Steering the metabolism of Bacillus subtilis under oxygen-limited conditions with anode assisted electro-fermentation

doi:10.21203/rs.3.rs-2199976/v1

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Steering the metabolism of Bacillus subtilis under oxygen-limited conditions with anode assisted electro-fermentation

https://doi.org/10.21203/rs.3.rs-2199976/v1

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published 10 Jan, 2023

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Background: Bacillus subtilis is generally regarded as a ubiquitous facultative anaerobe. Oxygen is the major electron acceptor of B. subtilis, and when oxygen is absent, B. subtilis can donate electrons to nitrate or perform fermentation. An anode electrode can also be used by microorganisms as the electron sink in systems called anodic electro-fermentation. The facultative anaerobic character of B. subtilis makes it an excellent candidate to explore with different electron acceptors, including an anode. This study aimed to optimise industrial aerobic bioprocesses using alternative electron acceptors. In particular, the change of metabolism and end product spectrum of B. subtilis with different electron acceptors, including anode from the electro-fermentation system, was investigated.

Results: B. subtilis was grown using three electron acceptors, i.e., oxygen, nitrate, and anode (poised at a potential of 0.70 V vs. standard hydrogen electrode). The results showed oxygen had a crucial role for cells to remain metabolically active. When nitrate or anode was applied as the sole electron acceptor anaerobically, immediate cell lysis and limited glucose consumption were observed. In anode assisted electro-fermentation with a limited aeration rate, acetoin, as the main end product showed the highest yield of 0.78 ± 0.04 mol_product/mol_glucose, 2-fold higher than without poised potential (0.39 ± 0.08 mol_product/mol_glucose).

Conclusions: Oxygen controls B. subtilis biomass growth, alternative electron acceptors utilisation and metabolites formation. Limited oxygen/air supply enabled the bacteria to donate excess electrons to nitrate or anode, leading to steered metabolic pathways. The anode assisted electro-fermentation showed its potential to boost acetoin production for future industrial biotechnology applications.

Bacillus subtilis

oxygen supply

anode assisted electro-fermentation

anode respiration

acetoin bioproduction

Oxygen is one of the vital features for many life forms to prosper, including microorganisms. In nature, many microorganisms can donate electrons to atmospheric oxygen to perform respiration and energy conservation. In industrial biotechnology, oxygen is essential throughout the entire aerobic process to promote rapid cell growth, and support microorganisms to maintain their metabolic functions and balance their metabolic redox state (Lange et al., 2017; Vassilev et al., 2021). The bioprocesses with high oxygen demand often face issues such as high operational expenses, poor liquid-gas mass transfer rates and fast foam generation (Junker et al., 1998; Lee et al., 2016; Delvigne and Lecomte, 2009; Humbird et al., 2017). In many oxygen-dependent systems, the overall yields are limited by unsolicited cell growth and unbalanced metabolisms. The present strategies to improve oxygen independence in aerobic processes include separating the cell growth and product formation into multiple steps, using synthetic biology and metabolic engineering tools, or substituting oxygen with alternative electron acceptors (Lange et al., 2017, Vassilev et al., 2021). However, many current practices are limited to certain anaerobic or facultative anaerobic microorganisms (Nealson et al., 2002; Nishimura et al., 2007; Tebo & Obraztsova, 1998), future studies should focus on how to demote oxygen dependency during the industrial aerobic process by balancing the production of biomass and end products with low operational costs and minimal microbial stress exposure.

Anaerobic processes include microorganisms performing fermentation or anaerobic respiration using different terminal electron acceptors other than oxygen. In fermentation, fermentable substrates, like polysaccharides or sugars, are converted into pyruvate and then further oxidized into organic acids and alcohols (Stanbury et al., 2013). In anaerobic respiration, some compounds such as nitrate, sulfate, fumarate, (III), manganese (IV) or CO₂ are used as the terminal electron acceptor (Lovley & Coates, 2000). Anaerobic processes are often preferred especially in industrial applications, for example, ethanol fermentation and anaerobic digestion, due to their low costs and relatively high volumetric production rates. However, compared to aerated systems, anaerobic respiration and fermentation come with less energy conservation, undesired by-product formation, and requirement for complex downstream processes (Weusthuis et al., 2011; Vassilev et al., 2021).

