Sawdust-derived biochar much mitigates VFAs accumulation and

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Sawdust-derived biochar much mitigates VFAs accumulation and improves microbial activities to enhance methane production in thermophilic anaerobic digestion Gaojun Wang, Qian Li, Xin Gao, and Xiaochang C. Wang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04789 • Publication Date (Web): 30 Nov 2018 Downloaded from http://pubs.acs.org on December 10, 2018

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ACS Sustainable Chemistry & Engineering

Sawdust-derived biochar much mitigates VFAs accumulation and improves microbial activities to enhance methane production in thermophilic anaerobic digestion Gaojun Wang, Qian Li**, Xin Gao, Xiaochang C. Wang* International Science and Technology Cooperation Center for Urban Alternative Water Resources Development; Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry of Education; Engineering Technology Research Center for Wastewater Treatment and Reuse, Shaanxi; Key Laboratory of Environmental Engineering, Shaanxi; Xi’an University of Architecture and Technology, No. 13 Yanta Road, Xi’an 710055, China

Corresponding authors: *E-mail: [email protected]; **

E-mail: [email protected]

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Abstract: Sawdust-derived biochar (SDBC) was added to an anaerobic digester at 15g/L for thermophilic co-digestion under semi-continuous operation for more than 130 days. With a stepwise increase of organic loading rate (OLR) from 1.6 to 5.4 gVS/L∙d-1, steady CH4 yields achieved between 462.3 and 500.1 mL/gVS. In contrast, for the control digester (without biochar addition) and another digester added with sewage sludge-derived biochar (SSBC), steady working condition could only be maintained for about 80 days with OLR and CH4 yields up to 2.7 gVS/L∙d-1 and 322.3-337.9 mL/gVS, respectively. The total volatile fatty acids (VFAs) accumulation up to about 30gCOD/L tended to be the threshold to restrain steady CH4 production but this occurred much earlier for the control and SSBC digesters at much lower OLR. By batch experiments of digestion in a methanogenesis inhibition condition and with propionate or butyrate as the sole substrate, it was found that with SDBC addition, the substrate was oxidized steadily in the 40-day experimental duration, while without SDBC addition substrate oxidation almost did not occur. SDBC apparently showed excellent electron accepting capacity for syntrophic oxidation of VFAs. High-throughput sequencing analysis identified that SDBC significantly altered the microbial community structure and brought about enrichment of Tepidimicrobium and Methanothermobacter – two microorganisms with potential capacity of extracellular electron transfer. It could thus be concluded that the main effects of SDBC would be the improvement of VFAs syntrophic oxidation and microbial activities which jointly enhanced CH4 production 2 ACS Paragon Plus Environment

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under thermophilic anaerobic condition. The high specific surface area might have provided favorable condition for microbial attachment growth and certain electroactive organic functional groups on its lignocellulosic structure might have stimulated direct interspecies electron transfer, which needs further study.

Key words: Sawdust-derived biochar; Thermophilic anaerobic digestion; Volatile fatty acids; Syntrophic oxidation; Interspecies electron transfer; Microbial community.

Introduction As an emerging sustainable and green technology, anaerobic digestion (AD) is 3 ACS Paragon Plus Environment


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drawing increasing attention for environment-friendly waste management and renewable energy production 1. Various types of bio-wastes, such as food waste (FW), sewage sludge (SS), municipal organic solid waste and animal manure, could be degraded in the AD process and most of the organic substrates could be converted into CH4 as a cleaner gaseous energy source 2. However, the AD process is often hindered by the accumulation of volatile fatty acids (VFAs), its intermediate metabolite products, especially under high organic loading rate (OLR). VFAs accumulation beyond a threshold concentration in an AD system may significantly lower the pH value and consequently inhibit the metabolic activity of methanogenic archaea for efficient CH4 production 3. Therefore, accelerating VFAs degradation in the AD process is a crucial issue for its widespread application in engineering. In principle, the degradation of VFAs, such as propionate and butyrate, is by syntrophic metabolism in which interspecies electron transfer (IET) occurs between VFAs oxidizing bacteria and methanogenic archaea 4. Under ordinary conditions, H2 and/or formate could act as IET mediators, but this biochemical reaction is theoretically energetically unfavorable (G’ 0), which means that this reaction could only occur under an extremely low H2 partial pressure condition, which largely limits the reaction rate during AD and subsequently enhances the accumulation of VFAs 5. Fortunately, it was recently identified that an alternative metabolic pathway, named as direct interspecies electron transfer (DIET), could occur between some electro-active microorganisms, such as Geobacter, Methanosarcina, and Methanosaeta

6, 7

. With

electrically conductive pili or/and some key outer-surface c-type cytochromes as 4 ACS Paragon Plus Environment

