Opening the black box of soil microbial communities · 2019-07-01 · Soil microbial community has...
Transcript of Opening the black box of soil microbial communities · 2019-07-01 · Soil microbial community has...
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Opening the black box of soil microbial communities
1
Jim HeProfessor of Molecular Soil Ecology
School of Agriculture and Food, FVASEmail: [email protected]
18-06-2019
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Soil is…..
• A very thin skin over the land;
• A mix of minerals, organic matter, organisms, water and air;
• A precious resource for producing crops (provides air, water, nutrients and physical support to plants).
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Soil is alive!
➢ Soil contains one of the most diverse groups of living organisms on Earth (a quarter of total biodiversity).
➢ A single gram of soil may contain billions (notmillions) of bacteria and thousands of bacterial species.
➢ Bacterial biomass can be 1-2 tons per hectare in the surface soil.
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Compositions of soil organisms
Bardgett & Putten, 2014
(a) Ectomycorrhizal fungi
(b) Decomposer fungi(c) Bacteria(d) Nematode(e) Tardigrade(f) Collembola(g) Mite(h) Enchytraeid worm(i) Millipede(j) Centipede(k) Earthworm(l) Ants(m)Woodlice(n) Flatworm(o) Mole
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http://eusoils.jrc.ec.europa.eu/library/themes/biodiversity/
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Biomass Microbes Plants M/P
Total C 350 – 545 562 ≈ 1:1
Total N 85 – 130 10 > 10:1
Total P 9 – 14 1.05 > 10:1
Data source: Whitman et al., PNAS, 1998
➢ The total amount of prokaryotic carbon is 60–100% of the
estimated total carbon in plants;
➢ The earth’s prokaryotic N and P are about 10-fold more of
these nutrients than do plants, and represent the largest
pool of these nutrients in living organisms.
Comparison of microbial and plant
biomass and nutrient elements (Unit: Pg)
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➢ Soil microbial community has long been treated as a black
box.
➢ Only ~ 1% soil microbes are culturable.
➢ Advances in molecular biology have made it possible to
develop culture-independent techniques to investigate the
structure, diversity and function of soil microbial communities.
Soils—The Final Frontier
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Methods for microbial
community analysis
• Culture-dependent
methods
• Microbial biomass
• BIOLOG plate
• PLFA (Phospholipid
Fatty Acid)
• Molecular methods
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Methods: 16S rRNA (amoA) gene analysis of
microbial communities
Lysis, DNA/RNA
extraction
DNA/RNA
purification
PCR amplification
DGGE, TGGE, RFLP, t-RFLP, SSCP
Cloning Sequencing
Identification
Phylogenetic analysis
Fingerprinting:
Quantitation qPCR
SIP, Metagenomics, Transcriptomics, Proteomics… (Multi-omics)
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➢Who are there?
➢How many are there?
➢Why they are there?
➢What are they doing?
➢How will they change?
➢……
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FastPrep®-
Instrument for
the lysis of
microbial cell to
isolate DNA &
RNA from soil
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YNF
Y2R
Y1R
Experimental site and treatments in Yarraman, QLD
He et al., 2004
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He et al., SBB, 2005
DNA Extraction. Pre-lysis washing procedure:
Washing the samples with the buffer solution to
remove those easily co-extracting soil components
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The exponential amplification of the gene in PCR
http://users.ugent.be/~avierstr/principles/pcr.html
PCR: Polymerase Chain Reaction
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Day 0471421354963
M M
PCP degradation RNA-DGGE analysis
35
Control PCPPseudomonas mandelii (94%)
Pseudomonas sp. IpA-93 (94%)
Pseudomonas sp. LAB-23 (94%)
Uncultured gammaproteobacterium clone
MB10gamma-k4 (97%)
Uncultured bacterium DGGE gel band
FW031-1(97%)
Uncultured gammaproteobacterium clone
LTUG07456 (97%)
Unidentified gammaproteobacterium (99%)
Uncultured bacterium clone ZZ15C16 (94%)
Uncultured bacterium clone LO13.5 (94%)
Uncultured Burkholderia sp. clone Ba04 (99%)
Uncultured eubacterium WD220 (98%)
Uncultured bacterium clone SG1-50 (88%)
What are they doing?
Pentachlorophenol (PCP) is an organochlorine compound used as a pesticide and a disinfectant
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PCP degradation
RNA-SIP-DGGE analysis
Day 0471421354963
12C13C1 2 3 4 5 6 7 8 9 10
M M
(courtesy Prof. Jim Prosser)
SIP--Stable
Isotope probing
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Case study example 1➢ Soil microbial distribution pattern (biogeography) and temporal succession
Microbes on map?
❖What is the distribution pattern of soil microbes? What factors are driving (governing) the pattern (ecological theories)?
