Extracellular Polymeric Substances from Geobacter sulfurreducens

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Extracellular polymeric substances from Geobacter sulfurreducens biofilms in microbial fuel cells Markus Stöckl, Natascha Caroline Teubner, Dirk Holtmann, Klaus-Michael Mangold, and Wolfgang Sand ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14340 • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 8, 2019

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ACS Applied Materials & Interfaces

Extracellular polymeric substances from Geobacter sulfurreducens biofilms in microbial fuel cells

Authors Markus Stöckl1, Natascha Caroline Teubner2, Dirk Holtmann3, Klaus-Michael Mangold1, Wolfgang Sand4*

Authors contact information 1

DECHEMA-Forschungsinstitut, Department of Electrochemistry, Theodor-Heuss-Allee 25,

60486 Frankfurt a. M., Germany, [email protected] 2

University of Duisburg-Essen, Biofilm Centre, Universitätsstr. 5, 45141 Essen,

[email protected] 3

DECHEMA-Forschungsinstitut, Department of Industrial Biotechnology, Theodor-Heuss-

Allee 25, 60486 Frankfurt a. M. Germany, [email protected] 4

Donghua University, College of Environmental Science and Engineering, Shanghai, 201620 ,

China; Technical Universtiy and Mining Academy, 09599 Freiberg, Germany; University of Duisburg-Essen, Universitätsstr. 5, 45141 Essen, [email protected] * Corresponding author

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Abstract Bioelectrochemical systems (BES) are hybrid systems using electroactive bacteria and solid electrodes, which serve as electron donor or acceptor for microorganisms. When forming a biofilm on the electrode, bacteria secrete extracellular polymeric substances (EPS). However, EPS excretion of electroactive biofilms in BES has been rarely studied so far. Consequently, the aim of this study was the development of a routine including the electrochemical cultivation, the biofilm harvesting, fractionation and the biochemical analysis of the EPS secreted by G. sulfurreducens under electroactive conditions. G. sulfurreducens was cultivated in microbial fuel cell mode on graphite based electrodes polarized

to

+400 mV vs. Ag/AgCl for 8 d. A maximum current density of 172 ± 29 μA cm-2 was reached after 7 d. EPS secreted from the biofilms, were harvested and fractioned into soluble, loosely bound and tightly bound EPS and biochemically analyzed. Electroactive cultures secreted significantly more EPS compared to cells grown under standard heterotrophic conditions (fumarate respiration). With 116 pg cell-1 the highest amount of EPS was measured for the soluble EPS fraction of G. sulfurreducens using anodic respiration, followed by the tightly bound (18 pg cell-1) and loosely bound (11 pg cell-1) fractions of the EPS. Proteins were found to dominate all EPS fractions of the biofilms grown under electrochemical conditions. To the best of the authors knowledge, these experiments are the first approach towards a complete analysis of the main EPS components of G. sulfurreducens under anode respiring conditions.


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Key words: bioelectrochemical systems, anodic respiration, extracellular polymeric substances, biofilm, surface attachment



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1 Introduction The combination of electroactive bacteria and electrochemical techniques offers a wide range of application opportunities. Electroactive bacteria are able to exchange electrons with electrodes and can function as self-reproducing biocatalysts in bioelectrochemical systems (BES). The latter are characterized by high reaction specificity, high reaction control and high coulombic efficiencies 1. Two of the most prominent examples of BES are the sustainable generation of electricity from (organic) waste (microbial fuel cell, MFC) and the consumption of current to produce valuable substances such as biofuels or process chemicals (microbial electrosynthesis, MES). Thereby, the interaction of the electroactive bacteria with the electrode interface is of great interest, because the extracellular electron transfer (EET) is one of the factors determining the performance of such systems. In many BES the electroactive bacteria are present as biofilms on the electrode surface, since the EET is a surface limited process and physical contact to the electrode is required for a direct electron transfer 2. A variety of studies already focused on the interplay between electroactive bacteria and electrode surfaces. The applied techniques range from microscopic and spectroscopic analyses of electroactive biofilms 3–8 over the genetic 6,9 and electrochemical analyses 10–12 to the combination of two or more monitoring techniques

