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Keywords:

  • Fermentation;
  • Homoacetogenesis;
  • Methanogenesis;
  • Hydrogen;
  • Gibbs free energy;
  • Radiotracer

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Washed excised roots of rice (Oryza sativa) produced H2, CH4, acetate, propionate and butyrate when incubated under anoxic conditions. Acetate production was most pronounced with a maximum rate (mean±standard error; four different root preparations) of 3.4±0.6 μmol h−1 g-dry weight−1 roots, compared to 0.45±0.13, 0.06±0.03, and 0.04±0.01 μmol h−1 g-dw−1 for propionate, butyrate and CH4, respectively. Hydrogen transiently accumulated to maximum partial pressures of >1 kPa after one day of incubation. Then it decreased and reached more or less constant concentrations of about 50–80 Pa after about 7–8 days. Hydrogen partial pressures were always high enough to allow exergonic methanogenesis (ΔG=−67 to −98 kJ mol−1 CH4) and exergonic homoacetogenesis (ΔG=−18 to −48 kJ mol−1 acetate) from H2 plus CO2. Radioactive bicarbonate/CO2 was incorporated into CH4, acetate and propionate. The specific radioactivities of the products indicated that CH4 was exclusively produced from H2/CO2 confirming a previous study. The contribution of CO2 to the production of acetate and propionate was 32–39% and 42–61%, respectively, assuming that each carbon atom was equally labeled. Propionate also became radioactively labeled, when the roots were incubated with either [1-14C]acetate or [2-14C]acetate accounting for 60–76% of total propionate production. Reductive formation of propionate was thermodynamically favorable both from H2 plus acetate plus CO2G=−15 to −38 kJ mol−1 propionate) and from H2 plus CO2G=−34 to −85 kJ mol−1 propionate). A substantial fraction of propionate was apparently reductively formed from acetate and/or CO2. In conclusion, our results demonstrate an intensive anaerobic dark metabolism of CO2 on washed rice roots with reduction of CO2 contributing significantly to the production of acetate, propionate and CH4. The CO2 reduction seemed to be driven by decay and fermentation of root material.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Flooded rice fields are an important source of atmospheric CH4 which significantly contributes to global warming [1,2]. Rice plants affect the emission of CH4 in three different ways. The aerenchyma system of the plants serves as a conduit for CH4 transport from the flooded soil into the atmosphere [3–5]. The aerenchyma system likewise allows the diffusion of O2 into the rhizosphere where substantial amounts of CH4 are oxidized by aerobic methanotrophic bacteria [5–8]. Methane production in the soil is enhanced by plant photosynthesis most probably by stimulating the microbial community in the rhizosphere with root exudates or products of decaying roots [9,10]. As a conceptual model rice field soils have been divided into two compartments, one which is adjacent to the oxic root surface and supports aerobic metabolism such as oxidation of CH4, sulfide, iron(II) and ammonium, and the other which is completely anoxic and supports anaerobic metabolism such as methanogenesis [11,12].

However, it has recently been shown that methanogenic bacteria are not necessarily restricted to the anoxic soil compartment but can also occur on washed rice roots which produce CH4 as soon as they are incubated under anoxic conditions [13,14]. This result was consistent with similar observations in other aquatic plants [15]. In fact, the roots of rice are populated with microorganisms which form novel euryarchaeotal main lines of descent [16]. Methanogenic enrichment cultures from root material contain methanogens that apparently belong to these novel groups of Archaea and utilize H2/CO2 or ethanol/CO2 as substrates [17]. Indeed, reduction of CO2 rather than cleavage of acetate has been shown to be the dominant methanogenic pathway on washed rice roots [17].

