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...
Opening the black box of soil microbial communities
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Jim HeProfessor of Molecular Soil Ecology
School of Agriculture and Food, FVASEmail: [email protected]
18-06-2019
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).
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.
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
http://eusoils.jrc.ec.europa.eu/library/themes/biodiversity/
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)
➢ 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
Methods for microbial
community analysis
• Culture-dependent
methods
• Microbial biomass
• BIOLOG plate
• PLFA (Phospholipid
Fatty Acid)
• Molecular methods
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)
➢Who are there?
➢How many are there?
➢Why they are there?
➢What are they doing?
➢How will they change?
➢……
FastPrep®-
Instrument for
the lysis of
microbial cell to
isolate DNA &
RNA from soil
YNF
Y2R
Y1R
Experimental site and treatments in Yarraman, QLD
He et al., 2004
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
The exponential amplification of the gene in PCR
http://users.ugent.be/~avierstr/principles/pcr.html
PCR: Polymerase Chain Reaction
Day 0471421354963
M M
PCP degradation RNA-DGGE analysis
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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
PCP degradation
RNA-SIP-DGGE analysis
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12C13C1 2 3 4 5 6 7 8 9 10
M M
(courtesy Prof. Jim Prosser)
SIP--Stable
Isotope probing
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)?
Community composition--class level
Wang et al., 2015
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
➢ 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.
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
The nitrogen cycle contains four main steps, i.e. biological nitrogen fixation,
ammonification, nitrification and denitrification, all of which are mainly
driven by microorganisms
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
• ….
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
Ammonia oxidizing archaea (AOA)
20 25 30
➔Quantifcation of target DNA by
measuring fluorescence in the log-
linear phase (kinetic quantification).
CK
NNP
NK PK NPK
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
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**
Alkaline and neutral soils with different fertilization treatments
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.
Establishing SIP method for AOA and AOB investigation
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
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
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.
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
Comammox
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
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
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
➢ 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
Acknowledgements
➢Funding agencies: UoM, Australian funding agencies (ARC,
ACIAR, ACJRF, CRCp), Chinese funding agencies (NSFC,
MoST, CAS), and industry partners.
➢My collaborators and students.