Facultative anaerobic bacteria are microorganisms that metabolise energy both aerobically and anaerobically when adapting to oxygen fluctuations (Lange et al., 2017). Many facultative bacteria are well-researched candidates that can fill the niche in both aerobic and anaerobic industrial processes, including Escherichia coli, Lactobacillus sp. and Bacillus subtilis. B. subtilis is a rod-shaped, gram-positive, catalase-positive, spore-forming bacterium that has been isolated from animals, soil, and marine habitats (Kovács, 2019). B. subtilis can have aerobic and anaerobic lifestyles by donating electrons to different terminal electron acceptors (Hoffmann et al., 1995; Sun et al., 1996; Nakano et al., 1997; Nakano & Zuber, 1998). Under oxygen-limited conditions, B. subtilis is reported to perform anaerobic respiration by nitrate ammonification or mixed acid fermentation where pyruvate is converted into various metabolites such as lactate, acetate, acetoin and 2,3-butanediol (Hoffmann et al., 1995; Nakano and Zuber, 1998).

B. subtilis is one of the most extensively exploited cell factories to produce enzymes, heterologous proteins, food additives, vitamins, antibiotics, amino acids, and insecticides (Schallmey et al., 2004; Gu et al., 2018; Su et al., 2020). Two fine chemicals naturally formed by B. subtilis, acetoin (3-hydroxybutanone or acetyl methyl carbinol) and its reduced form, 2,3-butanediol, have shown growing market share in food, cosmetics, and chemical and agriculture industries during the last decades (Xiao & Lu, 2014). Acetoin is a flavour and fragrance additive, biorefinery platform chemical and plant growth promotor. Similarly, 2,3-butanediol is used as a food additive, biofuel, anti-freezing agent, and precursor for butanone formation. At the industrial manufacturing level, bulk production of acetoin and 2,3-butanediol rely on chemical synthesis using fossil feedstocks (Xiao & Lu, 2014). Due to the increasing environmental and financial concerns, biotechnological acetoin and 2,3-butanediol production from biomass materials with microorganisms as biocatalysts are being explored. Dozens of bacterial strains with outstanding acetoin and 2,3-butanediol yields are recorded. Most of them, nonetheless, are pathogenic and cannot be used for industrial starter cultures, e.g., Serratia, Klebsiella, Enterobacter, Raoultella and Salmonella. (Bursac et al., 2017, Förster et al., 2017; Kim et al., 2017). B. subtilis, on the other hand, is considered safe by U.S. Food and Drug Administration (FDA) and European Food Safety Authority (EFSA) and has been widely used as probiotics and dietary supplements in the market (Hong et al., 2008).

The acetoin and 2,3-butanediol production in B. subtilis typically use oxygen as the electron acceptor and small molecule sugars (e.g., glucose, xylose or sucrose) as substrate, while the formation of by-products (e.g., lactate, acetate, and ethanol) has been observed (Renna et al., 1993; Härtig & Jahn, 2012). In various aerobic acetoin and 2,3-butanediol production processes, glucose is added and converted into pyruvate via oxidative phosphorylation, and then pyruvate is further metabolised into acetoin and 2,3-butanediol. During the production processes, oxygen can be one of the limiting factors since high dissolved oxygen concentrations are required to re-oxidise NAD(P)H or FADH₂ and to generate ATP effectively (Hu et al., 2008). One novel approach to demote oxygen dependency and steer the metabolism of B. subtilis is the use of anodic electro-fermentation systems (Vassilev et al., 2021). In anodic electro-fermentation, an anode of a bioelectrochemical system (BES) working as an electron sink accepts surplus electrons from microorganisms upon substrate oxidation, which can support a more balanced redox state with fewer undesired by-products (Lai et al., 2016; Vassilev et al., 2018). B. subtilis was proposed as an ideal starter culture for anodic electro-fermentation due to its ability to produce naturally a redox-active mediator and form a conductive biofilm (Nimje et al. 2009, Vassilev et al., 2021). Yet, the comprehensive information about B. subtilis metabolism using an anode as an electron acceptor, especially under oxygen-limited conditions, has not been fully revealed.

This study investigated the potential of utilising B. subtilis for acetoin and 2,3-butanediol production from glucose under different oxygen supplies and alternative electron acceptors. Three electron acceptors (with different states of matter), i.e., oxygen (gas), nitrate (liquid) and anode from BES (solid) were tested. Furthermore, the effects of anaerobic and oxygen-limited conditions on the use of two additional electron acceptors, i.e., nitrate and the anode, were examined by monitoring the glucose consumption, cell densities and end product formation.

