Geobacter sulfurreducens metabolism at different donor/acceptor ratios

Abstract Geobacter species have great application potential in remediation processes and electrobiotechnology. In all applications, understanding the metabolism will enable target‐oriented optimization of the processes. The typical electron donor and carbon source of the Geobacter species is acetate, while fumarate is the usual electron acceptor. Here, we could show that depending on the donor/acceptor ratio in batch cultivation of Geobacter sulfurreducens different product patterns occur. With a donor/acceptor ratio of 1:2.5 malate accumulated as an intermediate product but was metabolized to succinate subsequently. At the end of the cultivation, the ratio of fumarate consumed and succinate produced was approximately 1:1. When fumarate was added in excess, malate accumulated in the fermentation broth without further metabolization. After the addition of acetate to stationary cells, malate concentration decreased immediately and additional succinate was synthesized. Finally, it was shown that also resting cells of G. sulfurreducens could efficiently convert fumarate to malate without an additional electron donor. Overall, it was demonstrated that by altering the donor/acceptor ratio, targeted optimization of the metabolite conversion by G. sulfurreducens can be realized.

aquifers (Lovley et al., 2011). Furthermore, different Geobacter species can transfer electrons from the metabolism oxidation of organic compounds to an electrode. Direct interspecies electron exchange between Geobacter species and syntrophic partners appears to be an important process in anaerobic wastewater treatment (Lovley et al., 2011).
This article aims to investigate the donor/acceptor-pair acetate-fumarate in more detail. Here, acetate is oxidized to CO 2 via the tricarboxylic acid cycle (TCA), while fumarate is reduced to succinate (Equation 1 and Figure 1, Galushko & Schink, 2000). of the consumed acetate is dissimilated and the remaining acetate is used for cell synthesis (Galushko & Schink, 2000). Hence, the actual donor/acceptor ratio observed is approximately 1:2. Fumarate is entirely reduced to succinate, which is secreted to the medium, so the TCA as found in G. sulfurreducens metabolism is not closed.
F I G U R E 1 (a) Acetate conversion in the TCA cycle with fumarate as electron acceptor. TCA is not closed as succinate is secreted to the medium and external fumarate is continuously supplied to fuel the reaction (adapted from Galushko & Schink, 2000). (b) Fumarate is simultaneously reduced as input to the TCA and by FrdCAB, which is coupled to ATP synthesis via the menaquinone pool (adapted from Butler et al., 2006). TCA, tricarboxylic acid cycle, NAD/NADH, reduced/oxidized nicotinamide adenine dinucleotide, NAPD/NADPH, reduced/oxidized nicotinamide adenine dinucleotide phosphate Instead, externally added fumarate is converted to oxaloacetate by fumarase and malate dehydrogenase and continuously introduced to the TCA cycle (Galushko & Schink, 2000). Additionally, fumarate is reduced at the membrane-bound G. sulfurreducens fumarate reductase FrdCAB which is coupled to the menaquinone pool and thereby to ATP synthesis (Butler et al., 2006). This enzyme simultaneously acts as succinate dehydrogenase to close the TCA cycle when not fumarate but Fe(III) is the electron acceptor (Butler et al., 2006;Esteve-Núñez et al., 2005). ATP is solely synthesized by electron transport phosphorylation, fueled by NADH and NADPH delivered to the menaquinone pool (Galushko & Schink, 2000). Fumarate is frequently used as a soluble terminal electron acceptor in G.
sulfurreducens growth medium but is usually omitted in microbial fuel cell experiments to channel electrons exclusively to the electrode.
Malate can be used in different technical applications, for example, in the food and beverage industry, chemical synthesis, textile finishing, and pharmaceutical industries (Jiang et al., 2020;Kövilein et al., 2020). Besides the chemical synthesis by hydration of maleic anhydride generated from the oxidation of benzene or butane, malate or maleic acid can be produced enzymatically by using the fumarase activity or microbial synthesis from renewable substrates (Jiang et al., 2020). Data regarding the market volume of malic acid range between 60,000 and 200,000 tons per year (Kövilein et al., 2020). Succinate or 1,4-butanedioic acid is a four-carbon dicarboxylic acid. Besides different applications in the food industry, succinate is a precursor for the production of many high-value chemicals, for example, 1,4-butanediol, tetrahydrofuran, and polybutylene succinate. Due to its versatile applications, succinate is rising to a bulk chemical in recent years. Its annual global production is estimated at between 30,000 and 50,000 tons (adapted from Cao et al., 2013).
In the following experiments, acetate/fumarate conversion of G.
sulfurreducens was studied by varying donor/acceptor ratios. As mentioned above, the theoretical optimum ratio between acetate as donor and fumarate as acceptor is 1:2. In contrast, the ratio proposed in the often applied DSMZ medium recipe is 1:5. Therefore, in the present study, growth and metabolism were monitored at different ratios and by using growing and resting (stationary) cells. In all experiments, the resulting kinetics as well stoichiometry were evaluated.

