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

A simple model (termed the syntrophy model) for simulating thecontribution of coaggregation to interspecies hydrogen fluxesbetween syntrophic bacteria and methanogenic archaea is described.We applied it to analyzing partially aggregated syntrophic cocultureswith various substrates, revealing that large fractions of hydrogenmolecules were fluxed in aggregates.

INTRODUCTION

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Introduction

The syntrophic interaction between fermentative bacteria andmethanogenic archaea is an essential part of methanogenesis(18) and has been found in such ecosystems as rice paddy fields(5), freshwater sediments (4), mammalian digestive tracts (29),petroleum-contaminated soil (12), and anaerobic digesters fororganic waste treatment (6, 9, 18, 21, 24). In this process,reducing equivalents (i.e., H₂ and/or formate) produced by fermentativebacteria should be efficiently consumed by methanogenic archaeain order for the bacteria and archaea to grow actively (2, 3,24, 27). This is particularly important for syntrophic volatilefatty acid (such as butyrate, propionate, and acetate) oxidation,since this reaction is endergonic under standard conditionsand is thermodynamically feasible only when the H₂ partial pressure(or formate concentration) is kept very low (1, 6, 20, 24, 27).

It has been theoretically suggested that close physical contactbetween volatile fatty acid-fermenting syntrophic bacteria (syntrophs)and methanogenic archaea (methanogens) is important for efficientinterspecies electron transfer (2, 19, 24, 28). Our previousstudy has also indicated that coaggregation facilitated interspecieshydrogen transfer between a propionate-oxidizing syntroph, Pelotomaculumthermopropionicum SI, and a hydrogen-consuming methanogen, Methanothermobacterthermautotrophicus ${Delta}$ H (11). However, since we were unable toseparately determine hydrogen flux in aggregates and that betweendispersed cells, the contribution of aggregates to interspecieshydrogen transfer has not yet been quantitatively evaluated.In the present study, we developed and applied a model (namedthe syntrophy model) for simulating the contribution of coaggregationto interspecies hydrogen flux between syntrophs and methanogens.

Model development.

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Model development.

Hydrogen flux was estimated based on Fick's diffusion law (equation1).

Formula 1 (1)
In this equation,J is the interspecies hydrogen flux, D_H2 is the H₂ diffusionconstant in water (at 55°C), C_H2-syntroph is the H₂ concentrationimmediately outside a syntroph cell, C_H2-_H is the H₂ concentrationimmediately outside a ${Delta}$ H cell, and d is the average distancebetween the syntroph and ${Delta}$ H cells (for units of parameters, referto the tables). Total interspecies hydrogen flux (Q_H2) is stoichiometricallycorrelated with the methane production rate (four times themethane production rate) and calculated by multiplying the Jvalue by the total surface area of hydrogen-releasing syntrophcells.

Formula 2 (2)
In this equation,X_syntroph is the cell concentration of syntroph, V is the culturevolume, and A_syntroph is the surface area of a syntroph cell.

In order to apply this estimation method to partially aggregatedcocultures, Q_H2 between aggregated cells and that between dispersedcells were separately estimated as follows.

Formula 3 (3)

Formula 4 (4)

Formula 5 (5)
In these equations, Q_H2-agg andQ_H2-dis are the total hydrogen flux between aggregated cellsand that between dispersed cells, respectively, X_agg-syntrophand X_dis-syntroph are the concentration of aggregated syntrophcells and that of dispersed cells, respectively, and d_agg andd_dis are the mean interspecies distance between aggregated cellsand that between dispersed cells, respectively. This schemefor estimating a Q_H2 value was named "the syntrophy model."

Growth and coaggregation.

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Growth and Coaggregation.