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electron transfer mediators, these microorganisms could achieve DIET for accelerating the electron transfer efficiency of syntrophic metabolism. Meanwhile, previous research confirmed that some electrically conductive materials could act as IET mediators and assist DIET between the syntrophic electron-donating and electronaccepting partners 8, 9, thus indicating a promising alternative pathway for accelerating syntrophic metabolism. Following the DIET theory, many kinds of electrically conductive materials have been tested for promoting VFAs degradation and CH4 production in the AD process10. In one study, magnetite addition successfully promoted the syntrophic oxidation of propionate and the CH4 production, even under a high H2 partial pressure in the AD reactor, which suggested that DIET was achieved via magnetite as the electrical conductor between bacteria and archaea 11. In a magnetitecontaining digester, DIET between Syntrophomonas and Methanosaeta was considered the main reason for accelerated AD of waste sludge 12. Recently, Wang et al. found that magnetite addition mitigated VFAs accumulation and promoted CH4 production under decreased expression of both pili and c-type cytochromes, which suggested that magnetite could fulfill their role and enhance DIET 13. Besides the magnetite-based material, different carbon-based materials, such as granular activated carbon, biochar, carbon cloth, carbon nanotubes and graphene, have displayed remarkable effects in DIET promotion

14-18

. Of these materials, biochar can

be easily derived from various types of bio-waste and is thus considered to be a cheap and environment-friendly additive to AD reactors. The advantages of biochar addition have been demonstrated in many studies through various findings, such as the 5 ACS Paragon Plus Environment


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stimulated DIET between co-cultures of Geobacter and Methanosarcina

19

, the

significant shortening of methanogenic lag time and increase in CH4 production remarkable acidific buffering capacity

21

20

,

, promotion of in-situ CO2 cleanup and 22

pipeline-quality bio-methane production

, and effective stimulation of VFAs

syntrophic oxidation with complex substrates

23

. However, although most of these

studies provided evidences on the function of biochar in promoting VFAs degradation and CH4 production possibly due to DIET actions, many questions still remain to be answered. For example, although it has been confirmed that biochar could promote syntrophic methanogenesis via DIET, it is unclear whether electrical conductivity is a determinant factor for DIET

19

. Meanwhile, as redox-active materials and electron

shuttles, biochar has been applied extensively in many processes, such as pollutant degradation, nutrient transformation and metalloids immobilization

24

. However, the

mechanism of redox-active biochar between the electron donors (syntrophic oxidizers) and electron acceptors (methanogens) during AD still needs further clarification. Furthermore, because most existing studies were based on a batch feeding operation model and performed under mesophilic conditions, there is insufficient information on the actions of biochar under continuous feeding, varied OLR and thermophilic conditions. Meanwhile, in any sense, the AD process depends much on microbiological actions, but few studies have been conducted so far on the long-term dynamic transformation of microbial communities in AD reactors when biochar is added. In order to fill in the abovementioned gaps in knowledge, in this study, sawdustderived biochar (SDBC) was prepared and added to a thermophilic AD system under 6 ACS Paragon Plus Environment

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varied OLRs in long-term operation for the co-digestion of FW and SS. The research focused on the physio-chemical properties of SDBC for microbe attachment and VFA degradation enhancement, the redox-active role of SDBC in syntrophic oxidation of VFAs in the absence of methanogenesis, and the characteristics of functional microbes in the SDBC-assisted thermophilic AD system. For comparison, experiments were also conducted in AD reactors with the addition of sewage sludge-derived biochar (SSBC) and without biochar addition (the control reactor).

Materials and methods Preparation and characteristics of biochar The SDBC was produced at a pyrolytic temperature of 500℃ from raw sawdust, which was collected from a local timber mill. The method of SDBC preparation was based on our previous study

23

. The SDBC yield and proximate analysis were

determined by ASTM methods 22. The pH value of the SDBC was measured in a 5% (w v-1) suspension in deionized water, prepared by shaking at 100 rpm under ambient temperature for 24h, using a pH meter (PHS-3C, Dapu Instrument Co., Shanghai, China). These measurements were conducted in duplicate. BET (Brunauer-EmmettTeller) surface area of the SDBC was measured via the N2 adsorption multilayer theory by a V-Sorb X800 surface area analyzer (Gold APP Instrument Co., Beijing, China). Elemental (CN/OH) analysis was performed using an isotope ratio mass spectrometer (IRMS, IsoPrime100,Elementar,Germany). Organic functional groups of the SDBC were measured with Fourier transform infrared spectroscopy (FT-IR, ThermoFisher, 7 ACS Paragon Plus Environment


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USA), under an attenuated total reflectance (ATR) model (supplementary materials). A field emission Scanning Electron-Microscope-Energy Dispersive Spectrometer (SEMEDS, FEI, quanta F50, USA) was used to investigate the structure of the original SDBC ( Figure 2 and supplementary materials). The electrical conductivity of SDBC was determined with a four-probe method by a power resistivity tester (ST2722, Suzhou Jingge, China)25. To identify the significance of the physio-chemical properties of SDBC during the AD process, an SSBC derived with the dewatered activated sludge of a local wastewater treatment plant was used in comparison with SDBC, due to their different properties, as shown in Table 1. Table 1. Physio-chemical properties of sawdust-derived biochar (SDBC) and sewage sludge-derived biochar (SSBC) Analysis Parameter SDBC SSBC Proximate analysis Biochar yield(wt%) 22.6±0.4 49.8±0.3

Physio-chemical property

Fixed carbon(wt%)

68.5±0.5

19.9±0.2

Volatile carbon(wt%)

23.7±0.2

12.9±0.2

Ash content(wt%)

7.8±0.2

67.2±0.5

pH in solution

9.1±0.1

7.8±0.1

0.11±0.03

0.09±0.02

BET surface area(m /g)

