Eng. Life Sci. 2007, 7, No.

6, 541–564 541

A. H. Kaksonen1 Review
J. A. Puhakka1

1
Tampere University of
Sulfate Reduction Based Bioprocesses
Technology, Environmental for the Treatment of Acid Mine Drainage
Engineering and
Biotechnology, Tampere, and the Recovery of Metals
Finland.

Biological sulfate reduction is increasingly replacing chemical unit processes in

mining biotechnology. Sulfate reducing bacteria (SRB) can be used for treating
ground- and surface waters contaminated with acid mine drainage (AMD), and
for recovering metals from wastewater and process streams. Biologically produced
H2S precipitates metals as metal sulfides, while biogenic bicarbonate alkalinity
neutralizes acidic waters. This paper reviews various passive and active SRB-based
alternatives as well as some process design aspects, such as reactor types, process
configurations, and choices of substrates for sulfate reduction. The latest develop-
ments of using various low-cost substrates together with new bioprocess designs
are increasing the uses and applications of SRB-based bioreactors in AMD control
and selective metal recovery.

Keywords: Acid mine drainage, Bioremediation, Metal recovery, Sulfate reduction

Received: July 2, 2007; revised: September 13, 2007; accepted: September 24, 2007
DOI: 10.1002/elsc.200720216

1 Introduction FeS2 + 14 Fe3+ + 8 H2O → 15 Fe2+ + 2 SO42– + 16 H+ (3)

Mining results in the introduction of oxygen and water to the
deep geological environment leading to the oxidation of
minerals, which are in a reduced state [1, 2]. Oxidation also
occurs when reduced minerals are brought to the surface and
deposited in heaps. The most abundant family of reduced
minerals are the sulfides. The oxidation of the sulfides of the
type MS2 leads to the liberation of protons [1]. The oxidation
of pyrite (FeS2) can be described with the following reaction
[1]:
2 FeS2 + 7 O2 + 2 H2O → 2 Fe2+ + 4 SO42– + 4 H+ (1)
The oxidation of ferrous to ferric iron consumes protons ac-
cording to the following reaction [1]:
4 Fe2+ + 4 H+ + O2 → 4 Fe3+ + 2 H2O (2)
Ferric iron may act as an electron acceptor for further pyrite
oxidation, or hydrolysis may occur, both processes releasing
further protons [1]:
– metals and sulfate from wastewaters (for reviews, see [9–12]).
Correspondence: A. H. Kaksonen (anna.kaksonen@tut.fi), Tampere
University of Technology, Environmental Engineering and Biotechnol-
ogy, PO Box 541, FIN-33101 Tampere, Finland.
Fe3+ + 3 H2O → Fe(OH)3 + 3 H+ (4)
The overall sequence of reactions is acid-producing [1]:
4 FeS2 + 14 H2O + 15 O2 → 4 Fe(OH)3 + 8 SO42– + 16 H+ (5)
Other sulfide minerals are oxidized in a similar way as py-
rite, releasing metals and sulfate in solution. However, the oxi-
dation of sulfides of the type MS does not release acid, e.g.
sphalerite oxidation [1]:
ZnS + 2 O2 → Zn2+ + SO42– (6)
Acidic conditions result in a further dissolution of heavy
metals from metal oxides and carbonates [3]. The oxidation of
sulfide minerals leads to the formations of acidic metal- and
sulfate-containing wastewaters, often called acid mine drainage
(AMD) or acid rock drainage (ARD) [3–6]. The environmen-
tal impacts of AMD are severe and widespread in many coun-
tries [7]. Pollution control of AMD can be achieved by pre-
venting AMD formation, migration and/or collection and
treatment [8, 9]. Numerous physicochemical and biological
techniques are available for the neutralization and removal of
The most widely used active treatment process for AMD is
based on chemical neutralization and hydroxide precipitation
of metals [10, 11, 13]. The disadvantages of the traditional

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542 A. H. Kaksonen et al. Eng. Life Sci. 2007, 7, No. 6, 541–564

chemical treatment include high cost of the chemical reagents, tive bioreactors have been developed for AMD. More recently,
inefficient removal of sulfate, and the production of a bulky the use of biogenic H2S has been extended to the selective re-
sludge, which must be disposed of [14, 15]. Sulfide precipita- covery of metals from various biohydrometallurgical process
tion of metals has been demonstrated to have several benefits streams.
over the hydroxide precipitation, such as lower effluent metal This review is an overview of the various SRB-utilizing bio-
concentrations, better thickening characteristics of the metal process alternatives as well as some process design aspects,
sludge and the possibility to recover valuable metals [11, 13, such as reactor types, process configurations, and choices of
16–19]. Chemical sulfide precipitation has not been widely substrates for sulfate reduction.
used for AMD treatment, probably due to the high cost of
chemicals [3, 10].
Efforts have been made to develop biological alternatives for 2 Passive Treatment Applications
AMD treatment and metal recovery. Several biological process-
es can remove metals from wastewaters, including biosorption, Passive SRB-based applications for the treatment of AMD con-
intracellular uptake and accumulation, complexation, oxida- taminated groundwater include the enhancement of the mi-
tion and reduction, methylation combined with volatilization, crobial activity in groundwater aquifers through substrate in-
and extracellular precipitation (for reviews, see [20–22]). Also, jection [32] or permeable reactive barriers [33–36]. Passive
a number of biological processes can generate alkalinity or treatment applications for surface waters include infiltration
consume acidity and which, therefore, have potential use in beds [37, 38], anoxic ponds and wetland systems (see Fig. 1)
neutralizing AMD [9]. These include photosynthesis [9, 23, (for reviews, see [23, 39]).
24], denitrification 25, 26], ammonification, methanogenesis,
and reduction of iron and sulfate [9, 25–27].
Due to the combined removal of acidity, metals and sulfate,
sulfate-reduction appears to be the most promising bioprocess
for AMD treatment and metal recovery. Recently, the interest
in the application of sulfate reduction as the dominant step
of wastewater treatment has been growing (for reviews, see
[28, 29]). The process is based on biological hydrogen sulfide
and alkalinity production by sulfate-reducing bacteria (SRB)
(reaction 7):

2 CH2O + SO42– → H2S + 2 HCO3– (7)

where CH2O denotes the electron donor.

When hydrogen is used as an electron donor for sulfate
reduction, the reaction yields hydroxide ions (reaction 8):

8 H2 + 2 SO42– → H2S + HS– + 5 H2O + 3 OH– (8)

The biogenic hydrogen sulfide precipitates dissolved metals

as low solubility sulfides (reaction 9):

H2S + M2+ → MS(s) + 2 H+ (9)

where M2+ denotes the metal, such as Zn2+, Cu2+, Ni2+, Co2+,
Fe2+, Hg2+, Pb2+, Cd2+, or Ag+.
The metal precipitation reaction releases protons, thus add-
ing to the acidity of the water. Therefore, excess sulfate needs
to be reduced to compensate for the acidity. Bicarbonate alka-
linity or hydroxide ions produced in the sulfidogenic oxidation
of electron donors (reactions 7 and 8) neutralizes the acidity of
the water (reactions 10 and 11) [2, 30].

HCO3– + H+ → CO2(g) + H2O (10)

OH– + H+ → H2O (11)

The potential utility of the microbial sulfate reduction in
mining applications was proposed already in the late 1960s
[31]. Since then, SRB-based passive treatment systems and ac-
Figure 1. Passive, sulfate reduction based applications for acid
mine drainage (AMD) or AMD contaminated groundwater: (A)
injection of substrates into the subsurface, (B) permeable reac-
tive barriers, (C) infiltration beds, (D) anoxic ponds, (E) anae-
robic wetlands, adapted from [23, 34, 38–40].

