w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

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Investigation on thiosulfate-involved organics and nitrogen removal by a sulfur cycle-based biological wastewater treatment process Jin Qian a, Hui Lu b,c,**, Yanxiang Cui a, Li Wei a, Rulong Liu a, Guang-Hao Chen a,d,* a

Department of Civil and Environmental Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China b School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, China c Guangdong Provincial Key Laboratory of Environmental Pollution Control and Remediation Technology, Guangzhou, China d SYSU-HKUST Joint Research Centre for Innovative Environmental Technology, Sun Yat-sen University, Guangzhou, China

article info

abstract

Article history:

Thiosulfate, as an intermediate of biological sulfate/sulfite reduction, can significantly

Received 9 July 2014

improve nitrogen removal potential in a biological sulfur cycle-based process, namely the

Received in revised form

Sulfate reduction-Autotrophic denitrification-Nitrification Integrated (SANI®) process.

31 October 2014

However, the related thiosulfate bio-activities coupled with organics and nitrogen removal

Accepted 23 November 2014

in wastewater treatment lacked detailed examinations and reports. In this study, S2O2
3

Available online 3 December 2014

2
transformation during biological SO2
4 /SO3 co-reduction coupled with organics removal as

well as S2O2
3 oxidation coupled with chemolithotrophic denitrification were extensively Keywords:

evaluated under different experimental conditions. Thiosulfate is produced from the co-

Thiosulfate bio-transformation

reduction of sulfate and sulfite through biological pathway at an optimum pH of 7.5 for

Biological sulfate/sulfite reduction

may disproportionate to sulfide and sulfate organics removal. And the produced S2O2
3

Chemolithotrophic denitrification

during both biological S2O2
3 reduction and oxidation most possibly carried out by Desul-

Organics and nitrogen removal

fovibrio-like species. Dosing the same amount of nitrate, pH was found to be the more direct

Sulfur cycle-based wastewater

factor influencing the denitritation activity than free nitrous acid (FNA) and the optimal pH

treatment process

for denitratation (7.0) and denitritation (8.0) activities were different. Spiking organics significantly improved both denitratation and denitritation activities while minimizing sulfide inhibition of NO
3 reduction during thiosulfate-based denitrification. These findings in this study can improve the understanding of mechanisms of thiosulfate on organics and nitrogen removal in biological sulfur cycle-based wastewater treatment. © 2014 Elsevier Ltd. All rights reserved.

* Corresponding author. SYSU-HKUST Joint Research Centre for Innovative Environmental Technology, Sun Yat-sen University, Guangzhou, China. ** Corresponding author. School of Environmental Science and Engineering, Sun Yat-sen University, Guangzhou, China. E-mail addresses: [email protected] (H. Lu), [email protected] (G.-H. Chen). http://dx.doi.org/10.1016/j.watres.2014.11.038 0043-1354/© 2014 Elsevier Ltd. All rights reserved.

296

1.

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

Introduction

Biological sulfate reduction (BSR) and biological reduced sulfur (sulfide, elemental sulfur and thiosulfate) oxidation (BSO) play the two major roles in sulfur bio-conversionbased processes in wastewater treatment (Visser, 1995; Lens et al., 1998; Cardoso et al., 2006). By integrating BSR with BSO, we developed the Sulfate reduction-Autotrophic denitrification-Nitrification Integrated (SANI®) process for organics and nitrogen (N) removal (Wang et al., 2009) and Denitrifying Sulfur-cycle Enhanced Biological Phosphorus Removal (DS-EBPR) process for simultaneous removal of organics, N and P (Wu et al., 2013, 2014), both for treatment of municipal saline sewage resulting from seawater toilet flushing for the purpose of saving 740,000 m3/d of freshwater demand in Hong Kong (Leung et al., 2012; Chen et al., 2012). These novel sulfur (S) bio-conversion-driven biological nutrients removal (BNR) processes not only double the N removal potential, but more importantly minimize excess sludge production significantly, thereby saving one-third of the energy consumption compared with conventional BNR processes (Lu et al., 2012). These new treatment approaches open up an opportunity to utilize S as both an electron acceptor in BSR for efficient organics removal (or P release in DS-EBPR) and electron donor in BSO for autotrophic denitrification (or P uptake in DS-EBPR), through which an S cycle between sulfate and sulfide is achieved for eliminating any discharge of toxic sulfide to the receiving water body. This new treatment recipe changes century-old BNR technologies from carbon and nitrogen two cycles to carbon, nitrogen and sulfur three cycles-driven bio-treatment processes to achieve energy-efficient wastewater treatment (van Loosdrecht et al., 2012). In order to benefit wastewater treatment in freshwater supply areas through the S cycle-driven processes, brackish water or acid mining drainage has been proposed as the potential sulfate sources. Sulfate/sulfite/elemental S laden wastewater also offers low-cost S sources to drive SANI® or DS-EBPR for freshwater sewage treatment. For instance, flue gas desulfurization (FGD) liquid wastes (sulfate/sulfite-rich wastewater) via alkaline absorption of SO2 from coal-burning power plants may enable SANI®/DS-EBPR applications in freshwater sewage treatment as shown in Fig. S1 (Qian et al., 2013; Jiang et al., 2013). In this case, thiosulfate is most likely produced from co-reduction of sulfate and sulfite through biological pathways instead of chemical normalized reactions between sulfide and sulfite (Selvaraj et al., 1997; Brunner and Bernasconi, 2005; Qian et al., 2013; Jiang et al., 2013). It is further reported that thiosulfate accumulation in biological sulfite reduction is affected by temperature rather than by COD-to-SO2
3 ratio (Qian et al., 2013; Jiang et al., 2013). The lower temperature is more favored for thiosulfate accumulation in biological sulfite reduction as the reduction of thiosulfate to sulfide (Step II) is more sensitive to lower temperatures than the reduction of sulfite to thiosulfate (Step I) (see Fig. S2). This beneficial co-treatment of FGD liquid wastes with freshwater sewage brings in a different S cycle, 2
2
2
2
i.e. SO2
4 /SO3 /S /S2O3 / SO4 for wastewater treatment, which presents a completely new S conversion-based BNR