248.6±9.4

2.6±0.3

C (wt%)

74.45±1.5

20.44±1.7

N (wt%)

0.44±0.1

3.31±0.3

H (wt%)

4.8±0.2

3.89±0.2

O(wt%)

12.19±0.7

21.38±1.4

Electrical conductivity (μS/cm) 2

Ultimate analysis

Substrates and seed sludge A mixture of FW and SS was used as the substrates for Co-AD. The FW was synthetic, based on the composition of real Chinese kitchen waste 23. The SS had the same source 8 ACS Paragon Plus Environment

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as the SSBC, with 80% water content approximately. After mixing the FW and SS with a wet weight ratio of 4:1 26, the substrate was homogenized with a high-speed blender. To maintain their properties intact, the fresh substrates were stored in a 4℃ environment before use and prepared once every two weeks. The seed sludge was collected from the mesophilic AD unit of a local brewery and conserved in an anaerobic environment before use. The properties of the substrate and seed sludge are listed in Table 2. Table 2. Physio-chemical properties of seed sludge and substrate. Parameter TS (%) VS (%) Total COD (g/L) Soluble COD (g/L) pH Total VFA (g COD/L)

Seed sludge 5.2±0.2 3.9±0.2 / / 7.45±0.03 0.07±0.01

Substrate 9.2±0.4 8.1±0.3 138.2±17.4 48.1±8.4 5.01±0.24 1.58±0.85

Semi-continuous operation of T-Co-AD To explore the effects of biochar on thermophilic Co-AD, 15 g/L SDBC was added into a Co-AD reactor

23

, which was operated under the semi-continuous operation

model, with serum bottles of 150mL as working volume, this was named the SDBCcontaining reactor (SDBC-R). The appropriate mass of pristine SDBC was replenished daily into the SDBC-R to maintain the SDBC concentration constant in the reactor. The reactor was operated under a daily-feeding model. Specifically, at a certain time point during the day, as the biogas composition and volume were measured, the reactor was opened and the digestate was discharged. Then, an equal volume of fresh substrate was fed into the reactor, and the reactor was purged with high-purity N2 for 3 min, to maintain the anaerobic environment prior to being sealed with rubber stoppers and an 9 ACS Paragon Plus Environment


aluminum cap. Lastly, the reactor was put into a 55℃ water bath with 120 rpm stirring for thermophilic Co-AD. After a 20-day acclimatization process, the reactor was operated with a hydraulic retention time (HRT) of 50 days. The OLR was increased gradually as the HRT decreased, until the reactor was acidified completely. During the whole process, biogas production and CH4 content in biogas were monitored daily, whereas the pH and VFA concentrations of the reactor were measured at least three times a week. As two control reactors, an SSBC-containing reactor (SSBC-R) and a control reactor without biochar addition (CT-R) were operated in parallel to the SDBC-R, respectively.

Batch experiments of VFAs oxidation in the absence of methanogenesis Generally, the traditional VFAs syntrophic oxidation process only occurs with the involvement of VFAs oxidizing bacteria and hydrogentrophic methanogens, to maintain a low H2 partial pressure. Although the promoting effect of SDBC addition on CH4 production was confirmed in the continuous experiment described above, it is still unclear whether SDBC addition could stimulate VFAs syntrophic oxidation in the absence of methanogens. To elucidate this problem, batch experiments were designed as described below. For the experimental group, two typical VFAs, propionate and butyrate, were respectively added, as the sole substrate, into 120-mL serum bottles at a concentration of 10mM, (acetate was excluded here because it could be utilized directly by methanogens and did not accumulate in the reactors, as shown in the semi-continuous 10 ACS Paragon Plus Environment

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experiment in this study). At the same time, 20mL thermophilic seed sludge, 20mL 2bromoethanesulphonate (50mM), 15 g/L SDBC and appropriate nutrient solution were added to maintain the working volume of 90 mL. The control group was operated like the experimental group but without SDBC addition. Furthermore, an abiotic experiment was conducted to study the effect of physical adsorption of SDBC on VFAs. All the batch experiments were conducted in duplicate at thermophilic condition.

Microbial community analysis To explore the effect of biochar addition on microbial community variation, sludge samples from the SDBC-R and CT-R under different OLR were collected for highthroughput sequencing analysis. For DNA extraction, the sludge samples were centrifuged at 13,000 rpm for 10min, and then the pellets were rinsed twice with phosphate buffering saline. Subsequently, the PowerSoil® DNA Isolation Kit (MO BIO, USA) was used for DNA extraction, according to the manufacturer’s instructions. The extracted DNA samples were stored at -20℃ for conservation pending analysis. An Illumina platform (IlluminaMiseq PE250, Sangon Biotech, Shanghai, China) was employed for the high-throughput sequencing analysis of both bacterial and archaeal communities.