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Eng. Life Sci. 2007, 7, No. 6, 541–564
2.1 Placing or Injecting Substrates into the
Subsurface
AMD-contaminated groundwater has been remediated in situ
by enhancing the activity of sulfate-reduction by placing or in-
jecting substrates to the subsurface through boreholes (see
Fig. 1A) [32, 40] or constructing permeable reactive barriers
across the groundwater flow path (see Fig. 1B) [33–36]. Grou-
dev et al. [32] reported an increase in the numbers and activity
of SRB in the contaminated groundwater after injecting an ac-
etate-bearing waste product and ammonium phosphate into
the subsurface near the Burgas Copper Mines, Bulgaria. The bio-
genic H2S reduced U6+ to U4+ and precipitated heavy metals
(Cu, Zn Pb, Mo and Mn) as sulfides [32]. Also, Canty [40] re-
ported high removal efficiencies for metals (Al, Cd, Co and Zn)
and an increased pH in mine water flowing through organic sub-
strate placed in mine shafts. However, higher flow rates during
the spring and oxygenated surface water resulted in a pH de-
crease and resolubilization of the metal precipitates [40].
2.2 Permeable Reactive Barriers
Permeable reactive barriers consist of zones of reactive material
installed across the flow path of the plume of contaminated
groundwater [41]. As the AMD-impacted groundwater flows
through this zone, SRB reduces sulfate from the water by using
the electron sources present in the barrier. This results in the
generation of bicarbonate alkalinity and precipitation of met-
als as sulfides. In addition to organic materials, zero-valent
iron (Fe0) can be used to stimulate sulfate-reduction, since Fe0
corrosion depletes oxygen and produces cathodic H2, a poten-
tial energy source for SRB [42]. The selection of the reactive
mixture significantly affects the permeability and reactivity of
the barrier [34, 36]. Gravel can be mixed with organic substrate
to increase the permeability of the barrier, and limestone may
be added to provide additional alkalinity [33, 34, 36]. A pre-
requisite for successful in situ remediation is a thorough
hydrogeological characterization of the site. Reactive barriers
often rely on the natural groundwater flow to transport con-
taminants through the treatment zone, which results in long
treatment times. Depletion of organic substrates and clogging
of the barrier due to metal precipitation may deteriorate the
long-term effectiveness of the system [41].
2.3 Infiltration Beds
Infiltration beds are used for remediating AMD contaminated
surface waters in a similar manner as reactive barriers are used
for groundwaters [38]. Infiltration beds can be constructed
into the ditches of mining areas, so that the water flows
through the bed material. The bed contains organic materials
that support the growth and activity of sulfate reducers. The
organic material is covered with an impermeable liner that
helps to create anaerobic conditions (see Fig. 1C). The organic
material can be supplemented with nutrients and an inoculum
of sulfate reducers to enhance the efficiency of the infiltration
bed. The material should also have a suitable hydraulic con-
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SRB Applications 543
ductivity, so that water flows through the material in a desired
time. An ideal material combination should retain its treat-
ment capacity for years without getting clogged due to metal
precipitates. The structure of the infiltration bed should also
prevent the redissolution of already precipitated metals [38].
Riekkola-Vanhanen [37] studied the use of a peat and lime-
stone containing infiltration bed for AMD treatment in Pyhä-
salmi mine, Finland. The reductions obtained in the water
flowing through the bed were 94–99 % Cu, 76–97 % Zn, 85–
96 % Fe, 76–96 % Mn, and 72–94 % sulfate [37].
2.4 Anoxic Ponds
Anoxic ponds are water basins supplemented with organic
substrate (see Fig. 1D). They can be used, for example, up-
stream of anoxic limestone drains, aiming at decreasing dis-
solved oxygen, reducing Fe3+ to Fe2+ and precipitating Al3+
[39]. Riekkola-Vanhanen and Mustikkamäki [43] used a
flooded open pit (Ruostesuo open pit near Pyhäsalmi mine,
Finland) as a large-scale basin to treat AMD with SRB. Press-
juice from silage and liquid manure were added as sources of
electrons and SRB, respectively. Sulfate reduction activity was
observed as a slow increase in the water pH and a decrease in
the sulfate, zinc, iron and manganese concentrations and redox
potential [43].
2.5 Wetlands
Wetlands have been recognized for several years as low-cost
systems to improve the water quality of AMD [44, 45]. During
the last two decades, constructed wetland systems have been
developed from an experimental concept to full-scale field ap-
plication [39] and used for the removal of sulfate, metals and
radionuclides from mine waters [46]. The approach has be-
come particularly popular in the USA, where hundreds of wet-
land sites have been operating for the treatment of wastewaters
from coal mine areas in Appalachia alone [9, 39]. Wetlands are
highly complex ecosystems, where the water quality is affected
by a number of physical, chemical and biological processes in-
cluding dilution, filtering of suspended particles, adsorption,
complexation, ion exchange and uptake of metals, and precipi-
tation by oxidative and reductive mechanisms [9, 39, 47, 48].
The wetland systems can be classified as aerobic and anaero-
bic wetlands, with the latter using SRB. Constructed aerobic
wetlands are shallow and their major objective is to enhance
the oxidation and hydrolysis reactions of iron and other met-
als, and to retain the resulting metal precipitates by entrap-
ment [6, 23, 49]. The hydrolysis of metals produces acidity
and, therefore, aerobic wetlands are applicable to the treatment
of net alkaline waters [23, 39]. Macrophytes are planted for
aesthetic reasons, as well as to regulate water flow and stabilize
the accumulating precipitates against erosion [6, 39]. In long
term, the periodical removal of solids may be needed, since the
accumulation of metal precipitates will likely decrease the
retention time in the wetland [50].
Wetlands supplemented with submerged organic substrate
and limestone are called anaerobic wetlands or compost wet-
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544 A. H. Kaksonen et al.
lands (see Fig. 1E). Anaerobic wetlands generate alkalinity
through a combination of microbial activity and limestone
dissolution [39, 50]. The reduction of both iron and sulfate are
considered important in anaerobic wetlands [9]. Other biolog-
ical activities may also contribute to the neutralization of
AMD, including methanogenesis and ammonification [6]. The
biogenic hydrogen sulfide precipitates metals as sulfides [23,
49]. Vegetation growing on the submerged substrate can pro-
vide a continuous supply of carbon and energy for the under-
lying microbial community [39, 51] and protect against wind
erosion at periods when the water level drops below that of the
substrate [39]. However, dense plant growth can also cause
problems, such as preferential flow paths and decrease in re-
tention time due to litter accumulation [50] or diffusion of
oxygen from the roots into the surrounding substrate [39].
Dense vegetation may also attract muskrats, which can damage
berms and create swimming channels that cause channelized
flow through the wetland cells [50]. It is likely that well-vege-
tated anaerobic wetlands will require periodic maintenance in
order to maintain the designed retention times [50]. Disposal
of metal-laden precipitates from anaerobic wetlands may be
more difficult than from aerobic wetlands [50].
Aerobic and anaerobic wetland units are often used consecu-
tively and in combination with anoxic limestone drains [6, 51–
53]. Wetland systems remediate AMD with low cost and mini-
mal maintenance [39]. However, their treatment efficiency has
been variable [6, 49, 52, 53]. The challenges of wetland treat-
ment include the following: (i) toxic effects of the AMD on
wetland organisms [54, 55], (ii) disposal of nonviable or excess
biomass containing heavy metals [54, 55], (iii) seasonal varia-
tions [6, 53, 55], (iv) catastrophic system failures that may occur
due to insufficient utilization of the treatment area, (v) metal
overloading and inadequate alkalinity production [49] and (vi)
futile cycling of iron and sulfur [6]. Wetland treatment may not
be effective in arid and semi-arid climates [6, 46], and exposure
of the metal-sulfide sediments to oxygen in the periods of
drought can lead to the dissolution of the metals and the acidi-
fication of the system [56]. Moreover, wetland treatment does
not always result in effective removal of manganese [6].
Figure 2. Characteristics of passive and active biological treat-
ment methods for acid mine drainage treatment, adapted from
[9, 39].
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2007, 7, No. 6, 541–564
Passive biological treatment approaches offer low-cost and
minimal maintenance solutions for treating AMD, and, thus,
they are also suitable for remote mining areas. However, the
required treatment area may be large, metal recovery difficult,
and control and predictability poor due to seasonal variations
(see Fig. 2) [39]. To alleviate the weaknesses of passive treat-
ment, SRB can be selectively enriched and their activity used
in more controlled bioreactors.
3 Active Bioreactors
Numerous reactor designs for biological sulfate reduction have
been reported (for reviews, see [9, 12, 28, 29]), such as batch
reactors [2], sequencing batch reactors [57–60], continuously
stirred tank reactors [61, 62], anaerobic contact processes [63],
anaerobic baffled reactors [64], anaerobic filters [65], fluid-
ized-bed reactors [66–68], gas lift reactors [3], upflow anaero-
bic sludge blanket reactors [69–73], anaerobic hybrid reactors
[74], and membrane bioreactors [75, 76] (see Fig. 3).
3.1 Continuously Stirred Tank Reactor (CSTR)
and Anaerobic Contact Process (ACP)
The reactor configuration has implications for the ratio of
sludge retention time/hydraulic retention time (SRT/HRT) in
continuous flow reactors [77]. The loading rates of a process
are largely dictated by the biomass retention in the reactor
[82]. Maximal sludge retention or biomass retention is desir-
able for process stability and minimal sludge production.
Minimal HRT minimizes the reactor volume and thus reduces
capital costs [77]. Continuously stirred tank reactors (CSTR)
(see Fig. 3A) are subjected to washout of active biomass [77].
Biomass retention has been enhanced by employing internal
sedimentation systems and cationic flocculants [83]. An anae-
robic contact process (ACP) relies on the separation of bio-
mass from effluent and recycling the biomass back to the reac-
tor to increase the SRT/HRT (see Fig. 3B) [77]. Several
methods have been used for recovering biomass from the reac-
tor effluent, including sedimentation, flocculation, centrifuga-
tion [71] and magnetic separation of sulfate-reducing bacteria
[84, 85].
3.2 Anaerobic Filter Reactor (AFR)
Various immobilized biomass reactors have gained increasing
attention. In anaerobic filter reactors (AFR), biomass is re-
tained as a biofilm on packing material as well as unattached
in the packing interstices (see Figs. 3C and D) [77]. AFRs have
been operated in horizontal [86], upflow [65, 87–94] or down-
flow modes [95–97]. The downflow AFR allows the utilization
of gravity and, thus, passive operation [98]. Packing materials
used in AFRs include cobbles [97], polypropylene pall rings
[99], glass beads [100, 101] and alkaline minerals [92, 97]. Bio-
logical sulfate reduction has been enhanced with solid organic
materials [91–93, 95, 97, 102, 103] as well as liquid substrates
[87, 90, 100, 101, 104–107]. AMD treatment using AFRs has
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Eng. Life Sci. 2007, 7, No. 6, 541–564 SRB Applications 545

