British Columbia Mine Reclamation Symposium

Assessing and optimizing microbial processes impacting mine reclamation Friesen, Vanessa; Haakensen, Monique 2016

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24    ASSESSING AND OPTIMIZING MICROBIAL PROCESSES IMPACTING MINE RECLAMATION   Vanessa Friesen, Ph.D.1 Monique Haakensen, Ph.D., RP.Bio., P.Bio., EP.2  1Principcal Scientist,  Contango Strategies, Saskatoon, Canada.  2President and Principal Scientist,  Contango Strategies, Saskatoon, Canada.   ABSTRACT  Microbes influence a wide range of processes, acting as catalysts to accelerate reactions or enable them to occur in a wider range of conditions. In the context of mine reclamation, many aspects are impacted by microbes and understanding their role is critical to making informed decisions, optimizing processes, and improving reclamation success. Examples of microbes affecting mine reclamation processes will be presented, including: passive- and semi-passive water treatment (including metals, metalloids, ammonia, nitrate, acid-rock drainage, heap leach detoxification), soil bioremediation, and revegetation.  An overview of the tools currently being used at mines for microbial community profiling will be discussed, spanning from traditional growth-based techniques, through to modern day standardized genetic community profiling based on DNA-sequencing. This information will be provided in the context of how they have been used to guide and optimize mine reclamation strategies.   KEY WORDS  Innovation, environment, genomics, bioremediation, passive water treatment, MCP testing   INTRODUCTION  Microbes are the driving force in many processes, acting as catalysts to facilitate biogeochemical reactions that influence mining operations and remediation efforts.  Despite this influence, microbes have often been overlooked in mining-associated processes. Historically, this has largely been due to the inability to effectively test and interpret mining-associated microbiological samples and data in a way that is useful to inform decisions. However, technology has advanced dramatically over the past 5 years, and genetic (genomic) and growth-25  based microbial community profile (MCP) testing is now being applied to diverse mining processes and reclamation activities to inform and de-risk decision making.  This article will first discuss ways that microbes impact mining activities, then provide an overview of traditional microbial analysis techniques and how they have progressed to modern day standardized testing. Case studies of how they have been used to guide and optimize reclamation strategies will also be provided.    ROLES OF MICROBES IN THE MINING SECTOR  While small, microorganisms (such as bacteria, archaea, fungi, algae) impact a variety of processes in the mining industry, in both beneficial and detrimental ways (Figure 1).  For example, passive and semi-passive water treatment (e.g., constructed wetlands, bioreactors, permeable reactive barriers, pit lake treatment, and in situ mine pool treatment) are largely driven by geochemical and biological reactions, coupled into biogeochemical processes to remove constituents from water. Many biogeochemical reactions are catalyzed by microbes. The efficiency of these microbial catalysts depends on the types (identity), their abundance, (quantity), the microbial community composition (relative or percent abundances within a system), and metabolic rate (reaction rate).   Examples beneficial biogeochemical reactions include treatment of ammonia by bacteria or archaea through a process called nitrification (oxidation of ammonia to form nitrite and nitrate; Kowalchuk and Stephen, 2001), or treatment of selenium by organisms that can reduce soluble selenate and selenite to form insoluble elemental selenium (Lovley, 1993). Microbes play an important role in passive water treatment systems to treat metals and metalloids, as they can use organic carbon sources (e.g., wood chips, plant matter, methanol) to reduce sulphate and form sulphides (Figure 1).  These sulphides can then complex with metals as insoluble metal-sulphides that can then be more easily removed from water.     Figure 1 Example equation of sulphate reduction and metals treatment by microbes in passive treatment systems. The equation is not balanced, and will vary depending on carbon source and pH. The equation shows the creation of alkalinity (HCO3-) and sulphides (HS-), the latter which can then form insoluble metal sulphide complexes.  26  Other areas commonly recognized as being influenced by microbes include the exacerbation of acid-rock drainage, and facilitation of heap leaching. These processes are both catalyzed by the same groups of microbes that can oxidize pyrite and other reduced iron and sulphide compounds resulting in the generation of acid (iron- and sulphide-oxidizing bacteria).   Perhaps less commonly thought of in the context of mining, there are also microbes that degrade contaminants such as hydrocarbons (e.g., Leahy and Colwell, 1990) which can be used for reclamation efforts to clean any impacted soils, and yet others involved in nutrient cycling and that can confer stress tolerance to plants, aiding in revegetation efforts.   