process driven by thiosulfate rather than SANI® and DS-EBPR by sulfide. Chemically, thiosulfate (S2O2
3 ) has a mean oxidation number of þII, falling in the middle of the whole S oxidation states ranging from eII in S2
to þVI in SO2
4 , possessing an equal capacity for donating and accepting electrons. This implies that S2O2
3 may play some important roles in S cyclebased wastewater treatment that have not been reported in detail. Jiang et al. (2013) only reported the effect of temperature on S2O2
3 generation and accumulation during biological reduction and concluded that lower temperature SO2
3  (decreased from 15 to 2 C) can promote thiosulfate accumulation. Systematic studies on the effects of pH, sulfide, oron BSO of thiosulfate coupled with ganics and NO
3 chemolithotrophic denitrification and its detailed kinetic analysis are missing in the literature, though some factors, i.e. pH, NO
3 and organics, have been simply reported in both pure cultures and denitrifying sludge (Trouve et al., 1998; Oh et al., 2000, 2001; Campos et al., 2008). Besides, thiosulfate may not only act as an electron acceptor in BSR, but also disproportionate itself to sulfate and sulfide carried out by Desulfovibriolike SRB species (Hernandez-Eugenio et al., 2000; Baena et al., 1998; Sass and Cypionka, 2004; Saafield and Bostick, 2009). However, no in-depth investigations on the thiosulfate biological disproportionation in wastewater treatment have been found up to date. Moreover, we also actually found concerted accumulation of thiosulfate and sulfide (1:1 mg S/mg S) in a 2
reactor with organics added and SO2
4 /SO3 -reducing completely utilized for chemolithotrophic denitrification in a thiosulfate-oxidizing reactor with nitrate dosed (Qian et al., 2015). The aim of this study is therefore, to investigate the thiosulfate-involved organics and nitrogen removal during the sulfur cycle-based biological wastewater treatment. The specific focuses are placed on: 1) the effects of pH and electron availability (organics) on thiosulfate generation, reduction and disproportionation during the anaerobic BSR; 2) the effects of experimental conditions, such as pH, nitrate, organics as well as sulfide, on thiosulfate bio-transformation during the denitrification; and 3) the dominant microorganisms carrying out the thiosulfate reduction along with its possible disproportionation in wastewater treatment.

2.

Materials and methods

2.1. Experimental study of thiosulfate biotransformation in the S cycle bioprocess The experimental study was designed and conducted via a series of batch tests to investigate: 1) the thiosulfate accumulation during biological sulfate/sulfite reduction, thiosulfate reduction and disproportionation by sulfur-reducing bacteria (SRB); 2) thiosulfate oxidation in chemolithotrophic denitrification under different conditions. These make up the two major parts for organics and nitrogen removal in the S cycle-based wastewater treatment process. The respective sludge used was cultivated as follows.

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Table 1 e Experimental conditions for each Batch Reactor in all Batch Tests. Batch Test no. (Batch Reactor No.) 1

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19)

2 3

4

5

a b c

pH value

Organic source (conc.)a

S source(conc.)b

N source (conc.)c

6.5 7.0 7.5 8.0 7.5

Synthetic wastewater (420)

2
SO2
4 (230) & SO3 (115)

e

Synthetic wastewater (470)

S2O2
3

e

(700) (350)

7.0 7.5 8.0 8.5 9.0 7.5

e

S2O2
3 (200)

NO
3 (60)

e

S2O2
3 (205)

NO
3

7.5

Acetate (192) Acetate (72) Acetate (144) e

e S2O2
3 (133.3) S2O2
3 (69) S2O2
3 (205) & FSS (15)

NO
3 (60)

(35) (55) (72) (95)

The conc. unit is mg COD/L. The conc. unit is mg S/L. N source here excludes NHþ 4 /NH3 and its unit is mg N/L.

