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Ecological Engineering 52 (2013) 278–289

Contents lists available at SciVerse ScienceDirect

Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Review

Sulphur transformations in constructed wetlands for wastewater treatment:

A review
Shubiao Wu a,∗ , Peter Kuschk b , Arndt Wiessner b , Jochen Müller b , Rania A.B. Saad b , Renjie Dong a
a
College of Engineering, China Agricultural University, 100083 Beijing, PR China
b
Department of Environmental Biotechnology, Helmholtz Centre for Environmental Research – UFZ, Permoserstrasse 15, Leipzig D-04318, Germany

a r t i c l e i n f o a b s t r a c t

Article history: The sulphur-cycle processes inside constructed wetlands (CWs) and their role in the complex network of
Received 27 February 2012 transformation processes and the long-term stability of wetland systems are still not sufficiently under-
Received in revised form 28 July 2012 stood. A comprehensive review is accordingly necessary for better understanding to optimize the future
Accepted 13 November 2012
design and operation of wetland systems. In this paper, the concept of sulphur cycling in CWs in prin-
Available online 7 December 2012
ciple, transformations dynamics of sulphur compounds and their interactions with other element cycles
(such as carbon, nitrogen and phosphorus) and plants, as well as importance for treatment efficiencies are
Keywords:
reviewed. The abiotic and biotic transformations of sulphur are both spatially and temporally dynamic in
Constructed wetland
Dissimilatory sulphate reduction
CWs. Next to physico-chemical processes, e.g. mineral precipitation and dissolution, biologically catalyzed
Sulphide toxicity redox reactions such as assimilatory and dissimilatory sulphate reduction, as well as oxidation/reduction,
Nitrification and disproportionation of more reduced sulphur compounds also simultaneously occur in the rooted
Wetland plants zones of CWs. Moreover, the interactions between sulphur transformations and other processes includ-
ing microbial transformations of carbon, nitrogen and phosphorus, as well as the impact of plants are
discussed. Briefly, sulphide as a product of dissimilatory sulphate reduction may remove heavy metals
from wastewaters via the precipitation of metal sulphide and the use of CWs in treating heavy metal
containing waters (e.g. acid mine drainage) is increasing. However, high sulphide concentrations may
also negatively affect growth of wetland plants and microbial activities, especially nitrification, but also
influence the mobilization of iron-bound phosphorus. This information may be used to offer future design
and operational methodologies that might enhance the performance of contaminants removal and drive
a sustainable management of CWs for wastewater treatment.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction
Constructed wetlands (CWs) are engineered extensive systems
designed to remove pollutants from contaminated waters. Use of
these systems for wastewater treatment over the last recent years
has increased in the world, especially in the areas without pub-
lic sewage systems and lower economic development (Brix, 1999;
Gopal, 1999; Vymazal, 2005). Due to their low maintenance and
operational cost requirements together with high removal capacity
for both point and non-point sources pollution, the application of
CWs has significantly expanded from traditional tertiary and sec-
ondary domestic sewage treatment (Kivaisi, 2001; Werker et al.,
2002) to the treatment of agricultural effluents (Zhao et al., 2004;
Wood et al., 2007), industrial effluents (Mbuligwe, 2005), landfill
leachate (Justin and Zupancic, 2009), as well as urban and highway
run off (Scholes et al., 1999).
∗ Corresponding author. Tel.: +86 10 62737852; fax: +86 10 62737885.
E-mail address: wushubiao@gmail.com (S. Wu).
Inside the wetland bed, macro and micro gradients of redox
conditions enable the development of highly diverse micro-
bial community capable of different beneficial redox reactions
(Bezbaruah and Zhang, 2004; Wiessner et al., 2005). Particularly,
the rhizosphere is of high importance for contaminants removal
due to the release of oxygen and organic carbon from the plant
roots into the surrounding environment (Wei et al., 2003; Wiessner
et al., 2005, 2008). In the rhizosphere various microbiological trans-
formations, such as mineralization of organic carbon, nitrification,
denitrification, sulphate reduction and re-oxidation of reduced sul-
phur compounds occur simultaneously on a small spatial scale
(Liesack et al., 2000; Bezbaruah and Zhang, 2004; Wiessner et al.,
2005). This combination and proximity of different redox processes
are of importance for the overall removal effectiveness and are
amongst the key advantages of wastewater treatment in wetlands,
as compared to other technologies.
Currently, the effectiveness of CWs for wastewater treatment
is mainly determined by treatment performance for organic car-
bon, nitrogen, phosphorus, and suspended solids (Vymazal, 2007;
Kadlec and Wallace, 2009). However, as sulphate is a common

0925-8574/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ecoleng.2012.11.003
 Author's personal copy
S. Wu et al. / Ecological Engineering 52 (2013) 278–289
Table 1
Examples for sulphate concentrations in various wastewaters.
Type of wastewater Concentrations (mg/L)
Mining water 3110
1336
Ground water 113–2666
17–402
Food processing 35–83
2500–4300
Industry 298–2322
12,000–35,000
Landfill 80
225
Runoff 722
42–130
constituent of many wastewaters, sulphur transformations such as
microbial sulphate reduction and re-oxidation of reduced sulphur
compounds, e.g. sulphide, may have a significant impact on the
efficiency of CWs (Stein et al., 2007; Wiessner et al., 2008). In addi-
tion to input via the influent, various sulphur compounds are also
present naturally in CWs (Winter and Kickuth, 1989; Kalin, 2004;
Vymazal, 2007; Kadlec and Wallace, 2009; Sturman et al., 2008),
where they may impact C, N, and P transformations. The fate of sul-
phate, sulphide, and other sulphur compounds is largely governed
by the redox state dynamics in the root zone, with different pro-
cesses of sulphur cycling occurring depending on the availability of
organic carbon and oxygen (Sturman et al., 2008; Wiessner et al.,
2010). However, the knowledge of the dynamics of those sulphur
transformations in CWs has not been comprehensively considered
to date (Vymazal, 2007; Wiessner et al., 2010).
