A novel elemental sulfur reduction and sulfide oxidation integrated process for wastewater treatment and sulfur recycling

Accepted Manuscript A novel elemental sulfur reduction and sulfide oxidation integrated process for wastewater treatment and sulfur recycling Yiping Zhang, Liang Zhang, Lianghai Li, Guang-Hao Chen, Feng Jiang PII: DOI: Reference:

S1385-8947(18)30318-8 https://doi.org/10.1016/j.cej.2018.02.105 CEJ 18584

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

21 November 2017 21 February 2018 24 February 2018

Please cite this article as: Y. Zhang, L. Zhang, L. Li, G-H. Chen, F. Jiang, A novel elemental sulfur reduction and sulfide oxidation integrated process for wastewater treatment and sulfur recycling, Chemical Engineering Journal (2018), doi: https://doi.org/10.1016/j.cej.2018.02.105

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A novel elemental sulfur reduction and sulfide oxidation integrated process for wastewater treatment and sulfur recycling Yiping Zhang a,1 , Liang Zhang b ,1 , Lianghai Li a , Guang-Hao Chen c , Feng Jiang a, * a

School of Chemistry & Environment, South China Normal University, Guangzhou, China

b

Department of Bioscience, Aarhus University, Aarhus, Denmark

c

Department of Civil & Environmental Engineering, Chinese National Engineering

Research Center for Control & Treatment of Heavy Metal Pollution (Hong Kong Branch) and Water Technology Center, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China

*Corresponding author: Dr. Feng Jiang Email: [email protected] 1: Yiping Zhang and Liang Zhang contributed equally to this work

1

Abstract Sulfidogenic processes have been successfully used in saline wastewater treatment for sludge minimization, but are inapplicable in treating sulfate-deficit wastewater. This study reported a novel internal sulfur cycling (ISC) process for sulfate-deficit wastewater treatment. The process consisted of a sulfur-reducing reactor (SRR) for organics removal, followed by a sulfide-oxidizing reactor (SOR) and sedimentation tank for sulfur recovery. Under different hydraulic retention times and organic loading rates, the performance of ISC system was evaluated. The lab-scale ISC system removed 94% of chemical oxygen demand (COD), of which 81% were accomplished in the SRR by sulfur reducers without excessive sludge withdrawal throughout the 200 days of operation. The produced sulfide were mainly re-oxidized back to elemental sulfur in the SOR by sulfide-oxidizing bacteria, and sulfur balance demonstrates that 76% of sulfur recycling were achieved. Cost-benefit analysis reveals that the ISC process is a more cost-effective sludge-minimized biotechnology for sulfate-deficit wastewater treatment compared to conventional activated sludge processes.

Keywords: Organic removal, Sulfur packed-bed reactor, Sulfur reducers, Sludge reduction, Sulfide-oxidizing bacteria (SOB), Sulfur balance

2

1. Introduction Conventional activated sludge (CAS) processes, which are widely used for the treatment of municipal and industrial wastewater [1], generate a high amount of waste activated sludge. The treatment and disposal of sludge is one of the major challenges facing by wastewater treatment plants (WWTPs) worldwide, especially in developing countries. One such developing country is China, where the amount of sludge production had an average annual increased rate of 13 % from 2007 to 2013, and 6.25 million tons of dry sludge was produced in 2013 [2]. The cost of treating waste activated sludge is 30-60 % of a WWTP’s operational cost [3]. Biological sulfate reduction (BSR) driven processes can significantly reduce the amount of excess biosolids by 60-90% [4-7]. A typical example is the sulfate reduction, autotrophic denitrification, and nitrification integrated (SANI) process [7]. The SANI process has been demonstrated to be energy-efficient for the treatment of sulfate-laden sewage with 60-70% sludge reduction [7]. As a matter of fact, the dual water supply system of Hong Kong supplies 750 000 m3/d of seawater for toilet flushing; seawater contains sufficient amount of sulfate [8] for allowing the successful application of the BSR process in domestic wastewater treatment in Hong Kong. However, in most inland cities, the sulfate concentration (<250 mg/L) in domestic wastewater is not sufficient enough [9] to allow wide application of BSR processes. 3

