Regulating secretion of extracellular polymeric substances through

Subscriber access provided by AUBURN UNIV AUBURN

Article

Regulating secretion of extracellular polymeric substances through dosing magnetite and zero-valent iron nanoparticles to affect anaerobic digestion mode Shiyang Li, Yi Cao, Zhiqiang Zhao, and Yaobin Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.9b01252 • Publication Date (Web): 23 Apr 2019 Downloaded from http://pubs.acs.org on April 23, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 35 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

ACS Sustainable Chemistry & Engineering

Regulating secretion of extracellular polymeric substances through dosing magnetite and zero-valent iron nanoparticles to affect anaerobic digestion mode Authors: Shiyang Li, Yi Cao, Zhiqiang Zhao, Yaobin Zhang* Affiliations: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China. * Correspondence: Tel.: +86 411 8470 6460, Fax: +86 411 8470 6263; E-mail address: [email protected] Full postal address: Key Laboratory of Industrial Ecology and Environmental Engineering (Dalian University of Technology), Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, No.2 Linggong Road, Ganjingzi District, Dalian City, Liaoning Province.

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 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

Abstract Anaerobic digestion technology is a promising method to reduce the usage of fossil fuels by transforming organic waste into biogas. Nano zero-valent iron (nZVI) and nano iron oxide have been reported to affect metabolism modes of anaerobic digestion, i.e., interspecies hydrogen transfer (IHT) and direct interspecies electron transfer (DIET). However, the effects of the nanoparticles on extracellular polymeric substance (EPS) potentially capable of participating in the mass transfer or electron transfer of these two metabolism modes remains unclear. In this study, the addition of nano magnetite (nFe3O4) significantly enhanced the performance of anaerobic treatment, while adding nZVI led to a decline of the performance. nFe3O4 stimulated the secretion of proteins and humic substances in EPS, which were confirmed electroactive to serve as electron shuttles to enhance DIET pathway of anaerobic digestion. In contrast, the addition of nZVI increased EPS especially polysaccharide to resist cell disruption caused by nZVI, which resulted in an inefficient mass transfer to decrease IHT. These results were in agreement with the microbial community analysis and the functional gene prediction. Keywords: Nano ZVI; Nano magnetite; Anaerobic digestion; Extracellular polymeric substance; Direct interspecies electron transfer.


Page 2 of 35

Page 3 of 35 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


Introduction Over the past half century, the syntrophic metabolism mode during anaerobic digestion is traditionally believed as the process of interspecies H2 transfer (IHT) or interspecies formate transfer (IFT)1. In IHT process, H2 is produced from anaerobic oxidation of complex matters by H+, and then H2 is utilized by hydrogenotrophic methanogens to produce methane2. However, this transfer mode is of weak link because the partial pressure of H2 produced must be maintained at a quite low level to make H+ oxidation favorable thermodynamically3. Therefore, the low efficiency of anaerobic digestion is frequently observed in the case of operating impacts such as organic loading rates (OLR) increase, low temperature and so on4. In general, there are currently two ideas to enhance anaerobic digestion. One is to increase the efficiency of IHT, and the other is to replace IHT by another electron transfer mode. Considering that the reducibility of zero-valent iron (ZVI) is capable of decreasing Oxidation-Reduction Potential (ORP) of anaerobic systems and of enriching hydrogenotrophic methanogens5,6, the traditional anaerobic digestion based on IHT mechanism could be improved with supplementing ZVI7. Nanomaterials have been applied to improve anaerobic digestion recently. Nanoparticles with higher surface area further intensified their chemical and physical properties. Strongly reductive nZVI (𝐸0𝐹𝑒2 + /𝐹𝑒0 = ―0.447 V)

interacting

with

functional

groups

of

proteins

and

lipopolysaccharides of outer membranes might cause cell disruption8. Therefore the decrease of methane production was observed with the increase of nZVI dosage. However,



hydrolytic acidification of waste activated sludge was always improved even when increasing nZVI dosage to a relatively high level, which was because the destruction of cells of waste sludge with nZVI might enhance the decomposition9. An alternative anaerobic digestion pathway based on direct interspecies electron transfer (DIET) between two microbial species Geobacter and Methanothrix was reported recently10. In this mode, electrons producing from organic-oxidizing bacteria (Geobacter) are transferred via its conductive pili10 or extracellular cytochromes to methanogens, with reducing CO2 to produce methane. Theoretical analysis reveals that electron transfer via conductive materials could be around eight times higher than that with IHT11. Dang et al. proved that dosing carbon-based materials could help establish DIET to promote the performance of the anaerobic digestion12. Li et al. and Yang et al. also found that not only magnetite but also magnetite nanoparticles were able to improve DIET13,14. For nano magnetite (nFe3O4), the high surface area tended to be evenly distributed in the microbial community, which was helpful to efficiently serve as a conductive material to enhance DIET. Since IHT is a process of mass transfer or H2/H+ diffussion between acidogens and methanogens, while DIET is a process of electron transfer between Geobacter and methanogens. This difference meant that the changes of extracellular substances might lead to different effects on the transfer efficiency of these two modes. Although the performance of anaerobic digestion with nZVI and nFe3O4 has been investigated, the


Page 4 of 35

Page 5 of 35 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


effects of these two nanoparticles on the extracellular substances capable of participating in electron transfer or mass transfer remained unclear. In this study, nZVI and nFe3O4 were dosed into the two anaerobic reactors to investigate their effects on anaerobic digestion, respectively. Effects of these two nanomaterials on the digestion pathways and their relationships with extracellular polymeric substance (EPS) that potentially participated in IHT or DIET were explored. High-throughput sequencing and functional gene prediction were applied to further clarify the metabolic pathway of microorganisms affected by the two nanomaterials.

