Carbon and electron fluxes during the electricity driven 1,3-propanediol biosynthesis. from glycerol

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1 Supporting Information Carbon and electron fluxes during the electricity driven 1,3-propanediol biosynthesis from glycerol Mi Zhou,, Jingwen Chen, Stefano Freguia,, Korneel Rabaey #,,, Jürg Keller, * Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian , China Advanced Water Management Centre, The University of Queensland, Brisbane, QLD 4072, Australia Centre for Microbial Electrosynthesis, The University of Queensland, Brisbane, QLD 4072, Australia # Laboratory of Microbial Ecology and Technology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium Number of pages: 24 Number of figures: 14 Number of tables: 2 S1

2 Figures Figure S1. Simplified glycerol metabolic pathways during anaerobic fermentation by mixed populations Figure S2. Syntrophic processes that might happen in the glycerol-fed BESs. Figure S3. Schematic diagram of external recirculation and feed circuits of the BES cells. Figure S4. Gases tripped out by helium before batch tests. Figure S5. Metabolites during the start-up period. Figure S6. Gases (i.e. H 2, CO 2 and CH 4 ) production during the potential control batch tests. Figure S7. Yields of the main metabolites as a function of different redox conditions in BES Figure S8. Electron distributions of the products from glycerol fermentation BES cells with polarized cathode. Figure S9. Carbon and electron balance of the products from glycerol fermentation BES cells with open circuit condition. Figure S10. Formic acid formation during the glycerol batch tests. Figure S11. Acetoclastic methanogenesis batch tests results. Figure S12. Bioelectrochemical ethanol production batch tests results. Figure S13. Homoacetogenesis batch tests results. Figure S14. Carbon and electron flux tree in glycerol fermentation BES cells with open circuit condition and open circuit with hydrogen supply Tables Table S1. Possible products from glycerol fermentation and the respective NADH formation involved in each metabolic pathway. Table S2. Average acetate production during glycerol fermentation batches for both reactors. S2

3 Glycerol metabolic pathway and calculations for NADH and hydrogen production. (1) Based on the literature, the dissimilatory pathway for glycerol fermentation by mixed culture is in below: Figure S1. Simplified glycerol metabolic pathways during anaerobic fermentation by mixed populations. Different microorganisms could lead to distinct products: butyrate and butanol are the products from Clostridia, and 2,3-butanediol is only formed by Enterobacteria. Here 51 we demonstrate all the possibilities from glycerol fermentations based literature. 1-5 The pathways could be divided as the following directions: (1) glycerol reduction; (2) biomass formation and after the formation of pyruvate: (3) succinate and propionate fermentation; (4) acetyl-coa formation; (5) lactate fermentation; (6) 2,3-butanediol fermentation (2) Based on the glycerol metabolic pathways above, the products and the degrees of reduction are in Table S1. Table S1. Possible products from glycerol fermentation and the respective NADH formation involved in each metabolic pathway. S3

4 Y prod./glycerol Y NADH/glycerol Product (mol/mol) (mol/mol) Y NADH/prod. (mol/mol) 1,3-PDO Succinate Propionate Lactate ,3-butanediol Acetate Ethanol Butanol Butyrate All the data are consistent to the drawing for metabolic pathway above and the previous literature on mixed culture fermentation. 3 (3) Cumulative NADH flux calculation: Cumulative NADH flux from the non-reductive branch was calculated based on the stoichiometry of reactions. The concentration of the products and the net NADH gain of this route (Table S1) were used for the calculation: 63 In this equation, is the NADH yield from the product (mol NADH/mol product, i.e Y NADH/prod. (mol/mol) in Table S1), and product/mol glycerol). (4) Hydrogen flux calculation: is the yield of the respective products (mol We assume that hydrogen could be produced from both the current and fermentation, and the productions from both methods were evaluated based on the data of electron flux. a. Fermentative hydrogen production. It was evaluated assuming hydrogen evolution occurred from pyruvate to acetyl-coa (route 4, Figure S1). As the cumulative concentrations of the two intermediates pyruvate or acetyl-coa were not measured directly, therefore, we used the concentrations of the S4

