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Applied and Environmental Microbiology, February 2006, p. 1013-1018, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1013-1018.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

In Vivo Emission of Dinitrogen by Earthworms via Denitrifying Bacteria in the Gut

Marcus A. Horn,¹ Ralph Mertel,¹ Matthias Gehre,² Matthias Kästner,² and Harold L. Drake^1*

Department of Ecological Microbiology, University of Bayreuth, 95445 Bayreuth,¹ Department of Bioremediation, Center for Environmental Research Leipzig-Halle, 04318 Leipzig, Germany²

Received 15 September 2005/ Accepted 8 November 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Earthworms emit the greenhouse gas nitrous oxide (N₂O), and ingested denitrifiers in the gut appear to be the main source of this N₂O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N₂), the end product of complete denitrification. When [¹⁵N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N₂ (and also labeled N₂O) under in vivo conditions; emission of N₂ by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N₂ per h per g (fresh weight), respectively. Isolated gut contents also produced [¹⁵N]nitrate-derived N₂ and N₂O under anoxic conditions. N₂ is formed by N₂O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [¹⁵N]nitrate-derived N₂ by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N₂O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N₂ (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N₂ per h per g (fresh weight), respectively; these emission rates approximated that of N₂O. These collective observations indicate that (i) earthworms emit N₂ concomitant with the emission of N₂O via the in situ activity of denitrifying bacteria in the gut and (ii) N₂O is quantitatively an important denitrification-derived end product under in situ conditions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Earthworms occur in diverse terrestrial habitats, may have densities of up to 2,000 individuals per square meter of soil, and represent the dominant soil fauna (9, 24, 25). Earthworms are divided into three feeding guilds. (i) Epigeic species (e.g., Lumbricus rubellus) live above the mineral soil and feed preferentially on litter. (ii) Endogeic species (e.g., Aporrectodea caliginosa) live in the upper zone of the mineral soil, ingest high amounts of mineral soil, and feed in the rhizosphere. (iii) Anecic species (e.g., Lumbricus terrestris) live in deeper zones of the mineral soil, ingest medium amounts of soil, and feed on litter that they drag into their burrows (3, 7). Earthworms contribute to the aeration, pore volume, water-holding capacity, nutrient availability, and mineralization rates of soils by virtue of their feeding and burrowing habits (9, 22, 24, 25). Casts (i.e., the fecal discharge) of diverse earthworms contain higher concentrations of nitrate, ammonium, and organic carbon than does the surrounding soil (10, 13, 31, 32, 40, 41, 49). Earthworms thus contribute to the cycling of elements in terrestrial ecosystems.

Terrestrial habitats are responsible for approximately 70% of the greenhouse gas nitrous oxide (N₂O) that is produced globally (5, 6), and the ecophysiological activities of earthworms include the emission of this gas (19, 27). This activity can be important at the local level; up to 56% of the in situ emission of N₂O from certain soils might be earthworm derived (2, 19, 27) and might yield 3 x 10⁸ kg N₂O globally each year (8). The N₂O that is emitted by earthworms originates in the anoxic core of the earthworm gut, and emission of N₂O is likely due to the in situ activities of ingested nitrate- and nitrite-reducing bacteria that are activated during gut passage (14, 16). Denitrification appears to be the primary N₂O-producing process in the earthworm gut (14). Dinitrogen (N₂) is the terminal product of complete denitrification and is formed by nosZ-encoded N₂O reductase (56). However, the in vivo emission of N₂ by earthworms has thus far not been assessed. The emission of N₂ by earthworms is highly probable, based on their capacity to emit N₂O (19, 27) and the occurrence of a large number of phylogenetically diverse nosZ sequences in earthworm gut contents (15). In the present study, [¹⁵N]nitrate and mass spectrometry were utilized to determine if earthworms emit N₂ by gut-associated microbial processes and to likewise determine if N₂ is the dominant denitrification-derived gas that is emitted.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Collection, storage, and N₂O emission status of earthworms.
Specimens of A. caliginosa and L. terrestris were collected in late summer and early autumn 2004 from the meadow Hofmanns Wiese in Bayreuth, Germany (14). Worms were identified by standard protocols (4) and stored in the dark in soil at 5°C until used; worms were utilized within 1 week of collection. As in earlier studies (14, 16, 19, 27), most of the earthworms collected emitted N₂O. In a random sampling of 50 specimens, 2 emitted <0.05 nmol N₂O per h per g (fresh weight), and the average rate of emission was 1.5 nmol N₂O per h per g (fresh weight) The maximum rate observed was 11.1 nmol N₂O per h per g (fresh weight).

