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'l‘hic is M certify that the

dissertation entitled

PRODUCTION AND EMISSION OF METHANE FROM
EXPERIMENTAL PADDY SOILS

presented by

Shan Ney Huang

has been accepted towards fulfillment
of the requirements for

Ph.D. degreein Crops and Soil Science

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PRODUCTION AND EMISSION OF HETHANE FROM
EXPERIMENTAL PADDY SOILS

By

Shan Ney Huang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Crop and Soil Science

1989

ABSTRACT
PRODUCTION AND EMISSION OP METHANE FROM EXPERIMENTAL PADDY SOILS

By!
Shan Ney Huang

Greater methane concentrations in the atmosphere has received
increasing attention. Flooded rice culture is considered to be one of
the major biogenic methane sources. Because of this, the effects of
rice cultivation practices on the change of inorganics (NOBI, 8042-) and
organics (volatile fatty acids) were followed and the production and
emission of methane from experimental soil studied under glasshouse
conditions.

Initially, nitrate in pore water was high after the flooding of the
dry soil, and then declined rapidly. Sulfate concentrations followed
the same pattern; however, the rate of sulfate depletion was lower than
that of nitrate. The concentration of volatile fatty acids increased
gradually after dry soil flooding and reached a maximum concentration at
30 days after rice transplanting followed by a rapid decline.

Methane concentration in soils increased markedly during the first
20 days after flooding. The non-planted soil had higher methane
concentration than the planted soil. Highest methane production rates
were observed at 40 days after flooding, while the lowest rates of
production were observed at 20 days. Rates of methane emission from

2

planted soils ranged from 0.73 (0.13) to 15.69 (4.4) mg CH lm lhr. which

4
was significantly (52) higher than that observed from non-planted soils
0.11 (0.04) to 1.43 (0.6) mg CH4/m2/hr. The highest emission rates were

observed during heading and flowering stage, while the lowest during the

tillering and maturing stage of growth. Most of the methane was emitted
through the rice plant.

The addition of previous crop residues resulted in an increase in
methane production and subsequent emission in both planted and
non-planted treatments. However, no significant differences were
observed in the production or emission of methane from fertilized

treatments over the controls.

ACKNOWLEDGMENTS

I would like to thank Dr. Michael Klug for giving me the opportunity
to work in his lab. His enthusiastic attitude, helpful suggestions and
constructive criticism were responsible for much of the success of this
project. I would also like to thank Dr. James Tiedje, my advisor, for
allowing me to come back to study at MSU. I am grateful for his
dedicated guidance which introduced me to the field of microbial
ecology. I am also grateful to Drs. E. Erickson, 3. Ellis, and A.
Smucker for their helpful discussions and advice. Greg Walker provided
excellent technical assistance and help in the laboratory. Art Viest
helped me in the greenhouse experiments. The assistance of many others
both at the station and on campus is gratefully acknowledged.

Most of all I would like to thank my wife Shu-Chu and my children

Yaohsin and Shu-Anwen for their loving support.

iv

TABLE OF CONTENTS

LIST 0FTABLESOOOOOOOOOOOOOOOOOO0.0.0....OOOOOOOOOOOOOOOOOOO0.00... Vi

LIST OF FIG‘JRESOooooooeeeoooaooe0000000000000000.000000000000000... Viii

ImonumION...OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1

HATERIALSANDMETHODS.0......O0.00...OOOOOOOOOOO0.00000000000000000 11

RBSULTSANDDISCUSSIONOOOO0.0000000000000...0.00.00.00.00.0.0.0.... 22

SUMMARY AND CONCLUSIONS.......... ..... ............................. 65

LITERATURE CITBDOOO0.0.00......OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO... 70

Table 1.

Table 2a.

Table 2b.

Table 3.

Table 4.

Table 5.

LIST OF TABLES

Anaerobic microbial processes and their reaction

products (Tiedje et a1. 1984)...... ....... .. ........ ..... 6

Comparison of nitrate and sulfate concentrations
(um/1) in soil solutions which were taken by

different sampling methods ........... . ................... 23

Comparison of pore water acetate and propionate
concentrations (um/1) taken by different

sampling methods ......................................... 24

Percent distribution of root biomass at panicle

initiation and maturing stage of growth.................. 27

Depth distribution of dissolved and exchangeable

NH4+ in planted soils at day 40 of experiment 1 .......... 33
Depth distribution of rates of potential

nitrification in planted and nonplanted soils at

day 40 of experiment 1..... ..... ..... ..... ............... 34

vi

Table 6.

Table 7.

Table 8.

Table 9.

Table 10.

Depth distribution of rates of potential

1

denitrification (ng-NZO-N/g-hr' ) at days 20,

40 and 70 of experiment 1.. ...... ........................ 36

Depth distribution of rates of sulfate reduction

(nmoles/g-hr-l) at days 20 and 70 of experiment 1........ 41

Influence of rice plants on the rates of methane
emission at the flowering and heading stage of
growth. Data reported as the mean (n-6) followed

by the standard deviation...................... .......... SS

Methane concentration, rates of methane production,

emission and percent of methane production emitted

from paddies in the Taichung and Tayei regions of

Taiwan in July 1985. ..... .... ...... ...................... 62

Effects of N—fertilizer on rates of emission of

methane from planted soils .............. . ..... .... ....... 64

vii

LIST OF FIGURES

Figure 1. Sources of biogenic methane and production rates.

(Ehhalt 1985)............................. .............. 3

Figure 2. Microbial processes and plant interactions in

paddy soils.... ..... ......... ....... . .................. . 9

Figure 3. Experimental pots and sampling cylinders................ 12

Figure 4. Above and below ground plant biomass during the

growth period .......... . ........... . .................... 26

Figure 5. Depth-time distribution of nitrate in planted and
nonplanted soils at 0, 30, 60 and 84 days of the

experiment .............................................. 29

Figure 6. Depth-time distribution of dissolved ammonia in
planted and nonplanted soils at 0, 30, 60 and 84

days of the experiment .................................. 31

Figure 7. Depth-time distribution of volatile fatty acids

(VFA's) in planted and nonplanted soils at 0, 23,

43 and 64 days of the experiment ........................ 37

viii

Figure 8.

Figure 9.

Figure 10.

Figure 11.

Figure 12.

Figure 13.

Figure 14.

Depth-time distribution of sulfate in planted and
nonplanted soils at 0, 17, 34 and 52 days of the

experiment........ ..... .... ......... .................... 39

Depth-time distribution of methane in planted and
nonplanted soils at 20, 30, 40, 50 and 70 days

of the experiment... ...... . ....... ..... ................. 44

Depth-time distribution of the production of methane
in planted and nonplanted soils at 20, 30, 40, 50

and 70 days of the experiment.. ............. . ......... .. 46

Relationship between the mean concentrations of sulfate

and methane in pore waters of planted soils.. ......... .. 48

Depth distribution of the respiration index

[14cn4/(1‘c34+1‘c02)1 in planted and nonplanted soils
at 60 days of the experiment.. ...... ............... ..... 49

Methane emission from planted, nonplanted soils and
between plants in the planted treatment at 10 day

intervals during experiment 1 ............... ..... ...... . 52
Methane production in and emission from planted and

nonplanted soils at 10 day intervals during

experiment 1 ............................................ 57

ix

Figure 15. Methane production in and emission from planted
and nonplanted previously cropped soils at 10 day

intervals during experiment 2........................... 58

Figure 16. Methane production in and emission from planted
and nonplanted previously cropped soils amended
with rice straw at 10 day intervals during

experiment 2.. ......... .... ......... .. ........ .......... 61

INTRODUCTION

Importance of Methane Emission to the Atmosphere
Although methane (CH4) is a relatively minor component of the global
carbon cycle, its increased concentration in the atmosphere has recently

received considerable attention (Cicerone and Shetter 1981, Seiler 1984)

It is estimated that methane concentrations have increased 1.5 to 1.72%

over the last 10 years (Seiler 1984, Ehhalt 1985). Because of the

chemical and physicochemical properties of methane its increased
concentration could greatly impact the chemistry of the atmosphere

(Seiler 1984). Briefly:

(1) The increased concentrations of atmospheric methane would perturb
the earth’s radiation balance resulting in an increased
”greenhouse effect" (Vang et a1. 1976). Doubling the methane
mixing ratio from the current concentrations of 1.6 ppmv would
result in an increase in tropospheric temperature of 0.2 to 0.300.

(2) The increased oxidation of methane in the troposphere would result
in a higher production of a series of gaseous intermediates and
final products such as CO, H2, HCHO, O3 and other radicals (Levy
1971, McConnell et al. 1971, Vofsy 1972, Crutzen 1973). Changes
in the concentration of these compounds will have a significant
impact on the chemical composition of the troposphere.

(3) Methane is the predominant source of water vapor in the

stratosphere since the products of its oxidation are 002 and water

2

vapor. Changes in the CH4 mixing ratio will increase the flux of
methane into the stratosphere and the H20 mixing ratio. This will
result in an impact on the distribution of 0H and other odd
hydrogen species in the stratosphere.

(4) The increase of tropospheric CH4 will enhance the reaction between
CH4 and Cl (Stolarski and Cicerone 1974). The reaction CH4 + Cl 4
HCl + CH3 is the dominant sink mechanism for the stratospheric

chlorine radicals C1 and 010. These compounds act as catalysts in

the destructive reactions of stratospheric ozone.

14

Sources of atmospheric methane: From the C content of atmospheric

 

methane, Ehhalt (1974) and Ehhalt and Schmidt (1978) have estimated that
about 80% of the methane is produced biogenically. The view that
atmospheric methane is biogenic is strongly supported by Lovelock and
Margulis (1974) who demonstrated that a purely abiogenic thermodynamic
equilibrium would predict 29 orders of magnitude less CH4 in the
atmosphere than is currently reported. Ehhalt (1985) concluded that one
major biogenic source of methane is wetlands (e.g. rice paddies)

(Figure 1).

Methane is produced by a group of strict anaerobic bacteria in
highly chemically reduced habitats (Smith and Hungate 1958) i.e. rumen,
sludge digesters, swamps, and rice paddies. Methane produced in lake
sediments must diffuse through an oxic—anoxic interface either at the
sediment water interface or in the overlying water. Greater than 80
percent of the produced methane is oxidized to carbon dioxide at these
interfaces (Rudd and Hamilton, 1979). However, methane produced in

ruminants and rice paddies (Cicerone and Shetter 1981, Cicerone et a1.

Figure 1.

