Review

A review of biological sulfate conversions in

wastewater treatment
Tian-wei Hao
a
, Peng-yu Xiang
a
, Hamish R. Mackey
a
, Kun Chi
a
, Hui Lu
c
,
Ho-kwong Chui
a
, Mark C.M. van Loosdrecht
b
, Guang-Hao Chen
a,c,*
a
Department of Civil & Environmental Engineering, The Hong Kong University of Science and Technology,
Clear Water Bay, Kowloon, Hong Kong
b
Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands
c
SYSUeHKUST Joint Research Centre for Innovative Environmental Technology, Sun Yat-sen University,
Guangzhou, China
a r t i c l e i n f o
Article history:
Received 16 April 2014
Received in revised form
26 June 2014
Accepted 30 June 2014
Available online 10 July 2014
Keywords:
Sulfur conversion biotechnology
SRB operation condition
Sulfate reduction
Wastewater treatment
a b s t r a c t
Treatment of waters contaminated with sulfur containing compounds (S) resulting from
seawater intrusion, the use of seawater (e.g. seawater ushing, cooling) and industrial
processes has become a challenging issue since around two thirds of the world's population
live within150 kmof the coast. Inthe past, researchhas producedanumber of bioengineered
systems for remediation of industrial sulfate containing sewage and sulfur contaminated
groundwater utilizing sulfate reducing bacteria (SRB). The majority of these studies are
specic with SRB only or focusing on the microbiology rather than the engineered applica-
tion. In this review, existing sulfate based biotechnologies and new approaches for sulfate
contaminated waters treatment are discussed. The sulfur cycle connects with carbon, ni-
trogen and phosphorus cycles, thus a new platform of sulfur based biotechnologies incor-
porating sulfur cycle withother cycles can be developed, for the removal of sulfate and other
pollutants (e.g. carbon, nitrogen, phosphorus and metal) from wastewaters. All possible
electron donors for sulfate reduction are summarized for further understanding of the S
related biotechnologies including rates and benets/drawbacks of each electron donor. A
review of known SRB and their environmental preferences with regard to bioreactor oper-
ational parameters (e.g. pH, temperature, salinity etc.) shed light on the optimization of
sulfur conversion-based biotechnologies. This review not only summarizes information
from the current sulfur conversion-based biotechnologies for further optimization and
understanding, but also offers newdirections for sulfur related biotechnology development.
2014 Elsevier Ltd. All rights reserved.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
* Corresponding author. Department of Civil & Environmental Engineering, The Hong Kong University of Science and Technology, Clear
Water Bay, Kowloon, Hong Kong.
E-mail address: ceghchen@ust.hk (G.-H. Chen).
Available online at www.sciencedirect.com
ScienceDirect
j ournal homepage: www. el sevi er. com/ l ocat e/ wat res
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1
http://dx.doi.org/10.1016/j.watres.2014.06.043
0043-1354/ 2014 Elsevier Ltd. All rights reserved.
2. Biological sulfur conversion on different electron donors and acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Key organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
4. Sulfur conversion biotechnologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5. Factors affecting the efficiency of sulfate reducing bioprocesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
6. Summary and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Supplementary data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1. Introduction
In the natural environment, sulfur is mainly present in the
stable form of reduced pyrite (FeS
2
) and oxidized gypsum
(CaSO
4
) in sediment, and as the sulfate ion in seawater where
it was possibly generated photochemically from volcanic SO
2
and H
2
S (Farquhar and Wing, 2003) Seawater intrusion has
become a serious issue in many coastal areas with inuences
from both natural and anthropogenic processes, contami-
nating groundwater resources and agricultural production by
sulfate and salts (Bear et al., 1999). The Intergovernmental
Panel on Climate Change (IPCC 2001) predicts that by 2100,
global warming will lead to a sea-level rise of between 110 and
880 mm (Werner and Simmons, 2009). Such a rise would
severely increase seawater intrusion, thus intensifying water
scarcity in the coastal zones (<100 km from coast) where 40%
of the global population (UN Atlas of the oceans) dwells and
where about one third of total freshwater consumption reliant
upon groundwater (Bear et al., 1999). Therefore, the need for
cost-effective and functional purication technologies for
seawater contaminated groundwater resources is inevitable.
On the other hand, seawater also provides various alter-
native water resources, such as seawater desalination, cooling
water, and seawater toilet ushing (Chui et al., 2011).
Compared with desalination, replacement of freshwater by
seawater for toilet ushing presents an economic and sus-
tainable alternative water resource for water-scarce coastal
cities (Chui et al., 2011). For instance, Hong Kong has practiced
seawater toilet ushing for over 50 years, with a daily supply
of 750,000 cubic meters of seawater supplied for 80% of its 7
million inhabitants (WSD, 2010). This amounts to 20% of total
water demand of the city (Lee and Wu, 1997). Such large scale
seawater toilet practice results in saline sewage that contains
550 mg sulfate and 5000 mg chloride on average (van
Loosdrecht et al., 2012).
In addition to brackish and saline water, various sulfate-
laden wastewaters are also produced from industrial pro-
cesses, such as pulp and paper, fermentation, pharmaceutical
production, food production, tannery operations, petro-
chemical and mining processes (Omil et al., 1996; Lens et al.,
2003; Klok et al., 2012, 2013; Jarvis and Younger, 2000).
Comparatively, mining and metallurgical industries generate
the largest volumes of wastes containing high concentrations
of sulfate and/or dissolved metals. Acid mine drainage (AMD)
is currently one of the most serious water and soil pollution
sources in the world (Huisman et al., 2006). For instance, in
1989 ca. 19,300 km of streams and rivers, and ca. 72,000 ha of
lakes and reservoirs were heavily polluted by mine efuents
(Johnson and Hallberg, 2005). The atmosphere is also a large
source of sulfur-derived waste discharge. Sulfur-containing
fossil fuels account for approximately 90% of the anthropo-
genic emission of SO
2
(Brimblecombe et al., 1989). Although
wet ue gas desulfurization (FGD) has been practiced suc-
cessfully as one of the major control measures for atmo-
spheric S contamination in industrialized nations, cost-
effective disposal of the FGD by-products/wastes, which
include calcium sulfate and resultant wastewater high in
chlorides, heavy metals and dissolved solids, still remain a
challenging issue in developing nations including China (Wu
et al., 2004).
The root of these problems originates from complex and
interrelated sulfur conversions and transformations due to its
multiphase nature under common environmental conditions
(i.e. solid, liquid and gaseous state) and a wide range of redox
states (from 2 to 6), which current chemical and/or bio-
logical control processes are built upon, as summarized in
Fig. 1 (Muyzer and Stams, 2008; Wu et al., 2013; Vallero, 2003).
For instance, in microbe-mediated biological sulfur conver-
sions/transportations, there are typically three reactions
applied: 1) assimilation of sulfur (S), 2) desulfurization/
dissimilation of organic S, and 3) oxidation and reduction of S
compounds, during which macromolecular organic S com-
pounds are desulfurized/decomposed to simple inorganic S
compounds such as sulfate, sulde, and thiosulfate, etc.
(Starkey and Temple, 1956). A diverse range of microorgan-
isms take up inorganic S as a nutrient to synthesize crucial S-
containing organic compounds, e.g. cysteine (Na and Salt,
2011). On the other hand, inorganic sulfur compounds (e.g.
sulde, elemental sulfur and thiosulfate) serve as electron
donors in carbon dioxide xation by phototrophic sulfur
bacteria. These bacteria are divided into the purple and green
sulfur bacteria and the mechanisms were well reviewed by
Frigaard and Dahl (2008). Due to the incomplete understand-
ing of their metabolism and limited environmental bioreme-
diation application, the study of phototrophic sulfur bacteria
has to date focused mainly on microbiology rather than bio-
process development. On the contrary, S-reducing bacteria
(SRB) play a crucial role in many technologies for waste
treatment or bioremediation, which have been studied
extensively over the past decades (Muyzer and Stams, 2008).
The biological sulfur conversion based treatment pro-
cesses were developed mainly for: a) control of sulde for-
mation, b) volatilization of hydrogen sulde, c) chemical and
biological oxidation of sulde, and d) precipitation of metal
suldes (Zhang et al., 2008). Most of these processes combine
one single biological step (i.e. sulfate reduction) with a
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 2
chemical step (i.e. sulde chemical oxidation) as post-
treatment to eliminate the sulde hazard or recover
elemental sulfur as a resource. (Visser, 1995; Lense et al., 2000;
Hulshoff Pol et al., 2001). Often the process is used for metal
precipitation rather than control or removal of other pollut-
ants distinctly.
Overall the sulfur cycle connects with carbon, nitrogen and
phosphorus cycles, due to the analogous biochemical proper-
ties (Muyzer and Stams, 2008; Wu et al., 2013). To facilitate
further development and/or optimization of the existing S
conversion technologies, better understanding of the metabo-
lisms/mechanisms of the biochemical S conversions and mi-
crobial consortiums in the relevant bioprocesses are deemed
necessary. This motivates us to conduct a comprehensive re-
view of biological sulfur conversion reactions, sulfate reducing
microbial organisms, relevant bioprocesses and the key pa-
rameters in their application to wastewater treatment.
2. Biological sulfur conversion on different
electron donors and acceptors
Diverse carbon sources and electron donors have been shown
to be involved in the SRB metabolism according to the type of
growth (autotrophic, heterotrophic).
Hydrogen can serve as an efcient energy source (electron
donor) for many SRB that are able to grow on it with sulfate as
electron acceptor (Muyzer and Stams, 2008; Davidova and
Stams, 1996). When H
2
and CO
2
are co-utilized as the sub-
strates, high sulfate reducing rates can be achieved in both
mesophilic and thermophilic bioreactors within 10 days (van
Houten et al., 1994; van Houten et al., 1997). Synthetic gas
mixtures of H
2
, CO
2
and CO have been explored for possible
reduction of operational cost and optimization in uidized
bed bioreactors. Parshina et al. (2005) successfully isolated a
highly specic CO conversion specie Desulfotomaculum car-
boxydivorans, and Du Preez and Maree (1994) showed a sulfate
reduction rate of 2.4 g SO
2
4
/L/d on pure CO. However, the use
of synthetic gas has dual constraints, i.e. low fraction by CO (5
to over 50%) (Perry et al., 1997), and the toxicity against SRB by
CO with a concentration range of 2e70% vol. (Parshina et al.,
2010).
Methane can also be oxidized with equimolar amount of
sulfate, yielding carbonate and sulde respectively (Nauhaus
et al., 2002). Marine gas hydrate areas or even hypersaline
seep sediments (40225 Cl gL
1
) are the evident zones for
anaerobic oxidation of methane (AOM) with sulde produc-
tion (Avrahamov et al., 2013). Methane dependent specic
reduction rates of sulfate, ranging 1.4e41.3 g SO
2
4
day
1
g cell
dry mass
1
were observed with various pure cultures of SRB
(Rabus et al., 2000). Nauhaus et al. (2002) reported the optimal
temperature for the sulfate reduction is between 4

