In-situ biogas upgrading by a stepwise addition of ash additives: Methanogen adaption and CO2 sequestration

Bioresource Technology 282 (2019) 1–8

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In-situ biogas upgrading by a stepwise addition of ash additives: Methanogen adaption and CO2 sequestration Changkai Yina, Yanwen Shena, Yamei Yua, Haiping Yuana, Ziyang Loua, Nanwen Zhua,b, a b

T ⁎

School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, PR China

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Keywords: Waste activated sludge Methane production Sludge ash Stepwise addition Carbon capture

Biogas from anaerobic digestion (AD) of waste activated sludge (WAS) limited its utilization due to low valueadded. In this study, an innovative addition mode for ash known as stepwise addition was developed to enhance methane production and improve CO2 scavenge from AD of sludge. Experimental results confirmed stepwise addition of ash improved methane content to 79.4%, compared to control group (69.1%). Compared to Pulse addition and Control, the cumulative CH4 production was promoted by 39.2% and 35.4%, respectively. Investigation of the mechanism indicated that stepwise addition of ash could decrease hydrolytic and acidifying enzyme activities but increase activity of coenzyme F420, compared to pulse addition group. Furthermore, stepwise addition of ash not only increased the abundance of Methanomassiliicoccus (34.48%), but also promoted amounts of CO2 capture. This method ameliorate utilization availability of sludge ash for sludge anaerobic digestion through promoting cumulative methane production and increasing CO2 storage capacity.

1. Introduction Huge amounts of waste activated sludge (WAS) generated annually from wastewater treatment plants (WWTPs) poses serious secondary pollution issues due to the inherent organic matter, pathogens and heavy metals. WAS contained high level of organic constituents such as protein and carbohydrate is required stabilization (Shen et al., 2016; Zhang et al., 2016). Anaerobic digestion (AD) of sewage sludge has been considered as one of the most widely-used technologies for efficient stabilization of sludge and renewable energy recovery (Braguglia et al., 2018; Chen et al., 2016).

⁎

AD of sludge mainly involves four stages: hydrolysis, acidogenesis, acetogenesis and methanogenesis. The hydrolysis of solid sludge particles is the rate-limiting step of the conventional AD (Ding et al., 2017; Svensson et al., 2018). Many pretreatment methods including chemical (Wang et al., 2013; Wei et al., 2017), thermal (Bougrier et al., 2007), mechanical (Wang and Li, 2016), biological disintegration (Wang et al., 2014a), as well as combinations of these have been applied for improving methane production and shorting lag phase of AD. Among these pretreatment methods, chemical treatment require large amount of chemical reagents, while thermal and mechanical processes need lots of energy to maintain optimal conditions. Evidently, the

Corresponding author at: School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai 200240, PR China. E-mail address: [email protected] (N. Zhu).

https://doi.org/10.1016/j.biortech.2019.02.110 Received 21 January 2019; Received in revised form 21 February 2019; Accepted 23 February 2019 Available online 25 February 2019 0960-8524/ © 2019 Published by Elsevier Ltd.