¹⁵N experiments with living earthworms.
Earthworms were washed with water, dried slightly by being blotted with tissue paper, weighed, and then given three 50 µl injections of 20 mM sodium nitrate (10% ¹⁵N) into the gut; the three injections were along the first two-thirds of the gut. Three such earthworms having a collective fresh weight of approximately 3 g were placed in a 10-ml serum vial. The vial was crimp sealed, and the gas phase was adjusted to He:O₂ (80:20) (200-kPa overpressure). Vials were incubated at room temperature (21°C) in the dark, and the gas phase was analyzed periodically. The amount of [¹⁵N]nitrate injected and the number of worms per vial could theoretically yield a maximum of 450 nmol of [¹⁵N]N₂ per vial; this experimental design was necessitated because of the detection limits of the mass spectrometry analysis. For experiments with nitrite, earthworms received three 10-µl injections of 20 mM sodium nitrite (10% ¹⁵N) along the first two-thirds of the gut (note: the amount of nitrite injected was one-fifth that of nitrate, to avoid the potentially toxic effects of nitrite).

¹⁵N experiments with gut contents.
Gut contents were pressed out of washed earthworms (14). The gut contents (approximately 0.4 g [wet weight]) of three worms having a collective fresh weight of approximately 3 g were placed in a 10-ml serum vial and supplemented with 450 µl of 20 mM sodium nitrate (10% ¹⁵N). The vial was crimp sealed, and the gas phase was adjusted to 100% He (200-kPa overpressure); vials were incubated as described above.

Acetylene experiments.
Serum vials containing earthworms were prepared as outlined above. The He:O₂ gas phase of the serum vials was modified to 15% (vol/vol) acetylene, and the effect of acetylene on the production of ¹⁵N-labeled products was determined.

Earthworms were also evaluated for the effects of acetylene on the production of N₂O by standard gas chromatography. Washed earthworms were incubated in crimp-sealed vials (unless otherwise indicated, one worm, which was approximately 1 g [fresh weight], per 10-ml vial); the gas phase was air or air supplemented with acetylene (15% [vol/vol]) (50-kPa overpressure; the overpressure was less than that used in the ¹⁵N experiments, because the amount of gas sampled was only 0.2 ml). In some cases, earthworms were given a single 10-µl injection of 10 mM sodium nitrate into the gut behind the clitellum.

Comparative emission rates for the gut contents and gut wall were obtained from microcosms prepared as previously described (16). Supplemental acetylene was 1% (vol/vol) in the headspace, and values are means of three replicates incubated for 20 h.

Analytical methods.
The concentrations and the isotopic compositions (¹⁵N/¹⁴N ratio) of gaseous N₂ and N₂O were analyzed with a GC-C-IRMS system (Thermo Electron, Bremen, Germany) consisting of a Hewlett-Packard Co. 6890 gas chromatograph (Agilent, Palo Alto, CA) and a GC/C III combustion and reduction system (Thermo Electron, Bremen, Germany) coupled via open split to a Mat 253 mass spectrometer (Thermo Electron, Bremen, Germany). Two-milliliter gaseous samples were taken with gas-tight syringes prepurged with He and injected into a split-splitless injector (1:1 ratio). Gases were separated on a Poraplot Q-FS capillary column (25 m plus 2.5 m precolumn by 0.32 mm by 10 µm; Chrompack, Darmstadt, Germany) with He (carrier gas) flow set at 1.5 ml per min. The oven was isothermic at 40°C. The system was calibrated with certified reference gases (Linde, Lübeck, Germany), and the concentrations were measured with relative standard deviations of <3.5%. The detection limits were <10 nmol for N₂O and <5 nmol for N₂.

The isotopic compositions were determined by calculating the ratios of the masses 28 and 29 with relative standard deviations of <0.5% after the instrument was calibrated with N₂ reference gas (purity was 99.995%; Linde). This reference gas had an N-isotopic composition of 0.366 ¹⁵N atoms, which is nearly identical to that of air. The isotopic ratios were measured as the percentage of atoms and calculated to the number of moles [¹⁵N]; the preciseness of the isotope ratio measurement was < 2 x 10^–4 atom %. The resulting detection limits for [¹⁵N]N₂ and [¹⁵N]N₂O were <1 nmol ¹⁵N excess, which corresponded to approximately 1/900 and 1/450, respectively, of the ¹⁵N applied. Although mass 30 was not used for calculations (this was not possible, due to the small amount of this mass in the experiments), mass 30 was used for testing the consistency of the data and assessing nonstatistical distribution of the isotopomers.

