Simulating the Contribution of Coaggregation to Interspecies Hydrogen Fluxes in Syntrophic Methanogenic Consortia

Model development.

Hydrogen flux was estimated based on Fick's diffusion law (equation 1). $$mathtex$$\[J{=}D_{\mathrm{H}2}\ \frac{C_{\mathrm{H}2-\mathrm{syntroph}}{-}C_{\mathrm{H}2-{\Delta}\mathrm{H}}}{d}\]$$mathtex$$(1) In this equation, J is the interspecies hydrogen flux, D_H2 is the H₂ diffusion constant in water (at 55°C), C_H2-syntroph is the H₂ concentration immediately outside a syntroph cell, C_H2-ΔH is the H₂ concentration immediately outside a ΔH cell, and d is the average distance between the syntroph and ΔH cells (for units of parameters, refer to the tables). Total interspecies hydrogen flux (Q_H2) is stoichiometrically correlated with the methane production rate (four times the methane production rate) and calculated by multiplying the J value by the total surface area of hydrogen-releasing syntroph cells. $$mathtex$$\[Q_{\mathrm{H}2}{=}X_{\mathrm{syntroph}}\ {\cdot}\ V\ {\cdot}\ A_{\mathrm{syntroph}}\ {\cdot}\ J\]$$mathtex$$(2) In this equation, X_syntroph is the cell concentration of syntroph, V is the culture volume, and A_syntroph is the surface area of a syntroph cell.

In order to apply this estimation method to partially aggregated cocultures, Q_H2 between aggregated cells and that between dispersed cells were separately estimated as follows. $$mathtex$$\[Q_{\mathrm{H2-agg}}{=}X_{\mathrm{agg-syntroph}}\ {\cdot}\ V\ {\cdot}\ A_{\mathrm{syntroph}}\ {\cdot}\ D_{\mathrm{H}2}\ \frac{C_{\mathrm{H}2-\mathrm{syntroph}}{-}C_{\mathrm{H}2-{\Delta}\mathrm{H}}}{d_{\mathrm{agg}}}\]$$mathtex$$(3)$$mathtex$$\[Q_{\mathrm{H2-dis}}{=}X_{\mathrm{dis-syntroph}}\ {\cdot}\ V\ {\cdot}\ A_{\mathrm{syntroph}}\ {\cdot}\ D_{\mathrm{H}2}\ \frac{C_{\mathrm{H}2-\mathrm{syntroph}}{-}C_{\mathrm{H}2-{\Delta}\mathrm{H}}}{d_{\mathrm{dis}}}\]$$mathtex$$(4)$$mathtex$$\[Q_{\mathrm{H}2}{=}Q_{\mathrm{H2-agg}}{+}Q_{\mathrm{H2-dis}}\]$$mathtex$$(5) In these equations, Q_H2-agg and Q_H2-dis are the total hydrogen flux between aggregated cells and that between dispersed cells, respectively, X_agg-syntroph and X_dis-syntroph are the concentration of aggregated syntroph cells and that of dispersed cells, respectively, and d_agg and d_dis are the mean interspecies distance between aggregated cells and that between dispersed cells, respectively. This scheme for estimating a Q_H2 value was named “the syntrophy model.”

Growth and coaggregation.

We have reported that cells in coculture of P. thermopropionicum SI (8) and M. thermautotrophicus ΔH were partially aggregated (11). The present study also analyzed cocultures of strain ΔH with butyrate-oxidizing Syntrophothermus lipocalidus TGB-C1 (22) and acetate-oxidizing Thermacetogenium phaeum PB (7). They were grown in 100-ml serum vials containing 50 ml of a culture medium as described elsewhere previously (22). The culture medium was supplemented with 0.1% Bacto yeast extract (Difco) and a growth substrate at 17 to 20 mM. Cultivation was conducted at 55°C under an atmosphere of N₂ plus CO₂ (80/20 [vol/vol]) without shaking. Cultivation was initiated by inoculation with 5 ml of a preculture in the same medium. Hydrogen releases from acetate by strain PB and from butyrate by strain TGB-C1 were examined in monocultures inoculated with 5 ml of precultures grown on methanol (PB) and crotonate (TGB-C1), in which methanol or crotonate was completely lost. Microscopic analyses and gas chromatography were conducted as described previously (11).

We found that although cells in monocultures of PB and TGB-C1 (grown on methanol and crotonate, respectively) were fully dispersed, their cells in cocultures with ΔH were partially aggregated (see Fig. S1 and S2 in the supplemental material for growth curves and phase-contrast micrographs, respectively). Cells in their cocultures were also observed by field emission-scanning electron microscopy (10), revealing that aggregates were comprised of both syntroph and methanogen cells (see Fig. S3 in the supplemental material). In addition, extracellular filamentous appendages were found in these cocultures, which connected syntroph cells to methanogen cells (see Fig. S3 in the supplemental material).

