WO2020217188A1 - Process for controlling in situ groundwater pollution from seepage of tailings tank water - Google Patents

Abstract

The invention discloses an in-situ method for reducing hydraulic conductivity and generating the precipitation of insoluble minerals in an underground aquifer which is affected by seepage of surface water or by seepage of water from a mining tailings tank, comprising at least the following steps: (a) providing a sulphate-reducing bacteria-enriched microbial culture adapted to the underground water of the aquifer to be treated, (b) injecting an enriched microbial culture into the underground aquifer, (c) injecting an electron and nutrient donor for reducing bacteria-enriched microbal culture and one or more metal cations that favour the precipitation of insoluble minerals, and (d) allowing the microorganims to multiply and colonise the solid material of the underground aquifer, reducing the hydraulic permeability of the underground aquifer and causing the precipitation of insoluble minerals in the solid material of the underground aquifer.

Description

PROCESS FOR IN-SITU CONTROL OF THE POLLUTION OF GROUNDWATER BY INFILTRATION OF TAILING TRANQUES WATERS

DESCRIPTIVE MEMORY

FIELD OF APPLICATION OF THE INVENTION

The present invention refers to a process for the "in situ" control of groundwater contamination by infiltration of mining tailings dam waters.

DESCRIPTION OF WHAT IS KNOWN IN THE SUBJECT

All mining activities generate waste, such as waste rock (excavated rocks that are not minerals) and tailings (the residue from the mineral extraction or refining process). In mining development, the design, operation and closure of a tailings management facility is a key part of the mining process. Effective environmental management of tailings disposal during the life of the mine and after closure is an important factor in managing long-term environmental impacts.

The problems associated with tailings storage are increasing. Advances in technology allow low-grade minerals to be exploited, generating a greater volume of waste that requires safe storage. Environmental regulations are also advancing, placing more stringent requirements on the mining industry, particularly with regard to tailings storage practices. Despite the rigorous studies that are carried out to decide its location, there is always the problem of the seepage of the dam waters into the groundwater table. It is for this reason that it is of great importance to have technologies that allow to control effectively and economically the contamination of the water tables, avoiding that the solutions generated by mining have a negative effect.

i Within the waste materials generated by the mining industry, tailings correspond to a fine suspension of solids in liquid. These are fundamentally constituted by the same material present in-situ in the deposit, in addition to reagents and chemical elements used in the mineral obtaining processes. The tailings contain high concentrations of chemicals and elements that alter the environment, so they must be transported and stored in "dams or tailings deposits", where the contaminants slowly settle to the bottom and the water is recovered or evaporated. Due to the decrease in the ore grades of the deposits, it will be necessary to extract more and more tonnages of material to maintain their production levels, which will mean a proportional increase in the amount of waste that must be disposed of in the form of tailings.

Currently, active and inactive tailings dams are exposed to possible failures that can generate important incidents in the environment, corresponding to 5 million m ³ of tailings released to the environment (Azam, S., & Li, Q. (2010 Tailings dam failures: a review of the last one hundred years. Geotechnical news, 28 (4), 50-54). Unfortunately, the reported incidents indicate that the failures have been generated in both active and non-active tailings dams (Venegas F. (2011), which implies that the closure of a mining site does not mean the end of the problem. Long-term failure mechanisms in tailings dams include cumulative damage (eg, erosion or earthquakes), geological hazards (eg, landslides), liquefaction, and changing weather patterns (Chambers, DM, & Higman, B. (2011) . Long term risks of tailings dam failure. Center for Science in Public Participation, Bozeman, Montana) Although it has been described that the majority of tailings dam failures are due to combined causes (39%) (Rico , M., et al. (2008) "Reported tailings dam failures: a review of the European incidents in the worldwide context." Journal of hazardous materials 152.2: 846-852), studies that have differentiated the causes of failure incidents worldwide, have shown that e Between 8% and 20% of incidents (1990-2009) are caused by leaks and the channels generated by them. This is consistent with the lack of control of the hydrological regime of the jam is one of the most common causes of failure. It is important to mention that these types of failures are the most obvious or most easily detectable, since they have caused important incidents that have been recorded over time. It is important to take into consideration that it has been estimated that the risk of failure in tailings dams is increasing, where it is directly related to the number of tailings accumulation dams greater than 5,000,000 cubic meters of capacity, necessary for allow the economic extraction of low grade minerals. Additionally, at least 11 catastrophic failures are expected worldwide between 2010 and 2019, with a total cost of approximately USD $ 6 billion (Mining (2015). Catastrophic mine waste spills increasing in frequency, severity and cost world-wide. Http: //www.mining.com/web/catastrophic- mine-waste-spills-increasing-in-frequency-severity-and-cost-world-wide /). Another type of seepage, which is not evident or easily detectable, corresponds to that which is generated from the waters of the dam basin towards the groundwater tables impacted by the tailings dam, which leads to the contamination of groundwater. In this case, it is not a question of failures or disasters of great violence and notoriety. On the contrary, these infiltrations into groundwater are a problem that occurs permanently and invisibly, but which can cause great damage, negatively impacting the health of people and the environment with toxic doses of certain elements. The flow of seepage from a surface tailings impoundment is known to be unavoidable and zero discharge from seepage from a waste facility is noted to remain an elusive goal, even with complex liner systems (Tailingsinfo (2013). Water Management Considerations for Conventional Storage. Http://www.tailings.info/technical/water.htm).

There are innumerable studies that report leaks into the water tables that are impacted by the infiltration of the tailings dam, and that show that this phenomenon is indeed occurring worldwide (Duda, R. (2014). The Influence of Drainage Wells Barrier on Reducing the Amount of Major Contaminants Migrating from a Very Large Mine Tailings Disposal Site. Archives of Environmental Protection, 40 (4), 87-99; Brindha K, Elango L. 2014. Geochemical Modeling of the Effects of a Proposed Uranium Tailings Pond on Groundwater Quality. Mine Water Environ. Vol. 33: 110-120; Huang, X. et al. (2016). Hydrogeochemical signatures and evolution of groundwater impacted by the Bayan Obo tailing pond in northwest China. Science of the Total Environment, 543, 357-372; Martín-Crespo, T. et al. (2018). Geoenvironmental characterization of unstable abandoned mine tailings combining geophysical and geochemical methods (Cartagena-La Union district, Spain). Engineering Geology, 232, 135-146). These studies are carried out with measurements inherent to each dam and allow evidence of the existence of this type of leakage.

At present, tailings dams consider in their design and operation different monitoring and control measures of possible leaks or seeps to prevent them from contaminating groundwater. Among the monitoring measures, the use of monitoring wells downstream of drainage collecting pools, a system for measuring flows, groundwater levels and monitoring of surface waters can be considered. Currently, the treatment of infiltrations in subterranean layers impacted by tailings dams is carried out using seepage collection wells installed downslope from the reservoir (Duda, R. (2014). The Influence of Drainage Wells Barrier on Reducing the Amount of Major Contaminants Migrating from a Very Large Mine Tailings Disposal Site. Archives of Environmental Protection, 40 (4), 87-99). This well is equipped with hydraulic pumps that send the seepage back to the tailings dam. These units can be used in conjunction with shear walls or trenches to minimize seepage downhill. In these wells, the quality of the effluent is monitored to determine the possibility of leaks, since any leakage that occurs in the underground layers can cause complications in the operation of the mining site with the consequent associated economic loss. It is important to mention that depending on the quality of the effluent, the operation of the hydraulic pump can continue indefinitely (EPA, 1994). Consequently, the current treatment with the hydraulic pump does not completely solve the problem.

Regarding the control measures, the water that filters through the wall is recovered through drains and the channeling of the flow. The water from the wall can be returned to the dam, or it can be collected in a common pond with the water recovered from the lagoon for its recirculation to the beneficiation plant (SERNAGEOMIN (2003) Guide of good environmental practices for small mining; Levenick JL et al. (2009). Hydrogeological assessment of seepage through the Antamina tailings dam - Antamina copper / zinc mine, Peru, South America, International Mine Water Conference 19th - 23rd October 2009, Pretoria, South Africa). The water that infiltrates at the subsoil level is recirculated from a series of catchment wells located downstream and is monitored by independent drilling. However, according to the information available, there is no technology that allows to control the total of the leaks or losses that occur at the subsoil level of the dam.

The problem that arises is that identifying the location of the leak is complicated, the accessibility of the leak is poor, and the treatment costs are very high. Traditional methods to repair the leak in situ, such as the injection of chemical compounds (e.g. silicates, acrylates, acrylamides, polyurethanes or biopolymers) are expensive and have negative effects on health and the environment (Ivanov, V., & Chu, J. (2008). Applications of microorganisms to geotechnical engineering for bioclogging and biocementation of soil in situ. Reviews in Environmental Science and Bio / Technology, 7 (2), 139-153). In addition, it is necessary to inject a large quantity of these compounds underground in order to reduce the overall permeability, since the exact location of the leak is often not known.

Considering the exposed problem, it is necessary to develop an alternative that is profitable and efficient enough to control groundwater contamination with tailings dam seepage. This type of leakage could be controlled by a treatment system that allows reducing the hydraulic permeability at the level of underground aquifers contaminated with high levels of sulfate. Taking into account that the injection of chemical compounds is high cost and has negative effects on health and the environment, one could consider the infiltration of microorganisms into contaminated groundwater to promote their growth in situ and reduce the permeability of the aquifer. . From the analysis of the state of the art, it can be deduced that currently there is no biotechnological development that allows solving the problem of leaks from tailings dams to groundwater. However, the use of ureolytic bacteria to reduce hydraulic permeability by precipitation of calcium carbonate has been described (Eryürük et al. "Reducing hydraulic conductivity of porous media using CaCÜ3 precipitation induced by Sporosarcina pasteurii." Journal of bioscience and bioengineering 119.3 (2015): 331-336). Most of the research has been carried out with the ureolytic bacteria Sporosarcina pasteurii in a medium containing urea and calcium chloride (Whiffin et al., 2007; van Paassen et al., 2010; Al-Thawadi, 2011; Al Qabany et al., 2012; Ng et al., 2012; Keykha et al., 2012; Ivanov, et al. 2015). Due to this enzymatic reaction, the pH increases and bicarbonate is produced. The precipitation of calcium carbonate begins, which binds the soil particles (Ca ²⁺ + CC> 3 ² CaC03j). From the technological point of view, most of the applications that use ureolytic bacteria, especially Sporosarcina pasteurii, as precipitating agents of calcium carbonate, have been oriented to microbial cementation processes (EP1974031 application, WO 2008120979 patent application, WO patent 2009133312, US patent 8,182,604, US patent 8,210,776 and US patent 9,199,880, US 8,951,786, US 8,728,365, US patent application 20130196419, US patent application 20180119185A1, US patent application 20180072632 , US Patent Application No. 15 / 066,692, US Patent Application No. 15 / 066,692).

