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Impact of zero valent iron on blackwater anaerobic digestion
Bioresource Technology 285 (2019) 121351
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Short Communication
Impact of zero valent iron on blackwater anaerobic digestion a,b,c,1
Rui Xu a b c
a,1
, Shengnan Xu
a
a
b,c
a,⁎
, Lei Zhang , Anna Patricya Florentino , Zhaohui Yang , Yang Liu
T
Department of Civil and Environmental Engineering, University of Alberta, 7-263 Donadeo Innovation Centre for Engineering, Edmonton, Alberta T6G 1H9, Canada College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082, PR China
ARTICLE INFO
ABSTRACT
Keywords: Anaerobic blackwater treatment Nano-scale zero valent iron Micro-scale zero valent iron Methane production
The source diverted blackwater treatment is receiving growing attention as an alternative to conventional energy intensive wastewater management and treatment systems. Blackwater, containing concentrated organic materials, can be anaerobically digested to recovery bioenergy. However, the methane recovery from blackwater is often inhibited by the presence of high free ammonia (FA) in blackwater. In order to improve the methane production in blackwater, nano-scale zero valent iron (nZVI, 35 nm or 50 nm) or micro-scale zero valent iron (mZVI, 200 μm) at different dosages (i.e., 0.5, 1, and 10 g/L) were applied respectively in the anaerobic digestion (AD) reactor for blackwater treatment. The results demonstrated that low doses (0.5–1 g/L) of nZVI slightly improved methane (CH4) production, possibly due to a reduced oxidation–reduction potential (ORP) and improved hydrolysis-acidification in the nZVI supplemented systems. However, a lower biochemical methane potential (BMP) of blackwater was observed with high doses (10 g/L) of nZVI which induced a pH increase (> 8.5) in AD reactor leading to a higher FA inhibition of CH4 production. In contrast, the effect of mZVI on blackwater AD system was not significant. The study demonstrated the successful application of nZVI for improving AD of blackwater, however, which requires dosage control.
1. Introduction With the growing attention to resource recovery based wastewater treatment, anaerobic digestion (AD, i.e., a sustainable wastewater treatment technology by converting organics into methane in the absence of oxygen (Liu et al., 2015)) of source diverted blackwater (i.e., toilet wastewater, which is an alkaline wastewater flow consisting of flushwater, urine and feces, toilet paper, and with food waste residuals in some scenarios (Xu et al., 2019)) has been recognized as an attractive option to maximize energy and nutrient recovery and to replace energy intensive aerobic municipal wastewater treatment (Tyagi et al., 2018). Blackwater characteristics vary significantly with the collection sources. For instance, anaerobic digestion was compromised in the treatment of blackwater collected from vacuum toilets because blackwater consisted of high FA concentration which inhibited methane (CH4) production (Gao et al., 2019). Florentino et al. (2019) applied granular activated carbon to improve blackwater digestibility, and demonstrated that granular activated carbon enriches the conductive microbial culture, especially H2 utilizers that resist FA inhibition. Xu et al. (2019) applied H2 enriched culture to blackwater, and observed a direct enhancement of CH4 production.
The strong reductants zero valent iron (ZVI, e.g., micro-scale ZVI (mZVI) or nano-scale ZVI (nZVI)) have been applied as a means to improve anaerobic digestion of anaerobic sludge or wastewater activated sludge by increasing CH4 production, eliminating the emission of the odorous gas H2S, and enhancing phosphate recovery (Wei et al., 2018). Su et al. (2013) found that an anaerobic digester supplemented with 0.1% nZVI increased CH4 production by 40.4%. Suanon et al. (2017) observed 25% enhancement in CH4 production with 0.1% nZVI supplement. It has been reported that ZVI improves CH4 production in anaerobic digestion of waste activated sludge by influencing both methanogenesis and hydrolysis-acidification processes (Feng et al., 2014). ZVI affects methanogenesis through two mechanisms. First, ZVI maintains stable and favorable conditions for methanogens by lowering the oxidation–reduction potential (ORP) of the treatment substrate (Meng et al., 2013; Wei et al., 2018) or by increasing the pH of the aquatic environment to a neutral range (Suanon et al., 2017). Second, ZVI can stimulate the growth of hydrogenotrophic methanogen by providing electrons directly or by producing hydrogen through the iron anaerobic oxidation to facilitate CH4 production from the consumption of CO2 (Eqs. (1)–(3)) (Karri et al., 2005; Meng et al., 2013).
Corresponding author. E-mail address: [email protected] (Y. Liu). 1 Both authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.biortech.2019.121351 Received 28 February 2019; Received in revised form 12 April 2019; Accepted 13 April 2019 Available online 15 April 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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CO2 + 4Fe0 + 8H+
Three types of commercially available zero valent iron (ZVI) particles were used in this study, including nZVI35 (35 nm, > 99.5% Fe0) obtained from Sigma-Aldrich (St Louis, United States); nZVI50 (50 nm, NANOFER STAR, with an oxide shell and > 80% Fe0) obtained from NANORION (Rajhrad, Czech Republic); and mZVI (200 μm, > 99% Fe0) obtained from Fisher Scientific (Hampton, United States). These commercial particles were selected to represent widely used nZVI and mZVI particles in previous studies (Carpenter et al., 2015; Feng et al., 2014; Velimirovic et al., 2014). The specific surface area for nZVI and mZVI were in the range of 10–25 m2/g and 0.05–5 m2/g, respectively. nZVI and mZVI particles were used immediately after being received. Anaerobic reactors were purged with pure N2 for at least five minutes to deoxygenate the solution before being incubated at 35 °C.
(1)
CH4 + 4Fe2 + + 2H2 O
Fe0 + 2H2 O
Fe 2+ + H2 + 2OH
(2)
CO2 + 4H2
CH4 + 2H2 O
(3)
ZVI has also been applied to accelerate the hydrolysis step (i.e., the limiting step) of anaerobic digestion of sludge by converting particulate matter to soluble substrates (Liu et al., 2015; Liu et al., 2012; Wang et al., 2018). The supplementation of ZVI can optimize the acidification step of anaerobic digestion by directly promoting propionate degradation and subsequently raising the production of acetate to facilitate methanogenesis (Liu et al., 2015; Liu et al., 2012; Meng et al., 2013; Suanon et al., 2017). Accordingly, the addition of ZVI could increase the abundance of microbial communities, such as bacteria which convert propionate to acetate (Liu et al., 2011; Meng et al., 2013). Moreover, due to its conductive characteristics, ZVI is assumed to stimulate direct interspecies electron transfer, which can enhance CH4 production in anaerobic digestion of sludge by prompting electron transfer between certain acetogens and methanogens. However, these mechanisms have been reported only for Fe(III) oxides such as Fe3O4 or Fe3O4-ZVI particles; the role of ZVI is still unclear (Viggi et al., 2014; Zhao et al., 2018). Acetoclastic methanogens, especially Methanosaeta, were found to be dominant in conducting such processes (Zhao et al., 2018). The impact of ZVI on anaerobic digestion depends on factors related to both ZVI particle size (e.g., particle size, surface properties, and dosage) and anaerobic systems (e.g., presence of a microbial community in the system) (Wang et al., 2018). The significantly higher specific surface area of nZVI compared to mZVI leads to a significantly higher impact of nZVI on the improvement of anaerobic digestion. However, the superior reactivity of nZVI may also lead to the biocidal effect of nZVI in some scenarios, which has been reported to cause the reduction of microbial community diversity and biomass density (Su et al., 2013; Yang et al., 2013). For instance, Yang et al. (2013) reported that 1.68 g/ L of nZVI inhibited methanogenesis because of the potent reducing properties at the surface of nZVI particles can damage cell membranes through directly contact, although mZVI at the same dosage enhanced CH4 production of the target anaerobic sludge. Therefore, there is still a lack of consensus on the impacts of ZVI on AD process, especially in anaerobic microbial communities. Whether ZVI can improve anaerobic digestion in blackwater, and alleviate FA inhibition of AD, has not been investigated. This study investigates the impacts of ZVI size and dosage on methanogenesis and on hydrolysis-acidification processes during anaerobic digestion in blackwater. To the best knowledge, this is the first report that discusses the application of ZVI in AD of blackwater.
