British Columbia Mine Reclamation Symposium

Nitrogen and cyanide compound removal from gold mine impacted water using an anammox bioreactor Risacher, F. F.; Mancini, S.; Dollar, P.; Dennis, P.; Coffey, K.; Kennedy, C. 2019

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NITROGEN AND CYANIDE COMPOUND REMOVAL FROM GOLD MINE IMPACTED WATER USING AN ANAMMOX BIOREACTOR   F.F. Risacher, M.Sc1, S. Mancini, Ph.D1, P. Dollar M.Sc1, P. Dennis M.ASc3, K. Coffey1 , C. Kennedy, Ph.D2   1Geosyntec Consultants International, Inc. 1243 Islington Avenue, Suite 1201 Toronto, ON M8X 1Y9  2Agnico Eagle Mines Limited 145 King St. East, Suite 400 Toronto, ON, Canada M5C 2Y7  3 SiREM 130 Stone Road Guelph, ON, Canada N1G 3Z2  ABSTRACT The use of ammonium-nitrate fuel/oil (ANFO) explosives and cyanide in the gold mining industry can lead to elevated concentrations of nitrogen compounds in mine-impacted water, often requiring treatment before discharge. Recent limits on the concentration of ammonia added to the Canadian Metal and Diamond Mining Effluent Regulation (MDMER) further stress the need for cost-effective solutions to remove nitrogen from effluents. Nitrogen compounds are typically removed through two biological processes: 1) aerobic oxidation of ammonia, and 2) anaerobic reduction of nitrate. These processes can be costly due to the need for separate reactors, addition of a carbon source and aeration. Anaerobic ammonium oxidation (anammox) bacteria solve this issue by simultaneously converting ammonia and nitrite to nitrogen gas in a single anaerobic autotrophic process. Despite the successful application of anammox to wastewater treatment plants, little research has been done on its application to mine effluents. Here, we present an anammox-containing culture with an emphasis on its nitrogen removal capabilities as well as the microorganisms identified to carry out the metabolism. Results of our laboratory application of the culture to remove nitrate, ammonia and cyanide compounds from a gold mine effluent are presented.  KEY WORDS  Thiocyanate, Anammox, Deamox, Denitrification, Reclamation and Closure.   INTRODUCTION  Cyanidation of gold ore is an industry standard method for leaching gold into solution that can then be recovered (Cornejo and Spottiswood, 1984). This method uses cyanide (CN-) under highly basic conditions (pH>10) to form gold cyanide complexes (Na[Au(CN)2] (aq)) which are recovered from solution by adding zinc that binds to cyanide, leaving the gold to precipitate (Marsden and House, 2006). This process generates high concentrations of cyanide compounds in the effluent (300-500 mg/L) that are usually treated by adding hydrogen peroxide to oxidize cyanide to cyanate (OCN-) which is less toxic and can be further degraded by acid hydrolysis to carbon dioxide and ammonia (Carroll, 1990; Akcil and Mudder, 2003). Complicating matters, thiocyanate, which forms during the cyanidation of the ore by interaction with sulfur minerals, cannot be readily degraded and is much more persistent in effluents. Thiocyanate typically requires additional treatment such as biological oxidation (Ibrahim et al., 2015) in effluent treatment plants that use a series of reactors, in which air is injected, so that bacteria can oxidize thiocyanate to carbon dioxide, sulfate and ammonia (Ebbs, 2004; Dash et al., 2009; Naveen et al., 2011; Kumar et al., 2017). This biological process is widely used due to its rapid rate and lack of requirement for an external carbon source, but a downside is that it also produces large amounts of ammonia that must be oxidized to nitrate (Baxter and Cummings, 2006).  With the degradation of cyanide compounds to nitrogen in addition to the use of ammonium-nitrate fuel oil (ANFO) as a blasting agent, high nitrogen loads in mine impacted waters are probable. Moreover, because recent limits on the concentration of ammonia were added to the Canadian Metal and Diamond Mining Effluent Regulation (MDMER), further importance is placed on the cost-effective treatment of nitrogen that can be integrated into existing systems without compromising current water treatment processes. Recent research (Villemur et al., 2015; Mekuto et al., 2017; Tanabene et al., 2018; Luque-Almagro et al., 2018) pointed towards the use of aerobic reactors for nitrification in conjunction with anaerobic reactors for denitrification where the sole source of carbon was cyanate and thiocyanate. Anaerobic oxidation of thiocyanate using nitrate can likely provide cost-effective removal of nitrogen without the need for an additional carbon source. Moreover, Villemur et al. (2015) pointed out that a portion of additional nitrogen loss could be due to anaerobic ammonium oxidation (anammox) which could further improve the nitrogen removal of an anaerobic reactor.  