Journal of Hazardous Materials 269 (2014) 98–109
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Sulfate reduction at low pH to remediate acid mine drainage Irene Sánchez-Andrea a,b,∗ , Jose Luis Sanz a , Martijn F.M. Bijmans c , Alfons J.M. Stams b,d a
Departamento de Biología Molecular, Universidad Autónoma de Madrid, 28049 Madrid, Spain Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands c Wetsus, Centre of Sustainable Water Technology, P.O. Box 1113, 8900 CC Leeuwarden, The Netherlands d IBB – Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, 4710-057 Braga, Portugal b
h i g h l i g h t s • • • •
Acid mine drainage (AMD) is an important environmental concern. Remediation through biological sulfate reduction and metal recovery can be applied for AMD. Microbial community composition has a major impact on the performance of bioreactors to treat AMD. Acidophilic SRB are strongly inﬂuenced by proton, sulﬁde and organic acids concentration.
a r t i c l e
i n f o
Article history: Received 6 August 2013 Received in revised form 29 November 2013 Accepted 16 December 2013 Available online 26 December 2013 Keywords: Acid mine/rock drainage Sulfate reduction Heavy metals Reactors Acidophilic SRB
a b s t r a c t Industrial activities and the natural oxidation of metallic sulﬁde-ores produce sulfate-rich waters with low pH and high heavy metals content, generally termed acid mine drainage (AMD). This is of great environmental concern as some heavy metals are highly toxic. Within a number of possibilities, biological treatment applying sulfate-reducing bacteria (SRB) is an attractive option to treat AMD and to recover metals. The process produces alkalinity, neutralizing the AMD simultaneously. The sulﬁde that is produced reacts with the metal in solution and precipitates them as metal sulﬁdes. Here, important factors for biotechnological application of SRB such as the inocula, the pH of the process, the substrates and the reactor design are discussed. Microbial communities of sulﬁdogenic reactors treating AMD which comprise fermentative-, acetogenic- and SRB as well as methanogenic archaea are reviewed. © 2013 Elsevier B.V. All rights reserved.
1. The sulfur cycle and generation of acid mine drainage Sulfur is one of the most abundant elements on Earth. The largest sulfur reservoirs are in sediments and rocks (7800 × 1018 g) in the form of iron sulﬁdes, mainly pyrite (FeS2 ), and gypsum (CaSO4 ) or as sulfate in seawater (1280 × 1018 g) . Sulfur occurs in different oxidation states (from −2 to +6, see Fig. 1) and chemical forms (cysteine, sulﬁde, sulfate, etc.) in the environment. These compounds can be transformed both chemically and biologically. - Chemical sulfur processes: The environmental sulfur cycle comprises both atmospheric and terrestrial redox processes. In the terrestrial part, the weathering of rocks releases stored sulfur.
∗ Corresponding author at: Laboratory of Microbiology, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, The Netherlands. Tel.: +31 0317 48 3115; fax: +31 0317 48 3829. E-mail addresses: [email protected]
, [email protected]
(I. Sánchez-Andrea). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.12.032
Sulfate (SO4 2− ) is usually the ﬁnal oxidation product, which accumulates in minerals (e.g. CaSO4 ) and in the ocean. There is also a variety of sources that emit sulfur directly into the atmosphere. These sources can be either natural such as volcanic eruptions and evaporation of water or anthropogenic. For instance, burning of fuels releases large quantities of sulfur dioxide into the environment, contributing signiﬁcantly to air pollution and causing acid rain . - Biological sulfur processes: Microorganisms play an essential role in the sulfur cycle, catalyzing both oxidation and reduction reactions of sulfur compounds (Fig. 1). These reactions include: (1) dissimilatory sulfate reduction, the reduction of sulfate to sulﬁde is coupled to energy conservation and growth (see Section 3); (2) dissimilatory sulfur reduction, the electron acceptor is elemental sulfur; (3) assimilatory sulfate reduction, the reduced sulﬁde is assimilated in biomass, proteins, amino-acids and cofactors by plants, fungi and microorganisms; (4) mineralization of organic compounds with hydrogen sulﬁde release; (5) sulﬁde oxidation by O2 , NO3 − , Fe3+ or Mn4+ as electron acceptors by lithotrophic and phototrophic bacteria, producing sulfur and subsequently
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Fig. 1. Biological sulfur transformations.
sulfate; and (6) disproportionation, the coupled oxidation and reduction of sulfur compounds (thiosulfate, sulﬁte and sulfur) to sulfate and sulﬁde. Table 1 shows the equations for some of the relevant biological processes with their energy release per reaction. The generation of acid mine drainage (AMD), waters with low pH and high heavy metals content, is a combined physicochemical and biological process. It starts with the chemical attack (Fe3+ ) of the ores and continues due to the microbiological regeneration of the Fe3+ . The chemical oxidation of the minerals can follow two Table 1 Stoichiometry and Gibbs free energy changes of some of the relevant conversions in the biological sulfur cycle. Gibbs free energy changes were calculated from . G◦ [kJ mol−1 ]
Reaction equations Sulﬁde oxidation HS− + 3/2O2 + H+ → S0 + H2 O HS− + 2O2 → SO4 2− + H+ HS− + Fe3+ → S0 + Fe2+ + H+ HS− + 2/5NO3 − → S0 + 1/5N2 + 6/5H2 O
(1) (2) (3) (4)
−210 −709 −47 −214
Sulfur oxidation S0 + 1.5O2 + H2 O → SO4 2− + 2H+ S0 + 6/5NO3 − + 2/5H2 O → SO4 2− + 3/5N2 + 4/5H+
Disproportionation S2 O3 2− + H2 O → SO4 2− + HS− + H+ S0 + H2 O → 1/4SO4 2− + 3/4HS− + 5/4H+ SO3 2− + 2/3H+ → 2/3SO4 2− + 1/3S0 + 1/3H2 O
(7) (8) (9)
−22 9.5 −7.6
Sulfur reduction 1/4C2 H3 O2 −a + H2 O + S0 → 1/2HCO3 − + 5/4H+ + HS− (12) (13) H2 + S0 → HS− + H+
Sulfate reduction CH3 COO−a + SO4 2− → 2HCO3 − + HS− 4H2 + SO4 2− + H+ → HS− + 4H2 O
a Acetate is used as a representative organic compound, but other organic compounds may be used as well.
