New Application of Ethanol-Type Fermentation: Stimulating

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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 ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02581 • Publication Date (Web): 04 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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ACS Sustainable Chemistry & Engineering

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Author list

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Dr. Zhiqiang Zhao

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E-mail address: [email protected]

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Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering

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(Dalian University of Technology), Ministry of Education, School of Environmental

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Science and Technology, Dalian University of Technology, Dalian 116024, China.

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Address: No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province

First author

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Dr. Yang Li

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Prof. Xie Quan

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ACS Paragon Plus Environment


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Prof. Yaobin Zhang

Corresponding author

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New application of ethanol-type fermentation: Stimulating methanogenic communities with ethanol to perform direct interspecies electron transfer

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Authors:

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Zhiqiang Zhao, Yang Li, Xie Quan, Yaobin Zhang*

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Affiliations:

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Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian

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University of Technology), Ministry of Education, School of Environmental Science

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and Technology, Dalian University of Technology, Dalian 116024, China.

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* Correspondence: Tel: +86 411 8470 6460, Fax: +86 411 8470 6263;

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E-mail address: [email protected].

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Abstract

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Direct interspecies electron transfer (DIET) has been considered as an effective

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mechanism to proceed syntrophic methanogenic metabolism. However, up to now,

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this working mode has been still not widely established in the Geobacter-rare

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methanogenic digesters. In this study, a strategy that could enrich Geobacter species

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and stimulate the methanogenic communities to continuously perform DIET was

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proposed in a two-phase anaerobic digestion (AD) system with the aim to enhance

74

and stabilize the better AD. The results demonstrated that, under the conditions

75

employed, the ethanol-abundant acidogenic products could be produced via

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ethanol-type fermentation when acidogenic-phase pH was kept at 4.0-4.5.

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Enrichments in the methanogenic phase continuously stimulated with the

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ethanol-abundant acidogenic products presented a higher conductivity as well as more

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positive response to granular activated carbon (GAC) supplemented, compared with

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the enrichments without this stimulation, suggesting that DIET might be established.

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Microbial community analysis showed that Geobacter species were only detected in

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the methanogenic enrichments stimulated by the ethanol-abundant acidogenic

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products. Together with the significant increase of Methanosarcina species in these

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enrichments, the potential DIET between Geobacter and Methanosarcina species

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might be permanently established in the methanogenic digester to maintain the acidic

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balance as well as syntrophic metabolism stable.

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Keywords:

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Interspecies Electron Transfer (DIET); Syntrophic Metabolism

Anaerobic

Digestion

(AD);

Ethanol-type

4


Fermentation;

Direct

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Introduction

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An understanding of the effective mechanisms for syntrophic metabolism during

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anaerobic digestion (AD) is important because of its central importance in carbon and

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electron flow

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an alternative to interspecies hydrogen/formate transfer (IHT/IFT) for the long-range

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electron and energy transport

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connections involved in electrically conductive pili

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cytochromes 9, or a combination of biological and abiological electron transfer

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components, such as conductive carbon-based materials

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drawbacks of IHT (or IFT) that requires the essential hydrogen (or formate) as the

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electron carrier, as well as a low enough hydrogen partial pressure (or formate

1-3

. Direct interspecies electron transfer (DIET) has been considered as

4-6

. DIET can proceed via biological electrical 7, 8

or outer surface c-type

10-12

, compensating the

1, 3

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concentration) in thermodynamics

. For DIET, acetate-utilizing methanogens, such

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as Methanosaeta or Methanosarcina species, replace the hydrogen-utilizing

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methanogens to accept the electrons for the reduction of carbon dioxide to methane 13,

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14

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conversion of acetate to methane yields little energy, and the replacement may be not

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always a potential advantage, DIET (44.9 × 103 e−cp−1s−1) provides rapider electron

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transfer than IHT (5.24 × 103 e−cp−1s−1)

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methanogens are well-known as the dominant methanogens in most of traditional

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anaerobic digesters 15 and have the heavy duty for producing methane 16, 17. Therefore,

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enhancing DIET is expected to promote the better AD.

. Even though Methanosaeta or Methanosarcina species grow slowly since the

57

. Furthermore, acetate-utilizing

110 5



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However, the application of DIET to AD in response to the impacts from high organic

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loading rates (OLRs) is still limited. An important reason is that, up to now, only

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Geobacter species have been confirmed to proceed DIET in defined co-cultures

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18, 19

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not abundant in most of traditional digester communities 24-27, which thereby limits its

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role to maintain syntrophic metabolism stable, when AD is influenced by the

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high-OLR impacts. Some studies

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involved in some sulfur/Fe(III)-reducing microorganisms and methanogens might be

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established under the methanogenic conditions if conductive materials were provided

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as electrical conduits. However, DIET was only inferred from the faster conversion of

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organics to methane in the presence of conductive materials. Another potential

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limitation of DIET during AD is that the available substrates in current studies are

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only involved in some small-molecule alcohols 13, 14, 18, 19, 21, 22 and fatty acids 20-23, 28-33.

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It should be pointed out that the available substrates for Geobacter species with Fe(III)

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oxides as electron acceptors are varied, such as benzene, glucose, glycerol and yeast

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extract

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decompose the complex organics, such as carbohydrates or proteins, via DIET in

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defined co-cultures. A potential reason is that the energy yield from the metabolism of

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these complex organics with Methanosaeta or Methanosarcina species as electron

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acceptors for the reduction of carbon dioxide is insufficient for Geobacter species to

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produce biological electrical connections

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once acidification of complex organic wastes is limited, DIET cannot proceed well,

as well as in some mixed cultures

13, 14,

10, 20-23

28-31

. While Geobacter species are usually

suggested that DIET between syntrophs

9

, while no study has reported that Geobacter species could directly

19

. Therefore, it is usually observed that,

6


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even in the presence of conductive carbon-based materials 24, 56.

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Previous studies 20, 23 demonstrated that, initially feeding an up-flow anaerobic sludge

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blanket (UASB) reactor with ethanol, similar to those that were previously reported to

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support communities that metabolized ethanol with DIET in methanogenic digesters

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treating brewery wastes

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syntrophic metabolism of propionate and butyrate. This ethanol-stimulated strategy

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presented two potential advantages: 1) Ethanol as the substrate could enrich

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Geobacter species, compensating the shortage that Geobacter species were quite rare

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in most of traditional methanogenic communities. 2) The energy yield from the

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metabolism of ethanol could support DIET to overcome the thermodynamical

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limitations of syntrophic oxidation of propionate and butyrate as well as to compete

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these substrates with IHT

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Methanosarcina species known as the syntrophic partners of Geobacter species

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significantly improved

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supplemented to the traditional anaerobic digesters can enrich Geobacter species and

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stimulate the methanogenic communities to perform DIET for the enhancement and

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stabilization of AD in response to the high-OLR impacts. However, the continuous

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supply of ethanol will increase the considerable costs, and is obviously uneconomical

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in the application of DIET. In the absence of any amendments, gradually reducing the

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content of ethanol in the feedings with the extension of operating time could result in

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the succession of methanogenic communities and interspecies electron transfer from

34

, could stimulate the communities to perform DIET for

34

. As a result, the abundance of Methanosaeta or

23

. Based on these advantages, it is expected that ethanol

7



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DIET to IHT 20.

