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Protists
Conro
l bulk
Contr
ol drilo
Bio
char
bulk
Bio
char
drilo
MP
N g
-1 d
w s
oil
1e+4
1e+5
1e+6
Basal respiration
Contr
ol bulk
Contr
ol drilo
Bio
char
bulk
Bio
char
drilo
µg C
O2 g
-1 d
w s
oil
0.0
0.2
0.4
0.6
0.8
1.0
a
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.
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