❖How do they respond to environmental factors (modelling and prediction)?
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Community composition--class level
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Wang et al., 2015
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The different symbol represent different species of Eucalyptus based on the database of AVH
Eucalyptus camaldulensis Eucalyptus tereticornis Eucalyptus pruinosa Eucalyptus macrorhyncha
Sampling route
Sampling
12-31 May 2019
by Hangwei Hu,
Qinglin Chen,
Zhenzhen Yan,
Chaoyu Li
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➢ Soil microbial (resources) distribution patterns and driving factors
➢ Sync/co-evolution of soil and plant microbiomes (antibiotic resistance genes),
indigenous plants (crops) rhizosphere and bulk soil microbiomes/ survival
mechanisms—breeding, synthetic microbial ecology, soil remediation.
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Impacts
Impact Factor: 9.520
Journal Rank: 3/160 Ecology;
9/126 Microbiology
ISME (International Society of Microbial Ecology)
Session Chair and Invited Keynote Speaker:
Global and Continental Biogeography, ISME 18
Invited Keynote Speaker: The 9th International
Conference on Geochemistry in the Tropics &
Sub-Tropics, 28-31 July 2019, Gold Coast, QLD
Grants and Paper reviewers in the field of microbial
biogeography
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The nitrogen cycle contains four main steps, i.e. biological nitrogen fixation,
ammonification, nitrification and denitrification, all of which are mainly
driven by microorganisms
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Nitrification
NH3
NH3
NO2-
CO2
Groundwater pollution Fertiliser loss
DenitrificationLeaching
Decomposing
organic matter
Animal
excreta
Fertiliser
N
NO2-
CO2
biomass
NO2-
NO3-
CO2
biomass
NO3-
CO2Ammonia
oxidisers
Nitrite
oxidisers
Nitrosomonas
europaea
Nitrobacter winogradskyi
• Central role
in global
nitrogen
cycle
• Loss of
ammonia-
based
fertilisers
• Nitrate
pollution
• N removal in
wastewater
treatment
• Production of
greenhouse
gases, N2O
• ….
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Nitrofication potential0.051 ~ 0.804 mg N kg-1 soil day-1
Soil pH: 4.3 ~ 5.5
Zhang J et al. (2011) Plant Soil 342Booth et al. (2005) Ecol Monogr 75
15NH315NO3
-
Soil nitrification rates
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Ammonia oxidizing archaea (AOA)
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20 25 30
➔Quantifcation of target DNA by
measuring fluorescence in the log-
linear phase (kinetic quantification).
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CK
NNP
NK PK NPK
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CK0 CK N NP NK PK NPK NPK+OM10
4
105
106
107
108
109
am
oA
co
pie
s p
er g
of
soil
Treatment
AOB
AOA
3.52 6.56 2.99 1.93 7.47 1.87 1.42 1.27 Ratio of AOA to AOB
r=0.835**
Abundances of archaeal and bacterial amoA genes
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Pote
nti
al n
itri
fica
tio
n r
ate
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
CK0 CK N NP NK PK NPK NPK+OM
treatment
Potential ammonia oxidation
(nmol N g-1dry soil h-1)
PNR with AOB r = 0.91**
PNR with AOA r = 0.87**
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Alkaline and neutral soils with different fertilization treatments
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4.00
5.00
6.00
7.00
8.00
CK NP NK PK NPK 1/2OMN OM
Log
num
ber
of a
moA
gen
e co
pies
g -1
dry
soi
l
AOB AOA
127.8 7.7 8.5 276.6 18.9 28.5 61.9 AOA :AOB
Alkaline sandy loam (pH 8.1-8.6) in Fengqiu, Henan Province
Correlations of AOB (AOA) abundance and
potential nitrification rates
AOB:r= 0.773, n=7, P <0.05 *
AOA:r= 0.331, n=7, P >0.05
AOB play predominant role in these
alkaline sandy loams, although AOA are
more abundant than AOB.