5,13,14

. In contrast to these

techniques, the biochemical analysis of EPS of biofilms grown on electrodes is not yet a routine research tool. Generally, EPS summarize hydrated biopolymers (including polysaccharides, proteins, nucleic acids and lipids) that are secreted by biofilm cells from cytoplasma via periplasma to outside

15

. In BES, an important role of the secreted EPS is

most probably the establishment of physical contact to the electrode surface. Additionally, EPS are also supposed to have an influence on the EET of sessile microorganisms as it has been shown in a couple of studies. In a series of experiments Li and colleagues identified heme-binding proteins in the EPS from Shewanella oneidensis and Pseudomonas putida. They studied the redox properties of the EPS using cyclic voltammetry, and suggested that these are important redox components mediating electron transfer

16

. In another study,

Rollefson et al. studied the polysaccharide network in an electroactive biofilm of Geobacter sulfurreducens and highlighted their importance for extracellular anchoring, cell-cell agglutination, and localization of essential extracellular cytochromes


17

.

Furthermore,

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Kouzuma et al. focused on the polysaccharides in the EPS of S. oneidensis MR-1 biofilms and demonstrated that cell surface polysaccharides also the current generation in MFCs.18. The named studies indicate the importance of EPS in BES and the EET of electroactive microorganisms. However, approaches pointing towards a comprehensive identification and quantification of the EPS and its constituents from electrode grown cells in BES have not been published so far as it has exemplarily been for leaching bacteria 19–21. Wikiel published a comprehensive analysis of EPS (proteins, carbohydrates, humic substances, peptides and extracellular DNA)) secreted by the leaching bacterium Desulfovibrio alaskensis 22. In addition to the different EPS group components, the spatial organization of EPS embedding the cells and the consequent harvesting of the EPS have to be taken into consideration for analysis. As it has been illustrated by Hsieh et al. 23 and Nielsen and Jahn in: 24

, EPS might be classified into soluble (SOL), loosely bound (LB) and tightly bound (TB) EPS,

depending on the physical attachment to the cell wall and the subsequent extraction possibility. Consequently, the comprehensive analysis of EPS group components (proteins, carbohydrates, humic substances, peptides and extracellular DNA) of the electrode active model organism G. sulfurreducens

25,26

was the main goal of our work in order to establish

EPS analysis in further BES research.

2 Materials and Methods 2.1 Microorganism and growth media Geobacter sulfurreducens was supplied from the DSMZ (DSM-12127, Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultivated under heterotrophic and anaerobic conditions according to the recommendation of the DSMZ. Acetate was the sole electron and carbon source, whereas fumarate served electron acceptor. Detailed cultivation conditions are presented in the supporting information (S1–S4). G. sulfurreducens cells grown for EPS production as control employing fumarate respiration were cultivated in 1 L septum flasks containing 350 mL medium at 30 °C without shaking.



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2.2 Laboratory electrochemical H-cell For the bioelectrochemical cultivation of G. sulfurreducens, a modified H-cell set up was developed based on reactor systems described before 6,27–29. Two 100 mL glass bottles were connected via two flanges and separated by a circular proton exchange membrane (Nafion 117, Sigma Aldrich, St. Louis, USA) to form an H-shaped reactor with separated working electrode (WE) and counter electrode (CE) chambers. Furthermore, the WE chamber was modified by the addition of a second flange with an inner diameter of 2.5 cm for the working electrode (geometrical AWE = 4.9 cm2). The WE was pressed on the second flange from the outer side with a self-designed clamp system constructed by the house internal work shop and sealed with a 1 mm thick circular silicon mat. As material for the working electrode a graphite based material (PPG86, Eisenhuth GmbH & Co. KG, Osterode am Harz, Germany) was chosen. More detailed information and an image of a completely mounted H-cell is provided in the supporting information (Figure S1). Both chambers of the H-cell reactor were filled with growth media lacking disodium fumarate. Finally, for constant potential control an Ag/AgCl/KClsat reference electrode (Sensortechnik Meinsberg, Waldheim, Germany) was added to the system via a HaberLuggin capillary.