 

GLOBAL PRODUCTION RATES (1013 grams CHglyear)

 

100-
200

 

Total produotlon:
184-520 x 10‘2 grams Cflglyear.

 

25-
1 10

 

 

 

 

 

 

 

 

 

 

Sources of biogenic methane and production rates.

(Ehhalt 1985).

4-7 2-5
0
9/0” (’09 09¢
a o o
o a, o
60 r
I‘ O
‘04, ”Io ’0

I.
1983) is emitted directly to the atmosphere and is only partially
oxidized prior to reaching the atmosphere in rice paddies.
Official statistics (FAO Yearbook 1985) indicate that harvested rice
paddies occupied 147 x 1010 m2 in 1984. Therefore rice paddies are the
largest wetland controlled by man. Since rice harvested acreage has

increased only 2% annually since 1950, increased emissions of methane

must be due to changes in the cultivars used or cultivation practices.

Importance of rice production: On a global basis rice ranks second

 

to wheat in terms of the total harvested acreage. In terms of
importance as a food crop, rice provides more calories per hectare than
any other cereal crop (DeDatta 1981). At the average world yield, a
hectare of rice could sustain 5.7 persons per year compared to 5.3 for
maize and 4.1 for wheat (DeDatta 1981). It is estimated that 401 of the
world population uses rice as a major source of calories. Rice provides
more than half of the food for 1.3 billion people and 25-50% of the food
for 400 million people. Rice is the only major food crop which grows
primarily on the vast areas of flat, low-lying river basins and delta
areas in Asia that are flooded to various depths during the monsoonal
season. The significance of rice production is further emphasized by
the fact that its successful production has often been linked with the

stability of the world (Palacpac, 1982).

Wetland rice culture and its associated soil characteristics: Rice
produces greater yields when it is grown in flooded vs unflooded soil
(DeDatta 1981). Submergence results in greater suppression of weed
growth; higher efficiency in the utilization of added nitrogen: and

better insect and weed control with grannular chemicals. In south and

5
southeast Asia, 95 percent of cultivated rice areas are in wetland rice
culture (Barker and Herdt 1979). The general operations involved in
submerged rice cultivation are:
(1) Submergence of the soil, with or without puddling, for the
duration of the crop and with or without soil drying in midseason;
(2) Draining and drying the soils before harvest: and
(3) Reflooding for the next crop a few weeks to several months after
harvest.

These wetland practices result in greater than 952 of the rice
growth cycle being completed while it is submerged. Since oxygen
diffuses 10,000 times slower through water than through a gas phase
(Greenwood and Boodman 1964), oxygen availability in submerged soils is
very low. Oxygen that is trapped or diffuses into soil is rapidly
consumed by microbial respiration. Under the resultant anaerobiosis,
facultative and strict anaerobes predominate the microbial community in
submerged soils (Hayashi et a1. 1978). The major anaerobic microbial
processes occurring in submerged soils are illustrated in Table 1
(Tiedje et a1. 1984). Soil constituents N03' (and other nitrogenous
oxides), Mn4+, FeaI, 8042', C02 and 8+ ions serve as electron acceptors
and are reduced, sequentially according to their thermodynamic yield to
N2, Mn2+, Fe2+, 82-, CH4, and H2 respectively. However, even in a
permanently flooded soil, a range of reducing conditions exist with
depth in the soil (Sethunathen et a1. 1982). A flooded rice field is
characterized by four distinct zones: flood (standing) water; a thin
(few millimeters) oxidized surface soil layer; reduced soil layer: and

the rhizosphere (Yoshida 1975, Sethunathen et a1. 1982, Vatanabe and

Furusaka 1980). Major microbiologically mediated processes in flooded

Table 1. Anaerobic microbial processes and their reaction products

(Tiedje et al. 1984).

 

 

Process Reactiona
Fe3+, Mn4+ reduction OMb + Fe3+, Mn4+ 9 Fe2+, Mn2+
Denitrification OM + "03- 9 N20, N2
Fermentation OM 9 organic acids, principally acetate
and butyrate

Nitrate respiration 0M + N03' 9 N02-
Dissimilatory NO I

reduction to NH4+ OM + N03“ 9 NH4+
Sulfate reduction OM or H2 + 8042- 9 CH4, acetate
Acetate splitting Acetate 9 CO2 + CH4
Proton reduction Fatty acids and alcohols + H+ 9 H2 +

acetate + CO2

 

a The major reduction products are shown. Oxidized products are also
produced: this is usually CO2 if the electron donor is an organic
compound.

b OM . Organic matter.

rice soils are:

(1) Nitrogen fixation by algae and bacteria, and nitrification in the
flood water.

(2) Methane and ethylene oxidation, nitrification, sulfide oxidation,
ammonification, nitrogen fixation and methane oxidation in the
surface oxidized layer.

(3) Nitrogen fixation, ammonification, denitrification, Fe2+ and Mn2+
reduction, dissimilatory nitrate reduction, sulfate reduction, and
methane production in the reduced soil layer.

(4) Nitrogen fixation, sulfide oxidation, nitrification,
denitrification, and oxidation of Mn2+, Fe2+, and methane in the
rhizosphere.

These oxidation and reduction reactions in flooded rice soils are

responsible for organic matter mineralization and the recycling of

mineral nutrients.

Sources of organic matter in flooded rice soil: In rice growing

 

 

areas, soil organic matter is supplied by rice straw, husks, root
exudates, and senescent roots. A large proportion of organic matter in
rice fields of China are derived from green manures, mainly milk vetch
(Lin 1982). Bluegreen algal biomass also contributes to the total
organic matter reaching paddy soils. However, their major contribution
is through nitrogen fixation carried out alone or in symbiosis with the
water fern azolla. The amount of rice straw produced in paddy
conditions is a function of water regime, season, cultivar, soil
fertility and the grain-straw ration (Ponnamperuma 1984). Alleviation

of growth-limiting factors to rice increases the grain and straw yields.

8
Tall cultivars grown during the wet-season produce more straw than short
cultivars in unfavorable seasons (Palacpac 1982). The grain/straw ratio
ranges from 0.57 during the dry season to 0.89 during the wet season. A
straw production of 15.2 t-ha_1 was recorded with the Meeung Naung 62M
variety (Ponnamperuma 1984). Rice grain production was 470 million tons
in 1984 (FAO Yearbook 1985). If one assumes a grain/straw ratio of 0.7,
320 million tons of rice straw was produced in 1984. On the average
rice straw contains 41.32 total carbon, a C/N ratio of 51.0, a crude ash
content of 18.82, hot-water extractable organic matter of 11.6%, and
7.72 lignin (Inoko 1984). Among the organic compounds produced, the
carbohydrate fraction contains 20.6% hemicellulose and 24.7% cellulose.
In addition to rice straw, the plant roots, stubble, sloughed root cells
and exudates contribute to the organic matter of flooded rice soils
(Hard 1974: Dommergues and Rinaudo 1979). The contribution of these
various sources of organic matter to the total amount of microbially
processed carbon and minerals is however poorly documented in the

literature.

Organic matter decomposition in flooded rice paddies: The potential

 

 

microbial processes involved in organic matter decomposition in flooded
rice soils are illustrated in Figure 2. Due to the lowered availability
of oxygen in submerged soils rice straw is decomposed primarily by
fermentative bacteria. Fermentation metabolites, (e.g., acetate,
propionate, butyrate, lactate, hydrogen and carbon dioxide) can be
oxidized to CO2 through aerobic and anaerobic respiration, or to C84
either through CO2 reduction or acetoclastic methanogenesis. The rates

of the various processes are controlled by the availability of the

 

 

 

 

 

 

 

H 4
1

Rice Straw
W
'W'A W
w.--_A_A_A ‘4 M WW“

m ................ MM°r “3:. :"°4
Soil Watoi'lhi'dr'fi'éé """"

lnltlal Metabolism

i
+ DI..O,rI;:iilcs

Acetate, Proplonate,
C02 H2

Figure 2. Microbial processes and plant interactions in paddy soils.

 

02

 

 

10

appropriate electron acceptors such as 02’ N03- and 3042' or H003“. The
availability of electron acceptors depends on 1) the rate of diffusion
of electron acceptors to the site of consumption: 2) the rate of
diffusion of products out of the anaerobic zone and their subsequent
reoxidation: 3) the demand for electron acceptors. When the rate of
regeneration of electron acceptors or the rate of diffusion can not meet
the demand for electron acceptors, fermentation and methanogenesis
become dominant processes in organic matter metabolism in submerged
soils. In flooded rice soil the overlying water column, the oxidized
surface soil layer and the rhizosphere are sites of the generation or
regeneration of inorganic electron acceptors. However, rice plants also
utilize NO3' and $042- for growth and are therefore in competition with
microbial processes for these ions. Furthermore, rice plants contain
large contiguous internal air spaces which transport atmospheric O2 to
the roots (Arashi and Nitta 1955: Barber et a1. 1962; Armstrong 1969,
1978, 1979: Armstrong and Webb 1985). Potentially a portion of the O2
diffuses into the rhizosphere and functions as an electron acceptor.

Since anaerobic metabolism in natural systems is partially regulated
by the ability of the microbial community to consume reducing
equivalents released during the oxidation of organic matter, it is
important to understand the major anaerobic processes involved in
organic matter decomposition which relate to methane production in and
emission from flooded rice soil.

The goals of this study were to: (1) investigate the changes and
distributions of inorganic ions (NO3-’ 5042', NH4+) and short-chain
volatile fatty acids after dry soil was flooded and planted with rice:

(2) to measure the potentials for nitrification, denitrification, and

11
sulfate reduction in flooded soils, (3) to examine the influence of rice
plants on methane production and emission from flooded rice soils, and
(4) to investigate the influence of continuous rice cropping, straw
incorporation and N-fertilizer application on methane production in and
emission from flooded paddy soils.

To accomplish these goals the study was divided into two major
experiments. Experiment one was designed to examine the affect of the
plant and its phenology on the microbial processes in never planted
experimental paddy soils. Experiment two examined the same questions,
in harvested soils (containing roots) from experiment one. Other soils
used in experiment two were further amended with either fertilizer

and/or rice straw.