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A possibility is that the supplemented ethanol is not from the external supply but

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expected to be produced by the self fermentation of complex organic wastes. If so, it

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would not only solve the uneconomical problem, but also stimulate the methanogenic

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communities to permanently perform DIET as well as avoid the succession of

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methanogenic communities from DIET to IHT. Ren et al

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could be significantly produced via ethanol-type fermentation by adjusting the

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operating pHs at 4.0-4.5 in a high-OLR acidogenic digester. Under the conditions

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employed, ethanol was abundant in the acidogenic products, accompanied by the

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significant release of hydrogen since the balance between NAD and NADH+ was

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preserved

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fermentation only focused on hydrogen production 37-39. The purpose of this study was

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to propose a sustainable strategy to achieve the better application of DIET to the

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enhancement and stabilization of AD. Namely, in a two-phase AD system, ethanol

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was expected to be initially produced from the fermentation of complex organic

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wastes in the acidogenic phase via adjusting the operating pHs. Then

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ethanol-abundant acidogenic products were fed to methanogenic phase, which was

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expected to continuously stimulated the methanogenic communities to perform DIET.

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.

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Materials and Methods

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Experimental Setup. The continuous-flow experiments were conducted in four

36

35

had reported that ethanol

. Therefore, until now, most of studies involved in ethanol-type

8


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parallel two-phase AD systems. The schematic diagram of a continuous-flow

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two-phase AD system is shown in Fig. 1. The acidogenic phase was operated in a

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cylindrical glass completely-mixed digester (internal diameter of 70 mm and height of

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300 mm) with a working volume of 1000 mL. At the top of acidogenic digester, a

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stirring rod at a speed of 30-60 rpm connected with electric motor was placed into the

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suspended sludge. The methanogenic phase was operated in a cylindrical glass UASB

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reactor (internal diameter of 100 mm and height of 300 mm) with a working volume

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of 2000 mL. A three-phase separator was placed at the top of UASB reactor (Fig. 1).

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Both acidogenic and methanogenic digester were equipped with a gas sampling bag at

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the top of digester and a sludge sampling port at the bottom of digester and operated

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at a temperature of 37.0 oC.

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At the beginning of continuous-flow experiments, the fresh artificial wastewater

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stored in the feeding tank was firstly pumped into the acidogenic digester with a

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peristaltic pump (Lange, BT100-2J, China) (Fig. 1). After acidogenesis, the treated

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wastewater contained a small amount of suspended sludge was drawn out to the

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adjusting tank (Fig. 1). The settling sludge at the bottom of adjusting tank were

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returned to acidogenic digester (sludge recirculation) with a sludge peristaltic pump

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(Lange, BT300-2J, China), and supernatant liquid (fresh acidogenic products) at the

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upside of adjusting tank was pumped into the methanogenic digester (UASB) for

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methanogenesis with another peristaltic pump (Lange, BT100-2J, China) (Fig. 1). The

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hydraulic retention time (HRT) of acidogenic and methanogenic digester was 9.6 h 9



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and 19.2 h, respectively.

200 201

The four parallel two-phase AD systems were initially operated for 20 days of startup

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with a OLR of 13.25 KgCOD/m3/d (initial concentration of COD was 5300 mg/L).

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Then the influent OLR of the four two-phase AD systems gradually increased from

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13.25 to 79.5 KgCOD/m3/d over the next 100 days by increasing the influent

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concentration of COD to 2-fold, 3-fold, 4-fold, 5-fold and 6-fold of initial

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concentration of COD to investigate the effects of OLRs on the performances. Under

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each OLR, the four two-phase AD systems were operated for 20 days of experiments.

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When the influent OLR increased from 53 to 79.5 KgCOD/m3/d, in order to keep the

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acidogenic-phase pH at 4.0-4.5 rather than further lower than 4.0, with the aim to

210

achieve ethanol-type fermentation

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for buffering the excess acidity. The dosage of NaHCO3 powder is shown in Table S1.

35, 36

, NaHCO3 was supplemented into the feeding

212 213

Sludge and Wastewater. The initial seed sludge was obtained from an anaerobic

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digester of a municipal sludge treatment plant in Dalian (China). Total suspended

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solids (TSS) of initial seed sludge was 17400 ± 270 mg/L (mean ± standard deviation,

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n = 3) and the ratio between volatile suspended solids (VSS) and TSS was 0.74. It was

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anaerobically stored at 4 oC. At the beginning of continuous-flow experiments, each

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acidogenic digester received a 400 mL of seed sludge as inoculum and each

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methanogenic digester (UASB) received a 1000 mL of seed sludge as inoculum.

220 10


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An artificial dairy wastewater was used as feeding for the four parallel two-phase AD

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systems. The initial composition (per liter) of the artificial wastewater was as follows

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40

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China), 2.00 g; NH4Cl, 0.46 g; KH2PO4, 0.10 g; NaHCO3, 5.00 g; trace element

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solution, 10 ml; vitamin solution, 10 ml. The composition of the trace element

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solution and vitamin solution was described in our previous study

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oxygen demand (COD) and pH of this artificial dairy wastewater was about 5300

228

mg/L and 7.2, respectively.

: glucose, 2.40 g; yeast extract, 0.48 g; milk powder (Songhuajiang; taobao.com;

22

. The chemical

229 230

Batch Experiments with Granular Activated Carbon. To assess the effects of

231

granular activated carbon (GAC, 8-20 mesh, Sigma-Aldrich, USA) on syntrophic

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metabolism of potential acidogenic products in the methanogenic enrichments taken

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from one of the parallel four UASB reactors with a 50 mL centrifuge tube from the

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sludge sampling ports of UASB reactors at day 20, 40, 60, 80, 100 and 120,

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respectively, batch experiments were conducted in six 125 mL serum bottles in the

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dark at 37.0 oC

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three of the six serum bottles. As a control, another three of the six serum bottles were

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supplemented with the non-conductive glass (diameter of 10-15 mm, Dewei, China)

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with the same supplemented volume as GAC. All the serum bottles contained 5 mL

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sludge taken from the UASB reactors and 35 mL media. The composition of the

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media (per liter) used to simulate the potential acidogenic products was as follows:

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ethanol, 0.84 mL; sodium acetate, 1.32 g; sodium propionate, 0.59 g; sodium butyrate,

23

. Before batch experiments, 10 g of GAC were supplemented into

11



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0.74 g; glucose, 2.58 g; NH4Cl, 0.59 g; K2HPO4, 0.14 g; MgCl2·6H2O, 0.10 g;

244

CaCl2·2H2O, 0.05 g; NaHCO3, 4.00g, respectively. The whole carbon source in this

245

media amounted to an approximate COD of 6900 mg/L. The proportion of each

246

carbon source accounting for total COD in this media was as follows: 20% of ethanol;

247

15% of acetate; 10% of propionate; 15% of butyrate; 40% of carbohydrates, according

248

to the composition of acidogenic products under the influent OLR from 53 to 79.5

249

KgCOD/m3/d (Fig. 6A). Before the sludge supplemented to the serum bottles, the

250

media was flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 0.5 h. Upon

251

preparation, all the serum bottles were sealed with Teflon-faced butyl rubber stoppers

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and then flushed with nitrogen and carbon dioxide (80%/20%, v/v) for 0.5 h in the

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headspace 23.