We have reported that cells in coculture of P. thermopropionicumSI (8) and M. thermautotrophicus ${Delta}$ H were partially aggregated(11). The present study also analyzed cocultures of strain ${Delta}$ Hwith butyrate-oxidizing Syntrophothermus lipocalidus TGB-C1(22) and acetate-oxidizing Thermacetogenium phaeum PB (7). Theywere grown in 100-ml serum vials containing 50 ml of a culturemedium as described elsewhere previously (22). The culture mediumwas supplemented with 0.1% Bacto yeast extract (Difco) and agrowth substrate at 17 to 20 mM. Cultivation was conducted at55°C under an atmosphere of N₂ plus CO₂ (80/20 [vol/vol])without shaking. Cultivation was initiated by inoculation with5 ml of a preculture in the same medium. Hydrogen releases fromacetate by strain PB and from butyrate by strain TGB-C1 wereexamined in monocultures inoculated with 5 ml of preculturesgrown on methanol (PB) and crotonate (TGB-C1), in which methanolor crotonate was completely lost. Microscopic analyses and gaschromatography 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 ${Delta}$ H were partially aggregated (seeFig. S1 and S2 in the supplemental material for growth curvesand phase-contrast micrographs, respectively). Cells in theircocultures were also observed by field emission-scanning electronmicroscopy (10), revealing that aggregates were comprised ofboth syntroph and methanogen cells (see Fig. S3 in the supplementalmaterial). In addition, extracellular filamentous appendageswere found in these cocultures, which connected syntroph cellsto methanogen cells (see Fig. S3 in the supplemental material).

Our previous study also found that strain SI utilized flagellum-likefilaments for making contact with strain ${Delta}$ H (11). Together withthe results of the present study, we consider that the connectionof syntroph and methanogen cells with filaments is a widespreadphenomenon. In order to know more specifically how these filamentscontribute to coaggregation, molecular analyses of filamentsshould be done. It has been known that extracellular filamentsfunction as adhesins in many different types of bacteria (10,14, 15, 17, 23, 26); information in those studies will be usefulfor examining the role of extracellular filaments of syntrophs.

X_agg-syntroph and X_dis-syntroph.

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Xagg-syntroph and xdis-syntroph.

In order to estimate Q_H2 values, we first determined X_agg-syntrophand X_dis-syntroph values for strains SI, PB, and TGB-C1 in cocultureswith strain ${Delta}$ H. For this, we measured optical densities at 600nm (OD₆₀₀) of a coculture before and after gentle homogenizationwith a tissue grinder, as described previously (11), until theOD₆₀₀ no longer increased. Phase-contrast images of severalhomogenized cultures revealed that cell aggregates were fullydispersed (data not shown). The OD₆₀₀ values measured were convertedto a total cell concentration (a sum of syntroph and methanogencells, X_total) using equations 6 to 8; these equations wereproduced from standard curves obtained by measuring OD₆₀₀ andcell concentrations (DAPI [4',6'-diamidino-2-phenylindole] counts)(11) of several fully dispersed cultures.

Formula 6 (6)

Formula 7 (7)

Formula 8 (8)
The concentration of total aggregatedcells (X_agg) was determined using equation 9, while the concentrationof total dispersed cells (X_dis) was calculated from X_agg usingequation 10.

Formula 9 (9)

Formula 10 (10)
X_agg and X_dis values can alsobe estimated from DAPI microscopic counts of cells before andafter homogenization, while the values determined by the ODmeasurement agreed well with those determined by the DAPI counts(data not shown).

In order to estimate a ratio of the number of syntroph cellsto that of total dispersed cells, dispersed cells were observedusing phase-contrast micrography (see Fig. 1A, for example)and fluorescence microscopy. Syntroph cells and methanogen cellscould be discriminated according to the differences in cellshape and the F₄₂₀ autofluorescence (9). An X_dis-syntroph valuewas estimated from X_dis and the ratio of the number of syntrophcells. On the other hand, the ratio of the number of syntrophcells to that of the total aggregated cells was determined basedon data from thin-section images of coaggregates obtained bytransmission electron microscopy (TEM) (Fig. 1B). TEM imageswere obtained by a standard procedure (13) using an H7000 transmissionelectron microscope (Hitachi). As shown in Fig. 1B, syntrophcells could be distinguished from ${Delta}$ H cells by shape and darkness.An X_agg-syntroph value was estimated from X_agg and the ratioof the number of syntroph cells (n > 90 for all cases).

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FIG. 1. Determination of interspecies distances for dispersed cells and aggregated cells in a coculture of strains SI and ${Delta}$ H. (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 coculturesat mid-log growth phases, namely, SI plus ${Delta}$ H grown on ethanol,SI plus ${Delta}$ H grown on propionate, TGB-C1 plus ${Delta}$ H grown on butyrate,and PB plus ${Delta}$ H grown on acetate. Table 1 shows that SI/ ${Delta}$ H (propionate)and PB/ ${Delta}$ H (acetate) coaggregated at relatively high ratios, althoughthe X_agg values were not high compared to X_dis values in allcases. In addition, we found that methanogen cells occupiedaggregates more abundantly than did syntroph cells; this trendwas prominent in cocultures of SI/ ${Delta}$ H (ethanol) and TGB-C1/ ${Delta}$ H (butyrate).We deduce that syntroph cells should have more vigorously aggregatedwhen they were grown on energetically unfavorable substrates,i.e., propionate and acetate.