As an alternative sulfur source, elemental sulfur can substitute sulfate in the BSR process. Sulfur reducers can reduce elemental sulfur, and are widespread among bacteria and archaea, such as Desulfurococcus, Methanococcus, Clostridium, Geobacter etc. [10]. They can use elemental sulfur as the electron acceptor to oxidize many organic matter, such as formate, acetate, lactate, pyruvate and sugars etc. [10]. Florentino et al. also reported that sulfur reduction process can be applied for acid mine drainage (AMD) treatment [11], in which they found that Desulfurella strain TR1 can use glycerol, methanol, acetate as the electron donors to reduce sulfur to sulfide. The biogenic sulfide can further used to remove heavy metals from AMD. Therefore, sulfur reduction process may be employed to remove organic matter from domestic wastewater. Furthermore, in our previous study, a sulfur-reducing anaerobic fluidize-bed (SRAFB) reactor removed 81% of the organic matter from domestic wastewater with low sludge production [0.16 kg VSS/kg COD (chemical oxygen demand)] by using sublimed sulfur powder as the electron acceptor [12]. The sulfide produced in the sulfur reduction process can be further oxidized back to elemental sulfur by autotrophic sulfide-oxidizing bacteria (SOB), such as Thiomonas, Thiobacillus, Halothiobacillus [13, 14]. As such, sulfur used in this process can be recovered by sulfide oxidation process. Selective sulfide oxidation process for the recovery of elemental sulfur has been extensively studied to remove dissolved sulfide 4

using SOB under oxygen-limited conditions [15-19], and high sulfur recovery (> 80%) was obtained [20, 21]. Recently, Huang et al. reported 98% of sulfur recovery was achieved in a biofilm formed membrane filtration reactor when treating sulfide- and nitrate-contaminated wastewater [22]. Therefore, we proposed a novel internal sulfur cycling (ISC) process that consists of a sulfur reduction reactor (SRR) and a sulfide-oxidizing reactor (SOR), as shown in Fig. 1. In this novel process, sulfur is reduced to sulfide (Equation 1) [12, 23], which is then oxidized back to sulfur by a selective sulfide oxidation process (Equation 2) [20]. Sulfur is insoluble in water (5 µg/L at 25 °C) [24] and can therefore be separated from the effluent and recycled. The ISC process may enable the application of BSR processes in the treatment of wastewater that has low sulfate content with less sludge production because the yields of both SRB [25] and SOB [26] are very low. Moreover, the sulfide oxidation process under micro-aerobic conditions can further remove the residual organic matter in the SOR [20]. This implies that the organic removal of the ISC process may be high enough to obtain effluent of satisfactory quality. Acetate   4S 0  H   2H 2O  2CO2  4H 2 S

2HS   O2  2S 0  2OH 

ΔG 0  39 kJ/mol

ΔG 0   169.35 kJ/mol

(1) (2)

However, the organic matter entered the SOR from the SRR effluent could favor the growth of heterotrophs and influence the growth and activity of autotrophic SOB, 5

interfering sulfur recovery and the performance of the ISC process. Sulfur recovery is a determining factor of the operational cost of the ISC process. While sulfide cannot be completely oxidized to elemental sulfur, additional sulfur is definitely required to maintain the sustainability of the ISC process. Although the low sludge production in the ISC process can save sludge treatment cost, it still remains unknown whether the ISC process is more cost-effective than CAS processes. To fill up the knowledge gaps mentioned above, the laboratory-scale ISC system was continuously operated for approximately 200 days. The objectives of this study are: 1) to investigate the performance of the ISC process in terms of organics removal and elemental sulfur recovery; 2) to identify the main-functional bacteria and understand the interactions between them in the SRR and SOR, respectively; 3) to calculate the sulfur balance and evaluate the cost-benefit of the ISC process.