2.2. Impact of zero valent iron on anaerobic digestion The impacts of nZVI or mZVI on AD were determined by measuring the biochemical methane potential (BMP) in batch reactors, as described below. To 160 mL serum bottles (batch reactors), 50 mL of sludge inoculum and 50 mL of blackwater were added to create a 60 mL head space. Aliquots of nZVI35, nZVI50, and mZVI particles were then added to separate bottles to reach respective final concentrations of 0.5, 1, and 10 g/L in the blackwater AD systems, which were selected based on the effective dosage range in the literature (Feng et al., 2014; Karri et al., 2005). Each condition was prepared in triplicate. Triplicates of a negative control (distilled water replaced blackwater, iron free) and a positive control (blackwater only, iron free) were subjected to the same conditions and procedure as the samples. The head space in each serum bottle was purged with high purity N2 gas for five minutes to remove oxygen before sealing the bottle with a butyl rubber stopper and a crimp cap (Bellco Gass, Vineland, NJ). The bottles were shaken in an incubator shaker (ThermoFisher Scientific, USA) at 120 rpm and 35 °C until biogas production ceased (50 days). BMP recording and calculation were performed following the methods in previous research (Xu et al., 2019). 2.3. Impact of zero valent iron on hydrolysis-acidification To measure the impact of ZVI on hydrolysis-acidification during anaerobic digestion, a methanogenesis inhibition procedure was conducted in the batch anaerobic digestors prepared in Section 2.2. Briefly, the blackwater and seed sludge mixture was heated at 102 °C for 30 min, then sodium 2-bromoethanesulfonic acid (BESA) was added to the batch reactors to a final concentration of 30 mM to completely inhibit methanogenesis (Feng et al., 2014; Yang et al., 2013). The batch experiments were conducted for five days in an incubator shaker at 35 °C, before the water samples were collected for soluble chemical oxygen demand (CODs) and acetic acid measurements.
2. Materials and methods 2.1. Preparation of seed sludge, blackwater, and zero valent iron
2.4. Methanogenic population analysis after zero valent iron exposure to anaerobic digestion
Anaerobic sludge was obtained from an anaerobic digester in a local sewer treatment plant in Edmonton (Canada) and blended to homogeneity before seeding in blackwater. Blackwater (1L water per flush, vacuum toilet wastewater) was collected directly from campus toilets at the University of Alberta. The collected seed sludge and blackwater were utilized immediately or stored anaerobically at 4 °C for less than 7 days before being utilized. Characteristics of anaerobic seed sludge and blackwater used in the experiments are summarized in Table 1.
nZVI35 was selected as the representative ZVI for the quantitative polymerase chain reaction (qPCR) experiment, which was used to determine the presence of methanogenic communities and their population changes after ZVI exposure to anaerobic digestion, according to the methods described in previous research (Xu et al., 2019). Briefly, sludge samples in the presence of 0, 0.5, 1, and 10 g/L nZVI35 were collected
Table 1 Characteristics of seed sludge and blackwater. Parameters
pH
CODt (g/L)
CODs (mg/L)
TAN (mg/L)
TP (mg/L)
TSS (mg/L)
seed sludge blackwater
7.24 ± 0.3 8.82 ± 0.1
22.8 ± 1.45 18.7 ± 0.8
475 ± 38 5058 ± 105
1144 ± 27 1254 ± 26
/ 330 ± 11
/ 17140 ± 104
2
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from the anaerobic batch reactors after 50 days of incubating at 35 °C, from which genomic DNA was extracted using a QIAGEN PowerSoil Pro Kit (Hilden, Germany). Immediately after DNA extraction, qPCR analysis was performed targeting hydrogenotrophic methanogens (Methanobacteriales, Methanomicrobiales, Methanothermobacter and Methanoculleus), strictly acetoclastic methanogen Methanosaeta and versatile acetoclastic methanogen Methanosarcina. Detailed primer information and qPCR reaction conditions were summarized in the Supporting Information.
1, and 10 g/L mZVI, BMP values in blackwater did not change significantly; they were 37.1 ± 0.4%, 37.2 ± 0.7%, and 36.9 ± 0.6%, respectively (p = 0.9), indicating that the impact of mZVI on the BMP was negligible. Compared to the mZVI application group, a low dose of 0.5–1 g/L of nZVI led to a blackwater BMP increase to 42.7 ± 0.5%–44.8 ± 0.6% for nZVI35 and to 42.9 ± 0.7%–45.2 ± 0.6% for nZVI50 (p < 0.05 for all). The production of electrons or H2 in the presence of nZVI facilitated the growth of methanogens (Karri et al., 2005; Suanon et al., 2017) and/or increased favorable fermentation under decreased oxidation-reduction potential (ORP) conditions at the low nZVI dose, keeping methanogen activities at a high level. However, a high dose (10 g/L) of nZVI significantly reduced the BMP by 75–81% (p = 0.013); and BMP values were 7.1 ± 0.3% and 9.3 ± 0.3%, respectively, in the presence of 10 g/L nZVI35 and 10 g/L nZVI50. This inhibition was also observed in Yang et al.’s research (2013), where a 69% reduction in BMP occurred with the dose of 1.68 g/L of nZVI50. According to the authors’ explanation, serious cell membrane or respiratory activity damage occurred through direct contact between nZVI and methanogen cells due to the potent reducing conditions at the nZVI surface (Yang et al., 2013). This observation is also evident in the qPCR results (Fig. 4), where the gene copy concentrations of Methanomicrobiales and Methanoculleus were reduced significantly. Moreover, the high dosage of nZVI led to a significant increase in pH (p = 0.01) (i.e., 8.79 ± 0.05 with nZVI35 and 9.44 ± 0.03 with nZVI50), likely because of nZVI oxidation (Eq. (2)) in the anaerobic system (Suanon et al., 2017). The significantly elevated pH (p < 0.01) in blackwater treated with high doses of nZVI35 and nZVI50 significantly enhanced the FA concentration from 14.7 mg/L (control) to 516.1 mg/L and 949.9 mg/L, respectively, based on the theoretical FA concentration calculated from the TAN concentration measured in blackwater (Table 1). The FA molecule diffuses more easily than the ammonium ion, leading to a more facile contact of FA with methanogens, and thus significant inhibition of methanogen activity and a subsequent lower CH4 yield (i.e., a decreased BMP at high nZVI dose (Fig. 1)) (Wang et al., 2016; Yenigün & Demirel, 2013). The moderate increase (< 95.2 mg/L) in FA concentration caused by a slightly increased pH at low doses of nZVI and at all doses of mZVI did not result in further inhibition of methanogens by FA (Gao et al., 2019). With a BMP inhibition prevailed in the system exposed to
2.5. Chemical and statistical analysis Chemical and statistical analyses were performed with the methods described in previous research (Xu et al., 2019). Briefly, the recording of biogas volume was performed with a gas meter (GMH3111, GREISINGER) and biogas composition was analyzed with gas chromatography (GC-7890B, Agilent Technologies, USA). The chemical concentrations in the collected samples – i.e., total COD (CODt), soluble COD (CODs), total NH4+-N (TAN), total PO43−-P (TP) and total suspended solid (TSS) – were measured using HACH kits as reported previously (Xu et al., 2014); FA concentration was determined based on the TAN concentration (Xu et al., 2019); acetic acid concentration was analyzed by a Dionex ICS-2100 ion chromatograph equipped with an IonPac AS18 column (Dionex, Sunnyvale, CA). Oxidation-reduction potential (ORP) and pH were measured under anaerobic conditions using a conductivity ORP/pH meter with different electrodes (Mettler Toledo, USA). One-way ANOVA (p values < 0.05) was performed to identify the statistical significance between groups. 3. Results and discussion 3.1. Impact of zero valent iron on methane production in blackwater Under mesophilic conditions, organic substrates in blackwater are anaerobically digested to generate CH4 (Gao et al., 2019). The anaerobic digestibility of organic substrates can be evaluated by measuring the BMP (Yang et al., 2013). The control groups are the groups where no nZVI or mZVI was added. After 50 days incubation in the control groups, blackwater BMP was 36.8 ± 0.4% (Fig. 1). With doses of 0.5,
Fig. 1. BMP of blackwater anaerobic digestion treatment in the presence of nZVI35, nZVI50, or mZVI, each at concentrations of 0, 0.5, 1, and 10 g/L. 3
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Fig. 2. CH4 content in the produced biogas of the blackwater treatment in the presence of nZVI35, nZVI50, or mZVI, each at concentrations of 0, 0.5, 1 and 10 g/L. Table 2 Summary of the ZVI performance in different systems.
