Anaerobic treatment of sulfur-rich wastewater is challenging because sulfide greatly inhibits the activity of anaerobic microorganisms, especially methanogenic archaea. We developed an internal phase-separated reactor (IPSR) that removed sulfide prior to methanogenesis by gas stripping using biogas produced in the reactor. The IPSR was fed with synthetic wastewater containing a very high sulfide concentration of up to 6,000 mg S L−1 with a chemical oxygen demand (COD) of 30,000 mg L−1. The IPSR was operated at an organic loading rate of 5–12 kg COD m−3 day−1 at 35 °C. The results show that the sulfide concentration was reduced from 6,000 mg S L−1 in the influent to <700 mg S L−1 in the first-stage effluent. The second-stage effluent contained <400 mg S L−1. As a result of effective sulfide removal by its gas stripping function, the IPSR had a COD removal efficiency of >90% over the entire experimental period. High-throughput 16S rRNA gene sequencing revealed that the major anaerobic archaea were Methanobacterium and Methanosaeta, which are frequently found in high-rate anaerobic reactors. Thus, the IPSR maintains these microorganisms and achieves high-process performance even when fed wastewater with very high sulfide concentrations.

  • An internal phase-separated reactor (IPSR) was developed.

  • The IPSR was used to treat sulfide-rich wastewater (3,000–6,000 mg S L−1).

  • Sulfide was removed by gas stripping using biogas produced in the IPSR.

  • The IPSR was operated with 5 kg chemical oxygen demand m−3 day−1 and 6,000 mg S L−1.

  • The IPSR maintained highly efficient organisms even under high sulfide conditions.

Sulfur-containing wastewater is produced by the pharmaceutical industry (Li et al. 2015), petroleum refineries (Altaş & Büyükgüngör 2008), paper mills (Janssen et al. 2009), paper recycling plants (Damianovic et al. 2018), slaughterhouses (Yan et al. 2018a), vinasse production (Barrera et al. 2014), and other processes (Pol et al. 1998; Robles et al. 2020; Shao et al. 2024). Research on the treatment of wastewater with a high sulfur concentration has increased over the past three decades and has focused on biological methods (Ding & Zeng 2022). However, the implementation of anaerobic reactors for treatment of sulfur-rich wastewater is challenging because sulfide (HS) produced by sulfate () is an inhibitor of various microorganisms, including sulfate-reducing bacteria and methane-producing archaea (Harada et al. 1994; O'Flaherty et al. 1998; Pol et al. 1998; Chen et al. 2008; Lens et al. 2010; Hao et al. 2014). The level of sulfide inhibition depends on the sulfide concentration and other environmental conditions, such as the pH during anaerobic digestion (Koster et al. 1986; Omil et al. 1995; Chen et al. 2008; Lee & Hwang 2019).

Various technologies have been used to remove sulfur from wastewater before or during the anaerobic digestion process. These technologies can be categorized as physicochemical techniques (e.g., gas stripping), chemical reactions (e.g., precipitation), and biological conversion (Chen et al. 2008; Robles et al. 2020). However, these sulfur removal processes need additional equipment (e.g., gas-stripping devices) and/or materials (e.g., chemicals). For successful application, the treatment technologies should be environmentally friendly, simple, easy to manage, and inexpensive.

To fulfill the need for appropriate technology, we recently developed a new reactor configuration that we call an internal phase-separated reactor (IPSR) (Onodera et al. 2022). The IPSR removes H2S by gas stripping using biogas produced during anaerobic treatment (Onodera et al. 2022). Removal of H2S from wastewater by gas stripping is possible because the biogas has a relatively low H2S concentration compared with the wastewater. This function is attributed to the structure of the IPSR. The IPSR consists of a first stage for gas stripping in an upper section of the reactor and a second stage for methane production in a lower section. A gas–liquid partitioning (GLP) valve is installed between the stages as a phase separator. The GLP valve stops liquid from flowing between the first and second stages and supplies biogas from the second stage to the first stage.

Previous experiments using a laboratory-scale reactor verified that the IPSR showed superior performance for sulfide removal compared with a control reactor when the sulfate concentration was between 400 and 800 mg S L−1 (Onodera et al. 2022) and the sulfide concentration was between 400 and 600 mg S L−1 (Onodera et al. 2023). After using the IPSR for sulfide removal, the sulfide concentration in the final effluent from the IPSR was half to one-third that obtained with the control reactor (Onodera et al. 2023). However, the various types of industrial wastewater contain different concentrations of sulfides (Shao et al. 2024). After spent caustic, the type of wastewater with the highest sulfide concentrations is distillery spent wash, which contains 102–130 g chemical oxygen demand (COD) L−1 and 1,100 to 3,000 mg SO4 L−1 (360 to 1,000 mg S L−1) (Karhadkar et al. 1990). For stillage from ethanol production, the average COD and total sulfur concentration as SO4 for various feedstocks are 91.1 g COD L−1 and 3,716 mg SO4 L−1 for beet molasses, 30.4 g COD L−1 and 1,356 mg SO4 L−1 for cane juice, 84.9 g COD L−1 and 3,478 mg SO4 L−1 for cane molasses, and 61.3 g COD L−1 and 651 mg SO4 L−1 for cellulosic feedstocks (Wilkie et al. 2000). Thus, for successful application of the IPSR to various types of sulfur-containing wastewater, it is necessary to evaluate the process performance when treating wastewater with much higher concentrations of sulfide than in our previous studies.