Anammox metabolism is performed by autotrophic bacteria able to fix inorganic carbon by utilizing the energy from the oxidation of ammonia and using nitrite as an electron donor (Jetten et al., 2009). This reaction produces nitrogen gas and thus removes nitrogen from a system without the need for a source of organic carbon, unlike denitrifiers (Figure 1). Microbial taxa identified to carry out anammox metabolism are Kuenenia stuttgartiensis, Brocadia, Anammoxoglobus, Jettenia and Scalindua which are all strictly anaerobic (Kuenen, 2008).  Due to the simultaneous removal of ammonia and nitrite under anaerobic conditions, the anammox process presents an attractive option for municipal wastewater treatment plants (WWTP) to replace the two-step nitrification/denitrification. Indeed, this process removes the cost of carbon (i.e., electron donor) addition for denitrifiers as well as the cost of aeration (Szatkowska and Paulsrud, 2014). The first applications of anammox cultures were demonstrated at full scale for municipal WWTP in 2007 and have since been used widely under multiple configurations across the globe (van der Star et al., 2007; Huy Quoc Anh et al., 2015; Zhang et al., 2015).  The oxidized form of nitrogen in municipal WWTP effluents is comprised of nitrate instead of the nitrite essential to the anammox metabolism, thus requiring the reactor to carry a process to generate nitrite from effluent nitrate or ammonia. Two processes exist, 1) partial nitritation of ammonia and 2) partial denitrification of nitrate. Partial nitritation is the conversion of some of the ammonia to nitrite by ammonia oxidizing bacteria (AOB) under micro-oxic conditions requiring low aeration. Partial nitritation is the preferred method where high levels of ammonia and low levels of nitrate are present. Multiple examples of this process have been used to treat wastewater under different names: Completely Autotrophic Nitrogen-removal Over Nitrite (CANON, Zhang et al., 2008); ANITATM Mox (Lemaire et al., 2015) and SHARON (Volcke et al., 2006). Partial denitrification or denitrifying ammonium oxidation (DEAMOX, Masłoń and Tomaszek, 2009) is the conversion of nitrate to nitrite by denitrifiers. DEAMOX requires small amounts of organic carbon, but has the advantage of being fully anaerobic, and is preferable when nitrate and ammonia are both present in the effluent.   Based on the success of anammox in municipal WWTPs and the chemical composition of gold mine effluent water, the application of the anammox process in conjunction with thiocyanate and cyanate degradation has the potential to provide additional nitrogen removal from gold mine processing effluents. Partial denitrification-anammox is a good candidate process as effluents contain both nitrate and ammonia and would additionally provide easy implementation due to the presence of existing anaerobic bioreactors in which nitrate is recirculated for thiocyanate oxidation. Here we present an approach for the startup and operation of a lab-scale bioreactor for the removal of nitrogen and thiocyanate from gold mine water effluent. Figure 1: Nitrogen cycle. Major compounds are in bold while processes are in italics. Blue arrows indicate aerobic processes, red arrows anaerobic processes and green arrows fixation/assimilation processes. ANAMMOX CULTURE REACTOR  As part of a multi-year internal research and development initiative, SiREM a laboratory and product vendor division of Geosyntec Consultants, has developed and operated a membrane bioreactor (i.e., a retentostat) treating ammonium (NH4+) and nitrite (NO2-). The reactor was inoculated in 2015 with groundwater from sites containing nitrogen compounds which tested positive for anammox bacteria using molecular biological tests. The reactor design is based on van der Star et al. (2008) and the media recipe is as described by van de Graf et al. (1996). Both the media and reactor were designed to encourage planktonic growth of annamox bacteria (i.e., not attached growth).  