2.2.

Sludge cultivation

A lab-scale setup comprising an S reducing up-flow sludge bed (SRUSB) reactor and an anoxic up-flow sludge bed (AnUSB) reactor for denitrification was adopted, as shown in Fig. S1 in Supporting Information (SI). Approximately 99 mg S/L sulfite and 190 mg S/L sulfate together with 560 mg COD/L organics diluted from stock synthetic wastewater (Table S1 in SI), were supplied to the influent of SRUSB. The SRUSB provides a mixture of three electron donors, i.e. sulfide, thiosulfate and simple organics (mainly acetate-like volatile fatty acids degraded from relatively complex organics of yeast and glucose). These three electron donors resulted in a mixed denitrification in the subsequent AnUSB, i.e. two autotrophic denitrification reactions driven by sulfide and thiosulfate respectively and one heterotrophic denitrification by organics when 30 mg N/L NaNO3 was added to this anoxic reactor as the nitrate source. The setup achieved 80% COD removal and almost 100% nitrate removal under the steady-state condition when the sludge production was also in steady-state with a total sludge yield of 0.03 g VSS/g COD in the system (Qian et al., 2015).

2.3.

Batch tests

Two sets of batch tests were conducted with the SRUSB sludge for BSR (Batch Tests 1e2) and three sets of batch tests with the AnUSB sludge for BSO (Batch Tests 3e5). All sludge taken from these two reactors was washed with distilled water three times to remove background substrates prior to each assay. Each batch reactor in Batch Tests 1e2 was purged by nitrogen gas (analytical grade) to exclude dissolved oxygen and maintain an anaerobic condition before the test. In Batch Tests 3-5

helium gas (analytical grade) was purged to the reactor for the same purpose. The batch tests were conducted in glass serum flasks (2 L for Batch Tests 1e2 and 500 mL for Batch Tests 3e5). All flasks were sealed with butyl rubber stoppers and aluminum crimp seals. The temperature of each flask was kept at 23 ± 1  C in an air-conditioned room. The reactors were stirred by using a magnetic stirrer at 150 rpm. Batch Test 1 examined the effect of pH on S2O2
3 formation 2
and accumulation in BSR of SO2
4 and SO3 . A common range of pH 7.0e8.0 was used by other researchers (Vallero et al., 2003; Selvaraj et al., 1997; Weijma et al., 2000; Omil et al., 1997a, b; Dries et al., 1998; Jiang et al., 2013). Moreover, to reflect the pH in typical domestic wastewater treatment, also normally varied from 7.0 to 8.0, a pH range of 6.5e8.5 was selected in the four batch reactors in this Batch Test (see Table 1). An appropriate portion of the synthetic wastewater stock solution with Na2SO4 (230 mg S/L) and Na2SO3 (115 mg S/L) solutions was added together into each reactor to set its initial 2
COD at about 420 mg/L and the molar ratio of SO2
4 to SO3 at 2:1. Each test lasted for 32 h and biomass concentrations were 810 ± 11 mg VSS/L in each reactor. In Batch Test 2, BSR of S2O2
3 was investigated. Stoichio2
metrically BSR of SO2
4 to S2O3 consumes 50% less electrons 2
2
than does BSR of SO2
4 to sulfide, while BSR of SO3 to S2O3 2
requires 66% less electrons than does BSR of SO3 to sulfide. S2O2
3 can be substantially generated from BSR of sulfite (Qian et al., 2013; Jiang et al., 2013) or its mixture with sulfate (Qian et al., 2015) and be further reduced to sulfide in BSR and disproportionated to sulfide and sulfate (Sass and Cypionka, 2004; Saafield and Bostick, 2009). Therefore, organic removal in BSR is affected by transformation of S2O2
3 . To study the behavior of S2O2
3 in BSR, two batch reactors were carried out under a fixed pH at 7.5 (a typical pH value in wastewater