MATERIALS AND METHODS

Experiment One

 

Experimental desigg and pot preparation: Pots used in all

 

 

experiments were 10 gallon PVC nursery pots (Molema Nurseries, Akron,
Ohio) lined with 6 mil polyvinyl plastic bags. A subsoil irrigation
system for initial internal flooding and gas displacement consisted of a
perforated 1/2” PVC pipe on the bottom of the container connected to a
vertical nonperforated 1/2" PVC pipe to the surface. Four sampling
cylinders were placed vertically in the pots as illustrated (Figure 3).
The cylinders consisted of polycarbonate tubing (1.25 inch ID, 1.37 inch
O.D., 12 inch long) having holes (0.25 inch) drilled over the entire

length and circumference. The tubes were covered with Nitemesh (Nitex

 

      

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‘ .
. a ‘
. . . u o.
I . . ’ \.\\00\ ~
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.w
‘‘‘‘‘ ~ .
. . . On Q‘ 0.0-. O \
. m...m\.o.-\onoo s~\ . e c
. . t..\\o..\.m.e§.§ eeO. - e
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. .n .. u . a I .. I
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\ . .7. ;. ..... .o I, .
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5.... ...... ..... 3.... so I s . . .
. .I : e...?oa. i .

we so.
.? 031“". .

s

  

 

Experimental pots and sampling cylinders.

Figure 3.

13
mesh No. 305T) which had a mesh opening of 48 um and an open surface of
33X.

Kalamazoo sandy loam (pH. 7.4, organic matter content 3.0%, CEC 15
meq/lOOg soil) was used in all studies. The soil was initially crushed
and sieved through a 2-mm-mesh screen followed by complete mixing in a
rotary cement mixer to assure homogeneity. The processed soil was
placed in the pots and gently settled to a depth of 12 inches. The same
soil was placed in the sampling cylinders. The cylinders allowed for
the taking of soil core samples during the growth of the rice without
damaging the roots, which encircled but did not penetrate the mesh.

Soils were initially flooded through the sub-surface irrigation
system with deionized water to displace air from pore spaces with the
rising water. Subsequent irrigation consisted of the manual addition of
deionized water from the surface to maintain a water depth of 2.5
inches.

Rice seeds (Oryza sativa cv An-nan-chiao, type japonicum) were

 

germinated in petri dishes on moistened filter paper. Plates were
maintained at 80°F and exposed to a 14 hour light and 10 hour dark
cycle. Germinated seeds were placed in seedling trays containing the
previously described soil. The soil was flooded with deionized water
and the seedlings were maintained under the conditions described above
for 14 days.

Four weeks after dry soils were flooded, four hills (each hill
contains 3 plants) of 18—day old rice seedlings were transplanted into
the experimental pots with a spacing of 6.5" x 6.5" (Figure 3) in the
pot. No fertilizer application was made during the rice growing period;

however, 1.5 mmoles of EDDHA (Sequtrene 138) was applied to correct Fe

14
deficiencies during the seedling and early growth stages. Greenhouse
conditions were: a temperature 80—75°F Day-night cycle, relative
humidity 40-602, and supplemental lighting to provide a light cycle of
14h and a 10h dark cycle. Pots were placed 6 inches apart in rows which
were 3 ft. apart. Replicates of the various treatment were randomized

among the controls.

Soil solutions sampling: Soil pore water samples were taken using a

 

suction microlysimeter at various depths of soil, e.g. O, 5, 10, 15, and
25 cm every three days from the beginning of the flooding of dry soil to
the maturing stage of rice.

Microlysimeters consisted of a 25—cm-long, 18 ga. needle which had
0.25-mm-diameter holes drilled into the distal centimeter. The
perforated end was covered with Porous teflon tubing (PTFE, Gore
Manufacturing, Cleveland, Ohio). The hydrophobic membrane was "wetted"
in 95% ethyl alcohol rinsed in deionized water and connected to a
syringe which was used to withdraw the samples.

Soil pore water samples were also taken from cores obtained from the
sampling cylinders at two week intervals. Cores were removed by hand
coring in 1” O.D. x 7/8" ID. acrylic plastic core tubes. Cores were
extruded and sectioned at 2 cm. intervals, followed by centrifugation at
1,000 g.

All soil solutions taken from the experimental treatments were

filtered through 0.2 um Nucleopore membrane filters prior to analyses.

Measurement 2: methane emission: Rates of methane emission were

 

 

determined by measuring the increases of methane concentration in a dome

placed over the water surface of the experimental pot. The dome, a

15

48-liter bell jar with a septum in the top, was placed on the edge of
the pot to avoid disturbing the soil surface, which would potentially
release entrained gas bubbles. An additional enclosure consisting of a
wire frame 13 inches in diameter by 32 inches high covered with a Saran
plastic bag (Anspec Inc., Ann Arbor, MI) was used in the latter growth
stages and in field measurements. A twenty-five ml gas sample was taken
by using a glass 50-ml syringe and needle through a stopper fitted on
the top of the dome. The withdrawn gas sample was transferred into an
evacuated serum bottle stoppered with a butyl rubber stopper (Bellco
Glass Inc., Vineland, NJ) and brought back to the laboratory for
analysis.

A smaller 1—liter dome consisting of a 6" glass pipe cap with a hole
drilled through the top to accept a septum was used to measure methane

flux across the water-air interface between plants.

Plant biomass: Two weeks after transplanting, two rice pots (8

 

plants) were sacrificed each week in order to observe changes in plant
biomass over the experimental period. Plants were cut at ground level
and roots were collected and washed. Roots were sectioned at 4 cm.
intervals to determine the percent of the total root biomass found with
depth. The samples were dried at 60°C in a forced air oven until a

constant weight was obtained.

Chemical Analyses

 

Nitrate and nitrite determination: High pressure liquid

 

chromatography (HPLC) was used for the analyses. Separation of N02- and
N03- was accomplished by eluting the sample through a Whatman PX-lO

column with 50 mM phosphate buffer solution (pH 3.0) at a flow rate of

16
2.0 ml/min. Peak detection was on a Shimadzu SPD—6A U.V. detector
(Shimadzu Corp., Columbia, MD) at 210 nm. The retention times for
nitrite and nitrate were 3.2 and 3.9 min., respectively. The signal

from the detector was processed with a Shimadzu C-R 3A integrator.

Sulfate determination: Sulfate was analyzed using (HPLC) and was

 

separated on a vydac anion exchange chromatography column (Vydac 302-2C,
25 cm long), eluted with 0.002 M phthalic acid buffer adjusted to pH 4.9
with anhydrous sodium borate. A flow rate of 2.5 ml-min"1 yielded a
retention time for sulfate of 7 min. Sulfate was detected with a
conductivity detector (Anspec Inc., Ann Arbor MI). A Shimadzu C-R,3A

integrator was used to process the signal from the detector.

Qgganic acid determination: Organic acids were separated on a HPLC

 

Biorad HPX-87 column (30 cm x 4.1 mm) eluted with 0.007 N H 80

2 4

continuously purged with helium gas, at a flow rate of 0.6 ml-min'1

and
column temperature of 60°C. The fatty acids lactate, fumarate,
succinate, acetate, propionate, butyrate, and isobutyrate were separated
and detected at 210 nM with a Shimadzu SPD-6A U.V. detector. A Shimadzu
C-R,3A integrator was used to analyze the recorded signal from the

detector.

Ammonium determination: N84+ was determined spectrophotometrically,

 

using the indophenol-blue method of Koroleff (1972). Three reagents
were used to develop the indophenol-blue. Reagent A consisted of a
citrate buffer containing 66.7 g trisodium citrate dihydrate, 34 g boric
acid, 19.4 g citric acid and 3 g sodium hydroxide per liter of ammonium

free water (AFW). Reagent B contained 35.0 g phenol and 0.4 g sodium

17
nitro ferric cyanide per liter of AFW. Reagent C was prepared by
dissolving 20 g sodium hydroxide in 5 mls of AFW, followed by the
addition of 2.0 g sodium dichloroisocyanurate, and diluting to 1 liter
with AFW. The procedure for NH4+ determination was as follows: 10 ml
of sample (or sample dilute to 10 mls with AFW) were transferred into 11
x 18 tubes followed by the addition of 1.2 mls Reagent A, 0.3 mls
Reagent B and 0.45 mls Reagent C. Addition of the reagents was done
quickly and the samples shaken immediately. Samples were allowed to
stand in the dark for a minimum of 8 hours but not in excess of 48
hours. Absorbance was read at 630 nm in 1 cm cell on a Hitachi

spectrophotometer (Model 100-40).

Gas analysis: Analysis of carbon dioxide, oxygen, nitrogen, and

 

methane was achieved with a Carle EGG-8500 (Carle Industries, Sunnyvale,
CA), equipped with a 1 meter (1/8" dia) Poropak O and a molecular sieve
5A columns (Carle Industries, Sunnyvale, CA), connected in series to
facilitate separation of the component gases. Helium was used as

carrier with a flow rate of 30 ml-min-1

and an oven temperature of 60°C.
Carbon dioxide and H20 were separated from other constitute gases on the
(Porapak 0) column. A valving arrangement (Carle Industries) allowed
those gases to bypass the downstream column (molecular sieve 5A 80/100)
where 02/Ar, N2, CH4 were separated. The separated gases were passed
directly to the thermister detector. Gas mixtures (Linde Corp, Chicago,
Ill.) were used as standards. A Shimadzu CR-3A integrator was used to
record the signal from the detector. Concentrations of CH4 were

analyzed on a Varian 3700 gas chromatograph (Varian Inst., Sunnyvale,

CA.) equipped with a flame ionization detector (FID). Methane was

18
separated on a 2-m (1/8" dia.) poropak N Column at an oven temperature
of 50°C using helium as a carrier gas at a flow rate of 20 ml-min-l.
Methane standards from 5000 ppm to 2 ppm were prepared from 100% methane
using Helium as a diluent gas. A Shimadzu CR-3A integrator was used to
record the signal from the detector.
Nitrous oxide was analyzed on a Varian 3700 gas chromatograph

63Ni electron capture detector (ECD). Gas samples were

equipped with a
injected into a 250 ul loop and separated on a 2.5 m long (1/8" dia.)
Porpak 0 column using P-10 gas mixture (10% CE, in Argon) as carrier.
The flow rate was 12 ml-min'1 and oven temperature 50°C. The retention

time was 4.3 min., Standards were prepared from 55 ppm standard (Linde

Corp.) and pure N20 using Helium as the diluent gas.