2. Materials and methods 2.1 Experimental setup The laboratory-scale setup consisted of a SRR, SOR, and sedimentation tank (Fig. 1) with effective volumes of 2.3, 1.08, and 1.2 L, respectively. The SRR was a packed-bed reactor with sulfur lumps (packing ratio of 0.66) and mainly used for the removal of organic matter. In the SRR, 2770 g of sulfur lumps (99.99% purity, Damao Chemical Co., 6

China, Fig. S1) were used. The sulfur lump was identified by X-ray powder diffraction (XRD) (see Fig. S2). These sulfur lumps were classified into three groups based on its size (large for Ø = 3-5 cm, medium for Ø = 2-3 cm, and small for Ø = 1-2 cm). The large, medium, and small sulfur lumps were placed in successive layers in the reactor from the bottom to the top. The height of each layer was 150, 250, and 150 mm in order. The SOR was packed with plastic media (Ø = 1 cm, packing ratio of 0.26) and used for sulfide oxidation. The sedimentation tank was used for the precipitation of produced sulfur. A stock solution of synthetic domestic wastewater was prepared weekly by following the procedure described by Jiang et al. [6] and stored at 4 oC in a refrigerator. The stock solution was diluted with tap water in different ratios and mixed with a trace element stock solution (2 ml trace element stock solution per liter of synthetic domestic wastewater) (Table S1) prior to being fed into the SRR in order to obtain different COD concentrations (described below). 2.2. Reactor start-up and operation The seed sludge for the SRR was collected from Shatin Sewage Treatment Works in Hong Kong, and this seed sludge contained abundant sulfate-reducing bacteria (SRB) due to saline wastewater treatment [27, 28]. The SRR was fed with synthetic domestic wastewater and continuously operated for approximately 200 days. The entire operational period of the SRR was divided into three stages: start-up period (days 0-46), 7

stage A (days 47-122), and stage B (days 123-200). The internal recycle ratio was kept at 5:1 throughout the operation period. In the start-up period, stage A, and stage B, the SRR was fed with synthetic sewage containing different organic concentrations: 286 ± 26, 533 ± 36, and 302 ± 22 mg/L COD, respectively (the operational conditions are shown in Fig. S3). When the SRR was getting stable (day 120, stage B), we started to test the SOR. The seed sludge for the SOR was obtained from another sulfide-oxidizing reactor that had been operated for approximately one year in our lab. The SOR was directly fed with the effluent from the SRR to facilitate the growth and accumulation of SOB for 30 days (data not shown). The SOR was then connected with the SRR to complete the ISC system on the 150th day of SRR operation, followed by a sedimentation tank. To maximize the yield of elemental sulfur and minimize the formation of sulfate and thiosulfate, the oxidation-reduction potential (ORP) in the SOR was maintained at around -420 to -360 mV based on the study by Klok et al. [17]. The O2/H2S supply ratio was maintained at 0.6-0.8 mol/mol [21] by adjusting the air flow rate based on the measured sulfide concentration in the effluent from the SRR. In order to optimize the performance of the SOR, four different hydraulic retention times (HRTs) of the SOR (5.8, 5.2, 4.3 and 3.7 h) were investigated during days 150-167. Finally, the SOR was continuously operated at HRT of 5.2 h from day 168 to day 200. Based on the sulfide concentration in the effluent 8

of SRR, the volumetric sulfide loading rate was calculated as 1.39 ± 0.2 kg S/m3-d. The ISC system was placed in a temperature-controlled chamber (~25 oC) throughout the experiment. During the experiment, water samples were periodically collected to measure the pH, total organic carbon (TOC), sulfide, thiosulfate, and sulfate concentrations in the SRR and SOR. The ORP in the SOR was also measured. 2.3. Analytical Methods TOC was determined by using a TOC analyzer (Shimadzu TOC-5000A). COD was calculated according to a theoretical ratio of 2.67 g COD/g TOC [29] to avoid the interference due to dissolved sulfides (H2S, HS-, and S2-). In order to prevent volatilization and abiotic oxidation, dissolved sulfides were preserved by sodium hydroxide (NaOH) and zinc acetate (ZnAc) according to the Standard Methods and then measured immediately with the methylene blue method [30]. The methylene blue method is based on

the

reaction

of

sulfide,

ammonium

iron(III)

sulfate,

and

NN-dimethyl-p-phenylenediamine to produce methylene blue. Absorbance of the methylene blue was measured at 665 nm using a V-5000 spectrophotometer (Shanghai Precision Instruments Co., Ltd., China). Sulfate and thiosulfate in the liquid samples were analyzed by using an ion chromatograph (DIONEX-900) after 0.45 µm filtration. pH, ORP and dissolved oxygen (DO) were measured by a pH, ORP and DO meter, respectively (HQ40D). Sulfur selectivity cannot be measured directly because the formed S0 9

attached to the reactor wall and filter. Therefore, the sulfur selectivity was calculated based on the sulfur mass balance (Equation 3). 