Sulphur transformations do not occur isolated from other
microbial transformations, but are interconnected through their
common substrates, products, and intermediates. Sulphate reduc-
tion in wetlands can result in Eh and pH changes, C-transformation
and, indirectly, the mobilization of nutrients (Feng and Hsieh, 1998;
Lamers et al., 1998)—all these processes are of importance for C,
N, P-removal. Sulphide as a product of sulphate reduction can be
highly toxic to microorganisms and macrophytes, and is a competi-
tor for the consumption of oxygen as well (Armstrong et al., 1996;
Chambers et al., 1998; Lee et al., 1999; Koch and Erskine, 2001;
Pedersen et al., 2004). Accordingly, the transformations of carbon,
nitrogen and phosphorus in CWs are closely tied to the sulphur
cycling and various sulphur transformations in wetland systems
also has to be considered as a factor that may control performance.
However, the sulphur-cycle processes inside the rhizosphere and
their role in the complex network of transformation processes are
still not sufficiently understood, and a comprehensive review is
accordingly necessary for better understanding of the “black box”
rhizosphere and for optimum design and operation of wetland sys-
tems.
In this paper, we summarized the present knowledge on sulphur
cycling in CWs. Additionally, interactions of sulphur transforma-
tions with other processes, such as turnover of C, N, P, heavy metals,
and their possible inhibitory effects on plants and microorgan-
isms are also reviewed. Finally, some future research needs are
discussed.
2. The concept of sulphur cycling
The valence states in which sulphur can mainly occur in CWs
are −2 in H2 S, 0 in S0 , +2 in S2 O3 2− (thiosulphate) and +6 in SO4 2− .
Depending on the sulphur species they can function as electron
donors or electron acceptors in various dissimilatory microbial
reactions (Sturman et al., 2008). The sulphur in the influent to
CWs is typically in the form of sulphate in oxidized environments
279
Comments References
Whittle, United Kingdom Batty et al. (2005)
Kristineberg, Sweden Nyquist and Greger (2009)
West Delhi, India Adhikary et al. (2010)
Erode City, Tamilnadu, India Palanisamy and Kavitha (2010)
Winery production Grismer et al. (2003)
Citric acid production O’Flaherty et al. (1998)
Metallurgic production Hadad et al. (2006)
Organic peroxide production Silva et al. (2002)
Isanti-Chisago, Minnesota Kadlec (2003)
Tre Monti, Northern Italy Frascari et al. (2004)
Agricultural runoff, California Kadlec et al. (2010)
Road runoff, North of London Sriyaraj and Shutes (2001)
and sulphide in reduced environments such as anaerobic efflu-
ents. Other sulphur compounds representing intermediate valence
states, such as thiosulphate, polythionates, elemental sulphur, sul-
phite, and organic sulphur also occur in CWs, but mostly are of
subordinated importance. Sulphate is a common constituent of
many natural waters and wastewaters as shown in Table 1. Sul-
phate presence is not problematic in CWs since it is non-toxic.
However, high sulphate concentrations can unbalance the sul-
phur cycle and further lead to a sulphur pollution (Lens et al.,
1998). Although sulphur pollution in CWs has not received much
attention, it indeed can lead to severe deterioration (Holmer and
Storkholm, 2001) and poses a serious threat (see detail in Sec-
tion 4) to wetland ecosystem structure and functioning (Lamers
et al., 2002; Van der Welle et al., 2008). As shown the sulphate
concentrations in Table 1 from various origins, the main sources
being wastewaters from anthropogenic activities including efflu-
ents from mining of metalliferrous sulphide deposits, landfills, food
processing, industrial production, agricultural and road runoff as
well as groundwater. In general, the sulphate concentration in
domestic wastewater may vary in a range of 20–500 mg/L, while
industrial effluents may contain several thousands of milligrams
per liter (Lens et al., 1998).
In addition to the abovementioned sources of sulphur pollution,
there is a further possible source. When oxic and/or nitrate-
polluted groundwater passes through soil layers that are rich in iron
sulphides, oxygen and/or nitrate can be used in the chemolithoau-
totrophic oxidation of iron sulphides (Brunet and Garcia-Gil, 1996;
Molenat et al., 2002; Haaijer et al., 2006). This may lead to the
immobilization of sulphur along the hydrological pathway and
make a high sulphate concentration in the downstream (Van der
Welle et al., 2008). The input of sulphate by air pollution is quite low
and only of importance in natural wetlands (Vile et al., 2003). The
application of CWs for sulphide-containing wastewater treatment
is rarely reported except for some laboratory-scale experiments fed
with artificial sulphide water (Gonzalias et al., 2007).
The main biotic and abiotic sulphur transformations in CWs are
shown in Fig. 1, including physico-chemical processes, e.g. mineral
precipitation and dissolution, biologically catalyzed redox reac-
tions such as assimilatory and dissimilatory sulphate reduction,
as well as oxidation/reduction, and disproportionation of more
reduced sulphur compounds. The most important biotic reactions
influencing sulphur cycling in CWs might be those catalyzed by
dissimilatory sulphate-reducing bacteria (SRB), which reduce sul-
phate to sulphide using the energy generated from the transfer
of electrons from organic substrates (Sturman et al., 2008). The
produced sulphide can be transported by diffusion and water flow
from anoxic to more aerobic zones and then be re-oxidized to ele-
mental sulphur and/or sulphate through either abiotic processes or
via reactions catalyzed by sulphur-oxidizing bacteria. This process
could be facilitated by wetland plants due to the release of oxygen
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280 S. Wu et al. / Ecological Engineering 52 (2013) 278–289
Fig. 1. Main known sulphur transformation processes in surface-flow constructed wetlands.
from their roots (Wiessner et al., 2010). Under anoxic conditions in
CWs, sulphide can precipitate with heavy metals to form insoluble
metal sulphides, such as iron sulphide (FeS), to be immobilized in
wetlands soil matrix. Additionally, regarding that the form of sul-
phide in the wetland pore water can vary from S2− to HS− and
H2 S depending on the pH, a propensity to offgas of H2 S would
occur in low pH systems with accompanying rotten-egg odor. Ele-
mental sulphur (S0 ) was often observed in CWs (Sturman et al.,
2008), which is an intermediate product of sulphide re-oxidation.
It can be firstly stored in the wetland soil matrix, and further either
to be oxidized to sulphate or disproportionated to sulphate and
sulphide, depending on the environmental redox conditions and
interplay between various bacterial catalyzed reactions. In high-
redox environments such as CWs with aeration and tidal operation,
or environments with relatively low levels of available organic
carbon like treating acid mine drainage or groundwater, dissimila-
tory sulphate reduction would be limited. Under these conditions,
sulphate concentrations can be lowered mainly through abiotic
mineral precipitation (e.g. gypsum) or biological assimilation into
plant or microbial biomass as organic sulphur. The fate of different
sulphur species in CWs, such as sulphate, sulphide and elemental
sulphur, will be discussed in detail below.
