Ruolin Guan,Hairong Yuan*,Liang Zhang,Xiaoyu Zuo,Xiujin Li*

Department of Environmental Science and Engineering,Beijing University of Chemical Technology,Beijing 100029,China

Keywords:CaO-LFD pretreatment Direct interspecies electron transfer (DIET)Rice straw Anaerobic digestion (AD)Methane

ABSTRACTTo improve anaerobic digestion(AD)efficiency of rice straw,solid alkaline CaO and the liquid fraction of digestate(LFD)were used as pretreatment agents of rice straw.The results showed that AD performance of rice straw with CaO -LFD pretreatment was optimal in different pretreatment methods of the CaO+LFD,CaO -LFD,LFD+CaO,CaO,and LFD.The maximum methane yield (314 ml.(g VS)-1) and the highest VFAs concentration (14851 mg.L-1 on day 3) of the CaO -LFD pretreatment group were 81% and 118% higher than that of the control group,respectively.Under the action of solid alkaline CaO,the bacteria of Clostridium,Atopostipes,Sphaerochaeta,Tissierella,Thiopseudomonas,Rikenellaceae,and Sedimentibacter could build up co-cultures with the archaeal of Methanosaeta,Methanobacterium,and Methanosarcina performing direct interspecies electron transfer (DIET) and improving AD performance of rice straw.Therefore,the combined pretreatment using CaO and LFD could not only pretreat rice straw but also stimulate co-cultures of microorganism to establish DIET enhancing AD efficiency.

1.Introduction

Anaerobic digestion (AD) is an effective organic waste treatment method,which is a process of biodegradation of organic compounds through a series of complex biological interactions [1].Various organic wastes include wastewater,sewage sludge,food waste,forestry resources,living stock manure,and crop residues[2].However,low AD efficiency and system stability are two key problems affecting its practical application [3].Therefore,various pretreatment methods(such as ultrasound,microwave,high pressure and temperature),chemical or biological additives,operational conditions (such as temperature,organic loading,and pH),and co-digestion have been used to enhance AD efficiency and system stability [4].

During pretreatment process,two or more combined pretreatment methods were most commonly used,including physical and chemical combined pretreatment,chemical and chemical combined pretreatment,chemical and biological combined pretreatment method,etc [5,6].Zhao et al.[7] discovered that maize straw under combined pretreatment of 1% NaOH and enzyme achieved a methane yield of 300.85 ml.(g TS)-1,which was 20.24%higher than that of the control.The liquid fraction of digestate(LFD)used as a biological pretreatment reagent of corn stover to enhance the methane production of anaerobic co-digestion with cattle manure,and the methane yield of 41.78% increasing was obtained [8].Combined pretreatment using calcium oxide (CaO)and LFD has advantages of both biological and chemical pretreatments.Guan et al.[9]compared two different pretreatment methods,CaO-ammonia and CaO -LFD,and found that the methane yield of CaO -LFD pretreated rice straw was 57.56% higher than that of the control.The effect of pretreatment temperature (5 °C,20 °C,and 35 °C) and pretreatment time (1 day,2 days,3 days,and 4 days) on the AD performance of CaO -LFD pretreatment has also been studied;results indicated that the highest biomethane yield of combined pretreatment was 77.4% higher than that of the control [5].However,the additional modes of CaO and LFD have not been investigated in previous studies.

A key factor for AD is the cooperation between different types of microorganisms that depend on the effective interspecies electron transfer (IET) in the methanogenesis process [10].Generally,IET includes indirect IET and direct IET (DIET) [11].DIET was first put forward in co-cultures of Geobacter and methanogenic archaea through the biological electrical connections dependent on conductive pili and/or outer surface c-type cytochromes[4].Geobacter,an electron-donating bacterium,can only metabolize simple organic substances of alcohols,volatile fatty acids(VFAS),benzene and others to implement DIET[12].Viggi et al.[13]found that biochar can be responsible for accepting and donating electrons,thus enhancing the methane production of food waste in AD system.Jin et al.[14] showed that in the presence of magnetite,sludge conductance could increase about 3 folds,and the methane production of artificial wastewater improved about 3–10 folds.These were because DIET-based syntrophic partners could directly attach to the carbon-based material surface and exchange electrons via high conductance of metal-based material,thus no longer dependent on conductive pili or c-type cytochromes [15].Conductive materials could act as conductive pili or c-type cytochromes for DIET between syntrophic bacteria,however,the conductive material cannot be used to pretreat the raw material.Currently,conductive materials of carbon-based(granular active carbon,biochar,carbon cloth,graphene,and single walled carbon nanotubes) and metalbased(Fe3O4,Fe2O3,and iron minerals)have been certified to facilitate DIET [1].