In all cases, understanding how microbes are influencing a given system or process is imperative to making informed decisions to either enhance or mitigate these beneficial or detrimental processes.     Figure 1 Examples of how microbes impact the mining sector   27   MICROBIAL COMMUNITY PROFILING TOOLS  Microscopy and single organism studies Historically, microbial investigations have been confined to traditional methods such as microscopic observations and studying single isolated organisms that could grow in an imposed artificial laboratory setting in attempts at further characterization (Figure 2). While microscopy can provide some high level information about the types of microbes that are present (e.g., fungi, algae, gram-positive or gram-negative bacteria), its ability to provide accurate quantification and identification of microbes is limited (Table 1). The number (quantity) and type (identity) of microbes present are two of the most important foundational pieces of information needed when assessing microbial populations. These aspects greatly impact the extent and capacity of different biogeochemical processes, and the need for this microbiological information also applies to understanding microbial influences that could impact mine reclamation efforts.   Historically, single organism laboratory studies have been used to characterize microbes, such as the ability to reduce sulphate to form sulphides (e.g., review by Postgate, 1965), or to fix nitrogen. The isolation and characterization of single organisms was necessary for foundational scientific investigations into the possibility that microbes could be involved in what used to be thought of as purely geochemical functions, which formed the basis of our current understanding of biogeochemistry and associated technology development. However, microbes tend to survive and function better as part of a mixed community in their natural environment, interacting and growing on substrates provided by other organisms in their niche (e.g., commensal relationships). As such, single-organism studies in reclamation settings can be misleading, as biogeochemical processes are often multi-organism or community driven, and it is now generally believed that more than 99% of microbes are not capable of being grown as a single organism in a traditional laboratory setting. Additionally, the growth of an organism in a laboratory setting can result in the loss of functional abilities that are used in their natural environment.     28  Figure 2 Progression of tools for microbial analyses Community growth-based laboratory tests Growth-based laboratory tests have therefore progressed over time from single-organism tests to community-based assays. These growth-based assays can identify characteristics of organisms that are in a community and are sometimes paired with quantification. One such example is the BART test, which is a simple tool to detect the presence of organisms in a community that are capable of a certain metabolic function that is being screened for (e.g., sulphate-reducing or iron-reducing bacteria). While this test is very simple to do, has a relatively low cost, and can detect some types of microbes important to mine closure, these tests are unable to provide accurate quantification, and serve best as a pre-screen for more accurate test methods (Table 1).   A more modern quantification assay is based on the most-probable number (MPN) method.  The MPN test is another tool that uses growth of whole microbial communities and can be used to detect organisms that are capable of a certain metabolic function that is being screened for. However, in addition to determining if certain metabolic capacity exists in the community, the MPN method is also able to provide quantification (Colwell, 1979).  The MPN has a standardized method, that uses statistical analyses of dilution series (Garthright and Blodgett, 2003; Blodgett, 2010). Using this method, the most probable number of organisms per volume or area of a sample can be quantified, and depending on the type of growth media used, metabolic capacity can also be inferred. MPN assays can be customized for different subgroups of microbes, such as: heterotrophic bacteria, sulphate reduction, selenium-reduction, nitrogen fixation, denitrification, molybdenum-reduction, arsenic-reduction, manganese reduction, iron cycling, and many more (e.g., Kuai et al., 2001; Mandal, et al., 2007; Papen and von Berg, 1998).  The MPN assay is therefore an informative tool to assess the abundance of certain types of microbes in different mine reclamation settings (e.g., monitoring tool for passive treatment systems such as constructed wetlands and bioreactors).   Growth-based assays such as the MPN method and BART test are affected by the limitation that a very large proportion (often estimated at >99%) of environmental microorganisms cannot be grown in a standard laboratory setting. Growth-based methods are therefore influenced by how well suited the microbes of any environmental sample are to be able to grow in a given assay growth media.  Additionally, growth-based methods cannot identify the types of organisms in a sample, nor inform regarding which organisms are present that may be capable of performing a given biogeochemical function. It is therefore difficult to gather information needed for optimization of reclamation strategies using only growth-based laboratory microbiology studies.   