BIOREMEDIATION OF ACID MINE DRAINAGE

E-mail address: xyz@abc.com. DIVERSITY OF ACIDOPHILIC BACTERIA AND ARCHAEA AND THEIR ROLES IN 1 BIOREMEDIATION OF ACID MINE DRAINAGE 2 3 Fashola, Muibat Omotola, 1 Ngole, Veronica, 2 Babalola, Olubukola Oluranti 1 4 1. Department of Biological Sciences, Faculty of Agriculture, Science and Technology 5 North-West University, Mafikeng Campus, Private Bag X2046 Mmabatho 2735 6 2. Department of Crop Sciences, Faculty of Agriculture, Science and Technology 7 North-West University, Mafikeng Campus, Private Bag X2046 Mmabatho 2735 8 9 10 . 11 ABSTRACT 12 13 Aims: To show the potential of acidophilic bacteria and archaea in bioremediation of acid mine drainage. The Mining industry generates wealth, but its long term adverse effects, which include acid mine drainage (AMD), cannot be overlooked. Acid mine drainage occurs as a result of biological and chemical oxidation of sulphide containing minerals with consequent production of acidic metal rich effluents. AMD is a serious environmental pollution problem in both active and abandoned mines worldwide, resulting in continual contamination of surface and groundwater resources with heavy metals. Acidophilic bacteria and archaea have been known to contribute to the accentuation of this problem by speeding up the reaction time for biological oxidation of sulphide containing mineral waste rock. The dominant metal present in AMD is iron with high sulphate content; the iron may be present in either ferrous or ferric form or both depending on the water pH. Reduction of these two important constituents by generating alkalinity through chemical or biological means has been reported to have a significant effect in AMD impacted water. The metabolic activities of the acidophilic bacteria and archaea through ferric iron and sulphate reduction, a natural attenuation process, also help in remediating this pollution problem by generating alkalinity that immobilizes metals thereby reversing the reactions responsible for the genesis of AMD. This article reviews the various groups of the acidophilic prokaryotic microorganisms and their metabolic activities that help in remediating the problem of AMD in gold mines.


INTRODUCTION
Acid mine drainage, also known as acid and metalliferrous drainage (AMD) or acid rock drainage (ARD), is the 19 biochemical oxidation of sulphide bearing minerals, which results in the production of acidic water that contains high 20 concentrations of heavy metals sulphate and low pH. Earth disturbances such as construction activities and mining 21 processes in the rocks that contain abundance of sulphide minerals as well as natural rock weathering processes can also 22 contribute to the generation of AMD. This indicates that AMD is the generation of acidic water from sources other than 23 mining. Typically, AMD is characterized by low pH value, high sulphate content and, often times, elevated concentrations 24 of ferric iron and other metals such as copper, zinc, chromium, cadmium and nickel. The chemical composition of AMD 25 varies depending on the kind of sulphide mineral associated with coal and metal ores [1,2]. Acid mine formation is greatly 26 enhanced by the mining process which increases the surface area of the sulphide containing mineral exposed to air and 27 oxygen thereby increasing the rate of acid generation [3]. Bacterial activity is an important factor in acid mine generation 28 because it helps in accelerating the rate of decomposition and oxidation of sulphide minerals. 29

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Many factors are known to contribute to the development of AMD. Acid mine drainage can be generated as a result of 31 coal and metal mining activity. Some of these sources are : Mined materials like spent ore from heap leach operations, 32 spoil, waste dump or tailings, overburden material, mine structure such as pit walls in surface mining operation and 33 underground workings associated with underground mines and subgrade ore piles. All these are known to contain 34 sulphide minerals like pyrite, which is the most abundant of all sulphide minerals, and others such as galena, covelite, 35 chalcopyrite, realgar, and arsenopyrite whose oxidation leads to the formation of AMD [4]. After extraction of ores from 36 underground or open pit, 80-90% of the crushed ore is dumped as tailings waste which contains large amounts (between 37 10 and 30 kg/ton) of sulphide minerals. 38