The alternatives currently available for a biological treatment have different drawbacks, which is why they are not applicable to underground aquifers contaminated with seepage from tailings dams. For example, Sporosarcina pasteurii is unable to grow and its ureolytic activity is inhibited in the absence of oxygen (Martin et al., Inhibition of Sporosarcina pasteurii under Anoxic Conditions: Implications for Subsurface Carbonate Precipitation and Remediation via Ureolysis. Environmental Science & technology, 46 (15), 8351-8355, 2012). This is because the de novo biosynthesis of its urease enzyme, essential for the process, is repressed under anoxic conditions. For this reason, it is not feasible to apply this process in an underground aquifer that has low oxygen levels and that presents anoxic conditions when nutrients are injected to stimulate the growth of bacteria. The temperature in groundwater is characterized by being low, which hinders the enzymatic activity of ureolytic bacteria that have their optimum at 30 ° C (Kimet al. "Effect of Temperature, pH, and Reaction Duration on Microbially Induced Calcite Precipitation. "Applied Sciences 8.8: 1277, 2018). Another important disadvantage is that ureolytic bacteria are not capable of removing the sulfate ion, which is a pollutant found in high concentrations in the aquifer to be treated. To all this, one must add the special chemical characteristics of groundwater contaminated with infiltrations from tailings dams, which contains high concentrations of sulfate and other mineral salts and, especially, the presence of different toxic metals, which hinder the growth of many groups. of microorganisms. In natural environments, such as marine sediments and on microbial mats, the participation of sulfate-reducing bacteria in the precipitation of calcium carbonates has been suggested. However, the state of the art is discouraging regarding the true role of these microorganisms and their possible application. Thus, for example, Meiter in your work entitled "Two opposing effects of sulfate reduction on carbonate precipitation in normal marine, hypersaline, and alkaline environments" (Geology 41.4: 499-502, 2013) concludes that the biological reduction of sulfate causes a decrease in pH, which favors dissolution more than carbonate precipitation. The exact contribution of sulfate-reducing bacteria to carbonate mineralization remains highly controversial in the scientific literature (Gallagher et al. "Two opposing effects of sulfate reduction on carbonate precipitation in normal marine, hypersaline, and alkaline environments: COMMENT." Geology 42.1: e313-e314, 2014; Meister, "Two opposing effects of sulfate reduction on carbonate precipitation in normal marine, hypersaline, and alkaline environments: REPLY. "Geology 42.1: e315-e315, 2014). The use of carbonate precipitation by sulfate-reducing bacteria in an industrial process does not appear promising in the state of This is how Zhu and Dittrich ("Carbonate precipitation through microbial activities in natural environment, and their potential in biotechnology: a review." Frontiers in bioengineering and biotechnology 4: 4, 2016) concluded that “ureolysis is the most developed technology among other metabolisms, followed by photosynthesis. Although "sulfate reduction" has been studied extensively, it is less likely to be exploited in engineering projects. "

The solution described in the present patent application shows that bioprecipitation is technically possible under anoxic conditions, by sulfate reducing bacteria. This invention solves the problems of the state of the art by using a biological process to reduce the permeability of sectors of the aquifer affected by the leaks of the tailings dam waters, based on the injection into a subsoil with a nutrient solution and the inoculation of a microbial consortium that has a high capacity to biomineralize "in situ" (simultaneous processes of biocolmatación and bioprecipitation). This consortium has the ability to grow anoxic conditions, at low temperatures and in contaminated groundwater with seepage from a tailings dam. Due to the ability to reduce sulfate of the microbial consortium used, this process also allows to reduce the concentration of this contaminant.

Therefore, the proposed solution has the following advantages over the state of the art:

• A zone of low hydraulic conductivity is generated in the contaminated underground aquifer.

• The treated area is highly stable due to the organomineralization of calcium carbonate precipitation.

The process occurs even in the absence of oxygen • The process is designed to operate at low temperature (for example at 19 ° C)

• The microbial consortium is adapted to groundwater contaminated with seepage from a tailings dam.

The process allows additionally to reduce the sulfate concentration.

DEFINITION OF THE INVENTION

The main object of the present invention is a method to reduce the hydraulic conductivity and generate the precipitation of insoluble minerals in an underground aquifer, which comprises at least the steps of:

a) provide a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated,

b) inject the underground aquifer with an enriched culture of microorganisms, c) inject the underground aquifer with an electron donor and nutrients suitable for the culture of microorganisms enriched in reducing bacteria and one or more metal cations that favor the precipitation of insoluble minerals, and

d) allow microorganisms to multiply and colonize the solid material of the underground aquifer, reducing the hydraulic permeability of the underground aquifer and generating the precipitation of insoluble minerals in the solid material of the underground aquifer.

In one embodiment of the invention, the underground aquifer is affected by surface water seepage. A particular embodiment refers to an underground aquifer that is affected by water seepage from a mining tailings dam.

In one embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, is produced with a method that comprises at least the steps: a) inoculate a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated in a fixed-bed anaerobic bioreactor with a support material,

b) continuously feed with water extracted from the underground aquifer, an electron donor and adequate nutrients for the cultivation of sulfate-reducing bacteria,

c) allow microorganisms to multiply, colonize the bioreactor support material, and

d) producing an effluent from the bioreactor containing a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated to be injected into said aquifer.

In another embodiment of the invention, the bioreactor support material is selected from the group of sand, silica, glass and ceramics.

In another preferred embodiment of the invention, the injection of the underground aquifer with an enriched culture of microorganisms and the injection of the underground aquifer with an electron donor and nutrients suitable for the culture of microorganisms enriched in reducing bacteria, and one or more metal cations that favor the Insoluble mineral precipitation is done by one or more injection wells.

In yet another embodiment of the invention, the electron donor suitable for microorganism culture enriched in reducing bacteria is selected from the group of formate, formic acid, acetate and acetic acid.

In a further embodiment of the invention, said nutrients suitable for the culture of microorganisms enriched in reducing bacteria, comprise at least ammonium and phosphate.

In another embodiment of the invention, the nutrients suitable for the culture of microorganisms enriched in reducing bacteria, further comprise a Complex nutrient, rich in vitamins, selected from the group of yeast extract and strained corn water.

In one embodiment of the invention, the insoluble mineral that precipitates in the solid material of the underground aquifer is calcium carbonate.

In another embodiment of the invention, the insoluble mineral that precipitates in the solid material of the underground aquifer is iron sulfide.

In one embodiment of the invention, the method for reducing the hydraulic conductivity and generating the precipitation of insoluble minerals in an underground aquifer also allows the removal of sulfate from the underground waters of said aquifer.

In one embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Phylum Proteobacteria, Firmicutes and Bacteroidetes.

In another embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria from the Phylum Proteobacteria or Firmicutes.

In another embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Gammaproteobacteria, Clostridia, Deltaproteobacteria and Bacteroidia Classes.

In another embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Clostridia or Deltaproteobacteria Class. In another embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Desulfovibrionales Order.

In another embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Order Desulfobacterales.

In one embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains at least bacteria of the Desulfuromonadal Order.

In another embodiment of the invention, the culture of microorganisms enriched in sulfate reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Order Clostridiales.

In a preferred embodiment of the invention, the culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains at least one genus of sulfate-reducing bacteria selected from the group consisting of Desulfomicrobium, Desulfovibrio, Desulfonema , Desulfuromonas, Desufutomaculum and Desulfosporosinus.

In another preferred embodiment of the invention, the culture of microorganisms, enriched in sulfate-reducing bacteria adapted to the groundwater of the aquifer to be treated, is obtained from samples taken from live microbialites from lagoons or saline lakes. Samples of these live microbialites are found, for example, in live thrombolites from Lake Sarmiento, or in live stromatolites from Laguna Amarga, both in Torres del Paine National Park, Chile, in live microbialites from Laguna Interna and La Laguna. Laguna La Brava in the Salar de Atacama, Chile, in the Socompa lagoon, Salta, Argentina, in the Lagoa Salgada area of Rio de Janeiro, Brazil, in the Bacalar Lagoon and in the Alchichica Lagoon, Mexico or in the Clifton lake, Australia . In one embodiment of the invention, the culture of microorganisms, enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, is obtained with a method that comprises at least the steps:

a) inoculate a sample of said live microbialite from a saline lake or saline lagoon into a column filled with sand,

b) continuously feed with water extracted from the underground aquifer, an electron donor and adequate nutrients for the cultivation of sulfate-reducing bacteria,

c) allow microorganisms to multiply, colonize the bioreactor sand, and enrich the culture in sulfate-reducing bacteria, and

d) produce a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated.

In one embodiment of the invention, the living microbialite is a living stromatolite. In another embodiment of the invention, the live microbialite is a live thrombolyte.

Definitions:

Microbialites: in this invention, the concept of microbialites is understood as the "organo-sedimentary deposits that have increased as a result of a benthic microbial community trapped and attached to detrital sediments and / or forming the site of mineral precipitation such as calcium carbonate The structures of microbialites can range from well-structured laminated stromatolites to coagulated thrombolites. Microbialites have been found in different environments, for example, hot springs, dolomitic ponds, hypersaline and / or alkaline lakes, rivers and lakes, environments open marine and hypersaline marine environments.

Stromatolites: they are microbialites that are characterized by being laminated organo-sedimentary structures (typically CaCÜ3) that grow attached to the substrate and emerge vertically from it, producing structures of great morphological, volumetric and biogeographic variety. Thrombolites: these are microbialites related to stromatolites, but they lack lamination and are characterized by macroscopic coagulated tissue.

Living microbialites, stromatolites or thrombolites: they are organo-sedimentary structures that are currently found in lake or marine systems and that present an active microbial community, whose metabolism participates in the mineralization process of these structures.

Microbial consortium: in this invention, the concept of microbial consortium is understood as a group of different microorganisms that act together. In a microbial consortium, microorganisms with different metabolic capacities can be found. In the particular case of the sulfate-reducing microbial consortium, it is composed, for example, of fermentative, acetogenic and sulfate-reducing anaerobic microorganisms.

Bio-clogging: Bio-clogging is defined as the reduction in hydraulic conductivity and porosity of a saturated porous medium due to microbial growth.

Biologically Induced Mineralization: Biologically Induced Mineralization is the process in which the metabolic activity of microorganisms (for example, bacteria) produces favorable chemical conditions for the formation of minerals (for example, the precipitation of calcium carbonates induced by increased alkalinity).

Description of Figures

FIGURE 1 :

This figure shows a schematic diagram of the sand-filled bioreactor. The bioreactors (1) were made of glass. For temperature control, an outer glass jacket (2) was used, fed with water from a thermoregulated bath. The bioreactor was filled with sterilized sand (3). To control the internal pressure of the system, a pair of glass piezometers located one in the lower part (4) and another in the upper part (5) of the bioreactor. The feeding was done by peristaltic pumps from the lower part (6) to the upper part (7) of the bioreactor.

