Seasonal Methane Oxidation Potential in Manure Crusts

Abstract

Organic crusts on liquid manure storage tanks harbor ammonia- and nitrite-resistant methane oxidizers and may significantly reduce methane emissions. Methane oxidation potential (0.6 mol CH₄ m⁻² day⁻¹) peaked during fall and winter, after 4 months of crust development. Consequences for methane mitigation potential of crusts are discussed.

TEXT

Liquid manure (slurry) storages are hot spots for ammonia, methane, and odor emission. Floating organic layers (crusts) on the slurry can mitigate the problem (1). Crusts consume methane in laboratory incubations (2–4) and reduce methane emissions in situ (5, 6). However, methane mitigation capabilities vary considerably between studies (1), indicating strong variation in the presence or activity of methane-oxidizing bacteria (MOB). Three factors are likely to play a key role for the crusts' methane-oxidizing potential. (i) High concentrations of ammonia (80 to 200 mM total ammoniacal nitrogen [TAN], from urea and amino acid degradation) (7) may inhibit MOB, since ammonia is a competitive inhibitor of the particulate methane mono-oxygenase, the key enzyme of a broad range of MOB (8, 9). (ii) Nitrite, which is formed in the oxic part of the crust by ammonia-oxidizing bacteria (AOB) (4, 10), is also inhibitory to MOB (11), although the mechanism has not been fully resolved. (iii) The physical structure of the crust is likely to control the development of the MOB community (10). In Northern Europe, slurry tanks are emptied between March and May, after which the crust slowly reforms as slurry is reintroduced (11). The goal of the present study was therefore to assess the extent of ammonia and nitrite inhibition and the development of the methane oxidation potential during crust formation.

Methane oxidation potential.

Crust samples were collected at eight time points between January 2008 and August 2009 from slurry tanks of two piggeries near Tranbjerg (TBG1) and Ørsted (HR1), Denmark. Samples were dissected into 1- to 2-cm³ pieces; weighed; washed in sterile, deionized water; and suspended in NMS medium (12) (to 24 ml) containing dissolved methane and CO₂ (∼1 mM) (13). Methane oxidation rates were determined in 2- to 6-h incubations with dissolved methane and an O₂ headspace (2 ml) at room temperature in a rotator (Sb3 Stuart; Bibby Scientific Ltd.). At each sampling point, the O₂ headspace was replenished and 0.5 ml of medium was extracted and replaced with fresh medium without methane. The resulting change in methane concentration (∼2.0%) was accounted for in calculations. The sample was transferred to a nonevacuated 3.3-ml Venoject vial (Terumo Europe) and shaken vigorously for 1 min to equilibrate methane with the headspace. Headspace methane was measured by gas chromatography. Potential ammonia oxidation rates were determined as described previously (10), except that nitrite was measured by the VCl(III) reduction method (15).

Methane and ammonia oxidation potential was absent or very low between the spring emptying and September but high during fall and winter (Fig. 1), with higher rates in the deeper crust layer (1- to 2-cm depth). Assuming a crust density of 1 g cm⁻³, the maximum cumulated potential (top 2 cm) was 0.6 ± 0.1 mol CH₄ m⁻² day⁻¹ and 1.3 ± 0.04 mol NH₃ m⁻² day⁻¹, respectively. Reported and modeled in situ methane emission rates (see the supplemental material) are in a similar range (<0.2 to 2 mol CH₄ m⁻² day⁻¹), indicating that the crusts have the potential to drastically reduce methane emissions from slurry storage. However, while maximum in situ emissions occur during the warmer summer months (17; see also the supplemental material), crusts reach their maximum methane oxidation potential only in the fall after a maturation period of >4 months.

Fig 1.