2.3 Electrochemical cultivation of G. sulfurreducens Electrochemical cultivation of G. sulfurreducens was done in MFC mode, polarizing the WE as anode under an incubation hood (TH 30, Edmund Bühler GmbH, Hechingen, Germany) at 30 °C. The electrochemical cells were connected to a multi potentiostat (IPS Elektronik GmbH & Co KG, Münster, Germany) and placed on two magnetic stirrers. The medium in the WE chamber was degassed with a cannula connected to a 0.22 µm pore size PVDF syringe filter to avoid contamination. N2/CO2 (80/20 v/v) gas flow was kept constant throughout the whole experiment at 30 mL min-1. After 3 h of degassing, the WE was polarized to +400 mV vs. Ag/AgCl to stimulate fast interaction of the cells with the electrode. G. sulfurreducens cells were harvested in stationary phase from the preculture, washed with anaerobic medium and added 30 min after starting the polarization to reach an OD600 of 0.1 corresponding to


5 ∙ 107 cells mL-1.

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MFC mode was run for 8 d before biofilm harvesting and EPS extraction. The relatively high electrode potential was chosen to stimulate fast interaction of the cells with the electrode. H-cells were run always in triplicates, current density was referred to the geometrical WE surface (4.9 cm2).

2.4 Biofilm staining and electrode imaging To qualitatively characterize morphology and thickness confocal laser scanning microscopy (CLSM) images were taken from DAPI stained biofilms. Therefore, G. sulfurreducens was grown on graphite plate WEs in a MFC as described earlier. To qualitatively evaluate biofilm harvesting from the electrode surface with a cell scraper, both Epifluorescence Microscopy (EFM) and Scanning Electron Microscopy (SEM) images were made from biofilms attached to a WE. Therefore, G. sulfurreducens was grown on graphite plate WEs in a MFC as described earlier. After dismounting the WE, half of the biofilm was scratched off/harvested from the electrode, whereas the other half remained on the surface. Samples were washed with phosphate buffered saline (PBS, 136.89 mM NaCl; 2.68 mM KCl; 10 mM Na2HPO4 ∙ 2 H2O; 1.98 mM KH2PO4, pH = 7) three times and further processed for EFM or SEM imaging, respectively. More detailed information concerning fluorescence staining, CLSM, EFM and SEM imaging are provided in S6.

2.5 EPS harvest and extraction Due to the relatively small electrode surface and the, consequently, small mass of biofilm on the electrode surface, three electrochemical cultivations were run in parallel. Both, biofilms and supernatant of the WE-chamber of the three parallel cultivations, were then combined to one sample each at the end of experiments for the biochemical analysis. This was done to gain EPS concentrations within the detection range of the biochemical analysis. The harvesting and EPS extraction routine was developed on the results presented by Wikiel

22

and are graphically displayed in Figure 1. Detailed information describing the single steps are provided in S7.



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2.6 EPS harvest of fumarate respiring cells of G. sulfurreducens (non-electroactive control) Besides the EPS of anode-respiring cells cultivated in a MFC,

heterotrophically-grown

G. sulfurreducens cells respiring on fumarate were analyzed for their EPS composition as non-electroactive control. Therefore, cells grown to exponential phase (EXP) for 2 d as well as cells grown to stationary (STAT) phase for 8 d were harvested. Cultivation conditions were the same as previously described. Schematically, the EPS harvesting can be also seen in Figure 1, which was the same as for the MFC-grown cells, if not stated otherwise. Detailed information describing the single steps are provided in S8.