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treatment plants) in this Batch Test. Under the same initial COD concentration, different initial concentrations of S2O2
3 were applied in each reactor (see Table 1) to set 150 (sulfur sufficiency) and 75% (sulfur deficiency) of the stoichiometric level of thiosulfate against COD. To investigate the effect of transformation in BSR, two chemical reaction on S2O2
3 chemical control batch reactors were also conducted in parallel under the same conditions as in Batch Reactors 5 and 6 in Batch Test 2(see Table S2 in SI) but lacked biomass. Biomass concentrations in these two batch reactors were 690 ± 10 mg VSS/L. Each test including the control tests in Batch Test 2 also lasted for 32 h. Batch Test 3 focused on the effect of pH on BSO of S2O2
3 . In this test, approximately 10 mL of stock solution containing only the inorganic part, 1 mL of trace element solution (Table S1 in SI), 60 mg N/L of NaNO3, and 200 mg S/L Na2S2O3 were spiked into five batch reactors filled with the AnUSB sludge. The pH effect was investigated from 7.0 to 9.0. Each test lasted for 24 h and biomass concentrations were 590 ± 6 mg VSS/L. Batch Test 4 evaluated the effect of NO
3 concentration on
BSO of S2O2
3 . Different NO3 eN levels against the same Na2S2O3 concentration were prepared in four batch reactors (see Table 1) to set the respective initial S/N mass ratios from 6:1 (S more sufficient), 4:1 (S sufficient) and 2.9:1 (S stoichiometrically met) to 2.2:1 (S deficient). The pH 7.5 was adopted in this and the following batch tests. Each test lasted for 24 h and biomass concentrations were 705 ± 13 mg VSS/L. Batch Test 5 examined the effect of organics and sulfide on BSO of S2O2
3 as organics and sulfide from BSR could, together with thiosulfate, enter the anoxic reactor for N removal (see Fig. S1). Four batch reactors, including one for heterotrophic denitrification (HD) (Batch Reactor 16) using acetate only as the electron donor, were performed. Mixtures of acetate and thiosulfate at different mass ratios were added to another two reactors: Batch Reactors 17 and 18 (Table 1), setting the COD/S/ N mass ratios at 1.2:2.4:1 and 2.4:1.2:1. In Batch Reactor 19, besides 205 mg S/L Na2S2O3 used in Batch Test 4, 15 mg S/L Na2S was also spiked to detect the effect of sulfide. Approximately 60 mg N/L of NaNO3 was spiked into all four reactors. All the other conditions were kept the same as in Batch Test 4. Each test lasted for 4 h and biomass concentrations were 860 ± 18 mg VSS/L mg VSS/L.

2.4.

2.5.

Kinetic analysis

In Batch Tests 1 and 2, BSR activity in terms of specific organic 2
degradation, S2O2
3 reduction, and TDS/SO4 generation rates were calculated through linear regression of the biomass2
specific COD, S2O2
3 and TDS/SO4 concentrations (mg/g VSS) versus time (h) from Fig. 1 and 2 (R2 > 0.9), while the specific S2O2
3 /TDS, organic oxidation and nitrate reduction rates in Batch Tests 3e5 were determined through linear regression of 2
theS2O2
3 /TDS, COD and NO3 profiles (R > 0.9) versus time (day) in Fig. 3e5 divided by the biomass concentration (g VSS/ L) (Cardoso et al., 2006). In these batch tests, the specific nitrite reduction rates in the presence and absence of nitrate were determined from a simplified two-step denitrification model as shown in SI (Glass and Silverstein, 1998).

2.6.

Analysis of the microbial community

At the end of Batch Test 2, the sludge sample from Batch Reactor 6 (thiosulfate-reducing reactor) was collected. The diversity of the microbial community in this thiosulfatereducing sludge sample was analyzed by 454pyrosequencing of the 16S rRNA gene, according to Lee et al.

Sampling and chemical/physical analysis

In all the above batch reactors, mixed liquor was sampled regularly by using a 10-mL syringe. All these samples were then filtered through disposable Millipore filters (0.22 mm pore size) to analyze sulfate, thiosulfate, nitrite, nitrate and acetate with an ion chromatograph (HIC-20A super) equipped with a conductivity detector and an IC-SA2 analytical column. COD was measured based on the Standard Methods (APHA, 2005). Total dissolved sulfide (TDS) was preserved by NaOH and ZnAc according to the Standard Methods and then measured immediately by the methylene blue method in order to prevent volatilization and abiotic oxidation. Sludge samples were further taken from each reactor at the end of each test for VSS measurement in triplicate according to the Standard Methods. pH and temperature were monitored using a multi-meter electrode during each test (WTW multi 3420).

Fig. 1 e Results of Batch Test 1 (Batch Reactor 1eBatch Reactor 4): (a) S2O2¡ 3 profile; (b) COD profile.

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

299

Fig. 2 e Results of Batch Test 2 (Batch Reactor 5 and Batch Reactor 6): results of Batch Reactor 5 (a) and Batch Reactor 6 (b).

(2011) and Zhang et al. (2012). Details of the methods for DNA extraction, PCR amplification, pyrosequencing, and data analysis are provided in the SI.

3.