mechanisms depending on the structure of the mineral substrate . Three metal sulﬁdes: pyrite (FeS2 ), molybdenite (MoS2 ) and tungstenite (WS2 ), undergo through the so-called thiosulfate mechanism and the rest of the sulﬁdes undergo through the polysulﬁde mechanism . Pyrite (FeS2 ), the most abundant sulﬁde mineral in Earth’s crust, can serve as example of the thiosulfate mechanism. When pyrite is exposed, its chemical oxidation occurs. Eq. (14) describes the oxidation of pyrite in the presence of oxygen and water . FeS2 + 3.5O2 + H2 O → Fe2+ + 2SO4 2− + 2H+
A critical factor is that ferric iron is able to oxidize pyrite even under anoxic aqueous conditions at a much faster rate (18–170 times faster) than molecular oxygen [7–9], according to Eq. (15). FeS2 + 14Fe3+ + 8H2 O → 15Fe2+ + 2SO4 2− + 16H+
First, the ferric iron attacks the iron disulfur bonds, oxidizing it partially to thiosulfate (Eq. (16)), which will be later completely oxidized to sulfate again by the ferric iron attack (Eq. (17)). In this mechanism, the end chemical product of the overall reaction is sulfuric acid . FeS2 + 6Fe3+ + 3H2 O → S2 O3 2− + 7Fe2+ + 6H+ S2 O3
+ 5H2 O → 2SO4
(16, abiotic) +
The rest of the sulﬁdes, e.g. chalcopyrite (CuFeS2 ), sphalerite (ZnS) or galena (PbS), are oxidized through the other pathway, the polysulﬁde mechanism, with a combined attack from iron and protons . In this mechanism, polysulﬁdes are ﬁrst generated (Eq. (18)) and then partially oxidized to elemental sulfur (Eq. (19)). 8MeS + 8Fe3+ + 8H+ → 8Me2+ + 4H2 Sn + 8Fe2+
(n ≥ 2) (18, abiotic)
4H2 Sn + 8Fe3+ → S8 o + 8Fe2+ + 8H+
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For the complete oxidation of these minerals, the intervention of sulfur-oxidizing bacterial communities is needed. Microbes such as Acidithiobacillus ferrooxidans, At. thiooxidans, Leptospirillum ferrooxidans and L. ferriphilum  oxidize elemental sulfur to sulfate according to Eq. (20): S8 o + 12O2 + 8H2 O → 8SO4 − + 16H+
As pointed, the complete oxidation of the sulﬁdes via the thiosulfate mechanism is chemical, while in the polysulﬁde mechanism sulfur-oxidizing bacteria are required for a complete oxidation to sulfate. However, in both mechanisms, the acidophilic chemolithotrophic microorganisms play a key role. This process is limited by the slow ferrous iron oxidation rate at low pH (Eq. (21)), which is greatly increased (up to 5 orders of magnitude) through the action of Fe-oxidizing chemolithoautotrophic bacteria such as Acidithiobacillus spp. and Leptospirillum spp. . Acidophilic chemolithotrophic microorganisms play a key role maintaining a high concentration of ferric iron, the chemical oxidant responsible of the process . 4Fe2+ + O2 + 4H+ → 4Fe3+ + 2H2 O
(21, mainly biotic)
For many years, the mechanism by which microbes obtain metabolic energy from the oxidation of metal sulﬁdes was a subject of controversy . A mechanism for the electron transfer from insoluble sulﬁdes to the electron transport chain was proposed . The attack of the insoluble sulﬁdes takes place in the exopolymeric layer between the microbial cell and the metal sulﬁde. This attack is mediated by iron compounds present in that exopolymeric layer. After the oxidation of the sulﬁdes, the ferric iron generated will strongly buffer the pH of the system around pH 2.3 (Eq. (22)). Fe3+ + 3H2 O ↔ Fe(OH)3 + 3H+
When these waters interact with the metallic sulﬁde ores, the low pH and the present ferric iron facilitate the solubilization of other metals embedded in the ores such as Cu, Cd, Co, Ni, Zn, etc. resulting in acidic metalliferous waters . Pyrite and ferrous iron oxidation are both processes that occur naturally and non-anthropogenic acidic drainage has been identiﬁed at many locations . However, mining activities in these areas are known to dramatically increase the ferrous iron oxidation rates . Coal and metal mining operations expose sulﬁde minerals to the combined action of water and oxygen, which facilitate microbial development, generating AMD or acid rock drainage (ARD), which are the cause of important environmental problems . 2. Remediation of acid metallic waters When there is risk of AMD generation, the ﬁrst option should be avoiding the formation itself. As Johnson and Hallberg stated in their review , “prevention is better than cure”. They described various approaches, called “source control”, to prevent or minimize the generation of mine drainage waters. Brieﬂy, as long as the AMD formation is increased by the action of acidophilic microorganisms, these technologies are based on eluding their activity by avoiding either oxygen, water or both from contacting the ore [18–20]. Generally, these approaches are not completely met and AMD is generated. Downstream from the source, the geochemical evolution of AMD is usually controlled by all the different reactions that take place naturally: (a) the oxidation of Fe2+ to Fe3+ with the progressive pH decrease; (b) the reversible sorption of different trace elements (As, Pb, Cr, Cu, Zn, Mn, Cd) onto the solid surfaces of metal hydroxides/hydroxysulfates; (c) the dilution of metal concentrations by mixing with pristine waters; (d) the precipitation of different metal cations (e.g. Fe3+ , Al3+ ) as pH increases; (e) biological-driven metal precipitation, which will be discussed