FIGURE 2:

This figure shows the parameters in bioreactor effluent inoculated with microorganisms from a live thrombolite from Lake Sarmiento. (A) pH values, (B) sulfate concentrations (filled squares) and calcium concentrations (open squares). FIGURE 3:

This figure shows the parameters in the effluent of a bioreactor inoculated with microorganisms from a live stromatolite from Laguna Amarga. (A) pH values and oxidation-reduction potential, (B) sulfate concentrations (filled squares) and hydrogen sulfide concentrations (open squares), (C) calcium concentration.

FIGURE 4:

This figure shows schematically the design of the steel columns of 1.40 meters high and 25 centimeters in diameter, with lateral outlets at 8, 50, 90 and 130 centimeters from the base for the installation of pressure measuring instruments, used to measure the reduction of hydraulic conductivity in the sand bed by the enriched microbial consortia.

FIGURE 5:

This figure shows the parameters in the effluent from column A inoculated with the microbial consortium enriched from the sample of the microbialite from Lake Sarmiento. (A) Microbial ATP in the effluent (open squares) and oxidation-reduction potential (solid squares), (B) sulfate concentrations (open diamonds) and hydrogen sulfide in the effluent (solid diamonds). The line indicates the sulfate concentration in the added culture medium. FIGURE 6: This figure shows the parameters in the effluent from column B inoculated with the microbial consortium enriched from the stromatolites sample from Laguna Amarga. (A) Microbial ATP in the effluent (open squares) and oxidation-reduction potential (solid squares), (B) sulfate concentrations (open diamonds) and hydrogen sulfide in the effluent (solid diamonds). The line indicates the sulfate concentration in the added culture medium.

FIGURE 7:

This figure shows the parameters in the effluent from column C with biocide. (A) Microbial ATP in the effluent (open squares) and oxidation-reduction potential (solid squares), (B) sulfate concentrations (open diamonds) and hydrogen sulfide in the effluent (solid diamonds). The line indicates the sulfate concentration in the added culture medium.

FIGURE 8:

This figure shows the pH measurements in bioreactors B1, B2 and B3. Bioreactors B1 and B3 were fed with culture medium A, but bioreactor B1 was switched to culture medium B on day 170. Bioreactor B2 was permanently fed with culture medium B.

FIGURE 9:

This figure shows the determination of intracellular ATP and the bacterial count in bioreactors B1, B2 and B3. Bioreactors B1 and B3 were fed with culture medium A, but bioreactor B1 was switched to culture medium B on day 170. Bioreactor B2 was permanently fed with culture medium B.

FIGURE 10:

This figure shows the sulfur (hydrogen sulfide) measurements in bioreactors B1, B2, and B3. Bioreactors B1 and B3 were fed with culture medium A, but bioreactor B1 was switched to culture medium B on day 170. Bioreactor B2 was permanently fed with culture medium B.

FIGURE 11: This figure shows the remaining sulfate measurements in bioreactors B1, B2, and B3. Bioreactors B1 and B3 were fed with culture medium A, but bioreactor B1 was switched to culture medium B on day 170. Bioreactor B2 was permanently fed with culture medium B. FIGURE 12:

This figure shows an example of the aquifer model design built on the basis of acrylic chambers. Shown is an acrylic chamber with a removable lid packed with quartz sand. In the figure you can distinguish the ports for inoculation and stimulation of microorganisms, and for taking samples. At their ends, a pipe was installed internally for the flow water inlet and another for the effluent outlet.

FIGURE 13:

This figure shows the microbial sulfate reduction (S04 ^2- and H2S) and physicochemical (pH and oxide-reduction potential) parameters in the inoculated aquifer models. Measurements for hydrogen sulfide (A), sulfate (B), pH (C), and oxide reduction potential (D) are shown. The values correspond to the average of duplicates (n = 2) for the inoculated models (filled circles), and to a single value (n = 1) in the case of the non-inoculated control (empty circles).

FIGURE 14: This figure shows the intracellular ATP values in the effluent of the aquifer models. The values correspond to the average of duplicates (n = 2) for the inoculated models (filled circles), and to a single value (n = 1) in the case of the non-inoculated control (empty circles).

FIGURE 15:

This figure shows the relative retention times of the aquifer models during the experimentation time. The retention times relative to the initial retention time of the aquifer models are shown. The values correspond to the average of duplicates (n = 2) for the inoculated models (circles filled), and to a single value (n = 1) in the case of the non-inoculated control (empty circles).

FIGURE 16:

This figure shows the change in tracer flow around a zone of lower permeability in the porous medium of the aquifer models. An example of the tracer passage in one of the aquifer models is shown on day 55 of operation. Tracer deviation is observed when meeting the blackened area of the porous medium (o): inoculation point. FIGURE 17:

This figure schematically shows a pH map in the sand of the inoculated aquifer models at the end of the experiment (60 days). In all inoculated models, a zone of pH 8 is observed that is distributed from the inoculation zone and reaches the end where the effluent exits, as well as specific zones of pH 8.5 in one of the duplicates. In contrast, in the control model a pH 7.5 was observed in all areas of the model. The circle represents the point of inoculation and stimulation with culture medium.

FIGURE 18:

This figure schematically shows the microbial ATP maps in the sand from the aquifer models at the end of the experiment. The ATP value (in URL / g of sand) in the three zones of the sand of the inoculated aquifer models (n = 2) and the control model (n = 1) is shown. The black arrows indicate the direction of the hydraulic flow inside the aquifer models. The circle represents the point of inoculation and stimulation with culture medium.

FIGURE 19:

This figure is a photograph illustrating the biomineralization of quartz sand at the inoculation point of the aquifer models. A portion of biomineralized quartz sand that was located at the inoculation point of the inoculated models is shown. In areas more distant from the inoculation point, the formation of sand agglomerates similar to the one in the image was not observed. FIGURE 20:

This figure shows the microbial sulfate reduction (SCU ^2- and H2S) and physicochemical parameters (pH and oxide-reduction potential) in the aquifer models stimulated with the different formate concentrations. The measurements of sulfate (A), hydrogen sulfide (B), oxidation-reduction potential (C) and pH (D) of the aquifer models stimulated with the different formate concentrations (3, 6, 12 and 18 g / L). The values correspond to the average of duplicates (n = 2).

FIGURE 21:

This figure shows the remaining formate concentration in the aquifer models stimulated with the different concentrations of the electron donor. The solid lines represent the amount of formate added in the models considering the amount of formate in the culture medium and its dilution in the models. The values correspond to the average of duplicates (n = 2).

FIGURE 22:

This figure shows the retention times relative to the initial time in the aquifer models stimulated with the different formate concentrations. The values correspond to the average of duplicates (n = 2).

FIGURE 23:

This figure schematically shows the pH maps in the sand of the aquifer models stimulated with the different formate concentrations at the end of the experiment (60 days). In general, the pH values in sand tend to increase as the concentration of formate in the growing medium is increased. In the models stimulated with 12 and 18 g / L of formate, specific areas of pH 8.5 were observed. The black arrows indicate the direction of the hydraulic flow inside the aquifer models. The circle (o) represents the point of inoculation and stimulation with culture medium. FIGURE 24:

This figure schematically shows the microbial ATP maps in the sand from the aquifer models stimulated with the different formate concentrations at the end of the experiment. The ATP value (in URL / g of sand) is shown in the three zones of the sand of the aquifer models stimulated with the different formate concentrations. The black arrows indicate the direction of the hydraulic flow inside the aquifer models. The white circle (o) represents the point of inoculation and stimulation with culture medium. The values correspond to the average of duplicates (n = 2).

FIGURE 25:

This figure shows the biologically induced mineralization of the quartz sand from the aquifer models inoculated with the different formate concentrations at the end of the experiment. Aquifer models uncovered at the end of the experiment are shown. The red line delimits the mineralized zone (of greater hardness compared to other zones) of the porous medium of the aquifer models. All the models (duplicates) used are shown (central column). The bioreactor on the right shows the percentage of the area occupied by the mineralized zone with respect to the total surface of the upper face of the models (Calculated using the ImageJ program).

FIGURE 26:

This figure shows the surface area of biomineralized sand in the aquifer models as a function of the formate concentration in the culture medium. A direct dependence of the formate concentration and the area of the biomineralized sand is observed in the aquifer models, which reaches a maximum value at the highest concentrations tested (12 and 18 g of formate / L). The values correspond to the average of duplicates (n = 2). FIGURE 27:

This figure shows the percentages of total carbonates determined by acid-base titration in the porous medium of the aquifer models stimulated with the different formate concentrations at the inoculation point and downstream at the end of the experiment. Bars represent the mean value of duplicates (n = 2). FIGURE 28:

This figure shows the images obtained by scanning electron microscopy of the quartz sand from the aquifer models stimulated with the lowest and highest concentrations of formate (3 and 18 g / L) at the end of the experiment. A: Crystalline CaCÜ3 mineral and bacterial clumps in a quartz sand sample from one of the models stimulated with 3 g / L formate. B: CaCÜ3 crystalline mineral and bacterial clusters in a quartz sand sample from one of the models stimulated with 18 g / L of formate. ac: quartz sand carb: carbonate. Arrows: bacterial clusters.

FIGURE 29:

This figure illustrates the flow diagram of a particular application of the process to reduce hydraulic permeability and generate precipitation of insoluble minerals in situ from a sulfate-contaminated underground aquifer.

The following examples illustrate some specific applications of the invention, but are not intended to limit the scope or scope of the present invention.

EXAMPLES

Example 1

Enrichment of a sulfate-reducing microbial consortium from live thrombolites.

Samples of live thrombolites were taken from Lake Sarmiento de Gamboa (51 ° 03O0 "S, 72 ° 45'0 W), Torres del Paine National Park, XII Region, Chile. Sarmiento Lake has a maximum depth of 312 m and an approximate area between 83 and 84 km ^2. Lake Sarmiento de Gamboa is an alkaline lake with an average salinity of 1.9 mg / L, a pH between 8.3 and 8.7, and an average surface temperature of 6.2 ° C in winter and 12.2 ° C in summer. Lake Sarmiento has live microbialites, which have a non-laminated structure made up of carbonate clots which classifies them as thrombolites. These living thrombolites are found under the surface of the water mirror (Solari et al. Paleoclimatic significance of lacustrine microbialites: A stable isotope case study of two lakes at Torres del Paine, Southern Chile. Palaeogeography, Palaeoclimatology, Palaeoecology, 297 (1), 70-82, 2010).

The sampling process consisted of the manual extraction of live thrombolites located underwater on the shore of the Northeast sector of the lake, which were placed at 6 ° C in sterile plastic jars. Because it is a State Protected Wild Area, permission was obtained from the National Forestry Corporation (CONAF Authorization N ° 012/2014) to carry out the sampling.

By means of X-ray diffraction (D8 Advance X-ray diffraction equipment, Bruker, Germany), it was determined that the mineralogy of living thrombolites from Lake Sarmiento is mainly represented by magnesium calcite (Mgo, o6Cao, 94CC> 3).

