Biotechnology Advances 25 (2007) 452 – 463

www.elsevier.com/locate/biotechadv

Research review paper

Electron donors for biological sulfate reduction
Warounsak Liamleam, Ajit P. Annachhatre ⁎
Environmental Engineering and Management, Asian Institute of Technology, PO Box 4, Klongluang, Pathumthani 12120, Thailand
Received 21 February 2007; received in revised form 9 May 2007; accepted 10 May 2007
Available online 17 May 2007

Abstract

Biological sulfate reduction is widely used for treating sulfate-containing wastewaters from industries such as mining, tannery,
pulp and paper, and textiles. In biological reduction, sulfate is converted to hydrogen sulfide as the end product. The process is,
therefore, ideally suited for treating metal-containing wastewater from which heavy metals are simultaneously removed through the
formation of metal sulfides. Metal sulfide precipitates are more stable than metal hydroxides that are sensitive to pH change.
Theoretically, conversion of 1 mol of sulfate requires 0.67 mol of chemical oxygen demand or electron donors. Sulfate rich
wastewaters are usually deficient in electron donors and require external addition of electron donors in order to achieve complete
sulfate reduction. This paper reviews various electron donors employed in biological sulfate reduction. Widely used electron
donors include hydrogen, methanol, ethanol, acetate, lactate, propionate, butyrate, sugar, and molasses. The selection criteria for
suitable electron donors are discussed.
© 2007 Elsevier Inc. All rights reserved.

Keywords: Biological sulfate reduction; Sulfate reducing bacteria; Anaerobic treatment

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
2. Overview of biological sulfate reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
3. Electron donors for sulfate reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
3.1. Hydrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
3.2. Formate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.3. Methanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.4. Ethanol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
3.5. Molasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.6. Lactate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.7. Acetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.8. Propionate and butyrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.9. Sugar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.10. Hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.11. Organic waste . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

⁎ Corresponding author. Tel./fax: +66 2 524 5644.

E-mail address: ajit@ait.ac.th (A.P. Annachhatre).

0734-9750/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2007.05.002
 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463 453

