Magnetite Triggering Enhanced Direct Interspecies Electron Transfer

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Energy and the Environment

Magnetite Triggering Enhanced DIET: A Scavenger for the Blockage of Electron Transfer in Anaerobic Digestion of High-solids Sewage Sludge Tao Wang, Dong Zhang, Lingling Dai, Bin Dong, and Xiaohu Dai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00891 • Publication Date (Web): 21 May 2018 Downloaded from http://pubs.acs.org on May 21, 2018

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Environmental Science & Technology

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Magnetite Triggering Enhanced DIET: A Scavenger for the Blockage of Electron Transfer in

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Anaerobic Digestion of High-solids Sewage Sludge

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Tao Wang†, Dong Zhang†, Lingling Dai†, Bin Dong†, Xiaohu Dai†*

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† State key laboratory of pollution control and Resources reuse, School of Environmental Science and Engineering,

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Tongji University, 1239 Siping Road, Shanghai 200092, China

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*

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Xiaohu Dai, Phone: 86-21-65983868, Fax: 86-21-65986313, E-mail: [email protected]

Corresponding author

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ACS Paragon Plus Environment


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Abstract: At present, high-solids anaerobic digestion of sewage sludge has drawn great attention due to the

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superiority of its small land area footprint and low energy consumption. However, a high organic loading rate may

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cause acids accumulation and ammonia inhibition, thus leading to an inhibited pseudo-steady state in which electron

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transfer through interspecies hydrogen transfer (IHT) between acetogens and methanogens is blocked. In this study,

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adding 50 mg/g TS (total solid) magnetite clearly reduced the accumulation of short-chain fatty acids and accelerated

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methane production by 26.6%. As demonstrated, the individual processes of anaerobic digestion could not be

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improved by magnetite when methanogenesis was interrupted. Analyzing stable carbon isotopes and investigating

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the methanogenesis pathways using acetate and H2/CO2 as substrates together proved that direct interspecies electron

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transfer (DIET) was enhanced by magnetite. Metatranscriptomic analysis and determination of key enzymes showed

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that IHT could be partially substituted by enhanced DIET, and acetate-dependent methanogenesis was improved

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after the blockage of electron transfer was scavenged. Additionally, the expression of both pili and c-type

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cytochromes was found to decrease, indicating that magnetite could replace their roles for efficient electron transfer

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between acetogens and methanogens; thus, a robust chain of electron transfer was established.

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Graphical abstract

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Introduction

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Waste activated sludge (WAS) produced from wastewater treatment plants (WWTPs) may become secondary

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pollution if reasonable handling is lacking.1 As an effective technology for WWTPs, anaerobic digestion could

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simultaneously control pollution and produce renewable energy.2 Traditional low-solids anaerobic digestion systems

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have been applied and investigated worldwide; however, these systems are not always feasible in small-scale

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WWTPs or those located in developing countries. Compared with low-solids anaerobic digestion, high-solids

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anaerobic digestion (TS (total solid) > 10%) is more attractive because of the relatively high loading rate, small land

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footprint, low energy consumption, etc. Nevertheless, operational instability of these systems may be caused by high

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concentrations of potential inhibitory substances, especially acids accumulating due to high organic loading.

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Consequently, the system will achieve a steady state, albeit, at a low level (called “inhibited pseudo-steady state”). In

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this case, methane production, and the degradability of the organic matter in the WAS will decrease.3-5

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During anaerobic digestion, methane is mainly produced by acetoclastic and hydrogenotrophic methanogens,

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which utilize acetate and H2/CO2, respectively. Before methanogenesis, H2-producing acetogenic bacteria play a

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critical role in converting intermediate products, primarily ethanol and short-chain fatty acids (SCFAs, e.g.,

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propionate and butyrate), into acetate and H2/CO2. Unfortunately, this biochemical process is thermodynamically

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unfavorable under standard conditions, as illustrated by a positive change in the Gibbs free energy, unless

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H2-consuming methanogenic archaea continuously consume H2 to reduce CO2, thus maintaining an extremely low 3



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partial pressure of hydrogen. The manner by which syntrophic partners transfer electrons using H2 as a shuttle is

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known as interspecies hydrogen transfer (IHT).6-8 In high-solids anaerobic digestion, SCFAs readily accumulate due

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to a process imbalance caused by organic overloading, accompanied by a high partial pressure of hydrogen.9, 10 The

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reason is that overproduction of SCFAs may be toxic to anaerobic microorganisms, among which methanogens are

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more sensitive than fermentative bacteria. In turn, the accumulated H2 and SCFAs cannot be efficiently scavenged,

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thus blocking the IHT.11,12 In this case, the whole process of anaerobic digestion will fail.

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In recent decades, a relatively new electron-transfer pathway called direct interspecies electron transfer (DIET)

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was discovered as a potential alternative to IHT and it was confirmed in universal anaerobiosis (e.g., anaerobic

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digesters, paddy soils and bioelectrochemical systems).13-16 Through microbial nanowires, syntrophic bacteria can

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directly transfer electrons produced from intermediate products to methanogenic archaea, and methane can be

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generated by the reduction of CO2, without any electron shuttles.15,17 In addition to these microbial nanowires, some

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artificial conductive materials (e.g., iron oxides, activated carbon, carbon cloth, carbon nanotubes (CNTs) and

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biochar) can also perform DIET.17-20 Currently, magnetite has been well confirmed to promote DIET between

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syntrophic bacteria and methanogens. As a surrogate for microbial nanowires, magnetite can transfer electrons more

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efficiently than IHT and significantly increase the rate of methanogenesis.21-23 However, most direct evidence was

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obtained in pure cultures.24,25 Even though DIET has been studied in some complex anaerobic systems (primarily

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synthetic wastewater and paddy soil), studies on the promotion of DIET by magnetite in the anaerobic digestion of

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sewage sludge are rare, let alone in high-solids systems. Meanwhile, direct evidence is difficult to obtain in complex

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systems due to the large number of microorganisms and intricate metabolic activities.

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Based on the above considerations, added magnetite was expected to maintain a smoother electron transfer

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between the syntrophic bacteria and methanogenic archaea to relieve acid inhibition in the high-solids anaerobic

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digestion of sewage sludge. The primary aim of this study was to reveal the effects of nano-magnetite on methane

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production. Thus the role of magnetite during the anaerobic digestion of sewage sludge was explored from two 4


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aspects: 1) changes in the chemical composition of magnetite and the effects of the released iron ions on methane

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production; and 2) the shift in methanogenesis pathways after magnetite was added. Furthermore,

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metatranscriptomic evidence for DIET was discussed in depth.