Sand-filled bioreactors were used to enrich the consortium (Figure 1). The bioreactors (1) were made of glass and had 173 mL capacity, 18 cm in length and 3.5 cm in internal diameter. For temperature control an outer glass jacket (2) was used, fed with water from a thermoregulated bath at 18 ° C. The bioreactor was filled with sand (3). To do this, a filter was installed at the bottom of the bioreactor to prevent sand from running into the feed hoses. This filter consisted of a PVC tube covered with a plastic mesh, through the lower opening. The bioreactor was filled with sterile distilled water, taking care to remove all trapped air. Subsequently, a layer of 40 g of coarse sand (approximately 850 pm) was added, on which 120 g of a layer of fine sand (300 to 500 pm) was placed. Both types of sand were previously washed with 10% HCl, which was rinsed with distilled water until the rinse reached pH 6.

After washing, the sand was sterilized and dried in an oven at 180 ° C for 2 hours. To control the internal pressure of the system, a pair of piezometers of glass located one at the bottom (4) and one at the top (5) of the bioreactor. The difference in the water height of the piezometers allowed determining the hydraulic conductivity. The feeding was done by peristaltic pumps (Cole-Palmer, model 7557-14, 1-100 rpm, USA) from the bottom (6) to the top (7) of the bioreactor, with an upward flow of 0.2 mL. / min.

The external jacket of the bioreactor was connected to the thermoregulated bath with silicone hoses, while the other connections were made as follows: with silicone hoses, the inlets, outlets and installation of the piezometers; with Tygon hoses (impermeable to gaseous exchange) the feeding, recirculation and effluent outlet routes. All the hoses of the system, and their connectors, were previously sterilized in an autoclave. To generate the feed flow, a peristaltic pump (Cole-Palmer, model 7557-14, 1-100 rpm, USA) was used. To prevent changes in the internal temperature of the system, along with preventing the passage of light to the interior (simulating the conditions of the underground layer), the bioreactors were wrapped with a thermal insulator.

Once the bioreactor was assembled, a washing was carried out by pumping distilled water, until no turbidity was seen in the effluent. Finally, the bioreactor was fed with approximately 1 L of sterile PCM culture medium, the composition of which is indicated below: K2SO4 2.4 g / L; MgSO4 ^* 7H ₂ 0 0.061 g / L; NaHCOs 0.35 g / L; CaCl ₂ 1.3 g / L; K ₂ HPC> 4 0.01 g / L; Na ₂ (Si0 ₃ ) 0.009 g / L; 0.28 g / L sodium citrate; NhUCI 1 g / L; yeast extract 1 g / L; sodium acetate 1.3 g / L. The medium was adjusted to pH 7.9. The culture medium was prepared in water extracted groundwater hydraulic barrier of a tailings dam, whose main components are HCO3 ^· 217.7 mg / L, Ca ²⁺ 302 mg / L, Mg ²⁺ 76.6 mg / L and SCL ^2- 1192.3 mg / L. Groundwater extracted from a contaminated aquifer was used to prepare the culture medium to enrich a microbial consortium adapted to the environmental conditions of the site to be treated.

Once the entire system was filled with PCM medium, it was immediately inoculated. This process involved a stage of introduction of a live microbialite sample to the bioreactor and after a colonization period, which consisted of recirculation at 10 rpm (with a nominal flow rate 2.1 ml / min) for 24 hours, the column was fed with culture medium, without recirculation , at an average flow rate of 173 mL / day for 68 days, then at 80 mL / day for 26 and finally at 18 mL / day until the end of the experiment.

To determine the sulfate concentration in solution, the standard method based on the precipitation of the sulfate contained in the samples as barium sulfate (BaSCL) that is formed by adding the soluble salt of barium chloride (BaCh) in excess was used (American Public Health Association, American Water Works Association and Water Environment Federation. 1998a. "4500-S04-2 E". In: "Standard methods for the examination of water and wastewater". Ed. 20). The barium sulfate formed was determined by turbidimetry using a spectrophotometer at 450 nm (Dynamica spectrophotometer, model HALO RB-10, Great Britain).

The concentration of calcium in solution was performed by absorption spectrometry, using the following protocol: 1 mL of effluent was centrifuged for 10 minutes at 11,600 g (Microcentaur MSB010.Cx2.5 centrifuge; Sanyo, Great Britain), 500 pL of supernatant was diluted in distilled water, to bring the determination within the measurable range. From the mentioned solution, 1 mL of solution was taken and added to a test tube with 8 mL of distilled water and 1 mL of a 4,000 mg / L Sr ²⁺ solution with 0.8N HCIO4. Finally the solution is measured using a Perkin Elmer atomic absorption spectrophotometer, model 3110, USA (American Public Health Association, 1992a; American Public Health Association, 1992b).

The result of the enrichment of the microbial consortium is shown in Figure 2. In part (A) it is observed that the pH values in the effluent reach values equal to 8.5. The alkalinization generated by this consortium is a very important condition for biologically induced mineralization. This increase becomes more apparent as the feed volume decreases. For their part, sulfate concentrations, plotted in part (B), indicate a tendency to decrease associated with decreased feeding volumes. In this case, sulfate concentrations lower than 2500 mg / L were reached. This suggests the action of sulfate reducing bacteria in this consortium. For calcium concentrations, a progressive decrease is observed from the point at which the feeding volume was reduced, it should be noted that calcium concentrations were even registered below 200 mg / L. The reduction in calcium concentration is explained by the mineralization of calcium carbonate inside the bioreactor (biologically induced mineralization). The effluent from this column, which contains the sulfate-reducing microbial consortium from thrombolites from Lake Sarmiento, Torres del Paine, is used as an inoculum for a process that reduces hydraulic permeability and generates the precipitation of insoluble minerals in a contaminated aquifer. . In addition, this effluent is used to inoculate a new bioreactor of equal or greater size. Example 2

Enrichment of a sulfate-reducing microbial consortium from live stromatolites.

Live stromatolites were sampled from Laguna Amarga (50 ° 58'27 "S, 72 ° 44'55" W), Torres del Paine, XII Region, Chile. Laguna Amarga is a shallow endorheic mesosaline lagoon (maximum depth 4 m) with an approximate area of 1. 9 km ² . The average pH of the lake is 9.1, while the average salinity and temperature is 26.1 mg / L and 11.7 ° C, respectively. La Laguna Amarga presents live laminar microbialites or stromatolites, with bulbous, domed and elongated shapes (Solari et al. Paleoclimatic significance of lacustrine microbialites: A stable isotope case study of two lakes at Torres del Paine, Southern Chile. Palaeogeography, Palaeoclimatology, Palaeoecology , 297 (1), 70-82, 2010). Living dome-shaped stromatolites are composed of a sequence of white and gray sheets. The sampling process consisted in the manual extraction of stromatolites located near the eastern edge of the lagoon, which were placed at 6 ° C in sterile plastic jars. By means of X-ray diffraction (X-ray diffraction equipment D8 Advance, Bruker, Germany), it was determined that living stromatolites present carbonate minerals, particularly aragonite (Ca (CC> 3), in addition to halite (NaCl), quartz ( SiOz), Calcian albite ((Nao, 84Cao, i6) Ali, i6S¡2,8408) and anorthite (Nao, 25Cao, 7i (AÍ2S¡208)).

To carry out the enrichment of the consortium, bioreactors filled with sand were used, according to what was previously described in Example 1. The inoculation of the bioreactors with a sample of stromatolite from the Laguna Amarga and the subsequent colonization of the sand was also carried out. as already described in Example 1. The culture medium was then fed to the bioreactor, without recirculation, with a flow rate of 30-90 mL / week. The determination of the sulfate and calcium concentrations in solution were carried out according to Example 1.

The measurements of hydrogen sulfide (or sulfides) were carried out according to what is indicated in "Standard Methods for the Examination of Water and Wastewater" (American Public Health Association, American Water Works Association and Water Environment Federation. 1998b. "4500-S- 2 D ". In:" Standard methods for the examination of water and wastewater ". Ed. 20.), whose foundation is the measurement of the absorbance produced by the formation of methylene blue when the compound N, N-dimethyl- 1,4-phenyldiamino oxalate and the sulfur present in the sample.

The result of the enrichment of the microbial consortium is shown in Figure 3. In Figure 3A it is observed that the pH values in the effluent reach values close to 8.5 at 120 days and that the potential for oxide reduction tends to decrease. in time, which is suitable for the establishment of a sulfate reducing microbial consortium. For its part, Figure 3B shows a significant reduction in the sulfate concentration in the bioreactor effluent, compared to the concentration added with the culture medium. In this case, it was reached sulfate concentrations less than 1500 mg / L. In Figure 3B it can also be seen that from day 100 the hydrogen sulfide concentration increases significantly. This shows the activity of the sulfate reducing microorganisms in the enriched microbial pool. As in example 1, Figure 3C shows a progressive decrease in the concentration of calcium in the effluent compared to what is added in the culture medium.

The effluent from this bioreactor, which contains the sulfate-reducing microbial consortium enriched from stromatolites from the Laguna Amarga, is used as inoculum for a process that allows reducing hydraulic permeability and generating the precipitation of insoluble minerals in a contaminated aquifer. In addition, this effluent is used to inoculate a new bioreactor of equal or greater size.

Example 3

Decrease in hydraulic conductivity in columns

To demonstrate the decrease in hydraulic conductivity due to the growth of the microbial consortium in a sand bed, a laboratory prototype was designed and built, consisting of an AISI 304 stainless steel column. Figure 4 schematically shows the design of the columns. 3 columns of 1.40 meters high and 25 centimeters in diameter were used, with lateral exits at 8, 50,

90 and 130 centimeters from the base for the installation of pressure measuring instruments. Filters made of PVC mesh and stainless steel mesh with pores of 0.3 mm diameter were installed at the base of the column. In addition, stainless steel mesh filters were placed alongside the columns to protect the pressure measurement outlets.

For the columns, 70 kilograms of 35-50 mesh quartz sand was used, which was not sieved again to pack the columns. However, its particle size was characterized by sieving (Erweka AR 400 sieve, Germany) and subsequently weighing the sand that remained in each sieve (Table 1). The sand used in all the columns was washed with water, later it was treated with 20% hydrochloric acid to remove carbonates, it was neutralized by washing with distilled water and finally it was dried and sterilized in a Pasteur oven at 180 ° C for two hours.

Table 1. Characterization of the sand used in packing the columns.

All the columns were packed filling them with water until reaching 30 centimeters in height, and then adding the sand evenly until it decanted. The columns were packed on a stainless steel mesh placed on a bed of quartz stones between 0.64 cm and 2.5 cm in diameter. The latter in order to avoid the descent of the packed sand towards the feed hoses.