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Acid mine drainage usually contains a variety of microorganisms. As a result of the characteristics features of the acid 40 mine drainage, prokaryotic microorganisms have been found to be the predominant life forms existing in the environments 41 [25].These prokaryotes are found in the groups of bacteria and archaea domains with ability to thrive well in the extremely 42 acidic environments. Acidophiles have immense contribution to sulphur and iron biogeochemical cycle [25,26]. 43 Acidophilic microorganisms are a subdivision of the extremophiles which have been gaining a lot of research interest as a 44 result of their ecological and economic importance. The ecology and biodiversity of the acidophilic prokaryotes has been 45 reviewed by Hallberg and Johnson [27]. Acidophiles have been classified using many criteria. On the basis of mineral 46 solubilization, two groups are recognized. The first group is those that accelerate mineral dissolution by an oxidative route 47 (iron and sulphur oxidizers) while the second group uses the reductive route (iron reducers) [14]. Some species (mostly 48 acidophiles) can reduce ferric iron as well as oxidize ferrous iron, depending on the prevailing environmental conditions. 49 The iron and sulphur oxidizers are found in both bacteria and archaea domains and their metabolic activities have been 50 utilized in extraction of gold from refractory ores (biomining) [28]. The bacteria are found within the Proteobacteria and 51 life forms which are functional in AMD grow at an optima pH between 2 and 4 or acid-tolerant (pH optimal for growth 95 above that normally encountered in AMD), but can also function in very low pH environments [40]. 96 The concentration of dissolved organic carbon in the majority of extremely acidic environments has been found to be very 97 low (<20 mg L -1 ). Thus these environments can be characterized as oligotrophic environments. In abandoned deep mines 98 where light penetration is restricted, the nutrition type that exists will be mainly chemolitho-autotroph, which is the 99 oxidation of ferrous iron and reduced sulphur compounds [25]. The majority of iron and sulphur-oxidizing acidophiles are 100 regarded as autotrophic, but utilization of formic acid as carbon source has also been reported in some of them such as 101 At. ferroxidans and they have been found to be responsible for the production of ferric iron and acid [41]. 102 The chemolithotrophs are the first prokaryotes isolated from extremely acidic environments and At. ferroxidans was the 103 first iron-oxidizing acidophile to be isolated and characterized [

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The development of acid mine drainage is dependent on six factors, namely (a) abundance of sulphide minerals (b) water 111 content (moist environment) (c) oxygen and ferric iron (oxidant) and pH (hydrogen ion concentration), (d) surface area of 112 the exposed sulphide mineral, (e) activation energy and (f) presence of sulphur and iron oxidizing bacteria (biological 113 activity) [5]. 114 Sub-surface mining often progresses below the water table, so water must be constantly pumped out of the mine in order 115 to prevent flooding. However, when a mine is abandoned, the pumping ceases, and water floods the mine, which results 116 in the accumulation of contaminated water in the environment [6,7]. Introduction of water is the initial step in most acid 117 mine drainage generation. This results in the production of drainage water that is highly polluting because of low acidity 118 which increases mobility and heavy metal content. This acidity occurs as a result of dissolution of the acidic salts that 119 have built up in the pore spaces of the exposed walls and ceilings of underground chambers. Exposing pyrite to oxygen 120 and water leads to an oxidation reaction, where hydrogen, sulphate ions and soluble metal cations are created as shown 121 in the equation below: 122 Further oxidation of ferrous iron Fe 2+ to ferric iron Fe 3+ occurs as a result of availability of dissolved oxygen in water or in 124 the atmosphere 125 Ferric iron (Fe 3+ ) can also precipitate as ochre (Fe(OH) 3 ), the reddish-orange precipitate often observed in acid mine 127 drainage waters: 128 Fe 3+ that did not precipitate from (2) left in solution from (3) will precipitate additional pyrite as indicated in equation (4): 130 Pyrite oxidation occurs through either direct or indirect pathways and it is very difficult to determine which of the pathways 133 is important in a given situation. The direct pathway involves close proximity of the sulphide bearing mineral with 134 microorganisms such as acidophilic At. ferroxidans, L. ferroxidans which aids in the oxidation of the sulphide mineral [8-135