below in more detail; and (f) the redissolution of sulfate salts during rainstorm events, which reintroduce some trapped metals (especially Fe, Al, Cu and Zn) to the aqueous phase. The overall result of these processes might represent a mechanism of natural attenuation, as has been shown in AMD-impacted rivers of the Iberian Pyritic Belt (IPB) . Self-mitigation processes are often insufﬁcient. Other options are remediation or “migration control” approaches to minimize the impact on the environment. Both abiotic  and biotic methods have been applied. Abiotic treatments include reverse osmosis, evaporation, ion exchange, magnetical separation and addition of chemicals. The latter is the most widely used approach. Here, ferrous iron is oxidized to ferric iron by aeration. Then, an alkalizing chemical (such as CaCO3 ) is added to raise water pH and precipitate metals as hydroxides and carbonates [22,23]. This chemical precipitation yields large quantities of gypsum contaminated with heavy metals that has a limited re-use potential. The alkaline waste has to be disposed in a safe way that implies additional costs. Furthermore, these metal hydroxides are sensitive to pH variations, so they are less stable than other metal forms such as sulﬁdes. Biological treatment is based on enhancing the activity of certain microbial groups that generate alkalinity. Most of the anaerobic reductive metabolisms are alkali-generating such as iron- or sulfate-reduction. As a common feature, AMD contain low concentration of organic carbon (10 mg L−1 ) . Therefore, addition of extra electron donor is mandatory to enhance microbial activity. Some technologies are applied in situ such as anaerobic wetlands and permeable reactive barriers (PRBs) or ex situ such as passive compost bioreactors (PCBs) or active sulﬁdogenic reactors. In situ technologies are generally considered the preferable option because they are durable, clean and cheap. On the other hand, these technologies are not feasible at many locations , they yield less removal efﬁciency than bioreactors and they have limited metal recovery potential [17,25]. For in situ treatment, the election of the external organic compost added is done according to their local availability and to their previous proven effectiveness . In this kind of application, the degradability of the substrate is crucial for the success of the microbiological treatment . Composts are prepared by mixing biodegradable materials (mushroom compost or manure) with more recalcitrant materials (peat, straw, sawdust) . PRBs additionally imply digging a pit in the course-ﬂow of the water and ﬁlling it with reactive material [29,30]. Ex situ applications (PCBs and off-line sulﬁdogenic reactors) imply the use of reactors which allow a better control of parameters and performance. Different scales and conﬁgurations of PCBs have been tested, such as ﬁeld reactors [31–33], pilot-scale bioreactors [34–37], laboratory bioreactors [38–40], percolation columns [41–44] or batch reactors [45–50]. These technologies have been reviewed  and it was concluded that for an optimal performance, a mixture of substrates rather than single substrates is preferred. The off-line sulﬁdogenic reactors are engineered systems designed to optimize the production of sulﬁde over the rest of the processes with the main goal of precipitating heavy metals minimizing the electron donor costs. Due to their great potential, special attention will be given to them in the next section. 3. Off-line sulﬁdogenic reactors Sulﬁdogenic reactors rely on the activity of sulfate-reducing bacteria (SRB). The dissimilatory sulfate reduction (SR) is a process by which the reduction of sulfate (SO4 2− ) to sulﬁde (S2− ) with organic electron donors or H2 is coupled to energy conservation and microbial growth. It is done by a group of strict anaerobic microorganisms, both bacteria and archaea, often termed SRB . The reduction of sulfate leads to a consumption of protons which
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increases the pH, and to the formation of sulﬁde. The sulﬁde reacts with heavy metals such as Fe, Zn, Cu, Cd, Ni, and Pb, leading to the precipitation of insoluble metal sulﬁdes . In this case, the hydrolysis during the metal precipitation will lower the pH increment. The precipitated metallic sulﬁdes can be easily recovered and reused in further industrial processes. Indeed, application of SRB to clean up acid waters containing high metal concentrations and frequently accompanied by high sulfate concentrations is a well proven technology [1,17,52]. Off-line sulﬁdogenic reactors present multiple advantages over other technologies such as (a) less metal concentration in the efﬂuents; hydroxide precipitation of metal is even ineffective at low pH , (b) lower sulfate concentrations in the efﬂuents due to high removal efﬁciencies, (c) highly predictable performance, (d) low production of hazardous waste and (e) pH-controlled differential metal precipitation with the possibility of valuable metal recovery from the sludge . For instance, by operating a reactor at pH 4–4.5, metals as copper, nickel and zinc can be differentially precipitated while iron will remain in solution, enabling these metals to be separate from iron [54–56]. If differential metallic sulﬁde precipitation is the objective of sulﬁdogenic reactors, the survival of SRB communities is mandatory for sulﬁde formation. A good quality/size of the formed crystals is also of key importance to recover metals. Each of both factors will again depend on several parameters (Fig. 2) which are of importance for the different bioreactor designs. For instance, the pH will directly inﬂuence the microbial communities which thrive in the reactors. It will also directly affect the quality and size of the metal sulﬁdes. Therefore the decision about the pH of the inﬂuent or the inner pH of the reactor needs special attention. 3.1. Inﬂuent/efﬂuent pH values Most of the known SRB are neutrophilic growing optimally at a pH between 6 and 8 . For such SRB, low pH implies more energy investment in proton pumping across the cytoplasmic membrane (Box 1a). For this reason, and despite the low pH of AMD waters, bioreactors operate with a previously neutralized inﬂuent . However, the existence of acidotolerant and acidophilic SRB has been lately reported [59–67], offering the possibility to treat acid waters directly, without previous inﬂuent neutralization. As long as SR generates alkalinity itself, most of the reactors are operated without pH control, allowing the pH to increase [33,68–70]. Here, metals precipitate simultaneously, which is useful for bioremediation, but disadvantageous for metal recovery. For metal recovery, the production of a pure or highly enriched metal sulﬁde is a key factor. The formation of metal sulﬁdes depends on the concentration of metal-ions, sulﬁde-ions and especially the pH [54,71]. Therefore,
Fig. 2. Interconnection of the different factors that have impact on formation of SRB communities and formation of crystals. Line styles show which factors and reactor design parameters correlate.