Experimental Section Reactors, sludge and wastewater Three polypropylene plastic anaerobic reactors with a working volume of 2 L (Φ100 mm × 260 mm) were applied. Seed sludge inoculated in the reactors was obtained from a waste sludge treatment plant (Dalian, China). The initial TSS and VSS of the sludge were 65.8 and 25.6 g/L. 600 mL of the seed sludge was inoculated into each reactor. Synthetic sucrose wastewater was used as the influent. Sucrose, NH4Cl and KH2PO4 were added into this wastewater as the carbon, nitrogen, and phosphorus sources, respectively with a COD:N:P ratio of 200:5:1. 10 mL trace element and 1 mL vitamin solution was added in per liter wastewater. The trace element solution contained ZnCl2 at 0.13 g/L, MnSO4·H2O at 0.5 g/L, FeSO4·7H2O at 0.1 g/L, CuSO4·5H2O at 0.01 g/L, CoCl2·6H2O at 0.048 g/L, NiCl2·6H2O at 0.04 g/L, H3BO3 at 0.01 g/L, AlK(SO4)2·12H2O at 0.01 g/L and Na2MoO4·2H2O 0.025 g/L. The composition of the vitamin solution (in



grams per liter) was as follows: biotin 0.002; pantothenic acid, 0.005; vitamin B-12, 0.0001; p-aminobenzoic acid, 0.005; thioctic acid (alpha lipoic), 0.005; nicotinic acid, 0.005; thiamine, 0.005; riboflavin, 0.005; pyridoxine HCl, 0.01; folic acid, 0.002. The pH of the influent wastewater was adjusted to 7.5 with NaHCO3 solution. The chemical oxygen demand (COD) of the wastewater was maintained at 1000 mg/L at the initial time and then rose to 3000 and 5000 mg/L Experimental setup After being seeded, the three reactors were operated in parallel under a continuous mode with a ten-hour hydraulic retention time (HRT) at ambient temperature. In the initial 40 days, the three reactors were fed with an influent COD of 1000 mg/L for startup and no nanoparticles were added into the reactors. Then, the COD of the influent was raised to 3000 mg/L (day 41 to day 80) and 5000 mg/L (day 81 to day 120). At day 41, nano ZVI and nano magnetite (both purchased from Aladdin Industrial Corporation, Shanghai, China) were firstly dispersed in ultrapure water and then separately supplemented once into the reactors with a final total iron concentration of 50 mM in an anaerobic atmosphere. These two reactors were referred to as nZVI-reactor and nFe3O4-reactor，respectively. The control anaerobic reactor was operated in parallel, but no nanoparticles were dosed. Chemical analytical methods Total suspended solids (TSS) and volatile suspended solids (VSS) of the sludge in each reactor and chemical oxygen demand (COD) of the effluent were measured in accordance with the Standard Methods for the Examination of Water and Wastewater


Page 6 of 35

Page 7 of 35 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


(APHA, 1998). The biogas produced from the reactor was collected by gas sampling bags. The methane content of biogas was analyzed by a gas chromatograph with a thermal conductivity detector (TCD) (Tianmei, GC- 7900P/TCD, China)15. Volatile fatty acids (VFAs) in the effluent, including acetate, propionate and butyrate, were measured by another gas chromatograph with a flame ionization detector (FID) (Tianmei, GC7900P/FID, China)16. The EPS of the sludge were extracted followed by the strategy reported in the previous study17, utilizing cation exchange resin (CER) technique (20-50 mesh, 732, 001 × 7). The concentration of protein in EPS was estimated according to the Coomassie Brilliant Blue G-250 method18, using bovine serum albumin as the standard solution. The optical absorbance of the protein sample is measured at a wavelength of 595 nm by a spectrophotometer (Techcomp, UV-2301, China). The concentration of polysaccharide in EPS was measured by the phenol-sulfuric acid method19. The total organic carbon (TOC) of EPS was analyzed with a TOC analyzer (Analytikjena, multi N/C 2100s, Germany). Three-dimensional excitation-emission matrix (3-D EEM) spectra were obtained to further characterize the composition of EPS by using a fluorescence spectrophotometer (Hitachi, FL4500, Japan). The excitation range and the emission range were from 200 to 400 nm and from 200 to 500 nm in 5 nm sampling intervals with a scan rate at 1200 nm/min20. Electrochemical Measurements. To measure the conductivity of the suspended sludge in anaerobic reactors after the 120 days’ experiment, three-probe electrical conductance measurement was applied. The



pretreatment of samples was in accordance with Li et al21. Then, samples were tiled in two gold electrodes and filled in a 0.5 mm insulation gap between the gold electrodes. An electrochemical workstation (Chenhua, CHI650B, China) was introduced to measure the conductivity of the samples according to the previous study22 with equation (1): 1

𝜎 = 𝜌 (1) where ρ is the resistivity of the samples (Um), which was calculated following equation (2): 𝜌=

𝑅𝑆 𝐿

(2)

where R is the reciprocal of the slope in the current-voltage curve (U), S is the crosssectional area (2.54 × 10-6 m2) and L is the width of the gap (0.5 × 10-3 m). The electron donor capacities (EDCs) and electron acceptor capacities (EACs) of EPS separated from the sludge in three reactors were obtained by using the chronoamperometry methods with a conventional three-electrode cell. The working electrode was a vitreous carbon with a projected surface area of 2.25 cm2. A platinum wire electrode was used as the counter electrodes. The electrode was equilibrated to the desired potentials (i.e., Eh = −0.49 V in mediated electrochemical reduction (MER) and Eh = +0.61 V in mediated electrochemical oxidation (MEO), respectively). The potentials were measured against an Ag/AgCl reference electrode but reported here versus the standard hydrogen electrode (SHE). The experimental details of MER and MEO measurement are followed by the previous study23. Subsequently, the mediators Diquat


Page 8 of 35

Page 9 of 35 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


dibromide monohydrate (DQ) for MER and 2,2 ′ -azino-bis (3-ethylbenzthiazo-line-6sulfonic acid ammonium salt) (ABTS) for MEO were spiked, respectively. Then, 1mL of EPS samples were spiked to the cells, and the transferred amount of electrons was measured. The EAC and EDC of EPS were determined by integrating the current peak response according to equation (3) according to reports from Gorski et al24. 1