DIET has recently been discovered as a widespread phenomenon in anaerobic environments.However,not all microorganisms can perform DIET.Geobacter species have been identified to perform DIET via conductive pili or materials in methanogenic systems [12].However,no direct evidence for Geobacter species could directly degrade the complex organics.The complex organic materials of dairy wastes,kitchen wastes,and waste activated sludge have been used as substrates for DIET in laboratory scale digesters[16].Nevertheless,reports on DIET in AD of lignocellulose materials are rare.Corn straw using 4 mol.L-1NaOH solution pretreatment was used as a bioethanol source to promote anaerobic co-digestion of waste activated sludge (1:1 proportion) [15].Even if electroactive bacteria are not detected,other microorganisms have been suggested to participate in DIET [17].Syntrophomonas,

Clostridium,Thauera,Spirochaeta,Corynebacterium and Coprothermobacter species were identified to perform DIET.Moreover,Methanosaeta,Methanosarcina,Methanolinea,Methanospirillum,Methanobacterium,and Methanoregula species were identified to stimulate DIET in methanogenic systems [12].

Although previous studies have achieved good results,the technique of adding CaO and LFD,and microbial aspects have not been further reported.CaO is a solid alkaline and this substance has the tendency to accept protons or donate electron pairs[18].Whether combined pretreatment of CaO and LFD can promote DIET in the AD system of lignocellulose materials is unclear.Therefore,in this study a combination of CaO and LFD pretreatment was used to pretreat rice straw for improving AD performance.The main objectives of this study were to (1) investigate the effect of different addition methods of CaO and LFD on AD performance,(2)evaluate the effect of CaO and LFD combined pretreatment on the microbial community structures in the AD system.

2.Materials and Methods

2.1.Raw material and inoculum

Rice straw was collected from the Tianjin District,China and air-dried in an open field until the moisture content was less than 10%.Thereafter,using a hammer mill the rice straw was ground to≤0.85 mm.The inoculum was obtained from an anaerobic tank in Biogas Station in Beijing,China.The LFD was collected from an anaerobic digester of continuously stirred tank reactors,which had been operated for more than a year.The total solid (TS) and volatile solid (VS) of rice straw,inoculum,and LFD were 93.3%and 83.8%,7.0% and 3.9%,and 2.4% and 1.2%,respectively.

2.2.Experimental methods

2.2.1.Pretreatment process

In this study,the pretreatment of combined CaO and LFD,single CaO,single LFD were used.For CaO and LFD combined pretreatment,three pretreatment methods were designed:CaO -LFD,CaO+LFD,and LFD+CaO.(1) In CaO -LFD,CaO and LFD were simultaneously added to the rice straw for 2 days;(2) In CaO+LFD,CaO was added on the first day,and then LFD was added on the second day;(3)In LFD+CaO,LFD was added on the first day,and then CaO was added on the second day.Pretreatment of single CaO and LFD were also conducted for 2 days.For each group,during pretreatment,40 g of dry rice straw was first placed into sealed plastic bags.Then,10% CaO concentration was added to the rice straw based on the dry matter of rice straw and 90%moisture content was simultaneously adjusted by the LFD[5].Then,each plastic bag was rubbed by hand to ensure that rice straw was evenly mixed with CaO -LFD.Finally,all prepared group were held at 35 °C.

2.2.2.Anaerobic digestion process

During the AD process,the added amounts of rice straw and inoculum were 50 g TS.L-1and 15 g MLSS.L-1[8],respectively.The working volume of each digester was 0.8 L in a 1 L total volume anaerobic digester.All digesters were sealed using rubber septa and then maintained at 35 °C.The untreated raw rice straw was used as the control group.Moreover,the digesters of only inoculum and only LFD were performed to offset the effect of background values.The total AD time was 50 days and three replicates of each pretreatment condition were analyzed.