The current study
Treatment
ZVI concentration
CH4 production
CH4 content
CODs
Acetic Acids
BW BW + nZVI35
0 g/L 0.5 g/L 1 g/L 10 g/L 0.5 g/L 1 g/L 10 g/L 0.5 g/L 1 g/L 10 g/L 0 0.1 (wt% sludge)
36.8 ± 0.4% 42.7 ± 0.5% 44.8 ± 0.6% 7.1 ± 0.3% 42.9 ± 0.7% 45.2 ± 0.6% 9.3 ± 0.3% 37.1 ± 0.4% 37.2 ± 0.4% 36.9 ± 0.6%
80.1 ± 1.2% 81.2 ± 0.4% 83.9 ± 1.1% 99.2 ± 0.8% 79.3 ± 0.6% 80.5 ± 0.7% 99.1 ± 0.9% 80.9 ± 0.9% 81.6 ± 1.0% 93.4 ± 0.6% 70 ± 3.9% 73.4 ± 2.3%
5124 ± 114 mg/L 5367 ± 120 mg/L 5477 ± 122 mg/L 6438 ± 100 mg/L 5336 ± 121 mg/L 5634 ± 125 mg/L 7013 ± 115 mg/L 5158 ± 121 mg/L 5116 ± 133 mg/L 5258 ± 109 mg/L 3683 mg/L 3032.1 mg/L
864 984 962 945 960 948 932 890 863 852
0 0.1 (wt % sludge)
8.16L 13.7L
42.8–64.2% 48.2–70.6%
0 18.6 g/L in 3.0 g VSS/L anaerobic sludge 0 1, 4 and 20 g/L ZVI
2.87 mmol CH4/L 8.17 mmol/L
BW + nZVI50 BW + mZVI Suanon et al, 2017 Su et al, 2013 Karri et al., 2005
Sewage sludge Sewage sludge + 160 nm nZVI Anaerobic sludge Anaerobic sludge + 20 nm ZVI Sludge Sludge + 10 um mZVI
Feng, et al, 2014
waste activated Sludge waste activated sludge + 0.2 mm nZVI
Yang et al, 2013
Digested sludge Digested sludge + 55 nm nZVI
0 1, 10, 30 mM nZVI
192.6 mL/VSS 211.1. 233.8 and 276.4 mL/VSS, respectively 30.8% 24.5%, 24.5%, and 9.5%, respectively
a high dose of nZVI, the potential positive effect of the further reduced ORP was negligible.
± ± ± ± ± ± ± ± ± ±
10 mg/L 16 mg/L 13 mg/L 12 mg/L 17 mg/L 16 mg/L 17 mg/L 11 mg/L 10 mg/L 10 mg/L
1041.89 + 90.07 mg/kg 623.77 + 89.5 mg/kg
752.2 mg/L 971.0, 1373.2 and 1303.1 mg/l, respectively 645 mg/L 588–776 mg/L
0 0–24.9 mg/L
increase of 23.8%, 23.7% and 16.6% (Fig. 2). Such enhancement was significant (p = 0.007) compared to the control group, which is also higher than the reported values in the previous research (Su et al., 2013; Suanon et al., 2017). For example, Suanon et al. (2017) reported a 3–11% enhancement in CH4 content in the biogas produced when ZVI was added to an anaerobia digester. Su et al.(2013) observed a 5.7–13.2% increase in CH4 concentration in biogas produced from an anaerobic sludge AD system with 0.1% supplementation of nZVI20 (Table 2). The enhanced CH4 content in the biogas produced at a high nZVI concentration (10 g/L) during AD may be attributed to three major pathways: (i) CO2 was converted to CH4 by hydrogenotrophic methanogens when nZVI served as an electron donor (Eq. (1)); (ii) CO2
3.2. Impact of zero valent iron on methane content in the biogas The application of nZVI35, nZVI50, and mZVI at low doses of 0.5–1 g/L did not significantly affect the methane content in the biogas (p = 0.25), with the methane content being 79.3 ± 0.6%–83.9 ± 1.1% compared to an average CH4 content of 80.1 ± 1.2% in the control group (Fig. 2). When nZVI35, nZVI50, and mZVI were dosed at 10 g/L, the CH4 content were 99.2 ± 0.8%, 99.1 ± 0.9%, and 93.4 ± 0.6%, respectively, with a respective 4
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Fig. 3. Change in CODs (a) and acetic acid (b) concentration after 5-day reaction with nZVI or mZVI treatments.
was reduced for CH4 production through hydrogenotrophic methanogenesis when the addition of nZVI caused H2 to be produced, and H2 served as an electron donor (Eqs. (2)–(3)); and (iii) CO2 may be absorbed in the aqueous environment under high pH conditions (Xu et al., 2018). In the current study, the impact of CO2 consumption by hydrogenotrophic methanogens to produce CH4 with high nZVI dose was low, considering the inhibition of BMP observed at this condition (Fig. 1). On the other hand, the significantly increased pH at high doses of nZVI35 (8.79) and nZVI50 (9.44) might have contributed to CO2 adsorption in aqueous environment of blackwater.
the sodium 2-bromoethanesulfonic acid (BESA) addition). The CODs results suggest that nZVI effectively accelerated the hydrolysis of sludge, especially at high dosage. In detail, after the 5-day reaction, the CODs was 5124 ± 114 mg/L with no ZVI addition (control in Fig. 3(a)), which demonstrated that particulate organic matter was rapidly decomposed by microbes and converted to smaller soluble organic compounds (Razaviarani & Buchanan, 2014). When nZVI35 was applied from 0.5 to 10 g/L, after a 5-day reaction, the CODs increased to 5367 ± 120–6438 ± 100 mg/L. Likewise, when nZVI50 was applied from 0.5 to 10 g/L, after a 5-day reaction, the soluble increased to 5336 ± 122–7013 ± 115 mg/L. This result indicated that the hydrolysis rate of particulate organic matter in blackwater improved with applying nZVI from 0.5 to 10 g/L, in agreement with the results of waste activated sludge treatment in Wang’s research (2018). In contrast, the impact of mZVI on the production of CODs in blackwater was
3.3. Impact of zero valent iron on hydrolysis-acidification in blackwater Fig. 3(a) shows the change in CODs with and without ZVI addition to blackwater in the presence of hydrolysis-acidification only (due to 5
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Fig. 4. Change in 16S rRNA gene copy abundance of methanogenic population in the presence of 0, 0.5, 1, and 10 g/L nZVI35.
negligible, which was predictable considering the low reactivity of mZVI in these systems. As a rate limiting step in anaerobic digestion, the conversion of particulate matter to soluble substrates affected the production of precursors of methanogenesis (i.e., acetate) in the following acidogenesis and acetogenesis processes (Gao et al., 2019). An increase in CODs with nZVI treatment could lead to higher volatile fatty acids (VFAs) production, which could prompt acetolactic methanogenesis by providing more substrates. Changes in the concentration of acetic acid in anaerobic digestion systems are summarized in Fig. 3(b). Generally, the concentration of acetic acid was enhanced in the presence of nZVI and was not affected significantly by the addition of mZVI. The concentration of acetic acid was 864 ± 120 mg/L in the batch reactor with no ZVI application (control). In the reactors with 0.5–10 g/L nZVI35 and nZVI50 application, the concentration of acetic acid reached 945 ± 12–984 ± 16 mg/L and 932 ± 17–960 ± 17 mg/L, respectively, which was significantly higher (p = 0.0001) than the acetic acid concentration in the control reactors. However, the concentrations of acetic acid in batch reactors with 0.5, 1, and 10 g/L mZVI treatments were comparable (p > 0.05) to the concentrations of acetic acid in the control reactors, which were 890 ± 11, 863 ± 10, and 852 ± 10 mg/L, respectively. The addition of nZVI to the AD system created a more reductive atmosphere, enhancing hydrolysis and acidification which provided favorable substrates for methanogenesis (Wang et al., 2006). The ORP results also support this finding. An increase in such substrates could stimulate the growth and metabolism of acetolactic methanogens, resulting in more CH4 production, as observed at low nZVI doses in the current study (Fig. 1).