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Materials and Methods

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Magnetite and Sludges. Magnetite (nano-sized Fe3O4) (< 50 nm) was purchased from Sigma Aldrich (St. Louis,

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MO, USA). Dynamic light scattering (DLS) using a Malvern Autosizer 4700 (Malvern Instruments, UK) device was

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conducted to measure the average size of the magnetite particles (45 ± 12 nm). X-ray diffraction (XRD) analysis was

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conducted using a Rigaku D/Max-RB diffractometer equipped with a rotating anode and a Cu Kα radiation source

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(Rigaku Corporation, Japan).

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The WAS used in this study was dewatered sludge obtained from a municipal WWTP in Shanghai, China and

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was stored at 4°C. The inoculum was obtained from a mesophilic semi-continuous reactor with long-term stable

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operation. The primary characteristics of the dewatered sludge and inoculum sludge are listed in Supporting

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Information (SI) Table S2.

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Effects of Magnetite on Anaerobic Digestion of High-solids Sewage Sludge During Batch Experiments. An

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Automatic Methane Potential Test System (AMPTS II) (Bioprocess Control Sweden AB) was used to perform the

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batch experiments (SI Figure S1). According to a preliminary experiment, 50 mg/g TS was chosen as an appropriate

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dosage. The batch experiments were conducted in eight identical reactors, each with a working volume of 400 mL.

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These reactors were divided into two groups, a blank group and a group with 50 mg/g TS magnetite added. Each

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group included three parallel tests plus one for gas collection using an aluminum foil gas-collecting bag. In each

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individual reactor, 130 g of inoculum, 140 g of dewatered sludge and 130 g of ultrapure water were mixed to reach a

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TS concentration of 10% for the high-solid content, and the ratio of inoculum to substrate was 1:4.6 (calculated as

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volatile solid (VS)). After being flushed with N2 to create anaerobic conditions, the reactors were sealed and operated

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at 35 ± 1°C in a water bath with agitation at 120 rpm. The volume of methane was recorded on-line by an automatic

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gas volume measuring device, followed by the adsorption of acidic gases (e.g., CO2 and H2S) in the biogas with 3 M

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NaOH with thymolphthalein added as indicator. Thus, both the methane production and methane production rate

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could be obtained simultaneously. The effects of magnetite on the enrichment of microbial communities (second

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generation) was determined in almost the same manner as that of the above experiment for the first generation,

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except that the inoculum was the digestate from reactors in the first generation, and the ratio of inoculum to substrate

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was 1:2.2 (calculated as VS).

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Effects of Relative Released Fe2+ on methane production. The concentrations of released iron ions in tests with

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and without magnetite at different fermentation times are shown in Figure S7A. Considering that iron ions exist as

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Fe2+ under anaerobic methane-producing conditions, the effects of 0 mg/L, 1 mg/L, 5 mg/L, and 10 mg/L Fe2+ (added

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using FeCl2) on methane production were investigated according to the results of inductively coupled plasma optical

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emission spectrometry (ICP-OES). The ratio of inoculum to substrate was 1:4.6 (calculated as VS), and the

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experimental method was the same as that in the previous section, except the corresponding dissolved Fe2+ was used

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to replace magnetite.

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Methane Production Modeling Using the Modified Gompertz Model. A modified Gompertz model was used to

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obtain the quantitative delineation of methane production in all of the experiments above (eq. (1)).

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 R ×e  (λ − t) + 1  M (t) = P × exp − exp  max  P  

(1)

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where M(t) is the cumulative CH4 production (mL CH4/VSadded) at time t; P is the maximum CH4 potential (mL

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CH4/VSadded) at the end of anaerobic digestion; t is the time (d); Rmax is the maximum CH4 production rate (mL

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CH4/VSadded/d); λ is the lag phase (d); and e is 2.71828.26 Among these parameters, Rmax was the measured value,

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while the methane production curve and the other relevant parameters were simulated using OriginPro 9.0 (Origin

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Lab Corporation, MA, US).

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Effects of Magnetite on the Individual Steps Involved in Anaerobic Digestion. The experimental procedure

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remained almost the same as that in the “Effects of Magnetite on Anaerobic Digestion of High-solids Sewage Sludge

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During Batch Experiments”, and the ratio of inoculum to substrate was 1:4.6 (calculated as VS). However, 50 mmol

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of 2-bromoethanesulfonate (BES) was added to the mixture of substrate and inoculum at the beginning, followed by

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sampling to determine the hydrogen production and the concentrations of soluble substrates (i.e., soluble proteins,

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soluble carbohydrates and SCFAs) at different fermentation times. In this experiment, BES, as a structural analogue

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of coenzyme M, was added to explore the effect of magnetite on the processes before methanogenesis by blocking

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the methane production process.

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The metabolic activity of acetotrophic methanogenesis (AM) utilizing acetate and hydrogenotrophic

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methanogenesis (HM or IHT) utilizing H2/CO2 were investigated with synthetic wastewater (SI). As the inoculum,

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100 g of digestate was respectively obtained from the reactors of the blank group and the group with magnetite in the

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first generation until methane produced from the initial dose of substrate almost ceased. As shown in SI Figure S2,

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six identical reactors were divided into two groups, and the three reactors in each group were fed with inoculum

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from the blank group, inoculum from the magnetite group and inoculum from the blank group with 50 mg/g TS

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external magnetite, respectively. To investigate acetotrophic methanogenesis, 1 g of CH3COONa was added as the

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substrate, and after a fermentation time of 7 d, methane production and residual SCFAs were determined. To

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investigate hydrogenotrophic methanogenesis, an aluminum foil gas-collecting bag with a 400 mL mixture of H2 and

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CO2 (20%:80%) was attached to each reactor after being flushed with N2 to remove residual methane. After a

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fermentation time of 2 d, methane production was determined.27 The working volume was 300 mL each, and all

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other operations were the same as those described above.

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Stable Carbon Isotope and Metatranscriptomic Analysis. The biogas was collected from the reactors in the

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second generation every three days. Carbon isotope ratios of CH4 and CO2 in the biogas were determined through

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gas chromatography-combustion isotope ratio mass spectrometry (GC-C III-IRMS) analysis. The apparent 7



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fractionation factor αc that is explicitly defined by the measured δ 13CCH 4 and δ 13CCO2 (eq. (2)) was used to roughly

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estimate the proportions of acetate- and CO2-dependent methanogenesis.28

α c = (δ 13 C CO + 103 )(δ 13 C CH + 103 )

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2

4

(2)

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For metatranscriptomic analysis, microbial samples were withdrawn from the reactors in the second generation

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after enrichment for 6 d when the methane production rate was relatively high, indicating vigorous microbial

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metabolism and a favorable amount of mRNA expression. The samples were then preserved in liquid nitrogen as

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soon as possible.