C and
16

C at 0.1 MPa methane, while methane pressure positively
inuences the sulfate reduction rate (e.g. increasing the
methane pressure to 1.1 MPa results in a four to vefold in-
crease in the sulde production rate). However, the mecha-
nismof AOMis still unclear. Hoehler et al. (1994) proposed that
AOM is performed by archaea and SRB in a consortium in
which the former produce a free, extracellular intermediate
that is scavenged by the latter. Nevertheless, the type of the
intermediate shuttling between the methane-utilizing
archaea and the SRB is still unknown (Jagersma, 2009).
Heterotrophic SRB metabolize organic compounds as
electron donors and carbon sources through Acetyl CoA or a
Fig. 1 e Web of sulfur transformations showing (1) assimilatory process; (2) mineralization process; (3) desulfurization; (4)
dissimilatory sulfate reduction; (5) volcanoes, weathering and hot springs; (6) biological oxidation with O
2
/NO
3
-
; (7) anaerobic
oxidation by phototrophic bacteria; (8) industrial process; (9) chemolithotrophic oxidation; (10) sulfur disproportionation.
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 3
modied TCA pathway (Londry and Des Marais, 2003). Many
intermediate products originating from anaerobic fermenta-
tion/hydrolysis can be metabolized by SRB, such as amino
acids, sugars, long-chain fatty acids, aromatic compounds,
lactate, butyrate, propionate and acetate (Muyzer and Stams,
2008; Coates et al., 1996; Jacob, 2007). It has been estimated
that heterotrophic sulfate reduction accounts for more than
50% of the organic carbon mineralization in marine sedi-
ments (Jrgensen, 1982). Four main types of biological S
conversions are involved in heterotrophic growth of SRB
which include: a) complete oxidation of acidied in-
termediates to carbon dioxide, b) incomplete oxidation of
acidied intermediates to acetate; c) syntrophic degradation
of intermediates by acetogenic SRB that are associated with
hydrogen-utilizing bacteria; and d) fermentative growth of
SRB in the presence of propionate and ethanol (Colleran
et al., 1995). Depending upon the microbial strains and re-
action completion degree, organic substrate is converted to
either CO
2
or acetate (Miletto, 2007), due to the absence of a
mechanism for Acetyl-CoA oxidation (Widdel and Hansen,
1992). Concurrently, the sulfur compounds are reduced to
sulde and a small amount of thiosulfate, and thiosulfate can
be further reduced with sulde as the end product (Hao et al.,
2013a,b). The synergistic interaction between heterotrophic
and autotrophic bacteria has been applied to enhance deni-
trication efciency (Tong et al., 2013), where, the CO
2
generated by heterotrophic metabolism can be used as car-
bon source for autotrophic bacteria. However, an obvious
synergism between heterotrophic and autotrophic SRB has
not observed to date (Londry and Des Marais, 2003), which
may be because the heterotrophic SRB still possess the op-
erons that can be switched on to produce enzymes needed
for autotrophic growth (Odom and Singleton, 1993).
Table 1 summarizes the sulfate reduction rates, hydraulic
retention times (HRTs) and benets/drawbacks of using
different electron donors in biological sulfate reduction pro-
cesses. Obviously the electron donor has a substantial impact
on the rate of sulfate reduction, and reported HRTs vary in a
wide range of 1e480 h. High heterotrophic sulfate removal
rates are achieved when manure (40 SO
2
4
g/L d) (Gibert et al.,
2004), formate (29 SO
2
4
g/L d) (Bijmans et al., 2008) and acetate
(65 SO
2
4
g/L d) (Stucki et al., 1993) are applied. The benets and
drawbacks of these electron donor sources are variable, and
their choice is dependent on the particular reaction
requirements.
The competition between SRB and methanogens, and the
yield factor of SRB are the two common issues concerning the
application of sulfate reduction in wastewaters treatment.
SRB are believed to out-compete methanogens in the presence
of unlimited sulfate concentrations based on their kinetic
properties (K
s
and m
max
) for hydrogen (Colleran et al., 1995),
formate, acetate, propionate, butyrate (Omil et al., 1996),
ethanol, and sucrose (Greben et al., 2000), as well as syntro-
phic methanogenic communities for substrates like propio-
nate and butyrate (Muyzer and Stams, 2008; Stams, 1994). For
methanol utilization, SRB would out-compete methanogens
at a temperature above 65