Our previous study also found that strain SI utilized flagellum-like filaments for making contact with strain ΔH (11). Together with the results of the present study, we consider that the connection of syntroph and methanogen cells with filaments is a widespread phenomenon. In order to know more specifically how these filaments contribute to coaggregation, molecular analyses of filaments should be done. It has been known that extracellular filaments function as adhesins in many different types of bacteria (10, 14, 15, 17, 23, 26); information in those studies will be useful for examining the role of extracellular filaments of syntrophs.

X_agg-syntroph and X_dis-syntroph.

In order to estimate Q_H2 values, we first determined X_agg-syntroph and X_dis-syntroph values for strains SI, PB, and TGB-C1 in cocultures with strain ΔH. For this, we measured optical densities at 600 nm (OD₆₀₀) of a coculture before and after gentle homogenization with a tissue grinder, as described previously (11), until the OD₆₀₀ no longer increased. Phase-contrast images of several homogenized cultures revealed that cell aggregates were fully dispersed (data not shown). The OD₆₀₀ values measured were converted to a total cell concentration (a sum of syntroph and methanogen cells, X_total) using equations 6 to 8; these equations were produced from standard curves obtained by measuring OD₆₀₀ and cell concentrations (DAPI [4′,6′-diamidino-2-phenylindole] counts) (11) of several fully dispersed cultures. $$mathtex$$\[X_{\mathrm{total}\ (\mathrm{SI}/{\Delta}\mathrm{H})}({\times}\ 10^{7}\ \mathrm{cells\ ml}^{{-}1}){=}122\ {\cdot}\ \mathrm{OD}_{600}{-}0.92\]$$mathtex$$(6)$$mathtex$$\[X_{\mathrm{total}\ (\mathrm{TGB}-\mathrm{C}1/{\Delta}\mathrm{H})}({\times}\ 10^{7}\ \mathrm{cells\ ml}^{{-}1}){=}318\ {\cdot}\ \mathrm{OD}_{600}{-}3.40\]$$mathtex$$(7)$$mathtex$$\[X_{\mathrm{total}\ (\mathrm{PB}/{\Delta}\mathrm{H})}({\times}\ 10^{7}\ \mathrm{cells\ ml}^{{-}1}){=}158\ {\cdot}\ \mathrm{OD}_{600}{-}3.44\]$$mathtex$$(8) The concentration of total aggregated cells (X_agg) was determined using equation 9, while the concentration of total dispersed cells (X_dis) was calculated from X_agg using equation 10. $$mathtex$$\[X_{\mathrm{agg}}\ (\%){=}\ \frac{X_{\mathrm{total}}\ \mathrm{after\ dispersion}{-}X_{\mathrm{total}}\ \mathrm{before\ dispersion}}{X_{\mathrm{total}}\ \mathrm{after\ dispersion}}{\times}100\]$$mathtex$$(9)$$mathtex$$\[X_{\mathrm{dis}}{=}X_{\mathrm{total}}{-}X_{\mathrm{agg}}\]$$mathtex$$(10)X_agg and X_dis values can also be estimated from DAPI microscopic counts of cells before and after homogenization, while the values determined by the OD measurement agreed well with those determined by the DAPI counts (data not shown).

In order to estimate a ratio of the number of syntroph cells to that of total dispersed cells, dispersed cells were observed using phase-contrast micrography (see Fig. 1A, for example) and fluorescence microscopy. Syntroph cells and methanogen cells could be discriminated according to the differences in cell shape and the F₄₂₀ autofluorescence (9). An X_dis-syntroph value was estimated from X_dis and the ratio of the number of syntroph cells. On the other hand, the ratio of the number of syntroph cells to that of the total aggregated cells was determined based on data from thin-section images of coaggregates obtained by transmission electron microscopy (TEM) (Fig. 1B). TEM images were obtained by a standard procedure (13) using an H7000 transmission electron microscope (Hitachi). As shown in Fig. 1B, syntroph cells could be distinguished from ΔH cells by shape and darkness. An X_agg-syntroph value was estimated from X_agg and the ratio of the number of syntroph cells (n > 90 for all cases).

• Open in new tab
• Download powerpoint

FIG. 1.

Determination of interspecies distances for dispersed cells and aggregated cells in a coculture of strains SI and ΔH. (A) A representative phase-contrast micrograph showing aggregates and dispersed cells. Arrows indicate an interspecies distance between dispersed cells. Bar, 20 μm. (B) A representative cross-section TEM image of aggregated cells showing parameters in equation 11 for determining a d_agg value. Bar, 500 nm.