4. Selection of an electron donor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

1. Introduction reduction has been recognized as an efficient method

Sulfate is one of the most abundant anions found in
the environment. It is generated and discharged from
many industrial processes including production of
edible oil, molasses fermentation, tannery operations,
food production, coal burning power plants, and pulp
and paper processing (Austin, 1984; Shin et al., 1997).
Apart from sulfate, wastewaters generated from these
industries may contain high concentrations of other
sulfur species such as sulfide, sulfite, thiosulfate and
dithionite (Hulshoff Pol et al., 1998). Furthermore,
sulfuric acid and sulfite are commonly used for pH
adjustments and as bleaching agents, respectively,
during manufacturing processes; therefore, sulfate and
sulfite occur commonly in wastewaters. In addition to
industrial sources, sulfate, the most oxidized form of
sulfur, is also generated naturally, for example, through
oxidation of sulfide ores in acid mine drainage.
Despite large quantities of sulfate being released into
the environment, little attention has been given to the
mitigation of sulfate because of its relatively low direct
environmental risk compared with the other pollutants.
Sulfate pollution is, however, a concern as it can cause
several indirect environmental effects. Excessive quan-
tities of released sulfate can affect public water supplies
and pose health threat to lifeforms. In the absence of
dissolved oxygen and nitrate, sulfate acts as a source of
oxygen or electron acceptor and is converted to sulfide
(H2S). This phenomenon creates odor and corrosion
problems (Sawyer et al., 2003). In addition, H2S is
fatally toxic to humans, causing death within 30 min at
gaseous concentrations of only 800–1000 mg/L, and
instant death at higher concentrations (Speece, 1996).
The upper concentration limit of sulfide in water
intended for human consumption is recommended at
250 mg/L (Sawyer et al., 2003). Therefore, sulfate rich
wastewaters require treatment before being released into
the environment.
Anaerobic biological treatment is a useful technology
for converting organic matter to methane and carbon
dioxide. It has been widely used for treating several kinds
of industrial wastewaters. In anaerobic treatment, sulfate
reduction is generally not wanted because of production
of odorous sulfide. Nevertheless, biological sulfate
for removing sulfate from wastewater. Various aspects of
this anaerobic process, especially its effects and role in
anaerobic treatment, have been studied (Rinzema and
Lettinga, 1988). For example, the commercial THIO-
PAQ® technology makes use of biological reduction to
remove sulfate from wastewater (Weijma et al., 2002c).
Biological sulfate reduction processes may be intended
primarily for oxidizing organic matter, or removing
sulfate, or both. Applications have been developed for
removing organic chemical oxygen demand, sulfur,
nitrogen and heavy metals (Hulshoff Pol et al., 1998).
Heavy metal removal is one of the useful applications
of biological sulfate reduction. Sulfide generated by
sulfate reduction is used to chemically precipitate metals
as sulfides. Extremely low solubility of metal sulfide
formed allows the removal of heavy metals from the waste
stream (Metcalf, 2003). The production of sludge from
sulfide precipitation is also low in comparison to
hydroxide precipitation. In addition, metal sulfide
precipitates are much more stable than metal hydroxide
precipitates over a wide pH range. Moreover, valuable
metals can be recovered from the metal sulfide sludge
(Kaksonen et al., 2003). Biological sulfate reduction is of
particular interest in removing heavy metals from
wastewater such as acid mine drainage (AMD) (Bayoumy
et al., 1999; Glombitza, 2001; Gibert et al., 2003; Johnson
and Hallberg, 2002). Research has focused on maximiz-
ing sulfide production from sulfate to optimize both
sulfate and heavy metal removal (Hao et al., 1996;
Hulshoff Pol et al., 1998). In cases where sulfide remains
in the effluent after the metal sulfide has been precipitated,
the sulfide rich effluent can be partially oxidized to sulfur
in a sulfide oxidation reactor for sulfur recovery
(Annachhatre and Suktrakoolvait, 2001a; Johnson, 2001).
Biological sulfate reduction can be accomplished
under mesophilic (25–35 °C) as well as thermophilic
conditions (35–70 °C). Thermophilic sulfate reduction
is applicable for treating sulfate-containing wastewater
that is relatively warm. For example, the wastewater
discharged from pulp and paper industry, rayon man-
ufacturing process, and flue gas desulfurization (Austin,
1984). Thermophilic treatment is preferred over con-
ventional mesophilic treatment because it eliminates the
need for cooling (Sipma et al., 1999). Furthermore, the
454 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
conversion rate of thermophilic treatment is much
higher than that of mesophilic treatment. Therefore,
higher loading rates and lower excess sludge production
are feasible compared with mesophilic systems (Rintala
and Lettinga, 1992; Visser et al., 1992).
A minimum chemical oxygen demand (COD)-to-
sulfate mole ratio of 0.67 is required for achieving
theoretically possible removal of sulfate (Choi and Rim,
1991). Some sulfate rich wastewaters, e.g. acid mine
drainage, are usually deficient in electron donors and
external addition of electron donors is necessary in such
cases. This paper reviews various electron donors
employed in the biological sulfate reduction process.
The commonly used electron donors are hydrogen,
methanol, ethanol, acetate, lactate, propionate, butyrate,
sugar, and molasses. The selection criteria for suitable
electron donors are discussed.
2. Overview of biological sulfate reduction
In the environment, sulfur can be present in different
oxidation states and in various chemical forms. Under
aerobic conditions, sulfate is the thermodynamically
stable form of sulfur, whereas hydrogen sulfide is the
stable form under anaerobic conditions. Sulfate reduc-
tion reaction can proceed at appropriate reduction
potentials (E0). The reduction potential shows that
sulfate is a much less favorable electron acceptor than
oxygen (O2) and nitrate (NO3−). In order to maximize the
sulfate reduction in wastewater, the reduction potential
of the system should be negative (Madigan et al., 2003).
The biological sulfate reduction process is mediated
by a group of microorganisms known as sulfate re-
ducing bacteria (SRB). Sulfate reducing bacteria (SRB)
are differentiated into two heterotrophic SRB and auto-
trophic SRB. Heterotrophic SRB use organic com-
pounds as substrates. In contrast, autotrophic SRB use
CO2 as the carbon source and obtain electrons from
oxidation of H2 (Lens and Kuennen, 2001). Reduction
of sulfate (S6+) to sulfide (S2−) involves eight electrons
as shown below (Choi and Rim, 1991):
8Hþ þ 8e− þ SO2−
4 S
2−
þ 4H2 O ð1Þ
3. Electron donors for sulfate reduction
Electron donors are essential for the treatment of
sulfate-containing wastewater by biological sulfate
reduction process. Many sulfate rich wastewaters
contain high concentrations of COD, or organic matter,
that can be utilized as electron donors. As noted
previously, 0.67 mol of COD are needed per mole of
sulfate for its complete reduction. Some sulfate- and
sulfuric-acid containing wastewaters, are deficient in
COD and this leads to incomplete sulfate reduction.
Electron donors or carbon sources must be added to such
wastewater to achieve complete reduction of sulfate.
Electron donors that are oxidized by SRB are usually
low-molecular-weight organic compounds. Various types
of organic substances have been employed as electron
donors and carbon sources including sewage sludge, leaf
mulch, wood chips, animal manure, vegetal compost,
sawdust, mushroom compost, whey, and other agricultural
waste (Dvorak et al., 1992; Hammack et al., 1994;
Christensen et al., 1996; Waybrant et al., 1998). In
addition, synthetic organic compounds have also been
used as electron donors, especially small molecular weight
compounds, such as lactate, acetate, propionate, pyruvate
and butyrate (Okabe and Characklis, 1992; Visser et al.,
1993; Harada et al., 1994). Ethanol and other alcohols can
also be used. Nearly all of these compounds are known to
be fermentation products of anaerobic bacterial degrada-
tion of carbohydrates, proteins and other constituents of
dead biomass (Widdel, 1988). Molasses, which contains a
high amount of sucrose, has also been used as electron
donor (Annachhatre and Suktrakoolvait, 2001b).
Table 1 summarizes the sulfate reduction rates obtained
in biological sulfate reduction process using different
electron donors. From Table 1, it is obvious that the choice
of electron donor has a substantial impact on the rate of
sulfate reduction. High sulfate removal rates are achieved
by using H2/CO2 (30 g/L d), acetate (28.5 g/L d), and
ethanol (21 g/L d) (van Houten et al., 1994; de Smul and
Verstraete, 1999; de Smul et al., 1997). Different electron
donors and acceptors can result in different bacterial
biomass yields of SRB as shown in Table 2. For example,
the use of H2 as electron donor yields less biomass
compared with the use of acetate. Biomass yield is only
0.086 g cells/mol of electron donor, if propionate is used.
The substrate consumption rate of the sulfate reducers is
dependent on concentrations of both the electron donor
and electron acceptor. This in turn affects the competition
between SRB and methanogens. The most frequently used
electron donors in biological sulfate reduction are
discussed in detail in the following sections.
3.1. Hydrogen
Hydrogen is an attractive electron donor for sulfate
reduction because its free energy of sulfate reduction is
more favorable than that of methanogenesis. For
example, Desulfovibrio valgaris has been found to use
hydrogen and formate with favorable energetics
(Weijma et al., 2002a). In addition, besides sulfate
 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463 455