In this study, the process performance of IPSR for the treatment of wastewater containing much higher concentrations of sulfides (3,000–6,000 mg S L−1) than in our previous study (Onodera et al. 2023). The process performance was evaluated for sulfide removal, biogas production, and H2S removal by gas stripping. The prokaryotic microbial community under high sulfide concentrations was also determined.

Synthetic wastewater

Synthetic sulfide-rich wastewater was prepared with a fixed COD of 30,000 mg L−1. The organic compounds were 12,000 mg L−1 sucrose, 9,000 mg L−1 CH3COONa, 4,500 mL L−1 propanoic acid, and 3,000 mg L−1 peptone. Sodium sulfide (Na2S·9H2O) was used as a sulfide source (inhibitor) at concentrations between 0 and 6,000 mg S L−1. The following compounds were added to the wastewater: 320 mg L−1 Na2SO4, 2,000 mg L−1 NaHCO3, 150 mg L−1 KH2PO4, 150 mg L−1 CaCl2·2H2O, 400 mg L−1 MgCl2·6H2O, 300 mg L−1 KCl, 110 mg/L NH4Cl, 3.93 mg L−1 FeCl2·4H2O, 0.17 mg L−1 CoCl2·6H2O, 0.07 mg L−1 ZnCl2, 0.06 mg L−1 H3BO3, 0.5 mg L−1 MnCl2·4H2O, 0.04 mg L−1 NiCl2·6H2O, 0.03 mg L−1 CuCl2·2H2O, and 0.03 mg L−1 NaMoO4·2H2O. An anti-foaming reagent (Antifoam SI, FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) was also added. The wastewater composition was established in our previous study (Onodera et al. 2023). The synthetic wastewater was prepared freshly each week and kept at ambient temperature.

Reactor setup and process operation

A schematic diagram of the laboratory-scale IPSR is shown in Figure 1. The IPSR consisted of a first stage (upper section), a phase separator (GLP valve), and a second stage (lower section). The IPSR was cylindrical (diameter: 0.079 m) and had a total height of 2.30 m (1.10 m for the first stage, 0.15 m for the phase separator, and 1.05 m for the second stage). The working volumes in the first and second stages were 5.49 and 5.15 L, respectively. A water jacket connected to a hot water circulator was used to control the temperature of the reactor at 35 °C. These volumes were used to calculate the volumetric organic loading rate (OLR) and hydraulic retention time (HRT) in the second stage (Table 1).
Table 1

Operating conditions of the internal phase-separated reactor during the experiment

First periodSecond periodThird periodFourth periodFifth period
Operation period Day 14 
Flow rate L day−1 2.06 2.06 1.54 1.03 0.85 
Influent concentration 
 COD mg L−1 30,000 30,000 30,000 30,000 30,000 
 Sulfate mg S L−1 72 72 72 72 72 
 Sulfide mg S L−1 3,000 4,000 5,000 6,000 
Second stage 
 HRT 60 60 80 120 145 
 COD loading g COD L−1 day−1 12.0 12.0 9.0 6.0 5.0 
First periodSecond periodThird periodFourth periodFifth period
Operation period Day 14 
Flow rate L day−1 2.06 2.06 1.54 1.03 0.85 
Influent concentration 
 COD mg L−1 30,000 30,000 30,000 30,000 30,000 
 Sulfate mg S L−1 72 72 72 72 72 
 Sulfide mg S L−1 3,000 4,000 5,000 6,000 
Second stage 
 HRT 60 60 80 120 145 
 COD loading g COD L−1 day−1 12.0 12.0 9.0 6.0 5.0 
Figure 1

Schematic diagram of the internal phase-separated reactor (IPSR): (1) first stage (upper section); (2) second stage (lower section); (3) phase separator (gas–liquid partitioning valve); (4) water jacket; (5) water seal; (6) H2S trapping pellet; (7) gas meter; (8) influent tank; (9) pump; and (10) gas sampling port.

Figure 1

Schematic diagram of the internal phase-separated reactor (IPSR): (1) first stage (upper section); (2) second stage (lower section); (3) phase separator (gas–liquid partitioning valve); (4) water jacket; (5) water seal; (6) H2S trapping pellet; (7) gas meter; (8) influent tank; (9) pump; and (10) gas sampling port.

Close modal

The first stage, which was used for H2S gas stripping, was filled with synthetic wastewater without sludge before start-up. The second stage, which was used for methane production, was filled with mesophilic granular sludge derived from an upflow anaerobic sludge blanket (UASB) reactor operated at an OLR of 15 kg COD m−3 day−1 under mesophilic conditions (35 °C) to eliminate the start-up period. The UASB was fed with synthetic wastewater that had a similar composition to the wastewater used in the present study. The operating conditions and reactor performance are detailed in Supporting Information (Table S1 and Figure S1). The sludge was transferred to the reactor, and the operation was started within 30 min. The sludge contained 69.2 g L−1 total suspended solids (TSS) and 63.7 g L−1 volatile suspended solids (VSS) (VSS/TSS = 0.92). The total volume of the sludge was 4.0 L (277 g of TSS and 255 g of VSS). After the experiment, the sludge mass in the IPSR contained 248 g of TSS and 225 g of VSS.