2.7 Biochemical EPS analysis For biochemical EPS analysis, samples were dissolved in 2 mL nuclease free and sterile H2O. Determinations were conducted in 96 well microtiter plates and a Tecan microtiter plate reader. Methods were partially adapted and modified from Wikiel 22. Concentrations of the EPS compounds were referred to single cells. Therefore, G. sulfurreducens was cultivated electrochemically in a H-cell reactor in duplicate. Biofilms were harvested as described before and resuspended in 30 mL PBS. The total amount of cells was determined by cell counting with a Neubauer chamber. For the controls OD600 of the heterotrophically grown cultures in EXP and STAT phase was used to determine the total amount of cells. Standard deviations of the respective EPS components were calculated from the three biologically independent replicates. The total protein concentration in the respective EPS fractions was evaluated with a BCA protein assay (Pierce BCA Assay Kit 23225/23227, Thermo Fisher Scientific, Waltham, USA). Concentration of carbohydrates was measured according to Dubois determined Blumenkrantz

photometrically and

using

Na2B4O7 and

Asboe-Hansen31.

Lipids

30

. Uronic acids were

ß-hydroxydiphenyl

were

initially

according

extracted

with

to a

chloroform/methanol mixture from the water phase of the dissolved EPS and then analyzed by colorimetry with vanillin according to van Handel 32. As last parameter double stranded eDNA was determined with the PICOGREEN® dsDNA assay (Invitrogen, California, USA)


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according to the standard protocol provided by the manufacturer. More detailed information describing the respective assays are provided in S9. 3 Results 3.1 Electrochemical cultivation G. sulfurreducens was grown under anode respiring conditions in a microbial fuel cell to produce electroactive and anode respiring biofilms. Therefore, three H-cells were run in parallel always and biofilms were harvested and combined after approximately 8 d. The experiments were performed in triplicate (9 MFC cultivations in total). The mean current density curve of the MFC including standard deviation is presented in Figure 2. Directly after cell addition a current increase was observed. Development of the current density might be described by a lag phase (day 0–day 1), an exponential phase (day 1–day 6) and a stationary phase (day 6–day 8). The maximum current density was reached after approximately 7 d at a value of 172 ± 29 μA cm-2 (referred to geometrical WE surface of 4.9 cm2). A small additional current decrease was observed prior to the end of the electrochemical cultivation.

3.2 CLSM imaging, biofilm harvest and subsequent EFM imaging CLSM images were taken from DAPI stained cells to characterize morphology and thickness of the biofilms. As can be seen in figure 3 the biofilm is characterized by a wavy and dense structure covering the electrode surface. The thickness of the biofilm ranged between 5 and 10 μm. The procedure of harvesting the electrochemically cultivated biofilm from the dismounted anode is displayed by the series of images in figure S2. Scratching off the biofilm with a cell scraper resulted in a complete removal of the visible part of the attached cells from the surface. Additionally, EFM and SEM imaging were performed to qualitatively evaluate biofilm harvest from the electrode surface. Biofilms were partially scratched off to visualize both, intact electroactively grown biofilm and the electrode surface after harvest. Electrode samples for EFM imaging were stained with both, DAPI and ConA (TRITC-labelled), to visualize DNA (cells) and carbohydrates (EPS), respectively. EFM images with different magnification from



both types of staining can be found in Figure 4. Images indicate that electrochemically cultivated G. sulfurreducens forms a homogenous biofilm covering the electrode surface (left half of each image), as it was also observed optically after dismounting the electrode from the H-cell reactor. In general, DAPI and ConA signals are very strong at the intact biofilm and very low or not present on the electrode after harvest. Images A and C point out that the cells had been removed successfully from the electrode with the cell scraper, since no single cells remain visible on the harvested electrode area (right part of the images). Figure 4 E shows stained single cells at the border of the multilayered biofilm. As can be seen from images B and D, the EPS were mainly removed from the surface, however in image B the TRITC signal seems to indicate that the removal of the EPS from the electrode surface was not complete. Furthermore, EPS fragments or agglomerates can be detected on images B and D. Based on these findings, repeated steps of scratching with a cell scraper and taking up in PBS were implemented in the harvesting routine. Additionally, micro- and macro-scale SEM images (figure S3 and figure S4) taken from freshly polished graphite plate electrodes (images A) and an electrode after electrochemical cultivation and removal of G. sulfurreducens biofilm (images B) confirm the successful biofilm harvest. Both, micro- (1000x magnification) and macro-scale (50x magnification) SEM images, show that the original pattern and morphology of the graphite based electrode were well visible after biofilm harvest. It can be stated that the biofilm harvesting process with a cell scraper from the planar electrode resulted in a nearly quantitative recovery of EPS for the subsequent biochemical analysis.