Table 1 summarizes X_agg, X_agg-syntroph, X_dis, X_dis-syntroph, X_total, and X_{total-syntroph} values for four types of cocultures at mid-log growth phases, namely, SI plus ΔH grown on ethanol, SI plus ΔH grown on propionate, TGB-C1 plus ΔH grown on butyrate, and PB plus ΔH grown on acetate. Table 1 shows that SI/ΔH (propionate) and PB/ΔH (acetate) coaggregated at relatively high ratios, although the X_agg values were not high compared to X_dis values in all cases. In addition, we found that methanogen cells occupied aggregates more abundantly than did syntroph cells; this trend was prominent in cocultures of SI/ΔH (ethanol) and TGB-C1/ΔH (butyrate). We deduce that syntroph cells should have more vigorously aggregated when they were grown on energetically unfavorable substrates, i.e., propionate and acetate.

d_agg and d_dis.

We calculated a d_dis value by supposing that dispersed cells were randomly distributed in the liquid phase (2, 11, 16, 24). In contrast, a d_agg value was determined by analyzing thin-section TEM pictures of aggregates (see Fig. 1B, for example). In this analysis, we supposed that cells were cylindrical and determined a mean radius of syntroph cells (R_s), a mean radius of ΔH cells (R_d)b and a mean minimal interspecies distance (d_agg-min) by analyzing over 80 syntroph/ΔH pairs for each coculture. From these values, an interspecies distance at angle θ (Fig. 1B) was calculated using equation 11. $$mathtex$$\[d_{\mathrm{agg}}({\theta}){=}[(R_{\mathrm{s}}\ {\cdot}\ \mathrm{sin}{\theta}\ {-}\ R_{\mathrm{d}}\ {\cdot}\ \mathrm{sin}{\theta})^{2}{+}(R_{\mathrm{s}}{+}d_{\mathrm{agg-min}}{+}R_{\mathrm{d}}{-}R_{\mathrm{s}}\ {\cdot}\ \mathrm{cos}{\theta}{-}R_{\mathrm{d}}\ {\cdot}\ \mathrm{cos}{\theta})^{2}]^{0.5}\]$$mathtex$$11The θ value ranged from 0 to π/2. For estimating d_agg, π/2 was divided into n parts, and d_agg(θ) at each θ point was calculated. Equation 12 was used for estimating d_agg from the d_agg(θ) values. $$mathtex$$\[d_{\mathrm{agg}}{=}\ \frac{{{\sum}_{{\theta}\ {=}\ 0}^{{\pi}/2}}\ d_{\mathrm{agg}}({\theta})}{n}\]$$mathtex$$(12) In our analyses, n was arbitrarily set at 90. Table 2 presents parameters in equation 11 and d_agg and d_dis values determined for the cocultures used in the present study. It is shown that the d_agg values for the different cocultures were similar to each other and that the d_dis values were approximately 100-fold higher than the d_agg values.

Estimation of hydrogen fluxes.

In order to estimate Q_H2-agg and Q_H2-dis, parameters in equations 3 and 4, other than X_agg-syntroph, X_dis-syntroph, d_agg, and d_dis, were determined as follows. D_H2 was cited from data reported previously by Wise and Houghton (30). C_H2-ΔH was defined as the minimum H₂ concentration, above which an H₂-consuming methanogen can gain energy by carbonate respiration (6). A_syntroph was estimated by assuming that cells were cylindrical and measuring diameters and lengths of syntroph cells in the field emission-scanning electron microscopy photos (see Fig. S3 in the supplemental material) (11). C_H2-syntroph was determined to be the H₂ concentration at a time point of microscopic analysis in the exponential growth phase, which was obtained from an H₂ release curve (see Fig S1 in the supplemental material for strains PB and TGB-C1) (see reference 11 for strain SI). These values are summarized in Table 2.

Table 3 summarizes Q_H2-agg and Q_H2-dis values estimated as described above, in which we also present Q_H2 values and total H₂ fluxes experimentally determined from methane production rates (see Fig. S1 in the supplemental material) (11). The estimation revealed that although numbers of syntroph cells in aggregates (X_agg-syntroph) were relatively small in all cocultures, they contributed largely to total hydrogen fluxes (49 to 92%). Notably, coaggregation was found to contribute largely to syntrophic propionate and acetate oxidation. It is also shown that the estimated total hydrogen fluxes (Q_H2 values) agreed well with those experimentally determined, supporting the adequacy of the syntrophy model.

We show here that the simulation model was applicable to several different syntrophic methanogenic cocultures irrespective of coculture members and substrates, suggesting that the model is widely applicable for partially aggregated heterogeneous systems. Although previous studies have theoretically discussed the importance of close physical contact for propionate oxidation (2, 11, 25, 28), the model could quantitatively show that coaggregation was important not only for oxidation of propionate, butyrate, and acetate (representing thermodynamically unfavorable substrates) but also for ethanol oxidation. Based on the results, we suggest that aggregation is the key factor for engineering syntrophic methanogenesis, for which the simulation model described herein will be useful.