Microbial strain and aerobic cultivation conditions

B. subtilis 168 trpC xyl⁺ previously constructed by Averesch & Rothschild (2019) for para-aminobenzoic acid production was cultivated in M9 minimal medium amended with 27.8 mM (5 g/L) glucose as the sole carbon source in all experiments. For detailed medium composition, the reader is referred to the Additional File. The pH of the growth medium was adjusted to 7.1 using 1 M HCl. Agar plates were prepared with the same M9 minimal medium and 15 g/L agar-agar. Pre-cultures were prepared by picking and transferring single colonies from agar plates into baffled shake flasks and placed on a rotary shaker (IKA KS4000i Control, Germany) for aerobic overnight cultivation at 300 rpm and 35°C. Cells were harvested by centrifugation (at 8000 rpm, 4°C, 10 min), washed and resuspended in fresh M9 medium before transferring into the main cultivation vessels. Five times higher initial cell densities (OD₆₀₀ ≈ 0.50) were aimed for the nitrate and BES than the aerobic experiments (OD₆₀₀ ≈ 0.10). Aerobic experiments were accomplished by using 100 mL shake flasks with 20% liquid volume at 300 rpm and 35°C. In selected experiments, the real-time cell densities for different concentrations of K₃[Fe(CN)₆] (0, 0.5, 1.5 and 5 mM) were monitored using a cell growth quantifier (SBI, Germany).

Anaerobic and microaerobic serum flasks

Anaerobic and microaerobic cultivations with nitrate as electron acceptor were done by using 120 mL serum flasks with 80% liquid volume and incubated at 150 rpm and 35°C. Serum flasks were prepared by initially adding the buffer solution and 23.50 mM (2 g/L) sodium nitrate sparged with N₂ gas (Woikoski Oy, Finland) for 30 minutes to remove the dissolved oxygen. Before the autoclavation, the headspace was then filled with N₂ at 1.40 atmospheres monitored by a gas meter (LEO1, KELLER, Switzerland). Concentrated glucose and trace elements solutions were prepared in separate sterile and anaerobic serum flasks using the same technique. All needles and syringes used for serum flasks experiments were flushed with sterile N₂ gas before adding the concentrated solutions to reach the concentrations of the M9 medium. For microaerobic experiments, the headspace overpressure was released immediately after the inoculum was added and replaced with 4 mL sterile O₂ gas (Woikoski Oy, Finland).

Bioelectrochemical system and operation

Bioelectrochemical experiments were prepared using 300 mL H-type borosilicate reactors (Adams and Chittenden Scientific Glass, USA). Detailed anolyte and catholyte compositions can be found in the Additional File. K₃[Fe(CN)₆] (1.50 mM) was added to the anolytes of selected runs to act as a redox mediator. A circular cation-exchange membrane (19.60 cm² projected surface area, CMI-7000, Membranes International Inc. USA) was placed between the anodic and the cathodic chambers. BESs used a three-electrode setup. Anode was made of carbon felt (17.90 cm² projected surface area, 1.12 cm thickness, Alfa Aesar, USA) wrapped around a graphite rod (15 cm × 3 mm, Sigma Aldrich, USA). A platinum wire (10 cm × 0.4 mm, Research Material Ltd, UK) was used as the cathode, and an Ag/AgCl 3 M NaCl reference electrode (RE-5B, BASi, USA) was placed in the anode chamber. All potentials are reported with respect to the standard hydrogen electrode (SHE). The working electrode, counter electrode, and reference electrode were connected to a multi-channel potentiostat (VMP3, BioLogic, France) to apply the desired anode potential and record the current.

In advance of anaerobic BES inoculation, anolyte was sparged with N₂ for 30 minutes at a 1.50 L/min flow rate to remove dissolved oxygen. Afterwards, N₂ gas was switched to continuously sparge the headspace of the anodic chamber to assure anoxic conditions during the operation. In aerated BES experiments, the air was sparged through a peristaltic tube (L/S 16, Masterflex, USA) to a liquid medium using mass flow controllers (EL-FLOW FG, Bronkhorst, the Netherlands) at 5 mL/min (limited aeration) or 30 mL/min (moderate aeration) flow rate. N₂ or air inlet was first attached to sterile vent filters (0.20 µm 50mm Millex-FG PTFE, Merck, USA) and to gas washing bottles filled with sterile MQ water, then connected to the reactors. Table 1 shows the different gases supplied and some key parameters of the different runs.

