In the name of Allah (God), Most Gracious, Most Merciful

TEMPORAL AND SPATIAL MICROBIAL COMMUNITY

AND CARBON FLOW DYNAMICS IN PADDY SOIL:
GHGs MITIGATION SIGNIFICANCE

By

Qaiser Hussain
IREEA
 CHARACTERISTICS OF PADDY SOILS

• Any soil that is used for growing aquatic rice can be called a paddy soil.

• Submerged period:

- Decrease in oxygen partial pressure

- Lowering of redox potential (Eh)
- Reduction of chemical species [O2→H2O, NO3– →N2, Mn(IV) → Mn(II),
Fe(III)→Fe(II), SO4 2–→S2–, CO2→CH4]
- Elevation of soil pH
- Changes in biotic composition (aerobic to anaerobic)

* Enhanced nitrogen availability and its fixation, solubilization of soil

phosphates

• Drained period:

- Eh rises
- Chemical species are oxidized,
- Soil pH decreases,
- Aerobic soil biota recover.
 INTRINSIC MERITS OF THE PADDY SOIL/RICE SYSTEM

(2) A higher natural supply of nitrogen, bases, and silica.

(4) Higher availability of soil phosphorus.

(6) Relative indifference to soil physical properties.

(8) Detoxification of excessive nutrients.

(10) Detoxification of many agrochemicals.

(12) Perfect resistance to soil erosion.

(14) Relative ease of weeding.

(16) Tolerance for monoculture.

(18) Carbon sequestration

( Kazutake Kyuma, paddy soil science,2004 )
NTRINSIC DEMERITS OF THE PADDY SOIL/RICE SYSTEM

AIR POLLUTION

• Paddy fields emit three GHGs i.e. CH4, CO2 and N2O

• The CH4, CO2 and N2O emission accounted for 78.2, 16.0 and 5.8% of total CO2-
equivalent emission, respectively. ( Tsuruta et al., 1998 )

• N2O through denitrification ( minor in paddy soils)

- Negligible emission ( De Datta and Buresh,1986; Bronson and Singh, 1995).
- Paddy soils may act as sink for N2O under strongly reduced conditions ( Minami and
Fukushi, 1987 )

• Global warming potential (GWP) ?

“ Compare the ability of a gas to trap heat in the atmosphere relative to CO2 “
Based on a 100-year time frame:

GWP of CH44 : 21 times

GWP of N2O : 296 times higher than the reference value for CO2 (IPCC, 2007)
 What is C sequestration?

• Agriculture provides a potential sink for CO2, through building up soil

organic matter stocks, which incorporate CO2 taken from the
atmosphere by plants. This is called "carbon sequestration".

• It refers to the provision of long-term storage of carbon in the

terrestrial biosphere, underground, or the oceans so that the buildup
of carbon dioxide (the principal greenhouse gas) concentration in
the atmosphere will reduce or slow.
( Carbon transfer model showing organic and inorganic carbon
sequestration in soil )
Strategies for carbon sequestration in soil

Lal, R. et al., 1995.

 ORIGIN OF ORGANIC-C TO PADDY FIELDS

Cultivation period of rice plant

 Hydrophytes such as floating plants and algae

 Organic fertilizers (compost and green manure)
 Weeds
 Rice litter of dead old leaves
 Rhizodeposition from rice roots

Off-crop season

 Rice straw
 Stubble and dead roots of rice plant in autumn.
 Weeds grown in winter and early spring
 Rhizodeposition from weeds after spring plowing

* Stubble and weeds plow- in in spring (20% and 30% of the total organic input)
* Green manure and compost <20%
 C sequestration in paddy soils
 Paddy soils, developed under rice-based agriculture with irrigation, play an
important role in soil C storage.

 Area of paddy soils 3.4% of China’s land & 26% of total cropland

 The estimated total SOC pool in China’s paddy topsoils is 1.3 Pg

 0.85 Pg (plow layer) + 0.45 Pg (plow pan) = 1.3 Pg
 2% of China’s total storage in topsoil, 4% of total topsoil (plow layer+ plow pan)

 The current C sequestration rate is in the range of 0.13–2.2 t C ha-1 yr-1,

 contributing to an annual total SOC sequestration of 12 Tg.
 one-third of the estimated yearly sequestration rate of the total croplands.