Box 1: Factors affecting sulfate reduction at low pH. (a) Proton concentration: The pH scale is a logarithmic scale for the proton concentration. This means that at pH 4 there are 1000 times more protons than at pH 7. This causes a diffusion pressure on the cell membrane in which much more protons diffuse through it at low pH compared to neutral pH. These protons have to be actively pumped out of the cell destroying the proton motive force. So, as long as at low pH bacteria need to invest energy to maintain a higher/circum-neutral internal pH, less energy is available for growth. This fact could imply the inexistence of acidophilic SRB (aSRB), but on the other hand, thermodynamical calculations show that the Gibbs free energy of sulfate reduction is higher at a lower pH resulting in more energy gain . If this extra energy can compensate for the extra energy needed to export protons out of the cell, growth can be achieved. Extra mechanisms described for other acidophiles might be present and need to be described, e.g. harnessing the proton motive force for ATP production, expelling of H+ containing vesicles, etc. (b) Organic acids: Organic acids are inhibitory at low pH. Their toxicity depends on their dissociation constants because different concentrations of the acid form would be present at different pH. For instance, the pKa of lactic acid is 3.08 (Ka = 8.3 × 10−4 ) what means that at pH 3, 50% of the lactate would be in the acid form. The acid (undissociated) form can diffuse into the cells. Once inside the cell, the higher internal pH will produce the acids to dissociate, releasing protons and lowering the intracellular pH. This again implies energy lost to pump out the protons. That is the reason why most of the studies failed in isolating aSRB when using lactate as electron donor, the most common substrate for SRB enrichments at neutral pH. Lately studies on aSRB strongly conﬁrm that non-ionic substrates such as glycerol, hydrogen, alcohols or sugars are more convenient for low pH applications [66,67,81,82]. Additionally, SRB can be sub-divided into “complete oxidizers”, those that oxidize organic substrates completely to carbon dioxide, and “incomplete oxidizers” which oxidize them to acetate and CO2 . In solutions of pH <4.75, the pKa of acetic acid, the dominant form of this metabolite is acetic acid rather than acetate, which has been widely reported to be highly toxic to the majority of micro-organisms, including acidophiles [83–85]. Most of the isolated aSRB are incomplete oxidizers  so when these SRB are grown in axenic cultures, acetate accumulates in the media creating inhibition by product. In a mixed SRB culture at around pH 6.2, 50% growth inhibition occurred at 0.9 mM of undissociated acetic acid, which would correspond to a total acetic acid concentration of 25 mM . In natural environments, within a mixed microbial community, acetate would be metabolized by other anaerobes (such as methanogens) that would co-exist with SRB. (c) Sulﬁde: H2 S has a pKa of 7 at 30◦ C, so the products of sulfate reduction at neutral pH are equal amounts of H2 S and HS− . Whereas at lower pH (pH 5) about 99% of the product is H2 S. Sulﬁde has an inhibitory effect on the bioreactor microbial community which is species dependent (H2 S, HS− and S2− ) . It is suggested that undissociated sulﬁde (H2 S) is the inhibiting compound [88,89] as they may pass the cell membrane in the undissociated/acid form . Therefore, at low pH there is a higher potential of sulﬁde inhibition. Additionally, sulﬁde inhibition is also due to the fact that it combines with iron in cytochromes or any other metal-containing compounds  affecting functionality. In conclusion, proton, sulﬁde and organic acid toxicity could be commonly explained by their diffusion into the cell with the consequent energy lost in proton pumping across the membrane. The maintenance energy of the cell is increased and therefore, the energy left for growth is reduced.