hydrogenotrophic methanogens, including Methanomicrobiales, Methanobacteriales, and Methanoculleus, gradually increased from (5.13 ± 0.21)×107 to (1.07 ± 0.04)×108, (2.01 ± 0.08)×107 to (3.71 ± 0.2)×107, and (1.22 ± 0.01)×107 to (1.50 ± 0.0)×107 copies/mL, respectively, whereas the gene copy concentrations of the hydrogenotrophic methanogen Methanothermobacter decreased slightly from (3.54 ± 0.2)×105 to (2.27 ± 0.1)×105 copies/mL. However, gene copy concentrations of Methanomicrobiales and Methanoculleus were reduced to (3.14 ± 0.1)×107 and (6.24 ± 0.2)×107 copies/mL, respectively, when nZVI35 was applied at 10 g/L, while the gene copy concentrations of Methanobacteriales and Methanothermobacter continued to increase to (1.03 ± 0.05)×108 copies/mL and (1.29 ± 0.04)×106 copies/mL, respectively. These observations suggest that nZVI might have served as an electron donor in the production of methane by hydrogenotrophic methanogens (Karri et al., 2005; Meng et al., 2013) until the biocidal effect of nZVI35 at high dosage (10 g/L) became dominant for some hydrogenotrophic methanogens (i.e., Methanomicrobiales and Methanoculleus). These results are consistent with the BMP results, which showed an inhibition of methanogenesis at high nZVI35 concentrations. Likewise, inhibition of a versatile acetolactic methanogen, Methanosarcina, which can feed on either H2 or acetate, was observed when nZVI35 was applied at 10 g/L. However, the impact of nZVI35 on Methanosaeta, a strictly acetolactic methanogen, was inconclusive in this study. As the nZVI effects on methanogens might be controlled by the direct contact of nZVI with cell membranes or by the influence of nZVI on methanogen substrate (e.g., acetic acid) production, additional research will shed light on these results. 4. Conclusions
3.4. Microbial qPCR analysis
This study presents a first-time demonstration of the effects of zero valent iron in the form of micro-scale particles (mZVI) and nano-scale particles (nZVI) on methane production in blackwater. For blackwater treatment, the optimum ZVI size range was between 35 and 50 nm and the optimum dose ranges was from 0.5 to 1 g/L. In particular, both nZVI35 and nZVI50 at low dosage (0.5–1 g/L) accelerated the hydrolysis of organic waste in blackwater and improved methane production.
Considering the similar impact of nZVI35 and nZVI50 on AD methane production, nZVI35 was selected as the representative agent for the microbial analysis. The gene abundance dynamics of the methanogenic community at different doses of nZVI35 are reflected in quantitative PCR analysis (Fig. 4). In general, at low dosage (up to 1 g/ L), nZVI35 improved the gene copy concentrations of hydrogenotrophic methanogens, while at high dosage, nZVI35 inhibited the gene copy concentrations of hydrogenotrophic methanogens selectively. When nZVI35 dosage increased from 0 to 1 g/L, gene copy concentrations of 6
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Acknowledgement
anaerobic conversion of propionate to acetate. Biochem. Eng. J. 73, 80–85. Razaviarani, V., Buchanan, I.D., 2014. Reactor performance and microbial community dynamics during anaerobic co-digestion of municipal wastewater sludge with restaurant grease waste at steady state and overloading stages. Bioresour. Technol. 172, 232–240. Su, L., Shi, X., Guo, G., Zhao, A., Zhao, Y.J.J.o.M.C., Management, W. 2013. Stabilization of sewage sludge in the presence of nanoscale zero-valent iron (nZVI): abatement of odor and improvement of biogas production. 15(4), 461-468. Suanon, F., Sun, Q., Li, M., Cai, X., Zhang, Y., Yan, Y., Yu, C.-P., 2017. Application of nanoscale zero valent iron and iron powder during sludge anaerobic digestion: Impact on methane yield and pharmaceutical and personal care products degradation. J. Hazard. Mater. 321, 47–53. Tyagi, V.K., Fdez-Güelfo, L.A., Zhou, Y., Álvarez-Gallego, C.J., Garcia, L.I.R., Ng, W.J., 2018. Anaerobic co-digestion of organic fraction of municipal solid waste (OFMSW): progress and challenges. Renew. Sustain. Energy Rev. 93, 380–399. Velimirovic, M., Carniato, L., Simons, Q., Schoups, G., Seuntjens, P., Bastiaens, L., 2014. Corrosion rate estimations of microscale zerovalent iron particles via direct hydrogen production measurements. J. Hazard. Mater. 270, 18–26. Viggi, C.C., Rossetti, S., Fazi, S., Paiano, P., Majone, M., Aulenta, F., 2014. Magnetite particles triggering a faster and more robust syntrophic pathway of methanogenic propionate degradation. Environ. Sci. Technol. 48 (13), 7536–7543. Wang, H., Zhang, Y., Angelidaki, I., 2016. Ammonia inhibition on hydrogen enriched anaerobic digestion of manure under mesophilic and thermophilic conditions. Water Res. 105, 314–319. Wang, L., Zhou, Q., Li, F.T., 2006. Avoiding propionic acid accumulation in the anaerobic process for biohydrogen production. Biomass Bioenergy 30 (2), 177–182. Wang, Y.Y., Wang, D.L., Fang, H.Y., 2018. Comparison of enhancement of anaerobic digestion of waste activated sludge through adding nano-zero valent iron and zero valent iron. RSC Adv. 8 (48), 27181–27190. Wei, J., Hao, X., Loosdrecht, M.C.M.V., Li, J., 2018. Feasibility analysis of anaerobic digestion of excess sludge enhanced by iron: A review. Renew. Sustain. Energy Rev. 89, 16–26. Xu, R., Xu, S., Florentino, A.P., Zhang, L., Yang, Z., Liu, Y., 2019. Enhancing blackwater methane production by enriching hydrogenotrophic methanogens through hydrogen supplementation. Bioresour. Technol. 278, 481–485. Xu, R., Yang, Z.H., Zheng, Y., Liu, J.B., Xiong, W.P., Zhang, Y.R., Lu, Y., Xue, W.J., Fan, C.Z., 2018. Organic loading rate and hydraulic retention time shape distinct ecological networks of anaerobic digestion related microbiome. Bioresour. Technol. 262, 184–193. Xu, S., Wu, D., Hu, Z., 2014. Impact of hydraulic retention time on organic and nutrient removal in a membrane coupled sequencing batch reactor. Water Res. 55, 12–20. Yang, Y., Guo, J., Hu, Z., 2013. Impact of nano zero valent iron (NZVI) on methanogenic activity and population dynamics in anaerobic digestion. Water Res. 47 (17), 6790–6800. Yenigün, O., Demirel, B., 2013. Ammonia inhibition in anaerobic digestion: a review. Process Biochem. 48 (5), 901–911. Zhao, Z.S., Li, Y., Yu, Q.L., Zhang, Y.B., 2018. Ferroferric oxide triggered possible direct interspecies electron transfer between Syntrophomonas and Methanosaeta to enhance waste activated sludge anaerobic digestion. Bioresour. Technol. 250, 79–85.