The effluent was recirculated to the second-stage influent at twice the flow rate (recirculation rate: 200%). A pH sensor was placed in the upper portion of the first section to maintain the pH at <5.9 by feeding HCl solution (3 mol L−1) into the influent line. The operation was divided into five periods according to the sulfide concentration (0–6,000 mg L−1) and OLR (Table 1). The influent COD concentration was fixed, and the flow rate was changed to adjust the HRT and OLR. The operation was conducted for 1 week for each period, except for the fifth period, which was 2 weeks. Between each operation period from one to five, some of the wastewater in the first stage was replaced with new wastewater with a high sulfide concentration to ensure the desired high sulfide concentration in the second stage was reached quickly.

Chemical analysis

Liquid samples of the influent, first-stage effluent, and second-stage effluent were collected. The pH was measured immediately after sampling using a pH meter (9625-10D, D-74, LAQUA, Kyoto, Japan). The dichromate COD and sulfate (SO4) were analyzed using a Hach water quality analyzer (DR1900, Hach, Loveland, CO, USA). The COD was measured using Hach testing kits (TNT822 and TNT823, Hach) after sulfide removal by sulfuric acid addition, followed by purging with N2 gas. The SO4 concentration was analyzed using a SulfaVer 4 reagent powder pillow (Hach). Sulfide in solution was determined using a total sulfide detector tube (coefficient of variation: 5%; 201H, HYDROTEC-S 330, GASTEC, Kanagawa, Japan). To prevent volatilization of H2S, liquid samples were taken directly from the tubes using a syringe while preventing contact with air and then stored in the sealed syringes until required for analysis. The TSS and VSS were determined using standard methods (APHA 2005).

In the IPSR, biogas produced in the lower section moved to the upper section and was then released. The hydrogen sulfide in the gas was measured using a gas detector tube system (coefficient of variation: 5–10%; 4HP, GV-100S, GASTEC). The biogas was desulfurized in an H2S trap column packed with ferrous oxide pellets. After desulfurization, the volume of the biogas was measured with a wet-test gas meter (WS-1A, Shinagawa, Tokyo, Japan). The biogas was collected in a gas bag and the biogas composition (CH4, CO2, N2, O2, and H2) was measured using a gas analyzer (Biogas 5000, Geotech, Geotechnical Instruments Ltd, Leamington Spa, UK). The volume of methane produced was calculated in the standard state (0 °C, 1 atm). The H2S concentration in the liquid phase was determined using the pH and total sulfide (H2S and HS) concentration using Equation (1) (Speece 1996).
(1)
where the pKa is 6.83 (35 °C).

Microbial community analysis

Samples of 0.5 mL of seeding sludge (before the experiment) and retained sludge (on day 42 after the initiation of the experiment) were collected for microbial community analysis. The collected sludge samples were stored at −80 °C until required for DNA extraction and subsequent 16S rRNA gene sequencing. The DNA extraction and 16S rRNA gene sequencing were performed at the Bioengineering Lab. Co., Ltd (Kanagawa, Japan). Briefly, the collected sludge samples were initially freeze-dried (VD-250R freeze dryer, TAITEC Corporation, Saitama, Japan) and then mixed vigorously at 1,500 rpm for 2 min using a multi-beads shocker (Yasui Instruments, Osaka, Japan). Next, Lysis Solution F (a mixture of sodium dodecyl sulfate and EDTA-2Na; Nippon Gene, Tokyo, Japan) was added to each sample. The samples were incubated at 65 °C for 10 min for cell lysis and then centrifuged at 12,000 ×g for 2 min. The supernatant was collected. DNA was purified from the supernatant using a Lab-Aid824s DNA extraction kit (Zeesan, Xiamen, China). The amplicon libraries of the V4 region of prokaryotic 16S rRNA genes were prepared by a two-step-tailed polymerase chain reaction method with 515F/806R primers (Caporaso et al. 2010). The resulting libraries were sequenced using the MiSeq platform with MiSeq reagent kit v3 (Illumina, San Diego, CA, USA). The obtained reads were trimmed with FASTX-Toolkit (ver. 0.0.14) and the amplicon sequence variants were generated using DADA2 implemented in QIIME 2 (ver. 2022.8) (Bolyen et al. 2019). The taxonomic classification of each amplicon sequence variant was performed using the SILVA 138 database (Quast et al. 2013). The obtained raw 16S rRNA gene amplicon sequence data are available in the DNA Data Bank of Japan Sequence Read Archive (https://ddbj.nig.ac.jp) under accession numbers DRR527022–DRR527023.

Process performance

The operating conditions and water quality are shown in Figure 2. The reactor experiments verified that the IPSR gave a high COD removal efficiency of approximately 90% with effective sulfide removal of up to 6,000 mg S L−1 under an OLR of 5 kg COD m−3 day−1 at 35 °C. The IPSR was gradually fed with synthetic wastewater containing 3,000–6,000 mg S L−1 and the OLR was decreased from 12 to 5 kg COD m−3 day−1 (Figure 2(a) and 2(b)). Although the upflow velocity in the fifth period was low, with a low flow rate in the IPSR, mixing was maintained with 200% recirculation and high biogas production. Therefore, the effect of the flow rate on the process performance of the IPSR in each period was not considered in this experiment. The pH in the influent was high because a strongly alkaline solution of Na2S·9H2O was added as the sulfide source. The pH of the first stage was controlled at <5.9, which is the preferred pH for sulfide removal by gas stripping. The pH in the second stage was maintained at 7.5–8.0 (Figure 2(c)). With these parameters, the IPSR had a COD removal efficiency of >90%, and the effluent COD concentration was <3,000 mg/L (Figure 2(d)). In the second stage, the sulfate concentration in the influent was markedly reduced (Figure 2(e)). Although the influent sulfide concentration drastically increased from 3,000 mg S L−1 in the second period to 6,000 mg S L−1 in the fifth period, the first-stage effluent had a lower concentration (700 mg S L−1). Additionally, the second-stage effluent contained <400 mg S L−1 (Figure 2(f)). These results indicate that sulfide removal is mainly carried out in the first stage, and the effluent provided in the second stage has a low sulfide concentration.
Figure 2