Figure 3. Various continuous flow reactors used in anaerobic wastewater treatment, adapted from [75–81].

© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.els-journal.com

546 A. H. Kaksonen et al.
been studied on a laboratory [87], bench [107, 108], pilot
[30, 89, 95] as well as full scale [98]. The main shortcomings of
AFRs are the channeling of the flow and clogging of the bed by
precipitates [66].
3.3 Fluidized-Bed Reactor (FBR) and Downflow FBR
In the fluidized-bed reactor (FBR), channeling and clogging
are avoided by fluidizing the inert biomass carrier with
recycled water (see Fig. 3E) [66]. FBRs have been reported to
efficiently retain biomass and allow high mass transfer and
reaction rates [77, 109, 110]. The type of the biomass carrier
affects the biomass concentration and the achievable loading
rates of the FBR [111, 112]. Carrier materials used include iron
chips [66], synthetic polymeric granules covered with iron
dust [68], pumice particles [113], porous glass beads [114],
carbon powder [67] and silicate minerals [115]. The fluidized
carrier provides a large surface area for biofilm formation
[77]. Sulfate-reduction rates per reactor volume and carrier
surface area have been reported to be higher in the FBR com-
pared to the AFR [66]. FBRs are well suited for the combined
removal of metals, acidity and sulfate from wastewater, since
the recycle flow in the FBR dilutes high influent concentrations
[110, 115–118].
The downflow fluidized-bed reactor (DFBR), also known as
inverse FBR, is based on floatable carrier material which is flu-
idized downward with a downflow current of liquid (see
Fig. 3F). Synthetic materials, especially foamed plastic pellets,
are the most usual carriers in DFBRs [80]. The DFBR is a very
promising development as it allows the recovery of solid prod-
ucts, such as metal sulfides, at the bottom of the reactor [81].
3.4 Gas Lift Reactor (GLR)
High mass transfer and good mixing are also achieved in gas
lift reactors (GLR) (see Fig. 3G) [73] which typically consist of
a riser column and a downcomer column. Gas is sparged
through the bottom of the riser. The density difference created
by the difference in gas holdup between the riser and the
downcomer is the driving force for the liquid circulation in
the reactor [3]. The GLRs can be operated with or without a
biomass carrier [113].
3.5 Upflow Anaerobic Sludge Blanket (UASB)
Reactor
In upflow anaerobic sludge blanket (UASB) reactors, biomass
retention is based on good settling characteristics of granular
sludge (see Fig. 3H) [77, 82]. Due to the biomass granulation,
no packing or carrier material is needed which reduces the
start-up costs of the UASB compared to the AFR and the FBR
[77]. However, extensive biogas production may require addi-
tional instrumentation which increases capital costs. Moreover,
methanogens compete for substrates (acetate and H2 + CO2)
with sulfate reducers [119, 120], resulting in a decrease in the
yields of H2S and alkalinity per amount of substrate added.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2007, 7, No. 6, 541–564
Other problems encountered with UASB reactors include poor
or slow granulation [74] and the rapid disintegration of the
granular sludge under certain conditions [77]. Sulfidogenic
UASB reactors have been used for metal recovery on a bench
[70], pilot [72], demonstration [73, 121] and full scale [18, 69,
71, 122, 123].
3.6 Anaerobic Hybrid Reactor (AHR) and Anaerobic
Baffled Reactor (ABR)
The anaerobic hybrid reactor (AHR) is a combination of the
UASB and the AFR, where the granular sludge bed is in the
lower section of the reactor and packing material in the upper
section (see Fig. 3I) [74, 124]. Steed et al. [74] compared the
feasibility of the UASB, AFR and AHR for removing heavy
metals from AMD. The performance of the AHR was superior
based on effluent metal and sludge concentrations [74]. Al-
though the UASB reactor reduced soluble metal concentra-
tions, it did not operate as an effective clarifier, and hence the
concentration of total suspended solids in the effluent was
high [74]. Another modification of the UASB reactor is an
anaerobic baffled reactor (ABR) which is a staged reactor
where biomass retention is enhanced by forcing the water
through several compartments (see Fig. 3J) [64]. ABRs and
AHRs have been used for treating sulfate-laden and in some
case metal-containing wastewaters [64, 74, 124], but their po-
tential for concomitant removal of acidity, metals and sulfate
has not been extensively studied.
3.7 Membrane Bioreactor (MBR)
The coupling of a membrane to a bioreactor has attracted at-
tention in recent years [76]. Biomass separation membrane
bioreactors are the most common type of membrane bioreac-
tors (MBR). These MBRs consist of a suspended growth reac-
tor and a membrane reactor filtration device. The membrane
unit can be located in a sidestream (SMBR) (see Fig. 3K) or it
can be immersed in the bioreactor (IMBR) (see Fig. 3L). Un-
like the SMBR, the IMBR has no recirculation loop and the
biomass separation occurs within the bioreactor. Typically, the
IMBR has a substantially higher membrane area per unit of
volume compared to the SMBR. The IMBR is also capable of
operating at much lower transmembrane pressures and at a
lower liquid cross-flow velocity. Therefore, the IMBR opera-
tion requires a lower cost and energy input [76]. Extractive
membrane bioreactors (EMBR) (see Fig. 3M) have been used
to prevent direct contact between SRB and wastewater [75]. In
the EMBR, the metal-containing water is passed over one sur-
face of a selectively permeable membrane, while a microbial
culture is maintained on the other side. The membrane allows
H2S to permeate from the biological compartment into the
wastewater and to precipitate the metals [76]. The membrane
is impermeable to any charged species in the wastewater, there-
by preventing the SRB from having direct contact with the
toxic metals, extremes of pH or high salinity in the wastewater
[75]. EMBRs have been tested for removing Zn from synthetic
wastewater [75]. The deposition of ZnS on the wastewater side
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Eng. Life Sci. 2007, 7, No. 6, 541–564 SRB Applications 547

Table 1. Benefits and drawbacks of various reactor types.

