Nutrient Cycling in Agroecosystems 64: 79–85, 2002.© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

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Microbial aerobic oxidation of methane in paddy soil

H. Min∗, Z.Y. Chen, W.X. Wu & M.C. ChenCollege of Life Sciences, Zhejiang University, Hangzhou 310029, P.R. China (∗Corresponding author; e-mail:minhang@zju.edu.cn)

Key words: influencing factors; methane formation; methane oxidation; paddy rice soil

Abstract

The microbial aerobic oxidation activity of methane, the population of aerobic methane oxidizers and the factors in-fluencing the activity of methane oxidation were investigated in three types of paddy rice soil in Zhejiang Province,China. Methane oxidation activity was different among Huangsong paddy soil developed from fluvo-aquic soil,Old huangjinni paddy soil developed from quaternary red clay and Qingzini paddy soil developed from coastalsaline soil. The Huangsong paddy rice soil showed the highest activity of methane oxidation. Different methaneoxidation activity and populations of methane-oxidizing bacteria were found in various Huangsong soil samplesthat had different plant-cover. Methane oxidation returned to the same level after these soil samples were incubatedand induced by extra added methane. The population of methane oxidizing bacteria was at maximum within thepeak-tillering, heading and flowering stages, during which the largest population of methanogenic bacteria alsoappeared. Temperatures from 25 to 35 ◦C and pH from 6 to 8 were the optimum conditions for aerobic oxidationof methane in paddy rice soil. Soil particle size also affected the activity of methane oxidation.

Introduction

A considerable part of methane in the atmospherecomes biologically from flooded paddy rice soils thatare distributed widely in South East Asia, China andother areas in the world (Bouwman et al., 1990). Tostimulate the microbial oxidation of methane is of thesame importance as to inhibit the microbial formationof methane for control of the emission of methanefrom paddy rice soils to the atmosphere, as a part ofmethane is oxidized on the pathway from the anaer-obic site at which methane is formed to the surfaceof the soil and to the atmosphere. Little information,however, has been published on methane oxidationin paddy rice soil although some research focused onmethane oxidizers (Min et al., 1993; Hanson and Han-son, 1996), on methane oxidation in anoxic freshwaterand marine sediments (Reeburgh, 1976; Reeburgh andHeggie, 1977; Iverson and Blackburn, 1981; Lid-strom, 1983), in lake water sample (Panganiban etal., 1979), and in Skan Bay sediments (Reeburgh,1980). Murase and Kimura reported methane produc-tion and its fate in paddy fields, including anaerobicoxidation of methane in the plow layer soil, meth-

ane flux distribution and decomposition of methanein subsoil during the growth period of rice plants,electron acceptor responsible for anaerobic methaneoxidation, etc. (Murase and Kimura, 1994a,b, 1996).Their colleagues studied the oxidation of methane andthe coupled ferric oxide reduction in subsoil (Miuraet al., 1992). Bosse and Frenzel (1997) demonstratedthe activity and distribution of methane-oxidizing bac-teria in flooded rice soil microcosms and in rice plants(Oryza sativa). These results confirmed that there isreal oxidation of methane in anaerobic niches suchas sediments in freshwater and marine, as well as inpaddy rice soil.

This paper deals with the oxidation of methane aswell as the factors influencing the rate and activity ofmethane oxidation in rice paddy soil.

Materials and methods

Soil sample tested

Three types of paddy soil developed from differentparent materials were sampled from three sites of

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Table 1. The main physicochemical properties of soil samples tested

Organic Total C/N Total Total pH∗ Physical composition (mm)

matter nitrogen potassium phosphorus 2–0.02 0.02–0.002 <0.002

(g/kg) (g/kg) (g/kg) (g/kg)

Huangsong paddy rice soil 21.3 1.27 9.7 16.5 0.73 7.94 42.42 37.71 9.87

developed from fluic-aquic

soil

Old Huangjinni paddy rice 25.7 1.49 9.7 12 0.36 6.32 29.85 42.44 27.75

soil developed from

quaternary red clay

Qingzini paddy rice soil 37.3 2.19 9.9 20 0.52 7.61 19.49 42.8 37.71

developed from coastal

saline soil

Zhejiang Province based on the Zhejiang Soil Clas-sification (General Soil Survey Office of Zhejiang,1994). Some physical and chemical properties of thesesoils are presented in Table 1. The bigger particlesand larger fibre residues of plant were removed in thesoil samples. The soil samples were broken into smallpieces and air-dried in a laboratory room to about 15%water content (Water weight %). The soil sampleswere mixed well and put into large glass bottles andincubated at 30 ◦C for 15 days to remove the solubleand degradable organic matters.