5 74 75 downstream end products of acetyl-coa (i.e. acetic acid, ethanol, butyric acid and butanol) for the estimation: 76 In the equations, is the mole of electrons per mole products (for instance, k for acetic acid is 8 and for ethanol is 12), and is the electron flux toward this product. As 1 mole of hydrogen has two moles of electrons, the total value should be multiply by two to get the electron flux for hydrogen. b. bioelectrochemical hydrogen production. Although the electrons from the graphite electrode surface might be occupied by other reductive reactions, a theoretically maximum hydrogen production was calculated assuming all the electrons were used by the electrolysis of water (i.e. electrochemical hydrogen production). So the hydrogen productions from current should be equal to the portion of current in the electron inputs. c. the real hydrogen production. Methane was formed from hydrogen and carbon dioxide. As all the electrons in methane were derived from hydrogen, the hydrogen electron distributions should be the sum of the fluxes towards hydrogen and methane: The comparisons were made between hydrogen inputs (i.e. from current and fermentation) and output (i.e. gas measurements) under different conditions. If there were more inputs than output, we speculate that the hydrogen produced was reused or the electrons supplied by the electrode were not all consumed by electrochemical hydrogen production (i.e. alternative reduction reactions happened on the electrode surface). Reversely, if there were more output than inputs, as the calculated hydrogen production from current is theoretical maximum, we S5

6 assume that more hydrogen was produced from fermentation: aside from route 4 in glycerol fermentation, hydrogen could also be released for the consumption of excessive NADH, and it has been reported that there might be two moles more hydrogen evolution from the fermentation of 1 mole glycerol (5) Efficiency of external hydrogen or electron supply: Hydrogen could be produced from glycerol fermentation as another NADH sink, which therefore, adversely affect 1,3-PDO yield. 6, 7 The assumptions of 1,3-PDO production with external hydrogen or electron supply should be clarified. Considering the redox potential of the related reactions (H + /H 2, E 0 ph 5.5 = V), 106 (NADH/NAD +, E 0 ph 5.5 = V), (glycerol/1,3-pdo, E 0 ph5.5 = V), it is thermodynamically feasible that hydrogen is the electron donor of NAD + or glycerol. On the other hand, the cathodic potentials we applied in this study were -0.6 V or -0.9 V, therefore, it is also thermodynamically feasible that direct electron transfer from the electrode to the fermentative bacteria could happen. If both hydrogen and the electrode could behave as the direct electron donor, the theoretical conversion efficiency for external hydrogen and electron supply should be identical. Furthermore, hydrogen could function as the electron shuttle between the electrode and the bacteria. However, this direct electron transfer may not happen in reality. Based on our results, we found out that in the hydrogen tests (with non-polarized electrode and external hydrogen supply), 1,3-PDO yield was enhanced likely by supressing fermentative hydrogen production. Hence, the maximum 1,3-PDO production (i.e., the maximum NADH availability for 1,3- PDO) at this condition would be achieved with no fermentative hydrogen production and only acetate was formed as the by-product. One acetate fermentation will liberate 3 NADH if no hydrogen was formed, therefore, 3 mole 1,3-PDO can be produced out of 4 mole glycerol S6

7 (0.75 mol 1,3-PDO mol -1 glycerol). It should be mentioned that biomass formation is not considered in this hypothesis, which could also change the internal redox conditions. With a polarized electrode, the metabolism of glycerol was redirected. Based on our results, acetate fermentation was enhanced, which delivered more NADH to 1,3-PDO production (Figure 2). However, if this was the only mechanism for an enhanced 1,3-PDO production, there should be no significant different between 1,3-PDO production with external hydrogen supply or with polarized electrode. Another mechanism that may happen is through extracellular electron transfer, with integrates the external electrons to glycerol metabolism. From glycerol to 1,3-PDO is a two-electron reduction. Then, it is possible that glycerol or NAD + received electrons directly to produce 1,3-PDO. We infer that the significant difference between hydrogen batches and polarized batch tests were derived from this (6) Assumptions in the balance calculation: a. All the metabolites from glycerol were measured; b. Despite the variations during the batch tests, the adopted biomass C:N ratio was 4 and it is assumed to be constant among all the 3-hour batch tests. 137 S7