For

bacteria,

the

universal

primers

515F

(5’-

GTGCCAGCMGCCGCGGTAA-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT) were used for amplifying the V4 regions of the 16S rRNA gene via PCR. For archaea, the two-cycle nested PCR was used to amplifying the V3-V4 regions of the 16S rRNA gene. In the first cycle, primers 340F (5’-CCCTAYGGGGYGCASCAG-3’) and 1000R 11 ACS Paragon Plus Environment


(5’-GGCCATGCACYWCYTCTC-3’) were used whereas primers 349F (5’GYGCASCAGKCGMGAAW-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) were used for the second cycle. As the amplification process ended, the PCR products were examined via agarose gel electrophoresis to determine the quality of the amplification. After purification with Agencourt AMPure XP magnetic beads (Backman Coulter, USA), the PCR products were quantified with a Qubit 2.0 DNA detection kit (Sangon Biotech, Shanghai, China).

Analytical methods The biogas production volume was measured with a glass syringe. The biogas composition, including H2, CH4 and CO2, was monitored by a gas chromatograph (GC) (GC7900, Tianmei, China) equipped with a thermal conductivity detector and a molecular sieve packed stainless steel column (TDX-01, Shanghai Xingyi Chrome, China). The CH4 production was calculated by adding the CH4 volume in the glass syringe and injecting it into headspace. The pH of reactors was measured with a portable pH meter (Horiba, Kyoto, Japan). VFA was monitored by a GC (PANNO, China) equipped with a flame ionization detector and a DB-FFAP column (φ 0.32mm × 50m; Agilent, USA).

Results and discussion Performance of SDBC-assisted T-Co-AD in long-term operation CH4 production and VFAs degradation The CH4 production during the operation of all three reactors is shown in Figure 1(b). 12 ACS Paragon Plus Environment

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After a 20-days acclimation at a low OLR (1.1gVS/Ld-1), the CH4 production in the reactors tended to be stable, due to the microbial adaption. Then, the reactors were operated at an OLR of 1.6 gVS/Ld-1 (HRT of 50 days). There was no significant difference in CH4 production among the SDBC-R, SSBC-R and CT-R. However, as the OLR was increased to 2.7 gVS/Ld-1 by shortening the HRT to 30 days, the average CH4 production in the SDBC-R reached 171.3 mL/d, which was 25.2% and 31.3% higher than that in the SSBC-R and CT-R, respectively. When the OLR was further increased to 3.5 gVS/Ld-1 by shortening the HRT to 23 days, the CH4 production in the SSBC-R and CT-R decreased dramatically due to the pH drop caused by the excessive accumulation of VFAs (discussed in section 3.1.2). In contrast, the SDBC-R could perform robustly until the OLR was increased to 5.4 gVS/Ld-1 (Figure 1(b)). When comparing the CH4 yield of the three reactors at each

8

80

OLR (gVS/Ld-1 )

HRT

70

6

60

5

50

4

40

3

30

2

20

1

10

(a)

5000

CH4 production (mL/d)

HRT (day)

OLR

7

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 SSBC-R

SDBC-R

CT-R

400 300 200 100

(b) 0 8.5 8.3 8.1 7.9 7.7 7.5 7.3 7.1 6.9 6.7 6.5

SSBC-R

SDBC-R

CT-R

(c) 0

15

30

45

60

75 90 Time (day)

105

120

135

150

Figure 1. Variations of hydraulic retention time (a), CH4 production (b) and pH (c) as the organic loading rate increase. 13 ACS Paragon Plus Environment


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OLR, it was clear that the CH4 yield of the SDBC-R (462.3 mL/VS-500.1 mL/VS) increased by 18.8%-48.0% and 16.0%-55.2% than those of the SSBC-R and CT-R, respectively (Figure 4). Both the high CH4 yield and the doubled maximum OLR for stable CH4 production in the SDBC-R suggested the tremendous potential of high lignocellulosic waste-derived biochar for being employed as an ideal additive for CH4 production in the semi-continuous T-Co-AD systems. (b)

(a)

(c)

(d)

Figure 2. SEM images of original SDBC (a) and SSBC (b), and original SDBC (c) and microbial enriched SDBC in Co-AD system (d). After the successful start-up, the total VFA concentration in the three reactors showed increase trends as the OLR increased until the extremely high VFA accumulation led to the occurrence of acidification (Figure 3(a)). Nevertheless, both SDBC and SSBC mitigated the accumulation of VFAs to some extent. As shown in Figure 3(b), acetate did not accumulate before the reactors deteriorated, since it could be used directly by the acetotrophic methanogens via the acetoclastic pathway

27

. Among the accumulated VFAs which can only be degraded via the

syntrophic pathway, propionate accounted for the largest percentage, which was consistent with previous resports 28. The concentration of propionate was similar among 14 ACS Paragon Plus Environment

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the SDBC-R, SSBC-R and CT-R (Figure 3(c)). However, the accumulation of butyrate and valerate was dramatically mitigated in the SDBC-R. As illustrated in Figure 3 (d) and (e), butyrate and valerate started to accumulate in the SSBC-R and CT-R at the OLR of 1.6 gVS/Ld-1. However, the concentrations of butyrate and valerate maintained at a very low level in the SDBC-R. As the OLR increased to 2.7 gVS/Ld-1, the concentrations of butyrate and valerate reached 1904-2333 mg/L and 2875-3298 mg/L in CT-R, respectively, and although the SSBC seemed to mitigate the accumulation of butyrate and valerate, this did not stop the acidification of SSBC-R. Nevertheless, the concentrations of butyrate and valerate in the SDBC-R were acceptable until the OLR reached 5.4 gVS/Ld-1. By comparing the concentrations of VFAs in the reactors, it could be confirmed that syntrophic methanogenesis was the rate-limiting step during TCo-AD in this study. In addition, the dramatic SDBC-induced stimulation on the syntrophic oxidation of butyrate and valerate ensured a stable and efficient CH4 production even at higher OLRs, though the syntrophic degradation of propionate seemed to be a bottleneck in this system 29.