Easily reachable SOC sequestration potential of 0.7 Pg, expected to
sequester as much as 3.1 Pg C from the atmosphere in the long run.

 The total C sequestration potential in paddy soils, however, can reach 40% of
the total cropland SOC sequestration potential of China.

( Pan et al., 2003. Global Change Biology)

 CH4 Emission Sources/Sinks

• * Methane is primarily produced through anaerobic decomposition of organic matter in

biological systems.

* More than half of the current CH4 flux to the atmosphere is anthropogenic.

* CH4 increased by about 150% since pre-industrial times, rate of increase is declining.

Sources:
Agricultural processes:
( Wetland rice cultivation, enteric fermentation in animals, decomposition of animal wastes )
Other sectors
( production and distribution of natural gas and petroleum, by-product of coal mining and
incomplete fossil fuel combustion )

Sinks:
Photochemical oxidation of CH4 in the troposphere, the major sink.
Reaction with Cl in the marine boundary layer
Soil sink

(IPCC 2001)
Net Global Atmospheric CH4 Emission, Global Consumption and Gross Global Production
(Tg CH4 per year)

E+C=P

Atmospheric Source/Sink term Global Budget Global Consumption Global Production

Animals  80 0 80

Wetlands  115 27 142

 bogs/tundra (boreal)  35 15 50

 swamps/alluvial  80 12 92

Rice Production  100 477 577

Biomass burning  55 0 55

Termites  20 24 44

Landfills  40 22 62

Oceans, freshwaters  10 75.3 85.3

Hydrates  5 5 10

Coal production  40 18 58

Gas production  40 18 58

 venting, flaring  10 0 10

 distribution leaks 30 18 48

( Reeburgh et al.,
1993 )
 Mechanism of CH4 Production in paddy soils

 CH4 is produced in rice fields after the sequential reduction of O2, nitrate, manganese,
iron and sulphate, which serve as electron acceptors for oxidation of organic matter to
CO2.

 Methane is produced in the anaerobic layers of paddy soil by bacterial decomposition of

organic matter.

Methanogen Archaea
 Two dominant methanogenesis processes:

1- Acetoclastic methanogenesis ( acetoclastic methanogens: Methanosaetaceae,

Methanosarcinaceae )
Convert acetate to CH4+ CO2

2- Hydrogenotrophic methanogenesis ( Hydrogenotrophic methanogens: RC-1,

Methanomicrobiales, Methanobacteriales, Methanosarcinaceae )

Convert H2+ CO2 to CH4

( Conrad R et al., 2006 )

 Methane Oxidation

 Chemical and biological processes consume methane

 Oxidation in aerobic soils by methanotrophic bacteria ( only biological sink )
 10-20% to total CH4 destruction

Methanotrophs:

 Oxidize CH4 with help of methane monooxygenase (MMO) enzyme.

 03 types:
Type-I ( Methylomonas, Methylocaldum, Methylosphaera, Methylomicrobium
and Methylobacter )

Type-II ( Methylosystis and Methylosinus )

Type-X ( Methylococcus )
 well adopted to high or low temperature, pH and salinity

( S. K. DUBEY., 2005 )
file:///H:/Morphology of the Archaea.files/esterether.jpg
Chains and Linkage
Archaea

Bacteria and Eukaryote

( S. K. DUBEY., 2005 )
 Pathways of methane emission

 Plant transport ( 90% of the CH4 )

 Ebullition ( < 10% )

 Early period of the season

 High organic input
 Only at surface layer
 Rate regulating factors (CH4 conc., temperature, soil porosity, plant aerenchyma )

 Diffusion ( < 1% )

 Very slow process ( diffusion rate of gaseous CH4 is very low in Liquid phase )
 Hardly contribution to total CH4 flux
 Factors ( surface-water concentration of CH4, wind speed and CH4 supply to
the surface water )

( S. K. DUBEY., 2005 )
Production, oxidation and emission of methane in paddy soils

< 90%

< 1% < 10%

( Yagi, 1997 )
he Factors Controlling CH4 emissions from paddy fields on different scal

( S. K. DUBEY., 2005 )
MITIGATING GHGs EMISSION FROM RICE–
WHEAT

WASSMANN. R. et
al,2003
 Background research
 Methane emission rate from paddy field increases as the rice growth stage advances and
application of rice straw promotes additional methane emission in the early period of rice
cultivation, while the mid-season drainage decreases the methane emission drastically
(Schu ¨tz et al., 1989; Yagi and Minami, 1990; Kimura et al., 1991; Kaku et al., 2000).