Table 1

Different cultivation vessels and operational parameters tested in this study.
Brief name	Condition tested	Gas supplied	Liquid volume	Operating time	Addition of 1.5 mM K₃Fe(CN)₆
Shake flask	Aerobic	O₂, freely diffused	20 mL	12 h	No
Serum flask	Anaerobic amended with 24 mM nitrate	N₂ headspace, 1.40 atm	96 mL	170 h	No
Serum flask (+ O₂)	Oxygen-limited amended with 24 mM nitrate and 4% O₂	N₂ + O₂ headspace, 1 atm	96 mL	170 h	No
Anaerobic AP	Anaerobic BES, + 0.70 V	N₂ continuously purged headspace	300 mL (anolyte)	170 h	Yes
Anaerobic OC	Anaerobic BES, open-circuit control	N₂ continuously purged headspace	300 mL (anolyte)	170 h	Yes
Moderate AP	Oxygen-limited BES, aerated at 30 mL/min + 0.70 V	O₂, continuously purged medium	300 mL (anolyte)	66–75 h	Yes
Moderate OC	Oxygen-limited open-circuit control, aerated at 30 mL/min	O₂, continuously purged medium	300 mL (anolyte)	66–75 h	Yes
Limited AP	Oxygen-limited BES aerated at 5 mL/min, + 0.70 V	O₂, continuously purged medium	300 mL (anolyte)	170 h	Yes
Limited OC	Oxygen-limited open-circuit control, aerated at 5 mL/min	O₂, continuously purged medium	300 mL (anolyte)	170 h	Yes
Limited AP (-)	Oxygen-limited BES aerated at 5 mL/min, + 0.70 V	O₂, continuously purged medium	300 mL (anolyte)	170 h	No
Limited OC (-)	Oxygen-limited open-circuit control, aerated at 5 mL/min	O₂, continuously purged medium	300 mL (anolyte)	170 h	No

Analyses

Liquid samples were taken hourly from shake flasks, or daily from serum flasks and anodic chambers of the BESs. pH was measured using a pH meter (3110, WTW, Germany). Cell density was analysed photometrically using a spectrophotometer (UV-1800, Shimadzu, Japan) with absorbance at 600 nm wavelength. Each sample for the analysis of sugars, organic acids, alcohols, acetoin and 2,3-butanediol was determined using high-performance liquid chromatography (HPLC) after filtering through 0.2 µm syringe filters (CHROMAFIL® Xtra PET-45/25, Germany). An HPLC (SIL-20, Shimadzu, Japan) equipped with a refractive index (RI) detector and a sugar column (Rezex™ RHM-Monosaccharide H⁺, 300×7.80 mm) was used for glucose determination under the following conditions: column temperature of 40°C, injection volume of 50 µL, 5 mM sulfuric acid mobile phase at 0.50 mL/min flow rate, and 30 min retention time. Concentrations of lactate, succinate, acetate, acetoin, 2,3-butanediol, and ethanol were determined using the same HPLC and column under the following conditions: column temperature of 83°C, injection volume of 100 µL, 10 mM sulfuric acid mobile phase at 0.50 mL/min flow rate, and 30 min retention time. Nitrate (NO₃⁻) and nitrite (NO₂⁻) concentrations were determined using ion chromatography (ICS-1600, Thermo Fischer Scientific, USA) equipped with an IonPac AG42-SC (4 × 50 mm) guard column, an IonPac AS4A-SC (4 × 250 mm) analytical anion exchange column under the similar condition described by Määttä et al., (2022). The gas samples in the headspace (CO₂, O₂ and N₂) were measured by gas chromatography (GC-2014, Shimadzu, Japan) equipped with a thermal conductivity detector (TCD) using the same column and methods described by Khanongnuch et al. (2022).

Cyclic voltammetry

Cyclic voltammetry was performed before and after the experiments to characterise the mediator and the anode. The anodic chamber of the H-type reactors was filled with a defined M9 minimal medium, while the cathodic chamber contained a phosphate buffer solution. The same three-electrode setup as shown in Fig. 1, Additional File was used. For the CV measurements, current profiles were recorded by scanning the anode potential at a scan rate of 0.10 mV/s within a potential window from 0.20 to 0.90 V vs. standard hydrogen electrode (SHE), to measure oxidation and reduction reactions of the solution. During the measurements, the temperature was controlled at 35°C and stirring was switched off. Three cycles (repeats) were recorded at each CV measurement.