8 138 Syntrophic processes might happen in the glycerol-fed BES reactors Figure S2. Syntrophic processes that might happen in the glycerol-fed BESs. The two red rectangles denote the electrodes in the BESs, which were connected by an external electrical loop and the cathode potential was controlled by a potentiostat. At the cathode side, bacteria (yellow ovals) grow with glycerol as the substrate or with the products from glycerol fermentation as the substrates, depending on the type of the bacteria (labelled on the ovals). There might be some electroactive bacteria, which could uptake electrons directly and reduce glycerol to 1,3-PDO or ethanol to acetate. Glycerol fermentation could happen in the bulk solution, and acetic acid fermentation might induce the activity of acetoclastic methanogenesis. With a polarized cathode, H 2 could be produced both electrochemically and fermentatively, which might be taken up by hydrogenotrophic methanogenesis. In addition, the existence of CO 2 and H 2 might stimulate homoacetogenesis. S8

9 151 Schematic diagram of external recirculation and feed circuits of the BES cells Figure S3. Schematic diagram of external recirculation and feed circuits of the BES cells. The two reactors were identical. The chamber labelled A is the anode, whilst the other chamber with a port for the reference electrode and labelled C is the cathode. Tygon tubing (Masterflex, Cole-Parmer, U.S.), which has Low oxygen permeability, was used for all connections between vessels. ph probes were plugged at the cathode side, and acid dosing were required to keep the ph constant. Both the anode and cathode were continuously fed with fresh medium by a feeding pump with a hydraulic retention time of 8 hours. Both the anolyte and catholyte were recirculated by a recirculation pump at a rate of 100 ml/min. The anode side was flushed with nitrogen gas to get rid of oxygen. During the start-up period, gas bags were used to collect the gases from the biocathode. The biocathodes were then connected to TOGA for online gas measurements. In the hydrogen tests, an auxiliary graphite rod electrode was plugged into the cathode chamber for in situ hydrogen production. S9

10 HPLC method for glycerol, 1,3-PDO and formic acid. Sample preparation: 1. Immediately after sampling from the reactors, the liquid samples were filtered through a sterile 0.22um PES Millipore filter (Millipore Corporation, U.S.) 2. Prepare HPLC samples by adding 0.1 ml of 1.0% sodium azide solution to 0.9 ml sample HPLC method: HPLC system: Shimadzu HPLC system with autoinjector (SIL-10ADVP), degasser DGU- 14A), LC pump (LC-10ADVP), column oven (CTO-10ASVP), diode array detector (SPD- M10AVP), CLASS VP software and Shimadzu refractive index detector (RID-10A) Column: Phenomenex Rezex ROA Organic Acid H+ 300mm x 7.8mm, column for organic acid analysis (Cat. No ) Flow: 0.4 ml/min Eluent: N H 2 SO 4 Temperature of column: 35 o C Sample volume: 20 ul S10

11 Back-calculation of the side-reactions. Products that are not in glycerol metabolic pathways (i.e. methane, valeric acid and hexanoic acid) were detected, which indicates the occurrence of side-reactions. To get a whole flux of glycerol fermentation, these products were calculated back to their respective reactants. Reaction equations: a. condensation reactions: 8, b. hydrogenotrophic methanogenesis: Based on the stoichiometry of the above reactions, we calculated the yield of the respective reactants as following: In the equations, Y is the yield of the products. and are the yields calculated based on the GC and TOGA measurements, respectively. The carbon distributions for the respective reactants: S11

12 194 Cflux is the carbon flux toward this compound, and is the carbon flux calculated based on the concentrations derived from GC. Valeric acid has 5 carbons, among which two were obtained from ethanol and three were derived from propionate. So the fraction numbers 197 (i.e., ) denote the carbon contributions between the two reactants Similarly, electron distribution was also calculated based on the stoichiometry of the reactions and the electron contributions between the reactants. S12

13 200 Pre-existing gases removal during the 1 hour helium flushing before batch tests Figure S4. Gases tripped out by helium before batch tests. A) was derived from BES -0.9 and B) was from BES As illustrated in Figure S4, despite different levels of the pre-existing gases caused by different operation conditions, the gases were stripped out by helium within one hour. This ensures a same starting point for all the batches, or the gases produced during the batch test might be covered by the pre-existing gases, leading to errors in the balance calculation. 208 S13