Rate Measurements

 

 

Potential rates 2; nitrification: A modified technique of Belser

 

and May (1980) was used to examine potential rates of nitrification.
Soil cores were taken from the experimental treatments, sectioned at 3
cm intervals and homogenized with 50 ml of ammonium phosphate buffer (1
mM potassium phosphate, pH 7.2; 0.25 mM (NHA) 2804) in a 125 ml
Erlenmeyer flask. The flasks were covered with aluminum foil and
incubated at 22°C with vigorous agitation (200 revolutions per min) on a
gyrotory shaker (New Brunswick, Edison, NJ). Portions of the incubation
suspension were withdrawn at various time intervals, filtrated through
0.2 um Nucleopore filter and analyzed for N02” and NO3'. Increase in

concentration over time was used to calculate nitrification potential.

Potential rates 2; denitrification: Nitrous oxide reductase

 

 

activity was assayed and used as an estimate of denitrification capacity

l9

(Garcia 1973). Soil cores were taken from experimental pots as
described above, subcored through the center of the section with a 5-ml
syringe which had the needle hub cut off in order to minimize exposure
to oxygen. The subcored soil sample (4-ml) was transferred into 35-m1
serum bottles which were previously flushed with oxygen-free nitrogen
and sealed with a butyl rubber stopper (Bellco). The serum bottle was
again flushed with oxygen-free nitrogen for 3 min. and 50 ul of a N20
solution (prepared by applying 50 kPa of 102 N20 in the head space of
the dilution bottle) was added with a Hamilton microsyringe and needle.
The bottle was shaken vigorously for one minute and incubated at 22.5°C.
Change in the concentration of N20 in the head space was measured at 4
hour intervals using a Varian 3700 gas chromatograph equipped with 63Ni

electron capture detector described above. The rate of N20

disappearance was calculated as the denitrification potential.

Sulfate reduction: The rate of sulfate reduction was estimated by

 

directly measuring the depletion of sulfate concentration in pore water
of soils incubated in sealed serum bottles. Soil cores were obtained
from the various treatments and handled as described above. Pore water
was obtained over time with microporous lysimeters, and changes in

sulfate measured as described for sulfate determination.

Methane concentration and rates 9f methane production: Soil cores

 

 

were obtained from experimental pots as described above and subsamples
extruded into 35 ml serum bottles previously flushed with 02-free
nitrogen, and stoppered with butyl rubber stopper. Three bottles were
autoclaved for 15 min to stop methane production and drive methane from

the soil solution. The gases (0.5 ml) in the head space were withdrawn

20
and 0.3 ml injected into a Varian 3700 gas chromatograph equipped with
flame ionization detector (FID). Methane concentration was estimated by
head space volume, amount of soil used and water content. Bottles
containing soils for estimating methane production were evacuated and
filled with an 02-free gas mixture (83% N2, 72 002 six times to reduce
the ambient methane concentration and incubated (22.5°C). Methane
production rates were measured at one hour intervals for 8 hours, by

examining changes in methane concentrations in the head space.

14

Respiratory index: Radio labeled 2- C acetate (54 mCi/m-mole: New

 

England Nuclear Corp.) was used. The method used was after that
described in Lovley and Klug (1982). Briefly, soil cores were obtained
from an experimental treatment as described previously: subcored with
3—ml syringes with the needle hub cut off and the open end of the
syringe sealed with a butyl rubber stopper (Bellco). A 50 ul aliquot of
1-14C—acetate (containing 1 uCi of 14C) which had been preflushed with
oxygen-free nitrogen was directly injected into subcores with a 250 ul
Hamilton syringe. The syringe needle was inserted through the serum
stopper and the labeled acetate was injected as the needle was
withdrawn. The injected subcores were incubated at (22.5°C) for
appropriate time intervals. The incubation was stopped after injection
by immersing the subcores in liquid nitrogen (0 hour) followed by
freezing in -80°C freezer. Prior to analyses the surface of the
subcores was warmed to allow the cores to be extruded into 60 ml serum
bottles containing 2 ml of 4 N glutaraldehyde which had previously been
flushed with 02-free nitrogen. Samples were allowed to thaw in the

bottle, samples shaken and the head space sampled. Gas samples (1.0 m1)

21
were analyzed for specific activities of 14CH4 and 14C02 with a Varian
3700 gas chromatograph equipped with thermal conductivity detector that
was connected in series with a gas proportional counter. Gases were
separated at 50°C on a 3M, 1/8" dia. column packed with Porapak N
(100/120 mesh, Waters Associated) with a helium flow rate of 20 ml/min.
The injector and detector temperature were 120°C and 160°, respectively.
14CH4 and 14002 were eluted with retention times of 1.4 and 7.0 min. and
data recorded on a Shimadzu (C-R 3A) integrator. The Respiration Index

was calculated as: RI . 14CH4/(14CH4 + 14C02).

Experiment Two

 

Experimental desigp and pgt preparation: At the conclusion of

 

 

experiment one, one set of experimental pots used in experiment one were
drained and maintained at field capacity for one week. Rice straw
produced in experiment one was cut into 4 cm long pieces and
incorporated by raking into the top 3 cm of soil at a rate equivalent to
45kg ha'l. Pots were flooded and kept in a flooded condition until time
of planting. Another set was maintained in a flooded state (without
straw addition) until time of transplanting. Rice plants used,
transplanting, water management techniques, and environmental conditions
were the same as for experiment one. Rates of production in and

emission from experimental soils were measured every 1-2 weeks after

rice transplanting.

Effect 9f nitrogen fertilizer application: Ammonium sulfate or
1
t

 

ammonium chloride was applied at an equivalent rate of 50 kg N ha— 0
examine the affect of the application of nitrogen fertilizer on the

production and emission of methane. Four treatments and three

22
replications were involved in this experiment. Treatments were: A)
Ammonium sulfate addition: 8) Ammonium chloride addition: C) Ammonium
chloride plus ferrous sulfate addition; D) No-nitrogen addition.

In treatment C, the same amount of sulfate applied in Treatment A
was added by using 269 kg ha'1 of FeSOA. Rice transplanting and
management were the same as described in experiment one. The fertilizer
treatments were applied one day before rice transplanting. Methane
production and emission from experimental pot were determined at rice

flowering and maturing stages beginning 50 to 70 days after rice

transplanting.

RESULTS AND DISCUSSION

Sampligg techniques: In order to collect pore water without
disrupting conditions in the experimental pots, the micro-suction
lysimeter described in the materials and methods was used. In order to
validate the use of this method pore water concentrations of chemical
constituents such as nitrate, sulfate and short chained volatile fatty
acids at different soil depths were compared to those obtained with
conventional methods. The latter consisted of coring the soils,
sectioning, and centrifugation of sections at 1000 G to obtain pore
water for analyses. Results are illustrated in Table 2a and 2b.
Concentrations of NOB', $042— and short chain volatile fatty acids
(VFA's) in pore waters collected by the coring procedure were not
significantly (5% level) different from those taken by microporous

suction lysimeters and standard core sectioning method. Howes et a1.

23

000 O
was a a a 214

aofiumw>ov vmmeamum on» ma vosoaaom new: «

 

“Hm.~v m.mm Ao.HV o.n
Am.aaHv o.oa~ An.mv c.o
Am.amav c.4ae Aw.mv ~.e
Am.moev o.eaoa Am.mv c.aa
Aa.amav o.oama Au.m~v o.o~H
As.o~mV m.amam AN.oamv c.aamH

«on me:

 

venue- anaemumo>aou

Am.aav a.m~ Ae.ov n.m
«a «a
An.~mav ~.-~ An.av a.m
a~.¢wmv o.oon Am.ov ~.~H
Ae.~suv o.nma Ao.~sv m.~m
Aam.mv c.5oNH «Am.emav ~.eema
4cm mcz

 

mouoawmhaouomm aowuoam

«NICN
Dune“
wanna
Nana
w Is
a :0

l0

 

Hfiom mo suave

.ueoauu- mundane» aawuomuae an amass
onus nouns «newumaom Huom aw Aaxmnv anonymuuaooaoo oumuasm can wanna“: mo commune-co .mN wanna

24

o oo
o u use uozaa

aowumw>oe cummamum an vomoaaow amaze

 

 

 

 

 

 

 

Aa.o¢vawa Au.onsoH Ao.o~veon An.anmow c~no~

Am.mmVo~H «« Am.omvwwm «« ownea

Aa.navcoa Ao.o~vN¢H Ao.n¢vmo¢ Ac.¢Hv~¢m eHINH

An.c~vns AN.ovao AN.~mVoue Ao.o¢vN<¢ «Him

«a «« Am.0mvmow «Aw.o~v~m~ ate

«a «a «a «a «no
vogue- uouumwmhaouowa vogue- mouoawthouowa
Hmaofiuao>aom\. coflwme111 HmmOAuau>aow acmuosm ,

oumaomaouw oumuoo<

 

muscumnoou Hagan-mm amouomuwv up muse» Aaxaav
naofiueuuaoomoo oueaowaoun van uueuooa nouns anon mo acmwueaaou .aN manna

25
(1985) found the core sectioning technique in salt marsh soils to
significantly increase the concentration of total dissolved organic
carbon and dimethylsulfide, due to root damage, while other soil
constituents such as N03" and 8042' were not significantly different in
soil solutions obtained by different methods. The former would
presumably stimulate the rates of processes using dissolved organic
matter. In our experimental system, rice roots were separated from the
soil with the previously described porous cylinders which allowed core
removal without root damage. Pore water NO3-, 8042' (Table 2a) and VFA
concentrations (Table 2b) were not significantly (52 level) different in
samples taken by microporous suction lysimeters and from cores taken
from inside the cylinders by the standard core sectioning method.
Considerable heterogeneity occurred in the profiles of sulfate.
Jdrgensen (1978), heterogeneity such as this can be attributed to the

patchiness of sulfate reduction within sulfate containing zones.

Plant biomass: Changes in the above and below ground biomass at

 

various times after transplanting are illustrated on Figure 4. The same
general pattern of increase was observed for the above and below ground
biomass during various stages of growth with no increase in either after
flowering (50 days after transplanting). Above ground biomass was
greater than double that of the below ground biomass at any of the
sampling dates. Similar growth patterns have been reported for field
grown rice (Yoshida 1981). The standard deviation between plants was
small which probably reflects the homogeneity of the soil and conditions
associated with the experimental treatment.

The percentage of root biomass at various depths in the soil at

Dry Weight (g)

26

60 -
Above Ground
E Below Ground

mo-

20 9,

 

 

Days
Figure 4. Above and below ground plant biomass during the growth

period.

27
panicle initiation (30 days) and maturing phase (50 days) is illustrated
in Table 3. In both cases the maximum root biomass was observed in the
top 4 cm, and a steep decrease in biomass below this depth interval.

This relationship is also noted in field grown rice (Yoshida 1981).