The reactor operates in a continuous flow mode at a flow rate of 2 liters/day (L/day), with a residence time of approximately 10 days. The effluent is withdrawn through a membrane filter that maintains biomass in the reactor which is essential for slow growing anammox bacteria which have a doubling time less than 10 days. The nitrogen (N) compounds influent concentrations are currently 435 milligrams per liter as nitrogen (mg/L-N) of NH4, 402 mg/L-N of NO2- and 18 mg/L-N of nitrate (NO3). The media also contains calcium, magnesium and carbon dioxide (CO2; 5%) in argon which is continuously bubbled through the liquid to provide a steady long-term inorganic carbon source to autotrophic anammox bacteria. The reactor is maintained at 34°C and a pH of approximately 7. Wasting to remove biomass is performed once weekly with 1 L of volume being directly removed via the sludge pump resulting in a sludge age of approximately 20 weeks. A detailed listing of the media components is provided in Table 1.  Table 1: Media Composition in Nitrogen Reactor (Based on van de Graf et al., 1996).  Major Media Components Major Nutrient Supplied  Concentration in Prepared Media (mg/L) (NH4)2SO4 Ammonium/Sulfate  661 NaNO2 Nitrite  690 NaNO3 Nitrate  72 KHCO3 Carbonate  501 KH2PO4 Potassium/Phosphate  27 MgSO4.7H2O Magnesium/Sulfate 74 CaCL2.2H2O Calcium  44 EDTA Chelating agent  20 Yeast Extract  Vitamins  2 FeSO4.7H2O Iron  9 Trace Elements      ZnSO4.7H2O Zinc  0.43 CoCL2.6H2O Cobalt  0.24 MnCL2.4H2O  Manganese  0.99 CuSO4.5H2O Copper 0.25 NaMoO4.2H2O Molybdenum  0.22 NiCL2.6H2O Nickel  0.19 NaSeO4.10H2O Selenium  0.21 H3BO4 Boric Acid  0.014 CULTURE PERFORMANCE   The nitrogen compounds in the reactor are measured on average at 20% of the influent concentrations during steady state operations, indicating substantial nitrogen removal, presumably as nitrogen gas (N2), via anammox and possibly denitrification processes (Figure 2). Looking at the mean nitrogen concentration during steady operations (before April 2019) and given a flow rate of 2 L/day, a conservative nitrogen removal rate is : 2 L x (0.80 (removal fraction) * (176 mg/L NH4-N + 182 mg/L NO2-N)) – 6.5 mg/L NO3--N = 560 mg of nitrogen removal per day (mg N/d) for the entire reactor (20 L) or 28 mg N/L/d. Starting in April 2019, approximately twice the original concentration of ammonia and nitrite was added to the media to see if the anammox culture could sustain nitrogen removal during high loadings. Early results ( six weeks) indicate that nitrogen compounds in the reactor continue to be measured on average at 20% of the influent concentrations, consistent with lower nitrogen compound conditions. Using the same formula as above we can calculate a nitrogen removal rate of 1,092 mg N/d or 54.6 mg N/L/d, double of the removal rate under lower nitrogen loading. These results show that the anammox reactor can remove nitrogen at similar efficiency but at higher rates when nitrogen loading is increased. The ratio of ammonia to nitrite consumed in the reactor indicates an average of 1.43 (±0.22 SE), close to the theoretical 1.32 ratio (Eq. 1) expected as observed by (Szatkowska and Paulsrud, 2014). Another indication of an active anammox process is the production of small amounts of nitrate at a ratio of 0.26 for every mole of ammonia consumed. Production of nitrate in the reactor was observed but at an average ratio of 0.04 (±0.02 SE). The lower amount of measured nitrate is possibly due the activity of denitrifiers in the reactor.  Figure 2: Nitrogen compounds concentration in the anammox reactor and its influent. Eq. 1 NH4+ + 1.32NO2- + 0.066HCO3- + 0.13H+ →1.02N2 + 0.26NO3- + 0.066CH2O0.5N0.15 + 2.03H2O 010020030040050060070080090017-Oct-16 15-Apr-17 12-Oct-17 10-Apr-18 7-Oct-18 5-Apr-19Nitrogen compounds as N (mg/L)Influent nitrite Reactor nitrite Influent nitrate Reactor nitrateInfluent ammonia Reactor ammonia Influent nitrogen Reactor nitrogenMICROBIAL COMMUNITY COMPOSITION  The microbial community in the nitrogen reactor has been characterized using next generation sequencing (NGS) protocols targeting 16S rRNA gene amplicons and by quantitative polymerase chain reaction (PCR) methods targeting 5 common genera of anammox bacteria. NGS indicated a diverse microbial community in the reactor with almost 200 operational taxonomic units (OTUs) identified. Figure 3 shows the major microorganisms in the culture based on NGS. The data indicate that the dominant members of the culture belong to the Ignavibacteriaceae, Fimbriimonadaceae, Anaerolineae, Chlorobi and Thauera groups. Nitrogen removal is likely a combination of anammox and possibly denitrification processes. Denitrifying ability is sporadically distributed among taxonomically diverse groups of Bacteria, as well as some Archaea and Fungi; therefore, it is difficult to identify denitrifying organisms based only on their 16S rRNA gene sequences. Furthermore, it is unclear what electron donor would be used for denitrification processes, although biomass in the reactor could potentially provide a source of various reduced compounds which could act as electron donors for denitrification.  Fimbriimonadaceae20%Anaerolineae; o__envOPS1213%Thauera12%Ignavibacteriaceae21%Chlorobi6%Burkholderiales4%p__BRC12%Xanthomonadaceae2%Pseudomonas; s__pseudoalcaligenes1%Bacteria; p__WPS-21%Kuenenia; s__stuttgartiensis1%Other 17%Fimbriimonadaceae Anaerolineae; o__envOPS12Thauera IgnavibacteriaceaeChlorobi Burkholderiales p__BRC1 XanthomonadaceaePseudomonas; s__pseudoalcaligenes Bacteria; p__WPS-2Kuenenia; s__stuttgartiensis OtherFigure 3: Microbial composition of most abundant microbial members in SiREM nitrogen reactor. d=domain, p=phylum, c=class o=order, f =family, g =genus, s=species Organisms in the culture with a high likelihood of nitrogen metabolism include Ignavibacterium, a group of chemoheterotrophs with a versatile metabolism (Liu et al., 2012), that are routinely associated with nitrogen removal (Tian et al., 2015), and are a dominant organism in the nitrogen reactor (21% of OTUs). Thauera (12% of OTUs) are commonly associated with denitrification in wastewater treatment systems (Seviour and Nielsen, 2010). Other taxa possibly involved in nitrogen metabolism, but which comprise small proportions of OTUS (<1%), include Nitrosomonadaceae, Nitrospirales, which are putative nitrifiers that convert ammonium to nitrate. Kuenenia stuttgartiensis (1% of OTUs) is a known anammox bacterium and comprised 1% of OTUs in the NGS. Other genera of known anammox bacteria, Brocadia, Anammoxoglobus, Jettenia and Scalindua, were not detected indicating that Kuenenia are likely the key anammox bacteria. Interestingly, some strains of Pseudomonas pseudoalcaligenes (1% of OTUs) have been reported to degrade free cyanide, cyanate and metal cyanide complexes (Cobos et al., 2015) and; therefore, could be of potential benefit to cyanide compound treatment. Quantitative PCR tests for anammox bacteria have been performed regularly on the reactor and results are summarized in Figure 4. Overall, there has been a generally increasing trend in anammox bacteria concentrations over time particularly in 2017-2018, and despite some variation, anammox gene copies followed an exponential growth trend as indicated by the blue dashed line. Using linear regression, doubling time of anammox bacteria is calculated as approximately 78 days. Despite the long doubling time the addition of pre-grown anammox biomass will make for a faster startup of a new treatment system.     Figure 4: Quantification of anammox bacteria in reactor by quantitative PCR testing. y = 5E-159e0.0089xR² = 0.72571.00E+031.00E+041.00E+051.00E+061.00E+071.00E+081.00E+091.00E+1015-Jul-15 10-May-16 6-Mar-17 31-Dec-17 27-Oct-18 23-Aug-19Anammox 16S rRNA Gene Copies/LAnammox DNA GOLD MINE REACTOR STARTUP   Based on the successful demonstration of the anammox process in our initial reactor, a second reactor was designed to test the removal of nitrogen compounds from gold mine effluent. The function of this bioreactor is to test the feasibility of using the anammox process for treatment of ammonium and nitrate that originates from the thiocyanate and explosive residue from the tailing storage facility (TSF) at a gold mine site. The bioreactor design is represented in Figure 5.  