𝑡

q = 𝐹 × ∫𝑡2𝐼 × 𝑑𝑡 (3) 1

where q (mol e-) is the number of electrons transferred, F is the Faraday constant (96485 C/mol e-), I (C/sec) is the current, and t (sec) is time. EAC and EDC was the number of electrons per gram VSS sludge (μmol e-/g VSS). Microbial communities analysis and functional gene prediction. After 120 days’ experiment, the sludge samples (10 mL) of the two reactors were collected to analyze the microbial community via high-throughput 16s rRNA pyrosequencing. The pretreatment and the analysis procedure of samples were followed by the previous study25. The relative abundance is defined as the number of sequences affiliated with the taxon divided by the total number of sequences in each sample (%). The genus of each sample with the relative abundance less than 1.5% were defined as “others”. The composition of the functional genes of different samples was predicted through comparing the 16s rRNA sequencing results with Clusters of Orthologous Genes (COG) for gene function and Kyoto Encyclopedia of Genes and Genomes (KEGG) for metabolism pathways according to the previous studies26.



Results and discussion Performances of anaerobic digestion with nZVI and nFe3O4 After 40 days’ startup, the COD removal rate of the three reactors (Figure 1A) gradually increased to 85.1%, with no apparent differences among them. From day 41, two kinds of nanoparticles were added in nZVI and nFe3O4 reactors, respectively, and the influent COD of three reactors increased to 3000 mg/L and finally up to 5000 mg/L from day 81. After a fluctuation, the COD removal rate of the nFe3O4 reactor began to increase and finally reached to 84.9%, while the COD removal of the control reactor slightly increased as increasing COD on day 40. Reversely, the COD removal of the nZVI reactor declined over the operation. The profiles of methane production in the three reactors (Figure 1B) were similar to those of COD. A small addition of nZVI increased anaerobic digestion because ZVI was beneficial to decrease ORP, enrich hydrogenotrophic methanogens and improve enzyme activities 5,

which was reported to enhance anaerobic digestion especially methane production.

However, the positive influences might be outcompeted by the inhibition as increasing dosage of nZVI. It was previously reported that the reactive surface of nZVI with strong reducibility directly interacted with the cell membrane to destruct cell structure and decrease the activities of anaerobes27. The increase of H2 production with high nZVI dosage also impeded anaerobic digestion28. Accordingly, the inhibition of nZVI was observed when 2.8 g/L nZVI was dosed in this study. Comparatively, the low reactive activity of Fe3O4 did not cause the negative influences on the digestion.


Page 10 of 35

Page 11 of 35 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


On the contrary, as a conductive material, Fe3O4 was reportedly capable of participating in the interspecies electron transfer to improve anaerobic digestion29. The VFA removals of the three reactors were consistent with the COD removals and methane production. When the influent COD increased to 5000 mg/L, the total VFA (Figure 1C) with nZVI was 3292.48 mg/L, which was 2.3 and 5.1 folds of the control and the nFe3O4 reactor, indicating the inhibition effect of nZVI and promotion effect of nFe3O4, respectively. Electron transfer properties of sludge (1) Contents of protein and polysaccharide in EPS EPS contents of the suspended sludge of the three reactors was extracted for determination at the end of the experiment (day 120). The EPS extracted from the nFe3O4 reactor was 39.06 mgTOC/gVSS, slightly lower than that from the control (43.36 mgTOC/gVSS), while the EPS extracted from nZVI reactors was 1.58 folds as much as that from the control, up to 68.29 mgTOC/gVSS. Substrates including H+/H2 that were utilized for microbial metabolisms needed to pass through the EPS layer surrounding the outside of cells to enter inside. Therefore, increased EPS might decrease the efficiency of mass transfer into and out from the cell, which had been proved by both experimental data30 and model calculation31. The increased EPS around microorganisms with nZVI was a result from the microbial self-protection from the damage of nZVI. It undoubtedly impeded hydrogen transfer efficiency among microorganisms to decrease IHT efficiency. The concentration of protein and polysaccharide of EPS were further analyzed. As



shown in Figure 2, the protein of EPS from the nZVI reactors were 414.64 ± 22.57 mg/gTOC, slightly lower than that in the control (468.96 ± 40.5 mg/gTOC), while the protein with nFe3O4 was about twice of them, reaching 913.52 ± 10.92 mg/g TOC. The predominant component of proteins of EPS was reported to be exoenzymes32, most of which was proved electroactive, or to likely serve as electron shuttles. Ye et al.33 found that iron oxides facilitated the expression of redox active protein, such as c-type cytochrome to promote methane production. Thus, the increasing proteins with nFe3O4 might lead to more exoenzymes participating in extracellular electron transfer. On the other hand, the protein of EPS played a significant role in adhesion and aggregation of cells, which was beneficial for maintaining the stability of sludge structure34. Indeed, more protein in EPS potentially increased the diffusion difficulty of H+/H2 into or out from the cell, but the improved EET due to the increased electroactive protein outweighed its negative impacts on matter transfer with nFe3O4. Different from more protein detected with nFe3O4, around 2-fold polysaccharide content was detected in EPS from the nZVI reactor (470.41 ± 27.53 mg/gTOC) than the other two (229.07 ± 12.36 mg/gTOC for control and 229.35 ± 15.76 mg/gTOC for nFe3O4). Exopolysaccharides was reported as fine strands that were attached to the cell surface to form complex networks 34. More extracellular polysaccharide secreted around microorganisms might decrease the possibility of direct contact between reactive surfaces of nZVI and cells, which was a normal response of microorganisms to protect themselves