Table 1
Sulfate and sulfite reduction rates during biological removal of sulfate and sulfite with different electron donors under mesophilic conditions
Electron donor Temperature (°C) Bioreactor type SO2−
4 removal References
(g/L d)
Molasses 30 UASB a 4.3 (Annachhatre and Suktrakoolvait,
2001a,b)
Molasses 27 CSTR b 0.84 Maree and Hill (1987)
Molasses 35 Anaerobic RBC 0.35 Lo et al. (1990)
Molasses 31 Packed bed 6.5 Maree and Strydom (1985)
Molasses + mine water NA Pack bed 1.36 Maree et al. (1991)
Synthesis gas 30 Gas-lift 12–14 van Houten et al. (1995)
H2/CO2 30 Gas-lift 30 van Houten et al. (1994)
H2/CO 35 Pack bed 1.2 Du Preez and Maree (1995)
CO 35 Pack bed 2.4 Du Preez and Maree (1995)
Mixture of volatile fatty acids (VFA) 30 Baffled reactor 15 Vallero et al. (2003)
Acetate 35 Pack bed 15–20 Stucki et al. (1993)
Acetate 33–35 EGSB c 28.5 de Samul and Verstraete (1999)
Lactate RT d Plug flow 0.41 Hammack et al. (1994)
Glucose/acetate 35 Anaerobic digester 1.92 Polpresert and Haas (1995)
Sucrose/peptone 35 Baffled reactor 23.5 e Barber and Stuckey (2000)
Ethanol 33 EGSB 21 de Samul and Verstraete (1999)
High strength leachate 19–25 Anaerobic filter 0.02 Henry and Prasad (2000)
Wastewater from organic peroxide production + ethanol NA f Pack bed 15–18.8 Silva et al. (2002)
a
Upflow anaerobic sludge blanket (UASB).
b
Continuous stirred tank reactor.
c
Expanded granular sludge bed (EGSB).
d
Room temperature.
e
Average reduction rate of each compartment.
f
NA: Not available.

reducers and denitrifying bacteria, only a few other
types of anaerobes can grow with hydrogen or acetate as
a sole energy source (Widdel, 1988).
Using hydrogen as an electron donor, three groups of
microorganisms function in sulfidogenic reactors. These
groups are hydrogenotrophic sulfate reducers, homoace-
togenesis microbes and hydrogenotrophic methanogen-
esis microbes. A competition exists among these groups.
Hydrogenotrophic sulfate reduction, or sulfidogen-
esis, involves
þ −
4H2 þ SO2−
4 þ H →HS þ 4H2 O
substrate threshold, are often used to determine the
bacterial competition. The values of growth rate,
substrate affinity, and substrate threshold explain an
order of competitivity among the three microbial groups
at limited H2 concentrations in which hydrogenotrophic
SRB outcompete hydrogenotrophic methanogens and
the latter outcompete homoacetogens. (Weijma et al.,
2002b). In a laboratory-scale gas-lift reactor treating
sulfate-containing wastewater with hydrogen as the
electron donor, van Houten et al. (1995) found that the
biomass aggregates consisted predominantly of Desul-
ð2Þ fovibrio sp. and Acetobacterium sp.

The undesirable hydrogenotrophic methanogenesis

and homoacetegenesis reactions are the following: Table 2
Biomass yield of sulfate reducing bacteria on different electron donors
Methanogenesis : 4H2 þ HCO−3 þ Hþ →CH4 þ 3H2 O and acceptors (Speece, 1996)