Evolution of the influent sulfide concentration (a), organic loading rate (OLR) in the second stage of the IPSR (b), pH (c), COD (d), sulfate in first- and second-stage effluents (e), and sulfide in first- and second-stage effluents (f).

Figure 2

Evolution of the influent sulfide concentration (a), organic loading rate (OLR) in the second stage of the IPSR (b), pH (c), COD (d), sulfate in first- and second-stage effluents (e), and sulfide in first- and second-stage effluents (f).

Close modal

Although the sulfide concentration in the second-stage effluent in the present study was <400 mg S L−1, the concentration of free H2S was lower (39 mg S L−1) at pH 7.8 because the H2S/HS ratio was low. Free H2S is a stronger inhibitor than HS. The maximum free H2S concentration recommended to avoid severe inhibition is 125 mg S L−1 (Omil et al. 1995). In another study, the free H2S concentrations leading to 50% inhibition were 250 mg S L−1 at pH 6.4–7.2 and 90 mg S L−1 at pH 7.8–8.0 (Koster et al. 1986). Therefore, the pH in the IPSR could alleviate H2S inhibition even though the sulfide concentration was 400 mg S L−1 in the second stage of the IPSR.

Biogas production and H2S removal

The evolution of biogas production in the IPSR is shown in Figure 3. The biogas production decreased stepwise, with decreases in the OLR (Figure 3(a)). The CH4 content of the biogas before H2S removal was 60–70% (Figure 3(b)). The average CO2 content was 33.6%. The CH4 production was calculated from the quantity of biogas produced and its CH4 concentration (Figure 3(c)). The CH4 production per wastewater volume was relatively stable throughout the experiment (Figure 3(c)). These results suggest that CH4 production is associated with COD removal in the IPSR and indicate that the COD removed is converted to CH4.
Figure 3

Evolution of biogas production after H2S removal (a), CH4 concentration after H2S removal (b), and CH4 production as COD based on the wastewater volume (c).

Figure 3

Evolution of biogas production after H2S removal (a), CH4 concentration after H2S removal (b), and CH4 production as COD based on the wastewater volume (c).

Close modal
The H2S content in the biogas was measured before the desulfurization process (Figure 4(a)). With no sulfide addition to the influent wastewater (first period), the biogas contained <0.3% (3,000 ppm) H2S. The H2S content reached 10.5–11.2% in the second period and >15.0–16.8% in the third period. For the third and fourth periods, the biogas had a similar H2S content. In the fifth period, the H2S content of the biogas was >23.0%. The H2S contents found in this study are much higher than those from previous reports. In one earlier study, the H2S content was estimated at 0.1 to >17% from the feedstock carbon/sulfur ratio (Peu et al. 2012). In another study, the H2S content in biogas was typically between 50 and 5,000 ppm (0.5%) (Lupitskyy et al. 2018). Other research found that the H2S content was correlated with the influent sulfide concentration and inhibition level (Dykstra & Pavlostathis 2021).
Figure 4

Evolution of the H2S concentration in the biogas (a) and H2S removed from the IPSR using biogas (b).

Figure 4

Evolution of the H2S concentration in the biogas (a) and H2S removed from the IPSR using biogas (b).

Close modal

The H2S removal efficiency using biogas was calculated from the biogas production and H2S content (Figure 4(b)). During the experiment, the H2S removal efficiency increased with increases in the influent sulfide concentration. The IPSR provided better H2S removal when the influent sulfide concentration was high rather than low. In the IPSR, the use of biogas is a key feature, and the amount of biogas is an important factor affecting the gas stripping efficiency. The sulfide removal mechanism involves (1) the production of biogas with organic matter removal, (2) the removal of H2S by gas stripping using the biogas, and (3) the removal of organic matter under low sulfide conditions (Onodera et al. 2022).

Sulfur mass balance

The sulfur mass balance is shown in Figure 5. The sulfur input was from the synthetic wastewater (72 mg S L−1) and additional sulfide during the experiment. The sulfur output consisted of (1) residual or reduced sulfide in the effluent, (2) H2S removed from the wastewater via biogas, and (3) residual in the effluent. The H2S removal in the biogas was calculated from the wastewater flow rate, H2S gas content, and the biogas production rate. Most of the influent sulfur was derived from the additional sulfide after the second period. Gas stripping via biogas, which depended on the pH and biogas production, was the main mechanism for H2S removal from the wastewater. The results clearly showed that influent sulfide was removed as H2S via biogas in the IPSR, which decreased the sulfide concentration in the effluent. The content of unknowns in the effluent was approximately 19% in the fifth period. For sulfides, the content of unknowns will be partially influenced by the adsorption of sulfides by metals. Accurate measurement of the sulfide mass balance was difficult because of chemical and biological reactions, and loss with off-gassing (Damianovic et al. 2018). In another study, the unknown content was approximately 20% (Barrera et al. 2014).
Figure 5

Sulfur mass balance in the IPSR in the second period (a), third period (b), fourth period (c), and fifth period (d).