2

2

2

[ S 2 ]i  [ S 2 ]e  ([SO4 ]e  [ SO4 ]i )  2[ S 2 O3 ]e 100% [ S 2 ]i

(3)

Where,  represents sulfur selectivity (%); [ S 2 ]i and [SO4 2 ]i are the influent concentrations of sulfide and sulfate of the SOR, mg S/L, respectively; [SO4 2 ]e and 2 [S 2O3 ]e are the effluent concentrations of sulfate and thiosulfate of the SOR, mg S/L,

respectively. The XRD analysis used a Bruker D8-Advance X-ray diffractometer equipped with Cu kα radiation under operation conditions of voltage 40 kV, current 40 mA and scan speed 8.0000/min, and 10-700 (2θ) [20].

2.4. DNA extraction, PCR amplification, sequencing, and analysis The total genomic DNA in the sludge samples collected from the SRR and SOR at the end of experiment was extracted by using a FastDNATM SPIN Kit for soil (MP Biomedicals, Carlsbad, CA, USA) according to the manufacturer’s instructions. The 16S rRNA gene amplification was carried out according to Kozich et al. [31]. Briefly, the extracted gDNA was amplified with a primer set targeting the V4 and V5 hypervariable regions of both the Bacteria and Archaea domains. The primer set has been modified for Illumina Miseq platform (Illumina Inc., San Diego, CA) by using a 2-nt linker and a 10-nt pad sequences. Dual indexing strategy was used with the i7 index sequences. 10

Approximately 1 ng of template DNA was used for PCR amplification in a reaction volume of 50 μl. The PCR program used in Kozich et al. [31] was adopted in this study. The amplicons obtained after gel extraction were purified using a Wizard® SV Gel and PCR Clean-up System (Promega, Madison, Wisconsin, USA). An equimolar pool was prepared according to the quantification results of the Qubit dsDNA BR Assay (Life Technologies, Carlsbad, USA). The prepared pool was checked for quality and sequenced using the Illumina Miseq platform (Illumina Inc., San Diego, CA) at the Roy J. Carver Biotechnology Center of the University of Illinois at Urbana-Champaign (IL, USA). The obtained paired-end raw 16S rRNA gene sequences were aligned with Mothur [31]. The aligned sequences were checked for chimera by USEARCH 6.1 in QIIME and classified into operational taxonomic units (OTUs) within a 0.03 difference (97% similarity) by the de novo OTU picking workflow in QIIME [32].

3. Results 3.1. Organic removal During the start-up period (days 0-46), the organic loading rate (OLR) slightly increased from 0.33 to 0.41 kg COD/m3-d by gradually reducing the HRT from 22.4 to 17.5 h. The organics removal efficiency gradually increased and was maintained at 83 ± 4% at the end of this period (Fig. 2a). As protons were produced during the sulfur reduction 11

process (see Equation 1), the effluent pH of the SRR was decreased from 7.83 ± 0.33 in the influent to 7.08 ± 0.17 (Fig. S4). In stage A (days 47-122), the influent COD increased to 533 ± 36 mg/L and the HRT was gradually decreased from 17.4 to 8.5 h. The performance of the SRR was stable although the influent OLR increased from 0.41 to 1.66 kg COD/m3-d in this stage. Similar with the pH values in the start-up period, the effluent pH in this period decreased to 7.06 ± 0.17 from 8.21 ± 0.13 in the influent (Fig. S4). The organics removal efficiency in this stage was 72 ± 11% with 461 ± 75 mg S/L sulfide production. In stage B (days 123-200), the influent organic concentration was reduced to 302 ± 22 mg COD/L. The organics removal efficiency was around 80% when the HRT decreased from 7.2 to 4.3 h and finally reached 81 ± 7% with 342±32 mg S/L sulfide generation at an OLR of 1.66 kg COD/m3-d. The influent pH in this stage was similar with that in the stage A, and the effluent was declined to 6.97 ± 0.14 (Fig. S4).