Reactor type Benefits (+) and drawbacks (–) Reference(s)

Continuously stirred tank reactor + Consistent, reliable and rapid equilibrium conditions [61, 125]
(CSTR) – Poor retention of biomass [77]
Anaerobic contact + Better retention of biomass as compared to the CSTR [77]
process (ACP) – Pumping of biomass breaks down flocks and sludge [77]
Anaerobic filter + Low shear forces [61]
reactor (AFR) + Longer sludge retention time than in the CSTR [61]
+ Possibility to utilize gravitation in downflow mode [77]
+ Stripping of H2S effective in downflow mode [77]
– Channeling of the flow possible [61]
– Pressure gradients can be large [126]
Fluidized-bed reactor + Large surface area for biofilm formation due to fluidized carrier material [77, 126]
(FBR) + High retention of biomass on the carrier [110]
+ Efficient mass transfer [110]
+ Small pressure gradients [126]
+ No channeling of flow [126]
+ Influent concentrations diluted due to recycle flow [110]
+ No clogging [77]
+ Selects for microbes with low Km values [127, 128]
+ Possibility to recover solids from the bottom of the reactor in downflow mode [81]
– Energy needed for carrier fluidization [77]
– Shear forces can detach biomass [129]
– Less volume available for biomass compared to the UASB reactor due to the inert biomass carrier [130]
Gas-lift reactor (GLR) + Efficient mixing and mass transfer [73]
– High pressure drop of the water column that needs to be overcome when supplying the gaseous [29]
substrate
Upflow anaerobic + No channeling of flow [61]
sludge blanket + No compacting of sludge [61]
reactor (UASB) + No costs due to biomass carrier [77]
+ No clogging [77]
+ Possibility to obtain high treatment rates [77]
– Biomass flush out common during process failures [77]
– More susceptible to changes in influent quality compared to the AFR [131]
Anaerobic hybrid + Less susceptible to clogging compared to the AFR [74]
reactor (AHR) + Sludge removal easier than in the AFR [74]
+ Biomass retention better than in the UASB [74]
Anaerobic baffled + Long sludge retention time [77]
reactor (ABR) + No costs due to biomass carrier [77]
+ Good tolerance for shocks of hydraulic and organic loading [64]
Membrane bioreactors + Enhanced biomass retainment compared to other suspension bioreactors [76]
(MBR) + May prevent the SRB from having direct contact to toxic water (Extractive MBR) [75]
– Subjective to fouling (due to microbes or metal precipitates) of membranes → backwashing needed [75, 132, 133]

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548 A. H. Kaksonen et al.
of the membrane was observed to cause resistance to the H2S
mass transfer [75]. As the membrane does not allow charged
molecules, such as sulfate to pass through, the sulfate for the
SRB has to be added externally. Alternatively, the effluent from
the metal precipitation can be passed through the biological
compartment. However, in the latter case the SRB encounter
the acidity and possible salinity of the water.
Benefits and drawbacks of various continuous flow reactors
are listed in Tab. 1 (for a review, see [77]). Depending on the
reactor type and process configuration, the metal sulfide
sludge can be recovered from the bottom of the bioreactor,
Figure 4. Possible configurations of processes utilizing sulfate-reducing bioreactors for metal
precipitation, adapted from [12, 134].
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2007, 7, No. 6, 541–564
such as the AHR [74] and the DFBR [81], by back washing the
AFR at regular intervals [87] or with a clarifier downstream of
the precipitation unit [108].
4 Bioreactor Process Configurations
4.1 Single-Stage Processes
Biological sulfate reduction and metal precipitation using bio-
genic H2S can be applied in single or separated unit processes
(see Fig. 4) (for a review, see [12]). A
single-stage approach for sulfate reduc-
tion and metal precipitation (see
Fig. 4A) was used in the Palmerton pi-
lot plant installed to treat metal-con-
taminated drainage from a smelting
residues dump at the former New Jersey
Zinc Company plant in Palmerton,
Pennsylvania [30]. The system con-
sisted of two independent downflow
AFRs filled with loosely packed spent
mushroom compost [30]. Another ex-
ample of the single-state approach is a
full-scale plant at the Budelco zinc re-
finery in Budel-Dorplein, Netherlands,
that remediates metal-containing
groundwater [121]. This technology,
marketed under the trade name Thio-
paq®, consists of two biological pro-
cesses complemented with solid separa-
tion steps. The first biological step
utilizes SRB in an ethanol-fed UASB re-
actor to generate alkalinity and produce
H2S promoting the precipitation of
metals as sulfides within the bioreactor.
The second biological process involves
an aerobic filter in which the excess sul-
fide is oxidized to elemental sulfur by
aerobic colorless sulfur bacteria. The
solids are removed in a tilted plate set-
tler and a continuously cleaned sand
bed filter is used as a solids polishing
step before discharge. The elemental
sulfur can be used for agricultural
applications or to produce sulfuric acid
[18, 69, 71, 121–123]. The Thiopaq
technology has also been demonstrated
for the treatment of AMD at the former
Wheal Jane mine in Cornwall, UK [18].
Boonstra et al. [18] estimated that re-
placing the current active (lime) treat-
ment operation employed at the Wheal
Jane site with a biological plant would
reduce the annual discharge from the
site by > 600 kg iron, > 9900 kg zinc,
> 120 kg copper, 3600 kg aluminum,
9600 kg manganese, 21 kg cadmium
and 3895 tons of sulfate.
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Eng. Life Sci. 2007, 7, No. 6, 541–564 SRB Applications 549

The single-stage treatment process (see Fig. 4A) is a

low-cost solution for AMD treatment, but it may not
be viable if the wastewater is very acidic or contains
high concentrations of heavy metals [12]. Many single-
stage treatment systems have utilized alkaline materials
to generate additional alkalinity [86, 92, 95–98, 103]. In
some cases, several bioreactors have been used in series
to enhance sulfate reduction and metal precipitation
[30, 102]. Another approach is to recycle part of the
treated water to dilute the influent (see Fig. 4B) [67,
74, 87]. Recycling of the water requires additional
pumps and thus increases the investment and opera-
tional costs [12]. In single-state processes, the concen-
tration of dissolved sulfide has to be maintained at a
relatively high level to buffer the system against metal
shock loads. On the other hand, dissolved sulfide is
toxic to the SRB, and therefore, sulfide product inhibi-
tion may be expected in single-stage processes [135].