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2. Materials and methods

aforementioned methods have not been widely used due to their high cost. Sludge ash, a residual product from municipal solid waste incineration, has been proven to improve sludge disintegration and biogas production (Montalvo et al., 2017; Yin et al., 2018). Sludge ash contains high concentration of alkali metal oxide (CaO, MgO, Fe2O3, etc.) and trace metal elements (iron, nickel, zinc, et al.) (Lo et al., 2012b). Sludge ash can enhance the performance of methane production of WAS due to its buffer capability and microelement nutrition. Some authors have reported that the trace elements from ash are vital constituents of enzymes and cofactors in biogenic methane production (Linville et al., 2016; Thanh et al., 2016), and the high buffer capability of anaerobic system can efficiently prevent volatile fatty acids (VFAs) accumulation and provide a good environment for methanogens growth. The similar studies were also reported in application of biochar or activated carbon to improve AD. The sludge-based biochar and ash possessed the identical element composition. However, their mechanism for enhancement of AD were quite different. The conductive biochar could establish direct interspecies electron transport (DIET) between electroactive microorganisms and methanogenic archaea (Feng et al., 2018). For sludge ash, because of the ash were calcined at high temperature (> 900 °C), the component in ash were mainly metal oxide compound that extremely easy to be released into liquid phase due to adequate exposure. Hence, CO2 could be dissolved phase and further carbonated with alkali metal ions, and there is a linear relationship between CO2 sequestration and ash dosage. Application of sludge ash for enhancing AD performance will cut a lot cost for AD process and meanwhile provide a new approach for its disposal. However, according our previously study, a lower methane production was conversely obtained with excess dosage of sludge ash in AD of sludge, though there was high level of VFAs generated in AD process (Lo et al., 2012b). On the other hand, some authors have reported that dosage mode of additive or feeding mode of substrate will determine microbial adaption ability and system stabilization (De Vrieze et al., 2018; Zhao et al., 2018). Campo et al. (2018) reported that the stepwise increase of salinity (from 0.30 to 0.38 g NaCl/L) can promote a biological saltadaption. Park et al. (2018) reported that compared to stepwise feeding of undiluted food waste, continuous feeding of diluted food waste show more stable and efficient performance despite the increase of organic loading rate (OLR) up to 8.6 kg COD/m3/d, attributing to the insignificantly microbial distribution. Bearing in mind the above mentioned issues linked to dosage of sludge ash, a novelty stepwise addition mode for sludge ash was proposed to alleviate the inhibition for microbial and promote AD efficiency. To date, this novel hypothesis has not been experimentally verified for sludge ash addition and its specific influence on AD performance was still not well unknown. The main objective of this study is to analyze the anaerobic performance in batch AD reactor subjected to a stepwise addition of ashes additives. Firstly, the availability of stepwise addition mode was investigated. Secondly, the mechanisms for stepwise addition of ash improving biogas quality were explored by analyzing the variations of soluble chemical oxygen demand (SCOD), polysaccharide (PC), protein (PN) and volatile fatty acids (VFAs). Moreover, the acclimation of bacteria to the sludge ash addition environment was corroborated by analyzing the microbial community and key enzyme activities. The key hydrolytic enzymes (protease and a-glucosidase), acetate kinase and coenzyme F420, which related with hydrolysis, acetogenesis and methanogenesis, were analyzed to reveal the effect of addition mode for individual step during AD (Zhao et al., 2017). Finally, the effect of sludge ash on CO2 sequestration was investigated at various addition mode.

2.1. Substrate, inoculum and ash additives WAS used as the substrate was obtained from the secondary sedimentation tank of a local WWTP located in Shanghai, China. Prior to its use, the sludge was screened with a 2-mm sieve and then concentrated by settling at 4 °C for 24 h. The inoculum was obtained from a lab-scale bioreactor that had been carrying on semi-continuous anaerobic digestion of WWTP sludge for nearly 2 years. The characteristics of the substrate and inoculum were provided in the Supplementary Materials. The sludge incineration bottom ash was used as the additive in this study and its preparation and main constituents were previously reported (Yin et al., 2018).