ð3Þ Electron Electron donor Yield (g cells/mol electron donor)

acceptor
Homoacetegenesis : 4H2 þ 2HCO−3 þ Hþ →CH3 COO− SO2−
4 H2 1.9
þ4H2 O: S2O2−
3 H2 4.5
SO2− H2 4.0
ð4Þ 3
SO2− Acetate 4.8
4
S2O2−
3 Acetate 10.4
The kinetics of bacterial growth, quantified by a SO2−
3 Acetate 11.2
SO2− Propionate 0.086
maximum specific growth rate, substrate affinity and 4
456 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
Sulfate reducing bacteria are generally more efficient
in hydrogen utilization than methanogenic bacteria
(Davidova and Stams, 1996); therefore, using hydrogen
as an electron donor has an advantage over using
organic compounds. Considering the free energy ΔG of
sulfate reduction using hydrogen as electron donor, the
lower ΔG value for SRB is more favorable compared
with that for methanogens (MB):
MB : 4H2 þ CO2 →CH4 þ 2H2 O−32:7kJ=mol ð5Þ
SRB : 4H2 þ Hþ þ SO2− −
4 →HS þ H2 O−38:1kJ=mol:
ð6Þ
Hydrogenotrophic SRB (HSRB) have been found to
gain relatively more energy from the consumption of
molecular hydrogen (Oude Elferink et al., 1994).
In wastewater treatment systems, H2 can be directly
supplied to the reactor or generated on site from other
electron donors like propionate, methanol and glucose.
Consumption of hydrogen generates hydroxide as
follows:
8H2 þ − −
4 →H2 S
2SO2− þ HS þ 5H2 O þ 3OH ð7Þ
pH control may be necessary to neutralize the hydroxide.
When H2 is used as an electron donor, CO2 must also
be added to supply carbon for SRB. However, adding
CO2 normally leads to a decrease in the pH of the system
because of the formation of carbonic acid, especially
during the start up period. Attention is required to
prevent acidification (van Houten et al., 1994). Desul-
fovibrio species growing on hydrogen seem to require at
least an organic C2-compound such as acetate in
addition to carbon dioxide for cell synthesis (Widdel,
1988). Only about one-third of the cell material was
derived from carbon dioxide, whereas two-third was
derived from acetate (Widdel, 1988).
For free H2S concentrations of b450 mg/L, a
maximum sulfate conversion rate of 30 g SO42−/L d was
achieved under mesophilic conditions by van Houten
et al. (1994). Biological sulfate reduction using synthesis
gas (a mixture of H2, CO and CO2) as an electron donor
and carbon source allowed a sulfate conversion rate of
10 g SO42−/L d at low biomass concentrations (van Houten
et al., 1996). Presence of CO had a negative impact on
sulfate conversion. Under thermophilic conditions the use
of hydrogen is less efficient due to the formation of
methane as a by-product from H2 and CO2. Also, the
solubility of H2 declines with increasing temperature.
Thermophilic H2-utilizing microorganisms appear to
circumvent this challenge by an increased affinity and a
lower threshold concentration for H2 (Hansen et al.,
1999). Under thermophilic conditions (55 °C) sulfate
conversion rates of up to 7.5 g SO42−/L d have been
achieved using hydrogen. This is considerably less
compared with mesophilic conditions (van Houten
et al., 1997).
3.2. Formate
Most sulfate reducers that use hydrogen (e.g. De-
sulfobulbus propionicus, Desulfovibrio baarsii) are able
to grow on formate (Widdel, 1988). Indeed, formate
utilization is indicative of the presence of hydogen-
otrophic sulfate-reducing bacteria (de Samul and
Verstraete, 1999). The formate oxidation by sulfate
reducers is illustrated below:
− þ − −
4 þ 4HCOO þ H →HS þ 4HCO3 ðΔG-′
SO2−
¼ −146:7kJÞ ð8Þ
3.3. Methanol
Methanol is of particular interest as an electron donor
because it is readily available and cost effective
(Dijkhuizen et al., 1985; Glombitza, 2001; Weijma
et al., 2003). Methanol can be directly used by SRB and/
or indirectly used via involvement of other anaerobic
microorganisms. Weijma et al. (2000a) showed that the
degradation of methanol occurred via the intermediate
H2/CO2 and formate, whereas acetate was not found to
be an intermediate in this process. SRB can grow
syntrophically either with H2/CO2 or acetate producing
microorganisms (Vallero et al., 2003). This leads to a
loss of methanol as an electron donor. However, Paulo
et al. (2004) found that acetate was not used either by
methanogens or sulfate reducers. This makes the system
susceptible to its accumulation.
Sulfate reducers, Desulfotomaculum oreientis, De-
sulfoviobrio strains, Desulfobacterium catecholicum
and other microbes have been reported to oxidize
methanol (Widdel, 1988). The growth of the sulfate
reducers on methanol is slow, with a doubling time of
around a day or more, compared with the growth of
special methane bacteria or homoacetogens. In contrast,
under thermophilic conditions, Desulfotomaculum spe-
cies grow faster than methanogens and homoacetogens
(Weijma and Stams, 2001). Desulfotomaculum kuznet-
sovii is an example of thermophilic SRB that can degrade
methanol directly to carbon dioxide (Nazina et al., 1998).
When methanol is used, the fate of methanol in the
anaerobic reactor is determined by the outcome of
competition between methanogens, sulfate reducers and
homoacetogens for methanol. Methanol and its
 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
intermediate products are used as shown in Fig. 1. The
reactions carried out by the various bacteria are as
follows:
Sulfate reducers
− −
4CH3 OH þ 3SO2−
4 →4HCO3 þ 3HS þ 4H2 O
þ
þH ðΔG ¼ −364kJ=reactionÞ ð9Þ
Methanogens
4CH3 OH→3CH4 þ HCO−3 þ H2 O þ Hþ ðΔG
¼ −316kJ=reactionÞ ð10Þ
Homoacetogens
4CH3 OH þ 2HCO3− →3CH3 COO− þ Hþ
þ4H2 OðΔG ¼ −220kJ=reactionÞ: ð11Þ
Homoacetogenic bacteria can convert methanol to
acetate in the presence of bicarbonate (Diekert, 1992;
Weijma and Stams, 2001). The acetate produced can
serve as an electron donor for SRB (Widdle and Hansen,
1992). Hydrogen is a by-product of acetogenic bacteria
