Author list Dr. Zhiqiang Zhao First author E-mail address

Research Article pubs.acs.org/journal/ascecg

New Application of Ethanol-Type Fermentation: Stimulating Methanogenic Communities with Ethanol to Perform Direct Interspecies Electron Transfer Zhiqiang Zhao, Yang Li, Xie Quan, and Yaobin Zhang* Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China S Supporting Information *

ABSTRACT: Direct interspecies electron transfer (DIET) has been considered as an effective mechanism to proceed syntrophic methanogenic metabolism. However, up to now, this working mode has been still not widely established in the Geobacter-rare methanogenic digesters. In this study, a strategy that could enrich Geobacter species and stimulate the methanogenic communities to continuously perform DIET was proposed in a two-phase anaerobic digestion (AD) system with the aim to enhance and stabilize the better AD. The results demonstrated that, under the conditions employed, the ethanol-abundant acidogenic products could be produced via ethanol-type fermentation when acidogenic-phase pH was kept at 4.0−4.5. Enrichments in the methanogenic phase continuously stimulated with the ethanol-abundant acidogenic products presented a higher conductivity, as well as more positive response to granular activated carbon (GAC) supplemented, compared with the enrichments without this stimulation, suggesting that DIET might be established. Microbial community analysis showed that Geobacter species were only detected in the methanogenic enrichments stimulated by the ethanol-abundant acidogenic products. Together with the significant increase of Methanosarcina species in these enrichments, the potential DIET between Geobacter and Methanosarcina species might be permanently established in the methanogenic digester to maintain the acidic balance as well as syntrophic metabolism stable. KEYWORDS: Anaerobic digestion, Ethanol-type fermentation, Direct interspecies electron transfer (DIET), Syntrophic metabolism

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103 e− cp−1 s−1).57 Furthermore, acetate-utilizing methanogens are well-known as the dominant methanogens in most of traditional anaerobic digesters15 and have the heavy duty for producing methane.16,17 Therefore, enhancing DIET is expected to promote the better AD. However, the application of DIET to AD in response to the impacts from high organic loading rates (OLRs) is still limited. An important reason is that, up to now, only Geobacter species have been confirmed to proceed DIET in defined cocultures,13,14,18,19 as well as in some mixed cultures.10,20−23 While Geobacter species are usually not abundant in most of traditional digester communities,24−27 which thereby limits its role to maintain syntrophic metabolism stable, when AD is influenced by the high-OLR impacts. Some studies28−31 suggested that DIET between syntrophs involved in some sulfur/Fe(III)-reducing microorganisms and methanogens might be established under the methanogenic conditions if conductive materials were provided as electrical conduits. However, DIET was only inferred from the faster conversion of

INTRODUCTION An understanding of the effective mechanisms for syntrophic metabolism during anaerobic digestion (AD) is important because of its central importance in carbon and electron flow.1−3 Direct interspecies electron transfer (DIET) has been considered as an alternative to interspecies hydrogen/formate transfer (IHT/IFT) for the long-range electron and energy transport.4−6 DIET can proceed via biological electrical connections involved in electrically conductive pili7,8 or outer surface c-type cytochromes,9 or a combination of biological and abiological electron transfer components, such as conductive carbon-based materials,10−12 compensating the drawbacks of IHT (or IFT) that requires the essential hydrogen (or formate) as the electron carrier, as well as a low enough hydrogen partial pressure (or formate concentration) in thermodynamics.1,3 For DIET, acetate-utilizing methanogens, such as Methanosaeta or Methanosarcina species, replace the hydrogen-utilizing methanogens to accept the electrons for the reduction of carbon dioxide to methane.13,14 Even though Methanosaeta or Methanosarcina species grow slowly since the conversion of acetate to methane yields little energy, and the replacement may be not always a potential advantage, DIET (44.9 × 103 e− cp−1 s−1) provides rapider electron transfer than IHT (5.24 × © 2017 American Chemical Society

Received: July 28, 2017 Revised: August 24, 2017 Published: September 4, 2017 9441

DOI: 10.1021/acssuschemeng.7b02581 ACS Sustainable Chem. Eng. 2017, 5, 9441−9453

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ACS Sustainable Chemistry & Engineering

Figure 1. Schematic diagram of a continuous-flow two-phase AD system.

enrich Geobacter species and stimulate the methanogenic communities to perform DIET for the enhancement and stabilization of AD in response to the high-OLR impacts. However, the continuous supply of ethanol will increase the considerable costs, and is obviously uneconomical in the application of DIET. In the absence of any amendments, gradually reducing the content of ethanol in the feedings with the extension of operating time could result in the succession of methanogenic communities and interspecies electron transfer from DIET to IHT.20 A possibility is that the supplemented ethanol is not from the external supply but expected to be produced by the selffermentation of complex organic wastes. If so, it would not only solve the uneconomical problem but also stimulate the methanogenic communities to permanently perform DIET as well as avoid the succession of methanogenic communities from DIET to IHT. Ren et al.35 had reported that ethanol could be significantly produced via ethanol-type fermentation by adjusting the operating pHs at 4.0−4.5 in a high-OLR acidogenic digester. Under the conditions employed, ethanol was abundant in the acidogenic products, accompanied by the significant release of hydrogen since the balance between NAD and NADH+ was preserved.36 Therefore, until now, most of studies involved in ethanol-type fermentation only focused on hydrogen production.37−39 The purpose of this study was to propose a sustainable strategy to achieve the better application of DIET to the enhancement and stabilization of AD. Namely, in a two-phase AD system, ethanol was expected to be initially produced from the fermentation of complex organic wastes in the acidogenic phase via adjusting the operating pHs. Then ethanol-abundant acidogenic products were fed to methanogenic phase, which was expected to continuously stimulated the methanogenic communities to perform DIET.