Figure 5

Sulfur mass balance in the IPSR in the second period (a), third period (b), fourth period (c), and fifth period (d).

Close modal

Prokaryotic microbial community

The prokaryotic microbial community composition was analyzed at the genus level before and after the experiment (Figure 6). Eighteen prokaryotic genera were identified as dominant (>1% of total sequences), and there were few differences in their abundance ratios before and after the experiment. For archaeal genera, methanogenic Methanobacterium (14.8–15.1%) and Methanosaeta (8.9–9.7%) were dominant in the sludge. Methanogenic Methanolinea (1.2–2.3%) was also found in the sludge. For bacterial genera, Syntrophobacter (6.9–7.1%), Desulfomicrobium (5.5–6.4%), and Thermovirga (4.1–5.9%) were found. The other phyla showed almost the same proportions before and after the experiment. Methanobacterium is a hydrogenotrophic methanogen and Methanosaeta is an acetoclastic methanogen, and these archaea are dominant in most high-rate anaerobic reactors (Kuroda et al. 2015; Wu et al. 2018; Yan et al. 2018b; Lee & Hwang 2019). Because of its low tolerance to sulfur inhibition, the management of Methanosaeta is considered key to achieving stable operation of anaerobic digestion processes with high performance (Lee & Hwang 2019). Therefore, the maintenance of a prokaryotic microbial community similar to that of high-rate anaerobic reactors, even at high sulfide concentrations, is considered critical for the successful treatment of wastewater with a high sulfide concentration.
Figure 6

Microbial composition before (left) and after (right) the experiments (>1% relative abundance on the genus level). Phylum names are shown in the middle of the plot.

Figure 6

Microbial composition before (left) and after (right) the experiments (>1% relative abundance on the genus level). Phylum names are shown in the middle of the plot.

Close modal

Applicability of the IPSR for wastewater with high sulfide concentrations

The laboratory-scale experiments showed that the IPSR removed H2S without needing additional equipment for gas striping. This is an important feature of the IPSR compared with other technologies. In the conventional two-stage process, acidification and sulfate reduction are performed in the first stage and methanogenesis in the second stage, and sulfide removal can be performed before methanogenesis to reduce inhibition (Elferink et al. 1994; Hao et al. 2014). For instance, sulfide removal by gas striping has been achieved using a UASB reactor equipped with a sulfide-striping device (Yamaguchi et al. 1999). For gas striping, an additional operation, such as biogas or nitrogen purging, is required (Lens et al. 2010). A drawback of the two-stage process is the energy consumption of mechanical equipment for H2S stripping (Hao et al. 2014). By contrast, the IPSR requires only a pH control agent for the first stage, and this pH control agent is generally not needed when the pH of the influent wastewater is low.

A potential limitation of the IPSR is that sulfide removal depends on the quantity of biogas generated from the degradation of organic matter in wastewater. Thus, the sulfide concentration and sulfide/COD ratio need to be considered for the application of the IPSR. Sulfur-containing wastewater often has a high COD, for example, distillery wastewater has a COD of 38–56 g L−1 and a sulfate concentration of 1,100 to 5,000 mg S L−1 (Barrera et al. 2014). In the present study, the IPSR was applied to wastewater with a higher sulfide concentration (6,000 mg S L−1) and lower COD (30 g L−1) than distillery wastewater. Our results showed that the performance of the IPSR was sufficient, even with the high sulfide concentration.

This experiment showed that the IPSR achieved high COD removal efficiency. This high performance was attributed to the optimum pH conditions for H2S removal in the first stage and methane production in the second stage. The optimum pH was maintained because the GLP valve prevented the mixing of the liquid between the first and second stages. The simple structure of the GLP valve will allow for the construction of large IPSRs and full-scale reactors.

Although this experiment was conducted over a short period, long-term stability is important for anaerobic treatment technologies. The primary function of the IPSR is gas stripping, which will occur if biogas is produced. Therefore, long-term stability of the IPSR can be expected even under high sulfide concentrations because of the reduction of H2S by gas stripping. For real wastewater treatment, setting an appropriate OLR according to sulfide concentration and /COD ratio will ensure that the performance of the IPSR is stable over the long term.

The IPSR achieved high COD removal efficiency of >90% at an OLR of 5 to 12 kg COD m−3 day−1. Gas stripping was effectively performed and resulted in a low sulfide concentration of 700 mg S L−1 in the first-stage effluent and 400 mg S L−1 in the second-stage effluent. The maintenance of a prokaryotic microbial community, similar to that of high-rate anaerobic reactors, was observed even at high sulfide concentrations. Overall, this study shows that the IPSR can achieve high COD removal efficiency for wastewater with sulfide concentrations as high as 6,000 mg/L by its gas stripping function using biogas produced in the reactor.

We thank Mrs Naoko Okubo for her support with the experiments. We thank Gabrielle David, PhD, from Edanz (https://jp.edanz.com/ac) for editing a draft of this manuscript.

This study was supported by KAKENHI [grant number JP18K04416] from JSPS and the Steel Foundation for Environmental Protection Technology, Japan.

