Bioenergetics of aerobic and anaerobic growth of Shewanella putrefaciens CN32

Abstract

Shewanella putrefaciens is a model dissimilatory iron-reducing bacterium that can use Fe(III) and O₂ as terminal electron acceptors. Consequently, it has the ability to influence both aerobic and anaerobic groundwater systems, making it an ideal microorganism for improving our understanding of facultative anaerobes with iron-based metabolism. In this work, we examine the bioenergetics of O₂ and Fe(III) reduction coupled to lactate oxidation in Shewanella putrefaciens CN32. Bioenergetics were measured directly via isothermal calorimetry and by changes to the chemically defined growth medium. We performed these measurements from 25 to 36°C. Modeling metabolism with macrochemical equations allowed us to define a theoretical growth stoichiometry for the catabolic reaction of 1.00 O₂:lactate and 1.33 Fe(III):lactate that was consistent with the observed ratios of O₂:lactate (1.20 ± 0.23) and Fe(III):lactate (1.46 ± 0.15) consumption. Aerobic growth showed minimal variation with temperature and minimal variation in thermodynamic potentials of incubation. Fe(III)-based growth showed a strong temperature dependence. The Gibbs energy and enthalpy of incubation was minimized at ≥30°C. Energy partitioning modeling of Fe(III)-based calorimetric incubation data predicted that energy consumption for non-growth associate maintenance increases substantially above 30°C. This prediction agrees with the data at 33 and 35°C. These results suggest that the effects of temperature on Shewanella putrefaciens CN32 are metabolism dependent. Gibbs energy of incubation above 30°C was 3-5 times more exergonic with Fe(III)-based growth than with aerobic growth. We compared data gathered in this study with predictions of microbial growth based on standard-state conditions and based on the thermodynamic efficiency of microbial growth. Quantifying the growth requirements of Shewanella putrefaciens CN32 has advanced our understanding of the thermodynamic constraints of this dissimilatory iron-reducing bacterium.

Keywords: Gibbs energy consumption; Shewanella putrefaciens CN32; bioenergetics; dissimilatory iron-reducing bacterium; growth enthalpy.

Figure 1

Gibbs energy (ΔG_inc) and enthalpy (ΔH_inc) of incubation for O₂- and Fe(III)-based growth as a function of temperature. Error bars reflect standard deviation between replicate experiments.

Figure 2

Catabolic cycles to produce observed biomass (f_cat, defined in Equation 4) illustrated as a function of temperature. Error bars reflect standard deviation between replicate experiments.

Figure 3

Model fit to measured energy and growth parameters as a function of temperature. Solid curves indicate model predictions of specific heat evolution rate (A) and specific growth rate (B). Closed circles are data points for each experimental replicate. Predictions of growth associated costs (G×μ) and non-growth associated maintenance (m_ν) are shown by broken curves, while total the specific energy consumption rate is the solid curve (C). The standard deviations of parameters were used to show the error of the model (dotted curves) in all panels.

Figure 4

Biomass yield (C-mol/mol lactate) as a function of temperature for O₂- and Fe(III)-based growth.

Figure 5

Thermodynamic efficiency (η) as a function of electron donor uptake rate (μ_ED). O₂- and Fe(III)-based growth data are plotted along with compilation data from Smeaton and Van Cappellen (2018) and the power law scaling relationship between η and μ_ED from Calabrese et al. (2021).

Figure 6

Curves represent standard state values for $\Delta G_r^{°}$ and $\Delta H_r^{°}$ for both growth strategies. The curve thickness spans the variation in thermodynamic potentials with temperature from 25 to 36°C. ΔG_inc and ΔH_inc for O₂- and Fe(III)-based growth are plotted at as data points. Error bars reflect the standard deviation between replicate samples.