Results and discussion

3.1. Batch Test 1: effect of pH on S2O2
3 transformation in BSR of sulfate and sulfite 2
2
The trithionate pathway (SO2
3 / S3O6 / S2O3 / TDS) with 2
2
the main intermediate S2O3 in BSR of SO3 is widely reported

and confirmed by other researchers (Brunner and Bernasconi, 2005; Qian et al., 2013). As no trithionate was found during the biological sulfite reduction according to our previous study (Jiang et al., 2013), it was reasonable to simplify the two-step / S2O2
and ii) sulfite reduction pathway, i.e. i) SO2
3 3 2
S2O3 / TDS, as illustrated in Fig. S2. The peak specific thiosulfate concentration (i.e. S2O2
3 eS concentration (mg/L) per unit biomass concentration (1 g VSS/L) and the same scenario for other specific concentrations) achieved about 60 mg of S2O2
3 eS/g VSS at all tested pH levels except pH ¼ 6.5 when the test time reached 8 h (see Fig. 1a). The slower S2O2
3 generation 2
at pH 6.5 (peak time at 20 h) revealed that Step I (SO2
3 to S2O3 )

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¡ 2¡ 2¡ Fig. 3 e Results of Batch Test 3 (Batch Reactor 7e11): NO¡ 3 and NO2 profiles (a); S2O3 and SO4 profiles (b).

seems to be more sensitive to acid conditions than Step II to TDS), meaning neutral pH is favorable for BSR of (S2O2
3 2
SO2
4 /SO3 . In comparison with the thiosulfate profile, the organics degradation rate can be separated into two phases at 8 h (see Fig. 1b). Based on the our experimental results, the enzyme activity for organic oxidation in sulfate/sulfite reduction at pH 7.5 is 2.1, 1.3 and 1.2 times higher than those at pH 6.5, 7.0 and 8.0, respectively. The specific COD removal rates at pH 7.5 are 40.9 mg COD/g VSS/h before 8 h (fast) and 10 mg COD/g VSS/h after 8 h (slow), according to the slope of the specific COD concentration versus time in Fig. 1b. Interestingly both rates exceed that at pH ¼ 7 and 8. The reason for such a pHdependent rate is unclear, though it is common in most enzyme-driven biological reactions, as each enzyme has an optimal activity at a certain pH level and the biological activity will decrease while pH moves away from this pH level (Pan et al., 2012). And the higher specific COD removal rate in Phase 1 (or the thiosulfate accumulation period) than in Phase 2 (or thiosulfate reducing period) indicates that thiosulfate may decrease organic removal activity compared with sulfate/

sulfite (the details can be seen in the following results obtained from Batch Test 2). As discussed above, the highest organics degradation rate for achieving complete organics removal was obtained at pH 7.5 where thiosulfate formation and reduction were both remarkably observed. So thiosulfate, as the major intermediate in sulfate/sulfite co-reduction, possibly played an important role in organics removal in this co-reduction. Specifically, how the thiosulfate transformation (biologically and/or chemically) further proceeds in its biological reduction will be discussed in the following section results.

3.2. Batch Test 2: transformation of S2O2
3 in biological thiosulfate reduction As shown in Fig. 2a and b, the COD removal can be divided into three stages (see Fig. S3a and b): Stage I represents a rapid organics bio-degradation at the specific COD removal rates of 23 and 23.7 mg COD/g VSS/h in Batch Reactors 5 and 6 respectively before 4 h. They are only half of that in the biological sulfate/sulfite reduction in Batch Test 1 (before 8 h at

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

301

¡ 2¡ 2¡ Fig. 4 e Results of Batch Test 4 (Batch Reactor 12e15): NO¡ 3 and NO2 profiles (a); S2O3 and SO4 profiles (b).

pH ¼ 7.5); Stage II shows slow organic bio-degradation with the specific COD removal rates at 6.0 and 4.7 mg COD/g VSS/h in Batch Reactors 5 and 6 respectively between 4 and 16 h; and Stage III demonstrates no organic bio-degradation in both reactors during the time period from 16 to 32 h. In Stage I, thiosulfate was biologically reduced to TDS coupled with the rapid organic bio-degradation, possibly resulting in the accumulation of elemental sulfur and/or polysulfide because the ratio of the S2O2
3 reduction rate (mg S/ g VSS/h) to the TDS generation rate (mg S/g VSS/h) was 1.22 and 1.23 for Batch Reactors 5 and 6. Observation of white colloidal precipitates (insoluble nature of S0) in both reactors further supports this argument. Theoretically, the formation of elemental sulfur will decrease the COD removal efficiency; however, it is interesting to see that the specific COD removal rates were 1.4 and 1.3 times higher than the specific S2O2
3 reduction rates in Batch Reactors 5 and 6 though the stoichiometric ratio of COD removal to complete S2O2
3 reduction 2
(S2O2
3 to TDS) was 1 mg COD/mg S2O3 eS. As the sludge yield for BSR is as low as less than 0.1 g VSS/g COD (Lu et al., 2012; Jiang et al., 2013), this can be attributed to the fermenting biomass (see microbial analysis results below), such as Lacotococcus species which converts organics into CO2 through

