1. INTRODUCTION
4.3. Results and discussions
4.3.2. Fate of sulfur
To determine whether magnetite inhibited the formation or enhanced the decomposition of H2S, the existing forms of sulfur in the reactors were analyzed. The RM and RC digestates taken on day 346 (i.e., Phases M4 and C1) produced completely different Raman spectra (Fig. 4-3). The Raman spectrum of the RM digestate (at 6 mM Fe magnetite) entirely matched that of the S0 reference but not that of the FeS reference. In addition, the RC digestate (without Fe addition) exhibited a spectrum with no discernible peaks and high background signals. Such a strong background interference often occurs when analyzing biological samples containing a large amount of organic particles and impurities (Escoriza et al., 2006). These results suggested that S0 was likely the
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dominant form of inorganic sulfur in the reactors when magnetite was added. Correspondingly, the concentration of extracellular S0 was significantly higher (2.9–8.4-fold) in RM (at ≥8 mM Fe magnetite) than in RC (Table 4-3). The formation of S0 in the presence of magnetite was further supported by fluorescence-probing image (Fig. 4-4). All of these observations demonstrated that the addition of magnetite somehow promoted the generation of S0, not of FeS precipitation, thereby reducing the production of H2S in RM. As in RM, a notable amount of S0 was formed in RM1 and RM2. When magnetite was added (8–20 mM Fe), up to 69.2 and 69.9 mg/L of extracellular S0 were produced in RM1 and RM2, respectively, whereas negligible amounts of extracellular S0 (<1 mg/L) was observed in the absence of magnetite (Table 4-4). All the observations in the magnetite-added reactors (i.e., RM, RM1, and RM2) indicated that the magnetite altered the fate of sulfur as S0, which likely contributed to the decreases in H2S production in the presence of magnetite.
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Table 4-3 Process performance data of the RM and RC during the experimental phases.
Reactor Phase Magnetite or FeCl2a
(mM Fe)
COD removal
(%)
CH4
production rate (mL/L∙d)
CH4 yield (L/g COD fed)
H2S production rate
(mL/L∙d)
H2S content (ppmv)
Total dissolved sulfide (mg/L)
Extracellular S0 (mg/L)
RM M1 0 64.7 (4.0)b 60.3 (14.2) 0.22 (0.05) 0.51 (0.15) 6220 (840) 62.5 (4.3) n.d.c M2 2 64.1 (2.3) 61.3 (9.7) 0.22 (0.04) 0.49 (0.16) 6017 (1236) 59.4 (9.3) n.d.
M3 4 64.3 (2.6) 58.7 (8.9) 0.21 (0.03) 0.23 (0.11) 3194 (1594) 58.9 (10.1) n.d.
M4 6 58.7 (5.7) 61.1 (10.4) 0.22 (0.04) 0.11 (0.07) 1625 (569) 66.7 (21.8) n.d.
M5 8 63.2 (4.9) 64.7 (12.2) 0.24 (0.05) <0.01 <100 48.1 (8.3) 18.8 (5.2) M6 12 64.7 (2.5) 73.0 (15.6) 0.27 (0.06) <0.01 <100 50.9 (4.8) 18.4 (1.9) M7 20 66.6 (1.6) 57.0 (7.7) 0.21 (0.03) <0.01 <100 42.6 (4.8) 20.3 (2.2) RC C1 0 65.0 (3.8) 63.8 (12.8) 0.23 (0.03) 0.54 (0.24) 6936 (1185) 56.2 (10.7) 6.4 (0.0)
C2 2 64.8 (2.9) 58.0 (6.7) 0.21 (0.02) 0.01 (0.00) 151 (46) 47.4 (13.8) 3.1 (1.3) C3 4 62.3 (2.5) 64.9 (13.5) 0.24 (0.03) <0.01 <100 77.1 (11.6) 3.5 (2.0) C4 8 59.5 (2.9) 51.1 (8.7) 0.19 (0.02) <0.01 <100 55.4 (16.9) 5.8 (0.4)
a Magnetite for RM and FeCl2 for RC.
b Standard deviations are presented in parentheses.
c Not determined.
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Table 4-4 Process performance data of the RM1 and RM2 at different magnetite dose.
Magnetite dose 0 mM Fe 8 mM Fe 20 mM Fe
Process parameter Unit RM1 RM2 RM1 RM2 RM1 RM2
COD removal % 66.3 (11.4)a 64.1 (22.7) 62.6 (10.5) 63.2 (9.2) 66.0 (25.7) 65.9 (21.1) CH4 rate mL/L∙d 68.9 (7.0) 68.3 (4.2) 68.1 (8.9) 67.5 (7.8) 64.8 (2.6) 64.0 (3.1) CH4 yield L/g COD fed 0.25 (0.01) 0.25 (0.01) 0.25 (0.02) 0.25 (0.01) 0.24 (0.0) 0.23 (0.01)
H2S rate mL/L∙d 0.043 (0.01) 0.046 (0.0) 0.005 (0.0) 0.004 (0.0) n.d.b n.d.
H2S content ppmv 5283 (683) 5217 (161) 553 (49) 484 (42) n.d. n.d.