Table 3. Percent distribution of root biomass at panicle initiation and

maturing stage of growth.

 

 

 

Depth Panicle Initiation Maturing
_____ (30 days*) (50 days*)
0- 4 75 1 4 83 1 6
4— 8 15 1 2 10 1 3
8-12 41 1 0.8 3 1 2
12-16 31 1 1 2 1 2
16-20 1.8 1 2 1.2 1 2
20-25 1.2 1 1 0.8 1 3

 

*days after transplanting

Nitrogen Transformations

 

Nitrogen containing fertilizers are often added to paddy soils to
increase yields of rice. Nitrogen is considered to be the most
important nutrient of the normal fertilizer elements (N, P, K) added in
paddy soil (Dommergues et a1. 1979). In flooded rice soils the addition
of ammonium sulfate followed by ammonium chloride is recognized as the
most efficient N—fertilizer. Nitrate containing fertilizer is not
recommended in flooded soil conditions due to inevitable losses through

denitrification or leaching.

28
Ammonium produced through organic matter mineralization is either
consumed by the plant or is oxidized through nitrification at the root
surface or in the oxidized layer of flooded soils. The products of
nitrification NO3— and N02” are used by denitrifiers as electron
acceptors, and reduced to N2 or N20 depending on the species of

denitrifiers and concentrations of the reactants (Tiedje et al. 1984).

Distribution 2: N93 : In order to estimate the rate of

 

denitrification, pore water concentrations of NO3- were followed at 4
day intervals after the flooding of dry soil. The changes in the
concentration of N03- in the planted and nonplanted treatments over the
experimental period are illustrated in Figure 5. Initially nitrate
concentrations in pore water were high after the flooding of the dry
soil. This initial high concentration is thought to reflect the
concentration present in the agricultural soils at the Kellogg
Biological Station. It is also possible that NH4+ present in these
soils may have been nitrified during the period between when the soil
was dug, stored and sieved.

Nitrate concentration in planted treatments was higher, remained
longer and increased in concentration after planting as compared to that
in the nonplanted treatment. Nitrate concentration in nonplanted
flooded soil (Figure 5) rapidly declined following 12 days of flooding.
The profile of depletion with depth exhibited the same general pattern
over the first 40 days after which no N03” was detected. The results
agree with the data of MacRae et a1. (1968) who demonstrated that

15

59-1002 of added N03 disappeared with 6 weeks of its incubation in six

different submerged soils.

29

Planted Selle

m: '  
MAN 1

{Ti-N

25.677

IUOhI/L

9 moles

 

 

 

 

 

Nonplanted Solls

u moles nos-n.

Z: W‘
9

 

\\‘\\»

\\‘\\\\\\\

 

02. 0 “‘\\\\§‘
““\“‘

Figure 5. Depth—time distribution of nitrate in planted and nonplanted

soils at O, 30, 60 and 84 days of the experiment.

30

The higher N03- concentration observed in the planted soils may be
explained by increased ammonification driven by plant exudates or
senescent root cells followed by nitrification of the released ammonia
in the rhizosphere or surface soils. Interestingly the ammonia
concentrations (Figure 6) in the planted vs the nonplanted treatment are
very similar suggesting that nitrification may have been inhibited in
the nonplanted treatment by the lack of oxygen which would be produced
by the plants. This is supported by the highest root biomass (Table 3)
in this zone. Smith and Delaune 1984 report a similar effect of rice
plants on nitrification in the root zone. The process of 02 transport
through lysigenous channels in the roots and partial loss to the
rhizosphere has previously been shown to modify conditions in the
rhizosphere (Bristow 1975; Armstrong 1978: Trolldenier 1988). Another
factor which may be responsible for increasing oxygen concentrations in
surface sediments is downward movement of oxygenated surface waters due
to transpirational loss of water from the plant (Dacey and Howes 1984).
Although evaporation vs transpirational loss of water was not estimated,
it was noted that water addition to planted treatments was approximately
twice that added to the nonplanted treatment during the active growth
period. This suggests that downward water movement was occurring and
should be considered in future studies of this nature. The depth
profiles of NO3' depletion are steeper in the planted treatment and the
N03' remained in surface sediments 2 months after flooding. The
steepness of the profile in surficial soils also supports the suggestion
that increased N03- is produced in these surface soils due to the
presence of 02 which would increase rates of nitrification and decrease

rates of denitrification.

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

nsooi
'i' Planted Soils
+
i?
z:
e 1mmo-
.2
o
E
:
soo-
t.
0,, ..
’
4
z
9 10
o ‘ as
‘9 do '0
nsoo‘
_. Nonplanted Soils
\
+
:3
z 1,000-1
I
e
a N
E
3 500‘
°i.
O. a.
0,4 ______1
/- 1am ~
00 ‘14-'4"‘ "J‘iifJ'IJ 8‘
‘9 so

Days
Figure 6. Depth-time distribution of dissolved ammonia in planted and

nonplanted soils at 0, 30, 60 and 84 days of the experiment.

32

Distributions 2; dissolved N§4+: Distributions of dissolved NH4+ in

 

planted and nonplanted soils at different depths over the treatment
period is illustrated in Figure 6. The ratio of dissolved to total
ammonia in planted soils taken at 40 days after flooding (i.e. dissolved
plus adsorbed) is illustrated in Table 4. Dissolved ammonia levels
ranged from 9-221 of the total, indicating that the majority of NH4+ is
bound to soil particles. It is not known what the ammonia sorption
isotherms are for these soils when maintained under these conditions or
whether the ratio changes at various stages of growth. In both
treatments NH4+ concentrations increased during the first 40 days of the
treatment period. Following this time the concentration leveled off in
the planted treatment but continued to increase in the nonplanted
treatment. Concentrations were consistently higher in the deeper
portions of the profile, with the lowest concentrations being in surface
soils. These profiles are the inverse of those found for N03_ (Figure
5) suggesting a relationship between the processes controlling the
concentration of the two ions. The leveling off of the concentrations
of NH4+ in the planted treatment especially in the zones of highest root
biomass (0-10 cm) suggest that the more oxidized environment stimulates
nitrification in this zone and/or the plant is taking up NH4+ which is
the preferred nitrogen source of the plant. This type of relationship
was previously reported by Ando et al. 1983. As pointed out above this
is the zone where the concentration of N03” is the highest which would
correlate with increased nitrification in this zone. It would appear
that nitrification and plant uptake would be in competition with each
other, however it is not known what the rate limiting concentration is

for both of these processes (Broadbent and Reyes 1971).

33
Further studies should examine changes in the adsorbed to dissolved
ammonia ratio since changes in redox overtime could markedly change this
relationship (Broadbent and Nakashima 1970: Reddy and Patrick 1986).
The effect of redox differences is suggested by the lowest percentage of
dissolved ammonia (Table 4) in the deepest zone where the redox would be
expected to be the lowest.

Table 4. Depth distribution of dissolved and exchangeable NH + in

4
planted soils at day 40 of experiment 1.

 

Dissolved NH4+

 

 

Soil depth Dissolved NH4+ Exchangeable NH4+ + x 100
(cm) (uM) (uM) Total NBA
2 0.44 4.10 18.3
6 1.30 6.65 22.3
10 1.81 7.43 18.6
14 2.15 9.40 19.6
18 2.51 8.72 16.6
22 2.20 9.81 9.6

 

Nitrification pptential: Nitrification potentials were compared in

 

planted and nonplanted treatments 30 days after transplanting. This was
done to further examine the source of the observed NHA+ and N03—
profiles Figures 5 and 6, which could be as a result of differences in
rates of nitrification. Table 5 illustrates that the highest
nitrification rates occurred in the top 3 centimeters in both

treatments, however no nitrification was observed in depths below this

34
in the nonplanted treatment. Nitrification potential within the upper 3
cm of soil was 0.167 um NO3I-N/g soil/hr as compared to 0.04 um N03'-N/g
soil/hr in the nonplanted treatment. These results support the observed
highest N03“ concentrations in surficial soils of the planted treatment.
The greater depth distribution of nitrification in planted soils follows
the depth distribution of root biomass (Table 3) further suggesting the

involvement of roots in supplying oxygen to lower depths in the soil.

Table 5. Depth distribution of rates of potential nitrification in

planted and nonplanted soils at day 40 of experiment 1.

 

 

Treatment Depth (cm) umoles N03- g°hour-1

Nonplanted 0- 3 0.040 (0.021)*
12-15 ---**
21-24 ---**

Planted 0- 3 0.167 (0.084)
12-15 0.041 (0.008)
21-24 0.004 (0.006)

 

*Mean followed by standard deviation

**Not detectable

Denitrification potential: Denitrification potential was examined

 

by measuring the rate of nitrous oxide (N20) consumption. This assay of
denitrification potential has been previously used by Garcia and Tiedje

(1982) in paddy soils and Ormeland et a1. (1984) in marine intertidal

35
sediments. It is a useful method for measuring the activity of nitrous
oxide reductase, and therefore serves as a tool for assessing
denitrification potential.

Nitrous oxide consumption was measured during the tillering, heading
and flowering and mature stage of growth which respectively corresponded
to 20, 40, and 70 days after transplanting. No significant difference
was noted between the activities (Table 6) of nitrous oxide consumption
at any of the three growth periods. Differences were however observed
between activities in surface (0-2 cm) profiles vs those below the 10-12
cm profile. No significant difference was observed between the planted
and nonplanted treatment.

These results were somewhat unexpected since nitrate decreased in
concentration (Figure 5) after time of transplanting and was not
detectable in soils below 14 cm any time during the treatment period.
There also was a significantly lower concentration in surface soils of
the nonplanted treatment at all stages during the treatment period.

This is in marked contrast to the observed, significant effect of root
biomass on the rates of denitrification in agricultural soils by Smith

and Tiedje 1979.