Determination of the methane-oxidizing activity ofthe soil samples

Twenty grams of each of the three types of soil samplewas put into a 100-ml serum vial for experiments.A total of 0.5 ml of pure methane was injected intothe serum vial and the serum vials with different soilsamples were incubated at 30 ◦C. The concentration ofmethane in the headspace of the serum vial with soilsample was measured at fixed time interval.

Methane determination

Methane was measured using a 102G type gas chro-matograph with a hydrogen flame ionization detectorunder the following conditions: carrier: GDX-502, air:700 ml min−1; H2: 40 ml min−1; N2: 25 ml min−1,room temperature: 40 ◦C and the appearance of thepure methane peak was at 17 s. Pure methane gas,purchased from Beijing gas plant, was used as thestandard reference.

Measurement of the population of methane-oxidizingbacteria in soil samples

The population of methane-oxidizing bacteria wasmeasured using the anaerobic roll tube method byHungate’s anaerobic technique but aerobic mediumwas kept under aerobic condition in anaerobic tubewith specific rubber stopper to prevent escape ofmethane gas. The various anaerobic tubes contain-ing melted aerobic medium were inoculated withdifferent dilutions of soil sample and rolled, theneach tube was injected with 3 ml pure methane assubstrate for methane-oxidizing bacteria and incub-ated at 30 ◦C for 15 days. The number of coloniesformed in the rolled tube with methane was coun-ted after incubation. Medium for determination ofmethane-oxidizing bacteria was composed of (g L−1)KH2PO4 1.0, Na2HPO4·12H2O 2.9, MgSO4·7H2O0.32, (NH4)2SO4 3.0, trace element solution 10 mland distilled water 990 ml, pH 6.8. The medium wasaerobically prepared and 4.5 ml of medium was addedto each tube that was sealed with a rubber stopper, theninjected with pure methane using a syringe.

Measurement of methanogenic bacteria in soilsamples

The population of methanogenic bacteria was determ-ined using the anaerobic roll tube method by Hun-gate’s anaerobic operation technique. The mediumused for determination of methanogens was composedof (g L−1): NH4Cl 1, MgCl2 0.1, KH2PO4 0.4, yeastextract 1.0, cystein 0.5, HCOONa 5.0, CH3COONa 5,

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CH3OH 5 ml L−1, soil extract solution 300 ml L−1

and trace element solution 10 ml L−1. Before the me-dium was used, 0.1 ml mixed solution of anaerobicsterilized Na2S (10 g/kg)/NaHCO3 (50 g/kg) was ad-ded into each tube with 4.5 ml medium to decreasefurther redox potential of the medium and then 0.1 mlof 160 000 units ml−1 of penicillin was added to in-hibit eubacteria. After inoculation the headspace of thetube was flushed using anoxic hydrogen and injectedwith 3 ml high purity CO2. The number of coloniesformed in the rolled tube was counted after the tubeswere inoculated and incubated at 33 ◦C for 15 days.

Soil extract solution was prepared by mixing sev-eral kg of paddy soil and tap water (soil:water about1:1.5), stirring and then letting it stand for 24 h, filter-ing the supernatant liquid through filter paper, steril-izing the filtrate and storing it in a refrigerator beforeuse. Trace element solution was composed of (g L−1):N(CH2COOH)3 4.5, FeCl2·4H2O 0.4, MnCl2·H2O0.1, CoCl2·6H2O 0.12, ZnCl2 0.1, AlK(SO4)2 0.01,NaCl 1.0, CaCl2 0.02, Na2MnO4 0.01, H3BO3 0.01,and distilled water 1000 ml. Then the mixture wasstored in a refrigerator for later use.