organics to methane in the presence of conductive materials. Another potential limitation of DIET during AD is that the available substrates in current studies are only involved in some small-molecule alcohols13,14,18,19,21,22 and fatty acids.20−23,28−33 It should be pointed out that the available substrates for Geobacter species with Fe(III) oxides as electron acceptors are varied, such as benzene, glucose, glycerol, and yeast extract,9 while no study has reported that Geobacter species could directly decompose the complex organics, such as carbohydrates or proteins, via DIET in defined cocultures. A potential reason is that the energy yield from the metabolism of these complex organics with Methanosaeta or Methanosarcina species as electron acceptors for the reduction of carbon dioxide is insufficient for Geobacter species to produce biological electrical connections.19 Therefore, it is usually observed that, once acidification of complex organic wastes is limited, DIET cannot proceed well, even in the presence of conductive carbon-based materials24,56 Previous studies20,23 demonstrated that, initially feeding an up-flow anaerobic sludge blanket (UASB) reactor with ethanol, similar to those that were previously reported to support communities that metabolized ethanol with DIET in methanogenic digesters treating brewery wastes,34 could stimulate the communities to perform DIET for syntrophic metabolism of propionate and butyrate. This ethanolstimulated strategy presented two potential advantages: (1) Ethanol as the substrate could enrich Geobacter species, compensating the shortage that Geobacter species were quite rare in most of traditional methanogenic communities. (2) The energy yield from the metabolism of ethanol could support DIET to overcome the thermodynamical limitations of syntrophic oxidation of propionate and butyrate as well as to compete these substrates with IHT.34 As a result, the abundance of Methanosaeta or Methanosarcina species known as the syntrophic partners of Geobacter species significantly improved.23 On the basis of these advantages, it is expected that ethanol supplemented to the traditional anaerobic digesters can

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MATERIALS AND METHODS

Experimental Setup. The continuous-flow experiments were conducted in four parallel two-phase AD systems. The schematic 9442


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ACS Sustainable Chemistry & Engineering diagram of a continuous-flow two-phase AD system is shown in Figure 1. The acidogenic phase was operated in a cylindrical glass completely mixed digester (internal diameter of 70 mm and height of 300 mm) with a working volume of 1000 mL. At the top of acidogenic digester, a stirring rod at a speed of 30−60 rpm connected with electric motor was placed into the suspended sludge. The methanogenic phase was operated in a cylindrical glass UASB reactor (internal diameter of 100 mm and height of 300 mm) with a working volume of 2000 mL. A three-phase separator was placed at the top of UASB reactor (Figure 1). Both acidogenic and methanogenic digester were equipped with a gas sampling bag at the top of digester and a sludge sampling port at the bottom of digester and operated at a temperature of 37.0 °C. At the beginning of continuous-flow experiments, the fresh artificial wastewater stored in the feeding tank was first pumped into the acidogenic digester with a peristaltic pump (Lange, BT100-2J, China) (Figure 1). After acidogenesis, the treated wastewater contained a small amount of suspended sludge was drawn out to the adjusting tank (Figure 1). The settling sludge at the bottom of adjusting tank were returned to acidogenic digester (sludge recirculation) with a sludge peristaltic pump (Lange, BT300-2J, China), and supernatant liquid (fresh acidogenic products) at the upside of adjusting tank was pumped into the methanogenic digester (UASB) for methanogenesis with another peristaltic pump (Lange, BT100-2J, China) (Figure 1). The hydraulic retention time (HRT) of acidogenic and methanogenic digester was 9.6 and 19.2 h, respectively. The four parallel two-phase AD systems were initially operated for 20 days of startup with a OLR of 13.25 kgCOD/m3/d (initial concentration of COD was 5300 mg/L). Then the influent OLR of the four two-phase AD systems gradually increased from 13.25 to 79.5 kgCOD/m3/d over the next 100 days by increasing the influent concentration of COD to 2-, 3-, 4-, 5-, and 6-fold of initial concentration of COD to investigate the effects of OLRs on the performances. Under each OLR, the four two-phase AD systems were operated for 20 days of experiments. When the influent OLR increased from 53 to 79.5 kgCOD/m3/d, to keep the acidogenic-phase pH at 4.0−4.5 rather than further lower than 4.0, with the aim to achieve ethanol-type fermentation,35,36 NaHCO3 was supplemented into the feeding for buffering the excess acidity. The dosage of NaHCO3 powder is shown in Table S1. Sludge and Wastewater. The initial seed sludge was obtained from an anaerobic digester of a municipal sludge treatment plant in Dalian (China). Total suspended solids (TSS) of initial seed sludge was 17400 ± 270 mg/L (mean ± standard deviation, n = 3) and the ratio between volatile suspended solids (VSS) and TSS was 0.74. It was anaerobically stored at 4 °C. At the beginning of continuous-flow experiments, each acidogenic digester received a 400 mL of seed sludge as inoculum and each methanogenic digester (UASB) received a 1000 mL of seed sludge as inoculum. An artificial dairy wastewater was used as feeding for the four parallel two-phase AD systems. The initial composition (per liter) of the artificial wastewater was as follows:40 glucose, 2.40 g; yeast extract, 0.48 g; milk powder (Songhuajiang; taobao.com; China), 2.00 g; NH4Cl, 0.46 g; KH2PO4, 0.10 g; NaHCO3, 5.00 g; trace element solution, 10 mL; vitamin solution, 10 mL. The composition of the trace element solution and vitamin solution was described in our previous study.22 The chemical oxygen demand (COD) and pH of this artificial dairy wastewater was about 5300 mg/L and 7.2, respectively. Batch Experiments with Granular Activated Carbon. To assess the effects of granular activated carbon (GAC, 8−20 mesh, Sigma-Aldrich, USA) on syntrophic metabolism of potential acidogenic products in the methanogenic enrichments taken from one of the parallel four UASB reactors with a 50 mL centrifuge tube from the sludge sampling ports of UASB reactors at day 20, 40, 60, 80, 100, and 120, respectively, batch experiments were conducted in six 125 mL serum bottles in the dark at 37.0 °C.23 Before batch experiments, 10 g of GAC were supplemented into three of the six serum bottles. As a control, another three of the six serum bottles were supplemented with the nonconductive glass (diameter of 10−15 mm, Dewei, China) with the same supplemented volume as GAC. All the serum bottles contained 5 mL sludge taken from the UASB reactors