growing on methanol when hydrogen-consuming SRB
are presented.
Similar to other electron donors, the factors affecting
the competition between methanogens and SRB for
methanol are the growth kinetics, immobilization
properties, substrate diffusion in biofilms and environ-
mental conditions (e.g. pH, temperature and hydrogen
sulfide concentration). If methanol is used as electron
donor, temperature becomes a significant factor for the
competition (Weijma and Stams, 2001).
Methanol has been found to provide low rates of
sulfate reduction under mesophilic conditions. Weijma
et al. (2003) concluded that more than 90% of methanol
used was converted to methane, whereas only 5–10%
Fig. 1. Anaerobic methanol degradation (Weijma and Stams, 2001).
457
was used for sulfate reduction. Only 0.4 g SO42−/L d was
reduced under mesophilic conditions. Moreover, meth-
anol degradation to methane was quite stable in the
presence of sulfate.
Further investigation on the effect of temperature
under mesophilic and thermophilic conditions concluded
that by changing the temperature from 30 °C to 65 °C,
methanol conversion shifts predominantly from metha-
nogenic to sulfidogenic in an expanded granular sludge
bed (EGSB) reactor. Therefore, for methanol use as
electron donor, SRB would outcompete methanogens at
the temperature 65 °C or higher (Weijma et al., 2000b).
The stability of thermophilic methanol removal was
investigated by Vallero et al. (2003). Thermophilic sulfate
reduction is useful especially for flue gas desulfurization
(Weijma and Stams, 2001). Thermophilic processes are
known to be more effective than mesophilic process.
Although methanol is not suitable for completely
reducing sulfate under mesophilic conditions because
of excessive methane formation, it is the most efficient
electron donor under thermophilic conditions (Weijma
et al., 2000a; Weijma et al., 2000b; Goorissen et al.,
2004).
In addition to temperature, pH is a key factor in
suppressing methanogenic activity in sulfidogenic
reactor. Weijma et al. (2002b) reported that a relatively
short exposure to a slightly acidic pH in combination
with operating the reactor at a volumetric methanol-
COD loading rate close to the maximum volumetric
sulfide-COD formation rate favored sulfate reduction
over methanogenesis.
3.4. Ethanol
Ethanol is another attractive electron donor. A sulfate
conversion efficiency as high as 80% has been achieved
at high sulfate loading rate values while using ethanol as
electron donor (Barnes et al., 1991; Kalyuzhnyi et al.,
1997; de Smul et al., 1997). The complete oxidation of
ethanol to CO2 using SRBs alone was reported using
cultures of Desulfovibrio desulfuricans and Desulfo-
bacter postgatei (Nagpal et al., 2000). The various
microorganisms involved degrade ethanol as follows:
Acetogenesis
C2 H5 OH þ H2 O→CH3 COO− þ Hþ þ 2H2 ð12Þ
Methanogenesis
CH3 COO− þ H2 O→CH4 þ HCO−3 ð13Þ
4H2 þ HCO−3 Hþ →CH4 þ 3H2 O ð14Þ
458 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
Sulfidogenesis
− −
C2 H5 OH þ 0:5SO2−
4 →CH3 COO þ 0:5HS
þ
þ 0:5H þ H2 O ð15Þ
CH3 COO− þ SO2− −
4 →2HCO3 þ HS
−
ð16Þ
þ −
4H2 þ SO2−
4 þ H →HS þ 4H2 O: ð17Þ
Use of ethanol makes mass transfer limitations
insignificant as compared to the use of hydrogen,
carbon monoxide or synthesis gas. However, a draw-
back of using this electron donor is a rather low growth
rate of SRB on ethanol. Growth of SRB is inhibited by
undissociated H2S to a greater extent than the growth of
hydrogenotrophic (Stucki et al., 1993; van Houten et al.,
1994).
Similar to the use of other electron donors, the use of
ethanol produces acetate which results in an increase in
the COD of the effluent. This problem can be resolved
by incorporating acetate utilization by methanogens and
SRBs (Nagpal et al., 2000).
3.5. Molasses
Molasses is widely available in large quantities from
sugar producing processes (Hilton and Archer, 1988). It
has been used as an electron donor in sulfate reduction.
Because of its low cost and ready availability, molasses
is one of the most cost effective electron donors.
Molasses comprises mainly sugar. When molasses is
provided as an electron donor in sulfate reduction, it is
fermented by microorganisms such as Lactobacilli, to
products that are then consumed by SRB as electron
donors and carbon sources (Maree et al., 1986; Maree
and Hill, 1987). Molasses fermentation mainly involves
the following reaction:
C12 H22 O11 þ H2 O→4CH3 CHOHCOOH: ð18Þ
A satisfactory biological sulfate reduction has been
reported in an upflow anaerobic sludge blanket (UASB)
process that used molasses as an electron donor, but only
at COD:S ratio of b2. At higher ratios of COD-to-S, the
COD removal decreased due to accumulation of non-
biodegradable portion of molasses in the sludge
(Annachhatre and Suktrakoolvait, 2001a,b). Presence of
nonbiodegradable content is a disadvantage of molasses.
Nonbiodegradable material includes products of carame-
lization. Accumulation of these reduces activity of the
biomass and results in a high residual COD in the effluent.
The effectiveness of molasses in sulfate reduction can be
increased by using anaerobic reactors in series. This
allows better fermentative conversion of the molasses to
lactate which serves as the electron donor and carbon
source and is also much easier to degrade as compared to
unfermented molasses (Maree et al., 1991).
Another major drawback of using molasses is the
high volatile fatty acid (VFA; acetate, propionate, and
butyrate) content in the reactor. VFAs create a reactor
souring problem and negatively impact the growth of
both methanogens and sulfate reducers (Lo et al., 1990).
Addition of NaOH or NaHCO3 is necessary to prevent
acidification. Also, the quality of the effluent in terms of