3. Sulphur transformations in CWs
3.1. The fate of sulphate in CWs
3.1.1. Dissimilatory sulphate reduction
Sulphate from natural or anthropogenic origin is component of
nearly all water as well as most of the waste waters and microbial
dissimilatory sulphate reduction is highly important transforma-
tion within CWs. A variety of bacterially catalyzed redox reactions
can impact the fate of sulphur in CWs. The process of dissimilatory
sulphate reduction which is catalyzed by SRB in anaerobic zones
of CWs might be the most familiar one. By this process, energy is
gained by coupling the oxidation of organic compounds to sulphate
reduction and liberation of carbon dioxide and sulphide. As illus-
trated in the following stoichiometry with methanol as the electron
donor, SRB can utilize one mole of sulphate to generate one mole of
sulphide and also result in the generation of alkalinity which may
raise the pH of CWs with acidic conditions.
SO4 2− + 2CH2 O + 2H+ → H2 S + 2H2 O + 2CO2
Historically, SRB have been considered as strict anaerobes and
sensitive to low temperatures. However, the tolerance of low
concentrations of oxygen by some SRB species which have been dis-
covered to persist in oxic conditions and survive extended periods
of oxygen exposure has been shown in recent research (Brune et al.,
2000; Vasconcelos and McKenzie, 2000; Holmer and Storkholm,
2001). Sagemann et al. (1998) demonstrated that sulphate reduc-
tion is more dependent on substrate concentration and supply than
on temperature alone, since some SRB was shown to grow well
at low temperatures (−1.7 to 2.6 ◦ C). Moreover, Tsukamoto et al.
(2004) demonstrated in laboratory bioreactors treating acid min-
ing drainage that low temperatures can reduce the establishment
of SRB, but do not decline the already established communities
at room temperature. A few studies have demonstrated better
sulphate removal in winter and higher redox conditions than in
summer and lower redox conditions (Fortin et al., 2000), however,
results across most CWs seem reasonably conclusive that warmer
temperatures and lower redox potentials foster environmental
conditions ideal for a larger SRB population and therefore enhance
sulphate removal (Hsu and Maynard, 1999; Stein and Hook, 2005).
Stein et al. (2007) examined the effects of season, plant species and
organic loading on sulphate reduction in experimental subsurface
flow CWs fed in batch mode. Sulphate removal was found to be
lower in winter, higher in summer, and moderate in spring and fall.
Moreover, seasonal variations were more pronounced in planted
wetlands as compared to the unplanted control. The low removal
of sulphate in planted wetlands in winter has resulted from the
low temperature and the plant-mediated oxygen transfer which
increased the redox potential and inhibited the activity of SRB.
3.1.2. Abiotic mineral precipitation
In the absence of divalent cations, sulphate is highly soluble
in water. Sulphate concentrations can be lowered through abiotic
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S. Wu et al. / Ecological Engineering 52 (2013) 278–289
mineral precipitations (such as CaSO4 ) in CWs, particularly in some
special micro zones with deep sulphate concentration gradients.
The solubility of gypsum in the pore water of CWs is influenced
by the impurities in various wastewaters due to, for instance, non-
ideal solution behavior such as ion pairing and ion activity (Tait
et al., 2009). Taking an example, the solubility of gypsum in pure
water is approximately 2–2.5 g/L at 25 ◦ C (Bock, 1961), similar to
the solubility reported by Geldenhuys et al. (2003). In comparison,
in the experiments of Kabdasli et al. (1995) with a textile waste-
water, the solubility of calcium sulphate under this condition was
approximately 8 g/L. So, considering that the solubility of gypsum
varies depending on the characteristics of wastewaters, the pro-
portion of sulphate removal by mineral precipitation with metals
in CWs may also be varying.
In addition, the formation of iron(III) plaque on the root surface
of helophytes has been described by several authors as a result of
their oxygen input which oxidizes Fe2+ (Lucassen et al., 2000; Weiss
et al., 2005). Furthermore, it can also be assumed that other salts
like sulphates (calcium sulphate, iron hydroxy sulphate) precipi-
tate as a result of different root uptake rates of water and dissolved
ions which could cause steep micro-scale concentration gradients
nearby the root surface. This aspect could be a possible reason for
the decrease of the sulphate load in a laboratory-scale CW treat-
ing an artificial acid mine drainage under conditions unfavorable
for microbial dissimilatory sulphate reduction as low pH and high
redox potential in the pore water (Nguyen, 2011).
3.2. The fate of sulphide in wetlands
3.2.1. Emission of H2 S to the atmosphere
As an end product of dissimilatory sulphate reduction and pro-
tein degradation, hydrogen sulphide normally exists in the pore
water of wetlands in three forms as un-ionized (H2 S), singly (HS− )
and doubly ionized (S2− ), depending on the water temperature and
pH (Kadlec and Wallace, 2009). The dissociation reactions are:
S2− + 2H+  HS− + H  H2 S
At equilibrium, the unionized form is predominant at low pH,
and bisulphide is dominant at high pH in aqueous systems. In acidic
conditions like treating acid mine drainage in CWs with high con-
centration of sulphide and low pH in case of absence of heavy
metals, there might be a large fraction of unionized H2 S which is
volatile and could be lost to the atmosphere. The loss of H2 S to the
atmosphere represents a decrease in the acidity of the water and
an increase of pH (Machemer et al., 1993). Moreover, the emission
of hydrogen sulphide in the air gives an unpleasant odor and even
be toxic to human beings (Schiffman and Williams, 2005; Ahlborg,
1951). The emission of hydrogen sulphide could be of importance in
tidal operational wetlands, but its significance is almost unknown
and needs to be investigated.
3.2.2. Re-oxidation of sulphide
When the produced sulphide from anoxic zones of CWs is trans-
ported to the oxic zones with the presence of available electron
acceptors, it may be oxidized back to polysulphides, elemental sul-
phur, thiosulphate, tetrathionate or sulphate by either chemical or
biological pathways, or both (Wainwright, 1984; Sturman et al.,
2008). The exact mechanisms and magnitude of sulphur oxidation
within the sulphur cycle in CWs are not well understood (Holmer
and Storkholm, 2001). The known sulphide oxidation processes
may include chemical oxidation with oxygen, anoxic chemical oxi-
dation, and bacterial oxidation under oxic and anoxic conditions.