| MATERIALS AND METHODS
All chemicals were of at least analytical grade and purchased from Roth, Sigma-Aldrich, and Fluka.
2.1 | Strains and culture/growth conditions All methods are described in detail in  and are only briefly described here. G. sulfurreducens strain PCA (DSM 12127) was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures GmbH). All cultivations were done anaerobically in serum flasks sealed with a butyl septum (Glasgerätebau Ochs). Flasks were incubated shaking at 30°C and 180 rpm (Shaking throw 25 mm, Ecotron Infors HT shaker). The standard growth medium was DSM 826 and contained (per liter): 0.1 g KCl, 1.5 g NH 4 Cl, 0.5 g Na 2 HPO 4 , 0.82 g Na-Acetate as electron donor, 4.8 g Na-fumarate as an electron acceptor, 2.5 g NaHCO 3 , 10 ml of vitamin mix, and 10 ml of trace mineral mix. Vitamin mix contained (per liter): 2 mg biotin; 2 mg folic acid; 10 mg pyridoxine-HCl; 5 mg thiamine-HCl × 2 H 2 O; 5 mg riboflavin; 5 mg nicotinic acid; 5 D-Ca-pantothenate; 0.1 mg vitamin B 12 ; 5 mg p-aminobenzoic acid, and 5 mg lipoic acid. Trace element solution contained: was added and the medium transferred to an anaerobic chamber (Rigid Chamber, Coy Laboratory Products Inc.). Each 48 ml medium was aliquoted under N 2 /H 2 (95/5) atmosphere (forming gas) to 250 ml serum flasks, sealed with a butyl septum, and the septum secured with aluminum caps (Glasgerätebau Ochs). Then the forming gas atmosphere was exchanged by evacuating the flasks three times and refilling them with N 2 /CO 2 gas mixture. Subsequently, serum flasks were autoclaved. Before microbial cultivation 1.5 ml Nafumarate (160 g/L) (if not indicated differently) and 0.5 ml vitamin solution were added to each flask and degassed for another 15 min to remove any oxygen that might have diffused through the septum during storage of the flask. The final pressure in the septum flask was 1.8 bar. Before inoculation, medium and inoculum were prewarmed for 30 min and 1.5 ml of a stationary culture (maintenance culture) was used to inoculate a fresh culture. The maintenance culture was stored for a maximum of 2 weeks in the dark at 4°C and refreshed every 2 weeks from a cryo culture.

| Analytics
Growth experiments were carried out in triplicates and monitored by measuring OD 600 and metabolite concentration (acetate, fumarate, malate, and succinate) in the supernatant using HPLC analysis.
Growth and metabolite kinetics were calculated using the R package "growthcurver," which fits the optical density or concentration data to a logistic equation and allows the estimation of growth rate, respectively, production/consumption rate, as well as doubling time and other growth parameters (Sprouffske & Wagner, 2016). For sampling, shaking flasks were always transferred to the anaerobic chamber to avoid oxygen entering the flask when drawing a sample.
For each sample, 0.8 ml samples were drawn with a syringe and transferred to a cuvette to measure OD 600 . Afterward, the sample  In the following, the donor/acceptor ratio was altered to 1:5.
When fumarate is added in excess, malate accumulation is faster with a rate of 0.28 ± 0.01 h −1 and to a maximum concentration of 17 mM, in comparison to 5 mM when only 25 mM fumarate is available initially. Also, malate was accumulated continuously and not transiently ( Figure 3). After the culture reached the stationary growth phase, 10 mM acetate was added to monitor malate uptake by the cells when the electron source is refilled. The first growth phase (until 60 h) follows logistic growth as seen in Figure 3, the one after fresh acetate is fed seems to follow a limited growth model. The growth rate for the first growth term is 0.23 ± 0.01 h −1 with a maximum calculated doubling time of 3.05 ± 0.09 h −1 , which is slightly faster than growth with a 25 mM electron acceptor. Acetate and succinate metabolization were at similar rates compared to growth with a 1:2.5 donor/acceptor ratio, also showing that excess fumarate concentrations do not inhibit growth. Fumarate is metabolized at a faster rate with −1.23 ± 0.03 h −1 , which correlates with the faster malate accumulation. When acetate was available again, malate concentration decreased immediately and cell growth resumed, but only to 80% of the OD 600 that was expected possible with 20 mM acetate in total (theoretical OD 600 circa 0.84, actual OD 600 0.7).
To test the activity of fumarase and malate transporter of resting cells 35 mM fumarate was added to a stationary culture (OD 600 0.43) and incubated without carbon or electron source. In Figure 4, the conversion of fumarate to malate can be seen, following a classical   (2) When fumarate as a soluble electron acceptor is available in excess, it is constantly converted to malate, even by resting cells.
Transient malate accumulation was also observed in Butler et al. (2006) and Galushko and Schink (2000) and explained by the thermodynamically unfavorable oxidation from malate to oxaloacetate. To still shift the equilibrium toward oxaloacetate, malate is accumulated by the faster reaction of fumarase, converting fumarate to malate (Galushko & Schink, 2000). This effect is more pronounced the higher the fumarate excess. In chemostat studies in vivo flux analysis showed that when fumarate was used as the electron acceptor, fumarate was not only reduced to succinate but also converted to malate by fumarase and further to oxaloacetate via malate dehydrogenase (Yang et al., 2010). The malate dehydrogenase activity seems to be one limiting step in the conversion of the bioconversion using the redox pair acetate/fumarate (Muhamadali et al., 2015). In summary, the results underline that by using different acceptor/donor ratios, malate and succinate production by G.
sulfurreducens can be altered specifically. The results expand our knowledge of G. sulfurreducens metabolism and provide optimization possibilities for chemical synthesis as well as for the application of G.
sulfurreducens in electro-biotechnology and in remediation processes.