However, methanogenesis is only possible when methanogenic substrates are produced in the washed root samples. Production of H2 has been observed in washed rice roots by Kimura et al. [18] who proposed that this H2 may support methanogenesis in the adjacent anoxic soil compartment. Since anaerobic fermentation products other than H2 may as well be produced, we studied the potential of washed rice roots for anaerobic metabolism and found, besides H2 production, substantial production of acetate and propionate.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Rice plants were grown in the greenhouse using soil which was obtained from the rice fields in Vercelli, Italy [19]. The soil had been used for growing rice before. After harvest, the plants were removed and the soil was drained, air-dried, broken in a mechanical grinder and homogenized. The soil was filled into plastic containers (35×27×22.5 cm) and mixed with demineralized water so that the soil surface was covered with 3–10 cm of water. Rice seeds (Oryza sativa, var. Roma, type japonica) were germinated on water-soaked tissue paper and transplanted into the soil at a distance of about 5 cm to each other when the shoots had reached a length of 5–7 cm. The plants were then grown in the greenhouse at average conditions of 25°C, 60% relative humidity and 10–15 kLux light intensity during the day. After 70–85 days, the plants were carefully removed from the soil together with the roots. The soil was coarsely washed off the roots using tap water. Then, all remaining soil particles were carefully removed from the roots by several washing steps using demineralized and autoclaved water. During the washing procedure the water was permanently bubbled with N2. The roots were then cut off with a razor blade and permanently kept under N2. In one experiment, the surface of the roots was sterilized for 10 s in 5% NaOCl solution as described by Bosse and Frenzel [7].

The incubation experiments were done using glass bottles (150 ml volume; Müller and Krempel AG, Bülach, Switzerland) which were closed with latex stoppers. The bottles were filled with 50 ml anoxic sterile phosphate buffer (50 mM KH2PO4, 17 mM NaCl, 0.2 mM MgCl2) pH 7.0–7.2 and 10 g fresh roots (equivalent to 1.07±0.06 g dry weight), and then gassed with N2. The bottles were incubated at room temperature (about 25°C) without agitation usually for about 400 h. Gas samples (0.25–1.0 ml) were taken with a gas-tight pressure lock syringe (Dynatech, Baton Rouge, USA) after the bottles were vigorously shaken by hand, and analyzed immediately [20]. Liquid samples (0.5 ml) were taken with a syringe, membrane-filtered (0.2 μm; Minisart SRP15, Sartorius, Göttingen, Germany) and stored frozen (−20°C) until analysis by HPLC [21]. Some of the liquid samples were also analyzed for dissolved organic carbon by a commercial analytical laboratory (Wartig, Lahntal, Germany). In some experiments, CH3F was added to the gas phase at a concentration of 1.0%.

Gibbs free energies (ΔG) of the production of CH4, acetate and propionate from precursors were calculated from the respective standard Gibbs free energies (ΔG°) and the actual concentrations of reactants and products using Nernst's equation. The ΔG° were calculated from the standard Gibbs free energies of formation [22].

Radiotracer experiments were done by adding 50–100 μCi (1.8–3.7 MBq) of either NaH14CO3, Na-[2-14C]acetate or Na-[1-14C]acetate (Amersham, Braunschweig, Germany) in a volume of 0.5 ml to the bottles. The specific radioactivities of the added tracers were 54–56 mCi mmol−1 (2.0–2.1 GBq mmol−1). The radioactivity was added 20–70 h after gassing the bottles with N2. In a repetition of the experiment the radioactivity was added immediately after gassing. Gas samples were then taken as described above. At the end of the experiment, the radioactivity recovered in the gas phase, liquid phase plus roots was about 70–80% of the radioactivity that was initially applied. Total and radioactive CH4 and CO2 were analyzed in a gas chromatograph equipped with flame ionization detector, reduction column and a RAGA radioactivity detector [20]. Total and radioactive fatty acids were analyzed in the liquid phase in a high pressure liquid chromatography system equipped with refraction index detector and RAMONA radioactivity detector [21]. The fraction (f) of CH4 that was produced from the reduction of H14CO3 was calculated from the specific radioactivities of 14CH4 (SRCH4) and 14CO2 (SRCO2) measured in the gas phase using f=SRCH4/SRCO2[20]. The fraction of acetate that was produced from reduction of H14CO3 was calculated analogously from the specific radioactivities of the acetate carbon and the 14CO2 assuming that each of the carbon atoms of acetate was equally labeled. The fraction of propionate that was produced from reduction of H14CO3 was calculated from the specific radioactivities of the propionate carbon and the 14CO2 assuming that each of the carbon atoms of propionate was equally labeled. The fraction of propionate that was produced from reduction of radioactive acetate was calculated from the specific radioactivities of the propionate and acetate.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