Genetic microbial community profiling Genetic microbial community profiling (MCP) was developed to complement growth-based tools and fill in the limitations mentioned above. As there are many different technologies and names used for these genetic analyses, such as microbiome analysis, targeted metagenomics, and 16S profiling, the more general term of MCP testing is used here.  MCP testing can be used to directly identify all microbes in any type of mining-related sample based on their DNA sequences, 29  bypassing the need of growth in a laboratory or test bottle. Some regions of DNA are so important, they are conserved in all bacteria (or fungi, archaea, etc.). Over time, these conserved and universal regions of DNA have evolved, making it possible to use these sequences to identify and differentiate between microbes in a community.   Genetic MCP testing can be used with a broad range of sample types, including swabs, filters, biofilms, rocks, soil, and water, for example, and is not limited by the ability of organisms to grow in a laboratory setting. One major difference between the MCP testing and growth-based testing is the requirement of highly trained personnel within the testing laboratory (Table 2). This is because samples need to be aseptically processed to extract all DNA and sequence regions that are specific to the type of microbes that are of interest (e.g., all bacteria, archaea, or fungi). Once sequenced, bioinformatics analyses and quality control steps are performed to determine the identity and proportion of each organism in the community (Figure 3). This is necessary to provide standardized genetic sequencing and bioinformatics results, in order to have meaningful and interpretable data that can be used in reclamation strategies; however, because of the standardization of methods, application of algorithms, detection limits, and confidence levels, the reported data itself does not require a microbiology expert to interpret.    Figure 3 Process flow of Microbial Community Profiling (MCP) tools   Once the microbial community is profiled through genetic MCP testing, information on specific microorganisms that are of interest can be gathered to inform on environmental conditions and processes that may be ongoing (e.g., sulphide-producing bacteria, methanogens, iron-oxidizing bacteria). The ability to infer this type of information based on the community profile is entirely dependent on available literature (i.e., characterization of organisms) that is used to create databases, as well as how well the trait is conserved within a genus.  For example, sulphate-reducing bacteria are well-characterized in literature and the trait of producing sulphide from sulphate is conserved within the genus (i.e., different species of a genus all share this trait). Therefore, based on genetic MCP testing results, the proportion of sulphide-producing bacteria can be determined from organisms identified in the sample. This can be used to compare the distribution of organisms across sample types or over time (e.g., monitoring sulphide-producing bacteria in a bioreactor over time, or comparing distribution of iron-oxidizing bacteria at different acid-rock drainage locations).   30  While genetic studies are needed to identify the community composition and distribution of organisms, there are currently no universal genetic markers for certain types of characteristics such as selenium reduction, and it is difficult to identify selenium or nitrate-reducing bacteria based on their identities alone, as this ability is not conserved to a taxonomic level. That is to say, all organisms from a specific genus will not have the same ability to reduce selenium or nitrate, and likewise, not all organisms that can treat these constituents have been identified (nor the genetic basis for this ability). Additionally, genetic methods can provide information about the distribution of organisms (i.e., the percentage of a community that a given organism makes up), but do not inform regarding actual quantity in a given environment. For this reason, genetic and growth-based tests are best paired together to fully inform questions related to mine reclamation.  It should be recognized however, that microbiological data itself is of limited use in mine reclamation without the context of the environmental conditions in which the organisms exist.  For example, water and/or soil chemistry are fundamental aspects that needs to be reviewed to determine what microbiological assays are relevant in the context of biogeochemistry and treatability of the water by passive treatment systems. For every biogeochemical process, there are constituents and conditions within the water that can facilitate the reactions, while others can be inhibitory. It is therefore critical to identify these facilitating and inhibiting constituents and conditions in a site-specific manner for each reclamation strategy.   The following case studies provide examples of how standardized microbial community profiling tools have been paired with site-specific information guide and optimize reclamation strategies. 