3.3 Biochemical EPS analysis The harvested and extracted EPS fractions of both, anode respiring G. sulfurreducens in a MFC (MFC TB, MFC LB and MFC SOL) and of heterotrophically grown and fumarate respiring cells as control (C EXP TB, C EXP SOL, C STAT TB and C STAT SOL), were analyzed for their total amounts of proteins, carbohydrates, uronic acids, lipids and eDNA. Concentrations are referred to single cells and are summarized in Figure 5. Individual presentation of each analyzed EPS group compound including standard deviations and numerical concentrations can be found in figures S5–S9 and table S1.


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By far the highest amount of EPS was found in the soluble fraction of the EPS (MFC SOL) secreted by G. sulfurreducens under MFC conditions with a value of 116 ± 21 pg cell-1. The second highest EPS concentration was measured for the tightly bound EPS (MFC TB) of the electroactive biofilms with 18 ± 10 pg EPS cell-1. The lowest values were detected for the anode respiring cells in the LB EPS fraction at 11 ± 4 pg cell-1. All controls cultivated under non-electroactive conditions showed lower EPS concentrations compared to the electroactive fractions. The highest EPS concentration was measured in the SOL fraction of the controls harvested in EXP phase with 17 ± 1 pg cell-1. The SOL fraction of cells harvested in the STAT phase were found to have the second highest EPS concentration (6 ± 1 pg cell-1) followed by the TB fraction of the same cultures (STAT, 5 ± 1 pg cell-1) and the TB fraction of cells harvested in the EXP phase (2 ± 0 pg cell-1). Besides the total amount of EPS per cell, Figure 5 also illustrates the composition of the respective fractions and cultures. Under electrochemical conditions G. sulfurreducens mainly secrets proteins (97 ± 18 pg cell-1) into the electrolyte, followed by carbohydrates (14 ± 1 pg cell-1) and residual compounds. The same pattern can be observed for the TB fraction of the MFC cells, however, proteins (10 ± 7 pg cell-1) are not as dominant as in the SOL EPS. In the LB EPS, again protein (4 ± 2 pg cell-1) represents the biggest group followed by carbohydrates (3 ± 1 pg cell-1). In all samples eDNA is the smallest fraction of the EPS. Finally, it has to be mentioned that standard deviations presented in supporting information (figures S5–S9 and table S1) indicate a rather heterogeneous distribution of the analyzed EPS group compounds.

4 Discussion and conclusions The aim of the experiments presented in this work was the establishment of an EPS extraction routine for electroactive biofilm samples and the subsequent biochemical EPS analysis of electrochemically cultivated G. sulfurreducens biofilms.

4.1 Electrochemical cultivation in H-cells As already described before, G. sulfurreducens is one of the most applied model organisms in the field of microbial current production. Results of the MFC cultivation show a typical current density curve for a strain initially grown on fumarate as electron donor and then added to a polarized electrode 6. Analog to heterotrophic growth curves current production ACS Paragon Plus Environment