and 35 mL media. The composition of the media (per liter) used to simulate the potential acidogenic products was as follows: ethanol, 0.84 mL; sodium acetate, 1.32 g; sodium propionate, 0.59 g; sodium butyrate, 0.74 g; glucose, 2.58 g; NH4Cl, 0.59 g; K2HPO4, 0.14 g; MgCl2·6H2O, 0.10 g; CaCl2·2H2O, 0.05 g; NaHCO3, 4.00g, respectively. The whole carbon source in this media amounted to an approximate COD of 6900 mg/L. The proportion of each carbon source accounting for total COD in this media was as follows: 20% of ethanol; 15% of acetate; 10% of propionate; 15% of butyrate; 40% of carbohydrates, according to the composition of acidogenic products under the influent OLR from 53 to 79.5 kgCOD/m3/d (Figure 6A). Before the sludge supplemented to the serum bottles, the media was flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 0.5 h. Upon preparation, all the serum bottles were sealed with Teflon-faced butyl rubber stoppers and then flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 0.5 h in the headspace.23 Chemical Analysis. The TSS, VSS and COD were analyzed in accordance with the Standard Methods for the Examination of Water and Wastewater. To analyze the change of VSS concentration of methanogenic enrichments, the suspended sludge samples taken from the four parallel UASB reactors at day 20, 40, 60, 80, 100 and 120, respectively, with a 50 mL centrifuge tube from the sludge sampling ports of UASB reactors. Ethanol and volatile fatty acids (VFAs) (mainly including acetate, propionate, butyrate and valerate) were measured by a gas chromatograph with a flame ionization detector (FID) (Tianmei, GC-7900P/FID, China).25 Lactate was determined by a high performance liquid chromatography (HPLC) (Tianmei, LC2000, China) equipped with a UV−vis detector and a column Zorbax SB-Aq (Agilent, 150 mm × 4.6 mm, USA).59 The column temperature was maintained at 35 °C. The mobile phase was (99:1 v/ v) 20 mM sodium phosphate buffer (pH = 2)/methanol at a flow rate of 0.3 mL/min. Samples were detected by absorbance at 210 nm. The equivalent relationship between COD and substrates are as follows: 1.07 g-COD/g acetate, 1.51 g-COD/g propionate and 1.82 g-COD/g butyrate, 2.08 g-COD/g ethanol, 1.07 g-COD/g glucose, 1.07 gCOD/g lactate, 2.58 × 10−3 g-COD/mL methane and 0.645 × 10−3 gCOD/mL hydrogen. The produced biogas in each acidogenic/ methanogenic digester was collected by a gas sampling bag and the volume of biogas was measured by a glass syringe of 100 mL.22 The content of methane, hydrogen and carbon dioxide in the gas sampling bag and headspace of 125 mL serum bottles was analyzed by another gas chromatograph with a thermal conductivity detector (TCD) (Tianmei, GC-7900/TCD, China).21 pH was recorded using a pH analyzer (Denver Instrument; UB-10; Denver). Conductivity Measurement. To assess the conductivity of methanogenic enrichments, the three-probe electrical conductance measurement was performed with two gold electrodes separated by 0.5 mm nonconductive gap.8 The suspended sludge samples taken from the four parallel UASB reactors at day 20, 40, 60, 80, 100, and 120, respectively, with a 50 mL centrifuge tube from the sludge sampling ports of UASB reactors, were first collected by the centrifugation at 8000 rpm for 5 min and then washed three times by 0.1 M NaCl.41 After washing and centrifuging, the wet suspended sludge samples were placed on the gold electrodes and crushed with a cover glass to form a confluent film that spread across the nonconductive gap.23 An electrochemical workstation (Zhenhua, CHI1030C, China) was used to apply a voltage ramp of −0.3−0.3 V across split electrodes in steps of 0.025 V. For each measurement, after allowing the exponential decay of transient ionic current, the steady-state electronic current for each voltage was measured every second over a minimum period of 120 s. The time-averaged current for each applied voltage was recorded to create the current−voltage curve.8 Microbial Morphology. Field emission scanning electron microscopy (FESEM) (Hitachi, S-4800, Japan) was used to observe the microbial morphology of methanogenic enrichments taken from one of the four parallel UASB reactors with a 50 mL centrifuge tube from the sludge sampling ports of UASB reactors at day 20, 40, 60, 80, 100, and 120, respectively. For SEM observation, the suspended sludge samples were immobilized in a 2.5% glutaraldehyde solution, 9443