In addition to the removal of 81 ± 7% of the organic matter by the SRR, the SOR further removed 13 ± 4% of the organics from the wastewater within 5.2 h (Fig. 3). Therefore, the ISC system removed 94 ± 2% of the influent organics in total and the effluent COD concentration was only 19 ± 8 mg/L. This present results show that the ISC process can efficiently remove organic matter from domestic wastewater. 3.2. Contribution of sulfur reduction to organic removal

12

To evaluate the contribution of sulfur reduction to organics removal in the SRR, the C/S ratios (mg oxidized organic carbon/mg produced sulfide) were calculated based on Equation 1 (Fig. 2b). The C/S ratios in start-up period, stages A and B were 0.42, 0.35 and 0.25 on average, respectively (Fig. 2b), indicating that the C/S ratios approached the theoretical value (0.19) with time, and the contribution of sulfur reduction to organics removal increased along with time. In the stage B, approximately 76% of the removed organics were completely oxidized through sulfur reduction. Previous studies reported that sulfite/sulfate reduction contributed to 86 % of the organics removal in a sulfite-reducing UASB reactor [6] and 77% of the organics removal in a sulfate-reducing UASB reactor [33]. The present results suggest that sulfur reducers prevailed in the SRR.

3.3. Sulfide oxidation and S0 selectivity When the O2/H2S supply ratio was in the range of 0.6-0.8 mol/mol, the DO concentration in the SOR was less than 0.2 mg/L. Fig. 4 shows that the sulfide removal efficiency increased with decreased sulfide loading rates, and the SOR achieved a stable removal of 94 ± 6 % of the influent sulfide at a sulfide loading rate of 1.39 ± 0.2 kg S/m3-d with a HRT of 5.2 h during the stable stage (days 170-200). As the SOR was getting stable, the sulfur selectivity increased along with time. The main product generated by sulfide oxidation was elemental sulfur (81 ± 15%), and part of the influent sulfide was oxidized into sulfate and thiosulfate (Fig. 4 and Fig. S5). The oxidation of 13

sulfide to sulfur was attributed to two enzymes, flavocytochrome c-sulfide dehydrogenase (FCSD) and sulfide:quinone reductase (SQR) [14]. Klok et al. proposed a limited oxygen route (LOR) for sulfide oxidation under oxygen-limited conditions, in which the SOB are able to reduce NAD+ without direct transfer of electrons to oxygen, leading to the low yield of SOB and high selectivity of S0 as the end-product [17]. This mechanism could explain the high recovery of S0 in the SOR. These results indicate that the sulfide oxidation was not influenced by the presence of organic matter. As described in Section 3.1, the SOR further improved the organics removal, indicating that SOB and heterotrophs can coexist in the reactor under the low COD concentration and oxygen-limited conditions (supported by the microbial analysis in Section 3.4). Sahinkaya et al. [20] found that 64-89% of sulfide were converted to S0 in the presence of COD in a membrane biofilm reactor. Huang et al. [34] observed that SOB converted 77.9% of the influent sulfide into sulfur using nitrate as an electron acceptor in the presence of acetate. Wang et al. [35] reported that 100% of sulfur selectivity can be achieved in the treatment of sulfide and nitrate using waste activated sludge fermentation liquid as carbon source under micro-aerobic conditions. In addition, compared to the effluent pH of SRR (7.00 ± 0.15), the effluent pH of SOR was increased by 0.8 (7.83 ± 0.29) (Fig. S4). 3.4. Microbial community analysis As the performance of the SRR and SOR highly depends on the microbial 14