2.3.Analytic methods

2.3.1.Chemical and physical analysis

The water displacement method was used to record daily biogas production.The biogas components (H2,N2,CH4,and CO2)were measured using a gas chromatograph(SP-2100,Zhongkehuijie Co.,Beijing,China) equipped with a TDX-01 column and thermal conductivity detector.VS was evaluated based on the standard methods [19].The concentration of VFAs was measured using a gas chromatograph (GC-2014,Shimadzu,Japan) equipped with a flame ionization detector.The conductivity of the suspended solid was measured using a conductivity meter (DDB-303A,Leici,Shanghai,China).

2.3.2.Microbial community analysis

DNA analysis samples were obtained from the CaO -LFD and the control groups at their representative time points during AD process.Samples from the CaO-LFD group were collected during AD on day 0 (initial),day 3 (the first methane peak),day 12 (the second methane peak),and day 30 (the end of methanogenesis),which were recorded as CaO_LFD_1,CaO_LFD_2,CaO_LFD_3,and CaO_LFD_4,respectively.Samples obtained from the control group were collected during AD on day 0 (initial),day 3 (acidification),day 15 (the first methane peak),and day 30 (the end of methanogenesis),which were recorded as Control_1,Control_2,Control_3,and Control_4,respectively.

According to the manufacturer’s protocols,microbial DNA was extracted using the FastDNA Spin Kit (MP Biomedicals,USA).The PCR reactions were then conducted to amplify the 16S rRNA using ABI GeneAmp 9700 (ABI,USA),AxyPrep DNA Gel Extraction Kit(Axygen,USA) and QuantiFluor ST (Promega,USA) with primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGAC TACHVGGGTWTCTAAT-3′) for bacteria and primers 524F10extF(5′-TGYCAGCCGCCGCGGTAA-3′) and Arch958RmodR (5′-YCCGGC GTTGAVTCCAATT-3′) for archaea.PCR working program was performed as follows:95 °C denaturation for 3 min,27 cycles and 33 cycles for bacteria and archaea at 95 °C for 30 s,respectively,55°C annealing for 30 s,72°C elongation for 45 s,and 72°C extension for 10 min.[8].High-throughput 16S rRNA was analyzed at the Illumina MiSeq platform according to NCBI Sequence Read Archive database,UPARSE (version 7.1 http://drive5.com/uparse/)Silva (release 128 http://www.arb-silva.de) and RDP (release 11.1 http://rdp.cme.msu.edu/) databases.

3.Results and Discussion

3.1.Methane performance of rice straw

The daily methane production(DMP)of rice straw under different pretreatment condition was shown in Fig.1(a).The AD process was divided into three stages according to the DMP of CaO and LFD combined pretreatment groups:Stage I was from day 0 to day 9;Stage II was from day 10 to day 18;Stage III was from day 19 to day 50.The DMP of the LFD pretreated group and the CaO and LFD combined pretreated rice straw showed relatively similar change trends.In the CaO pretreatment group and the control group,the DMP of rice straw showed different change trends from the LFD pretreated group and the CaO and LFD combined pretreated group.The first and second peaks of DMP for the LFD pretreated group and the CaO and LFD combined pretreated rice straw appeared on days 3–6 (stage I) and 11–15 (stage II),respectively;the second peak of DMP appeared 3–7 days shorter than that for the CaO group.The highest DMP of the CaO+LFD and CaO -LFD pretreatment groups for the first methane peak were 1122 ml and 1041 ml on days 5 and 3,which were 7.2 and 6.7 folds higher than that of the control group,respectively.

The methane yield of combined CaO and LFD different pretreatment groups(CaO+LFD,CaO-LFD,and LFD+CaO)were all higher than that of other groups(Fig.1(b)).The maximum methane yield of rice straw was 314 ml.(g VS)-1under the CaO -LFD pretreatment condition,which was 9%,18%,36%,58%,and 81%higher than that of the CaO+LFD,LFD+CaO,LFD,CaO,and control group,respectively.The reason for the high methane yield of CaO -LFD pretreated rice straw,in which CaO and LFD were simultaneously added,was that solid alkali CaO reacts with waster to form calcium hydroxide(Ca(OH)2).Ca(OH)2exists in the form of Ca2+and OH-in water;OH-can selectively destroy the major linkages of ether and ester bonds in rice straw,leading to the degradation of effective cellulose and hemicellulose[9].Further,Ca is an essential nutrient for microbial growth;it is involved in the composition of cellular structural substances and is a key substance connecting cells with extracellular polymeric substances [20].Moreover,CaCO3is produced from Ca(OH)2and CO2during the AD process [21].CaCO3is an alkaline buffer,which reacts with the VFAs generated in the reactors,maintaining the neutral pH of AD system.Morever,Ca2+can influence methane production in a charge dependent manner because Ca2+possibly effects the Na+export channel,which is necessary for the final methane production [22].