 Wang et al., (1997, gric.Ecosys.Environ.) found high correlations between CH4 emission
rates and dry root weight , and between dry root weight and total carbon released from
roots ( root exudates ).

 Neue., (1997, Soil use and Management) mentioned that large variability of characteristics
of rice plants which affect CH4 emission provides an opportunity to breed cultivars with
high yield but low CH4 emission potentials.

 Jia et al., ( 2007, Bio Fertil Soils ) found that community structure of MOB in rice fields
remained largely unchanged and was independent of a wide variation of bulk soil water
content over a year round investigation period.

 Bodelier et al., (2000 , nature ) have reported that type-II methane oxidizers dominated
methane metabolism in unplanted soil and that type-I species were distinctly greater in the
rhizosphere soil than in non-rhizosphere soil, indicating that the presence of rice plant is
an essential factor for type-I methanotrophs to proliferate.

 Amaral and Knowles (1995, FEMS Microbiol. Lett.) concluded that type I methanotrophs
may be favored at low CH4 concentrations and high O2 concentrations, whereas type II
methanotrophs may be favored at high CH4 concentrations and lowO2 concentrations.
 Background research
 Bai et al., (2000, Microb Ecol ) suggested that paddy soil at the August sampling period
contained more abundant facultative anaerobic bacteria (ca. 36%) and archaea (ca.
37%), but the total microbial biomass was significantly lower than in the remaining
sampling periods. As the plant approached maturity, the microbial community
structure in the soil changed to contain more abundant Gram-negative bacteria and
methanotrophs.

 Watanabe et al., ( 2006, soil biology and biochemistry ) suggested that the difference in the
soil type (sampling region) largely influenced the community structures of
methanogenic archaea in paddy field soil, while the effects of sampling period and
different fertilizer treatments on them were small. Most of the sequences obtained from
theDGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae,
Methanosaetaceae and Rice cluster-I.

 Community structure of methanogenic archaea in paddy field soils did not change
throughout a year , which may be

 methanogenic archaea can tolerate higher oxidation–reduction potentials or oxygen (Fetzer and
Conrad, 1993; Kato et al., 1993),
 they are protected inside cysts of protozoa such as ciliates (Finlayand Fenchel, 1991)

 they have some enzymes to detoxify oxygen-related toxic compounds (Lead better and Breznak,1996;
Shima et al., 1999; 2001; Seedorf et al., 2004).

 Kru ger et al., (2005, FEMS Microbiology Ecology) showed that acetoclastic methanogens
on the rice roots were relatively less common than in soil. It has not yet been
understood well, what determined the community structures of methanogenic archaea
in paddy field.
 Framework Questions
 How sensitive is the CH4 and CO2 budget to climate
change?

 How sensitive are terms in the CH4 and CO2 budget to

changes in moisture, temperature and CO2/CH4?

 Which terms in the global CH4 and CO2 budget are

increasing?

 What steps can be taken to reduce anthropogenic

emissions?

 What characteristics of methane-producing and consuming

bacteria promote activity in the rhizosphere paddy soil ?
 Framework Questions
 How does microbial diversity affect CH4 and CO2 emissions?

 How does the diversity of microbes producing these gases vary in

paddy soil / rice system and in response to human disturbances?

 Will warming and elevated atmospheric CO2 concentrations lead to

greater methane production and lower methane consumption?

 Can microbes be managed to enhance organic matter sequestration in

soils and sediments to remove CO2 from the atmosphere and reduce
greenhouse warming?

 Can they be managed to reduce dependencies on synthetic fertilizers?

 Is it important or not to lose a population without affecting microbial

activity and how can we enhance the populations responsible for a
given activity of interest?
 Theme of research
Study Case 1: The analysis of the structure and the phylogenetic diversity
of the metabolically active methanotrophic and methanogens
archaea communities in paddy soil/ rice system.

Study Case 2: The spatial and temporal dynamics of the methanotrophic and
methanogens archaea communities.

Study Case 3: Determination of CH4 oxidation and production rates with elevated
CO2 concentration and temperature by incubation experiments.