2.2. Experimental design and operation 2.2.1. Biomethane production test For the 1st set of tests, a total of fifteen 2-L glass bottles with 1.5 L of working volume were used as the anaerobic digesters. One control group and four treatment groups were set, each of which was operated in triplicate. In the treatment groups, the total amount of sludge incineration bottom ash was the same (1.5 g/g dry matter of biomass), except that the dosage was split into one (Group A1), two (Group A2), three (Group A3) and four (Group A4) times. The varied strategy for ash addition among different treatment groups was illustrated in Fig. 1. As shown in our previous study (Yin et al., 2018), such a dosage was excessive to sequester the amount of CO2 generated in the digester. No ash was added to the control group during the experiment. An interval gradient of 0 h, 24 h, 48 h and 72 h for addition time was adopted after the experiment startup in light of reaction time of carbon capture. Substrate and inoculum sludge were mixed with an inoculum-to-substrate ratio of 1:4 based on total solids (TS) and eventually a final biomass concentration of 35 g·L−1 (as TS) was achieved by diluting the sludge slurry with distilled water. Each digester has a 2-port vent assembly for gas and liquid sampling, respectively. The gas outlet was connected to a multi-layer foil gas sampling bag, where biogas samples were taken for its composition analysis. The volume of biogas production was determined periodically using the water displacement method; the gas volume data reported in this study were normalized to standard condition (273 K, 1 atm). The AD experiment was operated under batch mode with temperature maintained at 55 ± 1 °C in an incubator at 100 rpm. Helium degassing was used to obtain an anaerobic

Fig. 1. Sludge incineration bottom ash addition strategy applied in the treatment groups (A1, A2, A3 and A4); no ash was added to the control group (A0). 2

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90

environment initially and all the digesters were maintained airtight throughout the experiment. The experiment was conducted for 25 days. Gas and liquid samples were extracted periodically from each digester for analysis every other day (day 0–10) and every 3 days (day 11–25).

Methane content (%)

(a)

2.2.2. Hydrolysis and acidogenesis test For the 2nd set of tests, anaerobic fermentation experiments were conducted under batch mode to investigate the impact of ash addition on the hydrolysis-acidogenesis phases during AD process. Nine 500-mL bottles with working volume of 450 mL were divided into three groups, each of which was operated in triplicate. To simulate the biomethane production test, each reactor contained 350 mL WAS and 100 mL seed sludge. The characteristics of the sludge and the inoculum were the same as those described above. In the pulse group, each reactor was dosed with 1.5 g/g of ash using single shot (labeled H1); while in the gradual group, incremental doses of 375 mg/g were given every day until the total ash content of 1.5 g/g was reached (labeled H4). No ash was added in the control group (labeled H0). To inhibit methanogenesis, the seed sludge was treated with heat shock at 102 °C for 30 min and later cooled down prior to inoculation (Oh et al., 2003). Moreover, 2-bromoethanesulfonate (BES) was mixed with sludge in each reactor at 50 mM (Feng et al., 2014). The operation of this test was the same as the 1st set, except that the experiments only lasted for 6 days. Liquid samples were extracted every day for analysis.

70

50

A0 A1 A2 A3 A4

30

10 2

4

6

8

10

13

16

19

22

25

Time (d)

2.2.3. Methanogenesis test For the 3rd set of test, batch experiments were conducted to investigate the impact of ash addition on the methanogenesis phase during AD process. Similar to the setup for the hydrolysis-acidogenesis test, nine 500-mL bottles with working volume of 450 mL were also divided into M0, M1 and M4. Each reactor contained 400 mL of substrate WAS and 50 mL seed sludge. Nevertheless, the substrate sludge was subjected to alkaline fermentation pretreatment at 37 °C for 8 days for enhanced hydrolysis, where pH 10.0 was maintained using 5 M sodium hydroxide (NaOH) (Zhang et al., 2011). The pH value was adjusted to neutral (7.0) by 6 M before the experiment. The characteristics of the pretreated sludge was presented in Supplementary Materials. 50 mL of seed sludge were collected from A0, A1 and A4 (day 6 after the experiment startup), respectively. To deduct the influence of seed sludge on CH4 production, same volume of seed sludge were also added into identical bottles. The setup and operation of the AD experiment followed the same procedures as described in the 1st set of test, and the experiment was conducted for 9 days.

55

(b)

CH mL/g COD-degraded Removal efficiency of TCOD

50

250

45 200 40 150 35 100

30

Removal efficiency of TCOD (%)

Methane production (mL CH /gCODdegraded)

300

25

50 A0

A1

A2

A3

A4

Fig. 2. Variables of (a) methane content and (b) methane production during ash-introduced AD.