Elemental sulphur as a product of sulphide re-oxidation
in sulphur cycling was frequently found in CWs proving the
281
abovementioned oxidizing processes. Winter and Kickuth (1989)
reported that about 36% of the removed sulphur in a horizon-
tal subsurface flow CW treating textile industry wastewater was
stored in the form of elemental sulphur. Significant concentrations
of elemental sulphur were also measured in the pore water of lab-
oratory scale CWs treating artificial sewage and were supposed to
be facilitated by the released oxygen from the roots of helophytes
(Wiessner et al., 2008, 2010). The presence of elemental sulphur
is also a proof of simultaneous reduction and oxidation of sulphur
compounds in the micro-gradient zones of wetland beds (Wiessner
et al., 2010).
3.2.3. Precipitation of metal sulphides
Dissimilatory sulphate reduction, its inherent proton consump-
tion as well as the subsequent precipitation of metal sulphides
has significant potential for the bioremediation of heavy metal-
contaminated waters in CWs, particularly for the treatment of acid
mine drainage (Kosolapov et al., 2004). A large variety of heavy
metals such as iron, zinc, copper, nickel, cadmium, mercury and
lead can be precipitated as metal sulphides (Sobolewski, 1999;
Sheoran and Sheoran, 2006). In addition, some metalloids can also
be precipitated as their sulphides in CWs. For instance, a sulphate
reducing bacterium reduces As(V) and sulphate resulting in the
precipitation of As2 S3 (Newman et al., 1997).
In general, sulphate reducers may create extremely reducing
conditions in CWs which promote also abiotic metal reduction,
such as Fe3+ to Fe2+ (Lovley and Phillips, 1987). In addition, low-
ering the acidity of a system due to bacterial sulphate reduction
to sulphide can prompt further precipitation of metals as hydrox-
ides (e.g. Al(OH)3 , Cr(OH)3 ) and carbonates (e.g. ZnCO3 ) (White
and Gadd, 1997). The removal of metals as a result of precipitation
with sulphide is considered as one of the most important processes
involved in the long-term retention of metals in CWs. Because of
the formation of metal sulphides, they may stay in the sediments
of wetlands permanently as long as they remain anoxic (Kosolapov
et al., 2004). However, in some CWs treating high sulphate and low
heavy metals contaminated waters, such as domestic sewage, the
considerable sulphide formation can easily fix the limited heavy
metals and lead to a deficiency of trace metals for microorganisms
and plants.
Besides the removal of heavy metals from contaminated waters,
the precipitation of metal sulphides also attributes to the detoxifi-
cation of sulphide. The addition of iron (Van der Welle et al., 2007a,
2008) and use of an iron-rich soil matrix (Wu et al., 2012a) were
reported to control the dissolved sulphide concentration. However,
these measures should be used with caution, since a surplus of iron
may also have toxic effects on plants if it is in its reduced form of
Fe2+ (Van der Welle et al., 2007a, 2008).
3.2.4. Sulphur fixation in organic carbon
Several studies have suggested that H2 S, formed as an end
product of dissimilatory sulphate reduction can react rapidly with
organic matter, producing carbon bonded S (Casagrande et al.,
1979; Altschuler et al., 1983; Brown, 1985; Rudd et al., 1986).
Carbon-bonded S is generally produced by nearly all plants and
microbes by assimilatory sulphate reduction via sulphite to sul-
phide, and incorporating it into some amino acids and some other
biomolecules (Jorgensen, 1977; Zinder and Brock, 1978; Fitzgerald
et al., 1984). Brown (1985) demonstrated that although SO4 2− can
be incorporated into organic S via assimilatory uptake by plants
or microorganisms, organic S formed by assimilatory reduction is
apparently relatively minor compared to organic S formation via
dissimilatory reduction of sulphate to H2 S and then subsequently
bonded to organic matter. The C-bonded S can be degraded to
biogenic compounds such as H2 S, CH3 SH and (CH3 )2 S which can
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282 S. Wu et al. / Ecological Engineering 52 (2013) 278–289
40
25 cm from inlet zone
Sulphur (mg/L) 30
20
10
0
40
75 cm from inlet zone
30
Sulphur (mg/L)
20
10
0
120 160 200
Time (d)
Fig. 2. Concentrations of elemental sulphur and sulphate sulphur in the pore water along the flow-path of a horizontal sub-surface-flow laboratory-scale CW fed with
artificial wastewater under conditions of limitation of organic carbon.
Adapted from Wiessner et al. (2010).
escape from the soil or be oxidized back to SO4 2− . However, the
turnover of the organic sulphur pools is relatively slow, as com-
pared to the reduced inorganic sulphur pool (Wieder and Lang,
1988).
3.3. The fate of intermediately reduced sulphur compounds
Depending on available electron acceptors or electron donors,
the produced intermediate sulphur compounds in CWs including
elemental sulphur, thiosulphate, tetrathionate and sulphite can be
either oxidized or reduced via chemical and biological pathways.
Compounds like thiosulphate, tetrathionate and sulphite are not
stable and can be relatively easily oxidized or reduced to sulphate
or sulphide, whereas elemental sulphur often accumulates both in
the pore water and in the soil matrix. Elemental sulphur can be gen-
erated from sulphide by abiotic oxidation, by chemolithotrophic
microbes using oxygen or nitrate as electron acceptor, as well as
by anoxygenic phototrophic bacteria which may couple sulphide
oxidation to CO2 reduction in some micro zones of CWs (Kadlec and
Wallace, 2009; Sturman et al., 2008). In both cases, the elemental
sulphur may be further deposited in the soil matrix under condi-
tions of continuous sulphide supply, or further oxidized to sulphate
when sulphide is limited. Under anaerobic conditions, elemental
sulphur may be also reduced back to sulphide by sulphur reducing
bacteria (Fuseler et al., 1996; Madigan et al., 2000; García-de-la-
Fuente et al., 2011). In most horizontal subsurface flow CWs with
50 cm from inlet zone
100 cm from inlet zone
240 280 120 160 200 240 280
Time (d)
Sulphate sulphur
Elemental sulphur
continuous inflow of sulphate containing wastewaters, the deposi-
tion of elemental sulphur could be the major pathway for the loss
of total sulphur when no significant metal sulphide precipitation
takes place.
