Pre-incubation conditions determine the fermentation pattern and microbial community structure in fermenters at mild hydrostatic pressure

Abstract

Fermentation at elevated hydrostatic pressure is a novel strategy targeting product selectivity. However, the role of inoculum history and cross-resistance, that is, acquired tolerance from incubation under distinctive environmental stress, remains unclear in high-pressure operation. In our here presented work, we studied fermentation and microbial community responses of halotolerant marine sediment inoculum (MSI) and anaerobic digester inoculum (ADI), pre-incubated in serum bottles at different temperatures and subsequently exposed to mild hydrostatic pressure (MHP; < 10 MPa) in stainless steel reactors. Results showed that MHP effects on microbial growth, activity, and community structure were strongly temperature-dependent. At moderate temperature (20°C), biomass yield and fermentation were not limited by MHP; suggesting a cross-resistance effect from incubation temperature and halotolerance. Low temperatures (10°C) and MHP imposed kinetic and bioenergetic limitations, constraining growth and product formation. Fermentation remained favorable in MSI at 28°C and ADI at 37°C, despite reduced biomass yield resulting from maintenance and decay proportionally increasing with temperature. Microbial community structure was modified by temperature during the enrichment, and slight differences observed after MHP-exposure did not compromise functionality. Results showed that the relation incubation temperature-halotolerance proved to be a modifier of microbial responses to MHP and could be potentially exploited in fermentations to modulate product/biomass ratio.

Keywords: anaerobic fermentation; halotolerance; mild hydrostatic pressure; piezotolerance; psychrotolerance.

Figure 1

Experimental design applied for the inoculum enrichment at atmospheric pressure and the non‐pooled biomass transfer for the glucose conversion experiments at mild hydrostatic pressure (MHP) of 5 and 8 MPa. Experiments with marine sediment inoculum—MSI were carried out at 10, 20, and 28°C, and the experiments with anaerobic digestion inoculum—ADI at 37°C. For all the experiments, triplicate incubation syringes (biological replicates) plus a negative control without substrate were placed inside the pressure reactors. The time points where samples from sacrificial pressure reactors were recovered are represented by t1, t2, and t3

Figure 2

Absolute change in the final cell concentration in the marine sediment inoculum (MSI) at 10 (a), 20 (b), and 28°C (c) and in the anaerobic digester inoculum (ADI) at 37°C (d) along the incubation at MHP of 5 and 8 MPa. The atmospheric control (0.1 MPa) was measured only at the end of the incubation. Bars represent the average of three biological replicates in pressure treatments and six replicates in the atmospheric controls. Bars with striped pattern differentiate the final time point for each experiment. Error bars correspond to the standard deviation. Dotted red line corresponds to the maximal absolute change in cell density measured during the enrichment before MHP incubation. MHP, mild hydrostatic pressure

Figure 3

Product spectrum (in mg COD L⁻¹) at three selected time points during anaerobic glucose conversion by temperature‐adapted halotolerant enrichments. The marine sediment inoculum (MSI) was evaluated at 10 (a), 20 (b), and 28°C (c) and the anaerobic digester inoculum (ADI) at 37°C (d). The pressure of atmospheric controls was 0.1 MPa (included in the subplots as reference) and the hydrostatic pressures were 5 and 8 MPa. Bars represent the average of three biological replicates in pressure treatments and six replicates in the atmospheric controls. Error bars correspond to the standard deviation. Dotted lines represent the average fraction of COD recovered with glucose as feed in the atmospheric controls (red) and in the experiments at 5 (blue) and 8 MPa (green). COD, chemical oxygen demand

Figure 4

Absolute abundances of bacterial top 13 OTUs in the different experimental treatments and calculated based on flow cytometry data. Columns represent biological replicates for the atmospheric controls (0.1 MPa) and the mild hydrostatic pressure (MHP) experiments at 5 and 8 MPa. (a) Marine sediment inoculum—MSI experiments for different temperature (10, 20, and 28°C)—MHP combinations. (b) Anaerobic digester inoculum—ADI experiments at 37°C and different MHP. OTU, operational taxonomic unit

Figure 5

Nonmetric distance scaling (NMDS) analysis of the Bray–Curtis dissimilarity index of the bacterial community at OTU level for the marine sediment inoculum at 10, 20, and 28°C and the anaerobic digester inoculum at 37°C. OTU, operational taxonomic unit

Figure 6

(a) Gibbs free energy available to carry out catabolism ($\Delta {G_{cat}}^{01}$) for each temperature incubation at 0.1 MPa (atmospheric control—AC) and mild hydrostatic pressure (MHP) of 5 and 8 MPa. (b) Gibbs free energy requirements for maintenance (m) to sustain the initial biomass density used to inoculate AC and the pressurized treatments. (c) Measured biomass yield (Y_X/S ) based on cell numbers converted to units of C‐mol biomass. (d) Theoretical biomass yield calculated based on proposed stoichiometries for the different incubations. Each subplot has a different scale and units for visualization purposes. Data correspond to the marine sediment inoculum at 10, 20, and 28°C and the anaerobic digester inoculum at 37°C

Figure 7

Graphical summary of foreseen effects of incubation temperature (T) (left) and T + mild hydrostatic pressure (MHP; right) on kinetic and bioenergetic parameters. Columns correspond to the experimental treatments with marine sediment inoculum (MSI) at 10, 20, and 28°C and anaerobic digester inoculum (ADI) at 37°C. Colored blue bars represent a qualitative indication of the expected range for the parameter (one = low, two = moderate, three = high, and four = highest). Upward green arrows in MHP column indicate an incremental effect whereas downward red arrows indicate a detrimental effect