Changes lg concentrations and distributions 2; volatile fatty acids:

 

 

The concentration of volatile fatty acids (VFA's) in planted and
nonplanted soil over the treatment period is illustrated in Figures 7.
Concentrations of VFA's increased gradually after dry soil flooding and
reached a maximum concentration at 30 days after rice transplanting (or
44 days after dry soil flooding). The VFA concentration declined

rapidly after this period in planted soils but maintained the same

36

uaomoummmom one haco

 

 

m
cowunw>ou pudendum N
umuumnanm:MHu momma when H
oo~.o Aofio.ov HNN.o Ammo.ov cm~.o Nun
ama.c Ammo.ov o¢~.o Aoeo.ov me.o man
Hm~.o “Ono.ov aH~.c AoHo.ov com.o «an
mam.o Ramo.ov mmw.c Ammo.ov mmu.o oHu
mnm.o Anno.ov n~m.o Anao.ov ¢e~.o on
vmuamHa
nnm.o Ammo.ov mnm.o Amao.ov oa~.c Ni 1:02
AHNo.ov so~.o Ammo.ov m¢H.o Aooo.ov w-.o mm:
Awuo.ov ¢o~.o Ammo.ov mm~.o Ammo.ov ~N~.o wan
Ammo.ov Hw~.o Ammo.ov nw~.c Ammo.ov mm~.o «an
Ammo.ov num.o Aeno.ov cem.c Aomo.ov o-.o can
Anwc.ov «mm.o Aamo.oV Nvm.o Ammo.ov own.o on
Ammo.ov oHe.c Aawo.ov ~mm.c Aneo.ov nmm.o Nu voucmam
Anzac oNV Amman owv Ammuv oNv Aauv mama

eumum unauaum: madam uauueaoam can Humvee: ouwum uawuoaafia gamma numoua

.a uaoauueaxo we as van co .ou when
an A u:.u\z-c 2 may coauaoamauuaaue Haasamuoa mo mused mo camsaaauumae guano .e wanna

ullolea VFA/L

uflolea VFA/L

37

3000+

Planted Solle

2300*

\A' \‘ ‘\\\“

\\\o§§\\ \\\>\ __

12004

 

0
=4—

 

 

 

ssoo
Nonplanted Solla

1200

 

as
0 Days

Figure 7. Depth-time distribution of volatile fatty acids (VFA's) in

planted and nonplanted soils at 0, 23, 43 and 64 days of the

experiment.

38
qualitative distribution of VFA's during the treatment period. A more
gradual decline was observed in the nonplanted soils. Spatial and
temporal distribution of VFA's during the treatment period in planted
soils is illustrated in Figure 7. Concentrations were consistently
higher in the deeper portion of the soil and low to undetectable in
surface soils. The same general pattern of VFA distribution was
observed in both planted and nonplanted soils. However, the nonplanted
soils appeared to have a higher VFA concentration, in the surface soils,
and the planted soils had higher concentrations in deeper soils and
during the first 60 days after transplanting. It is possible that these
increased concentrations are due to increased fermentation of dissolved
organic compounds derived from the rooting zone (Chandrasekaran and
Yoshida 1973). The vertical distribution of VFA's was inversely related
to soil profiles of N03” (Figure 5) and 8042' (Figure 8). Lovley and
Rlug (1986) demonstrated maintenance of low concentration of VFA's in
lake sediments when SO42— concentration was above 30 uM. Presumably
processes of denitrification and sulfate reduction utilize these VFA's
as carbon sources and control their concentrations in surface paddy
zone. This is also supported, especially in the top 10 cm of soil, by

the higher respiratory index (RI) (Figure 12) in nonplanted soil.

Rates of Sulfate Reduction

 

Sulfate is normally reduced after the depletion of nitrate and other
more energetically favorable reactions in anerobic paddy soils (Connel
and Patrick 1968, 1969, Ponnamperuma 1972). Sulfate is reduced to H28
which is toxic to rice at a concentration of approximately 0.07 ppm

(Mitsui et a1. 1951; Freney et a1. 1982). However, H25 seldom

39

 

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e 8. Depth-time distribution of sulfate in planted and nonplanted

40
accumulates at toxic concentrations in most paddy soils, since 828 is
either immediately precipitated as metallic sulfides, chiefly FeS, or is
oxidized to sulfate or elemental sulfur in the rice rhizosphere by
chemosynthetic microorganisms. Sulfide toxicity can occur in acid i.e.
high sulfate soils especially soils low in organic matter and in highly
degraded soils such as Akiochi soils of Japan where ferrous iron content

is nearly one fifth of normal paddy soils (Shiga and Suzuki 1964).

Distribution 2; sulfate 15 pore water: Distribution of sulfate at

 

 

different soil depths during the treatment period in planted and
nonplanted soils is illustrated in Figure 8, respectively.
Concentrations of sulfate were highest at the beginning of the treatment
period and gradually declined during the remainder of the period. The
depletion in sulfate in the soil profile was from the lower portion of
the profile upward over time of the experiment.

The depletion of 804' in both of the treatments was similar,
however, a general trend toward higher concentrations at 8 cm was noted
in the planted treatment. Since both the rice plants and microorganisms
were consuming 804' it would follow that considerable reoxidation of
reduced sulfur compounds must be occurring in the planted treatment in
order to account for the similar 5048 profiles in both treatments. This
is also supported by the higher sulfate reduction rates observed in the
planted treatments (Table 7). This observation suggests increased
oxidation of reduced inorganic compounds in the rooting zone of the
planted treatment. The rate of sulfate depletion was lower than that of
nitrate, supporting the previously discussed spatial and temporal

consumption of the most energetically favorable electron acceptor.

41

Table 7. Depth distribution of rates of sulfate reduction (nmole/goh'l)

at days 20 and 70 of experiment 1.

 

 

Treatments Depth Tillering Stage Maturing Stage
(cm) (20 days)* (70 days)*
4 4.7 1 1.16 8.6 1 0.91
8 3.8 1 0.47 5.9 1 1.34
Planted 12 2.4 1 0.81 2.7 1 0.14
Soil 16 2.5 1 0.74 2.5 1 0.49
20 2.6 1 1.69 1.9 1 0.78
24 1.1 1 0.79 0.5 1 0.42
4 4.9 1 0.21 4.9 1 0.42
8 4.5 1 1.13 3.7 1 0.91
Nonplanted 12 1.9 1 0.18 3.2 1 1.13
Soil 16 1.5 1 0.00 2.5 1 0.21
20 1.3 1 1.06 1.8 1 0.48
24 0.5 1 0.28 0.5 1 0.39

 

*Days after transplanting

42

Rates of sulfate reduction were assayed by measuring the depletion
of sulfate in pore waters of incubated soils sampled at 20 and 80 days
after transplanting. Soils sampled at 80 days were amended with Na2804
to a final concentration of 1 mm since sulfate was markedly depleted in
samples taken below 8 cm (Figure 8). Ingvorsen et a1. 1981 found no
affect of added sulfate on rates of sulfate reduction if sulfate was
above 30 um. In all treatments the concentration of sulfate was above
this value. In both treatments and sampling dates, rates of sulfate
reduction decreased with depth which corresponds to concentration
profile of 504' (Figure 8). Significant differences were observed
between the rates in samples taken at the depth of 0-8 cm at 20 and 80
days in the planted treatments. This is also the zone of the greatest
root biomass (Table 4) and the higher rate observed at 80 days is likely
due to the absence of NO3 (Figure 5) and the potentially higher
concentration of root exudates and microbial metabolites in this zone in
the later stages of growth. This is partially supported by the
increasing concentration of VFA's in soils at 64 days after
transplanting (Figure 7).

The finding of no significant difference in rates at any depth
between the 20 and 80 day samples in the nonplanted treatment supports
the concept that plant carbon stimulated the rates of sulfate reduction
in soils of the planted treatment. The lack of any significant
difference in rates observed in both treatments at 20 days suggests that
the high N03' levels observed (Figure 5) inhibits the rates in both of
the treatments regardless of the influence of plant carbon on sediment

microbial processes.

43

Methane Production and Emission from Planted and Nonplanted Soils

 

Chggges 19 methane concentration: Methane concentrations in soils

 

of planted and nonplanted soils sampled at various depths and times
during the treatment period are illustrated in Figure 9. Methane
concentration increased markedly during the first 20 days after flooding
especially in the deeper zones (16-24 cm). Methane concentrations were
generally higher in nonplanted soils at all depths and all times of
sampling. Differences in concentration could be explained by
differences in methane production (see below) or by differences in
oxidation especially in surface soils 0-4 cm and at all depths in
planted soils.

Root biomass was the highest in the 0-8 cm depth (Table 3), however,
root biomass was observed over the entire soil profile. Oxygen
diffusion from roots may be responsible for higher rates of methane
oxidation throughout the rooting zone, as compared to the nonplanted
soils. De Bont et al. (1978), using acetylene as a competitive
inhibitor of methane oxidation, demonstrated in situ oxidation of
methane in flooded soils. Analyses of methane in the atmosphere above
the pots of flooded soils showed significant increase in the amount of
methane evolved, but only in the presence of acetylene. In the absence
of acetylene, methane was evidently oxidized at the surface-oxidized
layer. Holzapfel-Pschorn et a1. (1985) reported that 801 of the methane
produced in planted paddy soils was oxidized in the rice rhizosphere.

Another explanation for the lower methane concentration in planted
soils is the potential for methane to diffuse into the roots and be
transported through the lacunae of rice plants to the atmosphere above

the soil and water environment. Transport such as this has been

44

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45
reported for other aquatic plants (Dacey and Klug 1979, Sebacher et a1.
1985) and in rice (Seiler et al. 1984). The relationship between these
various mechanisms is complex and both appear to have a major affect on

the methane concentration in planted paddy soils.

Rates 2; methane production: Rates of methane production increased

 

with depth in both planted and nonplanted soils (Figure 10). Highest
production rates were observed at 40 days after flooding, followed by
decreased production over the remainder of the treatment period. The
lowest rates of production were observed in samples obtained at 20 days
and in the 0-4 cm depth zones. Nitrate (Figure 5) and 804' (Figure 6)
was observed at varying depths at 20-40 days and in the 0-8 cm zones
during the first 60 days. Nitrate or 8043 would preferentially be used
as electron acceptors in zones where these ions are observed. The
distribution pattern of the rates of methane production is similar to
that shown for methane concentration, and exhibits an inverse
relationship with pore water nitrate and sulfate concentrations,
especially during the early period of rice growth (0-20 days). Koyama
(1963) indicated that Eh decreased to less than -200 MV before
methanogenesis was observed, and the presence of N03” and 8042'
inhibited the activities of methanogens (Royama, 1963).
Mutual exclusion of methane and sulfate in sediment interstitial
water has been reported (Martens and Berner 1974, Mah et a1. 1977).
Cappenberg (1974) suggested that the negative interactions between
sulfate-reducing and methane-producing bacteria in lake sediments was
due to the sulfide, produced by sulfate reduction, which inhibited

methane production. Martens and Berner (1974) suggested that methane

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47
production and sulfate reduction occur simultaneously, but that methane
is consumed by sulfate-reducing bacteria in sulfate-containing
sediments. However, Lovley and Klug (1986) demonstrated that at sulfate
concentration greater than 30 uM sulfate reducers maintain acetate
and/or H2 concentrations at concentrations too low for methanogens to
grow. The relationship of the distribution of methane concentration and
sulfate concentrations and indirectly to their respective role in carbon
metabolism at various times and depths in soils during this study is
illustrated in Figure 11. This profile demonstrates that at sulfate
concentrations above 250 um, methane concentration is maintained below
10 um. The lower RI in samples examined in soils sampled at 60 days
after flooding (Figure 12) further illustrates that anaerobic
respiratory metabolism dominates terminal carbon metabolism during this
period. Although there was considerable variance in these profiles a
significant difference between the two treatments in the intermediate
sample depths is observed and a clear trend toward lower values was
observed in the planted soil.