Wiessner et al. (2010) investigated the dynamics of sulphur
compounds inside planted (Juncus effusus) and unplanted hori-
zontal sub-surface-flow laboratory-scale CWs fed with artificial
wastewater. The results demonstrated the presence of elemental
sulphur as nearly 30% suspended in the pore water and however,
52% was immobilized in the soil matrix of the planted wetland.
Moreover, limitation of organic carbon causes continual decrease
of elemental sulphur and corresponding increase of sulphate in the
pore water along the flow-path to the outflow, as shown in Fig. 2.
Re-oxidation of dissolved elemental sulphur as well as parts of
immobilized reduced sulphur facilitated by oxygen released from
the plant roots was concluded. In contrast, surplus of organic car-
bon promoted the sulphate reduction and stability of deposed and
dissolved reduced sulphur compounds. The results demonstrate
the dynamics of sulphur redox processes within the CWs and indi-
cate possible correlations of sulphur and carbon cycles may result
in covered removal disturbances by, e.g. toxicity of intermediate
generated sulphide or competitions toward oxygen-supply.
In addition, bacterial disproportionation of sulphite, thiosul-
phate and elemental sulphur (as shown in the equation below), is
also an important process in the sulphur cycle of natural sediments
and wetlands, and leads to the formation of hydrogen sulphide and
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S. Wu et al. / Ecological Engineering 52 (2013) 278–289
Fig. 3. Schematic diagram of interactions of sulphur transformations with other
processes in constructed wetlands.
sulphate (Fuseler and Cypionka, 1995; Fuseler et al., 1996; Habicht
et al., 1998; Böttcher and Thamdrup, 2001).
4H2 O + 4S0 → 3H2 S + SO4 2− + 2H+
A common pathway of sulphide oxidation via elemental sul-
phur and coupled with sulphur disproportionation was shown to
be a pathway of complete oxidation to sulphate (Fuseler et al.,
1996). Wu et al. (2011) evaluated the sulphur transformations in a
pilot-scale CW treating a high sulphate-containing contaminated
groundwater using the stable isotope approach. The results indi-
cated the process of microbial sulphur disproportionation by a
considerable sulphur isotope fractionation and a significant iso-
topic difference between sulphate and sulphide.
4. Interactions of sulphur transformations with plants, C, N
and P in CWs
Considering the complexity of micro redox gradients inside CWs
developing highly diverse microbial consortia capable of differ-
ent beneficial microbial reactions, the sulphur transformations do
not generally occur isolated from other simultaneously occurring
microbial transformations, but are interconnected and interacted
through their substrates, products, and intermediates. As shown
briefly in Fig. 3, sulphur transformations in CWs are closely tied to
the dynamics of pH, wetland plants and the cycling of microbial C,
N and P transformations. Those interactions are discussed in detail
below.
4.1. Interactions with wetland plants
Sulphur is an essential element for the growth of wetland plants.
The functions of biological assimilation of sulphur into wetland
plants are closely related to those of nitrogen and the quanti-
ties required for plant growth is supposed to be equal to, and
sometimes exceeding those of phosphorus. In addition, it is also
essential in the formation of enzymes, vitamins such as biotin
and thiamine and a variety of other important compounds in the
wetland plants (Ceccotti, 1995). The sulphur content in wetland
plants is reported in a range of 0.1–0.6% of dry mass (0.35% of
dry mass in average) (Kadlec and Wallace, 2009). Moreover, the
sulphur content in belowground tissues of wetland plants is con-
siderably higher than aboveground plant part (Kadlec and Wallace,
2009). Based on a theoretical calculation using the parameters of
average sulphur content of wetland plants (0.35% of dry mass),
inflow sulphur concentration (about 20 mg/L), wetland load of 5
person equivalents/m2 d (approximately 100 L/person equivalent
in Europe) and a production of 3000 g/m2 year dry biomass of
Phragmites (Vymazal and Kropfelová, 2005), the percentage of sul-
phur removal by incorporation into wetland plant biomass is only
less than 0.3%.
283
Besides the assimilation of sulphur into wetland plants, the
interactions of sulphur cycling with wetland plants are also shown
as some negative effects from sulphide. Sulphide is a well-known
phytotoxin in wetlands, especially in sulphate-polluted wetlands
characterized by high sulphate reduction rates. High sulphide con-
centrations may lead to toxic effects to aquatic plants, such as
reduced growth (Koch and Mendelssohn, 1989; Koch et al., 1990;
Van der Welle et al., 2006), root decay (Armstrong et al., 1996;
Smolders and Roelofs, 1996), or even mortality (Smolders et al.,
1995; Lamers et al., 1998). Taking Phragmites australis as an exam-
ple of wetland plants, underground organs are sensitive toward
sulphide above 1 mM (Fuertig et al., 1996), and 1.4 mM sulphide
causes stunted adventitious roots and fine laterals, bud death, cal-
lus blockages of gas-pathways and vascular blockages (Armstrong
et al., 1996). 0.375 mM sulphide significantly lowered the ammo-
nium uptake (Chambers et al., 1998) and shoots were shorter in
marsh soil where sulphide is >0.4 mM, perhaps due to the sulphide
inhibition of nitrogen uptake for growth (Chambers, 1997). Fur-
thermore, inhibitory effects of elevated sulphide concentration on
the photosynthetic capacity of the leaves have been demonstrated
in several wetland plant species (Tretiach and Baruffo, 2001).
Wetland plants have evolved specialized adaptations to survive
in water-saturated anoxic sediments by detoxification of sulphide.
These plants have the ability to leak oxygen from their roots,
which oxidizes the sulphide in the rhizosphere and thus detoxi-
fies it (Armstrong, 1980; Mendelssohn et al., 1995; Sorrell, 1999).
However, when plants are growing in a permanently anaerobic
environment, this mechanism may not be powerful enough in case
the oxygen consumption around the roots is higher than what
the plant can supply to the apical parts, where growth and nutri-
ent uptake predominantly take place. Many plant species growing
in anaerobic environments have adaptations for oxygen loss by
their roots (Armstrong et al., 1996; Colmer et al., 1998; Connell
et al., 1999). Van der Welle et al. (2007a) describe how the differ-
ence in radial oxygen loss between Caltha palustris and J. effusus
results in large differences in sulphide concentrations in the pore
water, despite equal addition of sulphide and overall equal condi-
tions.