Once the sand was finished adding to the column, the column cover was placed and the hose connections made using Tygon hoses. Then a hose connected to a thermoregulated bath was wound along the columns and the structure was surrounded with a thermal insulator, in order to keep the interior of the columns at 18 ° C.

The following culture medium was used: K2SO4 4.6 g / L; MgSO4 * 7H ₂ 0 0.06 g / L; NaHCOs 0.035 g / L; CaCl ₂ ^* 2H ₂ 0 3.4; NhUCI 1 g / L; Na ₂ (Si0 ₃ ) 0.009 g / L; KH ₂ R0 ₄ 0.05 g / L; 0.279 g / L sodium citrate; sodium acetate 4.5 g / L; yeast extract 1 g / L; sodium thioglycolate 0.099 g / L. The culture medium was prepared with groundwater extracted from the hydraulic barrier of a tailings dam, whose main components are HCO3 ^· 217.7 mg / L, Ca ²⁺ 302 mg / L, Mg ²⁺ 76.6 mg / L and 1192.3 mg SCL ^2- / L. In addition, thioglycollate was also added, a reducing compound that gives the culture medium a suitable oxide reduction potential for the development of sulfate reducing bacteria.

Column A was inoculated with the effluent from the bioreactor of Example 1. Column B was inoculated with the effluent from the bioreactor of Example 2. Column C (control without microorganisms) was not inoculated and a biocide was added. For the inoculation, 50 liters of culture medium were passed through each stainless steel column, later columns A and B were inoculated with 450 mL of the effluent from the bioreactor of Example 1 and Example 2, respectively. Column C was not inoculated and 920 mg / L of cadmium sulfate was added to the culture medium that was fed to it to inhibit microbial development.

All the columns were started with a recirculation of the medium for 15 days, to favor the adherence of the microorganisms on the sand particles of the filling. Subsequently, culture medium was added, continuously, according to the following feeding regime for each of the columns:

- Column A: 816 mL / day 16 to 29, 302 mL / day from day 30 to 106, 907 mL / day of day

107 to 138, 302 mL / day from day 139 to 168 and 907 mL / day from day 169 to 182.

- Column B: 816 mL / day 16 to 29, 302 mL / day from day 30 to 126, 907 mL / day from day 127 to 172.

- Column C: 816 mL / day 16 to 29, 302 mL / day from day 30 to 126, 907 mL / day from day 127 to 172.

The determination of sulfate and calcium concentrations was carried out according to Example 1 and the measurement of hydrogen sulfide (or sulfides) in solution, according to Example 2.

The ATP concentration in the effluents was determined with the commercial ATP measurement system LixKit ^® , Biohidrica, Chile, which is based on the reaction of ATP-dependent oxidation of luciferin catalyzed by the enzyme luciferase. The amount of light produced by the reaction was determined using a Kikkoman luminometer, model Lumitester PD-20, Japan. For this, 5 mL of effluent from the models were taken, from which the microorganisms were concentrated by filtration on nitrocellulose membranes (0.22 pm). Subsequently, the filtered microorganisms were collected with the torula provided in the system, which is inserted into the reaction rod containing the cell lysis liquid and the other lyophilized components for ATP-dependent oxidation of luciferin. The luminescence value obtained (in Relative Light Units, URL), was divided by 5 to obtain the value of URL / mL.

In the case of column A, in Figure 5A, a decrease in the oxidation-reduction potential is observed throughout the experiment, which favors the establishment of the reducing sulfate metabolism in the column, since this metabolism is associated at low potential levels. Figure 5A shows that the intracellular ATP measurement in the column effluent is directly related to the microbial activity present, it remains high throughout the experiment, although with a downward trend at the end of the experiment. Figure 5B shows that during a period of 175 days of operation the column inoculated with the enriched consortium was able to reduce sulfate. Regarding the production of hydrogen sulfide in this column (Figure 5B), a tendency to increase in the values of the sulfide measurements can be observed over time, with a greater slope from the 120th operating day, when the sulfate reduction inside the column increases, reaching values close to 180 mg / L towards the end of the experiment.

For column B it was observed that the oxidation-reduction potential has a similar behavior to column A (Figure 6A), reaching negative values and remaining at the end of the experiment around -350 mV. This indicates the development of a sulfate reducing metabolism within the inoculated column. Figure 6A shows high values of intracellular ATP in the effluent of the column at the beginning of the experiment, which decrease over time. The sulfate measurements from the effluent, shown in Figure 6B, indicate that from the beginning of the experiment there was a decrease in the sulfate values in the effluent from column B compared to the sulfate concentration fed with the medium. cultivation. Figure 6B shows an active production of hydrogen sulfide from day 60, increasing its concentration in the effluent, to reach values of 450 mg / L on day 140.

As a comparative control, in Figure 7A it can be seen that unlike what happened in columns A and B, column C with biocide there is no reduction in the reduction oxide potential, maintaining the oxidative conditions with an average value of + 200 mV. Measurement of intracellular ATP in the effluent showed low levels of microbial activity. In the same way, this column did not show changes with respect to the sulfate concentration of the culture medium (Figure 7B). It was also not possible to detect the production of hydrogen sulfide in this column, which confirms the absence of sulfate-reducing microbial activity (Figure 7B).

To assess whether the growth and activity of the microbial consortium reduce the flow of a liquid through the sand, hydraulic conductivity was used. This parameter measures the ease with which an aquifer transmits water. The more difficult it is for the flow, the lower the hydraulic conductivity. Hydraulic conductivity was determined at the end of the experiment in the columns, performing pressure measurements with pressure transmitters (flush diaphragm pressure transmitters model C9000156, Veto, Chile). Hydraulic conductivity was calculated using Darcy's equation, presented below:

Where "Hsat" is the saturation hydraulic conductivity, "Q" is the flow rate (feed rate of the column), "L" is the distance between the measuring stations, "A" is the diameter area of the column and "DH "is the difference between the heights of the measured water columns. Subsequently, the value obtained was normalized with a measurement of Hsat at time zero, with which a dimensional value was obtained, both measurements being made at the same speed.

Table 2. Hydraulic conductivity in the columns. Values obtained in the calculation of the dimensionless hydraulic conductivity (Hsat).

It was found that the hydraulic conductivity generated during the test was lower in columns A and B inoculated with the enriched consortia in Example 1 and Example 2, compared to the hydraulic conductivity of column C, in which growth was prevented. of microorganisms by adding the biocide. The greatest reduction in hydraulic conductivity occurred in column B, inoculated with the sulfate-reducing microbial consortium enriched from thrombolites from Lake Sarmiento.

Example 4

Effect of culture medium on biologically induced mineralization and bio-clogging in bioreactors

In order to show the effect of the culture medium on the biologically induced mineralization and bio-clogging, three bioreactors filled with sand of 250 ml of internal volume were mounted according to Example 1 and were named: B1, B2 and B3. Each of the bioreactors contains quartz sand as internal support material (particle size 297-841 pm) and are maintained under anaerobic conditions at a temperature of 18 ° C and periodically fed with culture media A and B described in the Table 3. The media differ because medium A contains formate, while medium B has acetate as major electron donor. Furthermore, medium A lacks citrate and has a lower concentration of yeast extract. These culture media were prepared in water from the tailings dam, which had a pH of 7.9 and a sulfate concentration of 1500 mg / L. The three bioreactors were inoculated with the effluent from the bioreactor of Example 1, which contains the sulfate-reducing microbial consortium enriched from Lake Sarmiento thrombolites.

Two bioreactors were fed with culture medium A (B1 and B3), however, bioreactor B1 was switched to culture medium B on day 170. On the other hand, bioreactor B2 was permanently fed with culture medium B To each of the bioreactors, periodic measurements of physical-chemical parameters (pH, sulfate, sulfur,) and biological parameters (bacterial count, ATP) were carried out, in order to determine and establish possible conditions that occur in the assembled systems.

Table 3: Composition of culture media

The determination of the concentrations of sulfate, calcium, according to Example 1, ATP according to Example 3 and hydrogen sulfide (or sulfides) in solution were carried out according to Example 2. The microbial count was carried out using a Petroff Hausser chamber in a phase contrast microscope (Zeiss, Standard model 20, Germany).

With respect to the physicochemical parameters, the pH shows a clear increase (> 8.2) in the bioreactor B2, which contains formate as a carbon source (culture medium A). In the same way, the B1 bioreactor, when supplied with formate (from day 170 of operation), the pH increases considerably from 7.2 and exceeding 8.5. Bioreactor B3 maintains a constant pH, varying slightly between 7.1 and 7.6 (Figure 8).

The bacterial count (Figure 9), maintains a variable rhythm until day 125, however, then reaches a steady state until day 170, maintaining a count of the 3 bioreactors between 1, 8 and 3.0 x 10 ⁸ bacteria / mL. The same is observed in intracellular ATP measurements. A significant increase in the bacterial count (5.0 x 10 ⁸ bacteria / mL) is observed in bioreactor B1 when switching from electron donor to formate on day 200 (culture medium A).

An increase also occurs with the sulfide values (Figure 10), which amount to almost 700 mg / L in the B1 bioreactor when changing to formate and consequently a decrease in the sulfate values (Figure 11).

The results of this example show the importance of the culture medium in the generation of an alkaline environment, conducive to inducing the precipitation of calcium carbonate. It is concluded that formate is a suitable electron donor to increase the pH inside the bioreactor.

Example 5

Biologically induced mineralization and bio-clogging in model aquifers

To show the effect of the injection of nutrients and the microbial consortium in a contaminated underground aquifer, an aquifer model or (sandbox) was designed in which the water flows horizontally through a sand bed. East Aquifer model has an inlet (infiltration well) for the injection of nutrients and microbial consortium and sampling ports. For the construction of the aquifer models, transparent acrylic chambers (Plexiglas) with internal dimensions of 32 x 12 x 1 cm were used; length, width and height respectively (Figure 12). These were provided with a removable lid, which had holes where ports were installed for the inoculation and stimulation of microorganisms, and a sampling port located downstream with respect to the previous ones. To establish a homogeneous hydraulic flow front through the entire section of the models, an internal pipe was installed at one end, which spanned the entire width of the model, built with Tygon hose (MasterFlex) perforated every 1 cm, It was used for the water inlet that simulates the underground aquifer hydraulic flow. At the other end, a Tygon hose was installed for the effluent outlet of the models. Then, the internal volume of the model (384 mL) was packed with 545 g of quartz sand with a particle size between 200-800 pm, which was previously washed with 10% HCl (v / v), rinsed with distilled water. , and dried at 180 ° C for 4 hours to eliminate carbonates and microorganisms that may be found before the experiments. Once the systems were armed, distilled water was passed through them using peristaltic pumps, setting the pump outlet flow at 0.6 m / L. The standardization of the basic hydraulic parameters of the models was carried out using distilled water, which was replaced by hydraulic barrier water one day before the beginning of the infiltration of the microorganisms.