in the MFC is also characterized initially by a lag phase, followed by an exponential phase and a stationary phase, indicating the maturation of the anode respiring biofilm. Marsili et al. suggest that G. sulfurreducens must initially optimize attachment or electron transfer to a surface in an BES, which might also contribute to the initially low current production33. It is assumed that current production goes in parallel with the surface coverage as observed before13, but not tested in this study. The small current decrease at the end of the experiment is typical for a MFC and is often related to an acidification of the biofilm resulting from proton accumulation 34. Final current densities of around 172 µA cm-2 are comparable to values reported by Nevin et al. (215 µA cm-2) for G. sulfurreducens biofilms fed with acetate in a H-cell set-up 6. However, current densities up to 1000 µA cm-2 and higher have also been published for G. sulfurreducens biofilms growing in a flow through cell 35. The values indicate that the used MFC set-up was not designed to realize maximum current densities. The internal resistance resulting from the high distance between the WE and CE and the low electrode surface to electrolyte volume are assumed to be the main limiting factors. Additionally, electrochemical reactors providing an increased WE surface might be used in future studies in order to harvest an increased amount of biofilm. Nonetheless, the modified H-cell reactor provided an established electrochemical system and allowed precise electrochemical cultivation conditions. Furthermore, the main advantage of this system is the possibility to quickly dismount the WE by the designed clamp system in order to harvest the biofilms without loss due to mechanical disruption during dismounting. Finally, operation of the MFC in batch mode allowed the accumulation of the SOL EPS fraction during cultivation in the WE chamber.

4.2 Biofilm harvest Evaluation of the biofilm harvest with a cell scraper was made optically and monitored via EFM and SEM. Therefore, biofilms were cultivated electrochemically, harvested partially and examined optically. The biofilm imaging proved that the vast majority of the biofilms were harvested by the scratching process, especially if this process was repeated. Nevertheless, it has to be taken into account that cells or at least small amounts of the EPS may remain on the electrode surface, so that the biofilm could not be harvested completely in this way. To


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ensure a maximum EPS recovery from the WE surface a chemical extraction procedure from the electrode might be established in future studies. Another option might be the use of less structured and more planar electrode materials such as glassy carbon. In general, monitoring of biofilm formation on the electrode surface (optical and via EFM) illustrates a homogenously covered electrode surface, as it has been described for electroactive Geobacter biofilms before

6,7

. However, biofilm thickness appears relatively

thin, which might result from the relatively high anodic potential or stirring force of the magnetic stirrer.

4.3 Biochemical EPS analysis The biochemical EPS analysis was conducted in order to compare the major EPS constituents of anode respiring G. sulfurreducens cells with those of fumarate respiring cells. The results for the composition of the EPS fractions clearly indicate that anodic respiration of G. sulfurreducens strongly promotes the production of EPS. Proteins were secreted to the electrolyte (SOL EPS) almost tenfold more under electrochemical conditions as compared to the controls harvested in exponential phase and more than 30 times more as compared to the cells harvested in stationary phase. A similar pattern but with reduced differences was observed for the soluble fraction of the carbohydrates. They were secreted around 3.5 times more under MFC conditions as compared to EXP phase culture and almost 7 times more as compared to the STAT phase culture. Uronic acids and eDNA complete these findings with also following the same pattern. Again, the concentrations in the SOL EPS were around ten times higher in the anode respiring culture as compared to the controls. It might be assumed that the electrochemical cultivation leads on the one hand to an upregulation of genes responsible for the expression of the respective EPS compounds, as it already has

been

reported for cells of G. sulfurreducens on graphite electrodes 6. On the other hand, transport of substances from cytoplasm or periplasm beyond the outer membrane might be enhanced in the course of anodic respiration leading to an accumulation of EPS in the electrolyte (SOL EPS). The observation, that proteins dominate the composition of the SOL EPS fraction is presumably related to the extracellular electron transfer mechanisms of G. sulfurreducens, which mainly involve proteins such as cytochromes, e.g. via nanowires 36.