C (Weijma et al., 2000a,b). How-
ever, SRB will not compete with methanogens for utilization
of compounds like trimethylamine, or methionine (Oremland
and Polcin, 1982).
The lactate and propionate have been proven to be more
favorable substrates than hydrogen, methanol, ethanol, ace-
tate (Liamleam and Annachhatre, 2007a) and methane
(Nauhaus et al., 2007) in terms of biomass yield, energy release
and production of alkalinity. Lactate, especially, is superior to
many common organic substrates for heterotrophic biological
S conversion (van Kuijk and Stams, 1995; Das et al., 2013).
However, due to the diversity of SRB species, various sub-
stances serve as the respective electron donor for particular
metabolisms (Kleikemper et al., 2002). Hence, a mixture of
electron donors is recommended for SRB growth (Hao et al.,
2013; Waybrant et al., 1998).
Previous studies conrmed that a mixture of different
wastes, especially when containing both relatively easily
biodegradable (animal manure, compost, sludge) and recalci-
trant cellulosic materials (sawdusts or wood chips) (Neculita
et al., 2007, 2011; Thomas et al., 2010), can result in a better
SRB performance than with single waste (Song et al., 2012).
This implies locally available organic carbon sources are
usually preferred in application of S conversion bioprocesses
in carbon-decient sulfate wastewaters with a view to
lowering transport cost (Zaluski et al., 1999). As shown in
Table 1, various organic wastes can serve such electron donor
sources, i.e. food/seafood processing industries, animal
manure, sewage sludge, molasses, and composts providing
complex organic matters, while agricultural wastes, reed ca-
nary grass (Phalaris arundinacea), sawdusts, wood chips, rice
straws and leaf composts are suitable high cellulosic organic
matters (Kuyucak and St-Germain, 1994).
3. Key organisms
SRB are the key organisms in biological S reduction. The rst
evidence of SRB activity was identied in 1895 by Beijerinck
(Huisman et al., 2006) who discovered that sulfate could be
reduced to sulde by anaerobic respiration in sediments.
Various techniques have been applied for identication and
enumeration of the ubiquitously distributed SRB. Enumera-
tion methods can be classied into two categories: 1) direct
detection methods, and 2) culture methods (Vester and
Ingvorsen, 1998). Cultivation is one of the oldest techniques,
which underestimates true bacterial diversity. The direct
detection methods are recently developed molecular-based
techniques, i.e. 16S rRNA-based approaches including: poly-
merase chain reaction (PCR), uorescence in situ hybridiza-
tion (FISH), denaturing gradient gel electrophoresis (DGGE),
terminal restriction fragment length polymorphism (T-RFLP),
GeneChip

and pyrosequencing. With over a century's inves-

tigation, more than 120 species and 40 genera belonging to
three bacteria phyla and one archaeal phylum have been
recorded (Barton and Hamilton, 2007). These 40 genera, sum-
marized by Barton and Hamilton, were re-organized into two
divisions based on their physiological and ecological roles, i.e.
complete and incomplete organic oxidizers (Colleran et al.,
1995), and updated with all feasible electron acceptors and
morphology, as shown in Table 2.
Most SRB have rod, vibrio, or curved morphology. Among
these 40 genera, 16 genera belong to incomplete organic oxi-
dizers, 22 genera are complete oxidizers, and the remaining 2
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 4
Table 1 e Sulfate reduction rates, HRTs and benets/drawbacks of selected electron donor sources for biological sulfate
reduction.
Electron donor HRT (h) Sulfate
reduction rate
(SO
2
4
g/L/d)
Pro (), con () Reference
H
2
/CO 4e18 0.4e1.9 Low cost
Most of SRB can use H
2
as energy source
No organic residual in
the efuent
Sufcient supply
Toxicity of CO towards SRB,
Hydrogen mass transfer limits
the reaction rate
Induce competition with other
organisms (methanogens)
du Preez and Maree, 1994;
Sipma et al., 2007;
Colleran et al., 1995
H
2
/CO
2
4e12 4.5e30 SRB can outcompete methanogens
for hydrogen
No organic residual in the efuent
Induce competition with other
organisms (methanogens,
homoacetogenic bacteria)
Formation of methane lower
H
2
utilization efciency
Hydrogen safety requirements
van Houten et al., 1994;
Liamleam and Annachhatre,
2007a; Esposito et al., 2003;
Esposito et al., 2006
Synthetic gas
(H
2
CO
2
CO)
4.5 9.6e14 Low cost
Some SRB have a much higher
tolerance for CO than previous study
Availability may be limited
Colleran et al., 1995;
van Houten et al., 1996;
Dijkman et al., 1999;
Parshina et al., 2005
Methane 100.8 0.4 10
3
e0.24 Sufcient reserve
Low biomass growth rate
Lin et al., 2006;
Aharon and Fu, 2000;
Zhang et al., 2010
Methanol 3e10 0.4e20.5 Relatively low cost
Require a simple design reactor
SRB can outcompete methanogens
at high temperatures (55e70

C)
Methanogens dominates the
community under mesophilic conditions
Only a few SRB strains can
utilize methanol
Vallero et al., 2004;
Weijma et al., 2003;
Glombitza, 2001;
Weijma et al., 2000a,b;
Vallero et al., 2003
Ethanol 9.6e120 0.45e21 Relatively cheap reagent
Easily converted by SRB
Low biomass yield
Incomplete oxidation to acetate leading
to high efuent COD concentration
Colleran et al., 1995;
Liamleam and
Annachhatre, 2007a;
Nagpal et al., 2000a;
Liu et al., 2010;
Kalyuzhnyi et al., 1997;
De Smul and Verstraete, 1999
Formate 9.5e29 29 Produce less acetate during formate utilization,
Many SRB capable of growing on H
2
can
also grow on formate as a sole energy source
A safe alternative for hydrogen
e Methanogens outcompete SRB at 65e75