When excised washed rice roots were incubated in anoxic suspension, H2, CH4, acetate, propionate and butyrate were produced (Fig. 1). CO2 was also produced (not shown). In some experiments, small amounts (<0.5 mM) of lactate, succinate, formate, iso-valerate and 1-propanol were also detected. With exception of H2, all the compounds accumulated steadily for about 100–200 h and then approached a plateau. The maximum accumulation rates were highest for acetate, followed by propionate, butyrate and CH4. The results obtained in experiments using different microcosms and root material are summarized in Table 1. The accumulation of acetate, propionate and butyrate in experiment I (Table 1) accounted for 73% of the dissolved organic carbon detected at the end of the incubation. Hydrogen accumulated transiently to maximum partial pressures of 1.2–2.5 kPa after 1–2 days of incubation and then decreased until a constant level around 50 Pa was reached (Fig. 2). Root preparations that had been surface-sterilized produced no H2 during the first 75 h. Then, H2 steadily accumulated to partial pressures of >2 kPa and was not consumed again. The surface-sterilized roots produced no CH4 and produced acetate, propionate and butyrate at only 16–23% of the control. Incubation of the root preparations in presence of radioactive bicarbonate resulted in the formation of radioactive acetate, propionate and CH4 (Fig. 3A). Although the total amounts of 14CH4 produced were small, the specific radioactivity (dpm/mol) of the produced CH4 was rather high indicating that virtually all of the CH4 was produced from CO2 (Fig. 3B; Table 2). Methyl fluoride, a specific inhibitor of acetoclastic methanogenesis [23], did not affect the temporal change of H2 partial pressures and acetate concentrations (data not shown). The thermodynamic conditions for H2/CO2-dependent methanogenesis were very favorable (ΔG=−67 to −98 kJ mol−1 CH4; Fig. 4). The thermodynamic conditions for acetoclastic methanogenesis were also favorable (ΔG=−48 to −50 kJ mol−1 CH4; data not shown).

image

Figure 1. Accumulation of H2, CH4 and fatty acids in anoxically incubated rice root preparations (experiment I); mean±S.D. of n=3.

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Table 1.  Maximum accumulation rates (μmol h−1 g-dw−1 root) of methane, acetate, propionate and butyrate using different preparations of rice roots
ExperimentMethaneAcetatePropionateButyrate
I0.0645.10.370.08
II0.0082.80.670
III0.0582.50.660.12
IV0.0213.30.110.03
Mean0.0383.40.450.06
S.E.0.0130.60.130.03
image

Figure 2. Transient accumulation of H2 in anoxically incubated rice root preparations (experiment IV); the insert shows the H2 partial pressures on a magnified scale; mean±S.D. of n=3.

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image

Figure 3. Incorporation of radioactive bicarbonate into (A) CH4, acetate and propionate in anoxically incubated rice root preparations (experiment III); mean±S.D. of n=3; and (B) fraction of carbon of CH4, acetate and propionate derived from CO2.

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Table 2.  Fractions of acetate, propionate and methane produced by washed rice roots under anoxic conditions from 14CO2, [1-14C]acetate or [2-14C]acetate as precursors
ExperimentCH4 from CO2Acetate from CO2Propionate from CO2Propionate from [1-14C]acetatePropionate from [2-14C]acetate
II160±632±142±4 60±6
III107±739±161±660±876±1
Fractions (%mole carbon) are given as mean±S.E. determined from the specific radioactivity of precursors and products between 100 and 430 h of incubation. Experiment numbers are the same as in Table 1.
image

Figure 4. Temporal change of the Gibbs free energies of the conversion of H2 plus CO2 to either CH4, acetate or propionate in anoxically incubated rice root preparations (experiment I).

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Radioactive bicarbonate was also incorporated into acetate (Fig. 3A). The ratios of the specific radioactivity of acetate carbon and CO2 indicate that 32–39% of the acetate was derived from CO2 (Fig. 3B; Table 2). Obviously, chemolithotrophic homoacetogenesis contributed substantially to acetate formation. This interpretation is consistent with the thermodynamic conditions that were clearly exergonic (ΔG=−18 to −48 kJ mol−1 acetate) for acetate formation from H2/CO2 throughout the incubation (Fig. 4).