All relevant data are included in the paper or its Supplementary Information.

T.O. and K.S. have patent #Japanese Patent No. 6029081 issued to Licensee.

Altaş
L.
&
Büyükgüngör
H.
(
2008
)
Sulfide removal in petroleum refinery wastewater by chemical precipitation
,
J. Hazard. Mater.
,
153
,
462
469
.
https://doi.org/10.1016/j.jhazmat.2007.08.076
.
APHA
. (
2005
)
Standard Methods for the Examination of Water and Wastewater
.
Washington, DC, USA
:
American Public Health Association/American Water Works Association/Water Environment Federation
.
Barrera
E. L.
,
Spanjers
H.
,
Romero
O.
,
Rosa
E.
&
Dewulf
J.
(
2014
)
Characterization of the sulfate reduction process in the anaerobic digestion of a very high strength and sulfate rich vinasse
,
Chem. Eng. J.
,
248
,
383
393
.
https://doi.org/10.1016/j.cej.2014.03.057
.
Bolyen
E.
,
Rideout
J. R.
,
Dillon
M. R.
,
Bokulich
N. A.
,
Abnet
C. C.
,
Al-Ghalith
G. A.
,
Alexander
H.
,
Alm
E. J.
,
Arumugam
M.
,
Asnicar
F.
,
Bai
Y.
,
Bisanz
J. E.
,
Bittinger
K.
,
Brejnrod
A.
,
Brislawn
C. J.
,
Brown
C. T.
,
Callahan
B. J.
,
Caraballo-Rodríguez
A. M.
,
Chase
J.
,
Cope
E. K.
,
Silva
R. D.
,
Diener
C.
,
Dorrestein
P. C.
,
Douglas
G. M.
,
Durall
D. M.
,
Duvallet
C.
,
Edwardson
C. F.
,
Ernst
M.
,
Estaki
M.
,
Fouquier
J.
,
Gauglitz
J. M.
,
Gibbons
S. M.
,
Gibson
D. L.
,
Gonzalez
A.
,
Gorlick
K.
,
Guo
J.
,
Hillmann
B.
,
Holmes
S.
,
Holste
H.
,
Huttenhower
C.
,
Huttley
G. A.
,
Janssen
S.
,
Jarmusch
A. K.
,
Jiang
L.
,
Kaehler
B. D.
,
Kang
K. B.
,
Keefe
C. R.
,
Keim
P.
,
Kelley
S. T.
,
Knights
D.
,
Koester
I.
,
Kosciolek
T.
,
Kreps
J.
,
Langille
M. G. I.
,
Lee
J.
,
Ley
R.
,
Liu
Y.-X.
,
Loftfield
E.
,
Lozupone
C.
,
Maher
M.
,
Marotz
C.
,
Martin
B. D.
,
McDonald
D.
,
McIver
L. J.
,
Melnik
A. V.
,
Metcalf
J. L.
,
Morgan
S. C.
,
Morton
J. T.
,
Naimey
A. T.
,
Navas-Molina
J. A.
,
Nothias
L. F.
,
Orchanian
S. B.
,
Pearson
T.
,
Peoples
S. L.
,
Petras
D.
,
Preuss
M. L.
,
Pruesse
E.
,
Rasmussen
L. B.
,
Rivers
A.
,
Robeson
M. S.
,
Rosenthal
P.
,
Segata
N.
,
Shaffer
M.
,
Shiffer
A.
,
Sinha
R.
,
Song
S. J.
,
Spear
J. R.
,
Swafford
A. D.
,
Thompson
L. R.
,
Torres
P. J.
,
Trinh
P.
,
Tripathi
A.
,
Turnbaugh
P. J.
,
Ul-Hasan
S.
,
Hooft
J. J. J. v. d.
,
Vargas
F.
,
Vázquez-Baeza
Y.
,
Vogtmann
E.
,
Hippel
M. v.
,
Walters
W.
,
Wan
Y.
,
Wang
M.
,
Warren
J.
,
Weber
K. C.
,
Williamson
C. H. D.
,
Willis
A. D.
,
Xu
Z. Z.
,
Zaneveld
J. R.
,
Zhang
Y.
,
Zhu
Q.
,
Knight
R.
&
Caporaso
J. G.
(
2019
)
Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2
,
Nat. Biotechnol.
,
37
,
852
857
.
https://doi.org/10.1038/s41587-019-0209-9
.
Caporaso
J. G.
,
Kuczynski
J.
,
Stombaugh
J.
,
Bittinger
K.
,
Bushman
F. D.
,
Costello
E. K.
,
Fierer
N.
,
Peña
A. G.
,
Goodrich
J. K.
,
Gordon
J. I.
,
Huttley
G. A.
,
Kelley
S. T.
,
Knights
D.
,
Koenig
J. E.
,
Ley
R. E.
,
Lozupone
C. A.
,
McDonald
D.
,
Muegge
B. D.
,
Pirrung
M.
,
Reeder
J.
,
Sevinsky
J. R.
,
Turnbaugh
P. J.
,
Walters
W. A.
,
Widmann
J.
,
Yatsunenko
T.
,
Zaneveld
J.
&
Knight
R.
(
2010
)
QIIME allows analysis of high-throughput community sequencing data
,
Nat. Methods
,
7
,
335
336
.
https://doi.org/10.1038/nmeth.f.303
.
Chen
Y.
,
Cheng
J. J.
&
Creamer
K. S.
(
2008
)
Inhibition of anaerobic digestion process: A review
,
Bioresour. Technol.
,
99
,
4044
4064
.
https://doi.org/10.1016/j.biortech.2007.01.057
.
Damianovic
M. H. R. Z.
,
Godoi
L. A. G. d.
,
Saia
F. T.
&
Foresti
E.
(
2018
)
Horizontal-flow anaerobic immobilized biomass (HAIB) reactor for organic matter and sulfate removal from paper recycling plant wastewater with simultaneous conversion of sulfide into elemental sulfur