Fig.1.DMP (a) methane yield and T80 (b) of rice straw at different pretreatment conditions.

T80is the time when the methane production reaches 80%of the total methane production,which is regarded as a parameter to compare AD performance [23].T80for the CaO+LFD,CaO -LFD,LFD+CaO,and LFD pretreated rice straw was 14,17,17,and 17 days,respectively (Fig.1(b)),which was 39%–50% shorter than that of the control group.Thus,CaO and LFD combined pretreatment could enhance AD efficiency,which is consistent with our previous research result using the CaO and LFD combined pretreated rice straw [5].

3.2.Changes of VFAs during the AD process

VFAs are the key intermediate compounds whose generation and consumption maintain a dynamic balance by the acidogenic bacteria and methanogens in the AD system[23].VFAs concentration of rice straw under different pretreatment conditions in the AD process were showed in Fig.2.The total VFAs (TVFA) for the CaO and LFD combined pretreated rice straw groups accumulated rapidly and the peaked value appeared on day 3.Afterwards,VFAs concentration decreased quickly.In stage III,the total VFAs quickly declined to nearly zero for the CaO-LFD pretreated rice straw.The change tendency of VFAs was similar to that of added Fe3+to anaerobic digester using waste activated sludge as the substrate[24].Conversely,the total VFAs of other pretreated groups decreased slowly from day 9 to 21.Under different pretreatment conditions,the highest VFAs concentration for the CaO -LFD pretreatment group was 14851 mg.L-1appeared on day 3 of the AD process,which was 7%,66%,157%,35%,and 118%higher than that of the CaO+LFD,LFD+CaO,LFD,CaO,and control group on day 3,respectively.Generally,the range of VFAs concentrations was less than 1500 mg.L-1for a healthy AD system[25].Although the VFAs of the CaO -LFD group on day 3 was close to the inhibitory concentration,the AD system was not clearly suppressed and the methane production improved as shown in Fig.1.A likely reason was that the addition of CaO and LFD could improve the buffering capacity of the AD system.

Fig.2.VFAs of CaO+LFD (a),CaO -LFD (b),LFD+CaO (c),LFD (d),CaO (e) pretreated and untreated (f) rice straw during the AD process.

In particular,the main VFAs of the CaO+LFD and CaO -LFD pretreatment groups were found to be ethanol and acetic acid in VFAs components.Ethanol and acetic acid of the CaO -LFD pretreated rice straw accounted for 1%–3% and 45%–89%,12%–56%and 40%–58%,and 56%–81% and 12%–36% of the total amount of VFAs for stage I,stage II,and stage III of the AD process,respectively.The highest acetic acid concentration of the CaO-LFD pretreatment group was 11949 mg.L-1,accounting for 80%of the total VFAs on day 3.Then acetic acid concentration rapidly decreased from 5960 mg.L-1on day 6 to 152 mg.L-1on day 12.The propionic acid concentration of the CaO+LFD and LFD+CaO pretreatment groups during stage I and stage II were 7–2611 mg.L-1and 107–4276 mg.L-1,which accounted for 1%–29%and 3%–82%of the total VFAs concentration,respectively.Furthermore,the propionic acid concentration of control group accounted for 2%–62% of the total VFAs within 30 days.The conversion rate of propionic acid to methane was relatively low compared with that of ethanol and acetic acid.Propionic acid accumulation could affect the activity of methanogens,which has been considered as a parameter contributing to the failure of AD [26].Therefore,the high propionic acid content of the CaO+LFD,LFD+CaO and control group inhibited the generation rate of methane [27].

Interestingly,the highest total VFAs concentration with a high content of acetic acid (45%–89% at the stage I) for the CaO -LFD pretreatment group resulted in the highest methane production.This indicated that after CaO -LFD pretreatment,the accumulation of VFAs at the early stage could be rapidly degraded to methane.This phenomenon was consistent with the results obtained by Zhou et al.[27],which enhanced the methanogenic activity of high solid sludge with nano zero-valent iron in AD.Studies have shown that alkaline material and LFD could improve the buffering capacity of an AD system [9].Therefore,the improvement of VFAs conversion rate and methane performance was probably attributed to the relief of VFA accumulation by CaO and LFD.