Aerobic respiration and nitrate respiration of B. subtilis

Oxygen or nitrite as the electron acceptors for B. subtilis were first tested, and cell growth and formation of metabolites were measured. When oxygen was used as the electron acceptor, glucose was rapidly depleted within 10 hours with a consumption rate of 2.33 ± 0.10 mmol/L/h, at which time point the maximum cell density (OD₆₀₀ = 5.00) was reached and the pH dropped from 7.00 to 6.20 (Fig. 1a, 1b). Acetate (14.60 ± 0.20 mmol/L) was the main end product besides biomass, along with low titres of acetoin (2.24 ± 0.20 mM) and ethanol (1.97 mM, only measured in one replicate). Overall, aerobic growth gave approximately 61.50% carbon and redox balances (for equations, see Calculations, Additional File; for all carbon balances and redox balances calculated, see Table 1, Additional File), and acetate presented the highest product yield of 0.65 ± 0.20 mol_product/mol_glucose (for all production rates and yields calculated, see Table 1, Additional File).

Nitrate could not be used as the sole electron acceptor under anaerobic conditions. Immediate cell lysis (Fig. 1c) with no substrate consumption nor end products formation was detected in the fermentation broth. Nevertheless, B. subtilis performed nitrate respiration under the microaerobic condition-when oxygen (4%) was injected into the headspace of the serum flasks. After the headspace oxygen level dropped from 4% to below 1% in 8 h after the inoculation, the nitrate concentration started to decrease and completely consumed after 50 h, resulting in a 0.45 ± 0.10 mM consumption rate (Fig. 1d). Compared to aerobic growth, lower cell density (OD₆₀₀ = 1.71, Fig. 1c) was measured in serum bottles with 0.70 ± 0.17 mM glucose consumption rates. Main metabolites were acetate (11.86 ± 0.42 mM), acetoin (9.72 ± 1.24 mM) and 2,3-butanediol (3.21 ± 0.63 mM). By the end of the experiment, approximately 23 mL CO₂ was measured from the headspace (Fig. 2, Additional File), with 67% carbon and redox balances.

Anaerobic anode respiration of B. subtilis

To investigate whether an anode can be used as the sole electron acceptor, B. subtilis was inoculated in BESs operated under anaerobic conditions. In addition, K₃[Fe(CN)₆] was tested and added as a redox mediator to enhance the microbial extracellular electron transfer to the anode. The abiotic result of the cyclic voltammetry after adding the K₃[Fe(CN)₆] shows the oxidative and reductive peaks at 0.30 and 0.60 V, respectively, with a midpoint potential of 0.46 V (Fig. 2a). Briefly, in the biological experiments the added 1.50 mM K₃[Fe(CN)₆] boosted the overall current densities in BES and showed no significant inhibition on the aerobic growth of B.subtilis in shake flasks (for aerobic growth with different K₃[Fe(CN)₆] concentrations, see Fig. 6, Additional File). After inoculating the BES, the current density immediately increased to 0.05 mA/cm² and then gradually dropped to 0.01 mA/cm² after 50 h. In total, 1.62 mM of electrons were transferred to the anode over 170 h. The planktonic cell density decreased from OD 0.65 to 0.15 in 2 h after the inoculation and remained stable afterwards. A minor drop in pH from 7.10 to 6.90 was observed (Fig. 2b). Glucose uptake was hinged in the anaerobic BES with less than 0.01 mmol/L/h glucose consumption rate and 80% of the added glucose was left after 170 h (Fig. 2c). Lactate and 2,3-butanediol were the only metabolites detected (Fig. 2c) with end-point concentrations of 7.70 ± 0.99 mM and 1.12 ± 0.80 mM, respectively. The open-circuit reactors showed almost identical results with only lower end-point concentrations of lactate (4.93 ± 2.04 mM) and no 2,3-butanediol detected (Fig. 2c).

Anode assisted respiration of B. subtilis under moderate aeration

Based on the anaerobic serum flask results, oxygen is obligatory for B. subtilis. Thus, anode-assisted aerobic respiration was investigated by aerating the anolyte under a moderate aeration rate (30 mL/min). In total, three-fold more electrons (5.10 mM) were shuttled to the anode compared to the anaerobic condition with a three-fold higher current density reached (up to 0.17 mA/cm²) (Fig. 3a). The highest planktonic cell density (OD 1.26 ± 0.04) was measured after 44.70 h (Fig. 3b) under applied potential and open-circuit conditions. Similar concentrations of metabolites were detected under AP and OC (for statistical significance results, see Table 2, Additional File), and acetoin as the main metabolite reached an average end-point concentration of 19.22 mM, followed by 2,3-butanediol (12.68 mM, peak concentration), lactate (7.78 mM, peak concentration) and acetate (3.22 mM, peak concentration) (Fig. 3c; Fig. 3, Additional File). Lactate and 2,3-butanediol concentrations decreased after 26.60 h and 44.70 h under both conditions with average consumption rates of 0.28 and 0.41 mmol/L/h, respectively. Lactate and 2,3-butanediol as intermediate products were depleted completely at the end of the experiment. A slightly higher pH was measured in the AP (pH 6.90) compared to the OC reactors (pH 6.50). Overall, 82% carbon and redox balances were achieved, with an acetoin yield of 0.77 mol_product/mol_glucose under both conditions.