Potential methane (top row) and ammonia (bottom row) oxidation rates of crust samples collected in two storage tanks, TBG1 and HR1, in 2008 to 2009. Potential ammonia oxidation rates in July, August, and early September were too low to be visible in the graph (μmol g fresh weight [FW]⁻¹ h⁻¹): July, 0.011 ± 0.004; August, 0.012 ± 0.003; early September, 0.018 ± 0.07. Dashed lines indicate that the storage was emptied between the two sampling points. x, not measured; ↑, minimum rate estimate based on a few time points (all methane consumed before second time point). Error bars indicate standard deviations (n = 4).

Effect of inhibitors.

Crust samples (HR1, February 2009, 1- to 2-cm depth, 1- to 2-g fresh weight) were incubated in capped 117-ml serum vials with 40 ml NMS medium and 10% methane in the headspace. The medium was buffered with 100 mM NaHCO₃, and the pH was adjusted to that of the crust (pH 7.5 to 7.7). Each vial was amended with NaNO₂ (final concentration, 0, 0.5, 1, 5, and 10 mM) or NH₄Cl (final concentration, 0, 5, 10, 50, and 100 mM) and incubated for 3 to 4 days sideways on a rotary shaker (Edmund Bühler GmbH) at 160 rpm. Incubations with thiourea (300 μM) served as a negative control (22). Methane was measured in headspace samples by gas chromatography.

Both NaNO₂ and NH₄Cl reduced methane oxidation rates (Fig. 2A and B); however, >5 mM NaNO₂ at pH 7.7 (∼200 nM HNO₂) was required to completely inhibit methane oxidation (Fig. 2A). HNO₂ concentrations in old crusts are typically much lower (<3 nM) (2, 4), indicating that AOB activity is not a major inhibitor of MOB in old crusts. MOB activity was not affected by 10 mM TAN (0.25 mM free NH₃), and complete inhibition was not reached at the maximum concentrations tested (∼2.5 mM free NH₃). The initial lag period was not observed in a pilot experiment (see Fig. S4 in the supplemental material). Since many MOB are inhibited at much lower concentrations [e.g., inhibition constant K_i(NH3), 0.05 mM for Methylomonas methanica (14) and 0.01 mM for Methylosinus trichosporium OB3b (15)], these data suggest a specific adaptation to the high free ammonia concentrations (1 to 9 mM) (5, 7, 16) intrinsic to slurry.

Fig 2.

Depletion of methane (∼10% in headspace) by crust samples (A and B) and enrichment culture (C) suspended in media amended with different amounts of NO₂⁻ (A) and TAN (NH₃ + NH₄⁺) (B and C). Error bars indicate standard deviations (n = 4).

MOB diversity.

DNA was extracted in triplicate from crust samples by combining enzymatic lysis with the FastDNA Spin kit for soil (Q-Biogene) (17). The genes encoding particulate methane mono-oxygenase subunit A (pmoA) were successfully PCR amplified with primers A189f and mb661r (18), cloned (pGEM-T vector system; Promega), and sequenced (Macrogene). In contrast, primers A189f and A650r (19), which generally target high-affinity MOB, never yielded products. Other primers, targeting the phylogenetically distinct pmoA of Methylacidiphilaceae or the soluble methane mono-oxygenase-encoding genes of Methylocella/Methyloferula, were not tested since these MOB are generally found in acidic environments (pH < 5) and their occurrence in the neutral slurry (pH 7.0 to 7.7; see Table S1 in the supplemental material) was considered unlikely.

Sequences (n = 142) were translated to amino acids, aligned, and phylogenetically analyzed by neighbor joining and maximum likelihood methods (10,000 and 5,000 bootstraps, respectively) implemented in ARB (20), utilizing 157 amino acids (aa) (Fig. 3).

Fig 3.

Phylogeny of crust PmoA sequences by neighbor joining with bootstrap values from maximum likelihood indicated by circles on branches consistently found by both methods. Scale bar indicates 8% estimated sequence divergence. Crust (clusters C1 to C10) and enrichment culture sequences retrieved during this study are displayed in bold. Numbers in parentheses represent the numbers of clones in the cluster found in young and old crusts, respectively. The 16S rRNA gene sequence of the enriched MOB (accession number JN790630) was most closely related to the same Methylobacter strain as was its pmoA sequence (for phylogeny, see Fig. S3 in the supplemental material).