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The general trend that more EPS were produced by electroactive cells was valid also for the tightly-bound EPS. All EPS compounds occurred in increased concentrations with the MFC harvested cells than with the control cells. Again, proteins were most abundant, most probably resulting from their importance for the EET, followed by carbohydrates, which are connected to the surface attachment 17. When explaining the large differences between MFC grown cells and the control cells, anodic respiration and the consequent biofilm formation are assumed to have the biggest impact on EPS secretion. However, fumarate respiring cells were present partially as a biofilm on the glass wall of the septum flask, with the major part occurring as planktonic cells. In contrast, MFC cells were mainly sessile cells. The need to form a biofilm under fumarate respiring conditions is low, since fumarate is dissolved; hence, respiration is not a surfacebound process. Apart from the applied potential, the stirring of the WE chamber was an additional difference between MFC and septum flask (control) cultivation. Since stirring has been reported to influence biofilm formation

24

, its influence on G. sulfurreducens EPS

production should be tested in future studies. The relatively high standard deviations found for all major EPS compounds independent from cultivation and fraction are likely a result of the complex routine towards the biochemical analysis of EPS. The main steps were MFC cultivation

(1),

electrode

dismounting (2), biofilm harvest (3), resuspension (4), fractionation (5), dialysis (6), drying (7), resolving (8) and biochemical analysis (9). This multitude of steps towards the EPS analysis illustrates the high potential leading to quantitative differences between the individual samples due to a heterogeneous process. Comparing the data for EPS concentrations found in this study with data for bacteria from other lithotrophic biotopes for example indicates that G. sulfurreducens produces relatively high amounts of EPS (around 140 pg total EPS cell-1 summarizing TB, LB and SOL EPS). Wikiel for example found concentrations ranging at values lower than 1 pg EPS cell-1 for Desulfovibrio alaskensis under various corrosion conditions

22

. Castro et al. described

concentrations around 50 pg EPS cell-1 for Aeromonas hydrophila under reducing conditions 19

. However, significantly higher amounts of EPS have also been observed for example by

Gehrke et al. They found concentrations ranging between 0.2 and 2.8 μg EPS cell-1 for Acidithiobacillus ferrooxidans on different substrates 37.


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On the one hand side, the data presented in this study indicate that the electrochemical cultivation of G. sulfurreducens involving anodic respiration strongly promotes biofilm formation and EPS production. Furthermore, proteins dominate the secreted EPS, most probably because they are involved in the EET of G. sulfurreducens. On the other hand side, the comparison with other research reveal a lack of comparable studies focusing on electroactive biofilms and further research is needed. To the best of the authors knowledge, these experiments are the first approach towards a complete analysis of G. sulfurreducens EPS under electroactive conditions. The data indicate a strong promoting effect of an anodically polarized electrode on EPS production. However, since microbial EPS are very complex and the results strongly depend on the type of analysis, further studies are needed. A more detailed analysis of the major EPS compounds such as proteins or carbohydrate analysis 19,21 should be performed as well as a further evaluation of TB EPS extraction reagents 38. Furthermore, varying electrochemical parameters such as the applied anode potential might also influence the EPS composition and should therefore be tested in future studies. This will lead to a deepened understanding of electroactive biofilm formation and function, and finally might lead to the optimization of BES and contribute to the future energy management.

Acknowledgements We gratefully acknowledge the financial support from the German Federal Ministry of Education and Reseach (No. 031A226 and No. 031B0523). Furthermore, we would like to thank Prof. Dr. Rainer Meckenstock for the possibility and help analyzing the EPS of G. sulfurreducens.

Associated content The Supporting Information is available free of charge on the ACS Publications webside at DOI: Detailed cultivation and media conditions for G. sulfurreducens, detailed information on the electrochemical cell, detailed information on biofilm imaging, supporting information on EPS harvest



and extraction, experimental routines for biochemical EPS analysis, photographs and SEM images illustrating the biofilm harvest, individually illustrated concentrations of EPS compounds and numerical EPS concentrations

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Table of Contents/Abstract Graphic


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1

Heterotrophic Cultivation

MFC Cultivation +400 mV vs. Ag/AgCl Anodic respiration 30 °C 8 days

2

3

Harvesting of SOL EPS Supernatant

SOL EPS

4000 g 4 °C, 40 min Volume reduction at 55 °C 40 h

5

Supernatant

LB EPS

Supernatant

TB EPS

Extraction of TB EPS Resuspension cell pellet in 20 mL PBS 2.5 g DOWEX 4 °C, soft shaking 6h

