the residue pile identified the levels of C and N to determine the amount of sucrose needed to achieve the final C:N ratios. Each batch culture contained 95 ml of mineral salts solution (formula same as plates without agar) that also contained DI water (or treatment solution) and 10 g of freshly collected residue pile material (<2-mm fraction). The final CN and NO,-N concentrations in the batch cultures were within the range of concentrations reported within the residue pile. All cultures were incubated at 25 C on a shaker stand. At approximately 2-week intervals for a total of 8 weeks, 2 ml was removed from each flask for the determination of CN and NO,-N. D. Laboratory Simulation of Field Application: Column Experiment The experimental design of this portion of the study consisted of three replicated columns for three separate treatments: (1) sucrose amended, (2) sucrose amended to sterilized portions of the residue pile (as abiotic controls to demonstrate biological vs. chemical-physical processes), and (3) unamended controls. Another fresh sample was obtained from the residue pile and sieved to <1 cm prior to packing in to the column. A total of nine columns were packed with the residue pile material, each column was made from sections of clear Plexiglas tubing measuring 0.91 m by 10 cm i.d. and contained approximately 8 kg of residue pile material (shown in Figure 3). Material for the sterile columns was autoclaved for 45 min prior to packing into the columns. Column solutions were continuously recirculated via a peristaltic pump at a flow rate of 2 mllmin. The solution was pumped to the top of the column and allowed to flow by gravity through the column. The clarified solution was captured beneath a glass-wool filter supported on glass beads at the base of the column and recirculated. After construction of the columns, 1000 ml of DI water was added to each and 5 ml was taken after recirculation for 24 h to determine the initial CN and NO, concentrations. KCN was added to each of the nonsterilized columns to produce a starting concentration of 6 mg of free CN per liter. The sterilized columns had higher levels of free cyanide due to the effects of autoclaving. Sucrose was added to the top of three columns and to the three sterilized columns to produce a C:N ratio of 10:l in each (no phosphorous added). After 6 d of