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Chemical Analysis. The TSS, VSS and COD were analyzed in accordance with the

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Standard Methods for the Examination of Water and Wastewater. In order to analyze

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the change of VSS concentration of methanogenic enrichments, the suspended sludge

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samples taken from the four parallel UASB reactors at day 20, 40, 60, 80, 100 and

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120, respectively, with a 50 mL centrifuge tube from the sludge sampling ports of

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UASB reactors. Ethanol and volatile fatty acids (VFAs) (mainly including acetate,

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propionate, butyrate and valerate) were measured by a gas chromatograph with a

262

flame ionization detector (FID) (Tianmei, GC-7900P/FID, China)

263

determined by a high performance liquid chromatography (HPLC) (Tianmei, LC2000,

264

China) equipped with a UV-VIS detector and a column Zorbax SB-Aq (Agilent, 150 12


25

. Lactate was

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59

. The column temperature was maintained at 35 oC. The

265

mm × 4.6 mm, USA)

266

mobile phase was (99:1 v/v) 20 mM sodium phosphate buffer (pH = 2) / methanol at a

267

flow rate of 0.3 mL/min. Samples were detected by absorbance at 210 nm. The

268

equivalent relationship between COD and substrates are as follows: 1.07 g-COD/g

269

acetate, 1.51 g-COD/g propionate and 1.82 g-COD/g butyrate, 2.08 g-COD/g ethanol,

270

1.07 g-COD/g glucose, 1.07 g-COD/g lactate, 2.58 × 10-3 g-COD/mL methane and

271

0.645

272

acidogenic/methanogenic digester was collected by a gas sampling bag and the

273

volume of biogas was measured by a glass syringe of 100 mL

274

methane, hydrogen and carbon dioxide in the gas sampling bag and headspace of 125

275

mL serum bottles was analyzed by another gas chromatograph with a thermal

276

conductivity detector (TCD) (Tianmei, GC-7900/TCD, China)

277

using a pH analyzer (Denver Instrument; UB-10; Denver).

×

10-3

g-COD/mL

hydrogen.

The

produced

biogas

22

in

each

. The content of

21

. pH was recorded

278 279

Conductivity Measurement. To assess the conductivity of methanogenic

280

enrichments, the three-probe electrical conductance measurement was performed with

281

two gold electrodes separated by 0.5 mm non-conductive gap 8. The suspended sludge

282

samples taken from the four parallel UASB reactors at day 20, 40, 60, 80, 100 and

283

120, respectively, with a 50 mL centrifuge tube from the sludge sampling ports of

284

UASB reactors, were firstly collected by the centrifugation at 8000 rpm for 5 min and

285

then washed three times by 0.1 M NaCl

286

suspended sludge samples were placed on the gold electrodes and crushed with a

41

. After washing and centrifuging, the wet

13



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287

cover glass to form a confluent film that spread across the non-conductive gap 23. An

288

electrochemical workstation (Zhenhua, CHI1030C, China) was used to apply a

289

voltage ramp of -0.3-0.3 V across split electrodes in steps of 0.025 V. For each

290

measurement, after allowing the exponential decay of transient ionic current, the

291

steady-state electronic current for each voltage was measured every second over a

292

minimum period of 120 s. The time-averaged current for each applied voltage was

293

recorded to create the current-voltage curve 8.

294 295

Microbial Morphology. Field emission scanning electron microscopy (FESEM)

296

(Hitachi, S-4800, Japan) was used to observe the microbial morphology of

297

methanogenic enrichments taken from one of the four parallel UASB reactors with a

298

50 mL centrifuge tube from the sludge sampling ports of UASB reactors at day 20, 40,

299

60, 80, 100 and 120, respectively. For SEM observation, the suspended sludge

300

samples were immobilized in a 2.5% glutaraldehyde solution, dehydrated in graded

301

water-ethanol solutions, then lyophilized and sputter-coated with gold 42.

302 303

DNA Extraction, PCR Amplification and High-Throughput Sequencing. After

304

continuous-flow experiments the suspended sludge taken from one of the four parallel

305

acidogenic/methanogenic digester with a 50 mL centrifuge tube from the sludge

306

sampling ports at day 20, 40, 60, 80, 100, 120, respectively, were collected to analyze

307

the microbial communities via high-throughput sequencing. The suspended sludge

308

samples were firstly rinsed twice by phosphate-buffered saline (PBS; 0.13 M NaCl 14


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309

and 10 mM Na2HPO4 at pH 7.2) and then harvested by centrifugation (110 ×100 g for

310

15 min at 4 °C)

311

extract DNA from all the suspended sludge samples according to the manufacturer’s

312

protocols. The concentration and purity of the extracted DNA were determined by

313

analyzing its absorbance at 260 and 280 nm with a Nanodrop® ND-1000

314

spectrophotometer (Labtech International, UK).

23

. The FastDNA® SPIN kit for soil (Bioteke, China) was used to

315 316

Archaeal and bacterial 16S rRNA gene fragments were amplified via the polymerase

317

chain reaction (PCR) with the following primer sets: (Arch519F/Arch915R) and

318

(515F/806R). The following amplification cycling scheme was used: 94°C for 3

319

minutes, followed by 28 cycles of 94°C for 30 seconds, 53°C for 40 seconds and 72°C

320

for 1 minute, after which a final elongation step at 72°C for 5 minutes was performed

321

43

322

the success of amplification and the relative intensity of bands. Multiple samples were

323

pooled together (e.g., 100 samples) in equal proportions based on their molecular

324

weight and DNA concentrations. Pooled samples were purified using calibrated

325

Ampure XP beads.

. After amplification, PCR products were checked in 2% agarose gel to determine

326 327

Then the pooled and purified PCR products were used to prepare DNA library by

328

following Illumina TruSeq DNA library preparation protocol. High-throughput

329

sequencing was performed on an Illumina Hiseq 2000 platform (Illumia, San Diego,

330

USA) by Sangon Biotechnology Co., Ltd. (Shanghai, China). Sequences were placed 15



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331

into various operational taxonomic units with pipeline software. Final OTUs were

332

taxonomically classified using BLASTN against a curated database derived from

333

GreenGenes, RDPII and NCBI (www.ncbi.nlm.nih.gov, http://rdp.cme.msu.edu) 44.