C
Vallero et al., 2004;
De Smul and Verstraete, 1999;
Jansen et al., 1984;
Da Silva et al., 2011;
Bijmans et al., 2008;
Bijmans, 2008
Acetate 2e21 65 e Methanogens can outcompete SRB for acetate
e Only a few SRB can oxidize acetate
e Acetate inhibited sulfate reduction at
concentrations above 15 mmol L
1
e Low biomass yield
Colleran et al., 1995;
De Smul and Verstraete, 1999;
Muthumbi et al., 2001;
Koschorreck et al., 2004;
Widdel, 1988;
Stucki et al., 1993
Lactate 12e120 0.36e5.76 Wide spectrum of SRB can grow on lactate
Generate large amount of alkalinity
Relieve the sulde toxicity
Preferable carbon source for SRB
e High cost
Oyekola et al., 2010;
Kaksonen et al., 2004b;
Bertolino et al., 2011
(continued on next page)
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 5
genera, i.e. Desulfotomaculum and Desulfomonile, do not exactly
align in either of these two divisions as they appear to have
both complete and incomplete oxidizing species. The species
in genus Desulfotomaculum, such as Desulfotomaculum solfatar-
icum, Desulfotomaculum kuznetsovii, Desulfotomaculum
australicum and Desulfotomaculum thermoacetoxidans, can uti-
lize acetate as electron donor, however, the species Desulfo-
tomaculum luciae, Desulfotomaculum thermocisternum,
Desulfotomaculum geothermicum, and Desulfotomaculum thermo-
benzoicum subsp. Thermobenzoicum cannot metabolize acetate
Table 1 e (continued)
Electron donor HRT (h) Sulfate
reduction rate
(SO
2
4
g/L/d)
Pro (), con () Reference
Glucose/acetate 1e24 0.9e2.2 e Low pH of system due to fermentation Erdirencelebi et al., 2007;
Hao et al., 2013; Kuo
and Shu, 2004;
White and Gadd, 1996
Sucrose/peptone 3.6e48 0.6e12.4 Sucrose acidication is not inhibited
by sulde
Suitable carbon and energy sources
for SRB
e Acetate accumulated in the efuent
Lopes et al., 2007b;
Lopes et al., 2010;
Maree et al., 2004;
Greben et al., 2000
Molasses 2e9.5 1.2e7.22 Inexpensive and abundantly available.
The acidication products can easily
be used by SRB
e Partial complex compounds in molasses
are hardly decomposed resulting in
high COD efuent
e The accumulation of
un-biodegradable compounds
e Not suitable for SRB growth.
e Accumulation of volatile fatty acid
Liamleam and
Annachhatre, 2007a,b;
Annachhatre and
Suktrakoolvait, 2001;
Teclu et al., 2009
Fructose e e e Only few SRB use fructose
e The SRB growth on fructose is slow
Cord-Ruwisch et al., 1986
Benzene/benzoate 264 0.038 Can be completely oxidized to CO
2
without extracellular intermediates
e Long degrading time
e Cannot be used by some SRB species
Lovley et al., 1995;
Edwards and
Grbic-Galic, 1992;
Coates et al., 1996;
Musat et al., 2010
Algal extracellular
products/algal
biomass
12 0.003e0.0058 Low-cost carbon source
Easily utilized by SRB
No available limitation
e Cannot be used directly, need fermentative
bacteria collaborate together
e May cause high COD in the efuent
Molwantwa et al., 2000;
Boshoff et al., 2004
Cheese whey 192 0.34 Low-cost carbon source
No negative impact to the bacteria
e Cannot be used directly, need fermentative
bacteria collaborate together
e May cause high COD in the efuent
Borek et al., 1995;
Jimenez-Rodriguez et al., 2010
Watermelon rind 240e480 0.15e0.24 Low cost
e Availability may be limited
e May cause high COD in the efuent
Hussain and Qazi, 2012
Plant materials Low cost
Suitable for bioremediation application.
e May cause high COD in the efuent
Lakaniemi et al., 2010;
Zagury et al., 2006;
Johnson and Hallberg, 2002;
Song et al., 2012
Phalaris-arundinacea 10e16 2.2e3.3
Mixture of wood chips,
leaf compost and
poultry manure
e 0.01
Mushroom compost,
wood chips, sawdust,
and rice straw
72 0.33-0.57
Other waste products
Primary sewage sludge 23.5e14 2.4 Low cost
e Some organic matters cannot be used directly
e May cause high COD in the efuent
Whiteley et al., 2003;
Poinapen et al., 2009
Animal manure 216 40.3 Low cost
Efcient biodegradable substrate
e Availability may be limited.
Cocos et al., 2002;
Gibert et al., 2004
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 6
(Kaksonen et al., 2006). Sulfate, sulte, thiosulfate and
elemental sulfur are the common electron acceptors for most
SRB species. Nitrate, nitrite, ferric and other compounds (e.g.
fumarate, DMSO) can also serve as electron acceptor for some
SRB species (Table 2).
In conventional anaerobic bioreactors, SRB biomass is
either suppressed (Visser et al., 1993; Lens et al., 1998;
O'Flaherty and Colleran, 2000), or activated to suit specic
purposes. Intensive studies have also been conducted to
suppress SRB activity for methane generation from anaerobic
wastewater treatment processes, as well as avoidance/miti-
gation of sewer odor and corrosion problems (Zhang et al.,
2008). SRB biomass are mainly applied in treatment of sul-
fate, heavy metal or refractory organic (benzene, toluene
Table 2 e Main sulfate reducer genera and feasible electron donors.
Oxidation of organic
electron donors genus
Cell form Electron acceptors for
growth (other than SO
2
4
)
Reference
Incomplete organic oxidizers:
Desulfovibrio Vibro SO
2
3
/s
2
o
2
3
/Fumarate/Fe(iii)/
MnO
2
/NO

2
/NO

3
/O
2
Barton and Hamilton, 2007;
Krekeler and Cypionka, 1995
Desulfomicrobium Oval to rod SO
2
3
/s
2
o
2
3
/NO

2
/fumarate/DMSO Barton and Hamilton, 2007;
Dias et al., 2008
Desulfohalobium Rod SO
2
3
/s
2
o
2
3
/S
0
Ollivier et al., 1991
Desulfonatronum Vibro SO
2
3
/s
2
o
2
3
Barton and Hamilton, 2007;
Pikuta et al., 2003; Sorokin et al., 2011
Desulfobotulus Vibrio SO
2
3
Sorokin et al., 2010; Rees and Patel, 2001
Desulfocella Vibrio e Brandt et al., 1999
Desulfofaba Vibrio SO
2
3
/s
2
o
2
3
Knoblauch et al., 1999; Rees and Patel, 2001
Desulforegula Rod Desulfoviridin Rees and Patel, 2001
Desulfobulbus Lemon/onion SO
2
3
/s
2
o
2
3
NO

2
/NO

3
/
O
2
/Fe(iii)/Graphite
Dannenberg et al., 1992; Dilling and
Cypionka, 1990; Widdel and Pfennig, 1982;
Holmes et al., 2004
Desulfocapsa Rod SO
2
3
/s
2
o
2
3
/S
0
Finster et al., 2013
Desulfofustis Rod SO
2
3
/S
0
Barton and Hamilton, 2007; Brenner et al., 2005
Desulforhopalus Rod SO
2
3
/s
2
o
2
3
/NO

3
Lie et al., 1999
Desulfotalea Rod SO
2
3
/s
2
o
2
3
/S
0
/Fe(iii)-citrate Barton and Hamilton, 2007; Brenner et al., 2005
Thermodesulfobacterium Rod SO
2
3
/s
2
o
2
3
Brenner et al., 2005; Jeanthon et al., 2002
Thermodesulfovibrio Curved rod SO
2
3
/s
2
o
2
3
/Fe(iii)/Arsenate Sekiguchi et al., 2008; Haouari et al., 2008
Desulfosporosinus Straight/curved rod SO
2
3
/s
2
o
2
3
/S
0
/Fe(iii) Robertson et al., 2001
Desulfotomaculum
a
Vibrio SO
2
3
/s
2
o
2
3
/S
0
Spring et al., 2012; Kaksonen et al., 2006
Desulfomonile
a
Rod 3-chlorobenzoate/Fumarate
SO
2
3
/s
2
o
2
3
/S
0
/NO

3
Sun et al., 2001
Complete Organics Oxidizers:
Desulfothermus Rod to curved SO
2
3
Brenner et al., 2005; Nunoura et al., 2007;
Desulfobacter Rod to ellipsoidal SO
2
3
/s
2
o
2
3
Widdel, 1987
Desulfobacterium Oval to rod SO
2
3
/s
2
o
2
3
/Fumarate Brenner et al., 2005
Desulfobacula Oval to cured SO
2
3
/s
2
o
2
3
Kuever et al., 2001
Desulfococcus Sphere SO
2
3
/s
2
o
2
3
Brenner et al., 2005
Desulfofrigus Rod SO
2
3
/s
2
o
2
3
/Fe(iii)-citrate Knoblauch et al., 1999; Suzuki et al., 2008
Desulfonema Filaments SO
2
3
/s
2
o
2
3
/NO