Page 12 of 35

Page 13 of 35 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


from the destruction. Unlike the proteins of EPS serving as electron shuttles in literature 34-35,

no reports were documented about polysaccharide directly acting as the electron

donors and acceptors. The increase of polysaccharide was unlikely helpful for the electron transfer between microorganisms. Meanwhile, a thickened EPS layer of microorganisms with nZVI meant that the efficiency of traditional anaerobic digestion through hydrogen transfer reduced. If the new anaerobic digestion pathway was not established, the performances of the nZVI reactor might be whittled. (2) EEM analysis Three-dimensional EEM fluorescence spectra were applied to further analyze the content of dissolved organic material (DOM) of EPS. According to the previous study 20, EEM was delineated into five excitation−emission regions based on the fluorescence of model compounds, i.e. Ex < 250 nm/Em control>nZVI, which was in agreement with the protein content of EPS of the three reactors. The protein-like substances (PS),



including varieties of cytochrome, were proved to conduct mediated electron transfer between fermentative bacteria and methanogens37. The high intensity of humic-like substances (region V) was also observed in the nFe3O4 reactor (Figure 3C). HS are redoxactive organics which had been documented to act as electron shuttles among microorganisms38. The increased content of electroactive PS and HS with nFe3O4 predicted a more efficient extracellular electron transfer, which was beneficial to establish DIET mode in the anaerobic metabolism. This was the reason for the reactor to perform well as raising influent loads under the room temperature. (3) Electron transfer capacity of EPS and conductivity of sludge To a certain extent, EAC and EDC of EPS reflected the amounts of oxidative and reductive groups of EPS available to exchange electrons such as quinone and hydroquinone moieties. The highest EAC and EDC of EPS determined in the nFe3O4 reactor were up to 112.02 and 48.45 μmol e−/gTOC (Figure 4A), respectively, followed by the control reactor, which was 31.15 and 24.87 μmol e−/gTOC, respectively. The EAC and EDC of EPS of the nZVI reactor were the lowest (19.56 and 38.54 μmol e−/gTOC). Thus, the electron transfer capacities (sum of EDC and EAC) of the EPS with nFe3O4 was about 2.8 folds of that with nZVI. From Figure 4B, the sludge conductivity of nFe3O4 reactor was 22.2 ± 0.07 μS/cm, almost four folds higher than that of the nZVI reactor (5.6 ± 0.06 μS/cm). Notably, the sludge conductivity of the control reactor (8.0 ± 0.03 μS/cm) was higher than the nZVI


Page 14 of 35

Page 15 of 35 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


reactor. The conductivity of fresh sludge with newly dosing nFe3O4 was almost the same as that of fresh sludge (2.24 ± 0.54 vs. 2.05 ± 0.74 μS/cm), which eliminated the likelihood that nano magnetite itself was the reason for the increase of conductivity. In other words, the higher conductivity of the sludge with nFe3O4 was resulted from the changes of sludge composition, in agreement with the more electroactive components presented in the EPS of sludge with nFe3O4. Semiconductive mineral magnetite (Fe3O4) as conductor enabled cell-to-cell electron connection between microbial species or terminal electron acceptors such as CO2 for methanogenesis through DIET pathway. To accomplish this connection, microorganisms needed to secret functional components like humic-like substrates or proteins serving as electron shuttles to receive or release electrons, or increase the sludge conductivity by growing conductive pili to directly transfer electrons. In agreement, extracellular proteins and humic-like substances, both possessing electroactive groups like quinone/hydroquinone and carbonyl/hydroxyl, which was proved by the FTIR analysis (shown in Figure S1), might serve as electron shuttles to enhance electron transfer capacity. The increased electroactivity of EPS and sludge and less total EPS amount revealed that direct EET process could be the primary pathway of anaerobic digestion with nFe3O439. Nevertheless, the primary mode for anaerobic digestion in the nZVI reactor was still traditional IHT, which might be inhibited because increased EPS around the cells impeded the mass transfer, known as the primary way to exchange energy



Page 16 of 35

for IHT. Microbial Community and functional gene analysis In the seed sludge, the dominant methanogen was Methanothrix, accounting to 46.31% of the archaea (Figure 5A). At the end of the experiment, Methanobacterium became dominant to reach 74.06% in the control reactor and 82.37% in the nZVI reactor, while the proportion of Methanobacterium was less (23.91%) and Methanothrix (33.38%) still made up a considerable proportion. It has been reported that Methanothrix (formerly Methanoseta) and Geobacter could cooperate through direct interspecies electron transfer (DIET) through e-pili and c-type cytochrome in paddy soils. Holmes et al. found that Methanothrix highly expressed the gene for reduction of carbon dioxide to methane via DIET mode40. Thus, the high proportion of Methanothrix agreed well with the increase of EET efficiency with nFe3O4. Further classification of methanogens (Figure 5B) showed that hydrogenotrophic methanogens were the dominant archaea in the control (85.31%) and the nZVI (93.79%) reactor,

including

Methanobacterium41,

Methanospirillum42,

Methanolinea43,

Methanoregula44 and Methanosphaerula45. The hydrogenotrophic methanogens were responsible for consuming hydrogen produced from anaerobic oxidation of organics. Like ZVI, nZVI produced more H2 at the beginning of the dosage and maintained a low ORP environment, which are good for the growth of hydrogenotrophic methanogens6. However, with the increasing organic load and the inhibition of H2 diffusion by over secretion of EPS, the performance of nZVI reactor significantly dropped. Comparatively,