Anode-assisted respiration of B. subtills under limited aeration

When less oxygen was available by decreasing the air flow rate from moderate (30 mL/min) to limited (5 mL/min), B. subtilis showed different characteristics of growth and metabolism. In reactors under the poised anode potential, a current density of 0.15 mA/cm² was reached after 24 h (Fig. 4a) resulting in a total transfer of 9.60 mM electrons to the anode i.e., 4.50 mM more than under the moderate aeration. A steady planktonic cell density was measured (OD 0.61) and pH decreased over time from 7.10 to 6.10. Glucose was progressively consumed with an average rate of 0.22 mmol/L/h and depleted after 145 h (AP) and 169 h (OC) (Fig. 4c). Acetoin fermentation was dominant in AP reactors with a peak concentration of 17.77 ± 0.59 mM, as opposed to OC where the highest concentration of lactate (16.26 ± 2.08 mM) was measured. Both moderate and limited aeration led to similar amounts of 2,3-butanediol or acetate (Fig. 4d, e, f; Fig. 3, Additional File). Under limited aeration, lactate and 2,3-butanediol concentrations decreased after 47.80 h and 120.40 h, respectively, with 3-fold slower consumption rates than moderate conditions (0.10 mmol/L/h for lactate and 0.12 mmol/L/h for 2,3-butanediol). Overall, 85% carbon and redox balances were calculated under limited aeration, with the highest acetoin yield (0.71 ± 0.02 mol_product/mol_glucose) found in AP reactors.

The role of oxygen in B. subtilis growth and metabolism

In this study, the absence of oxygen restricted the metabolic activity of B. subtilis and resulted in abrupt cell lysis, although many studies have shown B. subtilis can grow without oxygen using alternative electron acceptors, including nitrate (Hoffmann et al., 1995; Priest, 1993; Clements et al., 2002). Among all the tested conditions, the highest cell density (OD = 5.09) was measured in aerobic shake flasks with an average doubling time of 1.60 h. A typical oxygen volumetric mass transfer coefficient (k_La) in similar aerobic shake flasks and growth conditions is up to 160 h^–1 (Hemmerich et al., 2011), nearly five times higher compared to the estimated 36 h^–1 in H-type reactors (Gowthaman et al., 1995) used with moderate aeration in anode assisted electro-fermentation in this study. When nitrate or anode was used as the alternative electron acceptor by B. subtilis, lower glucose consumption rates (< 0.70 mM/h) and cell densities (OD < 2.0) compared to aerobic shake flasks (2.33 mM/h) were observed, indicating that limited energy was available with restricted biomass formation when using the alternative electron acceptors. Oxygen as the electron acceptor facilitates sugar metabolism, energy conservation, and nicotinamide cofactors regeneration e.g., nicotinamide adenine dinucleotide (NAD⁺) in many microorganisms (Vassilev et al., 2021). In B. subtilis, reduced dissolved oxygen concentration during cultivations has also earlier shown enhanced fermentation products with 50% less biomass formed (Hu et al., 2017).

According to the results, low oxygen supply enabled B. subtilis to donate electrons to alternative electron acceptors. Anaerobically, neither nitrate nor anode was used as an electron sink resulting in no biomass growth and low concentrations of metabolites. Nitrate ammonification and anode-assisted respiration were only realised under oxygen-limited conditions. The results suggested that low concentrations of oxygen were essential during the initial stage of growth to activate anaerobic respiration. The anaerobic pathways of B. subtilis are controlled by the anaerobic regulatory network through the induction of enzymic systems which respond to low oxygen partial pressures (Härtig & Jahn, 2012). At the energy level, nitrate respiration compared to fermentation releases more free energy: ΔG⁰= -2870 kJ/mol_glucose for oxygen, -858 kJ/ mol_glucose for nitrate and − 218 kJ/ mol_glucose for fermentation, respectively (Tran & Unden, 1998; Unden & Bongaerts, 1997; Vassilev et al., 2021). Cell growth (OD₆₀₀ = 1.71) was also observed under the microaerobic condition with consumption of nitrate in contrast to fast cell lysis anaerobically in this study. However, the fundamental understanding of nitrate respiration in B. subtilis under the microaerobic condition remains to be further examined.