heterolactic fermentation (Anestis, 2006), thus increasing this ratio (Jiang et al., 2013). After 4 h, sulfate was generated in both Batch Reactors 5 and 6, when the specific COD removal rates decreased significantly in the both reactors. An explanation is that thiosulfate reduction and disproportionation (see Eq. (1)) were coexisting and competing for thiosulfate as a substrate in Stage II (4e16 h), supported by two pieces of evidence: (1) in the control batch tests without any biomass (see Fig. S4a and b), neither thiosulfate decrease nor sulfate/TDS generation was observed, implying thiosulfate disproportionation occurred in the biological reactors; and (2) Desulfovibrio-like SRB species could concurrently reduce thiosulfate to TDS and disproportionate thiosulfate to TDS and sulfate (the details can be seen in the microbial analysis results). However, different from Stage I, no elemental sulfur and/or poly sulfide accumulation were found in Stage II since the specific S2O2
3 removal rate (5.95 and 2.53 mg of S/g VSS/h for Batch Reactors 5 and 6) is just equal to the sum of the specific TDS generation rate and the specific SO2
4 generation rate (i.e., 3.13 þ 2.77 ¼ 5.9 mg S/g VSS/h for Batch Reactor 5, 1.21 þ 1.46 ¼ 2.67 mg S/g VSS/h for Batch 2
Reactor 6), indicating a good mass balance among S2O2
3 , SO4 and TDS. During Stage III (16e32 h), the thiosulfate reduction completely ceased, but the thiosulfate disproportionation still

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¡ 2¡ 2¡ Fig. 5 e Results of Batch Test 5 (Batch Reactor 16e19): NO¡ 3 and NO2 profiles (a); S2O3 and SO4 profiles (b).

existed (see Fig. 2a and b). The disproportionation in this stage is supported by the fact that the ratio of the SO2
4 generation rate to the S2O2
3 removal rate was 0.4 and 0.5 for Batch Reactors 5 and 6 respectively (determined from Fig. S3c and d), which is very close to the stoichiometric value (0.5) as shown in Eq. (1). Although this disproportionation has been reported in marine sediments and pure SRB (Desulfovibrio-like species) culture (Jørgensen and Bak, 1991; Hernandez-Eugenio et al., 2000), in the present study it was firstly investigated and revealed in anaerobic BSR for organic removal in wastewater treatment. Moreover, the disproportionation was only generation) and then observed after 4 h (indicated by SO2
4 gradually became dominant thereafter in this study. At a genus level 7.5% of SRB was detected in the batch reactor's sludge, of which Desulfovibrio-like species only accounted for 1.8% (see the microbial community analysis result section). This could explain why a 4-h lag phase for thiosulfate biological disproportionation was observed. 2

þ S2 O2
3 þ H2 O/SO4 þ 0:5H2 S þ 0:5HS þ 0:5H

DG00 ¼
25KJ=mol

(1)

In the thiosulfate bio-conversion-based wastewater treatment process as proposed in Fig. S1, thiosulfate can be

luxuriously accumulated through regulation of anaerobic BSR condition (i.e. HRT and SRT) to induce much higher chemolithotrohic denitrification activity than does TDS (Cardoso et al., 2006; Manconi et al., 2007) as discussed in the following section concerning the mechanisms and kinetics of thiosulfate-driven chemolithotrophic denitrification.

3.3. Batch Test 3: effect of pH on BSO of S2O2
3 in chemolithotrophic denitrification A pH range of 7.0e8.0 is reported to be optimal for denitratation activity in BSO of thiosulfate coupled with chemolithotrophic denitrification by Thiobacillus denitrificans isolated from soils, activated sludge, lagoons and surface water (Trouve et al., 1998). The higher pH inhibits denitratation activity and delays nitrite accumulation by, for instance, more than two hours at pH ¼ 9 in this study (see Fig. 3a). A pH from 7.0 to 9.0 substantially decreased the specific NO
3 reduction and S2O2
3 oxidation rates by 66 and 55% respectively (see Table 2). The chemolithotrophic denitrification proceeded in two stages (see Fig. 3a and b): (1) (from 0 to 6 h) both denitratation and nitrite accumulation rates significantly decreased as the reaction pH increased causing only little

172.0 ± 8.8 207.3 ± 9.6 253.4 ± 12.2 198.9 ± 8.9 144.0 ± 4.3 292.5 ± 11.7 82.7 ± 3.4 44.1 ± 1.9 20.1 ± 0.8 888.1 ± 32.3 161.3 ± 4.3 515.9 ± 12.7 NA 65.6 ± 3.8 36.2 ± 1.1 15.4 ± 0.4 16.7 ± 0.6 0 34.4 ± 1.3 46.4 ± 1.7 106.5 ± 3.6 118.9 ± 4.1 238.4 ± 10.5 257 ± 7.3 117.5 ± 2.2 NA 773.0 ± 23.2 681.1 ± 26.8 643.9 ± 11.3 547.7 ± 24.2 284.7 ± 9.8 699.2 ± 27.7 688.5 ± 21.8 564.3 ± 24.3 607.1 ± 13.7 625.7 ± 29.4 748 ± 34.3 613 ± 22.1 NA NA NA NA NA NA NA NA NA NA 1115.5 ± 46.8 739.4 ± 29.7 739.4 ± 22.4 NA 1389 ± 16.4 1287 ± 20.6 1086 ± 21.5 924 ± 15.5 630 ± 10.4 1202.6 ± 31.3 1391.9 ± 24.5 1566.4 ± 18.9 1768.9 ± 27.6 NA 774.6 ± 23.4 825.4 ± 14.7 421 ± 10.3 5