334 335

The 16S rRNA gene sequence data obtained in this study were deposited in the NCBI

336

(National Center for Biotechnology Information) GenBank databases under accession

337

MF573069 to MF573118 (for Acidogenic-phase Bacteria), MF573119 to MF573167

338

(for

339

Methanogenic-phase Archaea) on Jul 29, 2017, and the scheduled release date for this

340

submission was Aug 30, 2017.

Methanogenic-phase

Bacteria)

and

MF573168

to

MF573210

(for

341 342

Results and Discussion

343

Performances of Two-Phase AD Systems. The methane production rate in the

344

acidogenic digesters firstly increased from 2.11 ± 0.09 (mean ± standard deviation, n

345

= 4) to 3.37 ± 0.15 L/d, but then drastically declined to 0.92 ± 0.13 L/d as the influent

346

OLR increased from 13.25 to 26.5 and then to 39.75 KgCOD/m3/d (Fig. 2A). When

347

the influent OLR further increased from 53 to 79.5 KgCOD/m3/d, almost no methane

348

production was detected in the acidogenic digesters. Remarkably, the low-pH

349

acidogenic products only had the slight effects on the methane production in the

350

methanogenic digesters. Especially, when the acidogenic-phase pH was kept at

351

4.0-4.5 under the influent OLR from 53 to 79.5 KgCOD/m3/d, the methane production

352

rates still continuously increased from 9.09 ± 0.14 to 17.13 ± 0.15 L/d (Fig. 2A) and 16


Page 17 of 54

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353

the average effluent pHs were always kept at 6.9-7.1 (Fig. 2B) in the methanogenic

354

digesters.

355 356

The COD removal efficiencies of acidogenic and methanogenic digesters declined

357

(Fig. 2C) as the increase of OLR from 13.25 to 39.75 KgCOD/m3/d. In consistent

358

with the COD removal efficiencies, the methane conversion efficiencies of acidogenic

359

and methanogenic digesters also declined (Fig. 2D). The high-OLR impacts causing a

360

low-efficiency AD had been widely reported

361

that the balance between the production of organic acids and methanogenesis during

362

syntrophic metabolism was easily destroyed by the high-OLR impacts. The gradual

363

accumulation of organic acids could lead to the sour of anaerobic digester and then

364

inhibit methanogens. In this study, when the acidogenic-phase pH was kept at 4.0-4.5

365

under the influent OLR from 53 to 79.5 KgCOD/m3/d, the COD removal efficiencies

366

in the acidogenic digesters were still lower than 10%, similar to that before adjusting

367

the acidogenic-phase pH, but which in the methanogenic digesters gradually increased

368

from 60.0% to 77.2% (Fig. 2C). Also, the methane conversion efficiencies in the

369

acidogenic digesters still continuously declined from 36.1% to 6.7%, which however

370

in the methanogenic digesters were always kept at 85% and about 10 percentage

371

points higher than that before adjusting the acidogenic-phase pH (Fig. 2D). The

372

improvement in the rates of methane production as well as stabilization in the

373

efficiencies of methane conversion under the higher influent OLRs suggested that an

374

effective mechanism of interspecies electron transfer might be established in the

22-24, 45, 46, 56

. An important reason was

17



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Page 18 of 54

375

methanogenic phase to maintain the acidic balance as well as syntrophic metabolism

376

stable. As a result, the two-phase AD systems could be capable of withstanding the

377

high-OLR impacts.

378 379

Conductivity of Methanogenic Enrichments. DIET has been considered as an

380

effective mechanism to replace the traditional IHT/IFT to maintain syntrophic

381

metabolism stable

382

UASB reactors treating brewery wastes

383

conductivity of these aggregates was consistent with metallic-like conductivity

384

similar to that found in G. sulfurreducens electrically conductive pili and biofilms 18.

385

Consequently, the potential mechanism for the conductive aggregates was inferred

386

that Geobacter species facilitated DIET via their electrically conductive pili

387

To investigate the potential mechanism related to DIET in the methanogenic digesters,

388

the conductivity of methanogenic enrichments was measured in this study. Before the

389

pH adjustment of acidogenic phase, there was no significant difference in the

390

conductivity among these methanogenic enrichments (P > 0.05) (Fig. 3). The average

391

conductivity of these enrichments was 5.2 ± 0.7 uS/cm (mean ± standard deviation, n

392

= 4), which was nearly same with the conductivity of initial seed sludge (5.6 ± 0.2

393

uS/cm) (Fig. 3), suggesting that DIET might be not established to proceed syntrophic

394

metabolism. During this stage, the dominant working mode for syntrophic metabolism

395

was not DIET but the traditional IHT (or IFT) which was not an effective interspecies

396

electron link and easily limited by hydrogen partial pressure (or formate concentration)

20-23, 33.

It was for the first time demonstrated in the aggregates of 34

. The temperature dependence of

18


34

,

18, 34, 47

.

Page 19 of 54

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397

of anaerobic systems 1. Therefore, it was observed that the performances of syntrophic

398

metabolism in the methanogenic digesters presented a declining trend with the

399

increase of influent OLR from 13.25 to 39.75 KgCOD/m3/d (Fig. 2). However, when

400

the aciodgenic-phase pH was kept at 4.0-4.5 under the further increased OLR from 53

401

to 79.5 KgCOD/m3/d, the conductivity of methanogenic enrichments significantly

402

improved (Fig. 3). Especially, under the highest influent OLR of 79.5 KgCOD/m3/d,

403

the conductivity of methanogenic enrichments was 57.2 ± 2.6 uS/cm, about 10 folds

404

higher than that before adjusting the acidogenic-phase pH (5.2 ± 0.7 uS/cm),

405

suggesting that DIET had been established. It should be pointed that the VSS of these

406

methanogenic enrichments under the different influent OLRs had no significant

407

difference (P > 0.05) (Fig. S1), suggesting that the reason leading to the significant

408

improvement of conductivity of these methanogenic enrichments was not the different

409

biomass.

410 411

Effects of GAC on Syntrophic Metabolism in Methanogenic Enrichments. The

412

potential mechanism involved in DIET for syntrophic metabolism in the

413

methanogenic enrichments was further evaluated with GAC supplemented.