Different aeration schemes affected the metabolism of B. subtilis. In aerobic shake flasks, acetate was the main end product (15.18 mM), whereas in BES experiments with moderate and limited aeration one-fourth of acetate concentration was measured. In B. subtilis, acetate is typically formed from acetyl-CoA via a two-step reaction encoded by pta and ackA and the formation of acetate during aerobic respiration is explained by metabolic overflow, which activates fermentative pathways to allow faster ATP production per unit cell membrane area (Kabisch et al., 2013; Dauner et al., 2001; Szenk et al., 2017). When nitrate was used as an electron acceptor, acetate titre decreased to 11.87 mM. In addition, other fermentation products such as acetoin and 2,3-butanediol started to appear once oxygen was depleted. An increase in overall fermentative activities can be expected when oxygen is not available for the cofactors’ regeneration. In B. subtilis, increased NADH-coupled product secretion, i.e., lactate and 2,3-butanediol (up to 16.26 mM and 12.26 mM, respectively), was observed in this study under the oxygen-limited conditions in BESs. However, lactate was not excreted under nitrate respiration in this study. It has been reported that under nitrate respiration fermentation is less preferred by B. subtilis in terms of cofactors regeneration (Cruz Ramos et al., 2000; Härtig & Jahn, 2012) and that the presence of nitrate under oxygen-limited conditions can drastically decrease fermentative enzyme activities, including lactate dehydrogenase (Cruz Ramos et al., 2000)

Anode assisted electro-fermentation with B. subtilis

Previous anodic electro-fermentation studies demonstrated that the combination of poised anode potential (0.7 V vs. SHE) and K₃[Fe(CN)₆] as the redox mediator enhanced the anaerobic metabolic activities of Pseudomonas putida F1 and Corynebacterium glutamicum lysC (Kracke et al., 2015; Lai et al. 2016; Vassilev et al. 2018). In this study, B. subtilis cells showed incomplete glucose oxidation by donating an insufficient number of electrons to maintain metabolically active with rapid lysis during the anaerobic anode respiration. In contrast to the shake flasks, anaerobic BESs had almost zero glucose consumption, no planktonic cell growth and limited end product formations. Moderate aeration resulted in at least three-fold higher surplus electrons (5.10 mM) that were shuttled to the anode compared to anaerobic conditions. However, under moderate aeration, the anode did not effectively steer the metabolism and oxygen was likely still acting as the main electron acceptor. With moderate aeration, the anode was not fully functioning as an electron sink for cells to uninterruptedly balance the redox per se, or the number of electrons transferred to the anode was negligible compared to the number of electrons released from glucose, causing the almost identical end product concentrations with or without poised anode potential.

When the oxygen supply was switched to limited aeration in BESs, cells were forced to seek alternative electron acceptors and an increased number of electrons was shuttled to the anode (9.60 mM under limited aeration) with enhanced acetoin formation. Chen et al. (2019) suggested that NAD(H)⁺ played an important role during the extracellular electron transfer of B. subtilis, which likely explained the different NADH-coupled metabolites showed in this study: as the total amount of electrons shuttled to the anode increased over time, cofactor levels might have been altered by the anode, in which case less NADH-coupled metabolites are needed to balance the redox state (e.g., lactate and 2,3-butanediol). In B. subtilis, NAD⁺ regeneration is typically mediated by cytoplasmic lactate dehydrogenase (Romero et al., 2007). One hypothesis is that in anode assisted systems under limited aeration, NAD⁺ was regenerated in several ways, hence the lower lactate concentration was observed under the poised potential. However, anode did not significantly influence other fermentation steps which involved NAD⁺ regeneration, e.g., production of 2,3-butanediol from acetoin, as similar levels of 2,3-butanediol productivities were observed under limited aeration with poised potential (0.11 ± 0.02 mmol/L/h) and in open-circuit (0.09 ± 0.03 mmol/L/h). On the other hand, if anode helped to increase the cofactor levels such as NAD⁺, the conversion of lactate to pyruvate might likely also have been accelerated. Pyruvate can either re-enter the TCA cycle to potentially enhance the acetoin production or accumulate in cells to trigger the acetoin excretion as a strategy for cells to maintain the intercellular pH during the stationary phase (Härtig & Jahn, 2012; Tsau et al., 1992). Alternatively, the applied anode potential may also change the oxidation-reduction potential (ORP) of the fermentation broth or the intracellular redox state, the principle of which is still to be discovered (Virdis et al., 2022).