3
Barton and Hamilton, 2007; Icgen et al., 2007
Desulfosarcina Irregular
shape/Aggregate
SO
2
3
/s
2
o
2
3
/S
0
Arendsen et al., 1993; Poole, 2012
Desulfospira Curved SO
2
3
/s
2
o
2
3
/S
0
Brenner et al., 2005;
Desulfotignum Rod to cuverd SO
2
3
/s
2
o
2
3
/CO
2
Brenner et al., 2005; Kuever et al., 2001;
Schink et al., 2002; Simeonova et al., 2010
Desulfatibacillum Rod SO
2
3
/s
2
o
2
3
Barton and Hamilton, 2007;
Callaghan et al., 2012; Cravo-Laureau et al., 2004
Desulfarculus Vibrio SO
2
3
/s
2
o
2
3
Brenner et al., 2005; Kuever et al., 2001
Desulforhabdus Rod to ellipsoid SO
2
3
/s
2
o
2
3
Brenner et al., 2005
Desulfovirga Rod SO
2
3
/s
2
o
2
3
/S
0
Tanaka et al., 2000
Desulfobacca Oval to rod SO
2
3
/s
2
o
2
3
Oude Elferink et al., 1999
Desulfospira Curved SO
2
3
/s
2
o
2
3
/S
0
Finster et al., 1997; Kuever et al., 2001
Desulfacinum Oval SO
2
3
/s
2
o
2
3
/S
0
Sievert and Kuever, 2000; Rozanova et al., 2001
Desulfonauticus Cured rod SO
2
3
/s
2
o
2
3
/S
0
Mayilraj et al., 2009
Desulfonatronovibrio Vibrio SO
2
3
/s
2
o
2
3
/S
0
/O
2
Sydow et al., 2002
Thermodesulforhabdus Rod SO
2
3
Sievert and Kuever, 2000; Beeder et al., 1995
Thermodesulfobium Rod s
2
o
2
3
/NO

2
/NO

3
Mori et al., 2003
Archaeoglobus Irregular coccoid SO
2
3
/s
2
o
2
3
Mori et al., 2008; Hartzell and Reed, 2006
DMSO: Dimethyl sulfoxide.
a
Partial species in the genus metabolize organics completely.
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 7
xylene) contaminated industrial wastewaters (Kaksonen and
Puhakka, 2007; Laban et al., 2009). For municipal wastewater
treatment, the SANI

process (see Section 4) is the rst sulfur

conversion based biological wastewater treatment process. Its
merit is not only limited to the treatment of saline sewage
arising from seawater intrusion or toilet ushing, but also
applicable for continental cites after proper modications
(Jiang et al., 2013b; Wang et al., 2009). With a view to
expanding the application of sulfur conversion bio-
technologies in wastewater treatment and enhancing their
performance, identication of key SRB organisms commonly
detected in sulfate reducing bioreactors has been conducted
and is summarized in Table 3.
Other than full-scale applications (Hiibel et al., 2011; van
Houten et al., 2006; Wang et al., 2011) in which the tempera-
ture varied according to the ambient conditions, the majority
of the bioreactors were operated between 22

Ce35

C (Table
3). Desulfovibrio, Desulfobulbus, Desulfomicrobium and Desulfo-
bacter are the most commonly found SRB genera in these
sulfate reduction bioreactors. Desulfovibrio, Desulfobulbus and
Desulfomicrobium belong to the group of incomplete organic
oxidizers, while Desulfobacter is the only dominant complete
organics oxidizer identied.
So far, no apparent correlation between the substrates and
SRB genus has been identied. For individual bioreactors, the
SRB diversity is directly or indirectly inuenced or selected by
operational parameters such as HRT, sludge retention time
(SRT), temperature, pH, salinity etc. Besides these physico-
chemical parameters, the bacterial metabolic pathways also
play a signicant role in dictating organism diversity. Usually,
incomplete oxidizers would outcompete complete oxidizers
for substrates such as intermediates of anaerobic degradation
of organic matter (e.g. hydrogen and/or lactate) (Widdel, 1988;
Dar et al., 2008), and therefore incomplete oxidation becomes
the dominant metabolic pathway (Muyzer and Stams, 2008).
The growth kinetic parameters of complete/incomplete
organic oxidation by SRB used in Rodriguez's competition
model (Rodriguez et al., 2011) are listed in Table 4. The
maximum specic growth rate of both groups is close and
situates at the same order of magnitude, but the incomplete
organic oxidizers generally nd more favorable growth con-
ditions due to lower Monod half-velocity constants. Therefore
the metabolic pathway of SRB is a critical factor affecting SRB
competition and community diversity.
In sulfate reducing bioreactors dominated by the incom-
plete oxidizer metabolism remaining acetate in the bio-
reactors' efuent is a concern, as it could possibly cause a
breach of organic discharge standards. Under an electron
donor limited condition, lactate oxidation is mainly carried
out by incomplete oxidizing SRB, and when sulfate is limiting
acetogens and methanogenic archaea are the dominant mi-
crobial communities (Dar et al., 2008). As a consequence, more
stages or series reactors are required. Hence, complete or-
ganics oxidation SRB genera are theoretically preferred in real
Table 3 e Dominant SRB identied in various sulfate laden wastewater treatment bioreactors.
SRB genus/species Substrate Temperature Reference
Desulfovibrio spp. Lactate 35

C Okabe et al., 1995
Desulfovibrio spp. Ethanol/hay and pine wood
chips/corn stover and pine wood chips
13e36

C Hiibel et al., 2011
Desulfovibrio desulfuricans Lactate 22

C Beyenal and Lewandowski, 2004
Desulfovibrio desulfuricans
Desulfobacter postgatei
Ethanol 30

C Nagpal et al., 2000b
Desulfovibrio
Desulfomicrobium
Desulfotomaculum
Desulfobulbus
Phthalate and lactate 37

C Shabir et al., 2005
Desulfovibrio
Desulfobacter
Molasses 22

C Zhao et al., 2007
Desulfobulbus propionicus Propionate 37

C Roest, 2007
Desulfobulbus rhabdoformis
Desulfovibrio sulfodismutans
Desulfobacca
Ethanol/butanol
isopropanol/acetate
25-35

C/37

C Dar et al., 2007;
Oude Elferink et al., 1999
Desulfosarcina variabilis
Desulfoarculus baarsii
Pulp and ber producing wastes 35

C Dar et al., 2007
Desulfobulbus propionicus
Desulfobacter postgatei
Desulfovibrio gigas
Desulfosarcina variabilis
Desulfococcus multivorans
Lactate 35

C Oyekola
Desulfomicrobium Ethanol/acetate Ambient van Houten et al., 2006
Desulfonema
Desulfobulbus
Desulfobacter
Acetate 35

C Icgen et al., 2007
Desulforhopalus Saline municipal wastewater Ambient Wang et al., 2011
Desulfomicrobium Sulte glucose acetate 5 -30

C Jang et al., 2013
Desulfobulbus
Desulfobacter
Desulfomicrobium
Glucose acetate 22

C Hao et al., 2013
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 8
application. Yet, literature about the selection and develop-
ment of desired SRB genera (organics complete oxidizer) is
scarce. Studies on SRB species competition and preferable
genera enrichment are therefore deemed necessary in devel-
opment of the sulfur conversion biotechnologies.
4. Sulfur conversion biotechnologies
SRB can grow in a wide range of environments spanning a
temperature spectrum of at least 0100