The observed increase in methane production at 60-80 days in the
surficial 0—12 cm is at a point where the N03- has been depleted
throughout the soil profile and sulfate has been markedly reduced. This
is also a time when the above and belowground biomass is maximum.

During this stage of plant phenology decreased translocation of nutrient
and gaseous translocation occurs (Matsuo 1969, Yoshida 1981, Alberda
1953, Okajima 1964). Therefore, it would be expected that methane
concentration would increase since less methane would be oxidized and/or
translocated through the plant to the atmosphere. The combination of

these processes would result in an increase in methane concentration.

48

2000-
19004
1800*
17004
1600-
1500*
1400fl<>
1300+
1200~
1100-
1000*

900‘

uMOLE/L 0F SULFATE
‘0

800+

700-

500 ‘o
400.
300+ 0
2004

100 . °

 

0400 0 ° ° 001000 0
0 1000 2000 3000
uMOLE/L 0F METHANE
Figure 11. Relationship between the mean concentrations of sulfate and

methane in pore waters of planted soils.

49

 
   

 

 

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‘s \
‘s \
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Respiration Index

Planted sine-4- Nonplanted B-GE

Figure 12. Depth distribution of the respiration index

[140H4/C(14CHA+14C02)| in planted and nonplanted
soils at 60 days of the experiment.

50

Since the reported values are gross and not net rates of methane
production, it is not possible at this time to determine if the observed
increases are due to increased production or lower rates of oxidation.
In future studies it will be necessary to examine both methane oxidation
and production rates to obtain an actual net production value. This is
very important if one is to be able to relate net rates of methane
production in the rhizosphere to concentrations of methane transported
to the atmosphere.

A similar increase in production did not occur in the nonplanted
treatment. The concentrations of both N03” and 804' were also low in
the nonplanted treatment during this same time period. Therefore, the
general decline in methane production rates in nonplanted soils,
especially in the top 12 cm, may be due to organic matter depletion

since these were previously nonplanted soils.

Methane Emission

 

Static chamber methods were used for all emission studies. Cicerone
and Shetter (1981) and Seiler et a1. (1984) previously discussed the
problems associated with this approach. In summary, the use of static
chambers: (1) establishes an artificial environment of CH4, C02, and
H20 vapor and elevated temperatures; (2) blockage of air movement which
could influence flux; (3) possible alteration of flux due to lowered
eddy currents in the water column.

In order to minimize these problems flux measurements were kept
short to minimize high accumulations of CH4 and water vapor build up in
the chambers. Linear increases in the methane mixing ratios were

observed in 80 minutes, and rates were comparable using both the Saran

51
and glass sampling chambers. Sampling periods were always kept to an
intervals which assured less than a 10°C increase in temperature.

Rates of methane emission from planted and nonplanted soils and
between rice plants from experiment one (never planted soils) are
illustrated in Figure 13. Emission of methane was initially observed 20
days after transplanting. This coincided with the onset of methane
production (Figure 10) in these soils. Emission from both planted and
nonplanted soils increased, however were significantly different (5%
level) at nearly all sampling points. Rates of emission from planted

soils ranged from 0.73 (0.13) to 15.69 (4.4) mg CH /m2/hr, with the

4
highest rates of emission observed during the heading and flowering
stage, and the lowest during the tillering and maturing stage of growth.
This temporal pattern and total rate of emission is similar to that
reported by Holzapfel-Pschorn et a1. (1986) for paddy fields in
Vercelli, Italy. The temporal pattern is also similar to that reported
by Seiler et a1. 1984 from paddy soils in Andalusia, Spain, however,
their reported mean rate of emission was 2—fold lower. Comparison of
rates between sites is difficult without knowing the rates of
production, actual methane concentration in the soil pore water and
total soil volume measured at the time rates of emission are measured.
Both Seiler et a1. (1984) and Holzapfel-Pschorn et a1. (1986)
reported significant diurnal differences in methane emission during
their studies. Seiler et al. (1984) demonstrated that the diurnal
response was not dependent on light and concluded that the diurnal
differences were due to differences in soil temperature. Shifts in soil

temperature ranged up to 6°C over a diurnal cycle in their studies.

Koyama (1963) reported on the significant influence of temperature on

52

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

30‘
20-
H
a
,2 ‘ ..
I ll
I ‘ \
\ ’r \‘ I t‘
N I’ \ ' ‘
P \ l ‘ v
2 ‘ q l ‘
I, ‘ ' t
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O 10 20 30 40 50 50 70 30 90
Days
Between Planta— Planted ----- Nonplanted --—

Figure 13. Methane emission from planted, nonplanted soils and between
plants in the planted treatment at 10 day intervals during

experiment 1.

 

53
methane production, which would affect subsequent rates of emission.

No significant diurnal differences in rates of emission were
observed in our study, however changes in soil temperature would be
expected to be less, since air temperatures only fluctuated 5° during
the course of the study. This fluctuation would have had little effect
on soil temperature, in any of the treatments. Cicerone and Shetter
(1981) also reported small diurnal differences in the rates of emission
of methane in paddy soils in Davis, California. They did not record
emission during the whole growing season, however they had a maximum
diurnal air temperature of 10°C, which may have had little affect on
soil temperature.

Little fluctuation in rates of emission from between plants was
observed during the growth periods. The mean rate reported during the
growth period was 0.10 (0.02) mg CH4/m2/hr. Holzapfel-Pschorn et a1.

1986 reported rates of 1.7 (2.2) mg CH lmzlhr in paddy soils in

4
Vercelli, Italy, and concluded that the majority of the gas escaping
between plants was through ebullition.

Few bubbles were noted in our study and differences between the two
is most likely due to differences in methane production in the two
studies. Our study was conducted in never planted soils while the
Holzapfel-Pschorn et al. (1986) study was conducted on paddy soils
receiving rice straw. Increased organic matter content of the soil
would positively affect rates of methane production, and increase rates
of emission. This will be further discussed in later sections on the
effect of added organic matter on rates of methane production and

emission.

Emission of methane from nonplanted soils was significantly (5%)

54
different from that in the planted soils during the periods when maximum
emission was occurring. Rates ranged from 0.11 (.04) to 1.45 (0.6) mg
CH4/m2/hr. Although the rates are significantly different, the temporal
pattern of emission was quite similar.

Since these were never planted soils which were recently flooded one
would expect a slow development in the appearance of methane formation
which would coincide with the depletion of inorganic electron acceptors,
and appearance of precursors of methanogenesis. The rates and temporal
patterns of methane production were very similar (Figure 13), however
these are net rates of production which do not account for differences
in rates of methane oxidation which are expected to be greater in
planted soils due to the influence of the plant in providing oxygen in
the soil. The similarities in rates of methane production and the major
differences in rates of emission strongly suggest an influence of the
plants on rates of emission.

Table 8 illustrates the effects of cutting the plants off above and
below the water line on rates of methane emission. In the treatment
where the plant is cut off below the water line the rates of emission
are similar to those observed in nonplanted soils, while those cut above
the water line exhibit rates similar to those of the intact plant. The
observed inhibition of CH4 emission by cutting plants below the water
line demonstrates that the plants lacunal structure serves as a major
transport mechanism for methane from paddy soils. Approximately 942 of
the methane emitted is lost through the plant. Similar ranges were
reported in other studies with rice, Cicerone and Shetter (1981), Seiler
et al. (1984), and Holzapfel-Pschorn (1986), and in other aquatic plants
(e.g. Dacey and Klug 1979).

55
Table 8. Influence of rice plants on the rates of methane emission at
the flowering and heading stage of growth. Data reported as

mean (ns6) followed by the standard deviation.

 

CH4 Emission Rates mg/mzlhr

 

 

 

 

Plants cut above Plants cut below
Intact plants water level water level
11.7(2.1) 10.9(1.9) 0.43(0.10)

 

The direct relationship between changes in plant biomass and rates
of emission supports the above observed effect of the plant on rates of
emission. The decline in rates of emission during the maturing stage of
growth (> 70 days) is directly correlated to the observed increase in
methane concentration in planted soils (Figure 9). It is also noted
that rates of methane production also occur at this time (Figure 10)
which, as previously discussed, may be the effect of decreased methane
oxidation, due to less oxygen supplied to the soils from actively
growing plants. It is also possible that increased root exudation at
this time could be responsible for increased fermentation and subsequent
methane production.

Arashi and Nitta (1955) reported that roots of rice begin to senesce
following the flowering and heading stages of growth. This would most
likely lead to decreases in the internal structure of the lacunae
(Matsuo, 1969) and transport of methane through the plant. Root biomass
was only measured through the flowering and heading stage in our study

resulting in the inability to determine if root biomass decreased during

56
the maturing period of this study.

A summary of the temporal relationship between methane production
and emission from planted and nonplanted soils is reported on an areal
basis in Figure 14. Highest rates of emission from the planted soils
are observed when the production is actually shown to be decreasing. As
discussed previously this actually could be an artifact of our inability
to assess gross methane production. The percent of the methane
production emitted from these soils ranged from 1.3-69X with a mean of
27.0 (22.8)!. The highest percentages reported for the period of
maximum emission (50-60 days after transplanting). In the nonplanted
treatment the percentages ranged from 0.5-4X with a mean of 3.0 (1.4)X.
These results support the previous observations of the contribution of
the plant to emission rates from the soil, and emphasizes the importance

of this mechanism as a source of atmospheric methane.