,
J. Environ. Chem. Eng.
,
6
,
964
969
.
https://doi.org/10.1016/j.jece.2018.01.024
.
Ding
M.
&
Zeng
H.
(
2022
)
A bibliometric analysis of research progress in sulfate-rich wastewater pollution control technology
,
Ecotox. Environ. Safe
,
238
,
113626
.
https://doi.org/10.1016/j.ecoenv.2022.113626
.
Dykstra
C. M.
&
Pavlostathis
S. G.
(
2021
)
Hydrogen sulfide affects the performance of a methanogenic bioelectrochemical system used for biogas upgrading
,
Water Res.
,
200
,
117268
.
https://doi.org/10.1016/j.watres.2021.117268
.
Elferink
S. J. W. H. O.
,
Visser
A.
,
Pol
L. W. H.
&
Stams
A. J. M.
(
1994
)
Sulfate reduction in methanogenic bioreactors
,
Fems. Microbiol. Rev.
,
15
,
119
136
.
https://doi.org/10.1111/j.1574-6976.1994.tb00130.x
.
Hao
T.
,
Xiang
P.
,
Mackey
H. R.
,
Chi
K.
,
Lu
H.
,
Chui
H.
,
Loosdrecht
M. C. M. v.
&
Chen
G.-H.
(
2014
)
A review of biological sulfate conversions in wastewater treatment
,
Water Res.
,
65
,
1
21
.
https://doi.org/10.1016/j.watres.2014.06.043
.
Janssen
A. J. H.
,
Lens
P. N. L.
,
Stams
A. J. M.
,
Plugge
C. M.
,
Sorokin
D. Y.
,
Muyzer
G.
,
Dijkman
H.
,
Zessen
E. V.
,
Luimes
P.
&
Buisman
C. J. N.
(
2009
)
Application of bacteria involved in the biological sulfur cycle for paper mill effluent purification
,
Sci. Total Environ.
,
407
,
1333
1343
.
https://doi.org/10.1016/j.scitotenv.2008.09.054
.
Karhadkar
P. P.
,
Handa
B. K.
&
Khanna
P.
(
1990
)
Pilot-Scale distillery spentwash biomethanation
,
J. Environ. Eng.
,
116
,
1029
1045
.
https://doi.org/10.1061/(asce)0733-9372(1990)116:6(1029)
.
Koster
I. W.
,
Rinzema
A.
,
Vegt
A. L. d.
&
Lettinga
G.
(
1986
)
Sulfide inhibition of the methanogenic activity of granular sludge at various pH-levels
,
Water Res.
,
20
,
1561
1567
.
https://doi.org/10.1016/0043-1354(86)90121-1
.
Kuroda
K.
,
Chosei
T.
,
Nakahara
N.
,
Hatamoto
M.
,
Wakabayashi
T.
,
Kawai
T.
,
Araki
N.
,
Syutsubo
K.
&
Yamaguchi
T.
(
2015
)
High organic loading treatment for industrial molasses wastewater and microbial community shifts corresponding to system development
,
Bioresour. Technol.
,
196
,
225
234
.
https://doi.org/10.1016/j.biortech.2015.07.070
.
Lee
J.
&
Hwang
S.
(
2019
)
Single and combined inhibition of methanosaeta concilii by ammonia, sodium ion and hydrogen sulfide
,
Bioresour. Technol.
,
281
,
401
411
.
https://doi.org/10.1016/j.biortech.2019.02.106
.
Lens
P. N. L.
,
Visser
A.
,
Janssen
A. J. H.
,
Pol
L. W. H.
&
Lettinga
G.
(
2010
)
Biotechnological treatment of sulfate-Rich wastewaters
,
Crit. Rev. Env. Sci. Tec.
,
28
,
41
88
.
https://doi.org/10.1080/10643389891254160
.
Li
W.
,
Niu
Q.
,
Zhang
H.
,
Tian
Z.
,
Zhang
Y.
,
Gao
Y.
,
Li
Y.-Y.
,
Nishimura
O.
&
Yang
M.
(
2015
)
UASB treatment of chemical synthesis-based pharmaceutical wastewater containing rich organic sulfur compounds and sulfate and associated microbial characteristics
,
Chem. Eng. J.
,
260
,
55
63
.
https://doi.org/10.1016/j.cej.2014.08.085
.
Lupitskyy
R.
,
Alvarez-Fonseca
D.
,
Herde
Z. D.
&
Satyavolu
J.
(
2018
)
In-situ prevention of hydrogen sulfide formation during anaerobic digestion using zinc oxide nanowires
,
J. Environ. Chem. Eng.
,
6
,
110
118
.
https://doi.org/10.1016/j.jece.2017.11.048
.
O'Flaherty
V.
,
Lens
P.
,
Leahy
B.
&
Colleran
E.
(
1998
)
Long-term competition between sulphate-reducing and methane-producing bacteria during full-scale anaerobic treatment of citric acid production wastewater
,
Water Res.
,
32
,
815
825
.
https://doi.org/10.1016/s0043-1354(97)00270-4
.
Omil
F.
,
Mendez
R.
&
Lema
J.
(
1995
)