Iron addition can strongly reduce the concentration of dis-
solved sulphide through precipitation of iron sulphides. Van der
Welle et al. (2007a) showed clearly that the growth of C. palustris
was much higher when sulphide concentrations were decreased
as a result of iron addition. When there is little iron available,
increased sulphide concentrations will lead to phosphate mobiliza-
tion (Boström and Pettersson, 1982; Caraco et al., 1989; Smolders
et al., 1995), which may enhance vegetation development due to
better supply of nutrients (Gough et al., 2000; Güsewell et al., 2005).
However, experiments by Van der Welle et al. (2007a) show that
the effect of sulphide toxicity on C. palustris overrules any effects of
increased phosphate concentrations, as higher phosphate concen-
trations in the pore water along with high sulphide concentrations
did not result in increased growth of this species. In field situa-
tions, however, increased phosphate concentrations may favor the
growth of fast-growing species, like J. effusus (Lucassen, 2004; van
der Hoek et al., 2004).
Besides the influence of sulphur transformations on plants,
plants can also affect the biogeochemical dynamics of sulphur
in wetland sediments via evapotranspiration-induced advection,
which increases the loading of dissolved sulphate into the rhizo-
sphere (El-Shatnawi and Makhadmeh, 2001; Xu and Peter, 2006).
Furthermore, plants release organic carbon into the rhizosphere
via litter, root exudates and root turnover (Hale and Moore, 1980),
which subsequently drives dissimilatory sulphate reduction as the
organic carbon is degraded and sediments become more reduced
(Van Der Nat and Middelburg, 1998).
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284 S. Wu et al. / Ecological Engineering 52 (2013) 278–289
4.2. Interactions with microbial carbon transformations
Interest in sulphate reduction mainly in horizontal subsurface
flow CWs has grown in recent years because it was shown to con-
tribute significantly to the removal of organic matter (Garcia et al.,
2004, 2005; Aguirre et al., 2005). The relative contribution of sul-
phate reduction to dissolved organic carbon removal was examined
by Garcia et al. (2004) in different pilot-scale horizontal subsurface
flow CWs that were fed with the same urban wastewater with sul-
phate concentration of around 70 mg/L. The results demonstrated
that sulphate reduction caused 36–100% of the total removed dis-
solved organic carbon (measured as COD) in wetlands with a depth
of 0.5 m, while 17–40% in shallow wetlands with a depth of 0.27 m.
Moreover, varied sulphate concentrations along the length of hor-
izontal flow wetlands in the different periods and even in the
same bed, often resulted in variation of relative contribution of sul-
phate reduction to the removal of organic carbon depending on the
organic load and other environmental factors, such as temperature
and redox conditions (Garcia et al., 2005, 2010).
However, a negative correlation between BOD5 removal and
sulphate reduction was reported by Wiessner et al. (2005) in
laboratory-scale subsurface wetlands with complete mixing of the
pore water through permanent water recycling. Caselles-Osorio
and García (2007) compared the performance of COD removal in
two experimental horizontal subsurface flow wetlands with and
without sulphate load in the inflow and also found that the removal
of COD in the wetlands without sulphate present in the inflow was
higher.
Wu et al. (2012b) also examined the interactions between sul-
phur transformations and dissolved organic carbon removal in two
laboratory-scale horizontal subsurface flow constructed wetlands
fed with artificial sewage. In case of the inflow sulphate concen-
tration of 10 mg/L, the sulphide presented in the pore water was
in the range of 2–3.5 mg/L and the average removal of dissolved
organic carbon was achieved by 94% with inflow concentration
of 60 mg/L. However, with the increase of inflow sulphate to
30 mg/L, the increase of sulphide concentration to 8–10 mg/L and
the decrease of dissolved organic carbon average removal to 68%
were observed. These results indicate that the removal of carbon
depends on the inflow sulphate loads and also the sulphide concen-
tration in the pore water. In addition, some alternative processes
in high sulphate reduction systems may drive denitrification like
sulphur-driven autotrophic denitrification and the sulphide could
replace the organic carbon as electron donor to reduce nitrate and
accordingly lower carbon consumption (see Section 3.3). Therefore,
the relationship between microbial sulphate reduction and carbon
removal is still not sufficiently understood and should be further
investigated in more detail.
Efficient processing of acid mine drainage in CWs relies on
sufficiently available organic carbon. Plants can realize this sup-
ply by root exudates and dead plant matter. But the qualitative
and quantitative determinations of organic compounds originating
from plants and their corresponding efficacy for microbial sul-
phate reduction have not yet been achieved. Additional organic
matter and lime stone can be added to the soil body of the wet-
land to enhance efficiency of the neutralization. The selection
and efficient dosage of suitable electron donors (mainly organic
waste materials), facilitation of sulphidogenesis and efficiency
of simultaneous metal precipitation, and obtaining an improved
understanding of nature of the special microbial consortia are
aspects of future research (Faulwetter et al., 2009; Neculita et al.,
2007). Furthermore, despite a higher thermodynamic and kinetic
potential for sulphidogenesis, simultaneous methanogenesis was
often detected in natural and technical ecosystems (Leloup et al.,
2007; McDonald and Parkin, 2009). Different behavior of the
120
100
Ammonium removal (%)
80
60
40
20
0
0 5 10 15 20 25 30 35
Sulphide (mg/L)
Wiessner et al., 2007
Wu et al., 2012c
Wu et al., 2011
Fig. 4. Correlations of ammonium removal with generated sulphide concentration
investigated in different CWs.
bacteria concerning their retention in biofilms, flocs and micro-
niches can be assumed for this observation. This aspect needs more
profound understanding for biotechnological process optimization
in acid mine drainage treatment.
4.3. Interactions with nitrogen transformations
Several reports on the interactions between sulphur and
nitrogen transformations target the toxic effect of sulphide on nitri-
fication. A considerable inhibiting effect on the nitrification activity
was demonstrated by Aesoy et al. (1998) even at a sulphide con-
centration of 0.5 mg/L in a study describing a pilot-scale treatment
plant. In biofilters, it has been observed that the nitrification activ-
ity increased by about 10% as the concentration of sulphide was
reduced from about 5.5 to 2 mg/L (Bentzen et al., 1995). In addition,
clear correlations were shown between an increase in sulphate
reduction from 0 to 90% (with an inflow sulphate concentration
of about 148 mg/L) and a decrease in ammonium removal from
about 75% to 40% and from 50% to 20% (with an inflow concentra-
tion of about 48 mg/L) in two identical laboratory-scale subsurface
flow CWs with permanent water recycling (Wiessner et al., 2005).