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Each step in the stable carbon isotope and metatranscriptomic analyses is described in detail in the Supporting

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Information. All the raw metatransgenomic datasets have been deposited into the NCBI Sequence Read Achieve

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database (Accession Number: SRP117799).

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Other Analytical Methods. TS, VS/TS, TCOD, SCOD, carbohydrate, protein and total NH4-N (TAN) were

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analyzed using methods based on a previous publication.4 After microwave digestion, the released iron ions were

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detected by ICP-OES. The components of biogas were measured by gas chromatography (GC112A, INESA, China)

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with a thermal conductivity detector equipped with a GDX-102 packed column (2 m × 4 mm). The protocols to

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measure the SCFAs, dissolved organic carbon (DOC), protease, acetate kinase (AK) and coenzyme F420,

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hydrogenase and the scanning electron microscopy-energy dispersive X-ray (SEM-EDX) and sludge conductivity

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analysis protocols are described in detail in the Supporting Information.

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Statistical Analysis. All tests were performed in triplicate, and the significance of each of the results was determined

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using analyses of variance (ANOVAs). A p value < 0.05 was considered statistically significant.

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Results and Discussion

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Effects of Magnetite Exposure on the Anaerobic Digestion of Sewage Sludge. According to preliminary

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experiments on variations in methane production and the methane production rate caused by different concentrations

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of magnetite (SI Figure S3 and S4), 50 mg/g TS was chosen as the optimum concentration in this study, considering 8


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the promoting effect and practical applications. As seen in Figure 1A, 1B and SI Figure S4, and Tables S3 and S4,

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adding 50 mg/g TS magnetite could significantly accelerate methanogenesis in the anaerobic digestion of high-solids

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sewage sludge, and the maximum methane production rate (Rmax) increased by 26.6%. As calculated by the modified

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Gompertz equation, adding magnetite shortened the lag phase (λ) from 5.79 d to 3.75 d, implying a shorter time to

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reach the rapid methane production phase. This addition, however, could not improve the ultimate methane

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production (P) (p > 0.05). For the groups with magnetite, methane production almost stopped at day 27, while it

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tended to plateau at day 34 for the blank group. The biogas components were also influenced by the magnetite (SI

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Figure S5A). Before day 12, the methane content was relatively higher in the group with magnetite, while the CO2

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contents were higher in the blank group. Thereafter, these contents tended to change gradually.

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The production of SCFAs was observed to be clearly influenced by the addition of magnetite (Figure 1C).

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Overall, the concentrations of SCFAs were high due to abundant organic matter in the high-solids sewage sludge.

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Adding magnetite significantly decreased the concentrations of total SCFAs during the whole period of anaerobic

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digestion (p < 0.05). This result was consistent with other studies that reported propionate and butyrate more rapidly

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degraded due to magnetite.29-31 At day 4, the highest concentration of total SCFAs was 21,259.2 ± 493.5 mg-COD/L

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in the blank group, at which time the concentration of total SCFAs in the magnetite group was 13,494.3 ± 272.7

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mg-COD/L, when the reduction rate of total SCFAs (57.5%) was the maximum. Meanwhile, the highest

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concentration of total SCFAs in the magnetite group with addition appeared after 2 d, indicating that the process of

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methane production after enrichment was also accelerated. For individual SCFAs, acetic acid and propionic acid

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were the dominant types of SCFAs. Moreover, propionic acid accumulated during the later period of anaerobic

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digestion, and magnetite alleviated this accumulation. For DOC, the variation tendency was similar to that of SCFAs

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(Figure S5B). Specifically, DOC values were always lower in the magnetite group than those in the blank group, and

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the highest value appeared earlier. SCFAs and DOC are well known to be continuously produced and consumed

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under dynamic conditions. Low concentrations of SCFAs and DOC indicate more rapid consumption, which 9



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facilitates preventing the inhibitory effect of acids accumulation on anaerobic microorganisms, especially

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methanogens. In the earlier stage, the faster depletion of SCFAs and relatively faster release of TAN from the

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decomposition of organic matter may be the main reasons for the earlier increase in the pH in the magnetite group

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(SI Figure S5C and 5D).

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The First Generation:

A

250 200 150 100 50 0 0 4 8 12 16 20 24 28 32 36 40

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28000

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Acetic iso-butyric iso-valeric

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SCFAs (mg-COD/L)

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Magnetite

Methane Production Rate (mL / g VSadd / d)

Cumulative Methane Production (mL / g VSadd)

Blank

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20000 16000 12000 8000

5 4000 0

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BM BM BM BM BM BM BM BM BM BM

0d 2d 4d

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The Second Generation: Methane Production Rate (mL / g VSadd / d)

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Time (d)

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Acetic iso-butyric iso-valeric

7500

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Propionic n-butyric n-valeric

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6d 8d 10d 12d 18d 24d 40d

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Propionic n-butyric n-valeric

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Figure 1. Effects of magnetite (50 mg/g TS) on methane production (A), methane production rate (B) and

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concentrations of SCFAs (C) during the first generation of anaerobic digestion; methane production (D),

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methane production rate (E) and concentrations of SCFAs (F) during the second generation of anaerobic

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digestion. The error bars represent the standard deviations of duplicate tests. 10


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During the second generation, methane production rates were both notably improved with shorter lag phases

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and higher Rmax because of acclimation (shown in Figure 1D, 1E and SI Tables S3, S4). Nevertheless, methane

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production in the magnetite group (λ = 2.20 d, Rmax = 27.80 mL/(g VSadd · d)) was still 18.4% faster than that in the

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blank group (λ = 1.78 d, Rmax = 32.91 mL/(g VSadd · d)) (p < 0.05). The accumulation of SCFAs was not as severe as in

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the first generation, and the magnetite continued to alleviate acids accumulation (Figure 1F).