2.4. Statistical analysis An analysis of variance with p-values calculated at a 95% confidence level, was used to determine the significance of results obtained in this work.

3. Results and discussion 3.1. Methane content and production Biogas upgrading is determined by the improvement of methane production and CO2 fixation during anaerobic digestion. The daily methane content from the anaerobic digestion of WAS in the presence of ash additive were presented in Fig. 2a. Methane content during 6–12 d was promoted by introduced ash with the increase of addition frequency. The maximum methane content was enriched to 79.4% in biogas from the A4 reactor. It indicated that increasing the addition frequency could upgrade biogas efficiently. The cumulative methane production was fitted with Gompertz mode (Table 1). After 25 days of

2.3. Analytic methods Methane (CH4) and carbon dioxide (CO2) content in biogas were determined by using a gas chromatography (GC) equipped with a thermal conductive detector (Yin et al., 2018). Concentrations of volatile fatty acids (VFAs) were determined by using a different GC equipped with a flame ionization detector (Yu et al., 2015). Concentrations of TS, volatile solids (VS), total chemical oxygen demand (TCOD) and soluble COD (SCOD) were determined according to the standard method (APHA et al., 2012). The Coomassie Brilliant Blue method and the anthrone assay were used to determine proteins and carbohydrates concentrations, respectively (Bradford, 1976; Xu et al., 2018). Net CO2 sequestration amount in each digester during the AD process was quantified as described previously (Yin et al., 2018). Methods of other analyses carried out in this study, including the key enzymes associated with AD process (alkaline protease, α-glucosidase, acetate kinase and coenzyme F420), microbial communities (biomass sampling, preparation, DNA extraction, sequencing and data processing) and some specific metallic concentrations (calcium, magnesium and iron) were described in the Supplementary Materials.

Table 1 Kinetic parameters of methane production in AD of sludge at different addition mode of ashes.

3

Reactor

λ (d)

A0 A1 A2 A3 A4

1.60 1.98 2.02 2.40 2.34

± ± ± ± ±

0.26 0.21 0.20 0.23 0.15

M0 (mL·g−1 TCODadded)

Mmax (mL·g−1 TCODadded·d-1)

75.6 ± 1.2 74.2 ± 0.9 89.3 ± 1.0 97.7 ± 1.3 105.1 ± 0.9

15.3 13.8 14.4 16.8 17.1

± ± ± ± ±

1.5 1.0 0.9 1.6 0.8

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AD, the maximum CH4 production (PCH4, max ) was changed by −2.7% (A1), +17.5% (A2), +28.5% (A3) and +35.8% (A4), respectively, compared to A0 (75.6 ± 1.2 mL·g−1 TCOD-added). This showed that addition mode of ashes affected CH4 production (PCH4 ) largely. In addition, TCOD removal efficiency (Fig. 2b) for A1, A2, A3 and A4 were enhanced by 13.0%, 12.5%, 21.1% and 18.2%, respectively, indicated stepwise addition of ash could also accelerate organic matters degradation. Compared to A0, methane production based on per gram TCOD in A4 was increased by 17.8%. It inferred that the stepwise addition not only accelerate consumption of organics, but also enhance methanogenesis pathway. As we all known, the methane production involved sludge disruption, hydrolysis and acidogenesis and methanegenesis during AD, and these process was commonly affected by microbial environmental condition and nutrients. The enhancement of methane production could ascribe to the release of the metal cations (e.g. Ca2+, K+, Mg2+, Fe3+, etc.) from the ashes additive and simultaneously creation of an alkaline pH conditions (Lo, 2005). However, the enhancement efficiency of methane production highly depends on suitable environment and concentration of cations released (Inanc et al., 2007; Lo et al., 2012a). Thus stepwise addition mode of ash create a favorable substrate environment benefited to microbial growth and resulted in a maximum methane production. The promotion of methane content with supplementation of ash was attributed into the high methane yield and in-situ CO2 capture. The bottom ash from municipal solid waste incineration is reported as one of the most suitable objects to complete the carbonation with CO2 due to the alkalinity pH environment and released cations ions (Jung et al., 2014).