4.2 Precipitation of Metals prior to the Sulfate

Reduction Step

Metals can be pre-precipitated prior to the biological

step by recycling either sulfide-containing water (see
Figure 5. Used and/or recommended pH values for the selective precipita-
Fig. 4C) or H2S-containing gas (see Fig. 4D). Haas and
tion of metals as sulfides (or hydroxides) according to [108] (white bars),
Polprasert [63] recycled sulfide-rich water for precipi- [139] (grey bars) and [138] (black bars).
tating metals before a semi-continuous ACP process.
Separation of the chemical sulfide precipitation and
biological H2S production is also the basis of the patented Bio-
Sulphide process® [136]. The BioSulphide technology was
demonstrated at the Britannia Copper Mine, in British Colum-
bia, Canada [136]. Copper and zinc were selectively precipitat-
ed in consecutive steps of the chemical circuit by using sulfide–
and alkalinity-containing effluent from two bioreactors. Part
of the treated water from the chemical circuit was fed to the
bioreactors to provide sulfate for the SRB [136]. Based on the
pilot results, the capital costs of a full-scale treatment plant at
Britannia (with an average drainage flow of 12 000 m3/d) were
estimated to be 2.2 million U.S. dollars [136]. For comparison,
the capital costs of a lime treatment plant at Britannia were
estimated to be 3.5 million U.S. dollars, and the operating
costs over 900 000 U.S. dollars per year without consideration
of sludge disposal costs. The BioSulphide plant was estimated
to result in an annual net operating profit of 130 000 U.S. dol-
lars, resulting from the sale of CuS and ZnS concentrates to
smelters and mines [136].
Recycling of H2S-containing gas (see Fig. 4D) may assist the
selective precipitation of valuable metals (such as copper
which precipitates as sulfide already at low pH), since no alka-
linity is introduced to the precipitation step. However, the
metal sulfide precipitation produces protons adding up to the
acidity being fed to the bioreactor. Therefore, gas recycling has
often been used in combination with chemical neutralization
[88, 108] or water recycling [18, 72, 73, 122]. Bhagat et al.
[137] compared the precipitation of metals using either bio-
genic H2S gas or liquid supernatant from a sulfate-reducing se-
quencing batch reactor. The precipitation using liquid super-
natant resulted in higher metal removal efficiencies than the
sparging with H2S gas [137]. The precipitates produced are
comprised mainly of metal sulfides, but in the case of the liq-
uid supernatant precipitation, they also contained unidentified
complexes which may include nitrates, chlorides or carbonates
[137]. Hammack et al. [108] used biogenic H2S and NH4OH
to recover copper and zinc selectively in a bench scale system
treating acidic water from the Rio Tinto Mine, Nevada, USA.
The recycling of both H2S gas and bicarbonate-containing
water has been applied in a pilot plant treating water from the
Berkeley Pit, an abandoned open-pit copper mine in Butte
Montana, USA [72]. Hammack and Dijkman [72] estimated
that the costs of a full-scale treatment (19 000 m3 per day) of
Berkeley Pit water utilizing ethanol as a substrate in the UASB
reactor would be 2.0 U.S. dollars per m3 water treated based
on an annuity of 15 %. An alternative biological treatment
using a gas lift reactor and steam-reformed natural gas as the
substrate would cost 0.65 U.S. dollars per m3 water treated.
Hammack and Dijkman [72] estimated that plants using
steam-reformed gas are more economical for treatment appli-
cations requiring sulfate removal in excess of 10 tons per day.
Tabak et al. [138] developed a 6-stage process for precipitating
Cu, Zn, Fe(II) and Mn as sulfides using biogenic H2S, and Al
and Fe(III) as hydroxides using NaOH. Optimal solution pHs
for precipitating various metals have been suggested by Ham-
mack et al. [108], Govind et al. [139] and Tabak et al. [138]
(see Fig. 5).

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550 A. H. Kaksonen et al.
4.3 Combined Treatment of Multiple Water Streams
If the metals and sulfate to be removed are in different water
fractions, a treatment approach depicted in Fig. 4E can be
used. This idea has been applied in the demonstration plant at
Kennecott’s open pit copper mine in Bingham Canyon, Utah,
USA [18, 73, 119, 122]. Part of the H2S produced in the biore-
actor is used in a gaseous form to selectively recover copper
from a leach water stream. The bioreactor liquid effluent,
which is rich in sulfide and alkalinity, is further used to precip-
itate metals from sulfate-containing water, and to produce ele-
mental sulfur. The sulfate-containing effluent from the metal
precipitator is fed to the bioreactor to maintain biological sul-
fate reduction [18, 73, 119, 122]. De Vegt et al. [122] estimated
that the cost of a treatment system (15 % annuity) reducing
10 000 tons of sulfate per year would be approximately
330 U.S. dollars per ton of sulfate reduced when H2 and CO2
are used as an energy and carbon source for the SRB.
4.4 Off-line H2S Production from Sulfur
Some sulfate reducers are able to reduce elemental sulfur as an
alternative electron acceptor. If no sulfate-containing stream is
available for the treatment of metal-containing waters, elemen-
tal sulfur can be supplied to an offline bioreactor to produce
H2S gas that is fed to the metal precipitation units (see Fig. 4F).
Depending on the solution chemistry and the target metals,
pH adjustment of the water may be required after metal recov-
ery, since biogenic alkalinity is not introduced to the metal
precipitation circuit with the gas stream. Sulfur reduction has
been utilized, for example, in the treatment plant at Caribou
Mine, Canada. In this plant, lime is used to neutralize the
acidic solution after the metal recovery [134].
The separation of the biological sulfate or sulfur reduction
and metal sulfide precipitation with the biogenic sulfide allevi-
ates toxicity on the SRB. It allows selective metal precipitation
by the control of pH and H2S dosing, and reduces the amount
of biomass and organic substrates in the metal sulfide sludge
[108]. Drawbacks are the increasing investment and opera-
tional costs due to increased and more complex instrumenta-
tion.
Often, high metal removal rates from acidic wastewater have
been reached with systems where biomass retention is en-
hanced with biofilms [115–117], flocculants [121, 125], chemi-
cal neutralization of the wastewater [96], or metal precipita-
tion, and sulfate reduction takes place in separate reactor units
[72, 123]. The optimization of SRB-based processes includes
the consideration of several factors, such as application type
(wastewater or process water treatment), microbial composi-
tion, influent composition and operational conditions (see
Tab. 2). In the following, aspects related to substrate selection
and toxicity issues are discussed in more detail.
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2007, 7, No. 6, 541–564
5 Choice of Substrate for Sulfate Reduction
Many sulfate- and metal-containing waters are low in organic
matter. The cost-effectiveness of the sulfate reduction based
bioprocesses largely depends on the external electron donor
and carbon source that need to be added for the sulfate redu-
cers. The choice of the substrate is based on several criteria: (i)
the ability of SRB to utilize the substrate, (ii) the suitability of
the substrate for the particular application (passive vs. active,
reactor type, etc.), (iii) the sulfate load to be reduced and the
cost of the substrate per unit H2S produced, (iv) the availabil-
ity in sufficient quantities, and (v) the possible remaining
pollution load from the incompletely degraded substrate (see
Tab. 3) [73, 113, 168].
5.1 The Ability of SRB to Utilize Various Substrates
Sulfate reducers can oxidize various intermediate products
originating from the anaerobic degradation of complex organ-
ic compounds including H2, carboxylic acids, alcohols, some
sugars and aromatic compounds [119, 179]. Most of the sub-
strates are typical fermentation products and intermediate
breakdown products of larger molecules (for reviews, see [172,
180]). Direct utilization of biopolymers by SRB is very rare
[180]. The oxidation of organic substrates in SRB may be com-
plete, leading to the production of CO2, or incomplete, with
acetate usually being the end product. Incomplete oxidation is
due to the absence of a mechanism for Acetyl-CoA oxidation
[172]. The oxidation of acetate is important in AMD treat-
ment because it produces HCO3– which neutralizes acidic
water [117]. Some incomplete oxidizers are able to use acetate
as a source of carbon if H2 is used as the electron donor [181].
Furthermore, some SRB are autotrophs being capable of grow-
ing with CO or CO2 as a sole carbon source [182, 183].
Several studies have reported the potential of various com-
plex substrates, such as plant materials, and agricultural, in-
dustrial and municipal wastes to promote sulfate reduction.
The substrates include alfalfa, rye grass, hay bales, straw, rice
stalks, peat, spent mushroom compost, municipal and leaf
compost, molasses, sewage sludge, manure, paper products,
plant hydrolyzate, cellulose, sawdust and wood [9, 33–35, 39,
47, 87, 89, 91, 95, 97, 98, 103, 176, 184–187]. Also, the use of
poly(lactic acid) as a long-term source of lactic acid for bacter-
ial sulfate reduction has been demonstrated [188].
Prasad et al. [177] studied the suitability of activated sludge,
digested sludge and rabbit pellets (plant material) as carbon
and energy sources for SRB. Activated sludge was the most
suitable of these materials [177]. Hulshof et al. [189] reported
that wood chip material promoted less sulfate reduction in up-
flow AFRs than the waste solids derived from a pulp and paper
plant. Factors that could contribute to the lower rates include
insufficient concentrations of labile carbon or nutrients such
as phosphorous and nitrogen. The pulp waste contained signif-
icantly higher concentrations of phosphorous and nitrogen
than the wood chips [189]. If the substrate does not contain
sufficiently nitrogen, phosphorous and trace elements, these
must be supplemented to enhance SRB growth.
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Eng. Life Sci. 2007, 7, No. 6, 541–564 SRB Applications 551

Table 2. Factors affecting the efficiency of sulfate reducing bioprocesses.