3.3.Changes of pH and alkalinity during the AD process

Alkalinity and pH are important parameters to achieve AD system stability of the reaction system.Commonly,pH and alkalinity values have a significant effect on the activities of anaerobic microorganisms.The system stability indicators during the AD process under different pretreatment groups were shown in Fig.3.

Fig.3.pH (a) and alkalinity (b) of CaO+LFD,CaO -LFD,LFD+CaO,LFD,CaO pretreated and untreated rice straw during the AD process.

The pH values of CaO -LFD,CaO+LFD,LFD+CaO and LFD groups in the first three days showed a downward trend owing to the accumulation of VFA.In this stage,the alkalinity of these four groups could still regulate the accumulation of VFA.Therefore,in the late stage of Phase Ⅰ,the pH value of each group rapidly recovered to approximately 7 and DMP remained over 290 ml.The AD system of CaO-LFD,CaO+LFD,LFD+CaO and LFD groups did not show obvious acidification.In Phase Ⅱ,the alkalinity of CaO -LFD,CaO+LFD and LFD+CaO was 9000–9700 mg.L-1,8550–9750 mg.L-1and 8050–8350 mg.L-1,respectively.The alkalinity of CaO -LFD and CaO+LFD AD systems were higher than that of the LFD+CaO system,indicating that the addition method would affect the ability of the anaerobic system to resist acid shock.The pH of the CaO group and the control group rapidly dropped to 6.5 at the beginning of Phase Ⅰ.The methane production of these two AD systems stagnated,showing obvious acidification.After the 12th day of Phase Ⅱ,the pH value of the CaO group and the control group gradually recovered to approximately 7.The results showed that the stability of the AD system after CaO pretreatment or under no pretreatment was poor.The increase of buffer capacity in CaO -LFD,CaO+LFD and LFD+CaO AD systems was mainly attributed to the reuse of LFD.

3.4.VS removal rates in the AD process

The changes of VS can reflect the change of organic matter during the AD process [28].The removal rate of organic matter increased with the progress of AD time,which was consistent with the result of methane yield under different pretreatments.As shown in Fig.4,the highest VS removal rates were 15%–25%,31%–47%,and 56%–67%for stage I,stage II,and stage III of the different pretreated rice straw,which were 20%–97%,3%–54%,and 11%–34% higher than that of the stage I,stage II,and stage III of the control group,respectively.Among different pretreatment groups,the highest VS removal rate was 67%for the CaO-LFD pretreatment group.This result was similar to our previous research result for CaO -LFD pretreated rice straw [9].This indicated that after the CaO -LFD pretreatment,the higher VS removal rate of rice straw corresponded to higher methane yield.

3.5.Conductivity of the suspended solid

Fig.4.VS removal rate of rice straw under different pretreatment conditions.

Fig.5.Conductivity of the CaO -LFD pretreated and untreated rice straw under different pretreatment conditions.

Zhao et al.[29] reported that the conductivity of suspended sludge could be acted as potential evidence to indicate the establishment of DIET.The conductivity of the AD system in the CaO -LFD pretreatment group and control group were measured during the AD process every three days (Fig.5).At the stage I,the conductivity of the CaO-LFD pretreatment group and control group exhibited similar change trends.The electrical conductivity of the CaO -LFD pretreatment group began to increase from 5.9 to 8.1 mS.cm-1in the stage II and stage III,which was 8.3%–52.5%higher than that of the control group.The higher conductivity detected in the CaO-LFD pretreatment group than that in the control group suggested that DIET was likely established via high conductance of metal-based material or conductive pili of bacteria[15]in the AD system of the CaO-LFD pretreatment group.Moreover,the ethanol concentration of the CaO -LFD pretreated rice straw accounted for 12%–81%of the total VFAs in stage II and stage III,which also demonstrated a great possibility to fulfill DIET in the ethanol-stimulated enrichment [16].

3.6.Microbial community structure

The microbial communities of CaO -LFD pretreatment and untreated groups were investigated at their representative AD time points to explore the effect of pretreatment on the microbial community structure and provide a clue for the reason of enhanced methane production.