Total dissolved sulfide mg S/L 58.1 (4.4) 27.5 (3.2) 58.9 (6.2) 61.6 (1.2) 58.6 (1.1) 45.4 (4.3)
Extracellular S0 mg/L <1 <1 69.2 (1.3) 69.9 (0.5) 63.3 (2.0) 61.9 (8.0)
Residual magnetite mM Fe N.D. N.D. 8.5 (0.1) 8.2 (0.0) 19.7 (0.3) 23.4 (0.5)
a Standard deviation represents in parenthesis.
b Below the detection limit (<1 ppmv)
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Figure 4-3. Raman spectra of the digestates taken on day 346 from RM (A; 6 mM Fe magnetite) and RC (B;
no addition of iron compound) and reference S0 (C) and FeS (D) samples.
Figure 4-4. Microscopic image of extracellular S0 of RM1 digestate with fluorescent probe (SSP4). Green
70 color indicates the S0.
CV was conducted to probe the redox reactions occurring in RM (8 mM Fe magnetite), RC (2 mM FeCl2), and RM1 (20 mM Fe magnetite). The batch cultures of the RM, RC, or RM1 biomass on acetate with sodium sulfate as the sole source of sulfur exhibited different cyclic voltammograms (Fig. 4-5). Two anodic peaks corresponding to the oxidation of (poly)sulfide to S0 were observed at +0.4 and +0.6 V (vs. Ag/AgCl) in the cyclic voltammograms of the RM culture obtained after 24 and 48 h of cultivation (Lin et al., 2016; Vanysek, 2002), whereas no apparent oxidation or reduction peaks were produced by the RC culture. Correspondingly, a cathodic peak for the reduction of S0 to (poly)sulfide at –0.7 V (vs. Ag/AgCl) (Vanysek, 2002) was observed in RM only. Similarly, a couple of anodic peak for combined (poly)sulfide to S0 (+0.37 V vs.
Ag/AgCl) and cathodic peak for the reduction of S0 to combined (poly)sulfide to S0 (–0.02 V vs.
Ag/AgCl) (Vanysek, 2002) were observed in the cyclic voltammograms of the RM1 culture. These results further confirmed that S0 formation was promoted in the presence of magnetite. A peak corresponding to the reduction of sulfate to sulfide was not identified in all cultures probably because sulfate was rapidly exhausted primarily by SRBs within the first 24 h of cultivation (i.e., before the first scan). The dissolved sulfide concentrations in RM, RC, RM1 and RM2 were comparable across the experimental phases (Tables 4-3 and 4-4), demonstrating that dissimilatory sulfate reduction was active in both reactors, notwithstanding the addition of magnetite or FeCl2. It is therefore suggested that sulfate derived from the decomposition of sulfur compounds was first reduced to H2S and then oxidized to S0 in RM.
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Figure 4-5. Cyclic voltammograms measured in the batch cultures inoculated with the digestates taken on day 555 from RM (A; 6 mM Fe magnetite) and RC (B; 2 mM Fe FeCl2), and on day 511 from RM1 (C; 20 mM Fe magnetite) digestates. For RM and RC cultures, CV was measured after the first 24 and 48 hr of cultivation at a scan rate of 50 mV/s. For RM1 culture, CV was measured after the first 24 hr of cultivation at a scan rate of 30 mV/s.
It has been reported that chemical oxidation of H2S to S0 can be mediated by iron oxides, including magnetite (Poulton et al., 2004). However, this possibility was unlikely in magnetite- added reactors (i.e., RM, RM1, and RM2). The anaerobic batch tests for abiotic conditions using both autoclaved anaerobic sludge and DW and for biotic conditions using anaerobic sludge, with either Na2SO4 or Na2S as sulfur source revealed that measurable amounts of S0 was only detected in biotic conditions (Table 4-5). This observation indicated that if the S0 formed through the oxidation of H2S, microbial activity should have been necessary for the oxidation reaction. For biotic conditions, the batch test with Na2SO4 as a sulfur source demonstrated higher S0 concentration than
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that with Na2S. This suggests that the sulfate reduction by SRBs is likely necessary for sulfide oxidation to S0 in the presence of magnetite and deserves further investigation.
Table 4-5. Batch test result of sulfide oxidation by magnetite under abiotic and biotic conditions.
Extracellular S0 concentration (mg S/L)
In the presence of magnetite In the absence of magnetite
Sulfur source Sulfate Sulfide Sulfate Sulfide
Condition Incubation source
Abiotic DW N.D.a N.D. N.D. N.D.
Autoclaved sludge N.D. N.D. N.D. N.D.
Biotic Sludge 67.5 (11.0)b 11.2 (1.1) N.D. N.D.
a Below detection limit (1 mg/L)
b Standard deviations are in parenthesis
As discussed above, it is unlikely that the Fe2+/Fe3+ that leached from magnetite contributed to the H2S removal in RM by precipitating FeS or by serving as an electron acceptor/shuttle for sulfide oxidation. Accordingly, the XRD profile of the RM digestate on day 389 (at 8 mM Fe magnetite) matched that of pure magnetite (Fig. 4-6), indicating that chemical transformation of magnetite did not occur during the reactor operation. Consistently, previous studies have reported the limited transformation or dissolution of magnetite under circumneutral anaerobic conditions (Baek et al., 2017; Baek et al., 2016; Hu et al., 2019; Yin et al., 2018). In addition, the residual magnetite concentration in RM1 and RM2 (Table 4-5) excluded the possibility of magnetite dissolution in the reactors. Therefore, it is conjectured that magnetite stimulated the microbial oxidation of sulfide to S0 and hence reduced the production of H2S in RM. This inference is supported by the fact that direct biological reduction of sulfate to S0 hasnot yet been reported (Blázquez et al., 2017).
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Figure 4-6. XRD profile of the RM digestate taken on day 389 (at 8 mM magnetite).