community compositions, the microbial communities in the SRR and SOR were analyzed by the lllumina sequencing at the end of the experiment. More than 14000 sequences were obtained from each reactor. The number of OTUs in the microbial sequences obtained from the SRR and SOR were 361 and 134, respectively. Four bacterial phyla were observed in the SRR: Firmicutes (98.4 %), Proteobacteria (1.3 %), Chloroflexi (0.2 %), and Actinobacteria (0.1 %). Chlorobi (41 %), Bacteroidetes (39.4 %), Proteobacteria (19.3 %), and Firmicutes (0.3 %) were the bacterial phyla in the SOR (Fig. S6). The bacterial communities in the SRR and SOR also showed high diversity at the family level (Fig. S6). In the SRR, the main fermentation genera were Trichococcus and Leuconostoc with a relative abundance of 12.4% and 11.7%, respectively (Fig. 5). They can degrade complex organics that may be difficult for sulfur reducers to utilize into simple ones, such as short-chain VFAs, which can be readily used by sulfur reducers [36]. Clostridium was the identified sulfur-reducing genus, accounting for 15.9% (Fig. 5). The genus Clostridium was previously detected in a sulfur-reducing bioreactor for acid mine drainage treatment [37] . The high organics removal efficiency and sulfide production in the SRR was attributed to the high enrichment of sulfur reducer. In the SOR, both heterotrophs and SOB prevailed. Paludibacter (relative abundance of 39.4 %) (Fig. 5) degraded complex organics (28 ± 8 mg/L) into simple ones 15

[38, 39], which were subsequently converted into CO2 in the presence of oxygen [20]. Halothiobacillus (13.7%) and Thiomonas (5.2%) were the identified SOB (Fig. 5). Thus, we can speculate that Paludibacter can oxidize the organics in the SOR and consume excessive oxygen, creating a favor condition (oxygen-limited and organic-limited) for the growth and activity of SOB. Vannini et al. [40] reported that the genus Halothiobacillus was the dominant SOB in a membrane bioreactor treating leather tanning industrial wastewater. The genus Thiomonas is a typical SOB and was frequently observed in sulfide oxidation bioreactors [41-43]. In addition, lino et al. [44] claimed that Ignavibacterium album sp. in the Ignavibacteriaceae family, which was isolated from a sulfide-rich hot spring in Japan, is a green sulfur bacterium. Approximately 41% of the genera belonging to the Ignavibacteriaceae family are unclassified. We speculate that some of these unknown genera may have also contributed to sulfide oxidation in the SOR, which merits further investigation.

4. Discussion 4.1. Effect of sulfur particle size on sulfur reduction The SRR efficiently removed 245 mg/L COD from the domestic wastewater and generated 342 mg/L sulfide within 4.3 h. The organics removal and sulfide production 16

rates in the SRR were moderately lower than those in the SRAFB reactor [12]. In fact, the sulfur lumps (Ø = 1-5 cm) used in the sulfur packed-bed reactor in this study were much larger in size than the sublimed sulfur powder (Ø = 20-40 μm) used in the SRAFB reactor [12]. The large size of the sulfur lumps significantly reduced the specific surface area, which is essential for the direct attachment of sulfur reducers to solid sulfur. However, the organics removal rate did not decrease significantly when sulfur lumps were used as the biofilm carriers and electron acceptors in this study. This suggests that the direct contact between sulfur reducers and solid sulfur may not be main pathway for high-rate sulfur reduction. As polysulfide can be formed via abiotic reaction between sulfide and sulfur (Equation 4) [45], we speculate that the sulfur lumps were firstly converted into polysulfide and polysulfide was further reduced to sulfide within the cell by a polysulfide reductase-like complex (Equation 5, taking formate as an example) [46]. During the polysulfide metabolism, the Sud-His6 protein enhances the transfer of polysulfide to the active site of polysulfide reductase [47], allowing fast respiration of polysulfide even at low concentration [46]. This mechanism leads to a self-accelerating sulfur reduction process and ensures the high rate of carbon mineralization, which has been reported in our previous study [48]. Accordingly, large-sized sulfur particles or lumps, which are cheaper than sublimed sulfur powder, can be used in sulfur reduction processes. HS  

n 1 2 S8  S n  H  8

(4) 17



2



2

HCO2  S n  H 2 O  HCO3  HS   S n 1  H 

(5)