The reactor feed is a mixture of water from the TSF that contains ammonium, nitrate and thiocyanate and the post-aerobic treatment (PAT) that contains nitrate. Ammonium and nitrate in the water from the TSF and PAT were quantified and mixed so that the nitrate and ammonium are present in a ratio between 1:1 and 3:1 in the reactor feed to account for the anaerobic oxidation of thiocyanate. The empty reactor was sealed and purged for approximately 15 minutes with argon gas in order to remove atmospheric oxygen prior to filling with the mixed process water. Once filled with 10 L of water the argon gas was used to sparge the water to promote anoxic conditions. Dissolved oxygen (DO), oxidation reduction potential (ORP) and pH were measured, and small amounts of L-cysteine were added to stimulate reducing conditions.  After anoxic conditions were confirmed, the reactor was inoculated with 100 mL of inoculum from the anammox reactor. Following this, the reactor was mixed periodically via argon gas injection and the ORP/pH were monitored daily to ensure optimal growth conditions. Acetate was added depending on the concentration of nitrate (3:1 Chemical Oxygen Demand (COD) to NO3--N ratio) to help initiate the first step in the denitrification process (i.e., partial denitrification) which is needed to provide a source of nitrite Figure 5: Schematic of the gold mine reactor set up. to anammox bacteria. Acetate in these ratios has been shown to help provide the optimal amount of nitrite without further reduction to nitrogen gas (Oh and Silverstein, 2002; Du et al., 2017a ; Du et al., 2017b). After stable anaerobic conditions were confirmed, a second inoculation with anammox reactor culture was performed to further boost to the biomass and the flow in and out of the reactor was commenced, targeting a residence time of 10 days (1 L/day). The reactor is a retentostat due to a filter on the reactor effluent collection inlet that prevents any wasting of biomass to ensure slow growing anammox bacteria were not removed. Weekly measurements of the influent and reactor for ammonia, nitrate, nitrite, sulfate and bi-weekly measurement for thiocyanate/cyanate were taken. pH and ORP were monitored daily to ensure proper conditions are maintained for the development of the anammox culture. pH was maintained between 7 and 8 by addition of phosphoric acid. The startup of the reactor required five weeks with sampling commencing on week six after we ensured cysteine would not contribute to the generation of ammonia.  GOLD MINE REACTOR RESULTS  Nitrogen concentrations in the reactor and in the influent are presented in Figure 6. The results show that the reactor can either be nitrogen producing or nitrogen consuming. Under nitrogen producing conditions (week 9 and 10, Figure 6) the influent of the reactor has a nitrate:ammonia ratio close to 1 while the amount of thiocyanate oxidized is maximal (110 mg/L loss between influent and reactor, Figure 7). Equation 2 demonstrates that for each mole of thiocyanate oxidized, eight moles of nitrate are consumed (Sorokin et al., 2004). This would mean that the oxidation of 110 mg/L of thiocyanate would consume a total of 45 mg/L-N of nitrate which is the same as the observed loss of nitrate between the influent and reactor. If we consider the amount of ammonia produced from the oxidation of thiocyanate and cyanate (Figure 7), we expect to see a concentration of 60 mg/L-N of ammonia in the reactor which, when added to the influent concentration of ammonia, should equal to 97 mg/L-N. The measured concentration of ammonia in the reactor was 105 mg/L-N, indicating that the oxidation of cyanide products accounts for almost all ammonia production. These results suggest that under nitrate limitation, anaerobic oxidation of thiocyanate is the primary metabolism controlling the amount of nitrate consumed in the reactor.  