Interestingly, radioactive bicarbonate was also incorporated into propionate (Fig. 3A). The specific radioactivity of propionate indicated that 42–61% of the propionate carbon was derived from CO2 (Fig. 3B; Table 2). The thermodynamic conditions for reduction of CO2 to propionate were favorable (ΔG=−34 to −85 kJ mol−1 propionate; Fig. 4). Radioactive propionate was also formed with either [2-14C]acetate or [1-14C]acetate as precursor. The specific radioactivity of propionate indicated that 60–76% of the propionate were produced from [2-14C]acetate or [1-14C]acetate. Thermodynamic conditions (ΔG=−15 to −38 kJ mol−1 propionate) would allow the reductive formation of propionate from acetate, CO2 and H2 (Fig. 4).

4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

It has recently been demonstrated that washed rice roots contain methanogenic archaea that become active as soon as the roots are incubated under anoxic conditions [13,16,17]. Here we have shown that washed rice roots in addition produce H2, acetate, propionate and butyrate. These activities were greatly reduced in surface-sterilized roots indicating that the metabolic reactions observed were predominantly due to microorganisms living on the root surface. Some minor contribution of plant metabolism cannot be ruled out but is unlikely, since roots were excised from intact plants. Similarly as the methanogens, the microorganisms producing H2, acetate, propionate and butyrate became active as soon as the roots were incubated under anoxic conditions. Since no further substrates were added, we assume that the roots started to decay and supported anaerobic fermentation. Hence, the obtained results do not represent conditions that are characteristic for in situ, but only show the potential of the microbial community inhabiting the rice roots. Nevertheless, it is remarkable how easily anaerobic metabolism is initiated when incubating root preparations suggesting that the microbes colonizing the roots are highly vital.

In particular, the rates of acetate accumulation were remarkable, being about 100 times larger than those of CH4 production. Radioactive experiments indicate that 30–40% of the acetate was produced from H2/CO2. On the other hand, the amount of H2 consumed (51 μmol H2) in Fig. 1 between 24 and 143 h of incubation accounted for only 4% of the acetate produced (300 μmol). However, we have to assume that the H2 partial pressures in the incubations represented steady state values of H2 production and H2 consumption so that the actual H2 turnover was much faster than indicated from the net decrease of H2. The large rate of acetate production and the relatively large contribution of CO2 to acetate production indicate that the roots are colonized by homoacetogenic bacteria. Indeed, clone libraries of 16S rDNA extracted from washed rice roots showed that sequences closely affiliated with the genus Sporomusa, i.e. homoacetogenic bacteria, were abundant (Rosencrantz and Liesack, personal communication).

The processes involved in acetate production on roots may be compared to that in the lumen of termite hindgut where the carbon and electron flow to acetate is also much larger than that to CH4[24,25]. In the termite hindgut it is not fully understood why H2-utilizing methanogens are not able to outcompete homoacetogens although the former should have an advantage due to their intrinsically better kinetic and energetic properties [26]. In the root preparations, thermodynamic conditions were favorable for H2/CO2-dependent homoacetogenesis, since the ΔG values were low enough (i.e. ΔG<−20 to −23 kJ mol−1) to allow the synthesis of 1/3 ATP if metabolism was coupled to energy generation [27]. However, thermodynamic conditions were even more favorable for H2-dependent methanogenesis (i.e. ΔG<−60 to −70 kJ mol−1) and in fact, virtually all the CH4 was produced from H2/CO2 being consistent with earlier results [17]. Thus, the relatively high activity of H2/CO2-dependent homoacetogenesis cannot be explained by better energetics. Better kinetics is probably also not an explanation, as Sporomusa termitida, the prevalent homoacetogen in termite hindgut, exhibited similar affinities for H2 as methanogens [28]. It has been proposed that mixotrophic utilization of H2/CO2 together with a heterotrophic substrate may allow the homoacetogens to consume H2 at concentrations lower than useful for methanogens [26]. However, this hypothesis has not been supported by studies in pure culture [29].