Moreover, in two laboratory-scale horizontal subsurface flow CWs,
an inhibition of nitrification was indicated by a decrease of ammo-
nium removal after an increase in carbon loads and appearance of
sulphide of a concentration of about 3 mg/L (Wu et al., in press).
The reduced nitrification may be resulting from the competition
for oxygen between heterotrophic organic carbon degrading bacte-
ria and the autotrophic ammonium oxidizing bacteria with the
increase of biodegradable organic carbon, from an inhibitory effect
of sulphide toxicity, or from a combination of both. The negative
effect of organic carbon load and sulphide production on nitrifica-
tion normally occurs at the same time (Wu et al., in press). Under
low organic carbon loads, sulphate reduction is typically not very
significant as long as more energetically favorable electron accep-
tors, such as oxygen and nitrate are available. Consequently, an
increase in organic carbon load in the inflow may also initiate
the problem of sulphide toxicity and further hamper nitrifica-
tion (Aesoy et al., 1998; Wu et al., in press). Fig. 4 illustrates the
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S. Wu et al. / Ecological Engineering 52 (2013) 278–289
negative correlation between sulphide generation and the removal
of ammonium, as well as the sensitivity of ammonium removal
to sulphide toxicity in different investigated systems. The differ-
ence in resistance of ammonium removal to sulphide toxicity may
depend on many factors including influent nitrogen components
and loads, plants species, metal contents as well as oxygen input.
However, this aspect of knowledge is still not sufficiently known
and should be further investigated in detail.
Given the physiological inhibitory effects of sulphide on sev-
eral helophytes (Armstrong et al., 1996; Armstrong and Armstrong,
2005), a decrease of ammonium uptake and a decrease of oxygen
release from the roots into the rhizosphere can be easily imagined
if plants are living in sulphide rich conditions (Kadlec and Wallace,
2009). The decrease of released oxygen could be an additional
reason for the decrease of ammonium oxidation. Furthermore,
nitrifiers have to compete with the rapid chemical oxidation of sul-
phide with oxygen. However, compared to the direct toxic effects,
the decrease of ammonium removal due to a decrease of plant
uptake and less available oxygen released from roots seems to be
less important (Wu et al., in press).
Besides the interactions between sulphide and ammonium
removal, the process of sulphide oxidation coupled with the
reduction of nitrate or nitrite, which is known as sulphur-driven
autotrophic denitrification, may also be prevalent in CWs.
5HS− + 8NO3 − + 3H+ → 5SO4 2− + 4N2 + 4H2 O
3HS− + 8NO2 − + 5H+ → 3SO4 2− + 4N2 + 4H2 O
In anoxic zones of CWs, nitrate is usually rapidly consumed
by heterotrophic nitrate-reducing bacteria. However, given the
complex micro-gradients in rooted zones where biogenic sulphide
diffusing from sulphate reducing zone is present concurrently with
nitrate or nitrite, chemolithotrophic bacteria can couple the reduc-
tion of nitrate or nitrite with the oxidation of sulphide (Mahmood
et al., 2007, 2009; Zheng, 2007).
These sulphur-driven autotrophic nitrate-reducing bacteria are
typified by members of the genus Thiomicrospira, and their activ-
ity has been noted to inhibit sulphate reducing bacteria through
the production of the intermediate species nitrite during nitrate
reduction (Haveman et al., 2005). Moreover, although N2 is the
major end product of sulphur-driven autotrophic denitrification
in most cases, significant accumulation of N2 O often occurs and
attributes to the greenhouse gas emission (Shao et al., 2009; Zhang
et al., 2009). High nitrate concentration (Blackmer and Bremner,
1978) and low ratio of AVS (acid volatile sulphide)/nitrate in
microenvironments (Shao et al., 2011) can often lead to N2 O accu-
mulation/emission due to the inhibition of either nitrous oxide
reductase or denitrifiers with incomplete nitrogen reduction path-
way.
4.4. Interactions with phosphorus transformations
Interactions between sulphur transformation processes and
phosphorus removal also may exist in CWs, particularly in those
with a fraction of the accreted phosphorus bound by iron-
containing substances. Two possible effects have been reported:
(1) a reduction of phosphorus uptake by suffering plants which
are growing in sulphide rich environments; and (2) sulphide binds
iron and interferes with that component of phosphorus storage that
relies upon the iron-phosphorus link.
Previous reports on phosphorous removal from planted waste-
water treatment ponds via phosphine (PH3 ) (Devai et al., 1988) and
the production of volatile P-compounds like PH3 from phosphate
285
by sulphate reducing bacteria (Iverson et al., 1985) were not cor-
roborated in the more recent literature (Glindemann et al., 2005).
In experimental studies on sediment cores, Gunnars and
Blomqvist (1997) showed that the reduction of FeOOH in fresh-
water and brackish sediments and the subsequent formation of
iron sulphide were predominately responsible for the release of
phosphate to the overlying water column. Because iron sulphide
minerals have a low point of zero charge and do not adsorb
phosphate at neutral pH, these iron-sulphide reactions effec-
tively solubilize solid phosphorus and maintain phosphate in pore
waters, while removing ferrous iron from the solution (Rozan et al.,
2002).
Wu et al. (2012a) investigated long term and spatial dynam-
ics of pore-water sulphide, Fe(II) and phosphate concentrations
in a pilot-scale horizontal subsurface flow CW with an iron-rich
soil matrix for treating sulphate-containing contaminated ground-
water. The results demonstrated that the pore-water sulphide
concentrations gradually increased from less than 0.2 mg/L in 2005
to annual average concentrations of 13.5 mg/L in 2010, while
the pore-water Fe(II) concentrations decreased from 37.0 mg/L to
0.3 mg/L. At the meanwhile from 2005 to 2010, the phosphate
removal efficiency declined from 91% to 10% under a relative con-
stant inflow concentration of 5 mg/L. No significant response of
plants to high iron concentration and deficiency of phosphate in
the initial years were shown. The effect of the detoxification of sul-
phide and the immobilization of phosphate by the application of
the iron-rich soil matrix in the initial years was proven; however,
the life-span of this effect should be taken into consideration in
further design.