414

Conductive carbon-based materials, such as GAC, can promote DIET but have almost

415

no effect on IHT (or IFT) in defined co-cultures 11 as well as in some mixed cultures 21,

416

23

417

materials does not require the electrically conductive pili and associated c-type

418

cytochromes

. The primary mechanism is that DIET in the presence of conductive carbon-based

10

. Cells of both syntrophic partners directly attach to conductive 19



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

419

carbon-based materials for interspecies electron exchange via their high conductance

420

10-12

421

demonstrate the potential of DIET proceeded in the enrichments 23. From Fig. 4, there

422

was almost no significant difference in the performances of syntrophic metabolism in

423

the methanogenic enrichments with or without GAC before adjusting the

424

acidogenic-phase pH (Fig. 4A, B and C). The P value in terms of methane production

425

or effluent COD between the two groups was higher than 0.95. These results

426

suggested that, DIET might be not the predominant working mode for syntrophic

427

metabolism in these methanogenic enrichments, since the lack of positive response to

428

the performances in the presence of GAC. A potential reason was inferred that the

429

microorganisms capable of DIET, such as Geobacter species, were not sufficiently

430

enriched. However, when the aciodgenic-phase pH was kept at 4.0-4.5 under the

431

further increased influent OLR from 53 to 79.5 KgCOD/m3/d, the gap in terms of

432

methane production and effluent COD between the two groups both gradually

433

increased (Fig. 4D, E and F). Especially, under the highest influent OLR of 79.5

434

KgCOD/m3/d, the P value between the two groups was lower than 0.05, indicating

435

that there was a significant difference in the performances of syntrophic metabolism

436

in the methanogenic enrichments with or without GAC. These results were consistent

437

with the increase of conductivity of methanogenic enrichments (Fig. 3), suggesting

438

that the microorganisms involved in DIET might be enriched and DIET had been

439

established in the methanogenic communities to proceed syntrophic metabolism.

. Therefore, the conductive carbon-based materials can be used as an indication to

440 20


Page 20 of 54

Page 21 of 54

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441

Microbial Morphology of Methanogenic Enrichments. The microbial morphology

442

of methanogenic enrichments is shown in Fig. 5. It was observed a large number of

443

long and bamboo-shaped microorganisms possibly involved in Methanosaeta species

444

in the methanogenic enrichments under the influent OLR from 13.25 to 39.75

445

KgCOD/m3/d (Fig. 5A, B and C). As the influent OLR further increased from 53 to

446

79.5 KgCOD/m3/d, the dominant Methanosaeta species were replaced by the globular

447

microorganisms possibly involved in Methanosarcina species in the methanogenic

448

enrichments (Fig. 5D, E and F). The potential succession of dominant aceticlastic

449

methanogens with the increase of OLR might be associated with the concentration of

450

acetate in the acidogenic products, since it was well known that Methanosarcina

451

species were capable of utilizing the higher concentration of acetate than

452

Methanosaeta species to produce methane

453

required the acidogenic products together with the community analysis of

454

methanogenic enrichments. Both Methanosaeta and Methanosarcina species were

455

capable of directly accepting electrons via DIET for the reduction of carbon dioxide to

456

methane 13, 14. While, it was only observed that, some curved-rod bacteria (0.5-2.0 um

457

in length) closely attached to Methanosarcina species (globular) (see yellow arrows),

458

which seemed like syntrophs proceeding DIET with Methanosarcina species in the

459

methanogenic enrichments when the acidogenic-phase pH was kept at 4.0-4.5 (Fig.

460

5D, E and F). Conversely, these potential biological electrical connections were not

461

observed in the methanogenic enrichments before adjusting the acidogenic-phase pH.

15, 16

. However, the further clarification

462 21



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Page 22 of 54

463

Acidogenic Products Analysis. Multiple lines of evidence suggested that DIET as an

464

important working mode for syntrophic metabolism might be established in the

465

methanogenic enrichments to maintain the acidic balance as well as methanogenic

466

digesters stable, when the influent OLR further increased from 53 to 79.5

467

KgCOD/m3/d (Fig. 2). During this stage, the acidogenic-phase pH was kept at 4.0-4.5

468

known as the optimum pH range for the ethanol-type fermentation

469

the potential reason resulting in the methanogenic enrichments gradually performing

470

DIET was inferred that the acidogenic products might be favorable to support the

471

establishment of DIET in these enrichments.

35, 36

. Therefore,

472 473

Fermenting bacteria produce different distributions of reduced products in response to

474

environmental conditions, of which the fermentative pH is significant 36. For example,

475

lactate and propionate often are dominant products under the conditions close to

476

neutral pH 36. Ethanol is abundant at around pH 4.0-4.5, and butyrate is predominant

477

at slightly higher acidic pH than ethanol

478

characterized by the production of ethanol and acetate, and accompanied by the

479

significant release of hydrogen, since the balance between NAD and NADH+ is

480

preserved

481

products, was significantly produced only when the acidogenic pH was kept at 4.0-4.5

482

under the influent OLR from 53 to 79.5 KgCOD/m3/d (Fig. 6A). The ethanol

483

production accounted for about 20% of total acidogenic products, accompanied by the

484

significant production of hydrogen (Fig. 6B). However, before adjusting the

35, 36.

36

. The ethanol-type fermentation is

In consistent with these studies, ethanol, as the dominant aciodgenic

22


Page 23 of 54

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485

acidogenic-phase pH, almost no ethanol was detected in the acidogenic products

486

under the influent OLR from 13.25 to 26.5 KgCOD/m3/d, and the ethanol produced

487

only accounted for 1.7% of total acidogenic products under the influent OLR of 39.75

488

KgCOD/m3/d (Fig. 6A). During this stage, the dominant aciodgenic products were

489

involved in propionate, lactate and acetate, similar to the propionate- / lactate-type

490

fermentation occurring at a higher pH of 4.5 (Fig. 2B)

491

propionate can prevent the formation of Fdred and formate, both of which lead to the

492

hydrogen production, and also consume NADH2 (short for NADH + H+)

493

formation (at neutral pH) ought to lower hydrogen production. This was an important

494

reason resulting in the relativity lower hydrogen production in the acidogenic

495

digesters under the initial three influent OLRs (Fig. 6B).

36

. Since the production of

36

, its

496 497

Microbial Community Analysis. Microbial communities were analyzed to gain

498

insight into the microbial factors linked to the performances. From Fig. 7, the

499

dominant genus in the bacterial communities of acidogenic phase throughout the

500

whole experiments belonged to Mitsuokella and Bacteroides species with a relative

501

abundance of more than 50%. Mitsuokella and Bacteroides species are the typical

502

fermentative bacteria and their most likely role is to ferment carbohydrates and

503

proteins contained in the synthetic dairy wastes with the production of acetate and

504

succinate

505

ethanol/butyrate-type fermentative bacteria, Megasphaera species

506

about 10-15%, compared with that before adjusting the acidogenic-phase pH. The

48-50

. After the pH adjustment of acidogenic phase, the abundance of

23


48, 49

, increased by


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 24 of 54

507

fermentative products of Megasphaera species are mainly involved in ethanol and

508

butyrate, accompanied by the production of a small amount of hydrogen

509

Ethanoligenens and Acetanaerobacterium species, the well-konwn ethanol-type

510

fermentative genus, were only detected when the acidogenic-phase pH was kept at

511

4.0-4.5

512

detected should be responsible for the significant production of ethanol in the

513

acidogenic phase (Fig. 6A), which however were only abundant in the communities

514

when the acidogenic-phase pH was kept at 4.0-4.5.

