Protists

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ol drilo

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Bio

char

drilo

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oil

1e+4

1e+5

1e+6

Basal respiration

Contr

ol bulk

Contr

ol drilo

Bio

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bulk

Bio

char

drilo

µg C

O2 g

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0.0

0.2

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b

ab

Metagenomics of bacteria, fungi and protists

affected by biochar and earthworms in soil Anne Winding1, S.S. Santos1, P.D. Browne1, L.H. Hansen1, A. Johansen1, P.H. Krogh 2 1Department of Environmental Science - Aarhus University, Roskilde, Denmark

2Department of Bioscience - Aarhus University, Silkeborg, Denmark

Acknowledgments: This project was funded by AU-Danish Center for Ecology and Environment through the projects Bioash and eDNA center.

aw@envs.au.dk

Research Aims

Background

DEPARTMENT OF

ENVIRONMENTAL

SCIENCE AARHUS UNIVERSITY

Thermal gasification converts

biomass into a combustible gas in an

oxygen-poor environment, the bi-

product being biochar which can be

used as soil amendment to increase

pH, sequester carbon and supply

phosphate and potassium to crops.

However, the biochar may also affect

the soil organisms due to effects on e.g.

soil structure, water infiltration, potential

adsorption of nutrients and minerals.

To risk assess the potential effects of biochar amendment to

agricultural soils on soil ecosystem services especially biodiversity.

Results

Effect of biochar and earthworms on bacterial communities

Results

Earthworms

Conclusions

• Environmental variables explained 95.2% of total variation

16S rDNA diversity by Illumina MiSeq.

• Bioash amendment separated bulk and drilosphere soil

from unamended soils with pH and CFUoligo as driving

factors.

• Differences between drilosphere and bulk soil was limited.

• NGS analysis af Illumina MiSeq sequencing was more sensitive

compared to activity based assays.

Biochar:

• Tendency of higher abundance of earthworms

• No significant effect of biochar on functional diversity, CFU,

protists, however effects on bacterial genetic diversity

Earthworms:

Higher microbial activity in drilosphere, increase in oligotrophic

CFU in control drilosphere

Protists and fungi genetic diversity affected by earthworms

Generally, the addition of biochar according to the plant P demand

had limited effect on soil microorganisms and fauna in the tested

agricultural soil.

Effects of biochar and earthworms on protists and fungi

Control Drilo Control Bulk Bioash Bulk

Bioash Drilo

RDA 1 (26.8%)

RD

A 2

(1

0%

)

-0.8 0.8

-0.6

1

.0

pH

Drymatter

Respiration

MPNprotist

CFUeutro

CFUoligo

sulfatase

amylase

cellulase

xylolase glucosidase

phosphatase

glucosaminidase

proteinase

1

2 3

4

1

2

3

4

1

2

3 4

1 2

3

4

• Only one earthworm species increased in abundance in the

biochar amended soil.

• Earthworms had a priming effect on protist abundance and

basal soil repiration.

• Culturable oligotrophic bacteria responded positively to

earthworms while culturable eutrophic bacteria and

extracellular enzymatic activities were not significantly affected.

Basal respiration

Control Biochar

Abundance, Indv. m-2

A.

tuberculata 48.4a [29.1–67.6] 27 [2.9–51.1]

A. chlorotica 90 [45.0–135] 126 [76.3–176]

A. longa 9 [2.0–16.0] 10.1 [1.7–18.6]

Lumbricus

sp. 6.8 [-4.4–17.9] 3.4 [-2.2–9.0]

A. rosea 5.6 [0.0–11.2] 7.9 [0.4–15.3]

Total 161 [128–194] 176 [109–242]

MPN of soil protists

amylase phosphatase

Control Drilo

Control Bulk

Bioash Bulk

Bioash Drilo

-1.0 1.0

-0.8

0

.8

pH

Dry matter Respiration

MPNprotist

CFUeutro

CFUoligo

sulfatase

cellulase

xylolase glucosidase

chitinase proteinase

4

4

1

4 1

1 1

2

2

2 2

3

3

3

3

4

RDA 1 (14.7%)

RD

A 2

(1

2.1

%)

0% 20% 40% 60% 80% 100%

Alveolata

Apicomplexa

Centroheliozoa

Cercozoa

Chlorophyta

Ciliophora

Conosa

Eukaryota

Fungi

Lobosa

Ochrophyta

Opisthokonta

Stramenopiles

Unassigned

Others (< 1%)

CB2

CD2

CD1

100

100

96

81

50

55

47

25

26

56

30

81

94

51

96

0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1

BD2

CD4

BB3

BD1

BB4

BD4

CB3

BB1

CB4

BB2

CB1

CD3

BD3

Bray-Curtis Dissimilarity Index

Protists and fungi – Phyla level

Methods The effects on soil microorganisms and fauna (protists and earthworms) in

an agricultural clayey to sandy soil with SOM content of ca. 3% were

assessed with activity based assays and NGS. Crops were alternating oil

seed rape and winter wheat and biochar was added annually for 3 years.

Earthworms, bulk soil and soil from drilosphere were sampled from field

plots either left untreated, or amended with biochar.

Earthworms were determined to species by morphology. Culturable

eutrophic and oligotrophic bacteria were enumerated on TSA and water

agar, respectively, while culturable protist were enumerated by MPN.

Extracellular enzymatic activity was assayed using 7 different MUF-labelled

substrates (Hendriksen et al. 2016). Respiration (CO2 accumulation) was

measured by GC.

Metagenomics of protists, fungi and bacteria were assayed by 18S rDNA

and 16S rDNA sequencing by Illumina MiSeq.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CB

1

CB

2

CB

3

CB

4

CD

1

CD

2

CD

3

CD

4

BD

1

BD

2

BD

3

BD

4

BB

1

BB

2

BB

3

BB

4

Re

lati

ve

Ab

un

da

nc

e Others (<1%)

Verrucomicrobia

TM7

Proteobacteria

Firmicutes

Cyanobacteria

Crenarchaeota

Chloroflexi

Bacteroidetes

Actinobacteria

Acidobacteria

• Environmental variables explained 94.2% of total variation

of18S rDNA diversity by Illumina MiSeq.

• Drilosphere soil was separated from bulk soil irrespectively

of bioash amendment. This separation was driven by basal

respiration and MPN of protists.

Soil with signs of biochar and

drilosphere entrance