Page 17 of 35 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


in the nFe3O4 reactor the proportion of hydrogenotrophic methanogens dropped to 54.23%, while the relative abundance of acetotrophic methanogens, including Methanothrix46, Methanomassiliicoccus46 and Methanosarcina47, significantly ascended to 44.69%. These results further confirmed that although DIET process was established in the nFe3O4 reactor, IHT was gradually replaced by DIET. Taxonomic classification in Figure 5C revealed that most of the bacteria belonged to Clostridium in the nZVI reactor (67.87%) and the nFe3O4 reactor (83.55%). Clostridium is well-known as fermentative48 and electroactive anaerobe, which was capable of oxidizing many organic substrates like lactate and sucrose49 and then transports electrons to iron oxides or other microbes. With the addition of nano magnetite, Clostridium was able to directly transport electron to Methanothrix with magnetite as an electron mediator even without e-pili of Geobacter50. Analysis on the functional gene (Figure 6A) and the metabolism pathway prediction (Figure 6B)51 further implied the information about mass or electron transfer caused by nZVI or nFe3O4. As shown in Figure 6A, the high expression of functional genes of the nFe3O4 reactor including carbohydrate transport and metabolism, energy production and conversion, indicated that nano magnetite could facilitate the decomposition of complex organics52. The gene conducting inorganic ion transport and amino acid transport and metabolism were also up-regulated, which was consistent with the high proportion of electrical active microorganism Clostridium of the nFe3O4 reactor. As a result,



microorganisms could take advantage of nano magnetite-mediated dissimilatory ironreduction to secret electron shuttles (such as protein-like cytochromes and humic-like substances) for establishing a direct connection with others (such as Clostridium to Methanothrix). The metabolism pathways prediction (Figure 6B) showed that the gene involved in membrane transport expressed higher in the nFe3O4 reactor than that in other two, suggesting that nFe3O4 stimulated a more active electron transfer involved in anaerobic digestion. Amino acid metabolism and carbohydrate metabolism were also upregulated in nFe3O4 reactor, indicating that more protein was produced and better performance of COD removal was achieved in reactor with nFe3O4.

Conclusions Dosing nFe3O4 into the anaerobic digestion reactor significantly increased the COD removal rate and methane production whereas nZVI led to a dramatic decrease of the performances. The increased proteins and humic-like substances by dosing nFe3O4 promoted the electroactivity of the EPS and conductivity of the sludge, which benefited to establish the DIET mode that enhanced anaerobic digestion. Comparatively, the nZVI addition caused the increase of EPS secretion due to the microbial resistance of nZVI disruption, which might inhibit the mass transfer, resulting in negative impacts on IHT mode.

Associated Content


Page 18 of 35

Page 19 of 35 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


Supporting Information Available. Details about method for DNA extraction, PCR amplification, high-throughput 16S rRNA pyrosequencing and FTIR spectra analysis in the Supporting Information.

Acknowledgements The authors acknowledge the financial support from the National Natural Science Foundation of China (51578105). Conflict of interest statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.



References (1) Martins, G.; Salvador, A. F.; Pereira, L.; Alves, M. M. Methane Production and Conductive Materials: A Critical Review. Environ. Sci. Technol. 2018, 52 (18), 10241– 10253, DOI 10.1021/acs.est.8b01913. (2) Stams, A. J. M.; Plugge, C. M. Electron Transfer in Syntrophic Communities of Anaerobic Bacteria and Archaea. Nat. Rev. Microbiol. 2009, 7 (8), 568–577, DOI 10.1038/nrmicro2166. (3) Cruz Viggi, C.; Rossetti, S.; Fazi, S.; Paiano, P.; Majone, M.; Aulenta, F. Magnetite Particles Triggering a Faster and More Robust Syntrophic Pathway of Methanogenic Propionate Degradation. Environ. Sci. Technol. 2014, 48 (13), 7536–7543, DOI 10.1021/es5016789. (4) Zhao, Z.; Zhang, Y.; Li, Y.; Quan, X.; Zhao, Z. Comparing the Mechanisms of ZVI and Fe3O4 for Promoting Waste-Activated Sludge Digestion. Water Res. 2018, 144, 126–133, DOI 10.1016/j.watres.2018.07.028. (5) Liu, Y.; Zhang, Y.; Quan, X.; Li, Y.; Zhao, Z.; Meng, X.; Chen, S. Optimization of Anaerobic Acidogenesis by Adding Fe0 Powder to Enhance Anaerobic Wastewater Treatment. Chem. Eng. J. 2012, 192, 179–185, DOI 10.1016/j.cej.2012.03.044. (6) Feng, Y.; Zhang, Y.; Quan, X.; Chen, S. Enhanced Anaerobic Digestion of Waste Activated Sludge Digestion by the Addition of Zero Valent Iron. Water Res. 2014, 52, 242–250, DOI 10.1016/j.watres.2013.10.072. (7) Romero-Güiza, M. S.; Vila, J.; Mata-Alvarez, J.; Chimenos, J. M.; Astals, S. The


Page 20 of 35

Page 21 of 35 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


Role of Additives on Anaerobic Digestion: A Review. Renew. Sustain. Energy Rev. 2016, 58, 1486–1499, DOI 10.1016/j.rser.2015.12.094. (8) Kim, J. Y.; Lee, C.; Love, D. C.; Sedlak, D. L.; Yoon, J.; Nelson, K. L. Inactivation of MS2 Coliphage by Ferrous Ion and Zero-Valent Iron Nanoparticles. Environ. Sci. Technol. 2011, 45 (16), 6978–6984, DOI 10.1021/es201345y. (9) Wang, Y.; Wang, D.; Fang, H. Comparison of Enhancement of Anaerobic Digestion of Waste Activated Sludge through Adding Nano-Zero Valent Iron and Zero Valent Iron. RSC Adv. 2018, 8 (48), 27181–27190, DOI 10.1039/c8ra05369c. (10)

Rotaru, A. E.; Shrestha, P. M.; Liu, F.; Shrestha, M.; Shrestha, D.; Embree, M.;

Zengler, K.; Wardman, C.; Nevin, K. P.; Lovley, D. R. A New Model for Electron Flow during Anaerobic Digestion: Direct Interspecies Electron Transfer to Methanosaeta for the Reduction of Carbon Dioxide to Methane. Energy Environ. Sci. 2014, 7 (1), 408– 415, DOI 10.1039/c3ee42189a. (11)

Storck, T.; Virdis, B.; Batstone, D. J. Modelling Extracellular Limitations for

Mediated versus Direct Interspecies Electron Transfer. ISME J. 2016, 10 (3), 621–631, DOI 10.1038/ismej.2015.139. (12)

Dang, Y.; Holmes, D. E.; Zhao, Z.; Woodard, T. L.; Zhang, Y.; Sun, D.; Wang,

L.-Y.; Nevin, K. P.; Lovley, D. R. Enhancing Anaerobic Digestion of Complex Organic Waste with Carbon-Based Conductive Materials. Bioresour. Technol. 2016, 220, 516– 522, DOI 10.1016/j.biortech.2016.08.114.