14 209 The performance of BESs during start-up period Figure S5. Metabolites during the start-up period. A) was derived from BES -0.9 and B) was from BES The bar figures show the carbon balance of the fermentation products from glycerol, with glycerol as the sole carbon input. 214 S14

15 215 Gases (i.e. H 2, CO 2 and CH 4 ) production during the potential control batch tests Figure S6. Gases (i.e. H 2, CO 2 and CH 4 ) production during the potential control batch tests. BES cell with polarized cathode at -0.9 V (A) and -0.6 V (B). It is very obvious that elevated hydrogen production was obtained at -0.9 V. The batch tests lasted for 3 hours, but the online gas measurements lasted until 3.3 hours as there was lag time for the gases to diffuse out of the biofilm. S15

16 222 Yields of the main metabolites as a function of different redox conditions in BES Figure S7. Yields of the main metabolites as a function of different redox conditions in BES All the values are normalized with the consideration of the side-reactions V: batches with -0.6 V cathodic potential; Open circuit: batched with open circuit condition; hydrogen: batch with open circuit condition and hydrogen supply. 1,3-PDO production did not change significantly during different condition (p > 0.05). S16

17 Electron distributions of the products from glycerol fermentation BES cells with polarized cathode. A 1,3-PDO 47.9% B Unknown 12.5% 1,3-PDO 21.2% 231 H % Formate 0.03% Propionate 4.9% Butyrate 7.6% Biomass 12.7% Acetate Ethanol 7.8% 3.3% H 2 8.0% Formate 0.0% Propionate 20.7% Butyrate 8.9% Biomass 16.6% Acetate 4.3% Ethanol 7.8% Figure S8. Electron distributions of the products from glycerol fermentation BES cells with polarized cathode: at -0.9 V (A) and -0.6 V (B). The electron inputs were glycerol and current, with 83.7% of the inputs from glycerol for -0.9 V, whilst 98.2% of the inputs from glycerol for -0.6 V. Products that are not directly in glycerol metabolic pathways were back-calculated. 236 S17

18 Carbon and electron balance of the products from glycerol fermentation BES cells with open circuit condition 239 A Biomass 49.2% 1,3-PDO 24.8% B 1,3-PDO 28.3% Biomass 42.2% 1,3-PDO biomass acetate ethanol butyrate propionate valeric acid CO2 CH4 unknown Figure S9. Carbon and electron balance of the products from glycerol fermentation BES cells with open circuit condition: carbon balance (A) and electron balance (B). The sole carbon input was glycerol. 243 S18

19 244 Formic acid formation during the glycerol batch tests Figure S10. Formic acid formation during the glycerol batch tests V: with -0.9 V cathodic potential; -0.6 V: with -0.6 V cathodic potential; open circuit: without polarization; hydrogen: open circuit condition with hydrogen supply. There were no formic acid formation during the glycerol open circuit batches or the batches with the biocathode polarized at -0.6 V, and formic acid was formed as a transient compound for both -0.9 V cathodic potential batches and open circuit with hydrogen batches. S19

20 252 Acetoclastic methanogenesis batch tests results Figure S11. Acetoclastic methanogenesis batch tests results: acetate concentration and CH 4 production during acetate batches in BES -0.9 (A) and BES -0.6 (B). Both batch tests were running for 3 hours under open circuit condition with acetate injected at time zero as the only substrate. No CH 4 formation was detected during the 3 hours, which proves that there was no acetoclastic methanogenesis. This could also be verified by constant acetate concentrations among the batches. The initial acetate concentration was 0.7 mm. It was based on acetate fermentation from glycerol during the standard BES operations. The average acetate production during glycerol fermentation for both reactors was listed in Table S Table S2. Average acetate production during glycerol fermentation batches for both reactors. Δacetate (mm) polarized cathodes open circuit BES BES Although BES-0.9 tends to produce more acetate, 0.7 mm was chosen for both reactors as it would be useful to compare the two reactors in parallel. In addition, it is assumed that if there is acetoclastic methanogenesis, it should happen as long as there is acetate. S20