stepwise addition of ashes on the accumulation of VFAs. The trend of total VFAs productions were consistent with hydrolysis process. However, VFA production between H1 and H4 was gradually insignificant with the increase dosage of sludge ash in H4, especially for acetic acid (Fig. 3f), but the TVFA of H1 was 14250 ± 240 mg COD/L at 5th day, higher than that of H4 (13100 ± 110 mg COD/L). In comparison to H1, as shown in Table 2, the stepwise addition of ash in H4 decreased VFAs yield (Cmax) and specific rate (kVFAs). However, the difference of soluble organics composition (Fig. 3e) was insignificant between H1 and H4, implying that gradual addition could increase acidogenesis rate due to the promotion of hydrolysate generation, instead of acdiogenesis level during anaerobic fermentation. After all, the hydrolysis is the ratelimiting step in sewage sludge anaerobic digestion due to the hard cell and complex floc structure (extracellular polymeric substances) (Zhen et al., 2017). Alkaline sludge ash mainly enhance sludge disruption and hydrolysis of large molecular particulate matters. Pulse addition of ash can promote total alkalinity efficiently than stepwise addition at initial 3 days (Supplementary Materials). In conclusion, stepwise addition of ash could promote sludge disruption, hydrolysis process and acidogenesis rate, but not perform up to its superiority absolutely in AD when compared to ash-induced AD system with pulse addition mode. 3.2.2. Step of methanogenesis In this study, the methanogensis process was simulated by variation of PCH4 in M0, M1 and M4. The methane yield from pre-treated sludge, inoculum were listed in Table 3. As compared to M0, the cumulative methane production in M4 after 9 days operation time was enhanced by +44.2%, instead of −4.1% in M1. In comparison to A1 and A4, the decreased methane yield for M1 and M4 might ascribe to part of methane left in headspace of reactor. Considering VFAs accumulated rapidly by pulse addition of sludge ash, it can infer that methanogens activity was inhibited in M1 and adversely disinhibited with the stepwise addition for ash additives according to Yuan et al. (2016). Values of pH has also been an important contribution to those values, which derived from CaO, MgO and other alkaline metal oxides in ash that react with water to generate net alkalinity (Lo, 2005). However, the optimal pH for methanogens was around 7.5, and an alkaline condition can kill methanogens such as Methanobacterium sp. and Methanobrevibacter sp. (Saritpongteeraka and Chaiprapat, 2008; Yuan et al., 2016). Stepwise addition of sludge ash could be an ideal strategy, in practice, to stabilize the pH and prevent environmental shock for methanogen. Compared with control group, the hydrolysis and acidogenesis rate was enhanced by ash additives. However, pulse addition of ash can significantly promote rate of hydrolysis and acidogenesis process of sewage sludge. Interestingly, methane-producing ability could be increased through stepwise addition of ash into sludge digestion. And the process of hydrolysis, acidogenesis and methanation are biochemical reactions, which are related to the activities of anaerobic microorganisms and/or key biological enzymes. Thus it is necessary to further investigate the change of key enzyme activities and microbial communities in stepwise addition mode of ashes in sludge fermentation.