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pH controlled conditions inside the reactor are essential (see Fig. 2). Several reactor studies were successfully performed at a controlled pH [56,72–76] enabling selective metal recovery. Besides metal recovery, operating reactors at low pH has multiple advantages. Addition of neutralizing reagents is avoided to reduce costs. The produced sulﬁde is mostly in the gaseous phase at low pH, which simpliﬁes sulﬁde separation from the efﬂuent using well-known technologies, e.g. subsequent metal precipitation [54,55] or posterior elemental sulfur oxidation with oxygen . In addition, SRB will better outcompete methanogens at low pH [76,78] because methanogens are more sensitive to low pH. As a result, less of the costly electron donor is lost in other processes (acetogenesis, methanogenesis) than sulfate reduction, which increases the efﬁciency of the overall process. Substrate competition between different microbial groups has been broadly documented [79,80]. 3.2. Reactor conﬁguration The off-line sulﬁdogenic reactors are run in different ﬂow modes (batch, continuous or semicontinuous) being the continuous mode the most widely applied. In continuous mode, different reactor conﬁgurations are also used varying in the biomass type (suspended growth, bioﬁlm, etc.). Fig. 3 summarizes the main characteristic distinctions of the continuous off-line sulﬁdogenic reactors. In addition to that distinction, two main operational designs have been broadly used for sulﬁdogenic aims. The ﬁrst is based on two-stage reactors where SR occurs in one reactor and the produced sulﬁde is recirculated to another reactor to precipitate the metallic sulﬁdes at controlled conditions. The second device is an all-in-one reactor (one-stage), in which sulfate reduction and metal precipitation occur together. The chosen design inﬂuences the size and shape of the sulﬁde crystals, which has a direct effect on their settling abilities and subsequently on the posterior dewatering of the metal sulﬁdes and on the metal recovery (Box 1). When the sulﬁde concentrations are low, the nucleation rates (formation of new particles) are low as well resulting in growth of larger crystals instead of formation of new ones, which allows better settling qualities . In one-stage reactors where sulﬁde is produced by SRB, a homogeneous sulﬁde concentration will be reached and the crystal growth will be bigger. In this way, crystals with better settling properties will be obtained. In the two-stage reactors, when sulﬁde is dosed through the gas phase into the contactor of the ﬁrst reactor, local high sulﬁde concentrations exist around the injection [55,94,95] resulting in smaller particles. Based on that, one-stage reactor designs might be preferred for metal recovery. Other advantages of the one-stage reactor devices are the reduction of the investment costs, an easier process design, avoidance of transport of the corrosive sulﬁde to the ﬁrst reactor, and a lower potential of sulﬁde inhibition (Box 1c) due to instant metal precipitation. 3.3. Inoculum After analyzing all the factors that inﬂuence metal recovery, it appears that the best choice is a one-stage reactor with controlled pH. For differential metal precipitation, it is advantageous when SR takes place at low pH, which is performed by acidotolerant SRB communities or preferably by acidophilic SRB. In this context, the choice of the right inoculum is crucial. If a neutrophilic community is used as inoculum and forced to adapt to lower pH values, the process might work depending on the adaptability of the communities, but sooner or later it will fail at a certain low pH. For instance, some studies where a neutrophilic inoculum was used, achieved SR at pH 4 but failed at lower pH . In contrast, another study with a mixed
community of acidophilic SRB obtained from naturally extreme acidic environments, showed selective metal recovery operating at controlled pH as low as 2.2–2.5 . An important point of discussion is the difference in performance of acidophilic and neutrophilic SRB. In general, their nutritional requirements are similar, with the obvious exception of optimal pH and pH range for growth and the inﬂuence of the factors explained in Box 1 (sulﬁde and organic acid concentrations). The doubling time will strongly depend on the strain and the culturing conditions (electron donor and yeast extract concentrations, reducing agent used, etc.) and thus a good comparison is not always feasible. So far, just two truly acidophilic SRB have been characterized [66,96]. There is not enough information for comparison in terms of general kinetic coefﬁcients with neutrophilic strains. But as a reference, the growth rate of Desulfosporosinus acidiphilus on glycerol was 0.4 h−1 at pH 5.2 (doubling time of 1.7 h) . A novel proposed acidophilic species, Desulfosporosinus acidodurans, showed a growth rate of 0.046 h−1 (doubling time of 15 h) at pH 5.5 on glycerol (data not published). The closest neutrophilic SRB strains with available information are Desulfosporosinus lacus and Desulfosporosinus burensis. D. lacus presented at pH 7 on lactate a growth rate of 0.08 h−1 , equivalent to a doubling time of 8.6 h . D. burensis showed a growth rate of 0.095 h−1 , equivalent to a doubling time of 7.3 h  on fructose. Another comparison can be the sulfate reduction rate per cell (csSRR). The csSRR of the novel proposed acidophilic SRB D. acidodurans was 32.75 fmol cell−1 day−1 , which is in the range of different neutrophilic strains which varies from 0.9 to 434 fmol cell−1 day−1 . In conclusion, although more studies need to be performed at comparable conditions, preliminary data show that under optimal conditions acidophilic and neutrophilic SRB have a similar performance. 3.4. Substrate As aforementioned, AMD usually contain a low organic carbon concentration and addition of extra electron donor is needed. The electron donor will directly impact the reactor performance (Fig. 2). In passive reactors, complex organic sources are added to promote a long-term activity. The priority in that case, is mainly local availability to treat AMD and low costs of the added organic materials . However, in sulﬁdogenic bioreactors at low pH, the potential toxicity of the substrate (Box 1b) should be taken into consideration. Therefore, either hydrogen, glycerol, sugars or alcohols are preferred at low pH [56,59,72]. Many studies have been done at different reactor conﬁgurations, hydraulic retention times (HRT), substrates, inocula and running pH (Table 2). 4. Microbiology associated with bioremediation of metal-containing waters Although several factors have to be taken into consideration for an optimal reactor performance, the process will be essentially microbiologically driven. Therefore, the survival of the microbial communities, their activity and their growth are crucial. Indeed, some researchers have compared the inﬂuence of different aspects on AMD remediation such as different inocula , type of organic substrates [70,113], characteristics of inﬂuent AMD, conﬁguration and oxygen exposure  and the effect of bioaugmentation/biostimulation . In aerobic wetland mesocosms, different factors for Fe2+ removal rates were assessed: pH, Fe2+ concentration of the AMD, the wetland plants, and activity associated with the microbial community. Among them, the microbial activity had the largest inﬂuence on the removal rates .