21 267 Bioelectrochemical ethanol production batch tests results Figure S12. Bioelectrochemical ethanol production batch tests results: acetic acid, ethanol concentrations and CH 4 production in this batch. This test was operated with a polarized cathode of -0.9 V. Acetic acid was injected as the only substrate in the BES cells. Both the acetic acid injection and potential control started at time zero. No ethanol production was observed from acetate. In addition, no methane was detected, which also confirms that there were no acetoclastic methanogenesis activities. S21

22 275 Homoacetogenesis batch tests results Figure S13. Homoacetogenesis batch tests results: Carbon dioxide and hydrogen captured by TOGA from the biocathode (A) and acetic acid concentration (B). Carbon dioxide was supplied in the form of sodium bicarbonate, and was completely vented out within one hour, so the tests could only last for one hour. Hydrogen was supplied in situ with a bare electrode. There was no obvious increase for acetate concentrations (Figure S8 B), which tells that there was no homoacetogenesis happening in these glycerol-fed BESs. CO 2 was spike as bicarbonate at time zero, and the initial bicarbonate concentration was calculated based on the requirement of CO 2 for both homoacetogenesis and hydrogenotrophic methanogenesis. As CO 2 and H 2 are the reactants for both reactions and the amount of H 2 can be calculated based on the current, the demand for CO 2 was hence calculated based on the stoichiometry of the reactions. The CO 2 /H 2 ratio is 2/4 for homoacetogenesis, and hydrogenotrophic methanogenesis requires 1 mol CO 2 and 4 mol H 2 to generate 1 mol CH 4. Therefore, the total ratio between CO 2 and H 2 should be higher than 3/8. In this study, a ratio of 1/2 was employed. S22

23 291 Carbon and electron flux tree in open circuit and hydrogen condition Figure S14. Carbon and electron flux tree in glycerol fermentation BES cells with open circuit condition (the first row) and open circuit with hydrogen supply (the second row). In the latter case, H 2 shows here have deducted the amount provided directly. The carbon and electron inputs were 100% from glycerol for both cases. There were only 5 routes with these two conditions: (1) glycerol reduction, (2) biomass formation, (3) propionic acid fermentation, (4) acetyl-coa formation, and (5) methanogenesis. 299 S23

24 Literature cited (1) Celińska, E. Debottlenecking the 1,3-propanediol pathway by metabolic engineering. Biotechnol. Adv. 2010, 28 (4), (2) Saxena, R. K.; Anand, P.; Saran, S.; Isar, J. Microbial production of 1,3-propanediol: Recent developments and emerging opportunities. Biotechnol. Adv. 2009, 27 (6), (3) Temudo, M. F.; Poldermans, R.; Kleerebezem, R.; van Loosdrecht, M. C. M. Glycerol fermentation by (open) mixed cultures: A chemostat study. Biotechnol. Bioeng. 2008, 100 (6), (4) Barbirato, F.; Chedaille, D.; Bories, A. Propionic acid fermentation from glycerol: Comparison with conventional substrates. Appl. Microbiol. Biot. 1997, 47 (4), (5) Biebl, H.; Marten, S. Fermentation of glycerol to 1,3-propanediol: Use of cosubstrates. Appl. Microbiol. Biot. 1995, 44 (1-2), (6) Selembo, P. A.; Perez, J. M.; Lloyd, W. A.; Logan, B. E. Enhanced hydrogen and 1,3- propanediol production from glycerol by fermentation using mixed cultures. Biotechnol. Bioeng. 2009, 104 (6), (7) Biebl, H.; Marten, S.; Hippe, H.; Deckwer, W. D. Glycerol conversion to 1,3- propanediol by newly isolated Clostridia. Appl. Microbiol. Biot. 1992, 36 (5), (8) Barker, H. A.; Kamen, M. D.; Bornstein, B. T. The synthesis of butyric and caproic acids from ethanol and acetic acid by Clostridium Kluyveri. PNAS 1945, 31 (12), (9) Stadtman, E. R.; Stadtman, T. C.; Barker, H. A. Tracer experiments on the mechanism of synthesis of valeric and caproic acid by Clostridium Kluyveri. J. Biol. Chem. 1949, 178 (2), S24