1.6

SSBC-R

2.7

SDBC-R

3.5

4.5

5.4

6.7

CT-R

(a)

8 6 4 2

(b)

0 12 10 8 6 4 2 0 6

Butyrate concentration (gCOD/L)

Propionate concentration (gCOD/L)

Acetate concentration (gCOD/L)

Total VFAs concentration (gCOD/L)

OLR 1.1 35 30 25 20 15 10 5 0 10

(c)

5 4 3 2 1

(d)

0 Valerate concentration (gCOD/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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8 6

4 2

(e)

0 0

15

30

45

60

75 90 Time (day)

105

120

135

150

Figure 3. Concentration variations of total volatile fatty acids (a), acetate (b), propionate (c), butyrate (d) and valerate (e) of the three reactors during semicontinuous operation.

In the long-term experiment, the pH of all the three reactors was maintained in a suitable range for methanogens growth despite the heavy accumulation of VFAs, which was attributed to the intrinsic buffer capacity of the T-Co-AD system 30 (Figure 1(c)). However, the pH sharply declined as the total VFA concentration reached a similar concentration (approximately 30 gCOD/L) (Figure 3(a)), which should be the threshold for stable performance. It seemed that although biochar could enhance the buffering capacity due to the existence of alkali organics or ash contents as reported by 21, 31, the buffering capacity of T-Co-AD was not much improved after biochar addition in this study. Subsequently, the promoting effect of SDBC on the stable and efficient production CH4 is probably attributed to the enhancement of syntrophic 16 ACS Paragon Plus Environment

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methanogenesis, rather than the introduced buffer capacity of biochar. 600

30 CH 4 yield of SSBC-R

VFA con. of SSBC-R

CH 4 yield of SDBC-R

VFA con. of SDBC-R

VFA con. of CT-R

27

CH 4 yield of CT-R

24 21

400

18 300

15 12

200

9 6

100

Accumulated VFA concentration (g COD/L)

500 CH4 yield (mL CH4 /g VS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3 0

0 1.6

2.7 3.5 4.5 Organic loading rate (gVS/Ld-1 )

5.4

Figure 4. Variation of CH4 yield and accumulated VFAs based on OLR variation

Biochar properties and effects of process enhancement Compared to the performance of SDBC-R, the promotion efficiency of SDBC on CH4 yield and the maximum OLR of the system were significantly higher. To determine the underlying mechanisms of these observations, the structures and physicochemical properties of the SDBC and SSBC were compared. FT-IR results showed that although both SDBC and SSBC showed peaks in absorbance at wave numbers 1557-1567 cm-1 and 1416 cm-1, which likely corresponded to the quinone moieties

32

, SDBC also

presented an absorbance peak at wave number 1162 cm-1, suggesting the presence of phenazines 33. For SSBC, the broad region around the peak at 1005 cm-1 indicated the high Si-O and phosphate contents, which was consistent with the EDS results (supplementary materials). Moreover, as shown in Table 1, although the electrical conductivity of SDBC and SSBC was similar (0.11μS/cm vs.0.09μS/cm), the large difference in accumulated VFAs between SDBC-R and SSBC-R suggested that electrical conductivity is not a determinant factor for VFAs accumulation mitigation. In a previous study, it was reported that the crystalline form of phenazine stimulated DIET 17 ACS Paragon Plus Environment


in an AD system by redox potential modulation 34. Thus, it is believed that the redoxactive phenazine organic structure in SDBC likely promote the syntrophic oxidation of VFAs via DIET enhancement. In addition, the specific surface area (SSA) of SDBC was almost 100 times higher than that of SSBC (248.6 m2/g vs. 2.6 m2/g), due to its developed sheet-like structures and macro-pores, which are more beneficial for microbial attachment growth and enrichment (Figure 2(c, d)). Previous studies evaluated non-lignocellulosic derived biochar as a positive additive for CH4 production enhancement in batch experiments. Wang et al. pointed out that vermicompost-derived biochar enhanced the buffering capacity of an AD system, which was largely due to the presence of inorganic substances

21

. Jang et al. also mainly attributed the promoting

effect of dairy manure-derived biochar on CH4 production under different temperature to the alkaline property

31

. Interestingly, in a recently published article, it was found

that the SSBC could stimulate DIET in co-cultures between Geobacter metallireducens and Geobacter sulfurreducens. The authors attributed this to the surface functional groups of the SSBC 25. However, in the present study, although the FT-IR and proximate analysis results of SSBC revealed a high inorganic ash content, organic functional groups, and the high pH value of SSBC likely made the T-Co-AD operate steadily by enhancing the buffering capacity (Table 1 and supplementary materials). Nevertheless, the stimulation of CH4 production by SSBC addition was not significant in long-term semi-continuous experiments. The reason could be the lack of typical electro-active microorganisms (such as Geobacter) in the thermophilic AD system of our study. Thus, from the viewpoint of engineering applications, it was concluded that lignocellulosic18 ACS Paragon Plus Environment

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derived biochar could be a better additive for AD systems.