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Figure 2. Methane production rate (A), effluent pH (B), COD removal efficiency (C) and methane conversion efficiency (D) during the 120-day continuous-flow experiments. Error bars represent standard deviations of the four parallel experiments. dehydrated in graded water−ethanol solutions, then lyophilized and sputter-coated with gold.42 DNA Extraction, PCR Amplification, and High-Throughput Sequencing. After continuous-flow experiments the suspended sludge taken from one of the four parallel acidogenic/methanogenic digester with a 50 mL centrifuge tube from the sludge sampling ports at day 20, 40, 60, 80, 100, 120, respectively, were collected to analyze the microbial communities via high-throughput sequencing. The suspended sludge samples were first rinsed twice by phosphatebuffered saline (PBS; 0.13 M NaCl and 10 mM Na2HPO4 at pH 7.2) and then harvested by centrifugation (110 × 100 g for 15 min at 4 °C).23 The FastDNA SPIN kit for soil (Bioteke, China) was used to extract DNA from all the suspended sludge samples according to the manufacturer’s protocols. The concentration and purity of the extracted DNA were determined by analyzing its absorbance at 260 and 280 nm with a Nanodrop ND-1000 spectrophotometer (Labtech International, UK). Archaeal and bacterial 16S rRNA gene fragments were amplified via the polymerase chain reaction (PCR) with the following primer sets: (Arch519F/Arch915R) and (515F/806R). The following amplification cycling scheme was used: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed.43 After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of

bands. Multiple samples were pooled together (e.g., 100 samples) in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Ampure XP beads. Then the pooled and purified PCR products were used to prepare DNA library by following Illumina TruSeq DNA library preparation protocol. High-throughput sequencing was performed on an Illumina Hiseq 2000 platform (Illumia, San Diego, USA) by Sangon Biotechnology Co., Ltd. (Shanghai, China). Sequences were placed into various operational taxonomic units with pipeline software. Final OTUs were taxonomically classified using BLASTN against a curated database derived from GreenGenes, RDPII and NCBI (www.ncbi.nlm. nih.gov, http://rdp.cme.msu.edu).44 The 16S rRNA gene sequence data obtained in this study were deposited in the NCBI (National Center for Biotechnology Information) GenBank databases under accession MF573069 to MF573118 (for acidogenic-phase bacteria), MF573119 to MF573167 (for methanogenic-phase bacteria) and MF573168 to MF573210 (for methanogenic-phase Archaea) on Jul 29, 2017, and the scheduled release date for this submission was Aug 30, 2017.

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RESULTS AND DISCUSSION Performances of Two-Phase AD Systems. The methane production rate in the acidogenic digesters first increased from 2.11 ± 0.09 (mean ± standard deviation, n = 4) to 3.37 ± 0.15

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ACS Sustainable Chemistry & Engineering L/d, but then drastically declined to 0.92 ± 0.13 L/d as the influent OLR increased from 13.25 to 26.5 and then to 39.75 kg COD/m3/d (Figure 2A). When the influent OLR further increased from 53 to 79.5 kg COD/m3/d, almost no methane production was detected in the acidogenic digesters. Remarkably, the low-pH acidogenic products only had the slight effects on the methane production in the methanogenic digesters. Especially, when the acidogenic-phase pH was kept at 4.0−4.5 under the influent OLR from 53 to 79.5 kg COD/m3/d, the methane production rates still continuously increased from 9.09 ± 0.14 to 17.13 ± 0.15 L/d (Figure 2A) and the average effluent pHs were always kept at 6.9−7.1 (Figure 2B) in the methanogenic digesters. The COD removal efficiencies of acidogenic and methanogenic digesters declined (Figure 2C) as the increase of OLR from 13.25 to 39.75 kgCOD/m3/d. In consistent with the COD removal efficiencies, the methane conversion efficiencies of acidogenic and methanogenic digesters also declined (Figure 2D). The high-OLR impacts causing a low-efficiency AD had been widely reported.22−24,45,46,56 An important reason was that the balance between the production of organic acids and methanogenesis during syntrophic metabolism was easily destroyed by the high-OLR impacts. The gradual accumulation of organic acids could lead to the sour of anaerobic digester and then inhibit methanogens. In this study, when the acidogenicphase pH was kept at 4.0−4.5 under the influent OLR from 53 to 79.5 kgCOD/m3/d, the COD removal efficiencies in the acidogenic digesters were still lower than 10%, similar to that before adjusting the acidogenic-phase pH, but which in the methanogenic digesters gradually increased from 60.0% to 77.2% (Figure 2C). Also, the methane conversion efficiencies in the acidogenic digesters still continuously declined from 36.1% to 6.7%, which however in the methanogenic digesters were always kept at 85% and about 10 percentage points higher than that before adjusting the acidogenic-phase pH (Figure 2D). The improvement in the rates of methane production as well as stabilization in the efficiencies of methane conversion under the higher influent OLRs suggested that an effective mechanism of interspecies electron transfer might be established in the methanogenic phase to maintain the acidic balance as well as syntrophic metabolism stable. As a result, the two-phase AD systems could be capable of withstanding the high-OLR impacts. Conductivity of Methanogenic Enrichments. DIET has been considered as an effective mechanism to replace the traditional IHT/IFT to maintain syntrophic metabolism stable.20−23,33 It was for the first time demonstrated in the aggregates of UASB reactors treating brewery wastes.34 The temperature dependence of conductivity of these aggregates was consistent with metallic-like conductivity,34 similar to that found in G. sulf urreducens electrically conductive pili and biofilms.18 Consequently, the potential mechanism for the conductive aggregates was inferred that Geobacter species facilitated DIET via their electrically conductive pili.18,34,47 To investigate the potential mechanism related to DIET in the methanogenic digesters, the conductivity of methanogenic enrichments was measured in this study. Before the pH adjustment of acidogenic phase, there was no significant difference in the conductivity among these methanogenic enrichments (P > 0.05) (Figure 3). The average conductivity of these enrichments was 5.2 ± 0.7 μS/cm (mean ± standard deviation, n = 4), which was nearly same with the conductivity of initial seed sludge (5.6 ± 0.2 μS/cm) (Figure 3), suggesting