Once the aquifer models were built, the hydraulic flow was established using distilled water, at the previously mentioned speed. To achieve a constant flow speed inside the models, they worked for 48 hours only with water, after which the following hydraulic parameters were determined prior to inoculation of the models:

To determine the retention time of the model aquifer, 250 mL of a brilliant blue (AB) solution (as a tracer) at a concentration of 100 mg / L was passed through the water inlet of the models flow, and samples were taken from the sampling port every 30 minutes since its entry into the models was observed. This procedure was performed until the The concentration of the tracer in the sample port reached half the concentration in the initial solution (i.e., 50 mg / L), and that time was recorded as the time required for the tracer to travel through that model section (or time retention). The AB concentration was determined in a spectrophotometer at 630 nm (Dynamica spectrophotometer, model HALO RB-10, Great Britain).

For the flow of the liquid in the aquifer models, the effluent volume was collected during 5 min in a test tube previously tared. The value obtained was divided by 5 to obtain the effluent volume in mL per minute.

The effluent flow value measured in 13 aquifer models (without inoculating with microorganisms) was 0.61 ± 0.04 mL / min (mean ± standard deviation) reflecting the value established in the peristaltic pumps (close to 0.6 mL / min). The retention time determined in all the aquifer models was 228 ± 27 min. The variability observed in retention times is due to minor differences in packaging.

Once the hydraulic parameters were determined, the aquifer models were inoculated with the sulfate-reducing microbial consortium using the effluent from the bioreactor B1 of Example 4. The microbial composition of this consortium is detailed below in Example 6 (see Tables 4, 5, 6, 7 and 8). For this experiment, 3 aquifer models were used and they were infiltrated at 0.2 mL / min, (n = 2 models). In all experiments, each inoculum pulse contained T10 ⁹ microorganisms as calculated by microbial count. In addition, a non-inoculated control model was used (n = 1 model), in which hydraulic barrier water was infiltrated in order to simulate inoculation. After the inoculations, culture medium was supplied continuously at a rate of 0.2 mL / min. The composition of the culture medium per liter was: 0.035 g of NaHCC> 3, 0.03 g of MgSO7H ₂ 0, 2.4 g of K2SO4, 0.007 g FeSC> 4-7H ₂ 0.1 g of NH ₄ CI, with 4.95 g of sodium formate, 0.01 g of KH ₂ PC> 4, 1.72 g of CaCl ₂ -2H ₂ 0, 0.1 g of yeast extract and 0.099 g of thioglycolic acid. The culture medium was prepared with groundwater extracted from the hydraulic barrier of a tailings dam, whose main components are HCO3 ^· 217.7 mg / L, Ca ²⁺ 302 g / L, 76.6 mg Mg ²⁺ / L and S04 ^{2 to 1192.3} mg / L

In the case of the non-inoculated control, after water infiltration, hydraulic barrier water was continuously injected at 0.2 mL / min. The inoculated models were re-inoculated, at the corresponding speed, on days 15, 30, 45, 48, 52, 55 and 58, counted from the first inoculation.

Between 20-350 pL (depending on the parameter to be determined) of solution was obtained from the sampling port of the models using sterile syringes, taking care not to extract the volume quickly, in order to avoid sudden changes in the flow that result of the local pressure decrease as a result of the extraction. These samples were used for the determination of sulfate, hydrogen sulfide and retention time (when the tracer was used).

The effluent from the models was used to make the determinations of pH, potential, formate, microbial count and ATP.

The determination of the concentrations of sulfate, calcium, according to

Example 1, ATP according to Example 3 and hydrogen sulphide (or sulphides) in solution were carried out according to Example 2. Microbial counting was carried out using the methodology described in Example 4.

Once the experiment in the aquifer models was finished, they were opened and sand samples were taken for the following analyzes:

A protocol was used to determine the amount of ATP of the microorganisms adhering to the quartz sand of the aquifer models. For this, 200 mg of a sand sample was taken, which was washed with distilled water to remove the bacteria present in the pore volume, after which 1 mL of the cell lysis solution contained in the system was added. determination of ATP. Subsequently, the lysis supernatant was reacted with the other lyophilized components (luciferin-luciferase) to determine the amount of ATP by means of a Kikkoman luminometer, model Lumitester PD-20, Japan. The luminescence value obtained was expressed as URL / g of sand. Determination of pH in pore water: Once the aquifer models were opened, the pH in the sand pore water was immediately determined in different areas of the models, wetting pH indicator strips. The colors of the strips were compared to the standard provided on the product package. The pH readings were used to make a pH map corresponding to the upper plane of the porous medium of the models.

Determination of total carbonates in sand samples: Small sand samples (approx. 300 mg) were obtained from different areas of the models, which were placed under a magnifying glass and drops of concentrated HCI (1 M) were added in order to to observe bubbles that account for the possible presence of carbonates. The samples that were positive in this test were sent to the Soil Chemistry and Biochemistry laboratory of the Faculty of Chemical and Pharmaceutical Sciences, where total carbonates were determined by acid-base titration.

X-ray diffraction: The sand samples were ground in a mortar to obtain a sample with adequate granulometry to perform X-ray diffraction (XRD). They were then analyzed using a D8 Advance X-ray diffraction machine, Bruker, Germany. The diffractogram obtained was compared with those corresponding to standard mineral samples with CaCC> 3.

At the end of the experiment with the aquifer model and to quantify the percentage of the surface of the quartz sand that presented carbonate formation (formation of cemented sand aggregates), the analysis of the photographic images was carried out using the ImageJ program. To calculate a biomineralized area, the following equation was used:

Biomineralized area

where the area of the biomineralized porous medium and the total area of the porous medium correspond to the number of pixels that resulted from freehand selections on the photographs at the end of the experiment. The results of the aquifer models showed the sulfate-reducing activity of the inoculated microbial consortium and the changes in the hydraulic flow of the porous medium during 60 days.

As of day 10, the presence of hydrogen sulfide was detected in the aquifer models inoculated with the consortium enriched in sulfate-reducing bacteria (Figure 13A). This coincides with the time in which a reduction in the sulfate concentration was observed in the effluent of the models (Figure 13B). Sulfate values decreased to 1500 mg / L on average after day 14, fluctuating between 1400-2000 mg / L until the end of the experiment, while the hydrogen sulfide concentration increased to values between 60-80 mg / L, reaching between 130-200 mg / L on the final days of the experiment (Figure 13A).

In turn, acidification of the effluent was observed in the inoculated experiments compared to the non-inoculated control. This parameter fluctuated in the range 7.4- 7.6 in the inoculated models from day 20, while the control remained in the range of 7, 7-8.0 throughout the experiment (Figure 13C). In the inoculated models, there was a significant decrease in the oxidation-reduction potential, since from day 5 the value of this parameter reached between -170 to -190 mV, gradually decreasing to values lower than -250 mV in the final days of the experiment. These anoxic conditions are suitable for sulfate reducing bacteria. In contrast, the control model showed a lesser decrease in this parameter, averaging only -70 mV throughout the experiment (Figure 13D).

In the effluent of the inoculated and control models, the amount of intracellular ATP was periodically determined, finding high ATP values, which varied between 800-2500 URL / mL, which showed the microbial colonization inside the inoculated aquifer models ( Figure 14). In contrast, the intracellular ATP values in the non-inoculated control remained low throughout the experiment.

The decrease in the permeability of the porous medium of the aquifer models due to the reduction of the pore size caused by the bioclogging by microbial biomass and precipitation of carbonate minerals was demonstrated by changing the retention time of the tracer. From day 22, increases in the retention time of the tracer were observed in the inoculated models, which was not observed in the control model, except for a slight increase in this parameter on day 55 of experimentation (Figure 15) . In the inoculated models, a region of lower permeability located in the central zone was observed, which slows down the arrival of the tracer to the sampling port. Indeed, over the course of the experiment, a dark plume is observed in the inoculated models, emerging at the inoculation and stimulation ports, and extending towards the effluent outlet, following the direction of the water flow.

The analyzes of the experiments with the tracer on day 55 show that the blackened feathers represent an area of lower permeability compared to other areas of the porous medium, since it is observed that the dye deviates when it encounters this area, causing most of the hydraulic flow occurs through the non-blackened areas (Figure 16). The blackened areas of the plume are due to the formation of iron sulphide precipitates from the hydrogen sulphide generated in the inoculated models and the presence of traces of iron in the infiltrated culture medium.

After 60 days of testing, the aquifer models were disassembled to determine the pH, the microbial intracellular ATP, the relative abundance of the microorganisms and the carbonate minerals formed in the sand.

The pH value in the sand pore water was determined by means of pH indicator rods. This allowed making a map of this parameter (Figure 17). Three zones were distinguished in the sand of the models: a) zone 1: first third located in the vicinity of the flow water inlet zone, b) zone 2: second third located in the central zone, and c) zone 3: third third located towards the effluent outlet zone. In all the inoculated aquifer models, a pH 8 zone was observed in zones 2 and 3. Point zones of pH 8.5 were also observed in one of the duplicates, which are located within the pH 8 zones. A lower pH value (close to 7.5) was observed in zone 1. High local pH values are of great importance to induce the precipitation of carbonates. Instead, in the No alkalinization was observed in the control model, since a pH of 7.5 was detected in all areas.

Intracellular ATP measurements corresponding to the microorganisms adhered to the sand of the aquifer models were carried out. In the inoculated models, the highest amount of ATP was observed in sand in zone 2. In both zones 1 and 3, similar amounts of ATP were found in both experiments. These results confirm the microbial colonization of the sand and also demonstrate that the adhered microorganisms are metabolically active. The control model (not inoculated), showed considerably lower values in all zones compared to the inoculated ones (Figure 18).

At the time of taking sand samples from the inoculated models, a highly biomineralized quartz sand fraction was found, whose sand particles were found agglomerated, a product of biologically induced mineralization. This area was centered on the point of inoculation and stimulation of microorganisms, in the shape of an irregular circle of about 3 cm in diameter and occupying the entire internal height of the model (Figure 19).

To detect the formation of carbonates in the sand, sand samples were taken from different areas of the inoculated models, to which drops of concentrated HCl were added, in order to observe effervescence that reveals the possible presence of carbonates. From the models whose samples were generated effervescence, two samples were extracted for the quantification of total carbonates by acid-base titration: a) from the area located at the inoculation point, and b) from a point located downstream (12 cm) to the inoculation point. The highest amount of carbonates were generated in the inoculation zone (5.8% of total carbonates in the sand), where values up to 7 times higher were found compared to the downstream zone (0.8% of total carbonates in the sand ). In the carbonate test with HCl of the sand samples of the control model, no gas production was observed.

The XRD analyzes for the evaluation of the mineralogical composition of the sand samples of the inoculated models, confirmed the presence of the calcite, vaterite and aragonite minerals in different proportions depending on the sample (Table 9).

Table 9. Amount of CaCC> 3 (vaterite, aragonite and calcite) determined by XRD in sand from the aquifer models in the inoculation velocity evaluation experiment. The percentages of CaCÜ3 are shown at the inoculation point and downstream in one of the duplicates of the aquifer models inoculated at different speeds. N.D .: not detected.