Anaerobic treatment of saline wastewaters under high sulphide and ammonia content
,
Bioresour. Technol.
,
54
,
269
278
.
Onodera
T.
,
Takemura
Y.
,
Aoki
M.
&
Syutsubo
K.
(
2022
)
Anaerobic reactor with a phase separator for enhancing sulfide removal from wastewater by gas stripping
,
Bioresour. Technol. Rep.
,
20
,
101216
.
https://doi.org/10.1016/j.biteb.2022.101216
.
Onodera
T.
,
Takemura
Y.
,
Aoki
M.
&
Syutsubo
K.
(
2023
)
Enhanced sulfide removal by gas stripping in a novel reactor for anaerobic wastewater treatment
,
Water Sci. Technol.
,
87
,
2223
2232
.
https://doi.org/10.2166/wst.2023.120
.
Peu
P.
,
Picard
S.
,
Diara
A.
,
Girault
R.
,
Béline
F.
,
Bridoux
G.
&
Dabert
P.
(
2012
)
Prediction of hydrogen sulphide production during anaerobic digestion of organic substrates
,
Bioresour. Technol.
,
121
,
419
424
.
https://doi.org/10.1016/j.biortech.2012.06.112
.
Pol
L. H.
,
Lens
P. N. L.
,
Stams
A.
&
Lettinga
G.
(
1998
)
Anaerobic treatment of sulphate-rich wastewaters
,
Biodegradation
,
9
,
213
224
.
Quast
C.
,
Pruesse
E.
,
Yilmaz
P.
,
Gerken
J.
,
Schweer
T.
,
Yarza
P.
,
Peplies
J.
&
Glöckner
F. O.
(
2013
)
The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools
,
Nucleic. Acids Res.
,
41
,
D590
D596
.
https://doi.org/10.1093/nar/gks1219
.
Robles
A.
,
Vinardell
S.
,
Serralta
J.
,
Bernet
N.
,
Lens
P. N. L.
,
Steyer
J. P.
&
Astals
S.
(
2020
)
Environmental Technologies to Treat Sulphur Pollution: Principles and Engineering
, pp.
277
317
.
https://doi.org/10.2166/9781789060966_0277
.
Shao
X.
,
Huang
Y.
,
Wood
R. M.
&
Tarpeh
W. A.
(
2024
)
Electrochemical sulfate production from sulfide-containing wastewaters and integration with electrochemical nitrogen recovery
,
J. Hazard. Mater.
,
466
,
133527
.
https://doi.org/10.1016/j.jhazmat.2024.133527
.
Speece
R. E.
(
1996
)
Anaerobic Biotechnology for Industrial Wastewater Treatments
.
Nashvillee, TN, USA
:
Archae Press
.
Wilkie
A. C.
,
Riedesel
K. J.
&
Owens
J. M.
(
2000
)
Stillage characterization and anaerobic treatment of ethanol stillage from conventional and cellulosic feedstocks
,
Biomass Bioenerg
,
19
,
63
102
.
https://doi.org/10.1016/s0961-9534(00)00017-9
.
Wu
J.
,
Niu
Q.
,
Li
L.
,
Hu
Y.
,
Mribet
C.
,
Hojo
T.
&
Li
Y.-Y.
(
2018
)
A gradual change between methanogenesis and sulfidogenesis during a long-term UASB treatment of sulfate-rich chemical wastewater
,
Sci, Total Environ,
,
636
,
168
176
.
https://doi.org/10.1016/j.scitotenv.2018.04.172
.
Yamaguchi
T.
,
Harada
H.
,
Hisano
T.
,
Yamazaki
S.
&
Tseng
I.-C.
(
1999
)
Process behavior of UASB reactor treating a wastewater containing high strength sulfate
,
Water Res,
,
33
,
3182
3190
.
https://doi.org/10.1016/s0043-1354(99)00029-9
.
Yan
L.
,
Ye
J.
,
Zhang
P.
,
Xu
D.
,
Wu
Y.
,
Liu
J.
,
Zhang
H.
,
Fang
W.
,
Wang
B.
&
Zeng
G.
(
2018a
)
Hydrogen sulfide formation control and microbial competition in batch anaerobic digestion of slaughterhouse wastewater sludge: Effect of initial sludge pH
,
Bioresour, Technol,
,
259
,
67
74
.
https://doi.org/10.1016/j.biortech.2018.03.011
.
Yan
L.
,
Ye
J.
,
Zhang
P.
,
Xu
D.
,
Wu
Y.
,
Liu
J.
,
Zhang
H.
,
Fang
W.
,
Wang
B.
&
Zeng
G.
(
2018b
)
Hydrogen sulfide formation control and microbial competition in batch anaerobic digestion of slaughterhouse wastewater sludge: Effect of initial sludge pH
,
Bioresour. Technol,
,
259
,
67
74
.
https://doi.org/10.1016/j.biortech.2018.03.011
.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (CC BY 4.0), which permits copying, adaptation and redistribution, provided the original work is properly cited (http://creativecommons.org/licenses/by/4.0/).

Supplementary data