3.2. Effect of a stepwise addition of ashes on individual step involved in WAS anaerobic digestion 3.2.1. Step of disruption, hydrolysis and acidogenesis To discuss the effect of stepwise addition of ash, the process of disruption, hydrolysis and acidogenesis was simulated by variations of SCOD, PC, PN and VFAs in H0, H1 and H4. As can be seen from Fig. 3a, SCOD released from the sludge disruption was increased from 1860 ± 78 mg COD/(L∙d) (H0) to 2393 ± 289 mg COD/(L∙d) (H1). According the first-order mode, the specific hydrolysis rate is determined by the product of α and KH (Lin and Li, 2018), and the result were listed in Table 2. Both of α and KH were found to vary considerably with different addition mode of ash. The αKH for H4 was lower than that of H1 due to the lower KH, while an increase of αKH was found in terms of that of H0 (without ash). It implied that the sludge ash accelerate the decomposition and dissolution of particulate organics. However, stepwise addition did not further improve the hydrolysis of particulate organics. The results of soluble polysaccharide and protein in H0-H3 (Fig. 3c and d) also demonstrated the hydrolyzation was inhibited for H4 reactor. The particle organic matters was ulteriorly converted into soluble phase through the hydrolysis process of AD that commonly was treated as the speed limited step in AD of sludge. As ash introduced, the elevated alkalinity and salinity improve membrane permeability and benefit to related enzyme synthesis (Koyama et al., 2017; Zhao et al., 2017). Pulse addition group accelerated releasing of soluble organic matter than stepwise addition group because of the hydrolysis level is positively related with the ashes dosage and interaction time (Zhao et al., 2017). Additionally, the higher pH value in ash-induced AD with pulse addition mode (during initial 5 days) might contribute to sludge disruption and hydrolysis (Supplementary Materials) (Mancini et al., 2018). The acidogenesis process is a biological process, which requires the microbial and key enzyme catalysis. Substrates of hydrolysis stage are further bio-converted into simpler VFAs end products and secreted into extracellular due to the actions of acidification bacteria. It can be seen a certain amount of soluble proteins and carbohydrates have been consumed after 3 day (Fig. 3c and d), indicating acidogenesis rate was higher than that of hydrolysis process. Fig. 3b depicts the effect of

3.3. Effect of a stepwise addition of ashes on microbial community and key enzyme involved in WAS anaerobic digestion To estimate the microbial communities in the anaerobic reactors, the trimmed sequences were grouped into operational taxonomic units (OTUs) using a 97% identity threshold. The sufficient coverage found in each sample suggested that the Illumina MiSeq high-throughput sequencing method had captured the most frequently occurring organisms (Supplementary Materials). The circular taxonomic and phylogenetic trees (Supplementary Materials) illustrated that the bacterial sequences generated from anaerobic system were distributed among two major phyla: Firmicutes and Proteobacteria. It was mainly comprised of genus Defluviitage, Coprothemobactor, Anaerolina, Anaerobaculum and 4

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Fig. 3. Effect of a stepwise addition of ash on the variations of (a) SCOD, (b) TVFAs, (c) soluble protein, (d) soluble carbohydrate, (e) percentage of organic matter and (f) the individual VFA concentration (C2–C5) during 5 days of WAS digestion.

Anaerobaculum were responsible for hydrolysis and acid production (Kim et al., 2018; Tian et al., 2015). It seems that the bacterial diversity and relative abundance were influenced by the addition mode of sludge ash. The relative abundances of Archaea populations are also shown in (Fig. 4d–f). It can been seen that four genus Methanosarcina, Methanomassiliicoccus, Methanothermobacter and Methanospirillum dominated in anaerobic reactors. In the control check, the Methanosarcina, Methanomassiliicoccus, Methanothermobacter and Methanospirillum accounted for

Thermodesulfovibrio (Fig. 4a–c). In control group, the genus Defluviitage, Coprothemobactor, Anaerolina and Anaerobaculum accounted for 21.44%, 23.72%, 13.34% and 11.88%, respectively, of all the Bacterial sequences. In Pulse addition digesters, these values were 31.45%, 28.12%, 9.11% and 16.05%, respectively. It is obviously that incorporating of sludge ash increased the relative abundance of Defluviitoga, Coprothemobactor, and Anaerobaculum. However, the stepwise addition group showed the relative lower abundance of Defluviitoga and Anaerobaculum, compared to A1. It was documented Defluviitoga and

Table 2 Kinetic parameters for hydrolysis and acidogenesis steps during AD of sludge at different addition mode of ashes. Reactor