Factor Effect(s) Reference(s)

Application type
Means of retaining biomass Increases the potential populations of active SRB and the amounts of sulfate reduced [140, 141]
(biofilm/aggregation)
Microbial composition
Species Optimal growth conditions, substrates utilized and tolerance to toxic compounds [142–144]
vary among different species; Complex communities more robust to interferences
Attachment properties of The ability of microbial species to colonize sludge granules or surfaces is variable [145, 146]
microorganisms
Type and history of seed material Competition between SRB and other microorganisms [147–149]
Influent composition
Sulfate concentration Affects SRB growth and activity; SRB may be out-competed at low sulfate [62, 150, 151]
concentrations; Inhibition at high concentration
Metal concentrations High heavy metal concentrations toxic; Presence of Ca2+ and Mg2+ may enhance [12, 152]
the dominance of SRB over methanogens
pH Affects the growth and activity of SRB; pH decrease increases the free H2S concentration; [15, 145, 153-156]
High pH (> 8) can favor SRB over methanogens; pH affects the configuration and
diameter of biomass aggregates
Operational conditions
Substrate concentration and Affects SRB growth and activity; Sulfate reduction enhanced with increased substrate [62, 99, 147, 148,
loading rate concentrations; High SO42–/substrate ratio favors SRB over other microorganisms 155, 157]
N and P supplementation Nutrient addition may be needed to achieve the optimal C:N:P ratio; Ammonium [61, 99, 158]
salts and urea are ideal nitrogen sources, but high NH4+ concentrations are toxic to
bacteria; Nitrate has to be reduced before the assimilation which consumes electron
donor; The most suitable phosphorous source for SRB is phosphate
Trace elements The SRB need Fe, Ni, Se and Mo as cofactors for the enzymes responsible for electron [156, 159, 160]
donor oxidation and the electron transport chain in sulfate reduction
Flocculant addition Improves settling of solids [121]
Temperature Temperature increase may help the SRB to outcompete methanogens; The elevated [153, 155, 161-163]
temperature has advantages due to faster microbial metabolism and lower solubility
of toxic H2S; Decay rates are higher at elevated temperatures
Exposure to oxygen SRB may better outcompete methanogens after a short-term exposure to oxygen [145]
H2S concentration High H2S concentration can inhibit SRB growth or favor SRB over other [155, 156, 164]
microorganisms; N2 flushing can decrease the concentration of toxic H2S
Mixing conditions Increased agitation power can decrease substrate uptake [156]
Hydraulic retention time (HRT) Short HRT can lead to wash-out of biomass and affect sulfate reduction rate; [157, 165, 166]
at long HRTs, methanogens may outcomepete SRB
Upward liquid velocity (tup) High tup (4–6 m/h) in granular sludge reactors can result in biomass washout [155]

5.2 Suitability of Substrates for Various Applications
Depending on the SRB-based application, solid, liquid, or gas-
eous substrates may be preferred. For passive treatment appli-
cations, solid plant or waste materials are often required to
allow passive operation without pumping. However, solid sub-
strates have a limited lifetime and have to be replaced or sup-
plemented with liquid or gaseous substrates once the original
substrate has been depleted [176]. In active bioreactors, liquid
or gaseous substrates allow continuous operation.
Operational temperature, reactor type and pH may set some
limits to the choice of substrates. Sulfate reducing applications
usually utilize mixed cultures comprising of SRB and anaero-
bic fermentative microorganisms, methanogens and homoace-
togens. Therefore, the SRB have to compete for the substrate
with other microorganisms. Under mesophilic conditions,
SRB can outcompete methanogens on propionate, butyrate
[155], ethanol, sucrose [166] and H2 [167, 168], whereas
acetate [155] favors methanogenesis over sulfate reduction.
However, the operational temperature is crucial for the sulfi-

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552 A. H. Kaksonen et al. Eng. Life Sci. 2007, 7, No. 6, 541–564

Table 3. Benefits and drawbacks of selected electron and carbon sources for biological sulfate reduction

Energy and carbon source Benefits (+) and drawbacks (–) Reference(s)
H2 + CO2 + A large number of SRB can grow on H2 as a sole energy source [167]
+ Thermodynamically, sulfidogenic growth on H2 is more favourable than growth on acetate [167]
or other reduced 2, 3 or 4-C compounds
+ SRB can outcompete methanogens for hydrogen [167, 168]
+ Most economic for high sulfate loads [18]
+ Can be produced by cracking methanol or reforming natural gas [18]
– Reformer increases investment costs [73]
–Hydrogen mass transfer may be the rate limiting step since H2 is poorly soluble in aqueous solutions [3]
– Safety requirements stringent due to the explosive nature of H2 [169]
– Availability may be limited [73]
Synthesis gas (H2+CO2+CO) + Low cost [170]
– CO may inhibit some SRB [3]
– Availability may be limited [73]
Formate + Many SRB capable of growing on H2 can also grow on formate as a sole energy source [167]
+ Good alternative for H2 in laboratory studies due to less stringent safety requirements [169]
+ Low cost [171]
– Methanogens can outcompete SRB at high temperatures (65–75 °C) [171]
Acetate – Only a few SRB are capable of oxidizing acetate [167]
– Low biomass yield [172]
– Methanogens can outcompete SRB for acetate [146, 167, 168]
Lactate + Good electron and carbon source for many SRB [105]
+ High biomass yield [173]
+ High alkalinity production [117]
– High cost [105, 174]
– Incomplete oxidation may lead to the accumulation of acetate in the effluent [105]
Methanol + Low cost [105]
+ SRB can outcompete methanogens at high temperatures (55–70 °C) [171]
– Methanogens can outcompete SRB under mesophilic conditions [175]
– Only a few SRB can utilize methanol [105, 176]
Ethanol + The ability to utilize ethanol is very common among SRB [167]
+ Easily oxidized by SRB [177]
+ Lower cost than lactate [174]
– Operating costs are higher than for H2 [73]
– Incomplete oxidation does not produce alkalinity and leads to acetate accumulation [117]
Glucose – High cost compared to plant materials
– Fermentation may result in a decrease in the pH due to the accumulation of carboxylic acids [173]
Plant materials + Low cost [177]
– Use limited to remedial applications
– Fermentation may result in a decrease in the pH due to the accumulation of carboxylic acids [177]
– Competitive bioconversions possible [3, 6]
Waste products + Low cost [178]
– Competitive bioconversions possible [3, 6]
– Availability may be limited [178]
– May cause high COD in the effluent due to incomplete oxidation of organic compounds [105]