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In the indirect pathway, sulphide is reduced to ferrous as a result of its chemical oxidation by ferric iron and the ferric iron 137 is further regenerated by iron-oxidizing microorganisms, leading to continuous oxidation of the sulphide mineral. The 138 resultant effect of this reaction is the production of acidic water with characteristic corrosive patterns [11]. The acidity of 139 the medium which results from generation of the hydrogen ion makes the heavy metals contained therein highly soluble, 140 thereby preventing their precipitation out of the solution. Indirect production can also occur through the reaction of some 141 metal ions such as Fe 3+ and Al 3+ with water [12]. Further acidity can also be generated by dissolution of sulphide 142 containing minerals in the anoxic sediment of a constructed wetland or spoil heap as a result of the ferric iron 143 concentrated acidic water infiltrating through them [13]. 144 * Tel.: +xx xx 265xxxxx; fax: +xx aa 462xxxxx. E-mail address: xyz@abc.com.
Bacteria play a prominent role in the genesis of acid production because they act by accelerating the rate of 145 decomposition and oxidation of the sulphide minerals [14]. Evangelou and Zhang [15] stated that the major reaction which 146 ensures continuous oxidation of the sulphide mineral is continuous regeneration of ferric iron which is reduced to ferrous 147 upon reaction with pyrite. Hence, the primary oxidant is the ferric iron and not molecular oxygen as initially proposed in the 148 classical equation above. 149 Pyrite oxidation is a two-phase reaction. The first phase involves ferric iron attack on the sulphide mineral, while the 150 second phase is the reoxidation of ferrous iron to ferric, which is an oxygen dependent reaction. The reduced sulphur 151 compounds produced as intermediates in the reaction are also oxidized to sulphate [15]. Dissolution of the sulphide 152 mineral occurs after its attachment to oxygen and this result in the oxidation of the sulphide moiety which occurs in non-153 ferrous sulphides such as Cu 2 S or of both iron and sulphur in minerals such as pyrite (FeS 2 ) and pentlantite (FeNiS). Cd 2+ and Pb 2+ , which are not biodegradable. These metals accumulate in living organisms (bioaccumulation) and the 169 concentrations increase as they pass from lower trophic levels to higher trophic levels (a phenomenon known as 170 biomagnifications) causing various diseases and disorders [16]. It is, therefore, important to treat acid mine decants 171 effectively before allowing their release into the ecosystem. AMD. An Example of these technologies is the selective sequential precipitation (SSP) which precipitates metals using 202 solutions of sodium hydroxide (NaOH) and hydrogen sulphide produced by sulphate reducing bacteria. This approach 203 produces metals with a high degree of purity and is environmentally friendly [22]. 204

ACTIVE TREATMENT 206
The most widely used approach for remediating mine impacted waters is to aerate (to oxidize ferrous iron to ferric) and 207 add neutralizing chemicals such as calcium carbonate, calcium hydroxide or anhydrous ammonia to raise the pH. This

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The dominant metal present in AMD is iron, with elevated amounts of sulphate as a result of microbial oxidation of 214 sulphide containing minerals. The iron may be present in either ferrous or ferric form, or both, depending on the water pH. 215 Reduction of these two important constituents by generating alkalinity will have a significant effect in AMD impacted water 216 removing it from the solution will go a long way in preventing further oxidation of the sulphide minerals. 231 The acidophilic chemolithotrophic prokaryotes are known for their accelerated oxidative dissolution of pyrite and other 232 sulphide minerals in AMD using inorganic carbon. Other groups known as heterotrophic acidophiles also catalyse the 233 dissimilatory reduction of iron and sulphur using organic carbon as electron donor and carbon source, thereby reversing 234 the reactions involved in AMD formation. 235