All the data in this paper are the average values oftriplicates and the standard deviations and the stand-ard deviation error bars are showed in all tables andfigures, respectively.

Results and discussion

Effect of parent material on activity of methaneoxidation in paddy rice soil

Figure 1 showed the different activities of methane ox-idation among various paddy rice soils developed fromdifferent parent materials. It is obvious from Figure1 that in a closed system with 20 g of soil, air and0.5 ml of methane the rate of methane oxidation inHuangsong paddy soil (C1) was the highest one, OldHuangjinni paddy soil (B1) the second and Qingzinipaddy soil (A1) the lowest. The rate of methane ox-idation in Huangsong paddy soil reached up to themaximum for 24 h of incubation and decreased to 0after 48 h of incubation. However, the rate of methaneoxidation in the other soils decreased more slowly anddid not come to 0 in the period of the experiment.

Another interesting observation was that the samepaddy rice soil sample collected from various sitesplanted with different crops had similar maximumactivity of methane oxidation but the time required

Figure 1. The rates of methane oxidation in three types of paddyrice soils developed from different parent materials.

Figure 2. Effect of continuous addition of methane on methane ox-idation by Huangsong soil with 24.3% water weight % water contentand incubation at 28 ◦C.

to develop the maximum activity of methane oxida-tion was significantly different, as shown in Table 2.The time required was only 30 h–42 h for Huangsongpaddy soil planted rice crop, but more than 100 h forthe same soil planted with upland crops.

However, the lower rate of methane oxidation insoil could be induced and accelerated by increasingmethane concentration in the closed system, as shownin Figure 2. There was an obvious a lag phase formethane oxidation when the soil sample was first ex-posed to methane gas, but almost no lag phase whenmethane was reintroduced into the serum vial.

Effect of environmental factors on activity of methaneoxidation in paddy rice soil

The water content in the soil tested was an import-ant factor influencing methane oxidation activity. Theactivity of methane oxidation was the highest in soilwith about 25% (water weight %) of water contentunder aerobic condition, Figure 3. The activity ofmethane oxidation increased with increasing water

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Table 2. Effect of addition of methane on methane oxidation of soils with various plant-covera

Soil Soil Plant-cover Population of methane-oxidizing Reached the maximum methane

sample bacteria (107 cfu g−1 dried soil) oxidation rate and time

collected non-induced Soil after reached the The maximum rate Time needed to

from soil maximum rate of (×10−7 CH4 mol h−1 reach the

methane oxidation g−1 dried soil) maximum rate (h)

Huangsong Paddy soil Rice crop 6.67(±1.05) 21.31(±2.83) 7.25(±0.52) 30–42

paddy soil Corn soil Corn crop 8.42(±0.96) 18.65(±2.01) 7.00(±0.53) 124–148

Wheat soil Wheat seedling 3.48(±0.84) 16.01(±1.49) 6.67(±0.35) 148–160

Huangsong Grass soil Grass 14.3(±1.36) 15.84(±1.84) 6.68(±0.41) 100–124

upland soil Bamboo Covered 1.87(±0.28) 16.81(±1.58) 6.84(±0.37) 336–360

garden soil bamboo leaves

Mulberry soil 6.49(±0.94) 17.47(±1.47) 6.92(±0.46) 216–240

aThe data in the table were the average values and that in parentheses were the standard deviations of methane-oxidizing bacteria populationor methane-oxidizing rate.

Figure 3. Effect of various contents of water on methane oxida-tion in Huangsong soil at 28 ◦C during aerobic incubation for 24h (Water weight %: A 24.29%; B 65.72%; C 195.93%).

content when water content was lower than 25% anddecrease sharply once the water content was higherthan 25%. However, the activity of methane oxida-tion was lower in soil under anoxic and higher watercontent condition than under aerobic and lower watercontent condition (Figure 3).