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Effects of Dissolved Iron Ions on Methane Production. Considering that metal-oxide NPs may release metal ions,

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which could significantly affect microorganisms, the impact of iron ions potentially released from magnetite was

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investigated.32 The case for abiotic anaerobiosis was discussed, first combining the ICP results and XRD images (SI

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Figure S6). Iron ions could not be detected in the liquid phase. In addition, the XRD images of magnetite in the solid

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phase changed negligibly after 15 d of treatment, indicating that extremely few iron ions were discharged from the

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magnetite, even when the pH was adjusted to a value as low as 6.97, the lowest pH in the magnetite group (Figure

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S5D). Nonetheless, concentrations of dissolved iron in the anaerobic digestion of sewage sludge still differed after

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adding magnetite (Figure S7A). The concentrations were generally higher in the magnetite group than those in the

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blank group at different fermentation times, since anaerobic microorganisms (e.g., dissimilatory Fe(III)-reducing

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bacteria) may participate in the dissociation of iron ions from magnetite.33 The maximum difference in concentration

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was 6.0 mg/L, appearing at day 10.

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Based on the above findings, the effects of external Fe2+ dose on methane production were studied (shown in SI

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Figures S7B and S8, and Tables S5 and S6). A dose of 1 mg/L Fe2+ could slightly accelerate the process of methane

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production; however, Fe2+ was far less effective than magnetite. When the concentration of Fe2+ increased to 5 mg/L,

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the rate of methane production was slightly slower than that in the blank group. Moreover, 10 mg/L Fe2+ clearly

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depressed methane production (p < 0.05). Thus, excessive Fe2+ may harm anaerobic digestion. One possible reason

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for this detrimental effect was that an excessive concentration of Fe2+ is toxic to microbial cells. Another possible

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reason is that organic matter, especially protein, becomes difficult to biodegrade when it combines with Fe2+, thus 11



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decelerating methane production.34 To summarize, the promotional effect of magnetite on anaerobic digestion may

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not be due to Fe2+ release.

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Effects of Magnetite on the Individual Steps Involved in Anaerobic Digestion. BES is a classic specific inhibitor

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for methanogens that inhibits the methyl-coenzyme M reductase of the methanogens (SI Figure S9).35 Soluble

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proteins and soluble carbohydrates were the main components of the hydrolysis products, and SCFAs were the main

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components of the acidogenesis products. Additionally, the hydrogen content could reflect the activity of

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acedogenesis. To investigate whether the promotional effect of magnetite on methane production is derived from

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enhancing the processes before methanogenesis (i.e., hydrolysis, acidogenesis and acedogenesis), soluble proteins,

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soluble carbohydrates, SCFAs and hydrogen content were measured after BES was added to block the process of

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methanogenesis. As shown in SI Figure S10, at different fermentation times, the concentrations of soluble proteins,

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soluble carbohydrates, and SCFAs as well as the hydrogen content, the blank and magnetite groups were not

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significantly different (p > 0.05). In addition, in contrast with the reactors fed with the inoculum from the blank

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group in the first generation, adding external magnetite could not efficiently stimulate acetotrophic methanogens or

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hydrogenotrophic methanogens (SI Table S7, the two groups in the red box). These observations thus highlighted

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that the immediate cause for improvement of methane production was not related to the enhancement of any

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independent process of anaerobic digestion when the connection between methanogenesis and the processes before

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methanogenesis was cut off.

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The changes in the two main pathways caused by magnetite in already-established microbial relationships after

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enrichment were investigated (SI Table S7, the two groups in the green box). After 7 d of fermentation, the

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consumption of acetate and methane production were slower in the blank group than in the magnetite group when

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fed with acetate. However, after 2 d of fermentation, the uptake of H2/CO2 decreased, and less methane was

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generated in the presence of magnetite. These results indicated that acetate-dependent methanogenesis was

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significantly enhanced, whereas CO2-dependent methanogenesis, properly speaking, the pathway consuming 12


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molecular H2, was slightly reduced. In the future, the change in methanogenesis pathways will be explored in depth

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through multiple approaches.

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Methane Production Affected by Magnetite after Enrichment of Methanogenic Communities and

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Quantification of Methanogenic Pathways Using Stable Carbon Isotopic Signatures. The stable carbon isotopic

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signatures of CH4 (δ13CH4) and CO2 (δ13CO2) in the biogas were detected every 3 days during the second generation

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(Figure 2). The apparent fractionation factor (αc) was calculated to demonstrate the respective proportions of CH4

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derived from acetate-methyl and CO2 reduction. In general, αc > 1.065 and αc < 1.055 are characteristic for methane

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production dominated by CO2- and acetate-dependent methanogenesis, respectively. A decrease in αc means a shift to

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acetotrophic methanogenesis.36 In this study, overall trends were similar in both systems: αc first decreased (3-6 d),

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then increased (6-12 d) and finally decreased (12-15 d). In the preliminary stage of slow methane production (0-3 d),

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CO2-dependent methanogenesis accounted for a large part of the total methane production. Then, the process

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changed into a stage of rapid methane production (3-6 d). The pathway shifted to acetate-dependent methanogenesis

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as the system began to achieve the most favorable conditions for the methanogens. As organic matter continued to be

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consumed, the proportion of acetate-dependent methanogenesis decreased (6-12 d). In the final stage (12-15 d),

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acetate-dependent methanogenesis resumed slightly, probably because SCFAs were almost depleted except for a

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small amount of acetic acid. This trend was almost the same as that in studies on changes in pathways during the full

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process of mesophilic anaerobic digestion.37

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Notably, αc was slightly higher after magnetite was supplemented, indicating that CO2-dependent

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methanogenesis was slightly enhanced. Interestingly, the possibility that this enhancement derived from

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methanogenesis using H2 molecules as electron shuttles was ruled out by the results in “Effects of Magnetite on the

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Individual Steps of Hydrolysis, Acidogenesis and Methanogenesis”, which showed that adding magnetite failed to

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improve methane production by utilizing molecular H2. Collectively, these results suggest that CO2-dependent

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methanogenesis was enhanced not through IHT but rather through DIET. 13



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Figure 2. Effects of magnetite (50 mg/g TS) on stable carbon isotopic signatures of CH4 and CO2 in biogas

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during anaerobic digestion in the second generation after different fermentation times. Error bars represent

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the standard deviations of duplicate tests.

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Evidence for DIET and Changes in Microbial Community Compositions Revealed by Metatranscriptomic and

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Genetic Analysis. The sequence length distribution, a scatter plot of mRNA differential expression and the key

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KEGG pathway relative to the process of anaerobic digestion are shown in SI Figure S11 and Table S8 After

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magnetite was supplemented, the abundance of mRNA expression in methane metabolism was enriched by 72.7%.

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The metabolisms of carbohydrates and SCFAs were all promoted, whereas the effects of magnetite on the

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metabolisms of various amino acids differed. Moreover, after magnetite was added, the abundances of mRNA in

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relation to cellular activity (e.g., the bacterial secretion system and oxidative phosphorylation) were higher, implying

299

that the metabolism of organisms during the process of anaerobic digestion was more active. Notably, hydrolysis,

300

acidogenesis and acedogenesis were all promoted by magnetite when no BES was added to stop methanogenesis.