Dr Liu acknowledges financial support for this project provided by research grants from a Natural Sciences and Engineering Research Council of Canada (NSERC) collaborative research and development (CRD) project, an NSERC Industrial Research Chair (IRC) Program in Sustainable Urban Water Development, through the support of EPCOR Water Services, EPCOR Drainage Operation, Alberta Innovates, WaterWerx, the Canada Research Chair (CRC) in Future Community Water Services, and the China Scholarship Council (CSC) Ph.D. scholarship for Xu, R. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.121351. References Carpenter, A.W., Laughton, S.N., Wiesner, M.R., 2015. Enhanced biogas production from nanoscale zero valent iron-amended anaerobic bioreactors. Environ. Eng. Sci. 32 (8), 647–655. Feng, Y.H., Zhang, Y.B., Quan, X., Chen, S., 2014. Enhanced anaerobic digestion of waste activated sludge digestion by the addition of zero valent iron. Water Res. 52, 242–250. Florentino, A.P., Sharaf, A., Zhang, L., Liu, Y., 2019. Overcoming ammonia inhibition in anaerobic blackwater treatment with granular activated carbon: the role of electroactive microorganisms. Environ. Sci. Water Res. Technol. 2, 383–396. Gao, M., Zhang, L., Florentino, A.P., Liu, Y., 2019. Performance of anaerobic treatment of blackwater collected from different toilet flushing systems: Can we achieve both energy recovery and water conservation? J. Hazard. Mater. 365, 44–52. Karri, S., Sierra-Alvarez, R., Field, J.A., 2005. Zero valent iron as an electron-donor for methanogenesis and sulfate reduction in anaerobic sludge. Biotechnol. Bioeng. 92 (7), 810–819. Liu, Y., Zhang, Y., Ni, B.-J., 2015. Zero valent iron simultaneously enhances methane production and sulfate reduction in anaerobic granular sludge reactors. Water Res. 75, 292–300. Liu, Y., Zhang, Y., Quan, X., Chen, S., Zhao, H., 2011. Applying an electric field in a builtin zero valent iron - Anaerobic reactor for enhancement of sludge granulation. Water Res. 45 (3), 1258–1266. Liu, Y., Zhang, Y., Quan, X., Li, Y., Zhao, Z., Meng, X., Chen, S., 2012. Optimization of anaerobic acidogenesis by adding Fe0 powder to enhance anaerobic wastewater treatment. Chem. Eng. J. 192, 179–185. Meng, X.S., Zhang, Y.B., Li, Q., Quan, X., 2013. Adding Fe-0 powder to enhance the
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Short Communication
Impact of zero valent iron on blackwater anaerobic digestion a,b,c,1
Rui Xu a b c
a,1
, Shengnan Xu
a
a
b,c
a,⁎
, Lei Zhang , Anna Patricya Florentino , Zhaohui Yang , Yang Liu
T
Department of Civil and Environmental Engineering, University of Alberta, 7-263 Donadeo Innovation Centre for Engineering, Edmonton, Alberta T6G 1H9, Canada College of Environmental Science and Engineering, Hunan University, Changsha 410082, PR China Key Laboratory of Environmental Biology and Pollution Control (Hunan University), Ministry of Education, Changsha 410082, PR China
ARTICLE INFO
ABSTRACT
Keywords: Anaerobic blackwater treatment Nano-scale zero valent iron Micro-scale zero valent iron Methane production
The source diverted blackwater treatment is receiving growing attention as an alternative to conventional energy intensive wastewater management and treatment systems. Blackwater, containing concentrated organic materials, can be anaerobically digested to recovery bioenergy. However, the methane recovery from blackwater is often inhibited by the presence of high free ammonia (FA) in blackwater. In order to improve the methane production in blackwater, nano-scale zero valent iron (nZVI, 35 nm or 50 nm) or micro-scale zero valent iron (mZVI, 200 μm) at different dosages (i.e., 0.5, 1, and 10 g/L) were applied respectively in the anaerobic digestion (AD) reactor for blackwater treatment. The results demonstrated that low doses (0.5–1 g/L) of nZVI slightly improved methane (CH4) production, possibly due to a reduced oxidation–reduction potential (ORP) and improved hydrolysis-acidification in the nZVI supplemented systems. However, a lower biochemical methane potential (BMP) of blackwater was observed with high doses (10 g/L) of nZVI which induced a pH increase (> 8.5) in AD reactor leading to a higher FA inhibition of CH4 production. In contrast, the effect of mZVI on blackwater AD system was not significant. The study demonstrated the successful application of nZVI for improving AD of blackwater, however, which requires dosage control.
1. Introduction With the growing attention to resource recovery based wastewater treatment, anaerobic digestion (AD, i.e., a sustainable wastewater treatment technology by converting organics into methane in the absence of oxygen (Liu et al., 2015)) of source diverted blackwater (i.e., toilet wastewater, which is an alkaline wastewater flow consisting of flushwater, urine and feces, toilet paper, and with food waste residuals in some scenarios (Xu et al., 2019)) has been recognized as an attractive option to maximize energy and nutrient recovery and to replace energy intensive aerobic municipal wastewater treatment (Tyagi et al., 2018). Blackwater characteristics vary significantly with the collection sources. For instance, anaerobic digestion was compromised in the treatment of blackwater collected from vacuum toilets because blackwater consisted of high FA concentration which inhibited methane (CH4) production (Gao et al., 2019). Florentino et al. (2019) applied granular activated carbon to improve blackwater digestibility, and demonstrated that granular activated carbon enriches the conductive microbial culture, especially H2 utilizers that resist FA inhibition. Xu et al. (2019) applied H2 enriched culture to blackwater, and observed a direct enhancement of CH4 production.
The strong reductants zero valent iron (ZVI, e.g., micro-scale ZVI (mZVI) or nano-scale ZVI (nZVI)) have been applied as a means to improve anaerobic digestion of anaerobic sludge or wastewater activated sludge by increasing CH4 production, eliminating the emission of the odorous gas H2S, and enhancing phosphate recovery (Wei et al., 2018). Su et al. (2013) found that an anaerobic digester supplemented with 0.1% nZVI increased CH4 production by 40.4%. Suanon et al. (2017) observed 25% enhancement in CH4 production with 0.1% nZVI supplement. It has been reported that ZVI improves CH4 production in anaerobic digestion of waste activated sludge by influencing both methanogenesis and hydrolysis-acidification processes (Feng et al., 2014). ZVI affects methanogenesis through two mechanisms. First, ZVI maintains stable and favorable conditions for methanogens by lowering the oxidation–reduction potential (ORP) of the treatment substrate (Meng et al., 2013; Wei et al., 2018) or by increasing the pH of the aquatic environment to a neutral range (Suanon et al., 2017). Second, ZVI can stimulate the growth of hydrogenotrophic methanogen by providing electrons directly or by producing hydrogen through the iron anaerobic oxidation to facilitate CH4 production from the consumption of CO2 (Eqs. (1)–(3)) (Karri et al., 2005; Meng et al., 2013).
Corresponding author. E-mail address: [email protected] (Y. Liu). 1 Both authors contributed equally to this work. ⁎
https://doi.org/10.1016/j.biortech.2019.121351 Received 28 February 2019; Received in revised form 12 April 2019; Accepted 13 April 2019 Available online 15 April 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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CO2 + 4Fe0 + 8H+
Three types of commercially available zero valent iron (ZVI) particles were used in this study, including nZVI35 (35 nm, > 99.5% Fe0) obtained from Sigma-Aldrich (St Louis, United States); nZVI50 (50 nm, NANOFER STAR, with an oxide shell and > 80% Fe0) obtained from NANORION (Rajhrad, Czech Republic); and mZVI (200 μm, > 99% Fe0) obtained from Fisher Scientific (Hampton, United States). These commercial particles were selected to represent widely used nZVI and mZVI particles in previous studies (Carpenter et al., 2015; Feng et al., 2014; Velimirovic et al., 2014). The specific surface area for nZVI and mZVI were in the range of 10–25 m2/g and 0.05–5 m2/g, respectively. nZVI and mZVI particles were used immediately after being received. Anaerobic reactors were purged with pure N2 for at least five minutes to deoxygenate the solution before being incubated at 35 °C.