This example shows that it is possible to establish an active sulfate reducing microbial consortium in the porous medium of a continuous hydraulic flow model aquifer, which consists of hydraulic barrier water from a sulfate-containing tailings dam. The microbial reduction of sulfate, which is the product of the inoculation and stimulation of the microbial consortium, was able to biomineralize a portion of the quartz sand (porous medium), due to the accumulation of CaCÜ3 minerals, which according to the experiments carried out with the tracer corresponds to an area of less permeability to hydraulic flow.

Example 6

Relative abundance of inoculum microorganisms and sand-adhering microorganisms in aquifer models

In order to determine the microbial composition of the inoculum and the microorganisms adhered to the sand in the aquifers, the massive sequencing of the 16S rRNA gene was used. The microorganisms of the inoculum were concentrated by centrifugation from a sample of the effluent of the bioreactor B1 of Example 4, used for the production of the inoculum. To perform the analysis of the microbial community established in the sand of the inoculated aquifer models, it was taken a sand sample from the model aquifers inoculated in Example 5. From these centrifuged samples of the inoculum or sand samples, total genomic DNA was extracted using a commercial DNA extraction system (PowerSoil DNA Isolation Kit, MOBIO, USA). The PCR and sequencing of the amplifications of the V4 hypervariable region of the 16S rRNA gene was performed according to the protocol used by the sequencing service of the Center for Genomics and Bioinformatics of the Faculty of Sciences of the Universidad Mayor. The sequences obtained were processed and compared for identification with the SILVA database from 2018.

To perform the analysis of the microbial community established in the sand of the inoculated aquifer models, a sand sample was taken from the central zone (zone 2) of one of the inoculated duplicates. From these samples, the total genomic DNA of the microorganisms adhered to the sand was extracted for subsequent massive sequencing of the 16S rRNA gene by Illumin technology.

Tables 4, 5, 6, 7 and 8 show the relative abundance of the 16S rRNA gene sequences in the DNA extracts of the microorganisms of the inoculum used, at the level of Phylum, Order, Class, Family and genus. respectively.

The sequencing results showed that the main Phyla present in the inoculum microbial community were classified as Proteobacteria, Firmicutes and Bacteroidetes (Table 4). At the taxonomic Class level, the inoculum microbial community was composed mainly of Gammaproteobacteria, Clostridia, Deltaproteobacteria, Betaproteobacteria, Alphaproteobacteria and Bacteroidia (Table 5). The predominant Orders corresponded to Oceanospirillales, Clostridiales, Enterobacteriales, Desulfovibrionales, Burkholderiales, Pseudomonadales, Bacteroidales y Caulobacterales (Table 6). At the taxonomic level of Families, the main microorganisms present were: Oceanospirillaceae, Peptococcaceae, Enterobacteriaceae, Desulfomicrobiaceae, Pseudomonadaceae, Comamonadaceae, Porphyromonadaceae, Clostridiaceae, Caulobacteraceae and [Tissierellaceae] (Table 7). The sequencing results showed that the main genera of the microbial community of the inoculum were classified into: Family Oceanospirillaceae, Desulfosporosinus, Desulfomicrobium, Pseudomonas, Citrobacter, Family

Comamonadaceae, Family Porphyromonadaceae, Brevundimonas, Family Clostridiaceae, Family Enterobacteriaceae, Sedimentibacter, Family Rhodobacteraceae, Tissierella_Soehngenia and Stenotrophomonas (Table 8). The microorganisms that showed the highest relative abundance were Fam. Oceanospirillaceae (29.74%), Desulfosporosinus (14.42%) and Desulfomicrobium (8.36%). Desulfosporosinus and Desulfomicrobium stand out for their ability to reduce sulfate biologically.

Table 4. Relative abundance of the sequences of the 16S rRNA gene at the level of Filos in the DNA extracts of the microorganisms of the inoculum used and of the microorganisms adhered to the sand of the aquifer models at the end of the experiment.

Table 5. Relative abundance of the 16S rRNA gene sequences at the Class level in the DNA extracts of the microorganisms of the inoculum used and of the microorganisms adhered to the sand of the aquifer models at the end of the experiment.

Table 6. Relative abundance of the sequences of the 16S rRNA gene at the Order level in the DNA extracts of the microorganisms of the inoculum used and of the microorganisms adhered to the sand of the aquifer models at the end of the experiment.

Table 7. Relative abundance of the sequences of the 16S rRNA gene at the Family level in the DNA extracts of the microorganisms of the inoculum used and of the microorganisms adhered to the sand of the aquifer models at the end of the experiment.

Table 8. Relative abundance of the 16S rRNA gene sequences at the Genus level in the DNA extracts of the microorganisms of the inoculum used and of the microorganisms adhered to the sand of the aquifer models at the end of the experiment.

In the inoculated models, it was found that the similar microbial community adhered to the quartz sand at the end of the experiment is qualitatively similar to that of the inoculum used (Tables 4, 5, 6, 7 and 8).

The results of the sequencing showed that the main Phyla present in the microbial community adhered to the quartz sand were classified as Proteobacteria, Firmicutes and Bacteroidetes (Table 4). At the taxonomic level of Classes, the microbial community of the sand was composed mainly of Gammaproteobacteria, Clostridia, Deltaproteobacteria and Bacteroidia (Table 5). The predominant Orders corresponded to Clostridiales, Desulfovibrionales, Pseudomonadales and Bacteroidales (Table 6). At the taxonomic level of Families, the main microorganisms present were: Desulfomicrobiaceae,

Pseudomonadaceae, Porphyromonadaceae and [Tissierellaceae] (Table 7).

The sequencing results showed that the main genera of the sand microbial community were classified into: Desulfomicrobium, Pseudomonas, Family Porphyromonadaceae, Family Clostridiaceae, Sedimentibacter, Tissierella_Soehngenia and Order Bacteroidales (Table 8). The microorganisms that showed the highest relative abundance were Desulfomicrobium (58.37%), Family Porphyromonadaceae (19.04%) and Pseudomonas (4.53%). Desulfomicrobium stands out among them for its ability to reduce sulfate biologically.

Sequences corresponding to the sulfate reducing genus

Desulfomicrobium increased considerably with respect to the percentage composition of the inoculum community (Table 8). A significant increase in sequences corresponding to a genus of the Porphyromonadaceae family was also found. These differences can be attributed to the particular microenvironmental conditions of the sand.

The analyzes of this example show that the enriched microbial consortium used as inoculum in Example 5 exhibits high bacterial diversity, among which some classes can be found that include sulfate-reducing bacteria, such as the Deltaproteobacteria. Among them, the Desulfosporosinus and Desulfomicrobium genera stand out for their ability to reduce sulfate biologically. When studying the microbial community adhered to the quartz sand at the end of the experiment in the aquifer model, it was found that this was qualitatively similar to that of the inoculum used, with some microorganisms that significantly increased their proportion. This is the case of the genus Desulfomicrobium. Example 7

Effect of formate concentration on biologically induced mineralization and bio-clogging in model aquifers

To show the effect of the change in the formate concentration in the culture medium, 8 aquifer models were built, which were operated and monitored under the same conditions and methodologies as in Example 6. The formate concentrations were 3, 6 , 12 and 18 g / L. In this case, the models were inoculated with the same amount of microorganisms as in Example 6, and re-inoculated on day 15 of operation. The inoculation speed was 0.2 mL / min in all models. Then, in independent experiments (n = 2 models), the models were stimulated with the culture media containing the different concentrations of formate for 60 days.

To determine the concentration of the remaining formate in the effluent from the aquifers, a colorimetric method previously described by Sleat and Mah (Quantitative Method for Colorimetric Determination of Form in Fermentation Media. Applied and Environmental Microbiology. 47 (4): 884-885 was used. , 1984). The reaction between the components of the method and the sample was incubated at room temperature for 2.5 hrs, after which the reaction product was quantified in a spectrophotometer at 510 nm (Dynamica spectrophotometer, model HALO RB-10, Great Britain ).

During the experimentation time (60 days), in all the tests a reduction in the sulfate concentration was observed in the models from day 15 (Figure 20A). In turn, the production of hydrogen sulfide was verified in all models. However, a delay in the rise of this parameter can be seen in experiments carried out at higher concentrations of formate (12 and 18 g / L) (Figure 20B). In the models stimulated with a lower concentration of formate (3 and 6 g / L) (Figure 20C), a decrease in the potential for oxide reduction was achieved more rapidly compared to those with a higher concentration. Also can It can be seen that the pH values in the effluent of the models stimulated with the least amount of formate (3 g / L) follow a downward trend with respect to the values of the other experiments (Figure 20D).

In the effluent of the models stimulated with the highest concentrations of formate (12 and 18 g / L), an appreciable decrease in the concentration of this substrate was observed compared to the concentration of formate added during the experimentation time (Figure 21). However, in the tests carried out with the lowest formate concentrations (3 and 6 g / L), the same trend of formate consumption was not observed.

In all the formate concentrations tested, the retention time of the tracer was increased to a similar value in all models (Figure 22).

After 60 days of testing, the aquifer models were disassembled to determine the pH, the microbial intracellular ATP, the relative abundance of the microorganisms and the carbonate minerals formed in the sand.

The pH value in the sand pore water was determined by means of pH indicator rods. This allowed making a map of this parameter (Figure 23). Three zones were distinguished in the sand of the models: a) zone 1: first third located in the vicinity of the flow water inlet zone, b) zone 2: second third located in the central zone, and c) zone 3: third third located towards the effluent outlet zone. The pH values in sand tended to increase as the concentration of formate in the culture medium increased (Figure 23). On average, in the models stimulated with the highest concentrations of formate (12 and 18 g / L), this parameter reached values above 8 in zone 2 and 3, while a value lower than 8 was recorded in the same zones of the models stimulated with the lowest formate concentrations (3 and 6 g / L).

Intracellular ATP measurements corresponding to the microorganisms adhered to the sand of the aquifer models were carried out. In all the trials, ATP values in sand were higher in zones 2 and 3 of the models, while zone 1 (upstream to the inoculation point) was lower with respect to the other zones (Figure 24). Especially in zones 2 and 3, a dependent increase of formate concentration in the culture medium of this parameter was observed, within the range between 3 and 12 g / L. In the latter, a maximum value of ATP / g of sand is reached, since in the models with the highest amount of formate (18 g / L), these decrease to values similar to those of the test carried out with 6 g / L of formate.

The increase in the concentration of formate in the culture medium produced an enrichment of the sequences corresponding to the sulfate reducing genus Desulfomicrobium, which predominated in all samples (Table 10), reaching 35, 48, 52 and 57% in the sand from the aquifer models stimulated with 3, 6, 12 and 18 g / L of formate, respectively. The increase in the electron donor also produced the notorious enrichment of the sequences corresponding to the genus Pseudomonas. The opposite effect on other components of the microbial community was also observed, since the sequences of the predominantly aerobic family were reduced.