48, 49

.

48, 49

. Megasphaera, Ethanoligenens and Acetanaerobacterium species

515 516

Under the initial three OLRs, the dominant methanogenic genus in methanogenic

517

enrichments belonged to Methanosaeta species (Fig. 8A). Even though Methanosaeta

518

species could directly accept the electrons via DIET for the reduction of carbon

519

dioxide to methane

520

enrichments (Fig. 3) as well as the response of syntrophic metabolism to GAC (Fig. 4)

521

did not support that DIET was established. Therefore, during this stage, the most

522

likely role of Methanosaeta species was to consume acetate with methane production

523

(aceticlastic methanogenesis). The abundance of Methanosaeta species gradually

524

declined from 88.3% to 46.2% as the increase of influent OLR from 26.3 to 39.75

525

KgCOD/m3/d. Their decline was accompanied by the significant increase of

526

hydrogen-utilizing methanogens, such as Methanobacterium and Methanospirillum

527

species (Fig. 8A), suggesting that hydrogenotrophic methanogenesis might replace

528

aceticlastic methanogenesis to become the dominant methanogenic pathway in these

13

, multiple lines of evidence in terms of the conductivity of

24


Page 25 of 54

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529

enrichments. This might be ascribed to the low-efficiency acidification of acidogenic

530

phase resulting in the significant decline of acetate production but increase of

531

propionate and butyrate in the acidogenic products (Fig. 6A). During this stage, IHT

532

should be the predominant working mode for syntrophic metabolism due to the higher

533

abundance of hydrogen-utilizing methanogens in these enrichments, but which could

534

not maintain syntrophic metabolism function well as expected (Fig. 2). When the

535

acidogenic-phase pH was kept at 4.0-4.5 under the influent OLR from 53 to 79.5

536

KgCOD/m3/d, Methanosarcina species, another well-known aceticlastic methanogens,

537

replaced Methanosaeta species to become the dominant methanogens in the

538

methanogenic enrichments (Fig. 8A), as consistent as observed in the microbial

539

morphology of methanogenic enrichments (Fig. 5). Methanosarcina species are

540

capable of utilizing the higher concentration of acetate than Methanosaeta species for

541

methane production in traditional digesters

542

production only accounted for a small part of acidogenic products after adjusting the

543

acidogenic-phase pH (Fig. 6A). The potential reason for this succession of

544

methanogenic communities before and after adjusting the acidogenic-phase pH could

545

not be simply ascribed to the change of acetate concentration in the acidogenic

546

products.

15, 16

. However, in this study, the acetate

547 548

The bacterial communities of methanogenic enrichments provided a new insight into

549

the improved performances of methanogenic phase. From Fig. 8B, Geobacter species

550

known to proceed DIET with methanogens were detected in the methanogenic 25



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Page 26 of 54

551

enrichments with a relative abundance of 4%-9% after the pH adjustment of

552

acidogenic phase. However, almost no Geobacter species were detected in the

553

methanogenic enrichments before the pH adjustment of acidogenic phase. The

554

significant enrichment of Geobacter species should be related to the ethanol-abundant

555

acidogenic products, since ethanol could sustain the growth of Geobacter species as

556

observed in the aggregates of methanogenic digester treating brewery wastes

557

Multiple lines of evidence suggested that, under the conditions employed, DIET might

558

be established in the methanogenic enrichments to maintain the acidic balance as well

559

as syntrophic metabolism stable. Considering that Methanosarcina species capable of

560

directly accepting electrons via DIET were the dominant methanogenic genus in the

561

enrichments, the potential DIET might be established between the enriched Geobacter

562

and Methanosarcina species. Electron transfer via DIET as the primary mechanism

563

can provide energy to support the growth of syntrophic partners 6, which should be an

564

important reason resulting in the significant increase of Methanosarcina species that

565

were the syntrophic partners of Geobacter species under the low-concentration acetate

566

conditions.

34

.

567 568

The microbial communities of initial seed sludge taken from a municipal sludge

569

anaerobic digester were also analyzed (Fig. 7 and Fig. 8). The dominant

570

methanogenic genus belonged to Methanosaeta species that accounted for about 80%

571

of the communities (Fig. 8A), similar to that in the methanogenic enrichments under

572

the initial influent OLR of 13.25 KgCOD/m3/d. It was well-known that conversion of 26


Page 27 of 54

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573

acetate to methane by Methanosaeta species rather than Methanosarcina species was

574

the primary methanogenic pathway during AD of municipal sludge. Methanosarcina

575

species accounted for about 6% of the communities of initial seed sludge, which

576

however only accounted for less than 1% in the methanogenic enrichments under the

577

initial two OLRs (Fig. 8A). The dominant hydrogen-utlizing methanogens,

578

Methanomassiliicoccus species, only accounted for less than 5% of communities of

579

initial seed sludge (Fig. 8A). In the bacterial communities of seed sludge, the

580

dominant genus belonged to anaerobic fermentative microorganisms, such as

581

Caloramator (12.78%, relative abundance), Anaerolinea (11.8%), Capnocytophaga

582

(8.45%) and Fastidiosipila species (7.44%) (Fig. 8B), which however was almost not

583

detected in the bacterial communities of acidogenic digesters (Fig. 7) as well as

584

methanogenic digesters (Fig. 8B) after a long-term and stable culture with dairy

585

wastes. Their most likely role was to ferment the complex organic wastes contained in

586

the municipal sludge into simples

587

that were capable of fermenting carbohydrates and proteins contained in the dairy

588

wastes with the production of acetate and succinate were the predominant

589

fermentative bacteria in the acidogenic digesters (Fig. 7), and Brevundimonas,

590

Syntrophomonas, Desulfovibrio and Geobacter species that were capable of

591

converting the long-chain fatty acids, amino acids or alcohols to short-chain fatty

592

acids were the predominant syntrophic microorganisms in the methanogenic digesters

593

(Fig. 8B). Almost no ethanol-type fermentative genus, such as Megasphaera,

594

Ethanoligenens and Acetanaerobacterium species, was detected in the communities of

51, 58

. Instead, Mitsuokella and Bacteroides species

27



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Page 28 of 54

595

initial seed sludge (Fig. 7) since the fermentative pH of AD of municipal sludge was

596

usually close to neutral pH.

597 598

Implications. The results demonstrated that, under the conditions employed, an

599

UASB reactor served as mathanogenic phase continuously stimulated by the

600

ethanol-abundant acidogenic products could achieve the better performances in

601

response to high-OLR impacts. The acidification efficiency of acidogenic phase was

602

still lower (Fig. 6B), accompanied by a low-efficiency COD removal (Fig. 2C), even

603

if acidogenic-phase pH was kept at 4.0-4.5 to achieve the ethanol-type fermentation.