Figure 3. Conductivity of methanogenic enrichments taken from the four parallel UASB reactors during the 120-day continuous-flow experiments. “NaCl” means 0.1 M NaCl solution. “ISS” means the initial seed sludge (ISS). Error bars represent standard deviations of the four parallel experiments.

that DIET might be not established to proceed syntrophic metabolism. During this stage, the dominant working mode for syntrophic metabolism was not DIET but the traditional IHT (or IFT), which was not an effective interspecies electron link and easily limited by hydrogen partial pressure (or formate concentration) of anaerobic systems.1 Therefore, it was observed that the performances of syntrophic metabolism in the methanogenic digesters presented a declining trend with the increase of influent OLR from 13.25 to 39.75 kg COD/m3/ d (Figure 2). However, when the aciodgenic-phase pH was kept at 4.0−4.5 under the further increased OLR from 53 to 79.5 kg COD/m3/d, the conductivity of methanogenic enrichments significantly improved (Figure 3). Especially, under the highest influent OLR of 79.5 kg COD/m3/d, the conductivity of methanogenic enrichments was 57.2 ± 2.6 μS/cm, about 10fold higher than that before adjusting the acidogenic-phase pH (5.2 ± 0.7 μS/cm), suggesting that DIET had been established. It should be pointed that the VSS of these methanogenic enrichments under the different influent OLRs had no significant difference (P > 0.05) (Figure S1), suggesting that the reason leading to the significant improvement of conductivity of these methanogenic enrichments was not the different biomass. Effects of GAC on Syntrophic Metabolism in Methanogenic Enrichments. The potential mechanism involved in DIET for syntrophic metabolism in the methanogenic enrichments was further evaluated with GAC supplemented. Conductive carbon-based materials, such as GAC, can promote DIET but have almost no effect on IHT (or IFT) in defined cocultures,11 as well as in some mixed cultures.21,23 The primary mechanism is that DIET in the presence of conductive carbon-based materials does not require the electrically conductive pili and associated c-type cytochromes.10 Cells of both syntrophic partners directly attach to conductive carbon-based materials for interspecies electron exchange via their high conductance.10−12 Therefore, the conductive carbon-based materials can be used as an indication to demonstrate the potential of DIET proceeded in the enrichments.23 From Figure 4, there was almost no significant 9445


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Figure 4. Syntrophic metabolism of potential acidogenic products in the methanogenic enrichments taken from the four parallel UASB reactors at day 20 (A), 40 (B), 60 (C), 80 (D), 100 (E), and 120 (F), respectively, in the presence of GAC. Error bars represent standard deviations of the three parallel experiments.

Figure 5. Microbial morphology of methanogenic enrichments taken from one of the four parallel UASB reactors at day 20 (A), 40 (B), 60 (C), 80 (D), 100 (E), and 120 (F), respectively. Yellow arrow meant the potential biological electrical connections between syntrophis and methanogens. The magnification of all the pictures was 13 000-fold. The operating conditions of FESEM: accelerating voltage, 5000 V; emission current, 3.9−4.9 μA; working distance, 9.0−9.3 mm.

m3/d, the P value between the two groups was lower than 0.05, indicating that there was a significant difference in the performances of syntrophic metabolism in the methanogenic enrichments with or without GAC. These results were consistent with the increase of conductivity of methanogenic enrichments (Figure 3), suggesting that the microorganisms involved in DIET might be enriched and DIET had been established in the methanogenic communities to proceed syntrophic metabolism. Microbial Morphology of Methanogenic Enrichments. The microbial morphology of methanogenic enrichments is shown in Figure 5. It was observed a large number of long and bamboo-shaped microorganisms possibly involved in Methanosaeta species in the methanogenic enrichments under the influent OLR from 13.25 to 39.75 kgCOD/m3/d (Figure 5A− C). As the influent OLR further increased from 53 to 79.5

difference in the performances of syntrophic metabolism in the methanogenic enrichments with or without GAC before adjusting the acidogenic-phase pH (Figure 4A−C). The P value in terms of methane production or effluent COD between the two groups was higher than 0.95. These results suggested that DIET might be not the predominant working mode for syntrophic metabolism in these methanogenic enrichments, since the lack of positive response to the performances in the presence of GAC. A potential reason was inferred that the microorganisms capable of DIET, such as Geobacter species, were not sufficiently enriched. However, when the aciodgenic-phase pH was kept at 4.0−4.5 under the further increased influent OLR from 53 to 79.5 kgCOD/m3/d, the gap in terms of methane production and effluent COD between the two groups both gradually increased (Figure 4D− F). Especially, under the highest influent OLR of 79.5 kgCOD/ 9446