Table 2 Summary of research for sulﬁdogenic reactors under different conditions. pH
Temperature (◦ C)
Source of inocula
Carbon and energy source(s)
Sulfate volumetric reduction rate (g L−1 d−1 )
                        
8.0 7.5 7.3 7.0 7.0 6.8 6.5 6.2 6.0 5.0 5.0 5.0a 4.5 4.5 4.2 4.0 4.0 4.0 4.0 4.0 3.0a 3.0 2.5 2.5–3a 2.3
35 65 30 – 35 25 – (−13)−36 30 30 30 30 25 30 23–26 – 30 30 23 25 35 – 30 35 30
Waste-water treatment plant Sludge from a sulfate reducing reactor THIOPAQ granular sludge Mixed SRB Digested sludge Anaerobic digester ﬂuid Water from anaerobic zone of a creek Acidic enrichment culture Neutrophilic granular sludge Neutrophilic granular sludge Neutrophilic granular sludge Acidic sediment Water from the wetland ﬁlter of a mine site Neutrophilic granular sludge Spent manure Derelict mine sites Acclimated granular sludge Acclimated granular sludge SRB enriched from a wetland Disintegrated granular sludge Methanogenic sludge and mine sediments Water from a lignite mine – Methanogenic sludge and mine sediments Acidophilic microbial community
Acetate and peptone Methanol Synthesis gas-fed Ethanol Acetate Waste material Reactive mixture Ethanol or wood chips and corn stover Formate H2 /CO2 Formate Domestic water Lactate H2 /CO2 Methanol Ethanol, lactate and glycerol Formate H2 /CO2 Lactate Acetate, lactate (9:1) Ethanol Methanol Acetate and H2 /CO2 Lactate Glycerol
90–48 3.5 – 5.1 60a 480a – 72–216 24 18 12 24–48 16.2a
CSTR EGSB GLB FBR Packed-bed Packed-bed Packed-bed – MBR MBR MBR UASB Packed-bed MBR Packed-bed Packed-bed MBR MBR UASB Down-ﬂow FBR FBR Packed-bed Packed-bed UASB and FBR CSTR
0.17–0.40 15 15 6.336 0.312 0.12 0.12 0.03b 29 5 18 0.34 0.48 7.5 1.608 0.50 6.1 8 1.27 0.89b <4.3 3.12 4.8 <1.9 0.12b
6.6a 49.3a 12 12 35.5, 16, 10 24 21–6.1 12, 4.2 21.6a 16.0 47a
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Non controlled pH conditions, pH increment during the reactor performance. Calculated with data extracted from the referenced publication.
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Fig. 3. Reactor types used for sulfate reduction with: (a) continues stirred tank reactor (CSTR), (b) gas-lift bioreactor (GLB) with internal loop, (c) submerged membrane bioreactor (MBR), (d) ﬂuidized bed reactor (FBR), (e) upﬂow anaerobic granular sludge bed (UASB) bioreactor, and (f) expended granular sludge bed (EGSB) reactor. Source: Modiﬁed from .
The combination of culture-based techniques and cultureindependent molecular biological approaches during the last decades has greatly enhanced our understanding of the functioning of microbial communities in general and of bioremediation processes in particular [41,61,68,69,72,75,76,108,112,116–126]. In spite of this, sulfate-reducing microbial communities used for AMD treatment have not been extensively analyzed. Here, information on microbial communities associated with AMD treatment processes is presented. 4.1. Phylogeny of microorganisms detected in AMD treatment processes About 500 16S rRNA gene sequences detected in studies of AMD treatments have been analyzed for this review. The data were either present in general databases (NCBI or EMBL) or requested from authors of published papers. Sequences belonging to both Bacteria and Archaea domains have been identiﬁed. When methane was detected during the reactor operation, the Archaea domain was analyzed resulting in the detection of methanogens belonging to Euryarchaeota phylum. In the bacterial domain, different phyla (13) have been identiﬁed, including Chloroﬂexi, Chlorobi, Proteobacteria (classes Alpha-, Beta-, Gamma-, Delta- and Epsilon-Proteobacteria), Firmicutes, Actinobacteria, Planctomycetes, Spirochaetes, Acidobacteria, Bacteroidetes, Verrucomicrobia, Gemmatimonadetes, Synergistetes and Caldiserica. Most of the sequences retrieved from the literature could be identiﬁed at the genus level and showed a large diversity with around 100 genera. A detailed analysis of the phylogeny of all the sequences is presented in the supplemental information (Table S1). From these data, a condensed table is presented (Table 3) where more abundant detected genera were selected. It is generally assumed that acidic systems harbour a low bacterial, archaeal, viral and sometimes eukaryotic biodiversity [11,130–133]. That is indeed the case for AMD water bodies where an extreme low pH (<3) is maintained and buffered by high concentrations of iron (Eq. (22)). This results in a homogeneous
environment with a stable and well adapted community with iron and sulfur dependent metabolism, such as members of Leptospirillum, Acidithiobacillus or Acidiphilum genera . However, in sediments the situation is different. The existence of microniches, with different pH values and redox potential within the bulk media, different organic matter composition and concentration allows the development of complex and more diverse communities [134,135]. A similar high diversity has been observed in bioreactors treating AMD, where depending on the substrates added, the microbial communities can be very diverse with a whole range of microorganisms varying from cellulolytic fermenters, SRB, acetogens, and methanogens [41,70,114]. 4.2. Metabolic groups The pH, the type of inoculum and the substrate have a major effect on the microbial community composition. The pH directly affects the activity of different microorganisms. The inoculum may contain acidotolerant  or acidophilic communities [56,69,113,136]. The type of substrate(s) will inﬂuence the complexity of the communities . In sulﬁdogenic reactors, the major biological conversion processes are sulfate reduction, acetogenesis and methanogenesis , together with hydrolytic- and fermentative bacteria in case of complex substrates. Microbial communities can be considered as a reservoir of microbial strains with complementary ecological niches that is beneﬁcial for the stability of bioreactor performance . Koschorreck et al.  reported that the increase of microbial diversity stabilized the bioﬁlm’s function under ﬂuctuating and partly oxidizing conditions in a reactor treating acidic mine pit water. The role of different metabolic groups in AMD bioreactors will be brieﬂy highlighted in the following subsections. 4.2.1. Hydrolytic- and fermentative-bacteria SRB are not known to degrade biopolymers directly. When a complex polymeric substrate is used, SRB rely on the activity