4

3

(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19)

838.6 ± 21.2 717.3 ± 12.6 659.3 ± 13.5 564.4 ± 9.7 283.2 ± 8.8 733.5 ± 13.5 734.9 ± 20.3 670.8 ± 18.6 726 ± 14.7 864.1 ± 24.5 1005 ± 32.7 730.6 ± 18.3 106.6 ± 4.6

1001.7 ± 19.6 997.7 ± 31.2 896.0 ± 18.6 814.6 ± 21.9 559.2 ± 22.3 1058.4 ± 28.5 1147.3 ± 47.2 1266.7 ± 55.2 1436.5 ± 36.7 NA 427.7 ± 12.1 541.2 ± 10.7 347.5 ± 14.6

Biomass-specific organic oxidation rate (mg COD/g VSS/d) Biomass-specific Biomass-specific Biomass-specific NO
S2O2
SO2
3 reduction rate 3 oxidation 4 generation (mg N/g VSS/d) rate (mg S/g VSS/d) rate (mg S/g VSS/d) Batch Test no. (Batch Reactor no.)

Table 2 e Kinetic results in Batch Tests 3 & 4 & 5.

Biomass-specific Biomass-specific Biomass-specific NO
NO
NO
2 accumulation 2 reduction 2 reduction rate in the rate in the rate in the presence of NO
presence of NO
absence of NO
3 3 3

(mg NO
2 eN/g VSS/d) (mg NO2 eN/g VSS/d) (mg NO2 eN/g VSS/d)

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

303

nitrite reduction (see Table 2), and (2) (from 6 to 24 h) nitrite reduction rate in the absence of nitrate peaked when pH ¼ 8.0 (see the last column in Table 2), and this rate decrease as pH up to 9.0 or down to 7.0. This implies the different optimal pH values for nitrate and nitrite reductases and denitritation activity was mainly affected by the pH instead of FNA concentration. A similar phenomenon was reported by Chung et al. (2014) where thiosulfate and nitrite were used as the initial substrates.

3.4. Batch Test 4: effect of NO
3 concentration on BSO of S2O2
in chemolithotrophic denitrification 3 As the initial thiosulfate concentration was only 205 mg S/L, much lower than the possible inbitory value (2500 mg S2O2
3 eS/L, Campos et al., 2008), possible inhibition associated with high salinity of denitrifying bacteria can be neglected in this study. Although only 35 mg N/L NO
3 was spiked into Batch Reactor 12 to set a very high S/N ratio of 6:1 (the stoiwas completely oxidized chiometric ratio ¼ 2.9:1), all S2O2
3 after 24 h (see Fig. 4b). As the chemical thiosulfate transformation can be ignored (see Fig. S5a), S2O2
3 was consumed excessively by the possible biological thiosulfate disproportionation because of the sulfide generation in Batch Reactor 12 (Fig. S5b) during S2O2
3 bio-oxidation carried out by Desulfovibrio-like SRB (Jørgensen and Bak, 1991). The higher genus level of Thiobacillus-like denitrifiers (16.4%) than Desulfovibrio-like SRB (1.1%) in the AnUSB sludge (data will be reported on another paper) may be responsible for the 6-h delay of S2O2
3 disproportionation (indicated by TDS generation in Fig. S5b) compared with thiosulfate-based denitrification. The radiotracer tests will be conducted with four different 35 S-labeled 2
tracers (SO2
4 , TDS, and S2O3 with either an inner or outer S atom labeled) in a future study, to profile the detailed S2O2
3 biological disproportionation in the chemolithotrophic denitrification. The stoichiometric ratio for complete denitrification coupled with the thiosulfate full oxidation to sulfate is 0.35 mg 2
NO
3 eN/mg S2O3 eS as shown in Eq. (2). Although thiosulfate was sufficiently available for full denitrification in Batch Reactor 14 (S stoichiometrically meet), the nitrite reduction still ceased at the end of the test. This may be attributed to the fact that some of the electrons donated by thiosulfate were used for autotrophic biomass anabolism (Oh et al., 2000; Chung et al., 2014).
2
þ S2 O2
3 þ 1:6NO3 þ 0:4H2 O/2SO4 þ 0:8N2 þ 0:4H

DG00 ¼
765:7kJ=reaction

(2)