3.6.1.Bacterial community composition

Generally,bacteria are considered to exist in the AD system as fermentative bacteria [30].Bacterial community structures during the AD process of rice straw using CaO -LFD pretreatment and control group at genus level are shown in Fig.6.Clostridium,Terrisporobacter,Turicibacter,and Romboutsia have been shown to participate in all phases of AD.For the CaO-LFD pretreated rice straw and control group in all AD stages,the relative abundances of Clostridium (24%–59%)was the dominant species,the second most abundant was Terrisporobacter (5%–13%),followed by Turicibacter(3%–6%) and Romboutsia (2%–5%).Clostridium and Terrisporobacter had the highest proportion,because they were metabolically versatile and could degrade carbohydrates of rice straw to hydrogen along with the production of VFAs generating acetate,hydrogen and CO2[31].As shown in Section 3.4,DIET may be estabilished in the AD system of CaO -LFD pretreated rice straw.Geobacter is known to be the most common microorganism that exchanges electrons via DIET.However,Geobacter was not detected in these AD systems,indicating that some other types of microorganisms might be involved in DIET [11].Clostridium species are frequently found in the anodes of microbial fuel cells,suggesting that it is possible to transfer electrons across the cell membrane to the extracellular environment and donate electrons to conductive materials[12].

Fig.6.Bacterial community composition of the CaO -LFD pretreated and untreated rice straw during the AD process.

Some different acidogenic bacteria appeared in different AD stages.At stage I,the relative abundances of Clostridium and Terrisporobacter for the CaO-FLD group were 54%and 11%unchanged,whereas in the control group,the relative abundances of Clostridium and Terrisporobacter increased from 26% to 59% and from 5%to 13%,respectively.This is because the rice straw of the CaO -LFD pretreatment group had been pre-acidified before AD;the fermentative bacteria were domesticated during the pretreatment process.Anaerosporobacter and Solibacillus were also the main genera for the CaO-FLD group in Stage Ⅰ,the relative abundances of these two bacteria accounted for 6%and 5%,respectively.However,only a few Anaerosporobacter(0.01%)and Solibacillus(1%)were found in the control group.Anaerosporobacter and Solibacillus were found mostly involved in the hydrolysis and acidogenesis of the lignocellulose system [32]).Further,Anaerosporobacter is known to convert carbohydrates to H2.A higher abundance of Anaerosporobacter showed their additional contribution to H2production at the beginning of AD [33].

Fig.7.Archaeal community composition of the CaO -LFD pretreated and untreated rice straw during the AD process.

At the stage II of AD,the relative abundance of Clostridium decreased from 54% to 37% in the CaO -LFD pretreatment group;in the control group,the change of the relative abundance of Clostridium slightly decreased from 59% to 50%.The diversity of bacteria increased after CaO -LFD combined pretreatment in this stage.At the genus level,Fermentimonas,Rikenellaceae,Fastidiosipila,Proteiniphilum,Cellulosilyticum,Sedimentibacter,and Sphaerochaeta were detected on day 12 (the second methane peak),and their relative abundances were 6%,4%,3%,4%,2%,1%,and 1%,respectively.The Fermentimonas and Proteiniphilum abundances in the CaO -LFD pretreatment system were 3 and 4 folds higher than those in the control group at the Stage II,respectively.Fermentimonas can use different types of carbohydrate and cellulose to produce hydrogen,VFAs,and CO2[34].Proteiniphilum can use N-containing substrates as the energy source to generate acetic acid and propionate [35].Fastidiosipila and Sedimentibacter are hydrolyzing and fermenting bacteria and can produce VFAs by hydrolyzing and fermenting proteins [36].Fastidiosipila plays a key role in converting different compounds to acetic acid and CO2[37].Rikenellaceae was confirmed as potential syntrophic bacteria that can set up DIET during the VFAs degradation process forming methane with magnetite-mediated [37].Apart from Rikenellaceae species,Sphaerochaeta species could grow in defined co-cultures with Methanosarcina species,and have a great potential for participating in DIET[38].Moreover,Sphaerochaeta species can use more complex organic substrates than Geobacter,and these species have significant potential for enhancing substrates of DIET.Further,Zhao et al.[39]found that all epilina accessory proteins of proceeding interspecies electron and energy exchange were detected in Sphaerochaeta and Sedimentibacter.