H0 H1 H4

Hydrolysis

Acidogenesis

kH

α

kH × α

Cmax

kVFAs

0.08 ± 0.02 0.18 ± 0.03 0.10 ± 0.01

0.56 ± 0.11 0.68 ± 0.04 0.89 ± 0.15

0.045 ± 0.003 0.128 ± 0.003 0.089 ± 0.006

9790 ± 1028 13031 ± 1201 12292 ± 628

0.79 ± 0.11 1.01 ± 0.22 0.93 ± 0.11

5

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Table 3 Biogas and methane production for pre-treated sludge, inoculum and anaerobic digestate. Biogas production mL/L

Pre-treated sludgea Inoculum sludge Anaerobic digestateb a b

Methane production mL/L

M0

M1

M4

M0

M1

M4

2319 ± 105 460 ± 25 A0 457 ± 11

2458 ± 65 527 ± 21 A1 533 ± 10

3023 ± 85 917 ± 35 A4 810 ± 21

1140 ± 37 229 ± 16 A0 267 ± 25

1107 ± 146 295 ± 18 A1 321 ± 16

1647 ± 139 524 ± 21 A4 640 ± 29

P-pre-treated = P-gross − P-inoculum. Gas production from anaerobic digestate was calculated from batch reactor in Section 2.2, according to the biogas and methane production from day 6–15.

Fig. 4. Genus-level distributions of Bacteria (a–c) and Archaea (d–f) populations in the anaerobic digestion of WAS: A0 (a and d), A1 (b and e) and A4 (c and f).

To understand and exploit the impact of addition mode on microbial metabolism, the analysis of metabolic pathways of microbial communities was conducted. The functional composition of metagenome was predicted by PICRUSt approach using the 16 sRNA data obtained and KEGG pathway database (Langille et al., 2013). Metabolic pathway of microbial community in the category of “Metabolism” in the sample of A0, A1 and A4 was presented in Fig. 5a. It could see that and energy metabolism between A1 and A4 were significant. Amino acid, carbohydrate metabolism was improved by the incorporating sludge ash, especially in the pulse addition group. Furthermore, the enhancement of Energy Metabolism was mainly attributed to the pathway of Methane Metabolism (Zhang et al., 2017), in which methanogens obtain energy

35.11%, 16.43%, 14.79% and 8.35%; in the sample of A1 reactor, these values were 42.38%, 21.29%, 14.71% and 8.13%, respectively. Although the portion of Methanosarcina and Methanomassiliicoccus was enhanced with the incorporating of ash compared to control check, the corresponding quantity (or population) might be fewer because of the inhibitory effect on methanogens via high salinity (Yin et al., 2018). Abundance of Methanomassiliicoccus (34.48%) that produces methane only when methanol or hydrogen is used as substrates (Li et al., 2018), was further promoted with stepwise addition of ash in accordance with that of observed high methane yield. Apparent difference of Methanomassiliicoccus confirmed that overall susceptibility of methanogenic Archaea for ash addition mode. 6

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0.14

(a)

13500

A0 A1 A4

0.10

Zn Mn Mg Fe Ca

12000 Concentration (mg/L)

The relative abundance

0.12

0.08 0.06 0.04 0.02

10500 9000 3000

1500 0.00 s es sm sm sm sm sm ids sm ins tes sm lie tid oli oli oli oli oli oli oli oli Ac mi am ke Fa tab tab tab tab d Vit tab tab tab tab ino oly Me Me Me Me Me Me Me n me Am nd P a y y e e y d d d t i r z r g d n n s a a ra er da A he En Lip oti tor sa yd Ot on on En sis cle ino fac oid oh ec ati of the Nu rb en Co Am er S ad m yn Ca of gr lis erp h ios m de bo Ot fT B s o a i f i o t l o o an sB Me sm yc sis tab tic oli Gl Me the bio tab yn no Me ios Xe cid

Me

0

A0 A1 A4

EXC

CAR

FeMnOx

OMB

RES

Fig. 6. Effect of a stepwise addition of sludge ash on the distribution of main metal (Ca, Mg, Fe, Mn and Zn) in each anaerobic reactor at 25 day.