4.2. Organic removal When the HRT of the SRR was decreased from 22.4h to 4.3h during the entire operational period, the organic removal in the SRR seemed not to be influenced, indicating that the sulfur reducers had high activities to resist the impacts of the tested HRT range. During the final stage (stage B), the organics removal efficiency of the SRR was maintained at 81 ± 7%. In comparison, methane-producing UASB reactors achieved 65-85% COD removal under moderate temperature conditions [49, 50]. The present results show that the SRR outcompetes methane-producing UASB reactors. In addition to the high organics removal in the SRR, the SOR further enhanced the organics removal by oxidizing organic carbon into CO2 under micro-aerobic conditions (Equation 6) [20]. Therefore, the ISC system effectively removed organics from domestic wastewater. Nitrogen removal was not considered in this study but it has been well investigated in our previous studies on BSR processes, such as SANI and flue gas desulfurization-SANI (FGD-SANI) processes [6, 7]. Hence, the ISC process can be easily upgraded for nitrogen and phosphorus removal as well. CH 3COOH  2O2  2CO2  2H 2O

(6)

4.3 Sulfur balance 18

A small amount of sulfate (21 ± 12 mg S/L) (Fig. S3) was observed in the SRR effluent, indicating that biological sulfur disproportionation to sulfide and sulfate occurred to a small degree in the SRR. Subsequently, the produced sulfide were oxidized back to sulfur and undesired sulfate and thiosulfate (in small amounts) in the SOR. Considering sulfate generation in the SRR (6 % of conversion), the sulfate and thiosulfate produced in the SOR, and the residual sulfide in the SOR effluent, the sulfur loss in the ISC system during the stable operation stage was approximately 24% (83 mg S/L). Thus, the ISC system was able to recover 76 % of the sulfur consumed. 4.3. Cost-benefit analysis of the ISC process The G 0 value of the sulfur reduction process is as low as those of the sulfate and sulfite reduction processes based on Equations 1, 7 and 8 [6, 51]. This suggests that sulfur reduction processes also provide low energy for cell growth like the sulfate and sulfite reduction processes. In our previous study, we have reported a sludge production of 0.16 kg VSS/kg COD in the sulfur reduction process [12]. This value was used for the economic assessment of the ISC process in this study since sludge yield cannot be directly measured in sulfur packed-bed reactor.

Acetate   SO4

2



 2HCO3  HS 

ΔG 0  48 kJ/mol

4 5 1 4 2  2 Acetate   SO3  HCO3  CO3  HS  3 3 3 3 19

ΔG 0  80 kJ/mol

(7) (8)

Assessment of the sludge production in the ISC process should also consider the sludge yield in the SOR. Mora et al. [26] reported that the biomass (C5H7NO2) growth yields of sulfur and sulfate producers were 0.016 and 0.073 mol VSS/mol S, respectively. Since thiosulfate was mainly generated from the abiotic oxidation of polysulfide and the hydrolysis of nascent sulfur [21], it did not contribute to the sludge production in the SOR. Thus, the sludge production and sulfur balance were calculated and demonstrated in Fig. 6.

When one ton of COD was completely removed by the ISC process, it can be known that the SRR and SOR removed 0.86 ton and 0.14 ton of COD, respectively. The SRR would produce approximately 0.14 ton of sludge and reduce 1.37 ton of elemental sulfur based on the sludge production of 0.16 kg VSS/kg COD and C/S ratio of 0.25 mg C/mg S. Based on the measured selectivity of sulfur and sulfate formation in the SOR, we calculated that approximately 0.09 ton of sludge would be produced during the sulfide oxidation process according to the different yields of sulfate and sulfur producers (Fig. 6). Considering the biological oxidation of the residual organic matter and based on the sludge yield in CAS processes, it can be determined that the SOR produced 0.06 ton of sludge. The sludge yield coefficients in CAS processes used for municipal wastewater treatment range from 0.35 to 0.47 kg VSS/kg COD with an average value of 0.41 kg VSS/kg COD [52-54]. The total sludge production in the ISC system was calculated to be 20