At the stage III,30 days of AD,apart from the most relative abundant of Clostridium species,the dominant bacteria in the CaO -LFD pretreatment group were Atopostipes (13%),Ruminofilibacter (8%),Erysipelothrix (5%),Sporosarcina (3%),Amphibacillus(3%),Thiopseudomonas (3%),Rikenellaceae (2%),Cellulosilyticum(2%),Sphaerochaeta (2%),Ruminococcaceae (2%),and Tissierella(2%).Atopostipes,a fermentation bacterium that uses carbohydrates,was found on the anodic biofilm of microbial electrolysis cells;they can use electrodes as extracellular electron acceptors to generate electricity[40].This finding suggested that Atopostipes species had a great potential for participating in DIET.Ruminofilibacter is mainly responsible for the degradation of lignocellulosic materials [41].Rice straw was the lignocellulosic material,and it was reasonable that the relative abundance of Ruminofilibacter increased from 1%to 8%.Erysipelothrix was the typical fermentative acidogenic bacteria,leading to higher ethanol and acetate production in CaO -LFD pretreatment group.The results are similar to the addition of magnetite under suitable alkaline conditions with enhanced production of short-chain fatty acids from waste activated sludge [42].Sporosarcina can sequester CO2and simultaneously generate CaCO3precipitates [21].Moreover,Tissierella,belonging to Firmicutes,produces lactic acid and adapts to alkaline conditions[43].Tissierella is also found in bioelectroactive biofilms and is suspected to be electroactive [44].Studies have found that under anaerobic conditions,an alkaliphilic consortium dominated by Tissierella and Clostridium can use Fe3+as a final electron acceptor [45].Additionally,Thiopseudomonas species can use nitrate as an electron acceptor in the anaerobic sludge [44].Apart from Thiopseudomonas species,Rikenellaceae and Sphaerochaeta also had the ability to participate in sole DIET or conductive materialbased DIET [38,39].

3.6.2.Archaeal community composition

The predominant genera in the AD process were Methanosaeta,Methanobacterium,and Methanosarcina for the archaea community(Fig.7).Similarly,the archaea communities were reported to add both Fe3O4and zero-valent iron simultaneously in the AD system of waste activated sludge[24].The relative abundance of Methanosaeta,Methanobacterium,and Methanosarcina were 28%–67%,10%–34%,and 1%–24%,respectively,for CaO-LFD pretreatment group.

Throughout the AD process,the relative abundance of Methanosaeta gradually decreased from 67%to 28%for CaO-LFD pretreatment group,whereas for the control group,the relative abundance of Methanosaeta increased from 33%to 70%,and then decreased to 68%.Methanosaeta,a typical genus of acetoclastic methanogens,can convert high-concentration acetate to methane [46].The decreased abundance of Methanosaeta in the CaO -LFD pretreatment group suggested that the pathway of methane production by acetate cleavage gradually weakened during the AD process.

At the stage Ⅱ,the relative abundance of Methanobacterium and Methanosarcina in the CaO-LFD pretreatment system was 1.6 and 1.8 times higher than that of the control group,respectively.Methanobacterium,a hydrogen-utilizing methanogen,plays a key role in methane production,which could simultaneously grow on acetic acid and H2/CO2[47].The high abundance of hydrogenotrophic Methanobacterium in the CaO -LFD group indicated increased interspecies electron flux via H2or formate [48].Methanobacterium species seemed to be involved in DIET for maintaining stable syntrophic metabolism [16].Zhao et al.[29] found that Methanosarcina could directly accept the electron for reducing of CO2to CH4via DIET,particularly in the AD system using a conductive material,because of the additional conductive material that enhanced the electronic conductivity of the AD system.After CaO -LFD pretreatment,the increased relative abundance of Methanobacterium and Methanosarcina is likely ascribed to the Ca2+of solid alkaline CaO dissolved in water stimulating the DIET functional microorganisms,because the appropriate concentration of Ca can promote methanogenic activity [49].

4.Conclusions

After CaO -LFD pretreatment,the maximum methane yield of rice straw was 314 ml.(g VS)-1,which was 81%higher than that of the control group.The highest VFAs concentration in the CaO-LFD pretreatment group was 14,851 mg.L-1on day 3.Under the action of solid alkaline CaO,the bacteria of Clostridium,Atopostipes,Sphaerochaeta,Tissierella,Thiopseudomonas,Rikenellaceae,and Sedimentibacter could build up co-cultures with the archaeal of Methanosaeta,Methanobacterium,and Methanosarcina performing DIET and improving AD efficiency of rice straw.Therefore,CaO and LFD combined pretreatment could be used to pretreat rice straw and stimulate co-cultures of microorganisms for establishing DIET and enhancing AD performance.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Key Research&Development Program of Ministry of Science and Technology of the People’s Republic of China (grant number 2018YFC1900901).