FERRIC IRON REDUCTION 236
The rate of dissolution of most sulphide containing minerals is largely dependent on the availability of ferric iron, which 237 has been shown to be the major oxidant of these minerals in the environment [5,15]. Acid mine contaminated 238 environments, especially those associated with metal mining, contain high concentration of ferric iron whose solubility is 239 known to be greatly enhanced at acidic pH. Most prokaryotic acidophilic bacteria and archaea use this electron sinks to 240 oxidize organic matters in subsurface environments with high loads of organic matter [40]. 241 The ability to reduce ferric iron to ferrous has been reported in aerobic mesophilic chemoautotrophs (At. ferroxidans and 242 At.thioxidans) mesophilic heterotrophs (Acidiphilium spp. and jarosite and iron hydroxide formed as a result of iron oxidation has been reported in S. acidophilus which also oxidizes 249 sulphur compounds using ferric iron. Ferric iron reduction is also common in the Gram-positive mixotrophic iron-oxidizers 250 [45]. Although many neutrophilic microorganisms are known to have ability to reduce ferric iron, the ability to couple 251 organic matter oxidation exclusively to ferric iron reduction in order to conserve energy to support growth is lacking in the 252 majority of them [61]. 253 To remove soluble iron from AMD, the ferrous iron must first be oxidized to ferric. This will enhance the formation of ferric 254 minerals such as schwertmannite and ferrihydrite, which can then be easily precipitated out of solution by the ferric iron 255 reducers. This reduction results in mobilization of iron as well as other metals that may be associated with ferric iron 256 deposit. It is also an alkali generating reaction of high importance in passive treatment in wetland [13]. 257 The ability to reduce ferric iron has been reported to be affected by dissolved oxygen concentration in some of these 258 microorganisms. In a study by Martins et al. [62] to determine the effect of culture condition on the growth of two 259 acidophilic heterotrophic ferric iron reducers A.acidophilum and Acidiphilum SJH in fermenters, it was discovered that 260 growth of the A.acidophilum was affected by dissolved oxygen concentration whereas for Acidiphilum SJH the reverse 261 was the case. Also, the expression of the iron reductase system was found to be inducible in the A.acidophilum because it 262 was synthesized in the presence of very low concentration of dissolved oxygen while for Acidiphilum SJH it was 263 constitutive because it was able to reduce ferric iron irrespective of dissolved oxygen during growth. It has also been 264 The acidity of the system is reduced as a result of carbon metabolism and inherent ability of the bacteria to reduce the 285 sulphate. These fundamental properties make SRB useful in mitigating AMD [68,69], and this natural technology has been 286 considered the most promising approach of removing sulphate, acidity and heavy metals from AMD [4,70]. The ability of 287 the SRB to achieve the proposed sulphate standard of 500 ppm as well as 250 ppm required for drinking water has been 288 reported [71] as compared with the conventional chemical method that can only reduce it to 1500 mg/L. 289 Other advantages over chemical mitigation methods such as production of more compact sludge which settles faster and 290 is less subject to dissolution, selective precipitation of metal, high efficiency and low cost have been reported [67,72]. 291 Efficient sulphide production using SRB can be achieved by addition of a complementary carbon source because AMD is 292 deficient in carbon electron donors. Therefore, choosing an appropriate carbon source is very crucial in ensuring long time 293 usage, high efficiency, and economical viability of the system. Three factors are usually considered in selecting this 294 carbon source, namely, availability of the carbon source, its degradability which enhances its capacity to allow complete 295 sulphate reduction by the SRB and its cost per unit of sulphate converted [73,74]. 296 Despite the reported success of SRB in the treatment of AMD, the sensitivities of these bacteria to acidity and heavy 297 metals is the major setback in using them, hence there is a need for the addition of a remediation reagent to improve the 298 living conditions so as to enhance their activity [75]. The optimum pH of growth for SRB is between 7.0 and 7. 5  It has now been proven that microbial sulphate reduction with efficient heavy metals recovery can proceed in AMD 305 impacted environment. 306 307 The significant contribution of iron and sulphur oxidizing bacteria in the genesis of AMD has been confirmed by many 316 researchers [28, 34, 35], but the microbial diversity in the AMD sites is yet to be well characterized. [94]. The rate of 317 dissolution of sulphide mineral is a function of the population of iron oxidizing cells present and their level of activity in a 318 given environment. Information about the population of iron-oxidizers is important in deducing the microbial impact of 319 AMD [2] so that appropriate remediation approach(es) can be taken. 320