Influence g: pgevious cropping: Soils from experiment one were

 

maintained under flooded conditions for 90 days. At this time pore
water nitrate and sulfate concentrations were 1-2um and 3-4um
respectively. Active methanogenesis was noted shortly after time of
transplanting as illustrated in Figure 15. The absence of metabolites,
e.g. NOB', 804' for anaerobic respiratory processes and the presence of
senescent root biomass from the previous crop is thought to be
responsible for the observed rates of methane production at the
beginning of the experiment. A trend toward higher production in the
planted treatment could reflect the influence of root exudates or
sloughed new root tissue. In general the temporal pattern for maximum

rates of production are advanced approximately 15 days over those

57

 

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59
observed in the never planted soils treatment (Figure 14). Although
temporal differences exist, maximum production values for both
treatments don't appear to be significant. This is especially true in
the planted soils. The range in rates of production in the planted

2 2

soils were 25-58 mg CH lm /hr with a mean of 26 mg CH lm /hr. The

4 4
presence of the plant appears to have a much greater effect on
production rates in this treatment as compared to the never planted
soils (experiment 1). These differences might reflect the presence of
more readily available carbon in the fresh agricultural soils used in
experiment 1 which would have been depleted prior to initiation of
experiment 2 on these same soils.

Rates of emission were much more constant with time than was
reported for experiment 1. The maximum rate (15 mg CH4/m2/hr) was,‘
however, very similar to that obtained from the never planted treatment.
The percent of methane produced which was emitted ranged from 11-55%
with a mean of 29.2 (15.1)! in the planted treatment and 0.2-17X with a
mean of 5.3 (6.3)! in the nonplanted treatment. These values are quite
similar to those reported for the never planted soils. The mean hourly
rate of emission from the planted treatment was 9.8 mg CHAImZ/hr as
compared to 6.3 mg CH4/m2/hr for the planted treatment in never planted
soils. Therefore, although little differences existed between daily
maxima for emission (Figure 14,15) in the two different soils, the

longer period of emission in the previously planted soil leads to a

higher total emission from these soils.

The influence 9; previous cropping and straw addition: Rice straw

 

 

was added to soils previously planted and maintained as described in the

60
materials and methods.

Temporal patterns of methane emission and production from this
treatment are illustrated in Figure 16. The temporal nature of the
patterns is quite similar to that observed for previously cropped soils
with no straw addition (Figure 15). The major difference is in the
elevated rates of production and emission in the straw addition
treatment. Differences between the planted and nonplanted treatment are
the largest observed for any of the treatments. However, since only one
measurement was made at any time point, no significance can be placed on
these differences. Rates of production were the highest observed from
the three treatments ranging from 26 mg CHAImZIhr to 68 mg CH4/m2/hr
with a mean of 46 mg CH4/m2/hr. Rates of emission were similiarly the

highest observed ranging from 8.4 mg CH4/m2/hr to 24.4 mg CH4/m2/hr with

a mean of 16.8 mg CHA/mzlhr. The percentage of the methane produced
which was emitted ranged from 20-542 with a mean of 35.7 1 10.8.

The overall affect of the addition of previous crop root and straw
was an increase in methane production and subsequent emission in both
the planted and nonplanted treatments. The percentage of the methane
produced which is emitted in both the planted and nonplanted treatment
emission was, however, not significantly different. Holzapfel-Pschorn
1986 reported single point percent emitted values which fall within the
range of these measurements.

Huang (unpublished data) obtained single time point measurements of
methane production, emission, and pore water methane concentrations in
two rice growing regions in Taiwan. These values (Table 9) all fall

well within the range of our experimental treatments and are closer to

those for the previously cropped soils in the experimental treatments.

61

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63
Interestingly the highest rates of production and emission were in
poorly drained soils, which suggests a relationship between drainage
rate and rates of methanogenesis. To date no examples of the effect of
drainage on rates of methanogenesis exist in the literature. The
emission values from the well drained soil are slightly lower than those
reported by Cicerone and Shetter 1981 and higher than those reported by
Seiler et a1. 1984. They are, however, much closer to the range of
values reported by both Cicerone and Seiler than the values obtained
from the poorly drained soils or the experimental treatments.

Koyama 1963 pointed out the relationship of temperature and methane
production. Seiler et al. 1984 emphasized the importance of considering
the effects of growing seasonal mean temperature when comparing rates
from various sites. It is, however, difficult at this time to separate
out the effects of soil temperature, management techniques and the
actual rates obtained. These factors must be considered in future
studies if meaningful estimates of the effects of these various

parameters on methane emissions from paddies around the world.

Effect of nitrogen fertilization: Various combinations of nitrogen

 

fertilizer were added at a rate of 50 Kg N/ha to previously planted
soils prior to flooding. Soils were flooded and rice transplanted
within one week of flooding. The effects of various combinations is
illustrated in Table 10. No significant difference was observed in the
production or emission of methane from fertilized treatments over the
control values. Although there were no significant differences between
treatments the trend was toward higher rates of production and emission,

from the N8401, and control treatments, over those reported for the

 

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65
nitrogen additions which also included sulfate. As discussed
previously, the addition of 504' would be expected to inhibit methane
production since sulfate reducing bacteria would be expected to be more
competitive than methanogens for common substrates i.e. H2 and acetate.
The lower concentrations of VFA's observed in the sulfate containing
treatments supports this concept since the sulfate reducers have a lower
Km for VFA’s than methanogens.

Cicerone and Shetter (1981) reported significantly higher values of
methane emission from plots fertilized with 120 Kg N (as N34804) in
California rice paddies. Seiler et a1. (1984) found no increased
methane emission from rice paddies in Spain which were fertilized prior
to planting with 160 Kg N/ha (as urea) prior to flooding of the field,
and an additional application of 40 Kg N/ha (as NHANO3) which was
top-dressed after the tillering stage of rice growth. Seiler et a1.
1981 concluded that the relationship of mineral nitrogen to these
processes cannot be conclusively determined from these two studies. It
would be expected that a complex relationship exists between organic
matter additions, temperature, and mineral nitrogen which will affect
rates of methane production and emission, from various types of paddy

soils.

SUMMARY AND CONCLUSIONS

This study has demonstrated the influence of rice plants on the
production of methane in and emission from experimental paddy soils. It

was also shown how plants effect the concentrations of the major

 

66
inorganic species (N03-, NH4+, 804:), and volatile fatty acids (VFA's)
in the same soils.

The dynamics of the concentrations of both the inorganic species and
VFA's was followed by non-destructive sampling of pore water using
micro-suction lysimeters, followed by liquid chromatographic analyses.
This approach allowed for frequent sampling from the same treatment
which was considered as an advantage in establishing the temporal
dynamics of the measured parameters.

Initially nitrate concentrations in pore water were high after the
flooding of the dry soil. Nitrate concentration in planted treatments
was higher, remained longer and even increased in concentration after
planting as compared to that in the non-planted treatment.
Denitrification potential was slightly higher in planted soil, which
follows the trend towards higher N03- concentrations.

Major changes were recorded at depths where the highest root biomass
was observed. This suggested the influence of the roots on
nitrification and denitrifying activity, through increased oxygen and
root exudates in the root zone.

Concentrations of sulfate (804') were highest at the beginning of
the treatment period and gradually declined during the remainder of the
period. The depletion in sulfate in the soil profile was from the lower
portion of the profiles upward over time of the experiment. The rate of
sulfate depletion was slower than that of nitrate, which would
correspond to the sequential utilization of inorganic electron acceptors
i.e. 02, N03', 504‘.

The concentration VFA's increased gradually after dry soil flooding

and reached a maximum concentration at 30 days after rice transplanting

 

67
(or 44 days after dry soil flooding), and then declined rapidly, but
maintained the same qualitative distribution, during the treatment
period. The vertical distribution of VFA's was inversely related to the
profile of N03' and 804‘. This also suggests the sequential use of
inorganic electron acceptors, and the accumulation of VFA's following
their depletion. The profiles of methane concentrations supported this
concept; since the concentration of methane increased, following a
decrease in VFA concentration.

Methane concentration increased markedly during the first 20 days
after flooding especially in the deeper zones (16 - 24 cm). Methane
concentrations were generally higher in non-planted soils at all depths
and all times. Differences in concentration might be related to the
higher rates of methane oxidation and translocation throughout the
rooting zone, as compared to the non-planted soils.

Highest methane production rates were observed at 40 days after
flooding, followed by decreased production over the remainder of the
treatment period. The lowest rates of production were observed in
samples obtained at 20 days and in the 0-4 cm depth zones. Nitrate and
sulfate were observed at varying depths at 20-40 days and in the 0-8 cm
zones during the first 60 days. Those two ions would preferentially be
used as electron acceptors in these zones. A mutual exclusion of
methane and sulfate was observed. When the sulfate concentrations were
above 250 um, methane concentration was maintained below 10 um. Sulfate
reducers are known to have higher affinities for acetate and hydrogen
and thus can outcompete methanogens for these substrates in the presence
of $043. A lower respiration index was also observed at 60 days after

flooding which would support this relationship.

 

68

Emission of methane was initially observed 20 days after
transplanting. Rates of methane emission from planted soils ranged from
0.73 (0.13) to 15.69 (4.4) mg CHA/mzlhr. with the highest emission rates
observed during the heading and flowering stage, and the lowest during
the tillering and maturing stage of growth. Emission of methane from
non-planted soil was significantly (52) different from that in the
planted soil during the periods when maximum emission was occurring.
Rates ranged from 0.11 (0.04) to 1.45 (0.6) mg CHA/mzlhr.

The percent of the methane production emitted from these soils
ranged from 1.3-69X with a mean of 27.0%. The highest percentages
reported from the period of maximum emission (SO-60 days after
transplanting). Most of the emitted methane was observed through the
rice plant. These percentages are, however, based on net vs. gross
production values, since methane oxidation rates were not examined.
Therefore, these percentages may be an overestimate of the actual
methane production which is emitted through the plants.

The effect of the addition of previous crop root and straw was an
increase in methane production and subsequent emission in both planted
and non-planted treatments. No significant difference was observed in
the production or emission of methane from fertilized treatments over
the control values. However, ammonium sulfate which has 804' residue
significantly inhibited the production of methane in the experiment.

The contribution of wetlands to global atmosphere methane
concentrations is well documented. These studies have shown that rice
plants not only serve as a vehicle for transport of methane from the

site of production in the soils to the atmosphere, but stimulate methane

production in the soils, presumably due to root exudates. This

 

69
stimulation is, however, moderated by the expected increase in methane
oxidation in planted treatments due to increased oxygen in these soils
supplied through the plant.

Future studies should evaluate net vs. gross methane production
rates. They should also be considering manipulations which would
enhance oxidation rates i.e. increasing the percolation rate of water
through the soils, thereby increasing the potential for increased

methane oxidation.

10.

11.

12.

70

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