31  Table 1 Comparison of select microbiological assays for community analyses in environmental settings Assay Quantitative Strengths Limitations Microscope Poor • Low cost, almost immediate results if microscope available • Snapshot for high level information on types of organisms present (e.g., bacteria, fungi) which can be helpful for system upsets • Cannot accurately identify or quantify organisms • Influenced by sample preparation and requires training of personnel or submission of samples to microscopy laboratory BART Poor • Low cost, easy protocol • Anyone can perform the test, almost anywhere • Influenced by how much organisms like incubation conditions • +/- 30% accuracy using laboratory strains with known preferences MPN Strong • Most accurate growth-based quantification • Can be used for many different metabolic pathways involved in reclamation, as well as total heterotrophic bacteria • Complimentary to genetic methods • Influenced by how much organisms like incubation conditions  • Requires more training of personnel, statistical analysis, interpretation, and typically needs more time to conduct than BART • Laboratory required • Higher cost 32  MCP Relative abundances (i.e., proportion of community), with identification • Identification of organisms and their proportions within a sample • Does not rely on organism’s ability to grow in laboratory conditions, so provides better indication of organisms that are present in sample • Can be used on widest range of sample types • Complimentary to MPN methods • Requires more highly trained personnel, statistical analysis, interpretation • Few labs can perform test, fewer can interpret • Higher cost • Not absolute quantification 33  CASE STUDIES  Case study 1:  Design and testing of a passive treatment system for copper and selenium  Passive treatment was selected for evaluation and optimization for water treatment at closure for a mine in Northern Canada. Development of the passive treatment system was through a phased program, which included a site assessment and information gathering, technology selection and conceptual design, pilot-scale testing and optimization, and on-site demonstration-scale confirmation and optimization (Haakensen, et al., 2015). The first three phases were completed successfully, and an on-site at demonstration-scale system is currently being tested (Minto Explorations Ltd., 2016).  The system is being designed to improve water quality, including treatment of copper and selenium, which can be removed by constructed treatment wetlands.  Copper treatment can be achieved in several ways, through oxidizing or reducing geochemical and biogeochemical reactions. When treated from water under appropriately designed reducing conditions, copper will form insoluble copper sulphides, the creation of which is driven by microbes by reduction of sulphate. The mechanism for selenium treatment is more direct, through microbially catalyzed dissimilatory selenium reduction (Lovley, 1993). This is performed directly by bacteria acting on the selenium.  Through a site assessment, wetland plants were selected based on attributes and beneficial characteristics such as hosting microbes that drive selected treatment processes. Pilot-scale testing was performed with plants collected from site to select the optimal design from several options, and to test different predicted closure water chemistries (Minto Explorations Ltd., Appendix A2, 2016). Genetic and growth-based MCP testing was used to monitor the microbial populations in pilot-scale systems during maturation and through testing of different water chemistries and phases (Minto Explorations Ltd., Appendix A2, 2016). These results formed the basis for subsequent expectations for on-site implementation. Sulphide-producing bacteria (Figure 5), and selenium- and nitrate-reducing bacteria were profiled over time alongside in situ measurements such as soil redox in both pilot- and demonstration-scale wetlands. This confirmed maturation of the demonstration-scale system according to timelines defined in pilot systems, and provided important information that can be used to explain performance and make decisions (i.e., decision variables).      34   Figure 4 Microbial community profiling paired with soil redox measurements to assess demonstration-scale treatment wetland maturation.  Soil redox (right axis, diamonds), and sulphide-producing bacteria (left axis, bar chart) for pilot-scale (off site) and demonstration-scale (on site) systems. Horizontal dashed lines indicate targeted soil redox range, based on oxidation-reduction chemistry and refined through pilot-scale testing. From Minto Explorations Ltd., Appendix A4, 2016.    Case study 2: Selection of plants for ammonia treatment during operations A constructed wetland was being designed for treatment of ammonia (from blasting residues) during operations of an underground mine in Canada. Ammonia oxidation is a process performed by bacteria (nitrification), where ammonia is turned into nitrite and then to nitrate (Kowalchuk and Stephen, 2001).  In order to effectively select the best plant sources for the wetland (i.e., those which would bring along beneficial nitrifying bacteria with them), the microbial populations hosted by the roots of native Typha and Phragmites from different borrow sources near the construction site were compared with MCP testing.   