(13)

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. Microbiol. 2015, 17 (5), 1533–1547, DOI 10.1111/1462-2920.12576. (14)

Yang, Z.; Guo, R.; Shi, X.; Wang, C.; Wang, L.; Dai, M. Magnetite

Nanoparticles Enable a Rapid Conversion of Volatile Fatty Acids to Methane. RSC Adv. 2016, 6 (31), 25662–25668, DOI 10.1039/C6RA02280D. (15)

Zhao, Z.; Zhang, Y.; Woodard, T. L.; Nevin, K. P.; Lovley, D. R. Enhancing

Syntrophic Metabolism in Up-Flow Anaerobic Sludge Blanket Reactors with Conductive Carbon Materials. Bioresour. Technol. 2015, 191, 140–145, DOI 10.1016/j.biortech.2015.05.007. (16)

Zhao, Z.; Zhang, Y.; Wang, L.; Quan, X. Potential for Direct Interspecies

Electron Transfer in an Electric-Anaerobic System to Increase Methane Production from Sludge Digestion. Sci. Rep. 2015, 5 (April), 11094, DOI 10.1038/srep11094. (17)

Frølund, B.; Palmgren, R.; Keiding, K.; Nielsen, P. H. Extraction of

Extracellular Polymers from Activated Sludge Using a Cation Exchange Resin. Water Res. 1996, 30 (8), 1749–1758, DOI 10.1016/0043-1354(95)00323-1. (18)

Sedmak, J. J.; Grossberg, S. E. A Rapid, Sensitive, and Versatile Assay for

Protein Using Coomassie Brilliant Blue G250. Anal. Biochem. 1977, 79 (1–2), 544–552, DOI 10.1016/0003-2697(77)90428-6. (19)

Masuko, T.; Minami, A.; Iwasaki, N.; Majima, T.; Nishimura, S. I.; Lee, Y. C.


Page 22 of 35

Page 23 of 35 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


Carbohydrate Analysis by a Phenol-Sulfuric Acid Method in Microplate Format. Anal. Biochem. 2005, 339 (1), 69–72, DOI 10.1016/j.ab.2004.12.001. (20)

Chen, W.; Westerhoff, P.; Leenheer, J. A.; Booksh, K. Fluorescence

Excitation-Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci. Technol. 2003, 37 (24), 5701–5710, DOI 10.1021/es034354c. (21)

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. Microbiol. 2015, 17 (5), 1533–1547, DOI 10.1111/1462-2920.12576. (22)

Zhao, Z.; Zhang, Y.; Yu, Q.; Dang, Y.; Li, Y.; Quan, X. Communities

Stimulated with Ethanol to Perform Direct Interspecies Electron Transfer for Syntrophic Metabolism of Propionate and Butyrate. Water Res. 2016, 102, 475–484, DOI 10.1016/j.watres.2016.07.005. (23)

Aeschbacher, M.; Sander, M.; Schwarzenbach, R. P. Novel Electrochemical

Approach to Assess the Redox Properties of Humic Substances. Environ. Sci. Technol. 2010, 44 (1), 87–93, DOI 10.1021/es902627p. (24)

Gorski, C. A.; Klüpfel, L. E.; Voegelin, A.; Sander, M.; Hofstetter, T. B.

Redox Properties of Structural Fe in Clay Minerals: 3. Relationships between Smectite Redox and Structural Properties. Environ. Sci. Technol. 2013, 47 (23), 13477–13485, DOI 10.1021/es403824x.



(25)

Li, S.; Cao, Y.; Bi, C.; Zhang, Y. Promoting Electron Transfer to Enhance

Anaerobic Treatment of Azo Dye Wastewater with Adding Fe(OH)3. Bioresour. Technol. 2017, DOI 10.1016/j.biortech.2017.08.066. (26)

Wang, M.; Zhao, Z.; Niu, J.; Zhang, Y. Potential of Crystalline and Amorphous

Ferric Oxides for Biostimulation of Anaerobic Digestion. ACS Sustain. Chem. Eng. 2019, 7 (1), 697–708, DOI 10.1021/acssuschemeng.8b04267. (27)

Xie, Y.; Dong, H.; Zeng, G.; Tang, L.; Jiang, Z.; Zhang, C.; Deng, J.; Zhang,

L.; Zhang, Y. The Interactions between Nanoscale Zero-Valent Iron and Microbes in the Subsurface Environment: A Review. J. Hazard. Mater. 2017, 321, 390–407, DOI 10.1016/j.jhazmat.2016.09.028. (28)

Yang, Y.; Guo, J.; Hu, Z. Impact of Nano Zero Valent Iron (NZVI) on

Methanogenic Activity and Population Dynamics in Anaerobic Digestion. Water Res. 2013, 47 (17), 6790–6800, DOI 10.1016/j.watres.2013.09.012. (29)

Zhuang, L.; Tang, J.; Wang, Y.; Hu, M.; Zhou, S. Conductive Iron Oxide

Minerals Accelerate Syntrophic Cooperation in Methanogenic Benzoate Degradation. J. Hazard. Mater. 2015, 293, 37–45, DOI 10.1016/j.jhazmat.2015.03.039. (30)

Sheng, G. P.; Yu, H. Q.; Li, X. Y. Extracellular Polymeric Substances (EPS) of

Microbial Aggregates in Biological Wastewater Treatment Systems: A Review. Biotechnol. Adv. 2010, 28 (6), 882–894, DOI 10.1016/j.biotechadv.2010.08.001. (31)

Kim, A. S.; Chen, H.; Yuan, R. EPS Biofouling in Membrane Filtration: An


Page 24 of 35

Page 25 of 35 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


Analytic Modeling Study. J. Colloid Interface Sci. 2006, 303 (1), 243–249, DOI 10.1016/j.jcis.2006.07.067. (32)

Frølund, B.; Griebe, T.; Nielsen, P. H. Enzymatic Activity in the Activated-

Sludge Floc Matrix. Appl. Microbiol. Biotechnol. 1995, 43 (4), 755–761, DOI 10.1007/BF00164784. (33)

Ye, J.; Hu, A.; Ren, G.; Chen, M.; Tang, J.; Zhang, P.; Zhou, S.; He, Z.