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ACS Sustainable Chemistry & Engineering kgCOD/m3/d, the dominant Methanosaeta species were replaced by the globular microorganisms possibly involved in Methanosarcina species in the methanogenic enrichments (Figure 5D−F). The potential succession of dominant aceticlastic methanogens with the increase of OLR might be associated with the concentration of acetate in the acidogenic products, since it was well-known that Methanosarcina species were capable of utilizing the higher concentration of acetate than Methanosaeta species to produce methane.15,16 However, the further clarification required the acidogenic products together with the community analysis of methanogenic enrichments. Both Methanosaeta and Methanosarcina species were capable of directly accepting electrons via DIET for the reduction of carbon dioxide to methane.13,14 While it was only observed that some curved-rod bacteria (0.5−2.0 um in length) closely attached to Methanosarcina species (globular) (see yellow arrows), which seemed like syntrophs proceeding DIET with Methanosarcina species in the methanogenic enrichments when the acidogenic-phase pH was kept at 4.0−4.5 (Figure 5D−F). Conversely, these potential biological electrical connections were not observed in the methanogenic enrichments before adjusting the acidogenic-phase pH. Acidogenic Products Analysis. Multiple lines of evidence suggested that DIET as an important working mode for syntrophic metabolism might be established in the methanogenic enrichments to maintain the acidic balance as well as methanogenic digesters stable, when the influent OLR further increased from 53 to 79.5 kg COD/m3/d (Figure 2). During this stage, the acidogenic-phase pH was kept at 4.0−4.5 known as the optimum pH range for the ethanol-type fermentation.35,36 Therefore, the potential reason resulting in the methanogenic enrichments gradually performing DIET was inferred that the acidogenic products might be favorable to support the establishment of DIET in these enrichments. Fermenting bacteria produce different distributions of reduced products in response to environmental conditions, of which the fermentative pH is significant.36 For example, lactate and propionate often are dominant products under the conditions close to neutral pH.36 Ethanol is abundant at around pH 4.0−4.5, and butyrate is predominant at slightly higher acidic pH than ethanol.36 The ethanol-type fermentation is characterized by the production of ethanol and acetate and accompanied by the significant release of hydrogen, since the balance between NAD and NADH+ is preserved.35,36 In consistent with these studies, ethanol, as the dominant aciodgenic product, was significantly produced only when the acidogenic pH was kept at 4.0−4.5 under the influent OLR from 53 to 79.5 kg COD/m3/d (Figure 6A). The ethanol production accounted for about 20% of total acidogenic products, accompanied by the significant production of hydrogen (Figure 6B). However, before adjusting the acidogenic-phase pH, almost no ethanol was detected in the acidogenic products under the influent OLR from 13.25 to 26.5 kgCOD/m3/d, and the ethanol produced only accounted for 1.7% of total acidogenic products under the influent OLR of 39.75 kg COD/m3/d (Figure 6A). During this stage, the dominant aciodgenic products were involved in propionate, lactate and acetate, similar to the propionate-/lactate-type fermentation occurring at a higher pH of 4.5 (Figure 2B).36 Since the production of propionate can prevent the formation of Fdred and formate, both of which lead to the hydrogen production, and also consume NADH2 (short for NADH + H+),36 its formation (at neutral pH) ought to lower hydrogen

Figure 6. Composition of acidogenic products (A) and mass balance of acidogenesis (B) during the 120-day continuous-flow experiments. Each data was based on the standard deviations of the four parallel experiments.

production. This was an important reason resulting in the relativity lower hydrogen production in the acidogenic digesters under the initial three influent OLRs (Figure 6B). Microbial Community Analysis. Microbial communities were analyzed to gain insight into the microbial factors linked to the performances. From Figure 7, the dominant genus in the bacterial communities of acidogenic phase throughout the whole experiments belonged to Mitsuokella and Bacteroides species with a relative abundance of more than 50%. Mitsuokella and Bacteroides species are the typical fermentative bacteria and their most likely role is to ferment carbohydrates and proteins contained in the synthetic dairy wastes with the production of acetate and succinate.48−50 After the pH adjustment of acidogenic phase, the abundance of ethanol/butyrate-type fermentative bacteria, Megasphaera species,48,49 increased by about 10−15%, compared with that before adjusting the acidogenic-phase pH. The fermentative products of Megasphaera species are mainly involved in ethanol and butyrate, accompanied by the production of a small amount of hydrogen.48,49 Ethanoligenens and Acetanaerobacterium species, 9447


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Figure 7. Bacterial community structure of suspended sludge taken from one of the four parallel acidogenic digesters and initial seed sludge. The genus level with relative abundance lower than 2.00% was classified into group “others”.

enrichments (Figure 8A), as consistent as observed in the microbial morphology of methanogenic enrichments (Figure 5). Methanosarcina species are capable of utilizing the higher concentration of acetate than Methanosaeta species for methane production in traditional digesters.15,16 However, in this study, the acetate production only accounted for a small part of acidogenic products after adjusting the acidogenic-phase pH (Figure 6A). The potential reason for this succession of methanogenic communities before and after adjusting the acidogenic-phase pH could not be simply ascribed to the change of acetate concentration in the acidogenic products. The bacterial communities of methanogenic enrichments provided a new insight into the improved performances of methanogenic phase. From Figure 8B, Geobacter species known to proceed DIET with methanogens were detected in the methanogenic enrichments with a relative abundance of 4%− 9% after the pH adjustment of acidogenic phase. However, almost no Geobacter species were detected in the methanogenic enrichments before the pH adjustment of acidogenic phase. The significant enrichment of Geobacter species should be related to the ethanol-abundant acidogenic products, since ethanol could sustain the growth of Geobacter species as observed in the aggregates of methanogenic digester treating brewery wastes.34 Multiple lines of evidence suggested that, under the conditions employed, DIET might be established in the methanogenic enrichments to maintain the acidic balance, as well as syntrophic metabolism stable. Considering that Methanosarcina species capable of directly accepting electrons via DIET were the dominant methanogenic genus in the enrichments, the potential DIET might be established between the enriched Geobacter and Methanosarcina species. Electron transfer via DIET as the primary mechanism can provide energy to support the growth of syntrophic partners,6 which should be an important reason resulting in the significant increase of Methanosarcina species that were the syntrophic partners of Geobacter species under the low-concentration acetate conditions.