Besides to the application of the iron-rich soil matrix for the
detoxification of sulphide, the addition of iron into the inflow of
wetlands was also reported as an option to control the sulphide
concentration (Smolders et al., 1995; Van der Welle et al., 2007b).
If sulphide production is overestimated in the wetlands and a sur-
plus of iron is added, negative effects on plant growth could be
shown as iron toxicity as well as iron binding to phosphate lead
to a lower phosphorus availability to plants uptake (Boers et al.,
1994; Smolders et al., 1995). However, when there is no sufficient
iron available to control the continuously generated sulphide, the
performance of low phosphate removal may also be observed due
to the mobilization of phosphate (Boström and Pettersson, 1982;
Caraco et al., 1989; Smolders et al., 1995). Therefore, consider-
ing the complicated interactions of iron, sulphur and phosphorus,
the use of iron for the control of sulphide concentration in wet-
lands should be proper, and some other approaches may be further
investigated in the future studies and applications.
5. Conclusions and perspectives
In the past decades, the use of CWs as an ecological system
for wastewater treatment has received increasing popularity in
the world, and CWs have been successfully employed to remove
a diverse array of pollutants. However, despite the fact that sul-
phate is a common constituent of many contaminated waters
and considerable sulphur compounds are prevalent in CWs, the
understanding of sulphur transformations and their complex inter-
actions with other beneficial processes is still insufficient. The biotic
and abiotic sulphur transformations are determined by many fac-
tors like the dynamics of the redox conditions, temperature, and
hydraulic flow conditions. The redox dynamics are influenced by
the release of organic carbon and oxygen by the plants into the
rhizosphere, as well as the design and operation conditions of the
wetland. Redox micro-gradients in near-root environments of the
rhizosphere enable simultaneous anaerobic and aerobic processes,
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286 S. Wu et al. / Ecological Engineering 52 (2013) 278–289
particularly in horizontal subsurface-flow systems. Especially in
those systems a number of processes are still poorly understood
and further research and development are necessary, such as:
• The knowledge on the interactions between microbial carbon
transformations and sulphate reduction should be expanded.
The occurrence of dissimilatory sulphate reduction in wetlands
should theoretically enhance the removal of organic carbon as the
electron donor; however, some researches demonstrated nega-
tive correlation between sulphate reduction and organic matter
removal. It may depend on the inflow sulphate load, sulphide
concentration, redox potential and some other inflow compo-
nents such as nitrate which can drive autotrophic denitrification
in the presence of sulphide. The interactions between sulphur and
nitrogen transformations can also be involved and subsequently
influence the relations between microbial carbon and sulphur
turnover.
• The response of the generation of biogenic gases (e.g. H2 S, CH4 ,
N2 O, H2 ) and further transformations and/or emissions to the
varying influent components and loads should be investigated.
Considering the complicated interactions between simulta-
neously occurring microbial transformations of carbon, nitrogen
and sulphur, biogenic gases as the products of these processes
also do not occur isolated, but are interconnected and interacted
through their substrates, products, and intermediates.
• The abiotic precipitation of salts from sulphates with a low sol-
ubility as a result of steep micro-scale concentration gradients
nearby the root surface is assumed and needs further proofs and
understanding to use this principle in a more directed way in CWs
especially for waters with high inorganic ion loads.
• The understanding of the relationships between the transforma-
tions of deposited sulphur compounds (e.g. elemental sulphur,
metals sulphide) and the variations of overlying pore water envi-
ronmental conditions is still insufficient. The deposited sulphur
compounds can be oxidized or reduced to release more oxidizing
or reducing sulphur compounds depending on the availability
of more energetically favored electron donors and/or electron
acceptors.
• The formation of elemental sulphur in the pore water and exit
region under distinct conditions of CWs (Sturman et al., 2008) is
of special interest for strategy development to remove sulphate
from water and immobilize it in a relatively stable form.
• Little research has been carried out on the correlation between
the sulphide production and the removal performance of fecal
bacteria indicators in CWs, particularly for some specific bacteria,
viruses, protozoa, and helminths. The negative effect of sulphide
toxicity on helophytes, nitrifiers and even some heterotrophic
microorganisms was often shown, but in relation to pathogen
removal better detailed knowledge is still necessary.
• To minimize the toxic effects of sulphide as a product of dissimila-
tory sulphate reduction on wetland plants and activity of various
microbial processes, other control approaches than iron addition
should be investigated, such as some integrations of different
types of wetlands as well as aeration. Iron addition is an effec-
tive approach to control sulphide concentration, but it should be
handled properly. Surplus iron addition may easily not only lead
to phosphorus nutrient deficiency due to precipitation of iron
with phosphate, but also cause plants suffering since Fe2+ is also
a phytotoxin.
• Still in discussion is the selection of plant species for further pro-
cess intensification (Sturman et al., 2008; Brisson and Chazarenc,
2009). Especially the use of genetically modified plants with spe-
cial traits is difficult because of natural auto-selection of plants
in the CWs and also due to problems of acceptance and legal
restrictions in many countries. Ecologically well-adapted plants,
such as reed, rushes, and cattail, are dominant in uninfluenced
ecosystems and specially designed plants would be probably dis-
placed/outcompeted by the wildlife species in CWs.
• As considering the complex sulphur transformations in CWs
and their interrelated processes, an improved understanding of
microbial community structure of CWs is highly significant future
research direction to link process activities and microbial species
data (Faulwetter et al., 2009; Truu et al., 2009).
Acknowledgements
This work was supported by grants from the project of “Technol-
ogy and demonstration of cogeneration of gas, heat and electricity
by wet-dry fermentation in cold regions (2011BAD15B04)”, and
“key technologies on energy cycle regulation in typical agricultural
system (2012BAD14B03)”, by the Helmholtz Centre for Environ-
mental Research–UFZ within the scope of the SAFIRA II Research
Programme (Revitalization of Contaminated Land and Groundwa-
ter at Megasites, subproject “Compartment Transfer–CoTra”) and
by the German Federal Ministry of Education and Research (BMBF);
the Research Association Mining and Environment in Vietnam
(RAME, 02WB0957).
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