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Table 3 Selection of more abundant genera occurring in AMD remediation studies (complete Table in Supplementary Table S1). Phylum
BACTERIA Actinobacteria Bacteroidetes
Sphingobacteria Anaerolineae Alphaproteobacteria
Cellulomonas Bacteroides Paludibacter Proteiniphilum Unclassiﬁed Longilinea/Leptolinea/Uncultured Devosia/Pedomicrobium/Hyphomicrobium Uncultured Nitrobacter Rhizobium Sphingomonas Thiobacillus Albidiferax/Rhodoferax Enterobacter Desulfobacterium Desulfobulbus Desulfomicrobium Desulfovibrio Geobacter Desulfobacca Sulfuricurvum Spirochaeta/Treponema Uncultured Edaphobacter/Acidobacterium Clostridium sensu stricto Anaerofustis Anaerovorax Clostridium XlVa Uncultured Desulfosporosinus Clostridium XI Saccharofermentans Uncultured Uncultured
[33,56,113] [33,70,112,113] [33,67,70,72,75,113,127,128] [33,76,80,108] [33,68,70] [68,108,112] [33,112] [33,112] [33,70] [41,70] [33,70] [56,68,113,120] [33,70] [33,112] [108,112] [33,70,129] [70,80] [33,41,68–70,72,73,76,80,112,113] [68,70] [68,108] [33,68] [33,68,70,76] [80,108] [33,70] [33,69,76,120] [33,108] [70,112] [33,70,76,112] [69,72] [33,69,72,120,129] [33,68,69] [33,70] [33,70,112] [33,70]
Methanobacterium Methanobrevibacter Methanospirillum Methanosaeta Methanosarcina
 [76,113]   
Betaproteobacteria Gammaproteobacteria Deltaproteobacteria
Spirochaetes Synergistetes Acidobacteria Firmicutes
Epsilonproteobacteria Spirochaetes Synergistia Acidobacteria Clostridia
Negativicutes ARCHAEA Euryarchaeota
of anaerobic hydrolytic- and fermentative-bacteria. The breakdown of complex organic materials provide SRB with carbon and energy sources such as volatile fatty acids and hydrogen . Then, SRB catalyze the ﬁnal reaction of the digestion process. Efforts to improve microbiological design criteria must consider the entire microbial community and not merely SRB. For instance, Hiibel et al.  showed, based on 16S rRNA gene sequences, that SRB represented just 2–5% of the entire microbial community in lignocellulose-fed reactors, whereas SRB represented about 70% of the population in ethanol-fed reactors. Additionally, fermentative bacteria were estimated to be 25–30% of the lignocellulose-based communities and only 5% of the ethanol-based community. Within the potential hydrolytic communities, the cellulose degraders are the most signiﬁcant because the most common organic sources used are either simple substrates, or cellulosebased ones such as softwood dust, wood chips, stalks, hay, cereal straw and different mixtures of them [31,32,34–40,46–50]. The use of fat or protein based substrates such as whey  is less common. Cellulose degraders have been observed to perform a potentially rate-limiting step in substrate degradation . When cellulose is applied as substrate some phylotypes of microorganisms prevail, such as those belonging to the genus Bacteroides [33,70,112,113], Clostridium [33,70,112,120], Acetivibrio , Spirochaeta [33,68,70,76], Ruminococcus  and Cellullomonas [33,113]. Enterobacteria group is especially represented in cellulose fed reactors [33,70].
Some fermentative bacteria are more commonly found in AMD bioremediation systems such a bacterium 92% similar to Paludibacter propionicigenes (Bacteroidetes) [33,67,70,72,75,113,127,128] which interestingly, it has been also detected in several natural acidic environments [60,134,141]. The prevalence of this bacterium in acidic environments suggests its active role in both natural and engineered ecosystems, but thus far its physiological properties are not clear. Many representative of Clostridia group have been detected (Table S1). Clostridia are well-known sugar and protein fermenters using different fermentation pathways depending on the strain and the substrate. Their presence in reactors fed with easily degradable substrates such as sugars also suggests their active role in these kind of communities [68,70,76]. 4.2.2. Acetogenic bacteria Acetogens are a diverse group of organisms (over 100 species representing 22 genera)  which reduce CO2 , assimilating carbon into cell material through the acetyl-coA pathway. This pathway is also used for energy conservation. In some cases all the species of a genus are acetogens, as Moorella or Sporomusa genera, but in most of the cases acetogens are phylogenetically dispersed within genera that contain nonacetogenic species as it is the case for the Clostridium genus . Therefore, no conclusion about the acetogenic metabolism can be drawn based on 16S rRNA sequence analysis. However, some of the characteristic acetogenic genera appeared in the molecular analysis of reactors treating AMD such as
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oxic conditions for months . Interestingly, Desulﬁtobacterium is a genus with members that can use sulﬁte as electron acceptor, but not sulfate. The percentage of similarity of some of the detected sequences would imply their clustering in a new genus , therefore further characterizations are need to know their real metabolism. Some bacteria phylogenetically related to sulfur reducers such as Desulfurella  have been detected in AMD bioreactors as well in natural acidic conditions [134,135]. Desulfurella spp. are moderately thermophilic and are not known to reduce sulfate [153,154]. The ability to reduce sulfur is described for some cultured Desulfosporosinus strains such as strain M1, D and E [62,67].