This testing allowed for the selection of Typha from two different borrow sources for use in the constructed treatment wetland. Two sources were selected because the types of ammonia-oxidizing organisms were different in these borrow locations, and therefore inclusion of both increases the diversity of organisms that can perform the needed function in the wetland. In the context of treatment wetlands, the diversity of microbes present that can perform a desired function (in this case, ammonia 35  oxidation/nitrification) can be regarded as a measure of robustness to treat the water under a wider range of conditions and changes in water chemistry.  Case study 3: Assessment of passive water treatment potential and testing through freeze-thaw for permitting  A future mine project in the Northwest Territories is predicted to have seepage in closure that requires treatment. To assist in permitting, a site assessment was performed to delineate attenuation of constituents of concern that are occurring in a natural wetland on site, to inform site-specific design and testing of constructed treatment wetlands for closure.   Microbial communities were assessed along a watershed that receives seeps that are naturally high in arsenic, alongside other analyses for physicochemical parameters at the site. Using this information from the site assessment, a passive treatment wetland was designed and constructed at pilot scale to mimic natural conditions at the site that were found to improve arsenic treatment. The pilot-scale design successfully achieved targeted reducing and oxidizing conditions (in treatment cells designed for these respective conditions as part of a treatment train), and demonstrated the stability of key microbes through a freeze thaw cycle (Figure 5).  With historical tools, the microbial aspects of natural attenuation at the site and subsequent pilot-scale designs and testing through a freeze thaw cycle would have been poorly defined, leading to the inability to predict robustness and optimize performance.      Figure 5 Microbial populations in pilot-scale treatment wetlands through freeze-thaw testing. Multivariate statistical analysis can be used to suggest relationships between microbial populations in different wetland 36  designs over time and through a freeze-thaw cycle. Oxidizing and reducing conditions targeted in the treatment wetlands are outlined with dotted ellipses.   CONCLUSION  Microbes can be thought of as renewable catalysts that drive biogeochemical processes (e.g., changing of redox state, solubility, or chemical form) that influence mine reclamation. Advances in microbial technologies in the past 5 years have revolutionized the understanding of microbial systems in the mining sector.  While historically microbial analyses have relied on microscopy or single organism growth-based testing, community based tools are now available that can quantify, identify, and characterize microbes that impact a variety of mining processes.  Case studies in this manuscript described the application of MCP tools to inform decisions on designing and monitoring passive water treatment systems. Numerous other mine reclamation activities such as those involving acid-rock drainage, revegetation efforts, or other types of passive or active biological treatment systems (e.g., bioreactors, pit lake treatment, and in situ mine pool treatment) can also benefit from microbial community profiling tools, to gather information on microbial systems that historically have been considered a ‘black box’.     REFERENCES   Blodgett, R. 2010. US Food and Drug Administration, Bacteriological analytical manual, Appendix 2: Most probably number from serial dilutions. Colwell, R.R. 1979. Enumeration of specific populations by the most-probable number (MPN) method. Native Aquatic Bacteria: Enumeration, Activity, and Ecology, ASTM STP 695, J.W. Costerton and R.R. Colwell, Eds., American Society for Testing and Materials, 56-61. Garthright, W.E. and Blodgett, R.J. 2003. FDA’s preferred MPN methods for standard, large or unusual tests, with a spreadsheet. Food Microbiology, 20: 439-445. Leahy, J.G. and Colwell, R.R. 1990. Microbial degradation of hydrocarbons in the environment. Microbiological Reviews, 54: 305-315.  Lovley, D.R. 1993. Dissimilatory metal reduction. Annual Review of Microbiology, 47: 263-290. Minto Explorations Ltd. 2016. Minto Mine Reclamation and Closure Plan 2016-01. Prepared by Minto Explorations Ltd., Minto Mine, August 2016. Appendices A1 through A4.  Kowalchuck, G.A. and Stephen, J.R. 2001. Ammonia-oxidizing bacteria: A model for molecular ecology. Annual Review of Microbiology, 55: 485-529. Kuai, L., Nair, A.A., and Polz, M.F. 2001. Rapid and simple method for the most-probable-number estimation of arsenic-reducing bacteria, Applied and Environmental Microbiology, 67: 3168-3173. 37  Papen, H. and von Berg, R. 1998. A most probable number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil, Plant and Soil, 199: 123-130. Postgate, J.R. 1965. Recent advances in the study of the sulfate-reducing bacteria. Bacteriological Reviews, 29: 425-441.   

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