(1)
CH4 + 4Fe2 + + 2H2 O
Fe0 + 2H2 O
Fe 2+ + H2 + 2OH
(2)
CO2 + 4H2
CH4 + 2H2 O
(3)
ZVI has also been applied to accelerate the hydrolysis step (i.e., the limiting step) of anaerobic digestion of sludge by converting particulate matter to soluble substrates (Liu et al., 2015; Liu et al., 2012; Wang et al., 2018). The supplementation of ZVI can optimize the acidification step of anaerobic digestion by directly promoting propionate degradation and subsequently raising the production of acetate to facilitate methanogenesis (Liu et al., 2015; Liu et al., 2012; Meng et al., 2013; Suanon et al., 2017). Accordingly, the addition of ZVI could increase the abundance of microbial communities, such as bacteria which convert propionate to acetate (Liu et al., 2011; Meng et al., 2013). Moreover, due to its conductive characteristics, ZVI is assumed to stimulate direct interspecies electron transfer, which can enhance CH4 production in anaerobic digestion of sludge by prompting electron transfer between certain acetogens and methanogens. However, these mechanisms have been reported only for Fe(III) oxides such as Fe3O4 or Fe3O4-ZVI particles; the role of ZVI is still unclear (Viggi et al., 2014; Zhao et al., 2018). Acetoclastic methanogens, especially Methanosaeta, were found to be dominant in conducting such processes (Zhao et al., 2018). The impact of ZVI on anaerobic digestion depends on factors related to both ZVI particle size (e.g., particle size, surface properties, and dosage) and anaerobic systems (e.g., presence of a microbial community in the system) (Wang et al., 2018). The significantly higher specific surface area of nZVI compared to mZVI leads to a significantly higher impact of nZVI on the improvement of anaerobic digestion. However, the superior reactivity of nZVI may also lead to the biocidal effect of nZVI in some scenarios, which has been reported to cause the reduction of microbial community diversity and biomass density (Su et al., 2013; Yang et al., 2013). For instance, Yang et al. (2013) reported that 1.68 g/ L of nZVI inhibited methanogenesis because of the potent reducing properties at the surface of nZVI particles can damage cell membranes through directly contact, although mZVI at the same dosage enhanced CH4 production of the target anaerobic sludge. Therefore, there is still a lack of consensus on the impacts of ZVI on AD process, especially in anaerobic microbial communities. Whether ZVI can improve anaerobic digestion in blackwater, and alleviate FA inhibition of AD, has not been investigated. This study investigates the impacts of ZVI size and dosage on methanogenesis and on hydrolysis-acidification processes during anaerobic digestion in blackwater. To the best knowledge, this is the first report that discusses the application of ZVI in AD of blackwater.
2.2. Impact of zero valent iron on anaerobic digestion The impacts of nZVI or mZVI on AD were determined by measuring the biochemical methane potential (BMP) in batch reactors, as described below. To 160 mL serum bottles (batch reactors), 50 mL of sludge inoculum and 50 mL of blackwater were added to create a 60 mL head space. Aliquots of nZVI35, nZVI50, and mZVI particles were then added to separate bottles to reach respective final concentrations of 0.5, 1, and 10 g/L in the blackwater AD systems, which were selected based on the effective dosage range in the literature (Feng et al., 2014; Karri et al., 2005). Each condition was prepared in triplicate. Triplicates of a negative control (distilled water replaced blackwater, iron free) and a positive control (blackwater only, iron free) were subjected to the same conditions and procedure as the samples. The head space in each serum bottle was purged with high purity N2 gas for five minutes to remove oxygen before sealing the bottle with a butyl rubber stopper and a crimp cap (Bellco Gass, Vineland, NJ). The bottles were shaken in an incubator shaker (ThermoFisher Scientific, USA) at 120 rpm and 35 °C until biogas production ceased (50 days). BMP recording and calculation were performed following the methods in previous research (Xu et al., 2019). 2.3. Impact of zero valent iron on hydrolysis-acidification To measure the impact of ZVI on hydrolysis-acidification during anaerobic digestion, a methanogenesis inhibition procedure was conducted in the batch anaerobic digestors prepared in Section 2.2. Briefly, the blackwater and seed sludge mixture was heated at 102 °C for 30 min, then sodium 2-bromoethanesulfonic acid (BESA) was added to the batch reactors to a final concentration of 30 mM to completely inhibit methanogenesis (Feng et al., 2014; Yang et al., 2013). The batch experiments were conducted for five days in an incubator shaker at 35 °C, before the water samples were collected for soluble chemical oxygen demand (CODs) and acetic acid measurements.
2. Materials and methods 2.1. Preparation of seed sludge, blackwater, and zero valent iron
2.4. Methanogenic population analysis after zero valent iron exposure to anaerobic digestion
Anaerobic sludge was obtained from an anaerobic digester in a local sewer treatment plant in Edmonton (Canada) and blended to homogeneity before seeding in blackwater. Blackwater (1L water per flush, vacuum toilet wastewater) was collected directly from campus toilets at the University of Alberta. The collected seed sludge and blackwater were utilized immediately or stored anaerobically at 4 °C for less than 7 days before being utilized. Characteristics of anaerobic seed sludge and blackwater used in the experiments are summarized in Table 1.
nZVI35 was selected as the representative ZVI for the quantitative polymerase chain reaction (qPCR) experiment, which was used to determine the presence of methanogenic communities and their population changes after ZVI exposure to anaerobic digestion, according to the methods described in previous research (Xu et al., 2019). Briefly, sludge samples in the presence of 0, 0.5, 1, and 10 g/L nZVI35 were collected
Table 1 Characteristics of seed sludge and blackwater. Parameters
pH
CODt (g/L)
CODs (mg/L)
TAN (mg/L)
TP (mg/L)
TSS (mg/L)
seed sludge blackwater
7.24 ± 0.3 8.82 ± 0.1
22.8 ± 1.45 18.7 ± 0.8
475 ± 38 5058 ± 105
1144 ± 27 1254 ± 26
/ 330 ± 11
/ 17140 ± 104
2
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from the anaerobic batch reactors after 50 days of incubating at 35 °C, from which genomic DNA was extracted using a QIAGEN PowerSoil Pro Kit (Hilden, Germany). Immediately after DNA extraction, qPCR analysis was performed targeting hydrogenotrophic methanogens (Methanobacteriales, Methanomicrobiales, Methanothermobacter and Methanoculleus), strictly acetoclastic methanogen Methanosaeta and versatile acetoclastic methanogen Methanosarcina. Detailed primer information and qPCR reaction conditions were summarized in the Supporting Information.