Most sequences from both young and old (>4-month) crusts grouped in nine distinct clades of gammaproteobacterial type I MOB. Only five sequences from one young crust clustered with the alphaproteobacterial type II MOB Methylocystis (Fig. 3). This result is in agreement with other studies indicating that type I MOB dominate N-rich environments (13, 21). Half of the sequences (n = 71) clustered in group C5 with up to 97% similarity to a Methylomonas sequence from a landfill crust soil. The overall diversity, however, was dominated by sequences with 93 to 99% similarity to various Methylobacter groups; single sequences clustered with Methylocaldum and Methylococcus. Due to very low PCR yields in young crust samples (possibly indicating low MOB abundance) and consequently poor cloning success, no meaningful comparison of MOB communities in young versus old crusts was achieved.

High-ammonia MOB enrichment.

Ammonia-tolerant MOB were enriched by consecutive reculturing of the 100 mM NH₃ culture used in the inhibition test (see above); after 6 transfers, a 10-fold dilution series to extinction was prepared from the enrichment. As MOB often require specific growth factors or the removal of inhibiting products, an aerobic, rod-shaped non-methane-consuming heterotroph, isolated from the enrichment culture on general nutrient broth agar, was added to each dilution to promote MOB growth. The highest methane-consuming dilution (10⁻⁸) was challenged with NH₃ (50, 100, and 250 mM) as described above but in 12-ml Exetainers. The enrichment showed resilience against increased TAN similar to that of the crust samples (Fig. 2), with complete inhibition only at 250 mM TAN (∼7 mM NH₃). Incubations with 100 mM KCl did not reduce methane consumption, excluding inhibition from the increased salinity (data not shown).

The enrichment culture contained two distinct morphotypes, nonmotile rods (2.0 by 1.0 μm) that formed large aggregates and motile rods (1 μm long). Bulk DNA extraction, 16S rRNA gene-specific PCR with primers 8f and 1492r (22), cloning, and sequencing of 32 clones resulted in one sequence 96% identical to betaproteobacterial Methylophilus sp., probably representing the smaller rods (data not shown). The larger, aggregate-forming morphotype was identified by laser microdissection-PCR (LMD-PCR) of a single aggregate: 50 μl of paraformaldehyde (4%)-fixed enrichment was dried onto a polyethylene terephthalate (PET) membrane slide (Microdissect GmbH), and a single cell aggregate (diameter, ∼50 μm) was cut out with a Leica LMD7000 microscope, collected in a PCR tube, and directly used as the template for pmoA- and 16S rRNA gene-specific PCR, cloning, and sequencing. For both genes, only one sequence type was obtained from the 21 and 22 clones analyzed, respectively, with the highest similarity (97% for pmoA, 98% for 16S rRNA) being that to Methylobacter sp. strain BB5.1 (23). The pmoA sequence represents cluster C4, which was the dominant Methylobacter pmoA in young crusts in this study (Fig. 3), and has previously been retrieved from a wet, experimental straw crust (24). These Methylobacter spp. may therefore be particularly well adapted to high-TAN environments.

Conclusions.

Organic crusts have the potential to significantly reduce methane efflux to the atmosphere, and crust MOB show a remarkable resistance to ammonia and nitrite inhibition. Despite the initial presence of ammonia-tolerant MOB, methane oxidation potential requires up to 4 months of crust development, which under the current management practice decouples the period of high methane production and high potential methane consumption: production occurs primarily in the warm summer (5) whereas consumption potential is highest during winter. A simple measure to utilize more of the crusts' methane mitigation potential is to preserve them during emptying of the slurry tanks.

Nucleotide sequence accession numbers.

Sequences were deposited in GenBank under accession numbers JN790620 to JN790630.