3.5. Batch Test 5: effects of organics and sulfide on BSO of S2O2
3 in chemolithotrophic denitrification The experimental results from Batch Reactors 16, 17 and 18 show that the organics actually increased the specific NO
3 reduction rate to some extent (Table 2). Under a similar initial
NO
3 concentration, the specific NO2 reduction rates in the
absence of NO3 in Batch Reactor 18, where both heterotrophic and autotrophic reactions are involved, and in Batch Reactor16, where only HD existed, are both much greater than that of Batch Reactor 13, where only autotrophic

304

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

denitrification existed (see Table 2). This confirmed that organics enhanced the NO
2 reduction rate (Reyes-Avila et al., 2004; Chen et al., 2009), though Campos et al. (2008) and Oh et al. (2000) reported no positive effect of organics on denitrification activity. This difference may be due to the different communities in the seeding sludge. In their studies, the pure autotrophic denitrifying culture was adopted for lab analysis only, but the mixed heterotrophic and autotrophic denitrifying biomass was cultivated and seeded in this study to simulate the real wastewater treatment based on sulfur bioconversion, so that the organics can significantly stimulate denitrification. No TDS generation was observed in Batch Reactors 17 and 18 which were dosed with NO
3 , organics and S2O2
3 . So unlike BSR of thiosulfate (see the Batch Test 2 results), in the BSO of thiosulfate with organics, neither S2O2
3 biological reduction to TDS with COD as electron donor nor S2O2
biological disproportionation to TDS and SO2
were 3 4 found. Sulfide at a concentration as low as 10 mg S/L exhibits toxicity in relation to many microorganisms including both heterotrophic and autotrophic denitrifying bacteria (Sorensen et al., 1980; Brunet and Garcia-Gil, 1996; Cardoso et al., 2006), because sulfide can combine with iron from cytochromes to inhibit respiration (Visser et al., 1997). Therefore, in this study, only 15 mg S/L of Na2S was spiked into

Batch Reactor 19 to investigate the effect of sulfide on thiosulfate-based chemolithotrophic denitrification. Compared with Batch Test 4 without sulfide addition, the 2
oxidation were specific rates of NO
3 reduction and S2O3 decreased by 85 and 71%, respectively (derived from the results in Table 2).

3.6.

Analysis of the microbial community

The SRB group in Batch Reactor 6 (thiosulfate-reducing reactor) comprising Desulfobulbus, Desulfococcus, Desulfomicrobium and Desulfovibrio accounts for 7.52% at genus level (see Fig. 6). Among all SRB genera in the S2O2
3 -reducing reactor (Batch Reactor 6), Desulfobulbus-like species (most abundant SRB group in this study) could biologically reduce thiosulfate to sulfide (Suzuki et al., 2007); while Desulfovibrio-like species (second abundant SRB group), accounting for 1.83% (Fig. 6), could carry out biological thiosulfate reduction together with thiosulfate disproportionation (Cypionka et al., 1998; Brunner and Bernasconi, 2005; Hernandez-Eugenio et al., 2000; Baena et al., 1998), as shown in the Batch Test 2 results. Therefore, from a microbial point of view Desulfovibrio- and Desulfobulbuslike species should be responsible for biological S2O2
3 reduction and disproportionation in Batch Test 2 of our study. This disproportionation decreases organic removal, while

Fig. 6 e Distribution of phylogenetic taxa at phylum level, class level and genus level for the sludge sample from S2O2¡ 3 reducing reactor (Batch Reactor 6) in Batch Test 2.

w a t e r r e s e a r c h 6 9 ( 2 0 1 5 ) 2 9 5 e3 0 6

fermenting biomass, such as Lactococcus, that was observed at a genus level of 1.29%, converts organics into CO2 through heterolactic fermentation (Anestis, 2006), thereby resulting in the higher CODconsumption/Sreducing ratio than the theoretical value in Batch Test 2.

4.

Conclusions

This paper presents a novel study on thiosulfate biotransformation coupled with organics and nitrogen removal in a sulfur cycle-based wastewater treatment process. The main findings are summarized as follows: 1. Organic degradation in biological sulfate/sulfite coreduction is fastest at pH 7.5, during which large amount of thiosulfate was detected as the intermediary sulfur compound. 2. Thiosulfate disproportionation, carried out by Desulfovibrio-like SRB species, occurs in biological thiosulfate reduction and may decrease the organic removal capacity. 3. In the thiosulfate oxidation/denitrification process, pH 7 is optimal for denitratation while pH 8 is optimal for denitritation. 4. Organics could enhance both the denitratation and denitritation activities in the thiosulfate-based denitrification process while sulfide, as low as 15 mg S/L, could depress the denitratation activity by 85%.

Acknowledgments The research was partly supported by the Hong Kong Research Grants Council (611211) and by the Natural Science Foundation of China through projects No. 51308558 and 51278501. Hui Lu acknowledges the support from Fundamental Research Funds for the Central Universities of China (No. 13lgpy59).

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2014.11.038.

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