7

Supernatant

Extraction of LB EPS 9400 g 4 °C 15 min Combining with SOL EPS

Supernatant

Extraction of LB EPS 9400 g 4 °C 15 min

6

Cell harvesting

Supernatant

Biofilm harvesting, resuspension in 20 mL PBS

SOL EPS 5

11

Electrode dismounting

Electrolyte from MFC WE chamber 0.22 µm filtration Volume reduction at 55 °C 40 h

4

10

Control Fumarate respiration 30 °C 2 days (EXP phase) 8 days (STAT phase)

6

Extraction of TB EPS Resuspension cell pellet in 20 mL PBS 2.5 g DOWEX 4 °C, soft shaking 6h

Dialysis 3.5 kDa cut off 5 L deionized H2O 72 h

8

Drying 55 °C 48 h

9

Biochemical Analysis

Figure 1: Schematic route of the biofilm harvest and EPS fractionation of G. sulfurreducens for MFC grown anode respiring cells (left) and heterotrophically grown fumarate respiring cells. Fractionation in soluble (SOL 39 EPS), loosely bound (LB EPS) and tightly bound EPS (TB EPS) .



200 180 160 current density / µA cm-2

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

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140 120 100 80 60 40 20 0 0

1

2

3

4 time / d

5

6

7

8

Figure 2: Mean current density curve for 9 biologically independent cultivations of G. sulfurreducens in H-cell microbial fuel cells (minimal medium + acetate; electron acceptor: anode; 30 °C; anaerobic). Current density is referred to geometrical WE surface (4.9 cm2). 39 Standard deviations are shown in grey .


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Figure 3: CLSM images of G. sulfurreducens biofilm on a graphite electrode. The electrode was dismounted from a H-cell operated as MFC (minimal medium + acetate; electron acceptor: anode (graphite plate); 30 °C; anaerob). The upper image displays both structure and surface coverage by the biofilm, in the lower section a cross section indicated the biofilm thickness. Cells were stained with DAPI (1 μg mL-1 DAPI).



A

B

C

D

E

F

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Figure 4: EFM images of G. sulfurreducens biofilm on electrodes with the border after harvesting half of the biofilm. WE were dismounted from a H-cell operated as MFC (minimal medium + acetate; electron acceptor: anode (graphite plate); 30 °C; anaerob). Images A, C and E display the DAPI signals (1 μg mL-1 DAPI), images 39 B, D and F are ConA-TRITC (100 μg mL-1 TRITC-labelled ConA) signals .


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140 116

120 EPS components / pg cell-1

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


100

eDNA Lipids

80

Uronic acids

60

Carbohydrates

40 20

Proteins 18

17

11 2

0 MFC TB

MFC LB

MFC SOL

C EXP TB

C EXP SOL

5

6

C STAT TB

C STAT SOL

Figure 5: Summarized concentrations for EPS compounds per cell of EPS fractions (tightly bound: TB; loosely bound: LB; soluble: SOL) extracted from cells grown electrochemically (MFC) and heterotrophically (C) to exponential (EXP) and stationary phase (STAT). Summarized concentrations are given as number

39

.



Mean current density curve for 9 biologically independent cultivations of G. sulfurreducens in H-cell microbial fuel cells (minimal medium + acetate; electron acceptor: anode; 30 °C; anaerobic). Current density is referred to geometrical WE surface (4.9 cm2). Standard deviations are shown in grey 38. 951x729mm (144 x 144 DPI)


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CLSM images of G. sulfurreducens biofilm on a graphite electrode. The electrode was dismounted from a Hcell operated as MFC (minimal medium + acetate; electron acceptor: anode (graphite plate); 30 °C; anaerob). The upper image displays both structure and surface coverage by the biofilm, in the lower section a cross section indicated the biofilm thickness. Cells were stained with DAPI (1 μg mL-1 DAPI).



graphical abstract


Page 28 of 28

Extracellular Polymeric Substances from Geobacter sulfurreducens

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