Study Case 4: Influence of perturbations ( fertilizers, heavy metals, tillage, etc )

methane-producing and consuming bacteria promote activity
in paddy soil/ rice system.

Study Case 5: Study of carbon flow dynamics mediated by different microbial

communities in paddy soil/rice system.

Study Case 6: The cultivation and isolation of Acetoclastic methanogens in order

to enable further research on adaptation mechanisms and the
physiology of these methanogens in rhizosphere paddy soil.
 Objectives and significance
 To investigate how factors that determine the stability of
methanotrophs and methanogens archaea communities
influence the strength of the biological sink and source for
methane in paddy soil/rice system.

 To evaluate the potential for reducing the emission of

greenhouse gases and for increasing the sequestration of C
in paddy soil/rice system.

 To identify key methanotrophs and methanogens archaea

species at the community level and to elucidate their
particular functional role in the utilization of rhizodeposition.
Theme of microbial diversity studyQuestions to be addressed in analysi
analys
Linking structure with function
Methods and Approaches:

1- Biogeochemical study
2- Molecular biology study
Methods to elucidate plant-microbial interactions in rhizosphere
 Biogeochemical study
1- Soil Samplings
• Rhizosphere and bulk soil sampling in paddy field ( at two stages of
plant)
2- Physical and chemical soil parameters analysis
• Habitat characteristics influencing the methanotrophic and
methanogens
communities.
• Comprise grain size and pore volume distribution, organic carbon,
total carbon, and nitrogen content.
• Redox potentials, water level, soil temperature, methane production
and methane oxidation under in situ conditions.
3- Methane oxidation and production rates in dependence of the
temperature and elevated CO2 conc.
• Incubation study
4- Pulse-labeling 13C-PLFA approach
• This method uses stable carbon isotopes to label specific lipid
biomarkers, such as phospholipid fatty acids (PLFA).
 Molecular Biology study
Stability of methanogens
and methanotrophs communities
in paddy soil

Culture independent Culture dependent

Denaturing gradient gel electrophoresis Enrichment cultures

• Spatial and temporal comparisons of soil • Incubated under different
communities within and between locations conditions
or among treatments.
• Keystone acetoclastic
• Primers ( Functional markers ) methanogens archaea
- pMMO ( particulate methane monooxygenase ) Characterization (species-specific)
- A189-A650 • Physiology
- For methanotrophs detections • Function,
- mcr A ( methyl coenzyme-M reductase ) • adaptation
- M13F, M13R
- For methanogens archaea detections
Stable isotope probing-DGGE
Isotopic labeling principle
 Risk Assessment
Limitation and challenges
1- Soil sampling
 Sampling scale and spatial heterogeneity of bulk soils.
• Multiple soil samples
• Mixed thoroughly, and remove debris by sieving ( 2 mm sieve) and by manual
inspection.
 Sampling of rhizospheric soils
• Recovery of adherent soil from the roots by agitation (shaking) of root systems.
2- Transportation and storage conditions
 Cell lysis of microbes ( transported at 4 0C in sealed container or stored at -20 / – 80 0C )

3- DGGE
 Making the gradient gel is greatest challenge in DGGE.
• Broad gradient range ( 20-80% denaturant )
 Optimum resolution
• High voltage for shorter electrophoresis time
 Communication plan
 Who
 (personal, press, stakeholder…)
o Needs to know what, when, how often…

 How:
 Meetings and status report
o what has been done,
o what has not been done and why,
o what needs to be done…
 Research-Dissertation Schedule
 January- April 2008: Finish research proposal, outline procedure
 May- August 2008: Gathers necessary supplies and equipments, identification of
procedures I use, Rhizospheric/bulk soil samplings
 September-December 2008: possibly begin separating of DNA from soil
samples, physical and chemical analysis of soil.

 January- April 2009: Incubation experiments, PCR-DGGE, 13 C- PLFA analysis

 May- August 2009: Incubation experiments, PCR-DGGE, 13 C- PLFA analysis
 September-December 2009: Isolation of acetoclastic methanogens

 January- April 2010: Isolation of acetoclastic methanogens, writing of research

publication.
 May- August 2010: Thesis writing, submit first draft
 September-December 2010: Make revisions to thesis, give final presentation for
thesis defense.