During nitrogen consuming conditions (week 6 and 11 to 14) the amount of nitrate was higher as well as the ratio of nitrate to ammonia (Figure 6). Concentrations of oxidized thiocyanate and cyanate also decreased to 28 and 65 mg/L respectively (Figure 7). The observed amount of thiocyanate oxidized should consume 11 mg/L-N of nitrate in the reactor, however we observed a loss of more than 72 mg/L-N of nitrate between the influent and the reactor. The additional loss of nitrate can be accounted for by two mechanisms: 1) complete denitrification and/or 2) anammox. The difference between these two metabolisms is that complete denitrification should not consume ammonia, therefore if it were to occur the concentration of ammonia in the reactor should be equal to the ammonia in the influent plus the ammonia produced by the oxidation of thiocyanate and cyanate. The concentration of ammonia produced by the oxidation of thiocyanate and cyanate in the reactor should have been 28 mg/L-N, and totaling 82 mg/L-N if we account for the ammonia in the influent. This means that the reactor has 19 mg/L-N of ammonia less than predicted, suggesting that anammox could be partially responsible for the loss of nitrogen in the reactor. Eq. 2  5SCN- + 8NO3- → 5SO42- + 5NH4+ + 4N2 + 10CO2       Figure 6: Concentration of nitrogen compounds in the gold mine reactor and influent. Figure 7: Thiocyanate and cyanate loss in the gold mine bioreactor 0204060801001206 7 8 9 10 11 12 13 14Cyanide coumpound loss (mg/L)WeeksThiocyanate loss Cyanate loss0. 7 8 9 10 11 12 13 14 15Nitrogen compounds (mg/L as N)WeeksReactor Nitrate Influent nitrate Reactor nitrite Influent nitriteReactor Ammonia Influent ammonia Reactor total N Influent total NIMPLICATIONS AND NEXT STEPS  Quantification of anammox DNA via qPCR confirmed that an anammox population is present in the gold mine reactor at early stages at 9x104 anammox bacteria/L. Despite the low abundance we expect most of the biomass to be located on surfaces, such as the floating plastic carrier, in close association with denitrifiers. Moreover, some of the data presented here shows evidence of correlated nitrate and ammonia loss which is typical of the anammox process. To confirm this, 16S RNA amplicon (next generation) sequencing is being performed which should provide additional information on the overall bacterial community in the reactor that could be carrying out anammox or other forms of nitrogen metabolism. Actions to be undertaken to favor anammox bacteria are increasing the nitrate:ammonia ratio and reducing the concentration of acetate to encourage partial denitrification instead of full denitrification.  Anammox offers potential for cost-effective single stage removal of nitrogen as already demonstrated in the wastewater treatment industry. Based on a similar approach we have presented encouraging data that the anammox process has potential to remove ammonia and nitrate from gold mining effluent, despite the toxicity from thiocyanate and cyanate. Further optimization of our test reactor will be required to conclusively demonstrate the utility of anammox in this challenging environment. The next steps towards the development of a comprehensive nitrogen removal system would be a complete pilot system that uses TSF water with recirculation instead of using the water from the PAT for the additional nitrate. Scaling up the reactor would present some challenges, mainly associated with the temperature control since low temperature influents can slow down the anammox process, requiring higher residence time. Alternatively, modifications, such as separation of the anammox and thiocyanate oxidation process into two separate reactor zones, similar to the compartmentalization in Van Der Star (2007), could help create more favorable conditions. If successful, this would demonstrate in-situ the rates of nitrogen removal as well as possible configurations for full scale treatment systems.   ACKNOWLEDGMENTS   We would like to thank Rachel James and Brent Lazenby from Geosyntec Consultants, Rita Schofield and Evan Mayer from SiREM, Pierre-Olivier Gendron and Patrick Laporte from Agnico Eagle for their valuable help throughout this project. We would also like to thank our funding sources from Agnico Eagle as well as from the Technological Advisory Council from Geosyntec Consultants.    REFERENCES Akcil, A. and T. Mudder, 2003. Microbial destruction of cyanide wastes in gold mining: Process review. Biotechnology Letters. Kluwer Academic Publishers.  Baxter, J. and S. P. Cummings. 2006. The current and future applications of microorganism in the bioremediation of cyanide contamination. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology.  Carroll, J. E. 1990. A Look at Chemical Degradation vs. Biodegradation of Cyanide and Metal-Complexed Cyanides Found in Industrial Wastewater Generated by the Mining Industry Jason E. Carroll. 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