The temperature was another important factor formethane oxidation in paddy rice soil. The maximumtemperature was about 30 ◦C with a range from 10 to45 ◦C for methane oxidation, Figure 4. This temper-ature range for methane oxidation is consistent withthe fact of that there was no thermophilic speciesand strains among the methane-oxidizing bacteria isol-ated up date (Hanson and Hanson, 1996). The resultsshowed that the activity of methane oxidation was notdetectable if the tested soil was first incubated at 50◦C for 6 h and then incubated again at 30 ◦C, butthat the activity remained when the soil sample was

Figure 4. Effect of various incubation temperatures on the activityof methane-oxidation in tested soil incubated for 24 h.

Figure 5. Effect of different pH on the activity of methane oxidationin Huangsong paddy soil incubated at 28 ◦C within 24 h.

first incubated at lower than 50 ◦C and then incubatedagain at 30◦C.

Experiment also showed that the optimal pH was6.0–6.5 although the activity of methane oxidation

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Table 3. Effect of different types of soil on the populations of methane-oxidizing bacteria (cfu/g dried soil)a

Soil sample Huangsong paddy soil Old Huangjinni paddy soil Qingzini paddy soil

Non-submerged 1.55 (0.988–3.42)×107 3.57 (1.90–5.35)×106 6.38 (0.358–13.3)×106

(Apr 1–27)

Submerged and non-planted rice 5.70 (3.34–9.37)×107 9.00 (3.27–12.9)×107 4.59 (1.33–6.44)×107

(Apr 28–Nov 20

Submerged and planted 7.83 (3.51–26.0)×107 1.00 (0.387–3.86)×108 1.27 (0.244–2.12)×108

Earlier rice (Apr 28–Jul 22)

Submerged and planted 1.05 (0.592–11.41)×108 2.01 (0.387–3.86)×108 2.38 (0.445–3.47)×108

latter rice (Jul 22–Nov 20)

Submerged and planted rice 9.56 (3.51–26.0)×107 1.17 (0.141–3.86)×108 9.32 (2.44–34.7)×107

(Apr 28–Nov 20)

aThe data in the table were the average values and that in parentheses were the number ranges of methane-oxidizing bacteriafrom multiple determinations during various growth stages of rice.

Table 4. Annual variation of population of methane oxidizers in Huangsong rice soil planted withdifferent crops during various seasons (1999, cfu×107 g−1 dried soil)a

Determining No. 1 soil No. 2 soil No. 3 soil

Date (mon/day) Planted MOB Planted MOB Planted MOB

crop crop crop

Jan. 10 Wb 5.85(±0.61) V 4.03(±0.56) Ra 6.53(±0.96)

Feb. 10 W 7.80(±1.51) V 3.94(±0.49) Ra 5.82(±0.68)

Mar. 10 W 1.55(±0.29) V 1.81(±0.28) Ra 1.54(±0.36)

Apr. 10 W 6.28(±0.96) V 1.82(±0.24) Ra 13.6(±2.13)

May 10 W 9.41(±1.54) Ri 2.77(±0.38) Ra 8.33(±1.19)

Jun. 10 Wri 1.61(±0.15) Ri 4.62(±0.81) Ri 2.30(±0.32)

July 10 Ri 13.81(±2.8) Ri 8.47(±1.08) Ri 20.65(±2.97)

Aug. 10 Ri 12.67(±1.59) Ri 20.12(±3.42) Ri 12.36(±1.54)

Sept. 10 Ri 12.50(±1.78) Ri 12.90(±2.03) Ri 7.95(±1.09)

Oct. 10 Ri 7.21(±1.15) Ri 9.84(±1.37) Ri 5.73(±0.59)

Nov. 10 N 5.14(±0.64) N 8.31(±1.16) N 5.53(±0.7)

Dec. 10 N 6.18(±0.83) N 3.71(±0.23) N 5.82(±0.72)

aThe data in the table were the average values of triplicates and that in parentheses were the standarddeviations of population numbers of methane-oxidizing bacteria during various seasons.bW: wheat; Ri: rice; Wri: wheat- rice; V: Chinese milk vetch; Ra: rape; N: no crop.

could be detected in the range from 3.0 to 8.0 in paddysoil, indicating that pH was also an important affectingfactor, Figure 5.

