Opening the black box of soil microbial communities

1

Jim HeProfessor of Molecular Soil Ecology

School of Agriculture and Food, FVASEmail: jizheng.he@unimelb.edu.au

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

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

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

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.