Electron accepting capacity of SDBC in the syntrophic oxidation process Although the long-term experiment showed that SDBC addition could strongly promote CH4 production, likely via IET enhancement, it is necessary to further elucidate the role of biochar in the syntrophic VFA oxidation process. As a redox-active material, the capacity of biochar for electron accepting and donating has been reported with biochar being applied as a functional electrode or employed in some environmental remediation cases

35-37

, while only few studies have focused on the

evaluation of this capacity in the VFA syntrophic oxidation process. As introduced in Section 1, the energy barrier of traditional IET makes this process hard to achieve without the participation of methanogenesis 4. Therefore, it was important to determine whether SDBC could act as a substitute for H2 as the electron acceptor, to achieve successful syntrophic oxidation in the absence of methanogenesis.



14

(a)

16

BU-BC-acetate

14

BU-BC-butyrate

VFAs concentrations (mM)

VFAs Concentration (mM)

18

12

10 8 6

4 2 120

(b)

PR-BC-acetate

12

PR-BC-propionate

10 8 6

4 2 140

(c) 10 8 6

4 BU-CT-acetate

2

(d) VFAs concentrations (mM)

VFAs Concentration (mM)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 34

BU-CT-butyrate

12 10 8 6

4 PR-CT-acetate

2

PR-CT-propionate

0

0 0

5

10

15

20 25 Time (day)

30

35

40

0

5

10

15

20 25 30 Time (day)

35

40

Figure 5. Concentration variations of propionate (a) and butyrate (b) and the relative acetate (c, d) production in the absence of methanogenesis. For this purpose, a series of batch experiments were conducted, in which methanogenesis was inhibited. Less than 5mL of total CH4 were detected during the process. As illustrated in Figure 5, in the CT groups, only small amounts of propionate and butyrate were oxidized over 40 days. It was conceived that the intentional inhibition of hydrogentrophic methanogenesis resulted in the loss of electron acceptors for the IET process of syntrophy

38

. However, SDBC addition stimulated the oxidation of

propionate and butyrate successfully in the absence of methanogenesis, which highlighted the electron accepting capacity of SDBC in the syntrophic process. In a coculture environment, a study found that biochar could stimulate DIET between Geobacter metallireducens and Geobacter sulfurrenducens or Methanosarcina barkeri, which was attributed to the higher aromaticity of biochar. Furthermore, the authors speculated that part of the biochar was firstly reduced by the electron donating microorganism, and then oxidized by the electron accepting microorganisms 12. In this 20 ACS Paragon Plus Environment

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way, a bridge of electron flow was established between the syntrophic microorganisms and promoting DIET. In this study, the results suggested that SDBC acted as a temporary acceptor for the electrons released by the propionate and butyrate oxidation process, as simulated in Equations 1 and 2. The acetate produced in the butyratecontaining group was almost two-fold that in the propionate-containing group, which was consistent with the theoretical simulation in Equations 1 and 2. This is the first time that SDBC was confirmed as an electron acceptor for achieving VFAs oxidation in a thermophilic AD system. The electron accepting capacity was calculated according to Equations (1) and (2), which were 2.29 mM e-/gSDBC and 2.00 mM e-/gSDBC, respectively.

CH3CH2COO- + 2H2O + Biochar CH3COO- + CO2+ 6H+ + Biochar6-

(1)

CH3CH2CH2COO- + 2H2O + Biochar 2CH3COO-+ 5H+ + Biochar4-

(2)

To determine whether the reduced SDBC can be re-oxidized by methanogens, the reduced SDBC was cleaned and added into a CO2-rich methanogenic environment. Interestingly, no CH4 was detected over the two-month experimental period. One possible explanation is that the intrinsic charge differences were necessary for DIET, which is seen in the relationship between electric voltage and electricity generation 12, and the intrinsic mechanism needed to elucidate further.

Effect of SDBC on enriched functional microbes in syntrophic methanogenesis 21 ACS Paragon Plus Environment


Page 22 of 34

To analyze the effect of SDBC addition on the microbial community during a longterm T-Co-AD process, high-throughput sequencing was conducted in the SDBC-R and CT-R. The results showed that the addition of SDBC introduced great variation in both the bacterial and archaeal communities. In the SDBC-R, the lower Shannon index and higher Simpson index of bacteria and archaea than those of CT-R, implying that SDBC Table 3. Microbial community diversity analysis of bacteria and archaea of SDBC-R and CT-R under different OLR. Sample

OTU number

Shannon index

Simpson index

aB-CT-R-2.7

707 813 1028 697 736 653 267 304 261 360 288 264

2.34 2.72 2.60 2.04 2.16 1.70 1.78 1.75 1.52 1.51 1.16 0.97

0.23 0.19 0.24 0.42 0.38 0.48 0.30 0.31 0.42 0.41 0.59 0.67

B-CT-R-3.5 B-SDBC-R-2.7 B-SDBC-R-3.5 B-SDBC-R-4.5 B-SDBC-R-5.4 bA-CT-R-2.7 A-CT-R-3.5 A-SDBC-R-2.7 A-SDBC-R-3.5 A-SDBC-R-4.5 A-SDBC-R-5.4

a: “B” was short for “bacteria”; b: “A” was short for “archaea”.

