(<8 mL) (Fig. 1b). In contrast, the sludge treated with
30 mM ZVI powder did not show any significant hydrogen production, which was attributed to slow dissolution of iron powders and microbial consumption. Indeed, a water sample containing 30 mM NZVI only (the abiotic control without any sludge or substrate) produced a total of 314 ( 5) mL of H2 (Fig. 1b), which was consistent to the calculated value of
305 mL from the theoretical estimation of H2 release with complete reaction (1 atm, 37 C). In the presence of heat-killed sludge, the final hydrogen production volume was only 63% of that in abiotic control (Fig. 1b), likely due to slower hydrogen production because of microbial-induced nanoparticle aggre- gation, or coating of biomass with dissolved organic matter on the NZVI surface (Chen et al., 2011; Choi et al., 2010). However, at the same dose of 30 mM NZVI, the anaerobic sludge generated 12% less hydrogen (25 mL) than in the heat-killed sludge. Presumably some of H2 generated by NZVI or glucose was used in hydrogenotrophic methanogenesis, or other hydrogenotrophic bioprocesses by facultative bacteria in anaerobic sludge (Grady et al., 1999).
3.2. Impact of NZVI on anaerobic digestion under complete methanogenesis inhibition
In order to differentiate the impact of NZVI on different bio- logical processes such as methanogenesis and homoaceto- genesis in anaerobic digestion, BES was added to completely inhibit methanogenesis. As shown in Fig. 2, the groups of positive control, negative control, 1 mM NZVI, and 30 mM ZVI powder generated almost negligible, but the same amount of hydrogen gas 24 ( 1) mL. The group of 30 mM NZVI produced a total of 149 ( 1) mL of H2 while the heat-killed sludge control produced 188 ( 3) mL of H2. The amount of H2 produced was only 79% ( 1%) of that with heat-killed sludge, most likely due to bacterially controlled hydrogenotrophic processes. One of the hydrogenotrophic processes is homoacetogenesis (2CO2 þ 4H2 / CH3COOH þ 2H2O). However, the addition of
30 mM NZVI only increased the final acetic acid concentration
slightly (an average of 24 mg/L higher than that of the control group, Fig. S2b), which was lower than a theoretical value of
57 mg/L by homoacetogenesis assuming all the lost H2 was converted to acetate (at 1 atm, 37 C). It is possible that acetate can be converted to ethanol in the presence of hydrogen resulting in a dynamic change in acetate concentration (Dinamarca et al., 2011). Furthermore, other hydrogenotrophic bioprocesses may play an important role in converting H2 into other fermentation products such as methanol, acetaldehyde, and formate (Dinamarca et al., 2011).
3.3. SCOD, pH and VFA changes during anaerobic digestion
The dynamic changes in SCOD, pH and VFA during anaerobic digestion also reflected the negative impact of NZVI on methanogenesis and other important biochemical processes. The average SCOD concentration of the groups treated with glucose addition was 1542 ( 183) mg/L at the beginning of anaerobic digestion (Fig. 3a), while the negative control con- tained the SCOD concentration of 419 ( 35) mg/L. At the completion of anaerobic digestion, the SCOD concentration in
Fig. 2 e Cumulative hydrogen gas production profiles during mesophilic anaerobic glucose degradation in the presence of methanogenesis inhibitor BES, in the groups of negative control (no glucose, no iron, B), positive control (glucose only, no iron, C), 1 mM NZVI (D), 30 mM NZVI ( ), and 30 mM ZVI ( ), respectively. A water sample containing 30 mM NZVI only served as an abiotic control to show the hydrogen production trend in Fig. 1b ). A heat- killed sludge sample followed by the dose of 30 mM NZVI resulted in a different hydrogen production profile (,). Error bars represent the range of data from duplicate experiments. No methane was detected because of BES inhibition.
the negative control increased to 645 ( 87) mg/L possibly due to the continued endogenous biomass decay (Ekama et al.,
2007; Grady et al., 1999). The SCODs in the groups of positive control, 1 mM NZVI, 10 mM NZVI, and 30 mM ZVI powder generally had decreasing trends with final SCOD concentra- tions ranging from 588 to 776 mg/L due to anaerobic degra- dation (Grady et al., 1999). Surprisingly, the group of 30 mM NZVI showed much higher SCOD concentrations at an average of 2125 mg/L only after one day of anaerobic digestion, indi- cating cell lysis and a significant release of cellular compo- nents due to the disruption of cell membranes (Li et al., 2010) by NZVI at high concentrations, as can be visualized in Fig. S5.
The NZVI dissolution and VFA generation in anaerobic digestion were accompanied by the changes in mixed liquor pH, as the former process generates hydroxyl ions while the latter one produces hydrogen ions. The pH in the negative control was relatively stable at 7.9 ( 0.1) throughout the digestion (Fig. 3b). The group of 30 mM NZVI had a small pH drop from 7.8 ( 0.1) to 7.6 ( 0.1) on day 2, and recovered quickly to 7.9 ( 0.1) due to continued NZVI dissolution. In comparison, the pHs from the other groups dropped more rapidly from 7.8 ( 0.1) to 7.2 ( 0.1) on day 1 because of glucose fermentation, and then recovered to 7.6 ( 0.1), due to conse- quent VFA consumption (Rittmann and McCarty, 2001). Interestingly, the addition of 30 mM ZVI powder led to the gradual increase in pH to 7.9 at the end of digestion likely due to the slow dissolution of ZVI powder and the release of hy- droxyl ions.
In anaerobic degradation, glucose can be metabolized to produce CO2, H2, and VFAs including acetic acid and propi- onic acid (Zinder, 1986). Consistent with the SCOD profiles,
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