Enhancing Sludge Methanogenesis with Improved Redox Activity of Extracellular Polymeric Substances by Hematite in Red Mud. Water Res. 2018, 134, 54–62, DOI 10.1016/j.watres.2018.01.062. (34)

Flemming, H.-C.; Wingender, J. The Biofilm Matrix. Nat. Rev. Microbiol.

2010, 8 (9), 623–633, DOI 10.1038/nrmicro2415. (35)

Ross, D. E.; Brantley, S. L.; Tien, M. Kinetic Characterization of OmcA and

MtrC, Terminal Reductases Involved in Respiratory Electron Transfer for Dissimilatory Iron Reduction in Shewanella Oneidensis MR-1. Appl. Environ. Microbiol. 2009, 75 (16), 5218–5226, DOI 10.1128/AEM.00544-09. (36)

Watanabe, K.; Manefield, M.; Lee, M.; Kouzuma, A. Electron Shuttles in

Biotechnology. Curr. Opin. Biotechnol. 2009, 20 (6), 633–641, DOI 10.1016/j.copbio.2009.09.006. (37)

Shi, L.; Dong, H.; Reguera, G.; Beyenal, H.; Lu, A.; Liu, J.; Yu, H. Q.;

Fredrickson, J. K. Extracellular Electron Transfer Mechanisms between



Microorganisms and Minerals. Nat. Rev. Microbiol. 2016, 14 (10), 651–662, DOI 10.1038/nrmicro.2016.93. (38)

Aeschenbacher, M.; Sander, M.; Schwarzenbach, R. P. Novel Electrochemical

Approach to Assess the Redox Properties of Humic Substances. Environ. Sci. Technol. 2010, 44 (1), 87–93. (39)

Lovley, D. R. Live Wires: Direct Extracellular Electron Exchange for

Bioenergy and the Bioremediation of Energy-Related Contamination. Energy Environ. Sci. 2011, 4 (12), 4896, DOI 10.1039/c1ee02229f. (40)

Holmes, D. E.; Shrestha, P. M.; Walker, D. J. F.; Dang, Y.; Nevin, K. P.;

Woodard, T. L.; Lovley, D. R. Metatranscriptomic Evidence for Direct Interspecies Electron Transfer between Geobacter and Methanothrix Species in Methanogenic Rice Paddy Soils. Appl. Environ. Microbiol. 2017, 83 (9), 1–11, DOI 10.1128/AEM.0022317. (41)

Smith, D. R.; Doucette-Stamm, L. A.; Deloughery, C.; Lee, H.; Dubois, J.;

Aldredge, T.; Bashirzadeh, R.; Blakely, D.; Cook, R.; Gilbert, K.; et al. Complete Genome Sequence of Methanobacterium Thermoautotrophicum DeltaH: Functional Analysis and Comparative Genomics. J. Bacteriol. 1997, 179 (22), 7135–7155, DOI 10.1128/jb.179.22.7135-7155.1997. (42)

Ferry, J. G.; Smith, P. H.; Wolfe, R. S. Methanospirillum, a New Genus of

Methanogenic Bacteria , and Characterization of Methanospirillum Hungatei Sp . Nov .


Page 26 of 35

Page 27 of 35 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


Int. J. Syst. Bacteriol. 1974, 24 (4), 465–469, DOI 10.1099/00207713-24-4-465. (43)

Imachi, H.; Sakai, S.; Sekiguchi, Y.; Hanada, S.; Kamagata, Y.; Ohashi, A.;

Harada, H. Methanolinea Tarda Gen. Nov., Sp. Nov. a Methane-Producing Archaeon Isolated from a Methanogenic Digester Sludge. Int. J. Syst. Evol. Microbiol. 2008, 58 (1), 294–301, DOI 10.1099/ijs.0.65394-0. (44)

Bräuer, S. L.; Cadillo-Quiroz, H.; Ward, R. J.; Yavitt, J. B.; Zinder, S. H.

Methanoregula Boonei Gen. Nov., Sp. Nov., an Acidiphilic Methanogen Isolated from an Acidic Peat Bog. Int. J. Syst. Evol. Microbiol. 2011, 61 (1), 45–52, DOI 10.1099/ijs.0.021782-0. (45)

Cadillo-Quiroz, H.; Yavitt, J. B.; Zinder, S. H. Methanosphaerula Palustris

Gen. Nov., Sp. Nov., a Hydrogenotrophic Methanogen Isolated from a Minerotrophic Fen Peatland. Int. J. Syst. Evol. Microbiol. 2009, 59 (5), 928–935, DOI 10.1099/ijs.0.006890-0. (46)

Huser, B. A.; Wuhrmann, K.; Zehnder, A. J. B. Methanothrix Soehngenii Gen.

Nov. Sp. Nov., a New Acetotrophic Non-Hydrogen-Oxidizing Methane Bacterium. Arch. Microbiol. 1982, 132 (1), 1–9, DOI 10.1007/BF00690808. (47)

De Vrieze, J.; Hennebel, T.; Boon, N.; Verstraete, W. Methanosarcina: The

Rediscovered Methanogen for Heavy Duty Biomethanation. Bioresour. Technol. 2012, 112, 1–9, DOI 10.1016/j.biortech.2012.02.079. (48)

Jiang, L.; Wang, J.; Liang, S.; Cai, J.; Xu, Z.; Cen, P.; Yang, S.; Li, S.