residual COD and color, must be considered when
molasses is used.
3.6. Lactate
Lactate has been assessed as an organic substrate for
enrichment of sulfate reducers (Widdel, 1988). Several
species of sulfate reducers can use lactate as an electron
donor and carbon source. Sulfate reduction using lactate
as an electron donor can be described as follows:
CH3 CHOHCOOH þ 0:5H2 SO4 →CH3 COOH
þ CO2 þ 0:5H2 S þ H2 OΔG
¼ −34:2kJ=mole− : ð19Þ
Two moles of lactate are oxidized per mole of sulfate
reduced by D. desulfuricans, for example, and this
stoichiometric ratio is not temperature dependent
(Okabe and Characklis, 1992). However, complete
lactate oxidation is not achieved by most Desulfobac-
ter species and some Desulfobacterium species. Desul-
fonema magnum does not grow on lactate (Widdel,
1988).
3.7. Acetate
Acetate is a key intermediate in the breakdown of
organic substances in anaerobic processes. Acetate can
be used as an electron donor and carbon source in the
sulfate reduction process. Species of the genus Desul-
fotomaculum generally consume acetate, but Desulfoto-
vibrio does not use acetate. The latter genus only
degrades lactate to acetate and is commonly referred to as
an incomplete SRB.
In a sulfidogenic reactor, acetate-degrading sulfate
reducers must compete with Methanosaeta spp. for
acetate (Oude Elferink et al., 1998) and generally,
methanogens outcompete sulfate reducers due to their
higher growth rates (Yoda et al., 1987). SRB have a
thermodynamic advantage over methanogens and
 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
acetogens as indicated by the standard free energy
change of the acetate oxidation (Rinzema and Lettinga,
1988):
CH3 COO− þ H2 O→CH4 þ HCO−3 ΔG
¼ −28:2kJ=molC2 ð20Þ
CH3 COO− þ SO2− − −
4 →HS þ 2HCO3 ΔG
¼ −39:5kJ=molC2 : ð21Þ
Acetate is produced by microorganisms called
homoacetogenic bacteria that are always present in the
system and compete with methanogens and sulfate
reducers. Acetate generated can be used further as an
electron donor and carbon source by methanogenic
archaea (MA) and SRB. SRB are generally poor
competitors of MA for acetate. However, in a long-
term operation, SRB gradually out compete MA in a
sulfidogenic reactor due to their higher affinity for the
substrate and higher substrate removal rate (Harada et al.,
1994).
Acetate production during the biological sulfate
reduction is actually a major drawback of sulfate reducing
reactors because SRB cannot completely oxidize acetate
even with excess sulfate levels (Lens et al., 2002). The
acetate remaining in the effluent contributes largely to the
residual COD (Widdel, 1988; Omil et al., 1996; Lens
et al., 1998).
3.8. Propionate and butyrate
Propionate and butyrate are important fermentation
products in anaerobic sulfate reduction processes
(Speece, 1996). The mineralization of propionate and
butyrate occurs via two pathways. The first pathway
involves syntrophy between hydrogen-and acetate-
consuming sulfate reducers and hydrogen producing
acetogens. In the second pathway, both propionate and
butyrate are directly consumed by sulfate reducers
(Widdel, 1988). When propionate is converted to
acetate, the theoretical ratio of propionate to SO42− is
0.43. It was reported by Ghigliazza et al. (2000) that to
obtain complete sulfate reduction, the ratio between
propionate and SO42− should be 1.01. The stoichiometric
relationships for propionate and butyrate degradation
are as follows (calculated from Thauer et al., 1977):
− −
Propionate þ SO2−
4 þ H2 →HS þ HCO3
þ Acetate þ H2 O ð22Þ
− −
4 þ H2 →2HS þ 3HCO3 þ H2 O
Propionate þ 2SO2−
ð23Þ
459
− −
4 þ 2H2 →3HS þ 4HCO3
Butyrate þ 3SO2−
þ 5H2 O ð24Þ
−
Butyrate þ SO2−
4 þ 2H2 þ 6H2 O→HS
þ 2Acetate: ð25Þ
The degradation of propionate is substantially
enhanced by the presence of SRB (Speece, 1996).
SRB play an important role in the breakdown of
propionate either through direct utilization or through
interspecies transfer (e.g., H2). A propionate-degrading
SRB, D. propionicus, was reported to be able to
breakdown propionate efficiently to acetate (Harada
et al., 1994). Propionate oxidation by SRB becomes
more efficient at high sulfate concentrations. Visser et al.
(1993) found that only under sulfate-limiting conditions
syntrophic propionate oxidizers outcompeted propio-
nate-degrading sulfate reducers. However, syntrophic
butyrate oxidizers were well able to compete with
sulfate reducers for the available butyrate, even with an
excess of sulfate.
3.9. Sugar
Sugar is an effective electron donor that is easily
degraded under anaerobic conditions. The anaerobic
degradation pathway of sugar is also similar to that of
other organic compounds in which hydrogen is the
interspecies. Desulfotomaculum antarcticum has been
reported to use glucose, whereas Desulfovibrio and
Desulfotomaculum nigrificans are able to grow on
fructose (Klemps et al., 1985; Widdel, 1988). The
competition for the hydrogen substrate between the
hydrogenotrophic methanogens and sulfidogens has
been suggested to occur in biomass pellets in UASB
reactor (Sam-Soon et al., 1991). Hydrogen and a
mixture of lactate and VFA were reported to
accumulate during glucose degradation (Lens et al.,
2003).
3.10. Hydrocarbons
Hydrocarbons are regarded as inert under anoxic
conditions. However, enriched bacterial cultures have
been found to degrade hydrocarbons such as saturated
aromatic or unsaturated non-aromatic hydrocarbons in
combination with sulfate reduction. Occasionally,
sulfate reducers have been enriched to utilize hydro-
carbons in bioremediation processes in which hydro-
carbons serve as electron donors and carbon sources.
Thermodynamically, oxidation of hydrocarbons is
possible, but the free energy change would be mostly
460 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463