An alternative and more probable hypothesis is the heterogeneous distribution of microorganisms in both the hindgut and the root environment. In the hindgut, methanogens seem to be localized at the gut wall whereas the homoacetogens dominate the lumen [30,31]. Thus, methanogens are not present at the site where H2/CO2 is converted to acetate and vice versa. We assume that similar conditions are true for the rice root surface where homoacetogens and methanogens are probably localized at different sites (and in different abundance), each being juxtaposed to H2-producing fermenting bacteria that apparently also colonize the root surface, so that competition mechanisms as described for homogeneous suspensions do not apply. It is unclear whether the microorganisms on the root surface were able to proliferate in the phosphate buffer used for incubation of the root preparations. Obviously, the H2/CO2-utilizing methanogens did not outgrow the H2/CO2-utilizing homoacetogens during the initial phase of incubation (0–200 h). Acetate production as well as CH4 production eventually slowed down indicating that both processes became limited by H2. Acetate, on the other hand, became an abundant substrate. Acetoclastic methanogenesis was thermodynamically feasible (ΔG<−48 kJ mol−1). Nevertheless, acetoclastic methanogenesis was not observed in the present experiments and was only occasionally observed in earlier experiments when the roots were incubated in phosphate buffer for prolonged periods [17]. Although only relatively low numbers of acetoclastic methanogens were found on the rice roots [17], we presently do not understand why these methanogens did not become active upon accumulation of acetate.

Propionate was the second most abundant fermentation product of rice root incubations. Interestingly, a substantial fraction of the propionate was apparently derived from both CO2 and acetate. Incorporation of CO2 into propionate has been observed before in rice field soil [32]. In anoxic rice field soil, formation and degradation of propionate seems to be due to the randomizing succinate pathway [21,33]. In this pathway, up to 0.5 mol CO2 is transcarboxylated per mole propionate formed, depending on the equilibration of intracellular and extracellular CO2 pools. Therefore, we have to expect incorporation of CO2 when propionate is fermentatively produced.

However, the operation of the succinate pathway does not provide an explanation for the incorporation of acetate into propionate. Therefore, we conclude that propionate was at least partially produced by (1) reduction of radioactive acetate and perhaps (2) reduction of CO2. The latter reaction cannot be dismissed, since incorporation of H14CO3 accounted for 40–60% of the radioactive propionate formed:

  • image(1)
  • image(2)

Both reactions were thermodynamically feasible under the actual incubation conditions (Fig. 4). Vice versa, the oxidation of propionate by syntrophic microorganisms was endergonic under the conditions studied. To our knowledge, the reactions shown in Eqs. 1 and 2 have so far not been observed in a natural environment. The first reaction (Eq. 1) is the reverse of syntrophic propionate degradation [27,34]. This reverse reaction has been demonstrated to operate in Desulfobulbus propionicus[35]. Similarly, D. propionicus has been shown to produce propionate reductively from acetate by oxidizing alcohols [36]. D. propionicus is able to produce as well as to degrade propionate. It can grow as a fermenting bacterium by converting ethanol to propionate and acetate, or as a sulfate reducer by oxidizing propionate to acetate and CO2[37]. Although D. propionicus is unable to grow syntrophically on propionate (Widdel, personal communication), other phylogenetically related sulfate reducers do [38–40]. Hence, it is possible that bacteria with properties similar to D. propionicus and related syntrophic propionate oxidizers were colonizing the rice roots and catalyzing the reductive formation of propionate from acetate plus CO2.

The second reaction (Eq. 2) is a combination of Eq. 1 plus chemolithotrophic homoacetogenesis. If propionate-forming bacteria, such as those described above, could in addition exhibit homoacetogenesis, reductive production of propionate from H2+CO2 would be possible. Although an organism with such a metabolism has to our knowledge not yet been described, it would be energetically and mechanistically feasible. Sulfate-reducing bacteria with the acetyl-CoA pathway and the capacity to oxidize acetate may be candidates [41]. Some of these bacteria, e.g. Desulfotomaculum species [42], indeed exhibit a weak capacity for homoacetogenic growth.

In conclusion, our study has shown that rice roots are colonized with microorganisms able to reductively produce CH4, acetate and propionate from CO2. The root-colonizing methanogenic archaea have recently been phylogenetically characterized [16,17]. Obviously, the methanogenic microflora of the roots was completely different from that of the bulk soil [43]. The root microorganisms producing acetate and propionate have not yet been characterized. The root processes may be important for the ecosystem, since the root density in the upper 5 cm soil layer is relatively high (about 10 g-dw m−2; [44]). With an acetate production rate of 3.4 nmol h−1 g-dw−1 roots, the root-dependent acetate production in the upper 5 cm soil layer is about 17 nmol h−1 cm−3, or at a soil density of about 1.4 g-dw cm−3[45] equivalent to 12 nmol h−1 g-dw−1 soil. This value is >10% of the acetate turnover rates typically observed in methanogenic rice field soil [46–48].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was financially supported by the Fonds der Chemischen Industrie.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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