Enhanced Butyric Acid Tolerance and Bioproduction by Clostridium Tyrobutyricum Immobilized in a Fibrous Bed Bioreactor. Biotechnol. Bioeng. 2011, 108 (1), 31–40, DOI 10.1002/bit.22927. (49)

Niessen, J.; Schröder, U.; Scholz, F. Exploiting Complex Carbohydrates for

Microbial Electricity Generation - A Bacterial Fuel Cell Operating on Starch. Electrochem. commun. 2004, 6 (9), 955–958, DOI 10.1016/j.elecom.2004.07.010. (50)

Sydow, A.; Krieg, T.; Mayer, F.; Schrader, J.; Holtmann, D. Electroactive

Bacteria—molecular Mechanisms and Genetic Tools. Appl. Microbiol. Biotechnol. 2014, 98 (20), 8481–8495, DOI 10.1007/s00253-014-6005-z. (51)

Parks, D. H.; Tyson, G. W.; Hugenholtz, P.; Beiko, R. G. STAMP: Statistical

Analysis of Taxonomic and Functional Profiles. Bioinformatics 2014, 30 (21), 3123– 3124, DOI 10.1093/bioinformatics/btu494. (52)

Zhao, Z.; Li, Y.; Quan, X.; Zhang, Y. Towards Engineering Application:

Potential Mechanism for Enhancing Anaerobic Digestion of Complex Organic Waste with Different Types of Conductive Materials. Water Res. 2017, 115, 266–277, DOI 10.1016/j.watres.2017.02.067.


Page 28 of 35

Page 29 of 35

Figures A

Control nZVI nFe3O4

0.8

COD Removal Rate

Step 2

Step 1

1.0

5000

4000 0.6 3000 0.4 2000

Infuluent (mg/L)

0.2 1000 0.0 0

20

40

60

80

100

120

Time (d)

B 2500

Control nZVI nFe3O4

2000

CH4 (mL)

Step 2

Step 1

5000 4000

1500

3000

1000

2000

500

Infuluent (mg/L)

1000

0 0

20

40

60

80

0 120

100

Time (d) C 4000 3500

Concentration of VFAs (mg/L)

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


Isobutyrate Propionate Acetate

nZVI

3000 2500 2000

Control

1000 500 0

Control

nZVI

1500

nFe3O4 nFe3O4

Control nZVI nFe3O4

Day1-Day40

Day41-Day80

Day81-Day120

Figure 1. COD removal rate (A), CH4 production (B) and VFAs (C) of the reactors.



1000

Concentration (mg/g TOC)

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

Control nZVI nFe3O4

800

600

400

200

0

Protein

Polysaccharide

Figure 2. Polysaccharide and protein in the EPS secreted by the suspended sludge in the three reactors after 120 days’ experiment.


Page 30 of 35

Page 31 of 35

A 400

1300 1138

Ex (nm)

350

975.0 812.5

V 300

650.0

IV

487.5 325.0

250

II

I

162.5

III

0.000

200 200

300

400

500

Em (nm)

B 400

1300 1138

350

975.0

Ex (nm)

V

812.5

300

650.0

IV

487.5 325.0

250

III

II

I

162.5 0.000

200 200

300

400

500

Em (nm)

C 400

1300 1138

350

Ex (nm)

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


975.0 812.5

V

300

650.0

IV

487.5 325.0

250

II

I 200 200

III

162.5 0.000

300

400

500

Em (nm)

Figure 3. EEM contours of the EPS from the suspended sludge in control reactor (A) and reactors dosing nano ZVI (B) and nano magnetite (C).



electron donate and accept (μmol e- /g TOC)

A 120 Control nZVI nFe3O4

100 80 60 40 20 0

EAC

EDC

B

25 20 Conductivity (μS/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

15 10 5 0

0.1M NaCl

Fresh sludge

Fresh sludge + Nano nFe 3O 4 magnetite

Control

Nano ZVI nZVI

nFe Nano nFe33O O44 Magnetite

Figure 4. The conductivity of suspended sludge of the three reactors after 120 days’ experiment comparing with raw sludge, raw sludge with nFe3O4 and 0.1 M NaCl.


Page 32 of 35

Page 33 of 35

(A) 100

Relative Abundance (%)

80

others unclassified Methanobrevibacter Methanosarcina Methanosphaerula Methanoregula Methanosphaera Methanolinea Methanomassiliicoccus Methanospirillum Methanothrix Methanobacterium

60

40

20

0

Origin

Step1

Control

Nano nZVI nFe Nano 3O4 ZVI Magnetite

(B)

Relative abundance (%)

100 others unclassified Hydrogenotrophic Methanogen Acetotrophic Methanogen

80

60

40

20

0

Origin

Step1

Control

nFe nZVI Nano Nano 3O4 ZVI Magnetite

(C) 100

unclassified others Armatimonadetes_gp6 Anaerovorax Thermogutta Bellilinea Methanothrix Vampirovibrio Mariniphaga Smithella Lactococcus Sedimentibacter Paludibacter Treponema Ethanoligenens Thermoflavimicrobium Ornatilinea Syntrophomonas Desulfovibrio Levilinea Saccharofermentans Aminicenantes_genera_incertae_sedis Anaeroarcus Longilinea Clostridium sensu stricto

80

Relative Abundance (%)

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

40

20

0

Origin

Step1

nZVI Control Nano ZVI nFe Nano 3O4 nZVI Magnetite

Figure 5. Microbial community analysis of the suspended sludge for archaea (A) and bacteria (C) at genus level and the proportion of Hydrogenotrophic methanogens and Acetotrophic methanogens (B) in each reactor.



Figure 6. Heatmap of functional gene (A) and metabolic pathways (B) detected from the suspended sludge in the three anaerobic reactors. Different colors represent different expression level of a particular function or metabolic pathway. Green color means down-regulated expression and red color represents up-regulated expression.


Page 34 of 35

Page 35 of 35 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


TOC/Abstract Graphic

Synopsis Nano magnetite affected the anaerobic digestion mode from IHT to DIET by promoting electron transfer capacities of EPS, which significantly increased the methane production.


Regulating secretion of extracellular polymeric substances through

Recommend Documents