14


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Page 15 of 26


301 302

Figure 3. Changes in the electron transfer modes between acetogens and methanogens and the enzymes

303

involved in the carbon reduction or acetate decarboxylation pathways for methanogenesis with added

304

magnetite, as revealed by metatranscriptomics. Further details on the mRNA differential expression are

305

provided in Table 1.

306

The mRNA encoding enzymes involved in methanogenesis were almost all more abundant through both the

307

conversion of acetate-methyl and the reduction of CO2, implying these two pathways were both enhanced (Figure 3

308

and Table 1). During the methane production process, 5-methyl-THMPT is the co-intermediate that is catalyzed by

309

the acetyl-CoA decarbonylase/synthase complex, subunit beta in acetate-dependent methanogenesis and by

310

5,10-methylenetetrahydromethanopterin reductase in CO2-dependent methanogenesis. After magnetite was

311

supplemented, the relative abundance of the mRNA encoding acetyl-CoA decarbonylase/synthase complex, subunit

312

beta

313

5,10-methylenetetrahydromethanopterin reductase increased by a factor of 2.16. In accordance with the results of the

314

stable carbon isotopic signatures, CO2-dependent methanogenesis was slightly more enhanced than

315

acetate-dependent methanogenesis.

increased

by

a

factor

of

2.08,

while

the

relative

abundance

316

15


of

the

mRNA

encoding


Page 16 of 26

Table 1. Differential expression of key enzymes involved in the process of methane production after adding magnetite as revealed

by metatranscriptomics Sequence number

Definition

Blank-count

Magnetite-count

Log2 RPKM

Regulate

(1)

Acetate kinase

295

718

1.28

Up

(2)

Phosphate acetyltransferase

74

30

-1.30

Down

(3)

Acetyl-CoA synthetase

12

129

3.24

Up

468

2147

2.08

Up

150

942

2.65

Up

Acetyl-CoA decarbonylase/synthase complex, (4) subunit beta Tetrahydromethanopterin S-methyltransferase, (5) subunit A (6)

Methyl-coenzyme M reductase, alpha subunit

507

1921

1.63

Up

(7)

Formylmethanofuran dehydrogenase, subunit A

81

625

2.82

Up

42

66

0.65

Up

17

159

3.09

Up

211

1996

3.20

Up

632

3049

2.16

Up

Formylmethanofuran-tetrahydromethanopterin (8) N-formyltransferase Methenyltetrahydromethanopterin (9) cyclohydrolase Methylenetetrahydromethanopterin (10) dehydrogenase 5,10-methylenetetrahydromethanopterin (11) reductase (12)

Membrane-bound hydrogenase

-

-

-

-

(13)

Type IV pilus assembly protein pil(A,B,C,M,Q)

1308

952

-0.46

Down

(14)

C-type cytochromes

148

13

-3.51

Down

317

Previous studies reported that electrically conductive microbial nanowire, which were composed of pili and

318

c-type cytochromes, played an important role in facilitating long-range extracellular electron transfer. The pili are

319

metallic-like cell-cell nanowires transferring electrons by electron delocalization due to the overlapping π-orbitals of

320

aromatic amino acids, while the c-type cytochromes adorning the pili transfer electrons via two modes (hopping and

321

tunneling); thus, they could enable the conductivity of the pili.38, 39 Additionally, some c-type cytochromes free of

322

microbial nanowires can also participate in electron transfer 38. Rotaru et al. reported that adding granular activated

16


Page 17 of 26


323

carbon could promote DIET by substituting pili between Geobacter metallireducens and Methanosarcina barkeri.40

324

Liu et al. proved that magnetite could compensate for the role of c-type cytochromes in extracellular electron

325

exchange using the method of gene knockout.41 In this study, the expression of mRNA encoding pili and c-type

326

cytochromes was down-regulated, with a log2 RPKM of -0.46-fold and -3.51-fold, respectively. In addition,

327

magnetite significantly enhanced the conductivity of the sludge system (shown in SI Figure S12). The results showed

328

that magnetite facilitated the establishment of DIET between fermentative bacteria and methanogens, and it

329

alleviated the need for pili and c-type cytochromes in DIET. As a result, energy metabolism could be effectively used

330

for other cellular activities, rather than being dissipated on the growth of filaments.42 More importantly, the result

331

confirmed that the conductive connection was indeed formed by magnetite, acting in the role of either pili or c-type

332

cytochromes. The rapid establishment and consolidation of DIET accelerated the electron transfer; meanwhile, IHT

333

was partially substituted. Clearly, the blocking effect of acids accumulation on electron transfer could be relieved,

334

thus further promoting the process of anaerobic digestion. Unfortunately, membrane-bound hydrogenase, catalyzing

335

the synthesis of H2 from H+/e- and the cleavage of H2 to H+/e- in IHT,43 could not be precisely distinguished,

336

probably due to numerous types of hydrogenase in the complex sludge system.

337

The results of taxonomic analysis revealed by gene transcript abundance are shown in Figure 4 and Figure S13.

338

For microbial communities at the phylum level, Firmicutes, Euryarchaeota, and Bacteroidetes were the three most

339

abundant phyla in both anaerobic systems, which accounted for 51.82%, 9.37, and 6.91% in the blank group and

340

42.71%, 15.02%, and 4.99% in the magnetite group, respectively. After magnetite was supplemented, the abundance

341

of Firmicutes and Bacteroidetes decreased, while the abundance of Euryarchaeota (mainly methanogens) increased.

342

Then, the detailed expression differences at the genus level were discussed (Figure 4). For bacteria, the relative

343

abundance of Syntrophomonas increased notably from 0.9% to 1.8% after magnetite was added, and it was the only

344

genus related to fermentation among the genera with significantly increased relative abundance (p < 0.05). The

345

mRNA encoding Geobacter, which has been extensively investigated in both pure cultures and complex anaerobic 17



346

systems,15, 24, 44 was rarely expressed in this system. For the methanogens, the relative abundance of Methanoculleus,

347

Methanosarcina and Methanospirillum increased from 6.1%, 1.5%, and 0.7% to 8.1%, 2.9%, and 3.0%, respectively,

348 349

Figure 4. Heatmap based on taxonomic classification at the genus level.

18


Page 18 of 26

Page 19 of 26


350

meaning that they were the main genera involved in promoting methane production due to the addition of magnetite.

