Trees, typically large trees in upland forests, emit CH4 produced in their trunk by methanogenic archaea. In this case, the spatial variability of emissions can be more complicated than when tree trunks act as a passive conduit for CH4 produced in the soil. However, due to poor accessibility, CH4 emissions from the trunk above 3 m, where a researcher cannot reach without a ladder, scaffold, or crane, have not been well studied. In this study, we investigated vertical patterns of CH4 emissions, internal CH4 concentration, production, and copy number of the methanogenic archaeal gene mcrA at 6 different heights up to 12 m above ground, in six trees of three species in a cool-temperate upland forest.

We targeted 6 mature trees from 3 species in a cool-temperate upland mixed forest. We measured variables at 6 different heights per 1 individual.

Trunk CH4 flux was measured by the dynamic closed chamber method. A polypropylene chamber, with a measuring area of 37 cm2 on average and a volume of 695 mL, including the cell of the analyser and tubing, was attached to the trunk with neutral seal putty. A lid connected to a portable trace gas analyser (Li-7810, LICOR, Lincoln, USA) with two 6-mm-diameter, 2-m-long PTED tubes was used to close the chamber during the measurement. The trunk CH4 flux was calculated from the changes in the CH4 molar fraction in the closed system measured every second during four minutes of chamber closure. Trunk CH4 flux was expressed not only in per unit area (nmol m-2 s-1), but also in the entire trunk circumference per unit length of trunk (nmol 0.1 m-1 s-1) to compare it to internal CH4 production rate.

CH4 concentration inside the trunk was measured after the CH4 flux measurement. The chambers were removed, and the trunk at each measuring location was bored with an increment borer (5-mm internal diameter, Haglof Sweden) to the pith. After the removal of the wood core, the hole left by the borer was enlarged to a diameter of 10 mm with a drill. A stainless-steel tube (10-mm outer diameter) was inserted into the hole, driven by a hammer to stop 7.5 cm before the pit. The outside of the tube was covered with silicon sealant prior to insertion to prevent any air from leaking along the contact area between the tube and the wood. The tube was flushed with pure N2 to expel as much O2 as possible before the open end of the tube was capped with a rubber septum (Asone, Butyl W Plug, Osaka, Japan). As the inner 5 or 7.5 cm part of the hole was not covered with the stainless-steel tube, gases inside the central trunk can diffuse into the cavity of the tubing and reach equilibrium. Five days later, 0.1 mL of the gas inside the tube was sampled with a syringe penetrating the septum and immediately injected through the septum of the small volume sample kit (Li 7810-110, LICOR, Lincoln, USA) connected to the analyser. The molar fraction of gas injected was calculated from the volume of the closed system, including the sampling kit and injected sample, and the CH4 molar fraction in the system before and after the injection (Mochidome & Epron, 2024, Trees, 38: p625–636). 

CH4 production potential was measured by laboratory incubation of wood cores sampled. The cores were cut into up to three segments of 7 cm in length, depending on the radius at the sampling location. The borer was sterilized with 70% ethanol every time after boring. Within five minutes after sampling, each of the 7-cm segments was placed into 12-mL glass vials separately. Within two hours after the sampling, the vials were flushed first with N2 for three minutes to expel CH4 and O2 from the vial and then with the incubation mixture (N2, 10% CO2, and 1% H2) for 1.5 minutes to supply substrates to methanogenic archaea. Soon after flushing, 0.2 mL of the gas inside the vials was drawn from the vial through the septum into a syringe, and the CH4 molar fraction in the vials was measured as described above for trunk internal [CH4]. The second gas samples were collected six hours after flushing. The vials were kept inside an opaque, insulated box between gas samples. CH4 production potential was calculated as an increase in CH4 amount in the vial headspace divided by duration and dry weight of the wood core. The wood core was then weighed before and after drying and scaled before drying to calculate the bulk density and moisture content of the wood.

For DNA extraction and subsequent quantitative PCR to target the mcrA gene, one individual was selected from each of the three species that encode the methyl-coenzyme M reductase α subunit of methanogenic archaea. Only the innermost wood core segments at every sampling height, for a total of 18 cores (1 depth × 6 heights × 3 trees), were used. After the anaerobic incubation experiments, the wood cores were stored in a freezer at -20 °C. The samples were then freeze-dried for at least 48 hours in a freeze dryer (FDU-1200, EYELA, Tokyo, Japan) and ground via a ball mill (MM-400, Retsch, Düsseldorf, Germany) for 3 minutes. DNA was extracted from a 40-mg ground wood sample via the FastDNA SPIN Kit for Soil (MP Biomedicals, Santa Ana, USA) following the manufacturer’s instructions. The DNA samples were further purified with a OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, USA) before PCR amplification. DNA extraction was performed in duplicate, and the reported values are the average of the duplicate samples. Real-time PCR analysis of the mcrA genes was performed via the Thermal Cycler Dice Real Time System (TaKaRa, Kusatsu, Japan) and primers MLf/MLr (Luton et al., 2002). Each 25-µL mixture contained TB Green Premix Ex Taq (TaKaRa), MLf/MLr primers (0.2 µmol/L each), 0.25 µL of bovine serum albumin (BSA, 0.2 g/L), 2 µL of template DNA, and sterilized ultrapure water. The PCR thermal cycle was as follows: 95 °C for 10 s (denaturization); 45 cycles of 95 °C for 40 s (denaturization), 55 °C for 30 s (annealing), and 72 °C for 60 s (elongation). An increase in the PCR product was detected by the fluorescent signal, and its threshold cycles (Ct values) were determined via the second derivative maximum method. The amplification of nonspecific DNA fragments was checked via dissociation curve analysis. The copy number of the mcrA gene was calculated via a standard curve generated from a series of dilutions (1.14×108 to 1.14×101 copies/µL) of the standard sample.