1, and 10 g/L mZVI, BMP values in blackwater did not change significantly; they were 37.1 ± 0.4%, 37.2 ± 0.7%, and 36.9 ± 0.6%, respectively (p = 0.9), indicating that the impact of mZVI on the BMP was negligible. Compared to the mZVI application group, a low dose of 0.5–1 g/L of nZVI led to a blackwater BMP increase to 42.7 ± 0.5%–44.8 ± 0.6% for nZVI35 and to 42.9 ± 0.7%–45.2 ± 0.6% for nZVI50 (p < 0.05 for all). The production of electrons or H2 in the presence of nZVI facilitated the growth of methanogens (Karri et al., 2005; Suanon et al., 2017) and/or increased favorable fermentation under decreased oxidation-reduction potential (ORP) conditions at the low nZVI dose, keeping methanogen activities at a high level. However, a high dose (10 g/L) of nZVI significantly reduced the BMP by 75–81% (p = 0.013); and BMP values were 7.1 ± 0.3% and 9.3 ± 0.3%, respectively, in the presence of 10 g/L nZVI35 and 10 g/L nZVI50. This inhibition was also observed in Yang et al.’s research (2013), where a 69% reduction in BMP occurred with the dose of 1.68 g/L of nZVI50. According to the authors’ explanation, serious cell membrane or respiratory activity damage occurred through direct contact between nZVI and methanogen cells due to the potent reducing conditions at the nZVI surface (Yang et al., 2013). This observation is also evident in the qPCR results (Fig. 4), where the gene copy concentrations of Methanomicrobiales and Methanoculleus were reduced significantly. Moreover, the high dosage of nZVI led to a significant increase in pH (p = 0.01) (i.e., 8.79 ± 0.05 with nZVI35 and 9.44 ± 0.03 with nZVI50), likely because of nZVI oxidation (Eq. (2)) in the anaerobic system (Suanon et al., 2017). The significantly elevated pH (p < 0.01) in blackwater treated with high doses of nZVI35 and nZVI50 significantly enhanced the FA concentration from 14.7 mg/L (control) to 516.1 mg/L and 949.9 mg/L, respectively, based on the theoretical FA concentration calculated from the TAN concentration measured in blackwater (Table 1). The FA molecule diffuses more easily than the ammonium ion, leading to a more facile contact of FA with methanogens, and thus significant inhibition of methanogen activity and a subsequent lower CH4 yield (i.e., a decreased BMP at high nZVI dose (Fig. 1)) (Wang et al., 2016; Yenigün & Demirel, 2013). The moderate increase (< 95.2 mg/L) in FA concentration caused by a slightly increased pH at low doses of nZVI and at all doses of mZVI did not result in further inhibition of methanogens by FA (Gao et al., 2019). With a BMP inhibition prevailed in the system exposed to
2.5. Chemical and statistical analysis Chemical and statistical analyses were performed with the methods described in previous research (Xu et al., 2019). Briefly, the recording of biogas volume was performed with a gas meter (GMH3111, GREISINGER) and biogas composition was analyzed with gas chromatography (GC-7890B, Agilent Technologies, USA). The chemical concentrations in the collected samples – i.e., total COD (CODt), soluble COD (CODs), total NH4+-N (TAN), total PO43−-P (TP) and total suspended solid (TSS) – were measured using HACH kits as reported previously (Xu et al., 2014); FA concentration was determined based on the TAN concentration (Xu et al., 2019); acetic acid concentration was analyzed by a Dionex ICS-2100 ion chromatograph equipped with an IonPac AS18 column (Dionex, Sunnyvale, CA). Oxidation-reduction potential (ORP) and pH were measured under anaerobic conditions using a conductivity ORP/pH meter with different electrodes (Mettler Toledo, USA). One-way ANOVA (p values < 0.05) was performed to identify the statistical significance between groups. 3. Results and discussion 3.1. Impact of zero valent iron on methane production in blackwater Under mesophilic conditions, organic substrates in blackwater are anaerobically digested to generate CH4 (Gao et al., 2019). The anaerobic digestibility of organic substrates can be evaluated by measuring the BMP (Yang et al., 2013). The control groups are the groups where no nZVI or mZVI was added. After 50 days incubation in the control groups, blackwater BMP was 36.8 ± 0.4% (Fig. 1). With doses of 0.5,
Fig. 1. BMP of blackwater anaerobic digestion treatment in the presence of nZVI35, nZVI50, or mZVI, each at concentrations of 0, 0.5, 1, and 10 g/L. 3
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Fig. 2. CH4 content in the produced biogas of the blackwater treatment in the presence of nZVI35, nZVI50, or mZVI, each at concentrations of 0, 0.5, 1 and 10 g/L. Table 2 Summary of the ZVI performance in different systems.
The current study
Treatment
ZVI concentration
CH4 production
CH4 content
CODs
Acetic Acids
BW BW + nZVI35
0 g/L 0.5 g/L 1 g/L 10 g/L 0.5 g/L 1 g/L 10 g/L 0.5 g/L 1 g/L 10 g/L 0 0.1 (wt% sludge)
36.8 ± 0.4% 42.7 ± 0.5% 44.8 ± 0.6% 7.1 ± 0.3% 42.9 ± 0.7% 45.2 ± 0.6% 9.3 ± 0.3% 37.1 ± 0.4% 37.2 ± 0.4% 36.9 ± 0.6%
80.1 ± 1.2% 81.2 ± 0.4% 83.9 ± 1.1% 99.2 ± 0.8% 79.3 ± 0.6% 80.5 ± 0.7% 99.1 ± 0.9% 80.9 ± 0.9% 81.6 ± 1.0% 93.4 ± 0.6% 70 ± 3.9% 73.4 ± 2.3%
5124 ± 114 mg/L 5367 ± 120 mg/L 5477 ± 122 mg/L 6438 ± 100 mg/L 5336 ± 121 mg/L 5634 ± 125 mg/L 7013 ± 115 mg/L 5158 ± 121 mg/L 5116 ± 133 mg/L 5258 ± 109 mg/L 3683 mg/L 3032.1 mg/L
864 984 962 945 960 948 932 890 863 852
0 0.1 (wt % sludge)
8.16L 13.7L
42.8–64.2% 48.2–70.6%
0 18.6 g/L in 3.0 g VSS/L anaerobic sludge 0 1, 4 and 20 g/L ZVI
2.87 mmol CH4/L 8.17 mmol/L
BW + nZVI50 BW + mZVI Suanon et al, 2017 Su et al, 2013 Karri et al., 2005
Sewage sludge Sewage sludge + 160 nm nZVI Anaerobic sludge Anaerobic sludge + 20 nm ZVI Sludge Sludge + 10 um mZVI
Feng, et al, 2014
waste activated Sludge waste activated sludge + 0.2 mm nZVI
Yang et al, 2013
Digested sludge Digested sludge + 55 nm nZVI
0 1, 10, 30 mM nZVI
192.6 mL/VSS 211.1. 233.8 and 276.4 mL/VSS, respectively 30.8% 24.5%, 24.5%, and 9.5%, respectively
a high dose of nZVI, the potential positive effect of the further reduced ORP was negligible.
± ± ± ± ± ± ± ± ± ±
10 mg/L 16 mg/L 13 mg/L 12 mg/L 17 mg/L 16 mg/L 17 mg/L 11 mg/L 10 mg/L 10 mg/L
1041.89 + 90.07 mg/kg 623.77 + 89.5 mg/kg
752.2 mg/L 971.0, 1373.2 and 1303.1 mg/l, respectively 645 mg/L 588–776 mg/L
0 0–24.9 mg/L
increase of 23.8%, 23.7% and 16.6% (Fig. 2). Such enhancement was significant (p = 0.007) compared to the control group, which is also higher than the reported values in the previous research (Su et al., 2013; Suanon et al., 2017). For example, Suanon et al. (2017) reported a 3–11% enhancement in CH4 content in the biogas produced when ZVI was added to an anaerobia digester. Su et al.(2013) observed a 5.7–13.2% increase in CH4 concentration in biogas produced from an anaerobic sludge AD system with 0.1% supplementation of nZVI20 (Table 2). The enhanced CH4 content in the biogas produced at a high nZVI concentration (10 g/L) during AD may be attributed to three major pathways: (i) CO2 was converted to CH4 by hydrogenotrophic methanogens when nZVI served as an electron donor (Eq. (1)); (ii) CO2
3.2. Impact of zero valent iron on methane content in the biogas The application of nZVI35, nZVI50, and mZVI at low doses of 0.5–1 g/L did not significantly affect the methane content in the biogas (p = 0.25), with the methane content being 79.3 ± 0.6%–83.9 ± 1.1% compared to an average CH4 content of 80.1 ± 1.2% in the control group (Fig. 2). When nZVI35, nZVI50, and mZVI were dosed at 10 g/L, the CH4 content were 99.2 ± 0.8%, 99.1 ± 0.9%, and 93.4 ± 0.6%, respectively, with a respective 4
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Fig. 3. Change in CODs (a) and acetic acid (b) concentration after 5-day reaction with nZVI or mZVI treatments.
was reduced for CH4 production through hydrogenotrophic methanogenesis when the addition of nZVI caused H2 to be produced, and H2 served as an electron donor (Eqs. (2)–(3)); and (iii) CO2 may be absorbed in the aqueous environment under high pH conditions (Xu et al., 2018). In the current study, the impact of CO2 consumption by hydrogenotrophic methanogens to produce CH4 with high nZVI dose was low, considering the inhibition of BMP observed at this condition (Fig. 1). On the other hand, the significantly increased pH at high doses of nZVI35 (8.79) and nZVI50 (9.44) might have contributed to CO2 adsorption in aqueous environment of blackwater.