B

200

(b) 160

Relative activity (%)

by +249% and +291% in A1 and A4, respectively, in agreement with result of the net CO2 sequestration and difference of crystal phase (Supplementary Materials). Increasing dosing frequency might decrease each dosage of cations in ash and avoid other consumption path (e.g., bonded to organic matter). The enhancement of CO2 sequestration in ash-amended AD with stepwise addition mode could attribute into high pH (later period). The generated biogas diffuses from the microbial cells through the liquid environment where CO2 is dissolved and converted into carbonic acid. Thus the stepwise addition benefit CO2 transportation to liquid phase (Lindeboom et al., 2013; Wang et al., 2014b). Additionally, the organic binding metals (OBM) that defined as the combination of the EXC and the OMB in sludge. The OBM fractions of A1 was higher than that of A4, inhibiting the biogas conversion of sludge by reinforcing the stability of the sludge structure according to Xu et al. (2017). The reaction of metal ions in AD of sludge was complicated. Stepwise addition of ash might accelerate the carbonation reaction, improve sludge floc structure and thus come true biogas upgrading.

A0 A1 A4

120

80

40

0 a-GC

AKP

ACK

F420

Fig. 5. (a) Metabolic pathways of microbial communities in the category of “Metabolism” in A0, A1 and A4, respectively, according to gene KEGG pathway analysis; (b) Relative activities of key enzymes responsible for hydrolysis, acidification and methanogenesis at 3rd day during anaerobic digestion of WAS.

for growth by converting simple substrates to methane during AD process. This might be a possible biological reason for the higher methane yield in A4. To further quantificationally evaluate the activities of microbial cells, lots of enzymes involved during sludge AD (showed in SM) were tested. The data in Fig. 5b showed that the order of protease, a-GC and ACK was in the sequence of pulse addition group (A1) > stepwise addition group (A4) > control check, and coenzyme F420 relative activity in A4 was enhanced by 43.8% in comparison with A1, which was consistent with the result of sludge hydrolysis, acidogenesis and methane production. The results demonstrated that hydrolysis, acidogenesis and acetagesis could be enhance against ash supplement, but high dosage of sludge ash into system instantaneously lead to inhibition of methanogens activities, and the inhibition could be alleviated via stepwise addition mode.

4. Conclusion In-situ biogas upgrading using sludge ash with a stepwise addition mode was assessed in views of CH4 production and CO2 sequestration. The PCH4, max was increased by +35.8% in stepwise addition of ash, and corresponding maximum CH4 content was elevated to 79.4%, which attributed into enriched Methanomassiliicoccus (34.48%) and high relative activity of coenzyme F420. Stepwise addition of ash could not just promote methane production, but also increase CO2 storage capacity. These findings provide a substantial explanation for biomethane in-situ upgraded by stepwise addition of sludge ash, which could be treated a theory evidence for sludge ash utilization in practice.

Acknowledgements This study was financially supported by the National Natural Foundation of China (No. 51178261 and 21876110), Program of Distinguished Professor (Eastern Scholar) at Shanghai Institutions of Higher Learning, Shanghai Pujiang Program (No. 18PJ1406300).

3.4. Effect of a stepwise addition of sludge ash on CO2 sequestration involved in WAS anaerobic digestion Fig. 6 shows the metal distribution (Ca, Mg, Fe, Mn, Zn) of the sludge stream for all AD reactor. It could be seen that the Ca and Fe were the main metal ions in the all sludge stream, especially for the CAR fraction. In this work, variables of CAR fraction for main metals was used to quantify CO2 sequestration during anaerobic digestion with different addition mode. As incorporated with sludge ash, the bulk Ca and Fe cations was carbonated, and the total CAR content was increased

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.02.110. 7

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