addition reduced the diversity of microbial communities (Table 3). This was consistent with the results of our previous study

23

. Noticeably, as the OLR increased, the

microbial community diversity decreased, suggesting that certain predominant microorganism were enriched, especially at high OLRs. Principal component analysis (PCA) was performed based on the OTU results (Figure 6). For the archaeal community, principal component 1 and principal component 2 accounted for 67.0% and 31.0%, respectively. At the lowest OLR (2.7 gVS/Ld-1), both the CT-R-2.7 and SDBC-R-2.7 were in the 2nd quadrant. However, as the OLR increased, the SDBC-R tended to locate in the 3rd quadrant, while the CT-R showed little variation. In contrast, the bacterial 22 ACS Paragon Plus Environment

Page 23 of 34

community differed significantly between the SDBC-R and the CT-R. The communities of the SDBC-R clustered in the 2nd quadrant and those of the CT-R concentrated in the 3rd quadrant, even at the lowest OLR. Hierarchical clustering analysis based on the Bray-Curtis method revealed a similar trend when comparing the microbial diversity in the SDBC-R and CT-R (Figure 7). These results indicated that SDBC addition transformed both archaeal and bacterial communities greatly. Interestingly, the transformation of the archaeal community lagged compared to that of the bacterial community, which is likely due to the longer microbial doubling-time of archaea 39.

0.4 CT-R-3.5

0.6

SDBC-R-4.5

(a)

(b) SDBC-R-2.7

0.2 SDBC-R-2.7

0.4

CT-R-2.7

SDBC-R-5.4

0.2

PC2 (13.0%)

0

PC2 (31.0%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


SDBC-R-3.5

0

-0.2

SDBC-R-3.5

-0.2

-0.4 CT-R-2.7

-0.6

-0.4

CT-R-3.5

SDBC-R-5.4

-0.8

-0.6 -0.8

-0.6

-0.4

-0.2

0

-0.8

0.2

-0.6

-0.4

-0.2

0

0.2

PC1 (86.0%)

PC1 (67.0%)

Figure 6. Principal component analysis of archaeal (a) and bacterial (b) community. Thermotogae, Firmicutes, and Synergistetes were the three dominant phylum of bacteria in both SDBC-R and CT-R. However, the addition of SDBC changed their relative abundance in the bacterial community considerably (Figure 7(a)). In the CT-R, the abundance of Defluviitoga (Thermotogae phylum) was between 30.3%-38.4%, whereas in the SDBC-R, this genus increased to 48.4%-66.6% under the same OLR, and even increased further as the OLR increased. As an important hydrolytic bacterium in thermophilic biogas systems, Defluviitoga can metabolize numerous types of complex substrates and produce acetate, H2 and CO2, which are main substrates for 23 ACS Paragon Plus Environment


methanogenesis 40. Thus, the enrichment of Defluviitoga in the SDBC-R could enhance the syntrophic metabolism between Defluviitoga and methanogens, which could possibly explain the lower VFA accumulation in the SDBC-R. Coprothermobacter, a typical hydrogen-producing bacteria, dramatically decreased from 35.7%-36.9% to 3.20.1% after the addition of SDBC. However, the Tepidimicrobium was enriched in the SDBC-R., which was increased from 1.6%-2.8% to 6.0%-7.2%. According to a previous study, one metabolic type of Tepidimicrobium can reduce ferric oxide, with low molecular organics as the electron donors

41

. This characteristics of

Tepidimicrobium is similar to that of Geobacter metallireducens, a typical ferric oxide reducing microorganism which could achieve DIET with methanogens under mesophilic conditions 7. Although few DIET active microorganisms have currently been isolated in thermophilic AD systems to date, the exoelectrogenic capacity of Tepidimicrobium suggests that DIET possibly occurred in the SDBC-R. Further study is required to determine the role of Tepidimicrobium in thermophilic AD systems.


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similarity

Defluviitoga unclassified Coprothermobacter Anaerobaculum Tepidimicrobium Lactobacillus Garciella Syntrophaceticus Clostridium III Atribacteria Tepidanaerobacter Tetrasphaera others

CT-R-2.7

CT-R-3.5 SDBC-R-2.7 SDBC-R-3.5 SDBC-R-4.5

SDBC-R-5.4 0

10

20

30

40

50

60

70

80

90

100

(b) CT-R-3.5

Methanothermobacter Methanobacterium Methanolinea Methanothrix Methanosarcina Methanomassiliicoccus Methanospirillum Methanoculleus unclassified Methanosphaerula Methanobrevibacter others

CT-R-2.7 SDBC-R-2.7 SDBC-R-5.4 SDBC-R-4.5 SDBC-R-3.5 0

10

20

30 40 50 60 70 80 Relative abundance (%)