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Establishing SIP method for AOA and AOB investigation
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NH3
NH3
NO2-
13CO2
Groundwater pollution Fertiliser loss
DenitrificationLeaching
Decomposing
organic matter
Animal
excreta
Fertiliser
N
NO2-
CO2
biomass
NO2-
NO3-
CO2
biomass
NO3-
CO2Ammonia
oxidisers
Nitrite
oxidisers
Nitrosomonas
europaea
Nitrobacter winogradskyi
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AOB
Day 0
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73
CsCl buoyant density (g ml-1
)
% M
ax
imu
m
AOB
Day 14
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73
CsCl buoyant density (g ml-1
)
% M
ax
imu
m
AOB
Day 28
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73
CsCl buoyant density (g ml-1
)
% M
ax
imu
m
AOA
Day 14
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73 1.74
CsCl buoyant density (g ml-1
)
% M
ax
imu
mAOA
Day 0
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73 1.74
CsCl buoyant density (g ml-1)
% M
ax
imu
m
AOA
Day 28
0
10
20
30
40
50
60
70
80
90
100
1.65 1.66 1.67 1.68 1.69 1.70 1.71 1.72 1.73 1.74
CsCl buoyant density (g ml-1
)
% M
ax
imu
m12C incubation13C incubation
AOA AOB
Abundance of amoA gene in CsCl density gradients after 12C- /13C-CO2
incubation
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AOA/AOB amoA DGGE patterns under CsCl densities
Day 14
Day 28
Day 14
Day 28
AOB Buoyant density (g ml - 1 )
13 CO2
12 CO2
Day 14
Day 28
Day 14
Day 28
Buoyant density (g ml - 1 )
13 CO2
12 CO2
AOA Buoyant density (g ml -1 )
Day 14
Day 28
Day 14
Day 28
1
2
1
2
1
2
1
2
1
1
2
1
2 2
2
13 CO2
12 CO2
Buoyant density (g ml -1 )
Day 14
Day 28
Day 14
Day 28
1
2
1
2
1
2
1
2
1
1
2
1
2 2
2
13 CO2
12 CO2
Day 14
Day 28
Day 14
Day 28
11
22
11
22
1
2
1
2
1
1
2
1
2 2
2
13 CO2
12 CO2
1
AOA could grow by fixing 13C-CO2 and
metabolizing NH3, driving nitrification in the soil;for the first time, this study provided direct
evidence of AOA autotrophic ammonia oxidation.
Zhang et al. 2010, PNAS, 107, 17240-17245.
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Soil Sampling
pH (H2O)pH
(KCl)
HZ 4.20 3.29
YH 4.21 3.34
QJ 4.36 3.66
QY 4.43 3.87
TY 4.47 3.89
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Comammox
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Abundances of amoA genes from comammox Nitrospira, AOA, and AOB, and nxrB gene of NOB in 300 forest soil samples with soil pH ranging from 4.0~8.6. Quantitative PCR analysis of comammox amoA clade A and clade B was performed using the primer sets and thermal-cycling conditions as described previously.
Hu & He, 2017
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Singh et al., 2010
➢ >50% applied N lossed into environment;
➢ Nitrification process accelerates soil acidification;
➢ We need to develop measures to manipulate nitrification microbes
ARC DP & LP working on the processes
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He et al. 2007. Quantitative analyses of the abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea of a Chinese upland red soil under long-term fertilization practices. Environmental Microbiology, 9(9): 2364-2374. (SCI times cited: 595)
Shen et al. 2008. Abundance and composition of ammonia-oxidizing bacteria and ammonia-oxidizing archaea communities of an alkaline sandy loam. Environmental Microbiology, 10: 1601-1611 (Times cited: 342)
Chen et al. 2008. Ammonia-oxidizing archaea: important players in paddy rhizosphere soil? Environmental Microbiology, 10: 1601-1611 (239)
Di et al. 2009. Nitrification driven by bacteria and not archaea in nitrogen-rich grassland soils. Nature Geoscience, 2 (9): 621-624. (Times cited: 418)
Zhang et al. 2010. Autotrophic ammonia oxidation by soil thaumarchaea. PNAS, 107(40): 17240-17245. (Times cited: 205)
Zhang et al. 2012. Ammonia-oxidizing archaea play more important role than ammonia-oxidizing bacteria in ammonia oxidation of strongly acidic soils. ISME Journal, 6: 1032–1045. (Times cited: 300)
He et al. 2012. New insights into microbial mechanisms of nitrification in acidic soils. Soil Biology and Biochemistry, 55:146-154. (Times cited: 139)
Hu et al. 2015. Microbial regulation of terrestrial nitrous oxide formation: understanding the biological pathways for prediction of emission rates.FEMS Microbiology Reviews, 39(5):729-49. (IF13.244) (Times cited: 125)
Highly cited papers and invited reviews/nitrification microbes
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➢ A single gram of soil may contain billions of bacteria & archaea. However, only ~ 1% soil microbes are culturable under laboratory conditions.
➢ Molecular biological methods based on microbial DNA/RNA play a key role in understanding the soil microbial diversity and functions.
➢ The basic procedures of molecular soil microbial analyses include microbial DNA extraction, PCR amplification, finger-printing profiling and sequencing etc.
➢ Soil microbial ecology study plays an essential role in understanding soil processes and soil sustainable management.
Take home message
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Acknowledgements
➢Funding agencies: UoM, Australian funding agencies (ARC,
ACIAR, ACJRF, CRCp), Chinese funding agencies (NSFC,
MoST, CAS), and industry partners.
➢My collaborators and students.