C, salinity from
freshwater tohalitesaturatedsolutionsandapHrangeof 3e9.8
(Mackenzie, 2005). SRB even could be found in aerobic habitats
(Wieringa et al., 2000), despite their obligatory anaerobic
metabolism. This provides a wide range of opportunities to
developSRB-basedor relatedtreatment technologies, inwhich
anaerobic sulfate reduction has been recognized as the key
step in all sulfur related waste treatment processes (Huisman
et al., 2006). Sulde, generated from organic degradation by
sulfate reduction, can serve as an electron donor for nitrogen
removal via autotrophic denitrication or as an agent for sub-
sequent precipitation of heavy metals (Kieu et al., 2011). In
more cases, sulde is oxidized chemically/biologically to
elemental sulfur and recovered, which can be used as raw
material for sulfuric acid production or a substrate for the
bioleaching of metal-polluted soils and sediments (Vallero,
2003). But some issues accompanied with elemental sulfur
production such as local corrosion, clogging of pipelines and
valves etc. (Fang et al., 2008) still needed to be addressed.
The major sulfur conversion biotechnologies developed
over the past decades for industrial and municipal wastewater
treatment are reviewed and summarized in the present work
Table 5. The corresponding process schematics/principles are
shown in the supplementary information. To date, most sul-
fur conversion wastewater treatment biotechnologies are
applied in the industrial eld. The two most signicant factors
for this are 1) sulfuric acid is one of the world's largest in-
dustrial chemicals in terms of volume and its extensive utili-
zation in many industrial processes, 2) mining and metallurgy
or application of sulfur containing minerals all result in
sulfate-laden wastewaters. Some principles of these industry
wastewater-oriented processes have also been applied in do-
mestic wastewater treatment applications, for instance the
in-sewer sulde control trials via chemical inhibition (e.g.
ferrous salt, nitrite), electrochemical oxidation (Pikaar et al.,
2011; Mohanakrishnan et al., 2008) and biological denitrify-
ing sulde removal (DSR) process (Jiang et al., 2009, 2013a)
have been explored in municipal wastewater treatment. The
DSR process can be combined with urine source separation,
nitrication and subsequent sewer discharge (Jiang et al.,
2011), providing benecial use of the sewer as a bioreactor to
control sulde odor and reduce carbon demand at down-
stream treatment plants.
More recently in the past 5 years two new systems and a
third proposed system with signicant potential in the
wastewater eld have been developed. The two developed
systems are both designed around municipal sewage treat-
ment using sulfur conversion bioprocesses. These are 1) the
sulfate reduction, autotrophic denitrication and nitrication
integrated (SANI

) process (Wang et al., 2009; Lu et al., 2012)

and 2) the denitrifying sulfur cycle associated enhanced bio-
logical phosphorus removal (DS-EPBR) process. The third
proposed system is based on the analysis of an anaerobic
uidized-bed reactor (Frigaard and Dahl, 2008). These three
systems will be described in the following text.
The SANI

system has been developed for treatment of S-

laden saline sewage resulting from the city scale practice of
seawater toilet ushing in Hong Kong (van Loosdrecht et al.,
2012). The SANI

process makes use of sulfur as an electron

carrier for pollutants removal, as shown in Fig. 2.
The SANI

process comprises three biological reactors

(Fig. 2). Firstly, organic carbon is oxidized to carbon dioxide
through SRB mediated sulfate reduction to sulde. This pro-
cess is accompanied by pHincrease. Within the reactor sulde
is completely ionized to S
2
with its toxicity greatly reduced
due to the pH. In the second reactor, nitrate is reduced to ni-
trogen while the sulde is converted back to sulfate through
autotrophic denitrication. Finally, ammonia in the waste-
water is oxidized to nitrate through autotrophic nitrication
in the third aerobic reactor. As these three key biological
chemical processes all produce minimal sludge, as shown in
Equations (1)e(3) (Lu et al., 2012), the SANI

process can
reduce 90% sludge production, 35% energy consumption and
36% greenhouse gas emission as compared to conventional
biological treatment processes (Lu et al., 2012) when treating
municipal sewage.
Sulfate reduction
127:8gCOD192gSO
2
4
55:8gH
2
O/68H
2
S
2:4gSludge 244gHCO

3
(1)
Autotrophic denitrication
124gNO

3
7:32gHCO

3
44:54gH
2
S/28gN
2
125:76gSO
2
4
2:66gSludge
(2)
Nitrication
18gNH

4
1:32gCO
2
62:4gO
2
/0:94gSludge
62gNO

3
2gH

17:64gH
2
O
(3)
Although the SANI

process was originally developed for

treating Hong Kong's saline sewage arising from seawater
Table 4 e Growth kinetic parameters of complete/
incomplete organics oxidization SRB from the developed
model.
Parameter Annotation Value
m
maxSRBc
maximum specic complete
SRB growth rate, d
1
2.5 10
2
m
maxSRBi
maximum specic incomplete
SRB growth rate, d
1
1.9 10
2
K
SRBc-C
Monod half-velocity constant
to complete SRB using organics,
g l
1
6.1 10
1
K
SRBc-S
Monod half-velocity constant
to complete SRB using sulfate, g l
1
5.1 10
1
K
SRBi-C
Monod half-velocity constant
to incomplete SRB using organics,
g l
1
2.6 10
3
K
SRBi-S
Monod half-velocity constant
to incomplete SRB using sulfate,
g l
1
9.1 10
2
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 9
Table 5 e Major S conversion biotechnologies and their apply targets, operation conditions and benets and drawbacks.
Process Applied targets Operation conditions S conversion
efciency & SLR
Pro (), con () Reference
Passive treatment
permeable reactive barriers
(PRB)/Inltration beds
(SI-Figure 1)
AMD, AMD contaminated
groundwater
pH: 36; T: ambient
CS: complex organics (e.g. sawdust,
wood chips)/mixture organic matters
HRT: N/A
SO
2
4
/HS
-
(Around 30%e50%)
Low capital investment
Little operation cost
Demanding large space
Metal cannot be recovered
Extra carbon source is required
Lens et al., 2003; Gazea
et al., 1996; Younger et al.,
2003; Benner et al., 2002
THIOTEQ
(SI-Figure 2)
AMD, groundwater
contaminated with
heavy metals and
metal mining,
metallurgical industry
wastewaters
1.Sulfate reduction:
pH: neutral; T: ambient
CS/ED: Acetate ethanol/hydrogen
HRT: N/A
Reactor: Gas lift
2. Metal recovery
pH: 010; T:080

C
DMC: 50e5000
Typical sulde generation:
10020,000 kg/d
Recover valuable metals such as
copper, nickel etc. separately
In-situ sulfate reduction eliminates
the cost and avoids safety issues arising
from transportation of NaHS/H
2
S
Lower metal efuent concentrations
Extra carbon source/electron donor
is required
Sulfate in the wastewaters is
not removed
Muyzer and Stams, 2008;
Huisman et al., 2006
Biotechnological ue-gas
desulfurization (Bio-FGD)
(SI-Figure 3)
Alternative for
conventional
physicalechemical
processes for
the removal
of sulfur dioxide
from ue-gases
pH: ~7.5; T: Thermophilic
(5470

C)
CS: methanol
HRT: 34 h
Reactor: UASB
SO
2
3
/HS

(100%)
SO
2
4
/HS

(50%)
HS

/S
0
(95%)
SLR: 3.7e11.2
Metals in the ue gas such as Ni and V
could be removed by means of
precipitation
Elemental sulfur recovery
Extra carbon source is required
Efuent of Bio-FGD has pH of 8.5e9
and temperature of 35e55