90

100

Figure 7. Similarity and relative abundance variation of bacteria and archaea in SDBC-R and CT-R under different OLR. The archaeal community also varied dramatically between the SDBC-R and CT-R as the OLR varied (Figure 7(b)). At the OLR of 2.7 gVS/Ld-1, the Methanobacterium was predominant in both the SDBC-R and CT-R. As the OLR increased, Methanobacterium in the SDBC-R decreased dramatically (from 70.27% at 2.7 gVS/Ld-1 to 15.4% at 5.4 gVS/Ld-1), which was not observed in the CT-R. Meanwhile, Methanothermobacter increased from 15.97% to 78.94%, and became dominant in the SDBC-R. Obviously, SDBC addition enriched Methanothermobacter as the OLR increased, while eliminating the Methanobacterium, a strictly hydrogentrophic methanogen. Although H2 is considered as the main substrate for the activity of Methanothermobacter, some evidence suggests the potential for DIET achievement in its metabolism. In coaggregation co-cultures, an electron bridge was established between Pelotomaculum and Methanothermobacter via the conductive pilus-like filaments for propionate 25 ACS Paragon Plus Environment


degradation

42

. Moreover, in a thermophilic electro-methanogenesis system,

Methanothermobacter was the predominant archaea attached to the biocathode, where it utilized electrons for CO2 reduction

43

. These findings suggest that

Methanothermobacter has the capacity of accepting electrons directly from electron donors and can achieve DIET for syntrophic metabolism with other bacteria. Overall, based on the above-discussed results, it is plausible to attribute the synergistic promotion of VFAs syntrophic oxidation by SDBC addition to the following reasons. First, the high SSA of SDBC not only shaped the microbial community but also connected the syntrophic partners more closely (Figure 2(d)), which enables the promotion of syntrophic metabolism potentially via both traditional IET and DIET 44. Moreover, the redox-active property of SDBC might assist the electron transfer within the system potentially acting as an electron transfer mediator to promote DIET between VFA oxidizing bacteria (Tepidimicrobium) and methanogens (Methanothermobacter) 45

, however this needs further sophisticated investigation. The results of this study

demonstrated that the biochar-assisted thermophilic AD system could be an ideal alternative for achieving better bio-waste treatment efficiency and energy production performance in practical engineering applications.

Conclusions SDBC used in this study showed excellent efficiency in maintaining a long-term steady operation at increased OLR, and enhanced CH4 production when it was added to the thermophilic co-digester. Moreover, SDBC addition significantly slowed down VFAs accumulation and prolonged the duration of steady operation to over 130 days 26 ACS Paragon Plus Environment

Page 26 of 34

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until the total VFAs reached the threshold of 30 gCOD/L. The underlying reason is that any organic substrate, such as propionate and butyrate used in the batch experiments of digestion under inhibition of methanogenesis, could be steadily oxidized into acetate. This could be due to the role of SDBC as the electron transfer mediators to promote the syntrophic oxidation of VFAs rather than the traditional IET with H2/formate as mediators. Moreover, SDBC induced significant alterations in microbial communities, along with enrichment of Tepidimicrobium and Methanothermobacter which are typical bacteria with high capacity of extracellular electron transfer. All these advantages might have been originated from the low ash content and high specific surface area of SDBC, which facilitated microbial attachment and growth. As SDBC was derived from highlignocellulosic bio-wastes, certain electro-active organic functional groups on its macro matrices might have also contributed to DIET. Considering its low cost and outstanding performance, SDBC is a promising additive to anaerobic digesters for enhancing the syntrophic methanogenesis process.

Supporting information The supplementary materials of this article showed detailed FT-IR spectra of SDBC and SSBC (Figure S1) and the EDS analysis of SDBC and SSBC (Figure S2). This material is available at http://pubs.acs.org.

Acknowledgements: This work was supported by National Natural Science Foundation of China (Grant No. 27 ACS Paragon Plus Environment


Page 28 of 34

51608430), the National Natural Science Foundation of China (Grant No. 51778522), and the Scientific Research Program Funded by Shaanxi Provincial Education Department (Grant No. 17JS077).

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and

Methanothermobacter

thermautotrophicus. Appl. Environ. Micr. 2005, 71, 7838-45, DOI 10.1128/AEM.71.12.7838-7845.2005 43. Kobayashi, H., Bio-electrochemical analyses of the development of a thermophilic biocathode catalyzing electromethanogenesis. Environ. Sci. Technol. 2015, 49, 1225-1232, DOI 10.1021/es5052233 44. Lü, F.; Luo, C.; Shao, L.; He, P., Biochar alleviates combined stress of ammonium and acids by firstly enriching Methanosaeta and then Methanosarcina. Water Res. 2016, 90, 34-43, DOI



10.1016/j.watres.2015.12.029

45. Shanmugam, S. R.; Adhikari, S.; Nam, H.; Sajib, S. K., Effect of bio-char on methane generation from glucose and aqueous phase of algae liquefaction using mixed anaerobic cultures. Biomass Bioenerg. 2018, 108, 479-486 DOI 10.1016/j.biombioe.2017.10.034

Anaerobic co-digestion system Acetate

Sawdust-derived biochar (SDBC)

Volatile fatty acids

Methane H2

Low efficient IET via H2

Acetate Volatile fatty acids SDBC

SDBC

Methane

Electron

High efficient DIET

TOC

Synopsis: Sawdust-derived biochar-assisted AD system is beneficial for VFAs accumulation mitigation and microbial activity improvement, which shows a sustainable strategy for integrated bio-wastes management.


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Sawdust-derived biochar much mitigates VFAs accumulation and

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