351

As reported by previous studies, Syntrophomonas is an obligate syntrophic microorganism capable of generating

352

electricity and utilizing a variety of fatty acids, especially butyric acid.45 Adding CNTs and magnetite could enhance

353

the activity of Syntrophomonas and promote DIET between syntrophic communities in lake sediments and paddy

354

soil.30, 46, 47 Methanosarcina has been well proven to be able to perform DIET, even though its primary substrate was

355

acetate in this study.18, 21 Salvador et al. confirmed that the syntrophism of Syntrophomonas and Methanospirillum in

356

the presence of CNTs was enhanced 1.5 times, independently of possible mechanisms such as DIET.43 For

357

Methanoculleus, evidence of DIET was rare, except that this genus exclusively produced methane via the reduction

358

of CO2.44 Thus, enhancing extracellular electron transfer between these syntrophic partners by adding conductive

359

materials deserves further pure co-culture investigation.

360

Effects of Magnetite on Key Enzymes in Anaerobic Digestion. In IHT, the membrane-bound hydrogenase plays a

361

key role in catalyzing the evolution of H2 released from fermentative bacteria.48 Nevertheless, in DIET, electron

362

transfer does not require H2, as electron transfer and membrane-bound hydrogenase was unnecessary.49 Considering

363

these characteristics, the activity of membrane-bound hydrogenase could be used to distinguish the contributions of

364

DIET and IHT. As shown in SI Figure S14, the activity of membrane-bound hydrogenase in the blank group was

365

20.5% higher than that in the group with magnetite. Clearly, DIET was more efficient, while IHT decreased in

366

efficiency. In addition, the activities of protease, AK and coenzyme F420 were determined, which could represent the

367

capabilities of hydrolysis, acidogenesis and methanogenesis, respectively. The activities of these three key enzymes

368

were all improved by adding magnetite. Note that the intensification of DIET could induce a promotional chain

369

effect on every step of anaerobic digestion.

370

SEM Images of Microbial Morphology and Magnetite after Anaerobic Digestion. EDX analysis showed that

371

elemental Fe on cellular surfaces significantly increased after magnetite was supplemented (Figure 5), indicating that

372

magnetite was adsorbed on the cellular surface primarily due to electrostatic interactions and the high specific area of 19



373

the nano-sized magnetite.50 As shown in the SEM images, in the blank group, the microorganisms scattered

374

sporadically in the sludge, whereas in the magnetite group, microorganisms appeared with close connections and

375

with a larger number of distributed particles surrounding them. In addition, the SEM image of solids separated by a

376

magnetic field from the digestate in the magnetite group in the second generation showed that magnetite remained

377

close to its original morphology (SI Figure S15). This result further indicated that magnetite functioned as an

378

electron transporter, rather than being utilized for their physicochemical properties.

379 380

Figure 5. SEM images (B-C, E-F) and EDX analysis (A, D) of sludge in the reactors exposed to magnetite.

381

Blank (A-C); Magnetite (D-F).

382

Overall Understanding of the Methanogenesis Pathway Shift with Magnetite Addition. The addition of

383

magnetite alleviates the blockage of electron transfer in IHT due to acids accumulation induced by high organic

384

loading. The establishment of DIET can accelerate the consumption of SCFAs by reinforcing the electron transfer

385

between syntrophic bacteria and methanogens. Overall, a virtual chain of energetic electron transfer in the entire

386

anaerobic digestion process resumes from the accumulation of acids. In terms of the methanogenesis pathway, there

387

was no evidence that DIET could be a substitute for acetate-dependent methanogenesis. The results of stable carbon

388

isotopic signatures and metatranscriptomic analysis could demonstrate that magnetite only slightly increased the

389

proportion of CO2-dependent methanogenesis. In the anaerobic digestion of sewage sludge, acetate-dependent

390

methanogenesis is the main pathway for methane production, accounting for approximately 2/3 of its production.51

20


Page 20 of 26

Page 21 of 26


391

After magnetite was supplemented, acetate-dependent methanogenesis remained dominant rather than being

392

substituted by DIET. Thus, enhanced DIET, similar to a scavenger, created a favorable environment for

393

acetate-dependent methanogenesis. This study is the first comprehensive investigation of the shift in methanogenesis

394

pathways induced by conductive materials in the anaerobic digestion of sewage sludge. Compared to analyzing the

395

diversity of microbial communities by using conserved marker genes,52 metatranscriptomic analysis combined with

396

stable carbon isotopic signatures analysis is a more effective method to reveal the variation in methanogenesis

397

pathways and can be used to improve accuracy.

398

Even though the promotion rate induced by magnetite decreased following the alleviation of acids accumulation

399

in the second generation, the effect of magnetite on continuous-operation anaerobic digesters fed with high-solids

400

sewage sludge deserves to be further investigated for engineering applications in terms of the recyclability of

401

magnetite due to its magnetic property. This study may provide an approach to enhancing the ability to withstand the

402

inhibition of acids accumulation led by negative factors such as shock organic loading, ammonia inhibition, and

403

temperature variation. Thus, materials possessing better conductive properties and more microorganisms with the

404

potential ability of DIET must be investigated further.

405

Acknowledgments

406

This study was financially supported by the State Key Program of National Natural Science of China (Grant no.

407

51538008) and the 12th National Five-Year-Plan of Key Science and Technology (Grant no. 2014BAC31B01).

408

Supporting Information

409

This file contains additional details on the methods and other tables and figures for the Results and Discussion

410

section, namely, Section S1-S8, Tables S1-S8, and Figures S1-S15.

411 21



412

Literature Cited

413

(1) Clarke, B. O.; Smith, S. R. Review of “emerging” organic contaminants in biosolids and assessment of

414 415 416 417 418

international research priorities for the agricultural use of biosolids. Environ. Int. 2011, 37(1), 226−247. (2) Mao, C. L.; Feng, Y. Z.; Wang, X. J.; Ren, G. X. Review on research achievements of biogas from anaerobic digestion. Renew. Sust. Energ. Rev. 2015, 45(2015):540-555. (3) Guendouz, J.; Buffière, P.; Cacho, J.; Carrère, M.; Delgenes, J. P. High-solids anaerobic digestion: comparison of three pilot scales. Water Sci. Technol. 2008, 58(9):1757-1763.

419

(4) Dai, X. H.; Duan, N. N.; Dong, B.; Dai, L. L. High-solids anaerobic co-digestion of sewage sludge and food

420

waste in comparison with mono digestions: stability and performance. Waste Manage. 2013, 33(2):308-316.