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Eng. Life Sci. 2007, 7, No. 6, 541–564
dogenic oxidation of methanol. Methanol is a suitable sub-
strate for SRB at high temperatures [171], whereas methano-
gens can outcompete SRB under mesophilic conditions
[166, 175].
Also, the reactor type may affect the ability of SRB to com-
pete for the certain substrates. Experiments with CSTRs (no
biomass retainment) showed that acetate was consumed by
SRB at high influent sulfate concentrations, whereas in reactors
with biomass retention (UASB, AFR), acetate was preferential-
ly converted to methane [167]. This was explained by the supe-
rior capability of acetoclastic methanogens to colonize support
materials [167].
5.3 Scale of Use, Costs, Availability and Residual
Pollution
Lactate is a good substrate for enriching SRB [143] and, there-
fore, the use of lactate may speed up the process start-up due
to the relatively high growth yields of SRB [174]. However, in
large scale operations the use of lactate would result in high
operational costs [174]. A low-cost substrate is needed for
large scale operations, once a reasonable biomass yield has
been achieved [116, 174]. Among the low molecular weight
compounds, like acetate, lactate, propionate, oxalate, metha-
nol, ethanol, glycerol and glucose, ethanol seems to be the
most cost-effective carbon and electron source [101, 190].
For large-scale applications (more than 2.5 tons of H2S per
day), H2 gas is preferably used [73]. Hydrogen gas can be pro-
duced on site by cracking methanol or by steam reforming nat-
ural gas or liquefied petroleum gas. These processes convert
hydrocarbons to hydrogen and carbon dioxide, which are both
fed to the bioreactor. The CO2 is used as a carbon source and
supplies buffer capacity, which keeps the pH at acceptable lev-
els. When ethanol is used, the operating costs are higher than
with hydrogen gas, but the investment costs are lower since no
reformer is required [73]. The operational costs of SRB plants
would be greatly reduced if methane could directly be used as
an electron donor for biological sulfate reduction without low-
efficiency stream reforming which requires high temperatures.
Geochemical studies have shown that anaerobic oxidation of
methane (AOM) occurs in marine sediments, and based on
field and laboratory experiments, AOM is believed to be medi-
ated by a consortium of archaea and SRB [191]. These anaero-
bic methanotrophs have yet to be recovered in pure culture,
and key aspects of their ecology and physiology remain poorly
understood [191]. However, a methane-utilizing SRB reactor
is currently under development.
Also, synthesis gas (mixture of H2, CO2 and CO) has been
proposed as an inexpensive alternative for H2 + CO2 [170].
However, the CO of the synthesis gas can inhibit some SRB.
The toxicity of CO on SRB may be reduced by growing the
SRB together with other CO-utilizing or CO-converting bacte-
ria. For example, homo-acetogenic bacteria grow on CO as a
sole energy and carbon source [3]. Moreover, these bacteria
can convert CO to CO2 by the so-called water-gas shift reac-
tion [3]. Recent research shows that some moderately thermo-
philic SRB have much higher tolerance for CO than previously
thought, which may allow the direct application of synthesis
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
SRB Applications 553
gas in bioreactors [192, 193]. For example, Desulfotomaculum
carboxydivorans can convert CO to H2 and CO2 which are
further used for sulfate reduction [194].
Various waste materials may be a low-cost alternative for de-
fined substrates. However, their availability may be restricted
to certain areas. An innovative low-cost process is the Bio-
SURE Process, which links AMD treatment and sludge dispo-
sal. Sewage sludge serves as the electron donor for the SRB and
becomes simultaneously stabilized [28, 195]. Another steadily
increasing organic waste-stream is the glycerol-methanol waste
remaining after the production of biodiesel fuel. Zamzow et al.
[196] recently showed that this biodiesel production waste
(BDW) is a suitable substrate to be used for AMD treatment.
The accumulation of acetate in the effluent has been report-
ed to be the limiting factor of high-rate sulfate-reducing sys-
tems fed with simple substrates, such as H2 + CO2, ethanol,
methanol or carboxylic acids [29, 117]. The use of complex
plant materials and waste products may result in even higher
effluent COD due to the more recalcitrant compounds [105].
Therefore, the choice of the electron donor and carbon source
affects the quantity of residual pollution [29]. For some com-
plex materials, pretreatment such as hydrolysis, or post-treat-
ment may be necessary to achieve more complete biodegrada-
tion.
6 Toxicity Effects
Biological sulfate reduction is inhibited by low pH, hydrogen
sulfide, high metal concentrations and some anions.
6.1 Low pH
The optimum pH for the growth of the most known SRB is
around 7 [197], and many of the SRB are sensitive to even
mild acidity (most are inhibited at pH 5.5) [198]. Therefore,
most of the SRB bioreactor applications use engineering solu-
tions, such as water recycling or separated precipitation and
“off-line” bioreactor units, to prevent direct contact between
acidic water and the SRB. Efforts have been made to enrich
and isolate acidophilic SRB which could operate at lower pH
ranges. Sen and Johnson [59] isolated acidophilic SRB at pH
3.6 on solid growth medium. They reported alkalinity produc-
tion in a fermenter culture even at a pH of 1.73 [59]. The dis-
covery of acidophilic and acidotolerant SRB [9, 90, 142] have
led to the development of novel biosulfidogenic systems for
selective metal recovery [198]. Recently, Johnson et al. [198]
demonstrated the selective precipitation of Zn from a Zn and
Fe containing solution using a syntrophic sulfidogenic consor-
tium operating at pH 3–4. Also, interesting new reactor
systems for low pH sulfate reduction have been introduced by
Bijmans et al. [199]. The “in-line” low-pH bioreactor could
allow the use of simple engineering designs leading to savings
on both construction and operation costs [198]. However, the
growth rates of acidotolerant SRB are likely very low,
and therefore, an additional bioreactor would be needed for
supplying enough biomass for the metal precipitating bio-
reactor.
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554 A. H. Kaksonen et al.
Furthermore, in low-pH bioreactors potential substrate tox-
icity needs to be considered. In acidic liquors (with pH values
below the pKa’s of acids), organic acids are predominantly in
their undissociated, lipophilic forms. The undissociated acids
can pass through the cell membrane, dissociate in the circum-
neutral internal cell cytoplasm causing influx of further un-
dissociated acid. This leads to the acidification of cytoplasm
[200] and uncoupling of the proton motive force [201]. Or-
ganic acid concentrations at even < 1 mM are lethal to many
acidophilic microorganisms [200]. The use of alternative
(non-acid) substrates for acidophilic/acidotolerant SRB has
been more successful [200]. Alcohols can be used as substrates
for acidophilic SRB, especially if the accumulation can be
avoided by incorporating acetate-utilizing microorganisms in
the consortia [101]. Kimura et al. [200] and Johnson et al.
[198] reported sulfidogenesis with glycerol in low pH (3.6–
4.2) media by a mixed culture consisting of a SRB strain M1
which was related to Desulfosporosinus sp. and a non-sulfate-
reducing strain PFBC which was related to Acidocella aromati-
ca. In fermenter cultures, the sulfidogenesis resulted in the
selective removal of Zn as ZnS, whilst ferrous iron remained in
solution [198, 200].
As the pH in the AMD is often lower than that tolerated by
the SRB, the alkalinity produced in sulfidogenic reactions
should be utilized. An engineering solution to avoid the low
pH toxicity would be more feasible than the use of SRB with
limited acid tolerance.
6.2 Hydrogen Sulfide
Undissociated hydrogen sulfide is generally assumed to be the
main toxic form of sulfide because only neutral molecules
permeate the cell membrane [202–205]. The concentration of
undissociated sulfide depends on chemical equilibria [163]:
H2S(l) ↔ HS– + H+ pKa1 = 6.97 (25 °C) (4)
HS– ↔ S2– + H+ pKa2 = 12.9 (25 °C) (5)
As the first pKa value of hydrogen sulfide is close to 7, small
variations in the pH range 6–8 will significantly affect the con-
centration of undissociated H2S (see Fig. 6) [119].
Hydrogen sulfide affects the functioning of metabolic coen-
zymes [119] and denaturates proteins by precipitating metal
ions in the active sites [206]. H2S may also affect the sulfur
assimilation and intracellular pH [207]. Additionally H2S pre-
cipitates metals needed by SRB thereby reducing their bioavail-
ability [208]. Especially the bioavailability of iron may signifi-
cantly affect the growth of SRB [146].
An understanding of sulfide toxicity on populations of SRB
is difficult to extract from the literature, as the effect of the pH
has not always been taken into account in experimental
designs [209]. Moreover, various studies have addressed the
effect of sulfide either on SRB growth, substrate utilization or
sulfate reduction, and these are not always linearly correlated.
For example, Visser et al. [205] discovered that at high pH lev-
els, SRB growth is more strongly inhibited than the activity.
The sensitivity of SRB to sulfide toxicity is also reported to
© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eng. Life Sci. 2007, 7, No. 6, 541–564
Figure 6. Sulfide speciation as a function of the pH at 25 °C (the
pKa values were taken from [160]).
The pH range and optimum of neutrophilic sulfate-reducers are
shown with light and dark gray areas, respectively [197].
depend on bacterial species [210–212]. Maillacheruvu and Par-
kin [211] studied propionate oxidizing, acetate oxidizing and
hydrogenotrophic SRB and reported that especially aceto-
trophic SRB were highly sensitive to H2S. Similarly, Yamaguchi
et al. [213] reported that acetate utilizers were more suscepti-
ble to sulfide inhibition than the hydrogen utilizers. However,
Kaksonen et al. [117] reported that ethanol oxidation was
more affected by sulfide toxicity than the acetate oxidation.
Tab. 4 summarizes previous studies on the effects of sulfide on
SRB cultures.
6.3 Metal Cations and Oxyanions
Metal cations and oxyanions can be toxic to the SRB at high
concentrations. The H2S produced by the SRB, however, rapid-
ly reacts with many metals precipitating them as metal sulfides.
The toxicity of metals depends on many factors, such as bio-
mass quantity, temperature, pH, carbon source, sulfate con-
centration, type of metal and concentration, as well as the
presence and concentration of iron and complexing com-
pounds [12].
Environmental temperature and pH affect the physiological
state of SRB and thus their tolerance to metals. Additionally,
the pH affects the charge and binding characters of metals.
Heavy metals form complexes in the presence of hydroxide
ions and other inorganic anions, such as chloride. The com-
plexes have different toxicities and binding characters as com-
pared to non-complexed metals. The solution also affects the
form of organic ligands and, therefore, the formation of organ-
ic metal complexes [214]. Some SRB produce exopolymers,
which complex metals thereby reducing their toxicity [215].
The various oxidation states of elements, such as As(V) and
As(III) or Se(VI) and Se(IV), can cause different toxicity ef-
fects. Inorganic cations can affect heavy metal toxicity by com-
peting with heavy metals on the anionic sites on cell surfaces
[214]. For example iron [216], magnesium, and calcium [217]
reduce heavy metal toxicity. Tab. 5 lists selected examples of
SRB inhibitory heavy metal concentrations. The combined
toxicity effect of several heavy metals can be higher than the
sum of individual components (synergism) [12]. Additionally,
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Eng. Life Sci. 2007, 7, No. 6, 541–564 SRB Applications 555