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TABLE 1. Cell concentrations in syntrophic cocultures

d_agg and d_dis.

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dagg and ddis.

We calculated a d_dis value by supposing that dispersed cellswere randomly distributed in the liquid phase (2, 11, 16, 24).In contrast, a d_agg value was determined by analyzing thin-sectionTEM pictures of aggregates (see Fig. 1B, for example). In thisanalysis, we supposed that cells were cylindrical and determineda mean radius of syntroph cells (R_s), a mean radius of ${Delta}$ H cells(R_d)b and a mean minimal interspecies distance (d_agg-min) byanalyzing over 80 syntroph/ ${Delta}$ H pairs for each coculture. Fromthese values, an interspecies distance at angle ${theta}$ (Fig. 1B) wascalculated using equation 11.

Formula 11 11
The ${theta}$ value ranged from 0 to ${pi}$ /2. For estimatingd_agg, ${pi}$ /2 was divided into n parts, and d_agg( ${theta}$ ) at each ${theta}$ pointwas calculated. Equation 12 was used for estimating d_agg fromthe d_agg( ${theta}$ ) values.

Formula 12 (12)
Inour analyses, n was arbitrarily set at 90. Table 2 presentsparameters in equation 11 and d_agg and d_dis values determinedfor the cocultures used in the present study. It is shown thatthe d_agg values for the different cocultures were similar toeach other and that the d_dis values were approximately 100-foldhigher than the d_agg values.

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TABLE 2. Parameters used in the syntrophy model

Estimation of hydrogen fluxes.

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Estimation of hydrogen fluxes.

In order to estimate Q_H2-agg and Q_H2-dis, parameters in equations3 and 4, other than X_agg-syntroph, X_dis-syntroph, d_agg, andd_dis, were determined as follows. D_H2 was cited from data reportedpreviously by Wise and Houghton (30). C_H2-_H was defined as theminimum H₂ concentration, above which an H₂-consuming methanogencan gain energy by carbonate respiration (6). A_syntroph wasestimated by assuming that cells were cylindrical and measuringdiameters and lengths of syntroph cells in the field emission-scanningelectron microscopy photos (see Fig. S3 in the supplementalmaterial) (11). C_H2-syntroph was determined to be the H₂ concentrationat a time point of microscopic analysis in the exponential growthphase, which was obtained from an H₂ release curve (see FigS1 in the supplemental material for strains PB and TGB-C1) (seereference 11 for strain SI). These values are summarized inTable 2.

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

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TABLE 3. Interspecies hydrogen fluxes estimated using the syntrophy model and those experimentally determined

We show here that the simulation model was applicable to severaldifferent syntrophic methanogenic cocultures irrespective ofcoculture members and substrates, suggesting that the modelis widely applicable for partially aggregated heterogeneoussystems. Although previous studies have theoretically discussedthe importance of close physical contact for propionate oxidation(2, 11, 25, 28), the model could quantitatively show that coaggregationwas important not only for oxidation of propionate, butyrate,and acetate (representing thermodynamically unfavorable substrates)but also for ethanol oxidation. Based on the results, we suggestthat aggregation is the key factor for engineering syntrophicmethanogenesis, for which the simulation model described hereinwill be useful.

ACKNOWLEDGMENTS

We thank Yoichi Kamagata for providing T. phaeum PB and S. lipocalidusTGB-C1 and Hiroyuki Imachi for providing P. thermopropionicumSI. We also thank Masanori Arita for valuable advice for modeldevelopment, Kohei Nakamura and Miho Enoki for valuable suggestions,and Yasuo Igarashi and Hiroshi Ikenaga for continuous encouragement.We are grateful to Katsutoshi Hori and Mika Atsumi for helpin electron microscopy and Reiko Hirano for technical assistance.

This work was supported by the New Energy and Industrial TechnologyDevelopment Organization (NEDO).

FOOTNOTES

* Corresponding author. Mailing address: Marine Biotechnology Institute, Heita, Kamaishi, Iwate 026-0001, Japan. Phone: 81-193-26-6581. Fax: 81-193-26-6592. E-mail: shunichi.ishii{at}mbio.jp.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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