421

(5) Gehring, T.; Klang, J.; Niedermayr, A.; Berzio, S.; Immenhauser, A.; Klocke, M.; Wichern, M; Lübken, M.

422

Determination of Methanogenic Pathways through Carbon Isotope (δ13C) Analysis for the Two-Stage Anaerobic

423

Digestion of High-Solids Substrates. Environ. Sci. Technol. 2015, 49(7):4705-4714.

424

(6) Rasmus, Jakobsen; Hansjørgen, Albrechtsen; Mette, Rasmussen; Henrik, Bay; And, P. L. B.; Christensen, T. H.

425

H2 Concentrations in a Landfill Leachate Plume (Grindsted, Denmark): In Situ Energetics of Terminal Electron

426

Acceptor Processes. Environ. Sci. Technol. 1998, 32(14):2142-2148.

427 428 429 430 431 432 433 434 435 436 437 438 439 440

(7) Appels, L.; Baeyens, J.; Degrève, J.; Dewil, R. Principles and potential of the anaerobic digestion of waste-activated sludge. Prog. Energy Combust. Sci. 2008, 34(6):755-781. (8) Schink B. Energetics of syntrophic cooperation in methanogenic degradation. Prog. Energy Combust. Sci. 1997, 61(2):262-280. (9) Duan, N.; Dong, B.; Wu, B.; Dai, X. High-solid anaerobic digestion of sewage sludge under mesophilic conditions: feasibility study. Bioresource Technol. 2012, 104(104):150-156. (10) Luo C; Lü F; Shao L; He, P. Application of eco-compatible biochar in anaerobic digestion to relieve acid stress and promote the selective colonization of functional microbes. Water Res. 2015, 68:710-718. (11) Batstone, D. J.; Keller, J.; Newell, R. B.; Newland, M. Modelling anaerobic degradation of complex wastewater. I: model development. Bioresource Technol. 2000, 75(1):67-74. (12) Siegert, I.; Banks, C. The effect of volatile fatty acid additions on the anaerobic digestion of cellulose and glucose in batch reactors. Process Biochem. 2005, 40(11):3412-3418. (13) Storck, T; Virdis, B; Batstone, D. J. Modelling extracellular limitations for mediated versus direct interspecies electron transfer. Isme J. 2016, 10: 621-631. 22


Page 22 of 26

Page 23 of 26

441 442


(14) Mcglynn, S. E.; Chadwick, G. L.; Kempes, C. P.; Orphan, V. J. Single cell activity reveals direct electron transfer in methanotrophic consortia. Nature. 2015, 526(7574):531-535.

443

(15) Rotaru, A. E.; Shrestha, P. M.; Liu, F.; Shrestha, M.; Shrestha, D.; Embree, M; Devesh, Shrestha; Mallory,

444

Embree; Karsten, Zengler; Colin, Wardman; Kelly, P. Nevin; Derek, R. Lovley. A new model for electron flow

445

during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon

446

dioxide to methane. Energ. Environ. Sci. 2013, 7(1):408-415.

447 448 449 450 451 452

(16) Zhao, Z. Q.; Zhang, Y. B.; Wang, L. Y.; Quan, X. Potential for direct interspecies electron transfer in an electric-anaerobic system to increase methane production from sludge digestion. Sci. Rep-UK. 2015, 5:11094. (17) Kato, S.; Hashimoto, K.; Watanabe, K. Kato S, Hashimoto K, Watanabe K. Methanogenesis facilitated by electric syntrophy via (semi)conductive iron-oxide minerals. Environ. Microbiol. 2012, 14(7):1646-1654. (18) Liu, F.; Rotaru, A. E.; Shrestha, P. M.; Malvankar, N. S.; Nevin, K. P.; Lovley, D. R. Promoting direct interspecies electron transfer with activated carbon. Energ. Environ. Sci. 2012, 5(10):8982-8989.

453

(19) Liu, X. W.; Chen, J. J.; Huang, Y. X.; Sun, X. F.; Sheng, G. P.; Li, D. B.; Xiong, L.; Zhang, Y.Y.; Zhao, F.; Yu, H.

454

Q. Experimental and theoretical demonstrations for the mechanism behind enhanced microbial electron transfer

455

by CNT network. Sci. Rep-UK. 2014, 4(1):3732.

456 457 458 459 460 461 462 463

(20) Chen, S.; Rotaru, A. E.; Liu, F.; Philips, J.; Woodard, T. L.; Nevin, K. P.; Lovley, D. R. Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures. Bioresour. Technol. 2014, 173(1):82-86. (21) Barua, S.; Dhar, B. R. Advances towards understanding and engineering direct interspecies electron transfer in anaerobic digestion. Bioresource Technol. 2017, 244:698-707. (22) Lovley, D. R. Syntrophy goes electric: direct interspecies electron transfer. Annu. Rev. Microbiol. 2017, 71(1): 643-664. (23) Cheng, Q. Call, D. F. Hardwiring microbes via direct interspecies electron transfer: mechanisms and applications. Environ. Sci. Proc. Impacts. 2016, 18(8):968-980.

464

(24) Rotaru, A. E.; Shrestha, P. M.; Liu, F.; Markovaite, B.; Chen, S.; Nevin, K. P.; Lovley, D. R. Direct interspecies

465

electron transfer between Geobacter metallireducens and Methanosarcina barkeri. Appl. Environ. Microbio.

466

2014, 80(15):4599-4605.

467

(25) Holmes, D. E.; Shrestha, P. M.; Walker, D. J.; Dang, Y.; Nevin, K. P.; Woodard, T. L.; Lovley, D. R.

468

Metatranscriptomic Evidence for Direct Interspecies Electron Transfer Between Geobacter and Methanothrix

469

Species in Methanogenic Rice Paddy Soils. Appl. Environ.Microbio. 2017, 83(9):AEM.00223-17.

470

(26) Behera, S. K.; Park, J. M.; Kim, K. H.; Park, H. S. Methane production from food waste leachate in 23



471

laboratory-scale simulated landfill. Waste Manage. 2010, 30(8):1502-1508.

472

(27) Luo, J.; Chen, Y.; Feng, L. Polycyclic Aromatic Hydrocarbon Affects Acetic Acid Production during Anaerobic

473

Fermentation of Waste Activated Sludge by Altering Activity and Viability of Acetogen. Environ. Sci. Technol.

474

2016, 50(13):6921-6929.

475 476

(28) Whiticar, M. J.; Faber, E.; Schoell, M. Biogenic methane formation in marine and freshwater environments: CO 2, reduction vs. acetate fermentation—Isotope evidence. Geochim. Cosmochim. Ac. 1986, 50(5):693-709.