C
Muyzer and Stams,
2008; Vallero, 2003;
Weijma, 2000
Two-phase anaerobic
digestion process
(SI-Figure 4)
Wastewaters contain
sulfate and high
concentrations of
organic matter such
as molasses, seafood,
edible oil, starch,
pulp and paper
etc industries
pH: 47; T: Mesophilic to
thermophilic (55

C)
COD/SO
2
4
: 9e3.5
HRT: 6 10 h
Reactor: UASB
SO
2
4
/H
2
S
Normally can achieve
100% with sufcient
carbon source/Dependents
on organic loading rate,
pH, temperature etc.
SLR: 0.13e0.33
Removing sulde before
methanogenic reactor
Methane gas collection
Separate the SRB and methanogensis
reducing their competition
Relive hydrogen sulde inhibition for
methanogensis
Acidication may cause extensive low
pH efuent
Odor and energy consumption of
H
2
S stripping
Require subsequent H
2
S treatment
Reis et al., 1988; Lopes et al.,
2007a; Wei et al., 2006
Denitrifying sulde
removal (DSR) process
(SI-Figure 5)
Gaseous or liquids
wastes that are
contaminated with sulfate/
sulde and nitrite/nitrate
such as renery, oil
industry, petrochemical
wastewaters;
Post treatment of efuents
from anaerobic reactors
pH: 7.3e8.3: T: 2030
HRT: 10.7e48 h
CS: Acetate
Reactor: CSTR/EGSB
HS

/S
0
(>95%)
HS

/SO
2
4
(
N/A)
SLR: 0.5e6.0
Removal sulfur (e.g. elemental sulfur,
sulde and thiosulfate) nitrate/nitrite
and carbon simultaneously
Elemental sulfur recovery
High sulfate production, low nitrate
removal ratio
Alkalinity consumption
Show et al., 2013;
Batcheler and Lawrence,
1978; Manconi et al., 2007;
Kleerebezem and
Mendez, 2002
CSTR: Continuous stirred-tank reactor; UASB: upow anaerobic sludge blanket; EGSB: expanded granular sludge bed reactor; N/A: not available; SLR: sulfur loading rate (kg m
3
d
1
); DMC: Dissolved
metal concentration (mg/L); CS: carbon source; ED: electron donor; T: temperature.
w
a
t
e
r
r
e
s
e
a
r
c
h
6
5
(
2
0
1
4
)
1
e
2
1
1
0
toilet ushing, it can be conveniently adopted for treating
industry wastewater by addition of sulfate, seawater or some
sulfate-laden waste streams. For instance, integrating the
treatment of ue gas desulfurization (FGD) efuent with
municipal wastewater treatment, the FGD-SANI process can
provide an additional option to co-treat industry wastewater
with mainstream sewage treatment (Jiang et al., 2013b).
However, the SANI system has some potential limitations for
application. As mentioned above, the availability of a suitable
sulfur source is vital. Furthermore, due to the low energy re-
actions and slow growing biomass fast startup is a challenge,
which deserves a thorough study. Additionally, making the
SANI process more compact by integrating either the anaer-
obic and anoxic unit or anoxic and aerobic units via granular
sludge would be desirable.
The second more recent development resulting from the
SANI process researchhas beenthe sequencingbatchoperation
DS-EPBR process, which integrates biological phosphate
removal into the previous SANI process. Wu et al. (2013) pro-
posed the possible mechanism for the observed P uptake and
release using brackish municipal wastewater: carbon (PHA:
Polyhydroxyalkanoate, an energy source for metabolism) stor-
age and sulfate reduction occur simultaneously with P release
in the anaerobic phase; subsequently the stored PHA and poly-
S/S
0
areoxidizedwhilebulkliquidPuptakeoccurstogether with
the sulfate increase (Wu et al., 2013). Current limitation of the
DS-EPBR process is long cycle times of around 48 h under
microaerophilic conditions. Although cycle length has been
improved to 12 h using denitrication of nitrate rather than
oxygen under the P-uptake phase (Wu et al., 2014), further
efciency improvement is deemed necessary for application.
Meanwhile, the microbial mechanism of sulfur related phos-
phate uptake and release is not fully understand to date with
further studies of the microbial bioprocesses needed. The
overall DS-EPBRprocess is described inFig. 3 (Wuet al., 2013).
Apart from the SANI

and DS-EPBR processes, a sulfate

reduction deammonication process has been proposed or
reported in a number of studies (Fdz Polanco et al., 2001; Zhao
et al., 2006; Liu et al., 2008; Schrum et al., 2009; Zhang et al.,
2009; Yang et al., 2009; Cai et al., 2010) according to the
following thermodynamically possible reactions (Fdz Polanco
et al., 2001).
3SO
2
4
4NH

4
/3S
2
4NO

2
4H
2
O8H

(4)
3S
2
2NO

2
8H

/3S
0
N
2
4H
2
O (5)
NO

2
NH

4
/N
2
2H
2
O (6)
Overall
SO
2
4
2NH

4
/S
0
N
2
4H
2
O DG
0
47:8 kj=mole (7)
Evidence for this processes exists from monitoring of
treatment systems (Fdz Polanco et al., 2001b; Zhao et al., 2006;
Yang et al., 2009) and marine sediment zones (Schrum et al.,
2009) where both chemical proles and dynamic thermody-
namic equations were used. Isolated batch tests and enrich-
ments have also been conducted to conrm this biological
pathway (Liu et al., 2008; Yang et al., 2009; Cai et al., 2010). Liu
et al. (2008) found a signicant enrichment of Candidatus
Anammoxglobus sulfate as simultaneous ammonium and
sulfate removal steadied in their reactor, while Cai et al. (2010)
reported a Bacillus benzoevorans strain responsible for the
process by using serial dilution and subsequent cultivation in
their study. However, to date knowledge is limited regarding
the bacteria that can perform this reaction, and the mecha-
nisms behind their metabolism. Due to the lack of nitritation
required in the sulfate reduction deammonication process
energy and N
2
O emission reductions could be realized over
the conventional ANAMMOX process. It is therefore recom-
mended that this process is further studied.
5. Factors affecting the efciency of sulfate
reducing bioprocesses
In terms of engineering design and operations, enhancing and
optimizing bioreactor performance is a key target. Apart from
Fig. 2 e Schematic diagram of SANI

process.
Fig. 3 e The ow diagram of DS-EBPR Process.
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 11
Table 6 e Common factors affecting the performance of sulfate reducing reactors.
Factor Effect(s) condition(s) preferred Reference
Sludge type
(ocs/biolm/
aggregate)
Biomass concentration;
Reaction rates, local pH,
temperature, toxicity resistance etc
SRB granular sludge/biolm Hao et al., 2013a;
Tabak and Govind, 2003
Organism structure
SRB species Organics oxidation complete to
CO
2
or acetate (efuent COD
concentration);
Growth rate; aggregation
ability
Organics complete oxidizers
SRB species
Brenner et al., 2005;
Jhobalia et al., 2005;
Omil et al., 1997
Syntrophic structure Enhance sulfate reduction rate SRB collaborates with other organisms
to acquire electron or energy source
Hao et al., 2013b;
Mulopo et al., 2011
Competition model Compete with methanogens,
acidogenic/hydrogenotrophic
bacteria and acetoclastics for
the available substrates
SRB predominate the microbial
community with syntrophic bacteria
Muthumbi et al., 2001;
Koschorreck et al., 2004;
OReilly and Colleran, 2006
Inuent components
Sulfate concentration Affects SRB growth and activity;
May be out-competed at low
concentration;
High concentrations inhibit
SRB activity
Typical COD/SO
2
4
values range between
0.7 and 1.5 depending on the carbon source
Hao et al., 1996;
Rzeczycka et al., 2010;
Mohanty et al.,2000;
Raskin et al., 1996
Trace element Fe, Cu, Zn, Co, Mo, Ni are
needed in electron transport,
redox-active metalloenzymes and
composition of some protein and
enzymes;
High Mo level inhibits SRB
metabolism
High levels of Fe in culture media in order to
compensate for that precipitated by sulde;
a
Mo above 2 mM completely inhibits SRB
Bridge et al., 1999;
Postgate, 1984;
Biswas et al., 2009
Metal concentration Elevated heavy metal concentration
can reduce or terminate SRB activity
Desired concentration and the order of
decreasing toxicity. (mg/L) Cu < 4, Cd < 11,
Ni < 13, Zn < 16.5, Cr < 35, Pb < 80
Kaksonen and
Puhakka, 2007;
Utgikar et al., 2001;
Naz et al., 2005
Nitrate concentration Nitrite is a strong inhibitor in growth
and activity of SRB
The impact level:
70 mM NO