Higher acetoin yield with anode assisted electro-fermentation systems

Figure 5 Maximum production yields (x-axis) associated with production rates (y-axis) under different conditions tested with oxygen, nitrate, and anode as electron acceptors. In contrast to acetoin (neon green) and acetate (metallic violet), intermediate products 2,3-butanediol (crimson red) and lactate (light azure) concentrations declined during the experiments, therefore, only the highest production rates are shown.

Adaptation of B. subtilis to alternative electron acceptors revealed future perspectives of using anode assisted electro-fermentation system under oxygen-limited conditions for enhanced acetoin production. First, amongst all tested conditions, the high yield of acetoin (0.71–0.78 mol_product/mol_glucose) achieved under oxygen-limited conditions is comparable to the acetoin production of metabolically engineered B. subtilis strains (from 0.62 to 0.77 mol_product/mol_glucose, Zhang et al., 2013; Zhang et al., 2014; Bao et al., 2015). Although the highest production rate of acetoin (0.31 ± 0.08 mM/h) was found under nitrate respiration, a lower yield (0.32 ± 0.01 mol_product/mol_glucose) indicated the limitation for acetoin production under the anaerobic respiration. The results suggested that aerobic acetoin bioprocesses can be optimized with a very low air supply rate along with poised anode potential. A low aeration rate can bring financial benefits regarding operational costs and fewer foaming-related issues for industrial applications. Furthermore, another industrially related strain, P. putida KT2440, was reported to improve glycolipid surfactants production under similar oxygen-limited conditions with poised anode potential (Askitosari et al., 2020), highlighting the potential of anodic electro-fermentation to decrease the oxygen requirements of aerobic bioprocesses.

The biomass production, alternative electron acceptors utilisation and end product spectrum of the B. subtilis strain differed under different oxygen supplies. Both nitrate and anode assisted respiration of B. subtilis occurred under oxygen-limited conditions, while fast cell lysis was observed in strictly anaerobic environments. Overall, depending on the electron acceptor the end product spectrum shifted from acetate in aerobic conditions to acetate and acetoin under nitrate respiration, and further to mostly acetoin in BESs. Under limited aeration, poised anode potential at 0.70 V showed steered metabolism towards acetoin production (17.77 ± 0.59 mM at applied potential vs. 9.68 ± 1.99 mM at open-circuit), with lactate and 2,3-butanediol as intermediate products. In such anode assisted systems, a high acetoin yield of 0.71 mol_product/mol_glucose was achieved with more balanced energy distribution between biomass production and end product formation compared to systems without poised anode potential or aerobic shake flask experiments. This research highlighted the potential of anode assisted electro-fermentation to connect an industrial aerobic microorganism with bioelectrochemical systems with reduced oxygen dependency for biochemical production.

ADP: adenosine diphosphate; ATP: adenosine triphosphate; AP: applied potential; BES: bioelectrochemical system; CV: cyclic voltammetry; FADH₂: Flavin adenine dinucleotide; GC: gas chromatography; HPLC: high-performance liquid chromatography; IC: iron chromatography; NAD⁺/NADH: nicotinamide adenine dinucleotide (oxidised/reduced); NADP⁺/NADPH: nicotinamide adenine dinucleotide phosphate (oxidised/reduced); OC: open circuits; OD: optical density; SHE: standard hydrogen electrode; TCA: trichloroacetic acid.

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Not applicable

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Not applicable

Availability of data and materials

Not applicable

Competing interests

The authors declare that they have no competing interests

Funding

This work was supported by Tampere University Doctoral School

Authors' contributions

YS: Conceptualization, Methodology, Investigation, Visualization, Writing – original draft. MK: Conceptualization, Methodology, Supervision, Writing – review & editing. IV: Conceptualization, Methodology, Supervision, Writing – review & editing. All authors read and approved the final manuscript.

Acknowledgements

The authors hope to thank Dr. Nils J.H. Averesch (Stanford University) for providing Bacillus subtilis strains.

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Steering the metabolism of Bacillus subtilis under oxygen-limited conditions with anode assisted electro-fermentation

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