477

(29) Cruz, V. C.; Rossetti, S.; Fazi, S.; Paiano, P.; Majone, M.; Aulenta, F. Magnetite particles triggering a faster and

478

more robust syntrophic pathway of methanogenic propionate degradation. Environ. Sci. Technol. 2014,

479

48(13):7536-7543.

480 481 482 483

(30) Li, H.; Chang, J.; Liu, P.; Fu, L.; Ding, D.; Lu, Y. Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments. Environ. Microbio. 2015, 17(5):1533-1547. (31) Jing, Y.; Wan, J.; Angelidaki, I.; Zhang, S.; Luo, G. iTRAQ quantitative proteomic analysis reveals the pathways for methanation of propionate facilitated by magnetite. Water Res. 2017, 108:212-221.

484

(32) Wang, T.; Zhang, D.; Dai, L.; Chen, Y.; Dai, X. Effects of metal nanoparticles on methane production from

485

waste-activated sludge and microorganism community shift in anaerobic granular sludge. Sci. Rep-UK. 2016,

486

6:25857.

487

(33) Jiang, S.; Park, S.; Yoon, Y.; Lee, J. H.; Wu, W. M.; Phuoc, D. N.; Sadowsky, M. J.; Hur, H. G. Methanogenesis

488

facilitated by geobiochemical iron cycle in a novel syntrophic methanogenic microbial community. Environ. Sci.

489

Technol. 2013, 47(17):10078-10084.

490 491

(34) Dai, X.; Xu, Y.; Lu, Y.; Dong, B. Recognition of the key chemical constituents of sewage sludge for biogas production. Rsc Adv. 2017, 7(4):2033-2037.

492

(35) Xu, Ke Wei; L, He; C, Jian. Effect of classic methanogenic inhibitors on the quantity and diversity of archaeal

493

community and the reductive homoacetogenic activity during the process of anaerobic sludge digestion.

494

Bioresource Technol. 2010, 101(8):2600-2607.

495 496

(36) Conrad, R. Quantification of methanogenic pathways using stable carbon isotopic signatures: a review and a proposal. Orga. Geochem. 2005, 36, (5), 739–752.

497

(37) Vavilin, V. A.; Qu, X.; Mazéas, L.; Lemunier, M.; Duquennoi, C.; He, P.; Bouchez, T. Methanosarcina as the

498

dominant aceticlastic methanogens during mesophilic anaerobic digestion of putrescible waste. Anton. Leeuw.

499

Int J. G. 2008, 94(4):593-605.

500

(38) Malvankar, N. S.; Lovley, D. R. Microbial nanowires for bioenergy applications. Curr. Opin. Biotechnol. 2014, 24


Page 24 of 26

Page 25 of 26

501 502 503


27(6):88-95. (39) Polizzi, N. F.; Skourtis, S. S.; Beratan, D. N. Physical constraints on charge transport through bacterial nanowires. Faraday Discuss. 2012, 155(1):43-62.

504

(40) Rotaru, A. E.; Shrestha, P. M.; Liu, F.; Markovaite, B.; Chen, S.; Nevin, K. P.; Lovley, D. R. Direct interspecies

505

electron transfer between Geobacter metallireducens and Methanosarcina barkeri. Appl. Environ. Microbiol.

506

2014, 80(15):4599-4605.

507

(41) Liu, F.; Rotaru, A.; Shrestha, P. M.; Malvankar, N. S.; Nevin, K. P.; Lovley, D. R. Magnetite compensates for

508

the lack of a pilin-associated c-type cytochrome in extracellular electron exchange. Environ. Microbio, 2015,

509

17(3):648-655.

510 511 512 513

(42) Tremblay, P. L.; Angenent, L. T.; Zhang, T. Extracellular Electron Uptake: Among Autotrophs and Mediated by Surfaces. Trends Biotechnol. 2016, 35(4): 360-371. (43) Guss, A. M.; Kulkarni GMetcalf, W. W. Differences in hydrogenase gene expression between Methanosarcina acetivorans and Methanosarcina barkeri. J. Bacteriol. 2009, 191(8):2826-2833.

514

(44) Feng, Y. H.; Zhang, Y. B.; Chen, S.; Quan, X. Enhanced production of methane from waste activated sludge by

515

the combination of high-solid anaerobic digestion and microbial electrolysis cell with iron-graphite electrode.

516

Chem. Eng. J. 2015, 259:787-794.

517

(45) Mcinerney, M. J.; Struchtemeyer, C. G.; Sieber, J.; Mouttaki, H.; Stams, A. J.; Schink, B.; Rohlin, L; Gunslus, R.

518

P. Physiology, ecology, phylogeny, and genomics of microorganisms capable of syntrophic metabolism. Ann. N.

519

Y. Acad. Sci. 2010, 1125(1):58-72.

520

(46) Salvador, A. F.; Martins, G.; Mellefranco, M.; Serpa, R.; Ajm, S.; Cavaleiro, A. J.; Pereira, M. A.; Alves, M. M.

521

Carbon nanotubes accelerate methane production in pure cultures of methanogens and in a syntrophic coculture.

522

Environ. Microbiol. 2017, 19(7): 2727-2739.

523 524 525 526 527 528

(47) Zhang, J.; Lu, Y. Conductive Fe3O4 nanoparticles accelerate syntrophic methane production from butyrate oxidation in two different lake sediments. Front. Microbiol. 2016, 7: 01316. (48) Vignais, P. M.; Billoud, B. Occurrence, classification, and biological function of hydrogenases: an overview. Chem. Rev. 2007, 107(10):4206-4272. (49) Shrestha, P. M.; Rotaru, A. E.; Summers, Z. M.; Shrestha, M.; Liu, F.; Lovley, D. R. Transcriptomic and Genetic Analysis of Direct Interspecies Electron Transfer. Appl. Environ. Microbiol. 2013, 79(7):2397-2404.

529

(50) Nel, A.; Xia, T.; Mädler, L.; Li, N. Toxic potential of materials at the nanolevel. Science. 2006, 311, 622-627.

530

(51) Khanal, S. K. Anaerobic Biotechnology for Bioenergy Production: principles and applications. Wiley-Blackwell. 25



531 532 533

2008. (52) Wang, H.; Fotidis, I. A.; Angelidaki, I. Ammonia effect on hydrogenotrophic methanogens and syntrophic acetate-oxidizing bacteria. Fems Microbiol. Ecol. 2015, 91(11): fiv130.

26


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Magnetite Triggering Enhanced Direct Interspecies Electron Transfer

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