the well-konwn ethanol-type fermentative genus, were only detected when the acidogenic-phase pH was kept at 4.0− 4.5.48,49 Megasphaera, Ethanoligenens, and Acetanaerobacterium species detected should be responsible for the significant production of ethanol in the acidogenic phase (Figure 6A), which, however, were only abundant in the communities when the acidogenic-phase pH was kept at 4.0−4.5. Under the initial three OLRs, the dominant methanogenic genus in methanogenic enrichments belonged to Methanosaeta species (Figure 8A). Even though Methanosaeta species could directly accept the electrons via DIET for the reduction of carbon dioxide to methane,13 multiple lines of evidence in terms of the conductivity of enrichments (Figure 3) as well as the response of syntrophic metabolism to GAC (Figure 4) did not support that DIET was established. Therefore, during this stage, the most likely role of Methanosaeta species was to consume acetate with methane production (aceticlastic methanogenesis). The abundance of Methanosaeta species gradually declined from 88.3% to 46.2% as the increase of influent OLR from 26.3 to 39.75 kg COD/m3/d. Their decline was accompanied by the significant increase of hydrogenutilizing methanogens, such as Methanobacterium and Methanospirillum species (Figure 8A), suggesting that hydrogenotrophic methanogenesis might replace aceticlastic methanogenesis to become the dominant methanogenic pathway in these enrichments. This might be ascribed to the low-efficiency acidification of acidogenic phase resulting in the significant decline of acetate production but increase of propionate and butyrate in the acidogenic products (Figure 6A). During this stage, IHT should be the predominant working mode for syntrophic metabolism because of the higher abundance of hydrogen-utilizing methanogens in these enrichments, but which could not maintain syntrophic metabolism function well as expected (Figure 2). When the acidogenic-phase pH was kept at 4.0−4.5 under the influent OLR from 53 to 79.5 kgCOD/m3/d, Methanosarcina species, another well-known aceticlastic methanogens, replaced Methanosaeta species to become the dominant methanogens in the methanogenic 9448


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Figure 8. Archaeal (A) and bacterial (B) community structure of suspended sludge taken from one of the four parallel UASB reactors and initial seed sludge. The genus level with relative abundance lower than 2.00% was classified into group “others”.

anaerobic fermentative microorganisms, such as Caloramator (12.78%, relative abundance), Anaerolinea (11.8%), Capnocytophaga (8.45%), and Fastidiosipila species (7.44%) (Figure 8B), which, however, was almost not detected in the bacterial communities of acidogenic digesters (Figure 7) as well as methanogenic digesters (Figure 8B) after a long-term and stable culture with dairy wastes. Their most likely role was to ferment the complex organic wastes contained in the municipal sludge into simples.51,58 Instead, Mitsuokella and Bacteroides species that were capable of fermenting carbohydrates and proteins contained in the dairy wastes with the production of acetate and succinate were the predominant fermentative bacteria in the acidogenic digesters (Figure 7), and Brevundimonas, Syntrophomonas, Desulfovibrio, and Geobacter species that were capable of converting the long-chain fatty acids, amino acids or alcohols to short-chain fatty acids were the predominant syntrophic microorganisms in the methano-

The microbial communities of initial seed sludge taken from a municipal sludge anaerobic digester were also analyzed (Figures 7 and 8). The dominant methanogenic genus belonged to Methanosaeta species that accounted for about 80% of the communities (Figure 8A), similar to that in the methanogenic enrichments under the initial influent OLR of 13.25 kgCOD/m3/d. It was well-known that conversion of acetate to methane by Methanosaeta species rather than Methanosarcina species was the primary methanogenic pathway during AD of municipal sludge. Methanosarcina species accounted for about 6% of the communities of initial seed sludge, which however only accounted for less than 1% in the methanogenic enrichments under the initial two OLRs (Figure 8A). The dominant hydrogen-utlizing methanogens, Methanomassiliicoccus species, only accounted for less than 5% of communities of initial seed sludge (Figure 8A). In the bacterial communities of seed sludge, the dominant genus belonged to 9449


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Figure 9. Potential application of ethanol-type fermentation in establishment of DIET for the enhancement of AD of complex organic wastes.

metabolism but promoted this process, since the establishment of DIET could be capable of compensating the shortage of IHT (or IFT) to maintain the acidic balance as well as syntrophic metabolism stable. The possible cost of NaHCO3 supplemented into the feeding for buffering the excess acidity to achieve the ethanol-type fermentation was also evaluated (Table S1). For example, under the influent OLR of 53 kg COD/m3/d, the dosage of NaHCO3 was about 1 g/L-wastewater. The unit price of NaHCO3 powder (commercially pure) was 2−2.5 Yuan/500 g. Therefore, the cost of NaHCO3 supplemented was about 4−5.4 Yuan/t-wastewater. During this stage, the produced ethanol that stimulated the methanogenic communities accounted for about 15−20% of total acidogenic products. It was assumed that these ethanol were not produced from the selffermentation of complex organics via ethanol-type fermentation but external supply. Theoretically, the dosage of ethanol was about 4.7−5.3 mL/L-wastewater (corresponding to 15−20% of influent COD). The unit price of ethanol (commercially pure) was 18−25 Yuan/2.5 L (Table S1). Therefore, the cost of ethanol supplemented was about 33.9−53 Yuan/t-wastewater, about 8−10 folds higher than that of NaHCO3 supplemented. These results suggested that, even though the ethanol supplemented into the methanogenic digester could enrich Geobacter species and stimulate the communities to perform DIET as reported in our previous study,20,23 this strategy was obviously uneconomical, which should be developed within a short-term operation or in a small-scale anaerobic digester with a lower treatment capacity. However, the current study utilized the ethanol-type fermentation to ferment the complex organics to ethanol just via the adjustment of acidogenic-phase pH with NaHCO3, which could significantly decline the costs and be more suitable for further applications. Conductive carbon-based materials, such as GAC, biochar and carbon cloth, can promote DIET in Geobacter-abundant methanogenic enrichments for accelerating the syntrophic conversion of alcohols and VFAs to methane.10,20−23 Although