[¹⁵N]NaNO₃ and [¹⁵N]NaNO₂ were obtained from Cambridge Isotope Laboratories (CIL International, Promochem, Wesel, Germany) with an isotope enrichment of >98% ¹⁵N. A Hewlett-Packard Co. 5980 series II gas chromatograph equipped with an electron capture detector was used for the analysis of N₂O (14, 19).

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Production of [¹⁵N]N₂ from [¹⁵N]nitrate by earthworms and gut contents.
Specimens of A. caliginosa and L. terrestris emitted labeled N₂ when [¹⁵N]nitrate was injected into the gut; the emission of labeled N₂ was concomitant to the emission of labeled N₂O and continued for several hours postinjection (Fig. 1). Although the relative amounts of [¹⁵N]nitrate-derived N₂ and [¹⁵N]nitrate-derived N₂O varied from one experiment to the next, the amount of [¹⁵N]nitrate-derived N₂O tended to be slightly greater than that of [¹⁵N]nitrate-derived N₂ (Table 1, experiments A to C). Earthworms injected with [¹⁵N]nitrite, an intermediate in denitrification (56), also emitted labeled N₂ and N₂O (data not shown). The gut of the earthworm is anoxic (14), and isolated gut contents produced [¹⁵N]nitrate-derived N₂ and N₂O under anoxic conditions (Table 1, experiment D). This result indicated that the emission of [¹⁵N]nitrate-derived gases by living earthworms was linked to microbial processes in the anoxic gut. Neither [¹⁵N]N₂ nor [¹⁵N]N₂O was detected in [¹⁵N]nitrate-supplemented control vials that lacked earthworms.

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FIG. 1. In vivo emission of [15N]N2O (•, ) and [15N]N2 (, ) by L. terrestris (•, ) and A. caliginosa (, ) following the injection of [15N]nitrate into the gut. Values at 0 h are from control vials that lacked earthworms. Values are means of duplicate experiments.

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TABLE 1. Emission of [15N]nitrate-derived [15N]N2 and [15N]N2O by living earthworms (experiments A to C) and isolated earthworm gut contents (experiment D)

Acetylene enhances the production of [¹⁵N]nitrate-derived N₂O.
Acetylene is an inhibitor of N₂O reductase, the N₂-forming terminal oxidoreductase in complete denitrification (54, 56). The emission of [¹⁵N]nitrate-derived N₂ by living earthworms was not observed when they were incubated in the presence of acetylene. For example, in a 12-h incubation, A. caliginosa formed 0 and 23 nmol [¹⁵N]nitrate-derived N₂ and N₂O per g (fresh weight), respectively, in the presence of acetylene. The emission of [¹⁵N]nitrate-derived N₂ was also inhibited when earthworms were exposed to acetylene after several hours of emitting [¹⁵N]nitrate-derived N₂ (Table 1, experiment C).

Acetylene enhances the production of N₂O.
The results above demonstrated that living earthworms emit N₂. However, because these experiments necessitated the use of gut supplements (i.e., the injection of labeled nitrate into the gut), the results did not provide information on the status of natural emissions. The acetylene block technique and standard gas chromatographic analysis were used to circumvent this problem and verify the in vivo emission of N₂.

Exposing earthworms to acetylene increased the amount of N₂O they emitted (Fig. 2). Injection of 100 nmol of sodium nitrate into the gut of earthworms resulted in a slight increase in the amount of N₂O emitted (Fig. 3), a result that was consistent with the emission of [¹⁵N]N₂ from injected [¹⁵N]nitrate (above). The emission of N₂O was relatively linear during initial incubation periods both with and without acetylene, and the rate at which N₂O was emitted was enhanced when earthworms were exposed to acetylene after a period of actively emitting N₂O (Fig. 4). The relative linearity of emission suggested that denitrification in the gut was ongoing from the onset of incubation. These results provided additional evidence that earthworms emit N₂ and thus corroborated the results obtained in the ¹⁵N-labeling experiments. They also extended the previous observation that earthworms bathed in nitrate solution emitted more N₂O when exposed to acetylene (27).