Comamonadaceae and of the obligate anaerobic genus Proteiniclasticum.

The previous results show that there is an effect of the formate concentration in the culture medium on the extent of the biologically induced mineralization of the porous medium in the aquifer model used, since an increase in total carbonates downstream and in the biomineralized area in the models as a function of the increase of the electron donor. This is related to the formate values measured in the effluent, since the electron donor was used to a greater extent in the experiments that contained the highest amounts of formate, while in the models stimulated with the lowest concentrations of this, there was no considerable use. of the giver. Table 10. Relative abundance of the 16S rRNA gene sequences in the DNA extracts of the microorganisms adhered to the sand of the aquifer models at the end of the experiment evaluating formate concentrations.

The presence of a portion of biomineralized sand was verified in all the aquifer models (Figure 25). The calculation of the area comprised by the biomineralized sand, based on the image analysis using the ImageJ program, revealed a direct relationship between the area of the agglomerated sand and the amount of formate in the culture medium (Figure 26). This relationship is linear in the range of formate concentrations between 3-12 g / L, after which the biomineralized area tends not to increase at higher formate concentrations.

The quantitative analysis of total carbonates in the zones defined in the previous experiment (inoculation point and downstream point) confirm the increase in the amount of precipitated carbonates at the downstream point as a result of the increase in the concentration of formate (Figure 27). Also, it is interesting that the amount of carbonates at the inoculation point decreases with the increase of formate in the culture medium

Through XRD analysis, calcite formation was found in the model aquifers at all formate concentrations. The quantitative analyzes of the calcite content showed the same trend as that determined by the quantitative analysis of carbonates by acid-base titration, that is, the increase in the amount of precipitated carbonates at the downstream point of the models (Table 11).

Table 11. Amount of CaCC> 3 (calcite) determined by XRD in the sand of the aquifer models stimulated with the different concentrations of formate. The percentages of CaCÜ3 at the inoculation point and downstream of one of the duplicates of the aquifer models stimulated with different formate concentrations are shown.

At the end of the experiment, observations were made by scanning electron microscopy (SEM) of the mineralized sand. For this, approximately 100 mg of quartz sand sample was taken from the inoculation point of one of the models stimulated with the lowest and highest concentration of formate and a 2.5% cacodylate-glutaraldehyde fixing solution was added. After this, the samples were dried in a critical point dryer and coated with gold (Dentón Desk V gold / carbon coater, USA and Autosamdri - 815 critical point dryer, USA). Subsequently, the samples were observed in a High resolution scanning electron microscope (HR-SEM), model INSPECT-F50, The Netherlands.

In Figures 28A and 28B, corresponding to samples of quartz sand from the models stimulated with the lowest and highest concentration of formate, respectively, bacterial clusters can be seen composed mostly of bacillus-shaped microorganisms, adhered to the surface. from quartz sand and adjacent to crystalline minerals, possibly calcite, as previously determined by XRD. Example 8

Process to reduce hydraulic permeability and generate the precipitation of insoluble minerals in situ from an underground aquifer contaminated with sulfates

The process consists of a system to reduce hydraulic permeability and generate the precipitation of insoluble minerals in an aquifer with groundwater contaminated with sulfate and / or with sulfate and metals. The process consists of at least the steps of: a) providing a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, b) injecting the aquifer with an enriched culture of microorganisms, c) injecting the aquifer with an electron donor and nutrients suitable for the culture of microorganisms enriched in sulfate-reducing bacteria and one or more metal cations that favor the precipitation of insoluble minerals, and d) allow the microorganisms to multiply and colonize the solid material of the aquifer, reducing the hydraulic permeability of the aquifer and generating the precipitation of insoluble minerals in the solid material of the aquifer.

As shown in Figure 29, the process begins with the extraction of groundwater contaminated with sulfate and / or with sulfate and metals (1), from an underground aquifer, whose flow direction is indicated by arrows (2). One well or more wells are used to extract groundwater extraction (3), which are installed from the surface level of the ground (4) to extend below the water table (5). In the water table sector, the well (s) have grooves, perforations or other permeable sections (6), which allow the extraction of groundwater. The extracted groundwater enters through the conduit (7) to the pond (8). To the latter, through conduit (9), an electron donor and nutrients suitable for the cultivation of microorganisms enriched in sulfate-reducing bacteria and one or more metal cations that favor the precipitation of insoluble minerals are added. The added compounds are stirred in the pond to obtain a solution Through conduit (10) the solution is entered into the fixed-bed anaerobic bioreactor (11), which has been previously inoculated with an enriched sulfate reducing microbial consortium, of according to the previous Examples. The effluent from the bioreactor (11), which contains the microorganisms of the sulfate-reducing consortium, through the conduit (12), enters one well or more injection wells (13), which are installed from the surface level of the soil ( 4) to extend below the water table (5). In the water table sector, the well (s) have grooves, perforations or other permeable sections (14), which allow the injection of microorganisms and nutritive solutions into the groundwater. To stimulate the growth and metabolic activity of the sulfate reducing microbial consortium, the solution from the pond (8) is also added directly, through the conduit (15) to one well or more injection wells (13), said solution is injected to groundwater through slots, perforations, or other permeable sections (14). Microorganisms proliferate in the pores and on the surface of the particulate matter in the underground aquifer, impeding the flow of water. Furthermore, the metabolic activity of microorganisms, especially the biological reduction of sulfate, produces the partial removal of sulfate and the alkalization of the solution. As a result of the above, it is possible to generate a zone of reduction of hydraulic permeability and of precipitation of insoluble minerals in situ (16) in an area downstream of the injection point in an underground aquifer contaminated with sulfates. Insoluble minerals are primarily metallic carbonates and sulfides. Carbonates, such as calcite, aragonite, or vaterite, are formed in the environment alkaline, from bicarbonate produced by the anaerobic microbial degradation of organic matter and calcium added and / or present in groundwater. Additionally, metallic sulphides, particularly FeS, precipitate by the reaction between the biologically formed H2S and iron oxides, such as maghemite, magnetite, hematite or goethite commonly present in soils and in particulate matter of underground aquifers.

Claims

1. An in situ method to reduce the hydraulic conductivity and generate the precipitation of insoluble minerals in an underground aquifer that is affected by superficial water seepage or by water seepage from a mining tailings dam, CHARACTERIZED because it comprises at least the steps of : a) provide a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, b) inject the groundwater aquifer with an enriched microorganism culture, c) inject the groundwater aquifer with an electron donor and nutrients for the culture of microorganisms enriched in reducing bacteria and one or more metal cations that favor the precipitation of insoluble minerals, and d) allow microorganisms to multiply and colonize the solid material of the underground aquifer, reducing the hydraulic permeability of the underground aquifer and generating insol mineral precipitation ubles in the solid material of the underground aquifer.

2. The method according to claim 1, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, is produced with a method that comprises at least the steps: a) inoculating a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated in a fixed-bed anaerobic bioreactor with a support material, b) continuously feed with water extracted from the groundwater aquifer, an electron donor and suitable nutrients for the cultivation of sulfate reducing bacteria, c) allow the microorganisms to multiply, colonize the bioreactor support material, and d) produce an effluent from the bioreactor containing a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated to be injected into said aquifer.

3. The method according to claim 2, CHARACTERIZED in that said support material is selected from the group of sand, silicic stone, glass and ceramic.

4. The method according to claims 1 - 3, CHARACTERIZED in that said injection of the underground aquifer with an enriched microorganism culture and said injection of the underground aquifer with an electron donor and nutrients suitable for the culture of microorganisms enriched in reducing bacteria, and one or more metal cations that favor the precipitation of insoluble minerals is carried out through one or more injection wells.

The method according to claims 1-4, CHARACTERIZED in that said an electron donor suitable for the culture of microorganisms enriched in reducing bacteria is selected from the group of formate, formic acid, acetate and acetic acid.

6. The method according to claims 1 - 5 CHARACTERIZED in that said nutrients suitable for the culture of microorganisms enriched in reducing bacteria, comprise, at least, ammonium and phosphate.

7. The method according to claims 1-6, CHARACTERIZED in that said nutrients suitable for the cultivation of microorganisms enriched in reducing bacteria, also comprise a complex nutrient, rich in vitamins, selected from the group of yeast extract and corn strained water .

The method according to claims 1-7, CHARACTERIZED in that said insoluble mineral that precipitates in the solid material of the underground aquifer is calcium carbonate.

The method according to claims 1-8, CHARACTERIZED in that said insoluble mineral that precipitates in the solid material of the underground aquifer is iron sulfide.

10. The method according to claims 1 to 9, CHARACTERIZED in that said method also allows to remove sulfate from the groundwater of said aquifer.

11. The method according to claims 1-10, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Phylum Proteobacteria, Firmicutes and Bacteroidetes .

The method according to claims 1-11, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria from the Phylum Proteobacteria or Firmicutes.

The method according to claims 1-12, CHARACTERIZED in that said culture of microorganisms enriched in sulfate reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Classes Gammaproteobacteria, Clostridia, Deltaproteobacteria and Bacteroidia.

The method according to claims 1-12, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated is constituted, at least by bacteria of the Clostridia or Deltaproteobacteria Class.

The method according to claims 1-14, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains at least bacteria of the Order Desulfovibrionales.

16. The method according to claims 1-14, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Desulfobacterales Order.

The method according to claims 1-14, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Desulfuromonadal Order.

18. The method according to claims 1-14, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least, bacteria of the Order Clostridiales.

19. The method according to claims 1 to 14, CHARACTERIZED in that said culture of microorganisms enriched in sulfate reducing bacteria and adapted to the groundwater of the aquifer to be treated contains, at least one genus of sulfate reducing bacteria selected from the group consisting of Desulfomicrobium, Desulf ovibrio, Desulfonema, Desulfuromonas, Desufutomaculum and Desulfosporosinus.

20. The method according to claims 1-19, CHARACTERIZED in that said culture of microorganisms, enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, is obtained from samples taken from live microbialites from lagoons or saline lakes.

21. The method according to claims 1-20, CHARACTERIZED in that said culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated, is obtained with a method that comprises at least the steps: a) inoculate a sample of said live microbialite from a saline lake or saline lagoon into a column filled with sand, b) feed continuously with water extracted from the underground aquifer, an electron donor and nutrients suitable for the culture of reducing bacteria of sulfate, c) allow the microorganisms to multiply, colonize the bioreactor sand and enrich the culture in sulfate-reducing bacteria, and d) produce a culture of microorganisms enriched in sulfate-reducing bacteria and adapted to the groundwater of the aquifer to be treated .

The method according to claims 1-21, CHARACTERIZED in that said living microbialite is a living stromatolite.

23. The method according to claims 1-22, CHARACTERIZED in that said living microbialite is a living thrombolyte.