The activity of methane oxidation showed positiverelation to the ratio of soil granules with >2.0–0.02mm particle size and negative to that with <0.02 mmparticle size. The higher ratio of soil granules of <0.02mm might prevent, at least partially, the diffusion ofmethane and oxygen to micro-niches where methane-oxidizers live and oxidize methane. Therefore, all ofthe factors which affected the supply of methane andoxygen for methane oxidizers, influence the activity ofmethane oxidation in paddy rice soil.

Population of methane-oxidizing bacterial flora inpaddy rice soil

The population of methane-oxidizing bacteria was sig-nificantly larger in Huangsong paddy rice soil thanin old Huangjingni paddy soil and Qingzini paddysoil. The population of methane- oxidizing bacteriaincreased in the three types of soils after submergence,especially after planting the rice crop. The popula-tion of methane-oxidizing bacteria reached the largestnumber of colonies, up to 108 g−1 dried soil when soilwas planted with the latter rice (Table 3).

The annual variation of population of methane-oxidizing bacteria was investigated in Huangsong rice

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Table 5. Effect of different type of soils on population of methanogenic bacteria (cfu g−1 dried soil)a

Soil sample Huangsong paddy soil Old Huangjinni paddy soil Qingzini paddy soil

Non-submerged 3.94(1.83–5.92)×104 9.71(8.34–11.5)×103 4.62(2.37–7.52)×103

(Apr 1–27)

Submerged and non-planted rice 4.14(0.48–17.0)×106 1.60(0.425–6.05)×106 1.92(0.206–6.35)×106

(Apr 28–Nov 20

Submerged and planted 1.26(0.019–5.35)×107 3.61(0.108–17.7)×106 4.38(0.0755–7.07)×106

earlier rice (Apr 28–Jul 22)

Submerged and planted 7.22(0.163–17.0)×106 4.50(1.51–73.8)×105 1.52(0.0508–5.43)×106

latter rice (Jul 22–Nov 20)

Submerged and planted rice 8.57(0.163–53.5)×106 1.54(0.108–17.7)×106 2.17(0.508–7.07)×106

(Apr 28–Nov 20)

aThe data in the table were the average values of triplicates and that in parentheses were the standard deviations ofpopulation numbers of methanogenic bacteria in planted or non-planted rice and submerged or non-submerged soils.

soil planted with different seasonal crops. The res-ults listed in Table 4 indicate that the population ofmethane-oxidizing bacteria was smaller when the soilwas planted with upland crops, such as wheat, rape,or Chinese milk vetch (Astragalus sinicus) in winterthan when the soil was planted with rice in summer.Maybe the lower temperature in winter was one of thefactors which influenced the population of methane-oxidizing bacteria. However, the lack of methanewhich was formed by methanogens and was usedas substrate by methane oxidizers was undoubtedlythe main reason of smaller populations of methaneoxidizers in unsubmerged soil.

Effect of population of methanogens on methaneoxidation in paddy soil

The population of methanogenic bacteria was dis-tinctly increased in paddy soil after planting rice, Table5. The number of methanogenic bacteria increasedfrom 104 g−1 dried soil to 107 g−1 dried soil inHuangsong paddy soil, and from 103 to 106 g−1 driedsoil in Old Huangjinni and Qingzini paddy soils. Theactivity of methane oxidation also enhanced with theincreasing of population of methanogenic bacteria.

Analysis of the population of methane-oxidizingbacteria flora in soil during various growth stages ofearlier rice and later rice crops demonstrated that themaximum population of methane oxidizers appearedwithin the peak-tillering and the heading-floweringstages, during which the maximum population ofmethanogens also appeared (Min et al., 1996, 1997).This fact indicates that the population of methaneoxidizer and the activity of methane oxidation were

closely and positively related to the population and theactivity of methangensis of methanogenic bacteria.

Acknowledgements

Project supported by Chinese National Natural Sci-ence Foundation 30170030 and the Lab. of Mater-ial Cycling in Pedosphere, Institute of Soil Science,Chinese Academy of Sciences.

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