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FIG. 2. In vivo emission of N2O by A. caliginosa (A) and L. terrestris (B). Symbols: •, vials without acetylene; , vials with acetylene. Bars indicate the range of values obtained from four replicate vials, each containing four earthworms. Abbreviation: gFW, g (fresh weight).

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FIG. 3. In vivo emission of N2O by L. terrestris without gut supplement (A) or following the injection of 100 nmol sodium nitrate into the gut (B). Symbols: •, vials without acetylene; , vials with acetylene. Bars indicate the range of values obtained from four replicates. Abbreviation: gFW, g (fresh weight).

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FIG. 4. In vivo emission of N2O by A. caliginosa following an injection of 100 nmol sodium nitrate into the gut. Acetylene was injected into vials at 1.6 h (arrow). Bars indicate the range of values obtained from three replicates. Solid and broken lines correspond to the rates of emission prior to and after the addition of acetylene, respectively. Abbreviation: gFW, g (fresh weight).

Localization of N₂ production in the gut.
Microcosms prepared from gut contents and gut wall from A. caliginosa produced 66 ± 26 and 8 ± 2 nmol N₂O per g (fresh weight), respectively, under conditions that simulated the in situ conditions of the gut interior (14). Acetylene-supplemented gut contents and gut wall microcosms under such conditions yielded 92 ± 33 and 5 ± 3 nmol N₂O per g (fresh weight), respectively. Thus, gut wall-associated microbiota did not appear to be a significant origin of N₂.

N₂ emission rates.
A. caliginosa and L. terrestris emitted 1.2 and 6.6 nmol nitrate-derived N₂ per h per g (fresh weight), respectively, during initial incubation periods in the ¹⁵N-labeling experiments (these values are averages and are based on the amounts of N₂ formed during the first 6 and 7 h of incubation in experiments B and C [Table 1], respectively, and take into account the fact that the sodium nitrate injected into the gut was 10% ¹⁵N). Based on the assumption that the acetylene-dependent increase in emitted N₂O is equal to the N₂ emitted without acetylene, the calculated rates for the native emissions of N₂ (i.e., without gut supplement) by A. caliginosa and L. terrestris approximated 1.1 and 1.5 nmol N₂ per h per g (fresh weight), respectively (Fig. 2). Earthworms that did not receive a gut injection of supplemental nitrate emitted, on average, 1.5 (±2.1) nmol N₂O per h per g (fresh weight) (data are from 50 specimens) in the absence of acetylene. Thus, the amounts of N₂ and N₂O emitted by living earthworms appeared to be similar. The acetylene method underestimates the amount of N₂ formed by denitrification because acetylene may not totally block N₂O reductase (45); thus, the amounts of N₂ estimated with this method in the present study should be regarded as minimum estimates. In contrast to the apparent coemission of similar amounts of N₂ and N₂O by living earthworms, N₂O was the dominant [¹⁵N]nitrate-derived product of isolated gut contents (Table 1, experiment D), suggesting that the physical disruption of the gut decreased the relative amounts of N₂ formed by denitrifiers in gut contents.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The results of the present study demonstrate that earthworms emit N₂. The emission of [¹⁵N]nitrate-derived N₂ by these invertebrates is consistent with previous results that identified denitrification as an ongoing process in the earthworm gut (14, 27). That the nosZ sequences detected in gut contents are phylogenetically nearly identical to those detected in soil (15) supports the hypothesis that the N₂ emitted by earthworms is primarily derived from ingested soil denitrifiers during gut passage.

The results of this study indicate that complete denitrification occurs in the earthworm gut. The expression of nosZ, and therefore the reduction of N₂O to N₂, is under regulatory control and is influenced by anaerobiosis and the availability of substrates (1, 28, 56). The feeding habits of earthworms might have an effect on the in situ conditions of their gut and therefore influence the amounts of N₂ that they emit. When supplemental nitrate was injected into the gut, the litter feeder L. terrestris tended to emit higher amounts of N₂ than did the soil-rhizosphere feeder A. caliginosa. However, differences in the calculated native emission of N₂ between these worm species were minor. This observation is consistent with studies that indicated the feeding guilds of earthworms do not significantly affect the amounts of N₂O they emit (27).