low as estimated for the methane oxidation (Widdel,
1988):
− −
CH4 þ SO2−
4 →HCO3 þ HS þ H2 OΔG
¼ −16:6kJ: ð26Þ
Kniemeyer et al. (2003) found that isolated SRB were
able to anaerobically utilize ethylbenzene. In addition,
crude oil containing toxic alkylbenzenes (e.g., xylene) can
also be used as an organic substrate and electron donor
during the growth of sulfate-reducing enriched cultures
(Harms et al., 1999). Lin and Lee (2001) reported on the
feasibility of phenol degradation by SRB. The reaction
stoichiometry of phenol utilization with sulfate reduction
in an anaerobic biofilm reactor is as follows:
C6 H5 OH þ 5H2 O→3CH3 COOH þ 2H2 ð27Þ
CH3 COOH þ SO2−
4 →2CO2 þ S
2−
þ 2H2 O: ð28Þ
It should be noted that SRB only function during the
utilization of the acetate product as an electron donor to
convert sulfate to sulfide.
Anaerobic processes have been found to be effective
for wastewaters containing aromatic pollutants such as
phenol (Fang et al., 1996) and benzoate (Li et al., 1995).
Kobayashi et al. (1989) found that benzoate is an
intermediate for the degradation of some aromatic
compounds like phenol. Benzoate can also serve as an
electron donor for SRB (Fang et al., 1997). Complete
and incomplete degradation of benzoate by SRB are
described as follows:
C6 H5 COO− þ 0:75SO2−
4 þ 4H2 O→3CH3 COO
−
− −
þ 0:75HS þ HCO3 þ 2:25H þ
ð29Þ
C6 H5 COO− þ 3:75SO2− −
4 þ 4H2 O→7HCO3
− þ
þ 3:75HS þ 2:25H ð30Þ
3.11. Organic waste
Many organic wastes are cost effective electron
donors for sulfate reduction. Such wastes include
sewage sludge, animal manure, leaf mulch, wood
chips, sawdust and cellulose. Mixtures of various
organic wastes have provided high sulfate reduction
rates because of their high carbon contents (Waybrant
et al., 1998). Sulfide production only occurs when
significant amounts of organic matter are present.
Organic waste was successfully used as electron donor
and carbon source in the treatment of acid mine drainage
(AMD). Work in this area has been discussed by Gibert
et al. (2003). Discharge of sewage, sewage sludge and
garbage in the sea leads to dramatically increased rates
of sulfate reduction in marine sediments that are carbon
limited (Madigan et al., 2003).