3
inhibits growth signicantly;
Long term 0.25e0.33 mM injection inhibits
the number and activity
Zhang et al., 2008;
He et al., 2010;
Bdtker et al., 2008
pH Effect the growth and activity;
Inuence the SRB species diversity
and out-compete with methanogens;
Effects dissolved sulde quantity
pH range for SRB:5.5e10 Aerts, 2009; EPA, 1974;
Gormly, 2005
Salinity Inuence the species of SRB present;
Generally, sulfate reducing rate is
inversely correlated with salinity
Optimum salinity range 6e12%. Kerkar and Loka
Bharathi, 2007;
Sorensen et al., 2004
Operation Conditions
Substrate/Sulfate Effect growth and activity and
microbial diversity; Proper C/S ration
favors SRB out-compete with other
organisms
Optimal COD/SO
2
4
ratio for COD removal is
0.6e1.2; for sulfate removal is 2.4e4.8
Rzeczycka et al., 2010
Oxidation reduction
potential (ORP)
Effect the competition between
SRB and other organisms i.e.
mathanogens;
Effect the performance of SRB
Suitable ORP for SRB is 50 to 300 mV;
Optimal ORP readings of 270 mV using
standard hydrogen probe.
Gerhardt et al., 1994;
Khanal and Huang, 2006;
Huan et al., 2013.
Temperature Control the activity and growth;
Initial cultivation temperature
effects SRB diversity;
Lower H
2
S solubility at high
temperature
SRB tolerate temperatures
between 5 and 75

C
Optimum temperature for most
SRB ranges 28e32

C
Nevatalo et al., 2010;
Mara and Horan, 2003
Sludge retention
time (SRT)
Effect the reactor's performance
and sludge production;
Effect the competition between
SRB and methanogens/homoacetogenic
bacteria
Elevated SRTs delay the outcompetition
of methanogens, and methanogens could
be rapidly removed by applying a low SRT
Esposito et al., 2003;
Weijma et al., 2002
Hydraulic retention
time (HRT)
Inuence SRB activity; Biomass
concentration; Competition with
other organisms
Overall optimum HRT of 20e30 h for
SRB activity
Sipma et al., 2007;
Polo et al., 2006
wa t e r r e s e a r c h 6 5 ( 2 0 1 4 ) 1 e2 1 12
substrates (carbon and sulfur) and microbial community as
discussed previously, other factors that could affect the ef-
ciency of the bioreactor operation deserve attention as well.
One such example is during the SANI biological phosphorus
removal exploration (Wu et al., 2013) where, despite the
conrmation of a sulfur related phosphate release and uptake
cycle, this process development was limited by the long
operation time required (48 h). Table 6 summarizes all the
main inuencing factors on sulfate reduction bioreactors.
Among the above factors the sensitivity of most known
SRB to even mild acidity (pH < 5) (Kaksonen and Puhakka,
2007) and their low growth rate constrain the design and
application of sulfate-reduction based systems. While eleva-
tion of the pH through lime addition or use of sidestream SRB
reactors to avoid direct contact of SRB with acidic wastewater
are commonly adopted for solutions in low pH applications,
little literature on low pH SRB reactor operation is available
with the exception of a lab-scale (pH: 3e4) suldogenic system
with glycerol, acetic acid and hydrogen as the energy sources
reported by Johnson et al. (2006). Information about scale-up
issues, substrate limitation, temperature and seeding sludge
effects in the low pH suldogenic systems is lacking. Sys-
tematic exploration of SRB bioreactor cultivation, develop-
ment and operation of such bioreactors at low pH (pH < 5) are
necessary.
Granular sludge provides a solution for slowly growing
anaerobic biomass (Lettinga et al., 1980). Anaerobic sludge
granulation processes such as upow anaerobic sludge
blanket (UASB)/expanded granular sludge bed (EGSB) pro-
vide more effective retention of bacteria than a occulent
sludge process and also provide layered microenvironments
and niches. Therefore, self-immobilized SRB granules would
enhance SRB system efciency as it offers increased
biomass concentrations, reducing reactor volume, and in-
creases reactor resilience against uctuations in pH,
temperature and etc. Treatment of AMD and extremely sa-
line and unfavorable-temperature wastewaters has already
been explored by adopting SRB colonized methanogenesis
granular sludge to enhance sulfate reduction activity (La
et al., 2003; Vallero, 2003). Omil et al. (1996) were the rst
to use an enriched SRB culture in developing SRB granular
sludge while SRB granular sludge has also recently been
developed with anaerobic digestion sludge as seed (Hao
et al., 2013a,b). Nevertheless, mechanisms of the SRB
sludge granulation and the reactor scale-up issues demand
extensive studies.
6. Summary and perspectives
The past several decades' research and development drawed a
widely accepted conclusion that no practical methods exist to
prevent sulfate reduction (Lens et al., 1998), despite various
lab-scale trials with selective inhibition of SRB by molybdate,
transition elements, or antibiotics. Instead, enhancing and
engineering SRB (Table 6) for beneciary S bioconversion ap-
plications may present an energy-efcient opportunity for
upgrade of current industrial and municipal wastewater
treatment technologies. For instance, sulfur sources caneasily
be incorporated into municipal wastewater systems through
seawater toilet ushing or directly introducing highly sulfate-
laden waste streams such as desalination brine at the
wastewater treatment plant. Recent developments in SANI,
DS-EBPR and sulfate reduction deammonication processes
have opened up potential opportunities to apply S biocon-
version systems for simultaneous removal of carbon and nu-
trients in wastewater treatment processes while achieving
minimal biological sludge production and greenhouse gas
emissions. These advantages are not possible using conven-
tional carbon cycle-based conventional treatment
technologies.
Acknowledgments
This study was partly supported by the Natural Science
Foundation of China (No. 51278501 and 51178194), the
Fundamental Research Funds for the Central Universities (No.
13lGPY59) and the Specialized Research Fund for the Doctoral
Program of Higher Education of China (No. 20120171120021).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.06.043.
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Table 6 e (continued)
Factor Effect(s) condition(s) preferred Reference
H
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S concentration High H
2
S direct and reversible
toxicity effect on SRB, and inhibit
the activity
Nitrogen purging;
a
Decrease the activity when H
2
S is higher
than 60e70 mg/l
Reis et al., 1992;
Jin, 2010;
Kaksonen et al., 2004a
Mixing condition Mixing frequency signicantly
impacts SRB activity;
Effect the SRB distribution and
detachment and hydraulic loss
of biomass
y Gantzera and
Stefanb, 2003
a
Negative impact limitation.
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