Bathing earthworms in dilute concentrations of nitrate stimulates the emission of N₂O by earthworms (27), and the addition of nitrate to earthworm gut contents also stimulates the anaerobic production of N₂O by microbes in the gut (14). In the present study, [¹⁵N]nitrate injected into the gut was likewise reduced to [¹⁵N]N₂ and [¹⁵N]N₂O, as shown by their emission from earthworms. These observations indicate that the in situ source of reductant required for the formation of N₂ and N₂O via denitrification is not limiting in the gut. Indeed, supplemental organic carbon does not increase the rate at which nitrate is reduced to N₂O by gut contents in anoxic microcosms (16). The aqueous phase of earthworm gut content contains readily utilizable sources of organic carbon, including up to 80 mM glucose, 40 mM amino sugars, and 10 mM maltose, as well as up to 1 mM of combined nitrate and nitrite; thus, the in situ conditions of the anoxic earthworm gut make it ideal for denitrification (14). The large amounts of readily available organic carbon in gut contents are likely derived from (i) ingested plant- and soil-derived materials that are partially degraded by digestive enzymes (e.g., proteases, chitinases, N-acetyl-glucosaminases, and maltases) (38, 39, 48, 51, 55) and (ii) the initial breakdown products of the intestinal mucus that is secreted by the earthworm to aid the passage of ingested material through the gut (26, 52).

Many gut ecosystems, including that of mammals, e.g., humans (37, 47), and various invertebrates, e.g., termites (12, 46, 50), harbor indigenous, autochthonous microorganisms. However, cell densities of earthworm gut wall-associated microbes are low, and the gut of the earthworm appears to have a quantitatively insignificant indigenous microbial biome (11, 15, 17, 24, 34, 35, 42, 53). Nonetheless, the gut of the earthworm constitutes a unique microenvironment to which ingested microbes are transiently subjected. The activation of certain members of the soil microbial community appears to increase their culturability (8, 16, 18, 19), indicating that gut passage might affect the general life cycles and survival strategies of certain soil microbes. It has been postulated that the earthworm-secreted mucus in the gut might stimulate ingested soil microorganisms in a mutualistic digestive system (23).

Denitrification is catalyzed by facultative microbes under anoxic conditions and might therefore occur in many digestive tracts (which tend to have anoxic compartments or zones). However, little is known about the occurrence of this microbial process in gut ecosystems or the emission of denitrification-derived nitrogenous gases from animals. Humans exhale N₂O, and the amounts of N₂O exhaled may be (i) correlated with aging and the concentrations of nitrate and nitrite in gastric juices (21, 30) and (ii) affected by the amount of nitrate in ingested foods (29). The N₂O that is exhaled by humans might be derived from denitrifiers. However, nitrate dissimilators (i.e., anaerobes that dissimilate nitrate to ammonium) can give rise to N₂O as a side product of nitrate reductase (43, 44). Thus, nitrate dissimilators in the human gut (33) might also be a source of the N₂O exhaled by humans. Traces of N₂O are formed in the bovine rumen during the dissimilatory reduction of nitrate to ammonium (20). N₂O is not reduced further to N₂ in the bovine rumen, and denitrification appears to be insignificant in this gut ecosystem. With the exception of the earthworm, the exhalation and/or emission of microbially derived N₂ by other animals has not been reported.

Although earthworms are well recognized for their importance to the general fertility of soil (9, 22, 25), the potential importance of the earthworm gut to specific microbial processes that occur at the local level is less well understood. N₂ is classically considered the end product of denitrification at neutral pH, but the in situ production of similar amounts of denitrification-derived N₂ and N₂O by denitrifiers in the near pH neutral gut of the earthworm suggests that this process can be incomplete under in situ conditions considered favorable for complete denitrification. The loss of nitrogen from the terrestrial ecosphere via denitrification at a global level is estimated at 10¹¹ kg N per year (36). The results of the present and earlier (2, 19, 27) studies illustrate the potential impact that soil fauna might have on the turnover dynamics of soil nitrogen (e.g., the ratio of N₂O/N₂ emitted) via their transient hosting of ingested soil microorganisms.

 
We thank Anja Miltner for assistance in data aquisition and Andreas Schramm for evaluation of the manuscript.

Support for this study was provided by grants from the Deutsche Forschungsgemeinschaft (DFG DR310/2-1) and the Center for Environmental Research Leipzig-Halle (UFZ).

 
* Corresponding author. Mailing address: Department of Ecological Microbiology, University of Bayreuth, 95445 Bayreuth, Germany. Phone: (49) (0)921-55561. Fax: (49) (0)921-555793. E-mail: hld{at}uni-bayreuth.de.

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Applied and Environmental Microbiology, February 2006, p. 1013-1018, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1013-1018.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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