genic digesters (Figure 8B). Almost no ethanol-type fermentative genus, such as Megasphaera, Ethanoligenens, and Acetanaerobacterium species, was detected in the communities of initial seed sludge (Figure 7) since the fermentative pH of AD of municipal sludge was usually close to neutral pH. Implications. The results demonstrated that, under the conditions employed, an UASB reactor served as mathanogenic phase continuously stimulated by the ethanol-abundant acidogenic products could achieve the better performances in response to high-OLR impacts. The acidification efficiency of acidogenic phase was still lower (Figure 6B), accompanied by a low-efficiency COD removal (Figure 2C), even if acidogenicphase pH was kept at 4.0−4.5 to achieve the ethanol-type fermentation. However, the continuous self-production of ethanol not only significantly enriched Geobacter species (Figure 8B), but its metabolism could also sufficiently support Geobacter species to produce the biological electrical connections with Methanosarcina species as well as to utilize the more complex organics in the methanogenic digesters. Similar to this study, Wu et al.52 reported that the performances of AD of food wastes could be improved when food wastes were initially fermented by yeast under the acidogenic stage via ethanol-type fermentation, which, however, ignored the potential stimulation of methanogenic communities by the ethanol-abundant fermentative intermediates. This process improvement just via adjusting the aciodgenic-phase pH at 4.0−4.5 to achieve the ethanol-type fermentation held a great promising to break the limitation of application of DIET. However, up to now, a pH of higher than 6.0 has been recommended for acidogenic operation to avoid a high propionate production as well as to reduce the dosage of alkali for adjusting the pHs of acidogenic products.35 Because of this reason, very few researches had selected a pH of less than 5.0 for acidogenic-phase operation. Remarkably, in this study, the ethanol-abundant acidogenic products without acidic neutralization by any alkali were directly fed to the methanogenic digester, which did not break the balance of syntrophic 9450



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conductive carbon-based materials were relatively inexpensive, the design of conductive carbon-based materials incorporated as part of traditional anaerobic digesters to provide a permanent conductive conduit for syntrophic metabolism still adds the considerable costs. Furthermore, the size and type of conductive carbon-based amendments also need to be considered to avoid the increase of technical complexity. Some recent studies24,41,53,54,56 attempted to enhance AD of complex organic wastes via DIET in the presence of conductive carbon-based materials, but the improved performances were not well as expected. An important reason was the lack of Geobacter species known as the unique genus confirmed capable of DIET. Therefore, most studies just focused on the application of DIET to the ecological remediation in the rice paddy soils28,29,55 as well as sediments,30 in which Geobacter species were among the most metabolically active microorganisms. The study presented here proposed a sustainable strategy involved in the process improvement of a two-phase AD system (Figure 9), which could permanently establish DIET in the Geobacter-rare methanogenic digesters to continuously enhance and stabilize AD of complex organic wastes. The improved process included three systems as follows: feeding system, ethanol-fermentation system and methanogenic system (Figure 9). The core component of ethanol-fermentation system was responsible for the production of ethanol-abundant acidogenic products via adjusting the acidogenic-phase pH at 4.0−4.5. The stable ethanol production could continuously stimulate the methanogenic communities to enrich Geobacter species to proceed DIET for syntrophic conversion of ethanol and VFAs as well as the potential more complex organics to methane. An UASB reactor was served as the methanogenic system which had the heavy duty for the conversion of organic wastes to methane, since the long solid retention made the energetic investment required for producing biological electrical connections favorable.23

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.7b02581. Economical evaluation of NaHCO3 supplemented into wastewater and change of VSS concentration of methanogenic enrichments during the 120-day continuous-flow experiments (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86 411 8470 6460. Fax: +86 411 8470 6263. E-mail: [email protected]. ORCID

Xie Quan: 0000-0003-3085-0789 Yaobin Zhang: 0000-0001-6052-0508 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the financial support from the National Natural Scientific Foundation of China (51578105). REFERENCES

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CONCLUSIONS

This study proposed a strategy that could permanently establish DIET in the Geobacter-rare methanogenic digesters to enhance and stabilize AD in response to the high-OLR impacts. Namely, in a two-phase AD system, the acidogenic-phase pH was always kept at 4.0−4.5 with the aim to ferment the complex organic wastes to the ethanol-abundant acidogenic products via ethanol-type fermentation. The ethanol produced could continuously support the growth of Geobacter species and stimulate the communities to perform DIET in the methanogenic phase. The results demonstrated that, under the conditions employed, the produced ethanol accounted for about 20% of the total acidogenic products. The potential DIET from Geobacter to Methanosarcina species might be established in the methanogenic enrichments, which thereby presented a higher conductivity, as well as a more positive response to GAC, compared with that before the pH adjustment of acidogenic phase. This strategy involved in the process improvement just via adjusting the aciodgenic-phase pH to achieve the ethanol-type fermentation held a great promising to break the limitation of DIET to AD. 9451


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Author list Dr. Zhiqiang Zhao First author E-mail address

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