Fig. 4. Phylogenetic afﬁliation of 16S rRNA sequences of SRB detected in reactors treating AMD. Tree was generated by parsimony with ARB software .
Acetobacterium  and Clostridium [33,68–70,76,112,120], a genus which harbours the most known acetogenic species ; or others which contain some acetogenic species such as Ruminococcus  or Treponema [33,68,70,76]. Other acetogenic genera have been also detected in acidic sulﬁdogenic enrichments such as Oxobacter . 4.2.3. SRB In reactors fed with easy degradable substrates (lactate, hydrogen, ethanol), SRB are the major key players. Based on 16S rRNA sequence analysis, SRB are comprised by seven phylogenetic lineages, ﬁve within the Bacteria and two within the Archaea . SRB were also described clustering in 4 four groups : (1) the Deltaproteobacteria lineage, the Gram-negative mesophilic SRB (i.e. Desulfovibrio spp.); (2) the Firmicutes lineage of the Gram-positive mesophilic spore-forming SRB (i.e. Desulfosporosinus spp.); (3) three lineages of thermophilic bacterial SRB, Nitrospirae (Thermodesulfovibrio spp.), Thermodesulfobacteria (Thermodesulfobacterium spp.) and Thermodesulfobiaceae (Thermodesulfobium narugense); and (4) two lineages of thermophilic archaeal sulfate-reducers Archaeoglobus (Euryarchaeota) and Thermocladium and Caldirvirga (Crenarchaeota). Diverse SRB genera have been found in AMD bioreactors belonging to the mesophilic Gram-negative SRB (Deltaproteobacteria class) and Gram-positive spore forming SRB (Clostridia class, Firmicutes phyla) (Fig. 4). Sequences belonging to known neutrophilic genera were mainly detected such as Desulfobacca [68,108], Desulforhabdus , Desulfovibrio [33,41,68–70,72,73,76,80,112,113], Desulfobacterium [108,112], Desulfobulbus [108,112], Desulfomicrobium [70,80], and Desulfomonile . Nevertheless, some of the identiﬁed neutrophilic species also have been found in different natural acidic environments such as Desulfosarcina, Desulfococcus or Desulfobulbus  and Desulfovibrio . Their presence could be either due to the presence of microenvironments or to the existence of acidotolerant strains. Some of the cultured representative of the detected bacteria oxidize their substrates completely to CO2 such as Desulfobacca  and Desulforhabdus  but most of them such as Desulfovibrio and Desulfomicrobium spp. are known to oxidize their substrates incompletely to acetate , which will create the aforementioned acetate inhibition (Box 1b). SRB belonging to the genus Desulfosporosinus are commonly detected in reactors functioning at low pH values and inoculated with material from acidic sites. They are known to thrive in low pH environments [60,65,118,129,134,135,149–151] together with members of the closely related genus Desulﬁtobacterium [60,82,152]. Members of the last group have also been detected in reactors operating at low pH [33,56,69,72,120,129]. Both genera form spores, a property which enables them to survive dryness and
4.2.4. Methanogenic archaea At an excess of sulfate, according to the Gibbs energy yield, SRB generally should outcompete methanogens. The persistence of methanogenesis in reactors treating AMD at neutral pH was explained by the relatively similar Monod kinetics of SRB and methanogens and not by the hydrogen thresholds, which would have clearly favoured the SRB . Methanogens seem especially sensitive to low pH; so far, just one acidophilic methanogen has been isolated . Acidophilic SRB outcompeted methanogens at low pH [76,78]. However, methanogens have been detected in acid environments with an excess of sulfate [156–162] and in bioreactors treating AMD. From reactors treating AMD waters, some methanogens were detected in those reactors operating at neutral pH such as Methanobacterium and Methanospirillum , others appeared in reactors operating at acidic pH such as Methanosaeta , Methanosarcina  or Methanobrevibacter [76,113]. The latter one, Methanobrevibacter arboriphilus, which appeared in different studies has been detected also in enrichments inoculated with ARDrelated sediments  as well as in reactors operated at a pH 5 with formate as substrate . This fact could be attributed to three main reasons: (a) chemically, the Gibbs energy calculation for the hydrogenotrophic methanogenesis remains constant at different pH according to Dolﬁng et al. , but acetoclastic methanogenesis is energetically more favourable at pH below 4.5. If acetate inhibition could be overcome, the process would be thermodynamically possible and favourable in chemical terms, (b) survival of methanogens in bioﬁlms where the cells would be active but not dividing. In this way, several microorganism with different energetic efﬁciencies would coexist  and ﬁnally, the more feasible explanation, (c) methanogens would create microniches with higher pH where they can survive . 5. Concluding remarks Sulﬁdogenic reactors already passed the experimental phase and they have been satisfactorily applied at full-scale level to remove zinc at Nyrstar Budel Zinc reﬁnery (The Netherlands) with systems such as THIOTEQTM and SULFATEQTM developed by Paques. For industries with a load of mixed metals, new challenges have to be undertaken such as the application of low pH-controlled reactors at full-scale. Others technologies have been recently proposed such as the application of sulfur reduction instead of sulfate reduction. The reduction of sulfur to sulﬁde implies only 2 electrons instead of the 8 electrons needed for sulfate-reduction. Therefore, there is a reduction of electron donors supply with the concomitant saving of costs. Acknowledgements Research was ﬁnanced by grants of the divisions CW (Project 700.55.343) of the Netherlands Science Foundation (NWO) and ERC
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