604

However, the continuous self production of ethanol not only significantly enriched

605

Geobacter species (Fig. 8B), but its metabolism could also sufficiently support

606

Geobacter

607

Methanosarcina species as well as to utilize the more complex organics in the

608

methanogenic digesters. Similar to this study, Wu et al

609

performances of AD of food wastes could be improved when food wastes were

610

initially fermented by yeast under the acidogenic stage via ethanol-type fermentation,

611

which however ignored the potential stimulation of methanogenic communities by the

612

ethanol-abundant fermentative intermediates. This process improvement just via

613

adjusting the aciodgenic-phase pH at 4.0-4.5 to achieve the ethanol-type fermentation

614

held a great promising to break the limitation of application of DIET. However, up to

615

now, a pH of higher than 6.0 has been recommended for acidogenic operation to avoid

616

a high propionate production as well as to reduce the dosage of alkali for adjusting the

species

to

produce

the

biological

electrical

28


52

connections

with

reported that the

Page 29 of 54

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617

pHs of acidogenic products 35. Due to this reason, very few researches had selected a

618

pH of less than 5.0 for acidogenic-phase operation. Remarkably, in this study, the

619

ethanol-abundant acidogenic products without acidic neutralization by any alkali were

620

directly fed to the methanogenic digester, which did not break the balance of

621

syntrophic metabolism but promoted this process, since the establishment of DIET

622

could be capable of compensating the shortage of IHT (or IFT) to maintain the acidic

623

balance as well as syntrophic metabolism stable.

624 625

The possible cost of NaHCO3 supplemented into the feeding for buffering the excess

626

acidity to achieve the ethanol-type fermentation was also evaluated (Table S1). For

627

example, under the influent OLR of 53 KgCOD/m3/d, the dosage of NaHCO3 was

628

about 1 g/L-wastewater. The unit price of NaHCO3 powder (commercially pure) was

629

2-2.5 Yuan/500 g. Therefore, the cost of NaHCO3 supplemented was about 4-5.4

630

Yuan/t-wastewater. During this stage, the produced ethanol that stimulated the

631

methanogenic communities accounted for about 15-20% of total acidogenic products.

632

It was assumed that these ethanol were not produced from the self fermentation of

633

complex organics via ethanol-type fermentation but external supply. Theoretically, the

634

dosage of ethanol was about 4.7-5.3 mL/L-wastewater (corresponding to 15-20% of

635

influent COD). The unit price of ethanol (commercially pure) was 18-25 Yuan/2.5 L

636

(Table S1). Therefore, the cost of ethanol supplemented was about 33.9-53

637

Yuan/t-wastewater, about 8-10 folds higher than that of NaHCO3 supplemented. These

638

results suggested that, even though the ethanol supplemented into the methanogenic 29



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

639

digester could enrich Geobacter species and stimulate the communities to perform

640

DIET as reported in our previous study

641

uneconomical, which should be developed within a short-term operation or in a

642

small-scale anaerobic digester with a lower treatment capacity. However, the current

643

study utilized the ethanol-type fermentation to ferment the complex organics to

644

ethanol just via the adjustment of acidogenic-phase pH with NaHCO3, which could

645

significantly decline the costs and be more suitable for further applications.

20, 23

, this strategy was obviously

646 647

Conductive carbon-based materials, such as GAC, biochar and carbon cloth, can

648

promote DIET in Geobacter-abundant methanogenic enrichments for accelerating the

649

syntrophic conversion of alcohols and VFAs to methane 10, 20-23. Although conductive

650

carbon-based materials were relatively inexpensive, the design of conductive

651

carbon-based materials incorporated as part of traditional anaerobic digesters to

652

provide a permanent conductive conduit for syntrophic metabolism still adds the

653

considerable costs. Furthermore, the size and type of conductive carbon-based

654

amendments also need to be considered to avoid the increase of technical complexity.

655

Some recent studies 24, 41, 53, 54, 56 attempted to enhance AD of complex organic wastes

656

via DIET in the presence of conductive carbon-based materials, but the improved

657

performances were not well as expected. An important reason was the lack of

658

Geobacter species known as the unique genus confirmed capable of DIET. Therefore,

659

most studies just focused on the application of DIET to the ecological remediation in

660

the rice paddy soils

28, 29, 55

as well as sediments 30, in which Geobacter species were 30


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661

among the most metabolically active microorganisms.

662 663

The study presented here proposed a sustainable strategy involved in the process

664

improvement of a two-phase AD system (Fig. 9), which could permanently establish

665

DIET in the Geobacter-rare methanogenic digesters to continuously enhance and

666

stabilize AD of complex organic wastes. The improved process included three systems

667

as follows: feeding system, ethanol-fermentation system and methanogenic system

668

(Fig. 9). The core component of ethanol-fermentation system was responsible for the

669

production

670

acidogenic-phase pH at 4.0-4.5. The stable ethanol production could continuously

671

stimulate the methanogenic communities to enrich Geobacter species to proceed

672

DIET for syntrophic conversion of ethanol and VFAs as well as the potential more

673

complex organics to methane. An UASB reactor was served as the methanogenic

674

system which had the heavy duty for the conversion of organic wastes to methane,

675

since the long solid retention made the energetic investment required for producing

676

biological electrical connections favorable 23.

of

ethanol-abundant

acidogenic

products

via

adjusting

the

677 678

Conclusions

679

This study proposed a strategy that could permanently establish DIET in the

680

Geobacter-rare methanogenic digesters to enhance and stabilize AD in response to the

681

high-OLR impacts. Namely, in a two-phase AD system, the acidogenic-phase pH was

682

always kept at 4.0-4.5 with the aim to ferment the complex organic wastes to the 31



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

683

ethanol-abundant acidogenic products via ethanol-type fermentation. The ethanol

684

produced could continuously support the growth of Geobacter species and stimulate

685

the communities to perform DIET in the methanogenic phase. The results

686

demonstrated that, under the conditions employed, the produced ethanol accounted for

687

about 20% of the total acidogenic products. The potential DIET from Geobacter to

688

Methanosarcina species might be established in the methanogenic enrichments, which

689

thereby presented a higher conductivity as well as a more positive response to GAC,

690

compared with that before the pH adjustment of acidogenic phase. This strategy

691

involved in the process improvement just via adjusting the aciodgenic-phase pH to

692

achieve the ethanol-type fermentation held a great promising to break the limitation of

693

DIET to AD.

694 695

Associated Content

696

Supporting Information. The economical evaluation of NaHCO3 supplemented (Tab

697

S1), and change of VSS concentration of methanogenic enrichments during the

698

120-day continuous-flow experiments (Fig. S1).

699 700

Conflict of interest statement

701

The authors declare that the research was conducted in the absence of any commercial

702

or financial relationships that could be construed as a potential conflict of interest.

703 704

Acknowledgments 32


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705

The authors acknowledge the financial support from the National Natural Scientific

706

Foundation of China (51578105).