Table 3
Free energy of methanogenic reaction and sulfidogenic reaction with different electron donors (calculated from Thauer et al., 1977)
Equation no. Substrate Products ΔG°' (kJ/reaction)
Methanogenic reaction
1 Acetate− + H2O CH4 + HCO−3 −31.0
2 4H2 + H+ + HCO−3 CH4 + 3H2O −135.6
3 4Methanol 3CH4 + HCO−3 + H2O + H+ −316

Sulfidogenic reaction
Carboxylic acid
4 4Formate− + SO2− 4 HS− + 4HCO−3 −146.7
5 Acetate− + SO2−
4 HS− + 2HCO−3 −47.3
6 Propionate− + SO24− + H2 HS− + HCO3− + Acetate− + H2O −75.8
7 Propionate + 2SO2− 4 + H2 2HS− + 3HCO−3 + H2O −122.7
8 Butyrate− + 3SO2−4 + 2H2 3HS− + 4HCO−3 + 5H2O −198.4
9 Butyrate− + SO2−4 + 2H2 + 6H2O HS− + 2Acetate− −103.8
Alcohol
10 4Methanol + 3SO2−4 3HS− + 4HCO−3 + 4 H2O + H+ −361.7
11 2Methanol + SO2−
4 HS− + 2 Formate− + H+ + 2H2O −108.3
12 2−
2Ethanol + SO4 2Acetate− + HS− + 2H2O + H+ −132.7
Sugar
13 Glucose + SO2−
4 HS− + 2Acetate− + 2HCO−3 + 3H+ −358.2
14 Glucose + 3SO2−
4 3 HS− + HCO−3 + 3 H+ −452.5
Unsaturated acids
15 2Lactate− + SO2−
4 HS− + 2Acetate− + 2HCO−3 + H+ −159.6
 W. Liamleam, A.P. Annachhatre / Biotechnology Advances 25 (2007) 452–463
4. Selection of an electron donor
The selection of a suitable electron donor for
biological sulfate reduction is based on two major
considerations: (1) the treatment efficiency or ability of
electron donor to completely reduce and remove sulfate
while minimizing the occurrence of other pollutants in
the effluent; and (2) the cost of electron donor per unit of
sulfate converted to sulfide (van Houten et al., 1994).
Thermodynamic or kinetic parameters are important
in the selection of electron donors because they affect the
competition between SRB and MA, and hence the
treatment efficiency. The free energies of methanogenic
and sulfidogenic reactions with different electron donors
are shown in Table 3. Hydrogen (with carbon dioxide) is
a preferred electron donor for many SRB. The
competition between SRB and other microbial activities
may be minimized by controlling the operating condi-
tions so that SRB are dominant. For example, thermo-
philic metal-utilizing SRB are able to grow at b 65 °C,
whereas methanol-utilizing MA cannot grow at this
temperature (Weijma et al., 2000a). In this case, if
methanol is used as electron donor, MA activities can be
eliminated by operating at 65 °C.
A preferred organic electron donor and carbon source
should stimulate the SRB activity (Gibert et al., 2003). It
may be necessary to use more than one type of electron
donor to obtain best performance in sulfate reduction. For
example, Polpresert and Haas (1995) found that during
anaerobic sulfate reduction with dual substrates (glucose
and acetate) the mixed substrate enhanced COD removal
through both methane formation and sulfate reduction.
Mixtures containing multiple organic substrates have been
reported to show higher sulfate reducing rates compared
with those containing a single organic substrate (Waybrant
et al., 1998). A mixture of sewage sludge, leaf mulch,
wood chips, sheep manure and sawdust has been used to
treat sulfate rich wastewater to removal complete removal
of sulfate within 20 days. Similarly, a mixture containing
vegetal compost and cow manure gave sulfate reduction
rates from 5 to 300 mg/L d (Christensen et al., 1996).
The cost and availability are other factors that need to
be considered in selecting electron donors. Cost of some
common electron donors is given in Table 4. Complex
organic substrates such as molasses, are the most cost-
effective but they offer low treatment efficiency. Complex
organic substrates such as molasses, lactate, and hydrocar-
bons are not completely oxidized by sulfate reducers and,
therefore, produce relatively high COD levels in the eff-
luent. Complex organic compounds should be avoided
unless the effluent is further treated to remove residual
COD.
461
Table 4
Cost of various electron donors in Thailand
Electron donor Cost (Baht/kg)
Ethanol 80
Methanol 28
H2 + CO2 4500 (for H2), 60 (for CO2)
Acetic 240
Glucose 150
Molasses 2
Ethanol is reportedly the most cost-effective electron
donor and carbon source in the United Kingdom
(Kolmert and Johnson, 2001; White et al., 1996).
Although the operating cost with ethanol are higher
than that with hydrogen gas, for low sulfate loadings
(b200 kg SO42−/h), the use of ethanol is preferred and
more economical than using hydrogen. Furthermore, less
safety measures are needed when ethanol is used. For
large sulfate loads (more than 200 kg SO42−/h), hydrogen
is the most suitable electron donor at mesophilic
temperature (25–40 °C) (Weijma et al., 2002c).
5. Conclusions
Wastewater generated by certain processes contains
high concentrations of sulfate that can be removed by
biological reduction. A sufficient amount of a satisfactory
electron donor is needed to ensure complete removal of
sulfate. With an acceptable organic electron donor, 1 g of
sulfate requires 0.67 g of COD (i.e. a COD:sulfate ratio of
0.67) to assure complete reduction of sulfate. Selecting a
suitable electron donor requires consideration of cost,
availability, reduction efficiency and any residual COD.
Acknowledgements
Warounsak Liamleam wishes to acknowledge a
Japanese Schlolarship that supported his doctoral study.
This research was part of the “Industrial Hazardous Waste
Treatment and Management" project under “Asian
Regional Research Programme on Environmental Tech-
nology (ARRPET)" funded by the “Swedish Internation-
al Development Cooperation Agency (Sida)".
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