Hallberg and Johnson [34] isolated eight acidophilic moderate iron oxidizers from two abandoned mines in the United 321
States and a pilot-scale constructed wetland at one of the sites with pH 3-6. Analysis of the 16S rRNA gene sequences of 322 these isolates showed that they were previously undescribed residents of the acidic waters. Three of the isolates showed 323 greater than 99% genetic relatedness and have 97% gene identity to a clone deposited in the public data base [95]. The 324 closest recognized strain was Frauteria aurantia, a neutrophilic acetogenic iron-oxidizer with 93% gene identity, two of the 325 other three sets were found to have 99.6% gene identity and 97.5% to the third isolate. When their gene sequences were 326 compared to the gene sequences in the data bases, Thiomonas thermosulfata, a neutrophilic thiosulphate-oxidizer was 327 found to be the closest relative with 96% genetic relatedness. The remaining two isolates had 99.7% gene relatedness to 328 Propionibacterium acnes, an unknown anaerobic microbe to inhabit acidic waters. 329 Auld, et al. [94], in a study using direct sequencing of the 16S rRNA, also isolated three previously unidentified genera in 330 AMD: Legionella, a neutrophilic heterotroph, Alicyclobacillus pohliae, a Gram positive, aerobic, acidophilic bacterium and 331 Halomonas ventosae, a Gram negative, high salt tolerant, halophilic proteobacteria, from AMD tailing ponds. 332 An enormous diversity of ferrous iron oxidizing prokaryotes exists in the acidic environment [27] with different affinities to 333 the prevailing environmental conditions. Variation in environmental conditions such as pH, temperature and oxygen 334 contents exist in acid mine drainage worldwide. This lead to great differences in physiological properties of the acidophilic 335 microorganisms that can be found in these environments. This metabolic variation can be exploited in remediation 336 strategies of various AMD which is the principle behind the emerging strategies of using these acidophiles for oxidation 337 and precipitation of iron from acid mines of different water chemistry [58]. Since environmental factors are not uniform in 338 the mine waters. It will be appropriate to use different consortia to speed up iron oxidation rate in different situations. 339 * Tel.: +xx xx 265xxxxx; fax: +xx aa 462xxxxx. E-mail address: xyz@abc.com.

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Though acidophilic microorganisms are known to play a prominent role in the genesis of AMD, various advantages have 341 been derived from their metabolic activities which have been harnessed in biomining and bioleaching processes in the 342 mining industries [28]. 343 The diversities of this group of microorganisms are being shaped by geographical conditions prevailing in the various 344 environments which are not uniform. Due to this reason, there is need for more studies on the bacteria diversity, function 345 and the factors affecting the distribution of these microorganisms in the acid mine environments. This will help in 346 designing the appropriate bioremediation strategy for the contaminated sites. 347 Knowledge of these as well as the various metabolic processes and interactions that exist among these microorganisms 348 will help in identifying the various groups with potential to ameliorate the problem of acid mines. More groups with 349 unknown potentials are being detected every day, and the discovery of acidophilic anaerobic sulphate reducing bacteria 350 has greatly helped in the recovery of metals from polluted AMD.