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925 926 927 928 929 930 931 932 933 934 935 936 937 938 939 940 941 942 943 944

Figure:

43



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

945 946

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

947 948 949 950 951 952 953 954 955 956

44


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Page 45 of 54

Acidogenic-phase digester Two-phase AD system

(A) 20

Methanogenic-phase digester Influent OLR

100

80

3

16 14 12

60

10 8 40

pH adjustment of acidogenic-phase digester at 4.0-4.5 with NaHCO3

6 4

Influent OLRs (KgCOD/m /d)

Methane production rate (L/d)

18

20

2 0 0 0

20

40

60

80

100

120

Time (days)

957 Acidogenic phase Methanogenic phase

(B) 9

Influent OLR 100

3

80


8

7

Effluent pHs

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


60 6


5

40

20 4

3

0 0

20

40

60

80

Time (days)

958

45


100

120


(C) 100

Acidogenic phase Two-phase AD system

Methanogenic phase

COD removal efficiency (%)

90 80 70 60


50 40 30 20 10 0

13.25

26.5

66.25 53 39.75 3 Influent OLRs (KgCOD/m /d)

79.5

959

(D)100 Methane conversion efficiency (%)

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 46 of 54

Acidogenic phase Two-phase AD system

Methanogenic phase

80


60

40

20

0

13.25

26.5 39.75 53 66.25 3 Influent OLRs (KgCOD/m /d)

79.5

960 961

Figure.2. Methane production rate (A), effluent pH (B), COD removal efficiency (C)

962

and methane conversion efficiency (D) during the 120-day continuous-flow

963

experiments. Error bars represent standard deviations of the four parallel experiments. 46


Page 47 of 54

80

pH adjustment of acidogenic phase at 4.0-4.5 with NaHCO3

70

60

Conductivity (uS/cm)

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


50

40

30 20 10 0

NaCl

ISS

13.25

26.5

39.75

53

66.25

79.5

3


964 965

Figure.3. Conductivity of methanogenic enrichments taken from the four parallel

966

UASB reactors during the 120-day continuous-flow experiments. ‘NaCl’ means 0.1 M

967

NaCl solution. ‘ISS’ means the initial seed sludge (ISS). Error bars represent standard

968

deviations of the four parallel experiments.

969 970 971 972 973 974 975 976 977 47



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

978 979

Figure.4. Syntrophic metabolism of potential acidogenic products in the

980

methanogenic enrichments taken from the four parallel UASB reactors at day 20 (A),

981

40 (B), 60 (C), 80 (D), 100 (E) and 120 (F), respectively, in the presence of GAC.

982

Error bars represent standard deviations of the three parallel experiments.

983 984 985 986 987 988 989 990 991 48


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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


992 993

Figure.5. Microbial morphology of methanogenic enrichments taken from one of the

994

four parallel UASB reactors at day 20 (A), 40 (B), 60 (C), 80 (D), 100 (E) and 120 (F),

995

respectively. Yellow arrow meant the potential biological electrical connections

996

between syntrophis and methanogens. The magnification of all the pictures was 13000

997

folds. The operating conditions of FESEM: Accelerating voltage was 5000 V;

998

Emission current was 3.9-4.9 uA; Working distance was 9.0-9.3 mm.

999 1000 1001 1002 1003 1004 1005 1006 49



Composition of acidogenic products (%)

(A) 100

Others Valerate Propionate Ethanol

Lactate Butyrate Acetate


90 80 70 60 50 40 30 20 10 0

12.35

1007

39.75 26.5 66.25 53 3 Influent OLRs (KgCOD/m /d)

Others VFAs Hydrogen

Lactate Ethanol Methane

(B) 100

79.5


90

Mass balance of acidogenesis (%)

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 50 of 54

80 70 60 50 40 30 20 10 0

12.35

1008

26.5 39.75 53 66.25 3 Influent OLRs (KgCOD/m /d)

79.5

1009

Figure.6. Composition of acidogenic products (A) and mass balance of acidogenesis

1010

(B) during the 120-day continuous-flow experiments. Each data was based on the

1011

standard deviations of the four parallel experiments. 50


Page 51 of 54

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


79.5 pH Adjustment of Acidogenic Phase at 4.0-4.5 66.25 53 39.75 26.5 3

13.25 (Influent OLR [KgCOD/m /d]) Initial Seed Sludge 0

10

20

30

40

50

60

70

80

90

100

Relative abundance (%)

1012

Others Unclassified Caloramator Cytophaga Phaeodactylibacter Candidatus Cloacamonas Capnocytophaga Anaerolinea Fastidiosipila Propionispora Oscillibacter Phascolarctobacterium Holdemania Acetanaerobacterium Desulfovibrio Ethanoligenens Macellibacteroides Olsenella Clostridium Intestinimonas Stenotrophomonas Acetobacter Megasphaera Bacteroides Mitsuokella

1013

Figure.7. Bacterial community structure of suspended sludge taken from one of the

1014

four parallel acidogenic digesters and initial seed sludge. The genus level with relative

1015

abundance lower than 2.00% was classified into group ‘others’.

1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 51



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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(A) 79.5 pH Adjustment of Acidogenic Phase at 4.0-4.5 Others Methanoregula

66.25

Methanolinea Methanosphaerula

53

Methanomassiliicoccus Methanofollis

39.75

Methanospirillum Methanobacterium

26.5

Methanosarcina Methanosaeta

3


10

20

30

40

50

60

70

80

90

100


1026

(B)

Brevundimonas Levilinea Acinetobacter Aminicenantes_genera_incertae_sedis Cloacibacillus

79.5 pH Adjustment of Acidogenic Phase at 4.0-4.5 66.25 53 39.75 26.5 3


1027

10

20

30

40

50

60

70

80

90

100


Others Unclassified Caloramator Clostridium Cytophaga Anaerolinea Phaeodactylibacter Candidatus Cloacamonas Capnocytophaga Fastidiosipila Geobacter Dialister Megasphaera Bellilinea Mucinivorans Aminivibrio Meniscus Lactivibrio Aminobacterium Desulfovibrio Petrimonas Ornatilinea Thermogutta Prevotella Syntrophomonas

1028

Figure.8. Archaeal (A) and bacterial (B) community structure of suspended sludge

1029

taken from one of the four parallel UASB reactors and initial seed sludge. The genus

1030

level with relative abundance lower than 2.00% was classified into group ‘others’. 52


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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


1031 1032

Figure.9. Potential application of ethanol-type fermentation in establishment of DIET

1033

for the enhancement of AD of complex organic wastes.

1034 1035 1036 1037 1038 1039 1040 1041 1042 1043 1044 53



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

1045 1046

TOC graphic:

1047 1048

For Table of Contents Use Only

1049 1050

A Brief of TOC graphic: Stimulating methanogenic communities to permanently

1051

perform DIET by adjusting acidogenic-phase pH at 4.0-4.5 to achieve self-produced

1052

ethanol in a two-phase AD system.

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New Application of Ethanol-Type Fermentation: Stimulating

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