the sodium 2-bromoethanesulfonic acid (BESA) addition). The CODs results suggest that nZVI effectively accelerated the hydrolysis of sludge, especially at high dosage. In detail, after the 5-day reaction, the CODs was 5124 ± 114 mg/L with no ZVI addition (control in Fig. 3(a)), which demonstrated that particulate organic matter was rapidly decomposed by microbes and converted to smaller soluble organic compounds (Razaviarani & Buchanan, 2014). When nZVI35 was applied from 0.5 to 10 g/L, after a 5-day reaction, the CODs increased to 5367 ± 120–6438 ± 100 mg/L. Likewise, when nZVI50 was applied from 0.5 to 10 g/L, after a 5-day reaction, the soluble increased to 5336 ± 122–7013 ± 115 mg/L. This result indicated that the hydrolysis rate of particulate organic matter in blackwater improved with applying nZVI from 0.5 to 10 g/L, in agreement with the results of waste activated sludge treatment in Wang’s research (2018). In contrast, the impact of mZVI on the production of CODs in blackwater was
3.3. Impact of zero valent iron on hydrolysis-acidification in blackwater Fig. 3(a) shows the change in CODs with and without ZVI addition to blackwater in the presence of hydrolysis-acidification only (due to 5
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Fig. 4. Change in 16S rRNA gene copy abundance of methanogenic population in the presence of 0, 0.5, 1, and 10 g/L nZVI35.
negligible, which was predictable considering the low reactivity of mZVI in these systems. As a rate limiting step in anaerobic digestion, the conversion of particulate matter to soluble substrates affected the production of precursors of methanogenesis (i.e., acetate) in the following acidogenesis and acetogenesis processes (Gao et al., 2019). An increase in CODs with nZVI treatment could lead to higher volatile fatty acids (VFAs) production, which could prompt acetolactic methanogenesis by providing more substrates. Changes in the concentration of acetic acid in anaerobic digestion systems are summarized in Fig. 3(b). Generally, the concentration of acetic acid was enhanced in the presence of nZVI and was not affected significantly by the addition of mZVI. The concentration of acetic acid was 864 ± 120 mg/L in the batch reactor with no ZVI application (control). In the reactors with 0.5–10 g/L nZVI35 and nZVI50 application, the concentration of acetic acid reached 945 ± 12–984 ± 16 mg/L and 932 ± 17–960 ± 17 mg/L, respectively, which was significantly higher (p = 0.0001) than the acetic acid concentration in the control reactors. However, the concentrations of acetic acid in batch reactors with 0.5, 1, and 10 g/L mZVI treatments were comparable (p > 0.05) to the concentrations of acetic acid in the control reactors, which were 890 ± 11, 863 ± 10, and 852 ± 10 mg/L, respectively. The addition of nZVI to the AD system created a more reductive atmosphere, enhancing hydrolysis and acidification which provided favorable substrates for methanogenesis (Wang et al., 2006). The ORP results also support this finding. An increase in such substrates could stimulate the growth and metabolism of acetolactic methanogens, resulting in more CH4 production, as observed at low nZVI doses in the current study (Fig. 1).
hydrogenotrophic methanogens, including Methanomicrobiales, Methanobacteriales, and Methanoculleus, gradually increased from (5.13 ± 0.21)×107 to (1.07 ± 0.04)×108, (2.01 ± 0.08)×107 to (3.71 ± 0.2)×107, and (1.22 ± 0.01)×107 to (1.50 ± 0.0)×107 copies/mL, respectively, whereas the gene copy concentrations of the hydrogenotrophic methanogen Methanothermobacter decreased slightly from (3.54 ± 0.2)×105 to (2.27 ± 0.1)×105 copies/mL. However, gene copy concentrations of Methanomicrobiales and Methanoculleus were reduced to (3.14 ± 0.1)×107 and (6.24 ± 0.2)×107 copies/mL, respectively, when nZVI35 was applied at 10 g/L, while the gene copy concentrations of Methanobacteriales and Methanothermobacter continued to increase to (1.03 ± 0.05)×108 copies/mL and (1.29 ± 0.04)×106 copies/mL, respectively. These observations suggest that nZVI might have served as an electron donor in the production of methane by hydrogenotrophic methanogens (Karri et al., 2005; Meng et al., 2013) until the biocidal effect of nZVI35 at high dosage (10 g/L) became dominant for some hydrogenotrophic methanogens (i.e., Methanomicrobiales and Methanoculleus). These results are consistent with the BMP results, which showed an inhibition of methanogenesis at high nZVI35 concentrations. Likewise, inhibition of a versatile acetolactic methanogen, Methanosarcina, which can feed on either H2 or acetate, was observed when nZVI35 was applied at 10 g/L. However, the impact of nZVI35 on Methanosaeta, a strictly acetolactic methanogen, was inconclusive in this study. As the nZVI effects on methanogens might be controlled by the direct contact of nZVI with cell membranes or by the influence of nZVI on methanogen substrate (e.g., acetic acid) production, additional research will shed light on these results. 4. Conclusions
3.4. Microbial qPCR analysis
This study presents a first-time demonstration of the effects of zero valent iron in the form of micro-scale particles (mZVI) and nano-scale particles (nZVI) on methane production in blackwater. For blackwater treatment, the optimum ZVI size range was between 35 and 50 nm and the optimum dose ranges was from 0.5 to 1 g/L. In particular, both nZVI35 and nZVI50 at low dosage (0.5–1 g/L) accelerated the hydrolysis of organic waste in blackwater and improved methane production.
Considering the similar impact of nZVI35 and nZVI50 on AD methane production, nZVI35 was selected as the representative agent for the microbial analysis. The gene abundance dynamics of the methanogenic community at different doses of nZVI35 are reflected in quantitative PCR analysis (Fig. 4). In general, at low dosage (up to 1 g/ L), nZVI35 improved the gene copy concentrations of hydrogenotrophic methanogens, while at high dosage, nZVI35 inhibited the gene copy concentrations of hydrogenotrophic methanogens selectively. When nZVI35 dosage increased from 0 to 1 g/L, gene copy concentrations of 6
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Acknowledgement
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Dr Liu acknowledges financial support for this project provided by research grants from a Natural Sciences and Engineering Research Council of Canada (NSERC) collaborative research and development (CRD) project, an NSERC Industrial Research Chair (IRC) Program in Sustainable Urban Water Development, through the support of EPCOR Water Services, EPCOR Drainage Operation, Alberta Innovates, WaterWerx, the Canada Research Chair (CRC) in Future Community Water Services, and the China Scholarship Council (CSC) Ph.D. scholarship for Xu, R. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.121351. References Carpenter, A.W., Laughton, S.N., Wiesner, M.R., 2015. Enhanced biogas production from nanoscale zero valent iron-amended anaerobic bioreactors. Environ. Eng. Sci. 32 (8), 647–655. Feng, Y.H., Zhang, Y.B., Quan, X., Chen, S., 2014. Enhanced anaerobic digestion of waste activated sludge digestion by the addition of zero valent iron. Water Res. 52, 242–250. Florentino, A.P., Sharaf, A., Zhang, L., Liu, Y., 2019. Overcoming ammonia inhibition in anaerobic blackwater treatment with granular activated carbon: the role of electroactive microorganisms. Environ. Sci. Water Res. Technol. 2, 383–396. Gao, M., Zhang, L., Florentino, A.P., Liu, Y., 2019. Performance of anaerobic treatment of blackwater collected from different toilet flushing systems: Can we achieve both energy recovery and water conservation? J. Hazard. Mater. 365, 44–52. Karri, S., Sierra-Alvarez, R., Field, J.A., 2005. Zero valent iron as an electron-donor for methanogenesis and sulfate reduction in anaerobic sludge. Biotechnol. Bioeng. 92 (7), 810–819. Liu, Y., Zhang, Y., Ni, B.-J., 2015. Zero valent iron simultaneously enhances methane production and sulfate reduction in anaerobic granular sludge reactors. Water Res. 75, 292–300. Liu, Y., Zhang, Y., Quan, X., Chen, S., Zhao, H., 2011. Applying an electric field in a builtin zero valent iron - Anaerobic reactor for enhancement of sludge granulation. Water Res. 45 (3), 1258–1266. Liu, Y., Zhang, Y., Quan, X., Li, Y., Zhao, Z., Meng, X., Chen, S., 2012. Optimization of anaerobic acidogenesis by adding Fe0 powder to enhance anaerobic wastewater treatment. Chem. Eng. J. 192, 179–185. Meng, X.S., Zhang, Y.B., Li, Q., Quan, X., 2013. Adding Fe-0 powder to enhance the
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