Table 4. Inhibitory effect of dissolved sulfide (DS) and undissociated H2S on the growth, sulfate reduction and substrate utilization of eth-
anol- and acetate-utilizing sulfate-reducing bacteria (SRB).

Substrate Biomass Vessel T [°C] pH DS [mg S/L] H2S [mg S/L] Inhibition Reference
c
Ethanol Anaerobic sludge Serum bottle 37 6.8 500–561 262–294 50 % [212]
7.2 788–880 241–269
7.6 878–990 131–147
8.0 1019–1130 66–74
8.5 1004–1164 22–25
Ethanol Desulfococcus multivorans Serum bottle 37 6.8 498 261 50%c [212]
7.2 851 250
7.6 1383 206
8.0 1488 97
8.5 1570 34
a
Ethanol UASB sludge UASB 35 7.1–8 1700–9951 570–610 100 %d [157]
b e
Ethanol Mixed SRB culture in FBR FBR 35 6.9–7.3 225–248 76–84 Ki [117]
c
Acetate UASB sludge Batch reactor 30 7 521 217 50 % [205]
7.5 569 107
8 921 64
9 943 7
Acetate Desulfonema magnum, Serum bottle 30/37 6.8 443–583 233–306 50 %c [212]
Desulfotomaculum acetoxidans
7.2 671–926 205–283
or Desulfobacter postgatei
7.6 660–1360 98–203
8.0 659–1500 43–98
8.5 708–1450 15–31
Acetate Anaerobic sludge Serum bottle 37 6.8 374 196 50 %c [212]
7.2 550 168
7.6 867 129
8.0 990 65
8.5 1011 22
Acetate UASB sludge Serum bottle 35 7 699 270 5 %d [213]
e
Acetate UASB sludge Serum bottle – 7.2-7.4 615 161 50 % [205]
8.1–8.3 1125 54
Acetate Mixed SRB culture Serum bottle – – 35 8 Kie [211]
e
Acetate Mixed SRB culture in FBR FBR 35 6.9–7.3 338–356 118–124 Ki [117]

a
UASB = upflow anaerobic sludge blanket reactor, bFBR = fluidized-bed reactor, cInhibition of growth, dInhibition of sulfate reduction activity,
e
Inhibition of substrate utilization activity.

the tolerance of the individual species in microbial consortia
may differ from that of a pure culture [226]. The SRB may also
adapt to metal-containing environments. Therefore, metals
may cause more adverse effects in batch systems compared to
continuous processes [227]. Lateral gene transfer is the likely
mechanism by which SRB acquire genes conferring to metal
resistance [228].
Many metal oxyanions affect the activity of SRB. Selenate
ion (SeO42–) is a competitive inhibitor of sulfate reduction,
but does not affect the reduction of sulfite or thiosulfite.
Monofluorophosphate acts in a similar way to selenate [229].
Molybdate (MoO42–) has been observed to inhibit sulfate re-
duction in sediments [230] and anaerobic wastewater treat-
ment, but at the same time other microbes may also become

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556 A. H. Kaksonen et al. Eng. Life Sci. 2007, 7, No. 6, 541–564

Table 5. Examples of metal concentrations inhibitory to sulfate- and surface water, and for the recovery of metals from process
reducing bacteria (SRB) [mg/L]. waters. SRB-bioprocesses have several advantages over tradi-
tional active chemical treatment. Bioprocesses offer low-cost
Cd Cr(III) Cr(VI) Cu Pb Ni Zn Reference
alternatives and facilitate the recovery of valuable metals as sul-
6 23 6 25 13 25 [218]1 fides. Moreover, sulfidogenic bioprocesses result in more stable
metal precipitates, and lower effluent metal and sulfate con-
40 80 [219]2
centrations than the conventional chemical treatment. Com-
>4 60 4 > 80 10 40 [220]1 pared to the passive biological treatment, active bioreactors are
100 [221]3
more compact, and offer consistent performance and control.
Bioreactor plants require significant start-up capital and con-
35–70 [222]4 tinuous monitoring. The overall costs of a biological treatment
112 64 59 65 [223]5 plant depend on the engineering design and location of the
plant, the characteristics of the wastewater stream, the selec-
10 13 [224]6 tion of the substrate for the SRB, the profit obtained from
>100 [216]7 metal recovery and the discharge criteria. Recently discovered
low-cost substrate alternatives such as the use of various waste
32 [225]8
streams may further increase the uses of SRB-processes.
1) Complete inhibition of SRB activity in test tubes during 3 weeks.
2) 50 % inhibition of growth of a pure culture.
3) 50 % growth inhibition in a serum bottle. Acknowledgements
4) Minimum inhibition of two Desulfobacterium strains;
concentration depended on strain and electron source. This work was supported by the European Commission (Bio-
5) Complete inhibition in a SRB culture originating from the manure MinE contract 500329), the Finnish Funding Agency for Tech-
digester. nology and Innovation, Outokumpu Oyj, the Finnish Gradu-
6) Inhibition of Desulfovibrio desulfuricans in serum bottles. ate School of Environmental Science and Technology, the
7) Delay of growth of Desulfotomaculum sp. DF-1 strain. Academy of Finland, the Scientific Foundation of the City of
8) Complete growth inhibition of an SRB-mixed culture in a Tampere, Land and Water Technology Foundation, The Foun-
fermenter.
dation of Technology and Konkordia Foundation.

inhibited [231–234]. Molybdate ions consume the ATP reserve

of the cell. On the other hand, some SRB strains can reduce
MoO42– to MoO2(S). Similarly some strains can reduce SeO42–
and SeO32– to Se0 or Se2– [12].
High Na+ and Ca2+ concentrations may inhibit sulfate re-
duction. Many marine SRB require high Na+ concentrations
for growth, but other SRB may be inhibited at high salt con-
centrations. Vallero et al. [235] achieved high sulfate reduction
rates (up to 3.7 g SO42–/L d) with halotolerant SRB at salinities
exceeding 50 g NaCl/L and 1 g MgCl/L using ethanol or pro-
pionate as electron sources in a UASB reactor. Ca2+ ions do
not cause direct toxicity, but CaCO3 and Ca3(PO4)2 precipi-
tates can coat biomass, which hinders the substrate assimila-
tion. Moreover, the precipitation of phosphate as Ca3(PO4)2
can cause phosphorous deficiency in cells [119].
Immobilized cells tolerate higher concentrations of toxic
compounds than free cells [236]. The outer layers can protect
the inner layers of a biofilm from the inhibitory concentrations
due to mass transfer resistance [237]. Moreover, Teitzel and
Parsek [238] showed that biofilms were more resistant to cop-
per and lead stress than planktonic cells. They suggested that
the extracellular polymeric substances of the biofilm may pro-
tect cells by binding heavy metals and retarding their diffusion
within the biofilm [237].
7 Concluding Remarks
Various passive and active SRB-based applications have been
developed for the treatment of AMD contaminated ground-
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