0.28 kg VSS/kg COD (Fig. 6), which is significantly lower than that in CAS processes. In China, the sludge treatment costs through incineration, used as building materials, and land application are 240-360, 240-360 and 240-480 $/ton dry sludge, respectively [2]. Even when the chemical cost of the sulfur supplement (61 $/ton) is considered [11], the total operational cost required for the ISC process to remove 1 ton of COD from wastewater is 15% less than that of activated sludge processes (in the case that incineration is used for sludge treatment) (Fig. 6). However, this benefit would be higher if we take the energy consumption for aeration into account. This is because that the energy cost for aeration would be much lower than in the aerobic reactor of CAS processes (micro-aeration vs. aeration). Although this is a notable benefit, it is not attractive enough. Minimization of sulfur loss in the ISC process is essential for further reduction in the operational cost of the ISC process.

5. Conclusions The ISC process achieved 94% of COD removal, and 81% of which was accomplished by sulfur reducers in the SRR, without excessive sludge withdrawal throughout the 200 days of operation. The SOR not only oxidized the sulfide back to S0 by SOB (81% of selectivity), but also removed the residual COD by coexisting heterotrophs. The sulfur balance reveals that 76% of the sulfur consumed can be 21

recycled in the ISC system, which ensures the feasibility of the ISC process. According to the sulfur balance calculation and cost-benefit analysis, the ISC process is a cost-effective sludge-minimized biotechnology for domestic wastewater treatment.

Acknowledgements The authors acknowledge the support from the National Natural Science Foundation of China (51178914 and 51638005), the Guangdong Provincial Science and Technology Planning Project (2016A050503041 and 2017B050504003), and the Hong Kong Innovation and Technology Commission (ITC-CNERC14EG03).

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Figure captions Fig. 1. Schematic diagram of the internal sulfur cycling system: (1) feeding tank, (2) sulfur-reducing reactor, (3) buffer tank, (4) sulfide-oxidizing reactor, (5) sedimentation tank, and (6) effluent tank. Fig. 2. The performance of the SRR during the 200-day operation period: (a) COD removal and (b) Effluent sulfide production and the corresponding C/S ratios. Fig. 3. COD removal efficiency in the SRR and SOR of the ISC system during the integrative operational period (the operational days of the ISC system were determined based on the operational days of the SRR). Fig. 4. Elemental sulfur recovery, sulfide removal, and sulfide loading rate in the sulfur-reducing reactor (the operational days of the SOR system were determined based on the operational days of the SRR). Fig. 5. Taxonomic classification of the bacterial 16S rRNA gene sequences retrieved from the SRR and SOR at the genus level using RDP classifier with a confidence threshold of 97 %. Fig. 6. The sulfur balance and cost-benefit analysis of the ISC process compared to that of conventional activated sludge processes.

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Fig. 1. Schematic diagram of the internal sulfur cycling system: (1) feeding tank, (2) sulfur-reducing reactor, (3) buffer tank, (4) sulfide-oxidizing reactor, (5) sedimentation tank, and (6) effluent tank.

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Fig. 2. The performance of the SRR during the 200-day operation period: (a) COD removal and (b) Effluent sulfide production and the corresponding C/S ratios.

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Fig. 3. COD removal efficiency in the SRR and SOR of the ISC system during the integrative operational period (the operational days of the ISC system were determined based on the operational days of the SRR).

30

Fig. 4. Elemental sulfur recovery, sulfide removal, and sulfide loading rate in the sulfur-reducing reactor (the operational days of the SOR system were determined based on the operational days of the SRR).

31

Fig. 5. Taxonomic classification of the bacterial 16S rRNA gene sequences retrieved from the SRR and SOR at the genus level using RDP classifier with a confidence threshold of 97 %.

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Fig. 6. The sulfur balance and cost-benefit analysis of the ISC process compared to that of conventional activated sludge processes.

33



A novel sludge minimized internal sulfur cycling (ISC) process was proposed.



The ISC process removed 94% of organics, 81 % of which was due to the SRR.



The ISC process recovered approximately 76% of the sulfur consumed.



The main sulfur-reducing and sulfide-oxidizing bacteria were identified.



Cost-benefit of the ISC process was assessed.

34