Microbial communities in root zones ofPennisetum setaceum (African fountain grass) at aheavy metal contaminated site, Kabwe, Zambia
Chiba, A.1*, Mweetwa, A.2 and Uchida, Y.3
1 Research Faculty of Veterinary Medicine, Hokkaido University, Japan
2 Department of Soil Science, University of Zambia, Zambia
3 Research Faculty of Agriculture, Hokkaido University, Japan

Email: chiba@chem.agr.hokudai.ac.jp

Brief view of this study
1. Microbes in the root zone of African fountaingrass in Kabwe, Zambia
2. A new portable sequencer for microbial DNAanalysis

A waste dump at the center of Kabwe city

Kabwe waste dump

Pennisetum setaceum(African fountain grass)1km

Why can they grow in such a harsh environment?
(1) Morphology
Seeds

Roots

The unique characteristics protect African fountain grassfrom several stresses (e.g. heat and drought).

(2) Microbes in a root zone
Suppressing pathogens

CCMetCC CPbCZnMet

Pb2+Zn2+
MetN

Detoxification of heavy metals

NO3-/NH4+

Solubilizing nutrients

Mendes et al. 2013

Previous technique• Isolation of single microbialspecies• 1% of soil microbes can beisolated

New technique• Multiple microbial species
can be identified at the sametime• Non-culturable microbes areincluded

DNA level studies for microbial community
Iontrrent
PGM

DNA samples
from any environments

Reference database
Esherichia coli
GGTCATTAAATACTA

Obtained sequence data
GGTCATTAAATACTA••High running cost
Clean and well-equippedenvironments

AGCCTTCCCATATAGCGTTTCCTTCCATAAACTTCATATAGTTGAGGCCTTATAGCCCAG

New sequencing device “MinION” (Oxford Nanopore)
DNANanopore
Membrane• USB-like device• Reusable

We have to evaluate the usability of MinION sequencer
for analyzing microbial community in Zambia

The purposes of the study
1. To identify microbes in the root zone ofAfrican fountain grass in Kabwe, Zambia
2. To evaluate the usability of MinION
sequencer for analyzing microbialcommunity

Sampling in June, 2017

• African fountain grass• Different colored soils

Site A500 m

•

Black

Site B

Site C••Light brown

Dark brown

Reference:Kabwe scoping and design study phase 1 complement
report by Water Management Consultants Ltd, UK (2006)

Soil properties
Site A

Site BSite CpH(H2O)

8.73

6.39

7.22

Total C[g kg dry soil]

148

50.8

9.43

Total N[g kg dry soil]

5.30

3.20

0.38

Total Pb
[mg kg dry soil]

3732

11573

19319

Microbial analysis• Step 1: DNA extraction
PowerSoil DNA
isolation Kit (MO BIO)
Taking roots andsoils in the rootzone• Step 2: PCR-free preparation
Rapid Barcoding Kit (Oxford Nanopore)
DNA extraction

Fragmentation

Ligation

DNA

Step 3: Sequencing
Nanopore• MinKNOW software• To collect sequencing dataset in Fast5 format• Signal data• Sequencing reads (e.g. TAACG…)• Quality scores (e.g. 19,20,22,18,20)*It shows reliability of the sequencing read data.

Sequencing
reads

SignalMembraneStep 4: Sequence classification• What’s in My Pot (WIMP) workflow
1) Filtering
To remove sequences with low
quality (QS)
[Averaged QS < 10]
Sample 1
G C C C A T T … Further
15, 13, 16, 7, 10, 13, 10… analyzed
Sample 2
G C C C A T T …
5, 9, 10, 7, 10, 9, 10 … Removed2) Classification

Sequencedataset

NCBLdatabase

Sample 1GGTCATTAAATA…

Escherichia coli
GGTCATTAAATA…

Result and discussion: Classification

No. of reads
No. of classified
reads
No. of identified
species
Taxa(QS>10)

A897

B
220

C618

106

32

86

68

11

Root
Root
Lentzea
Escherichia
Escherichia
Root
Lentzea
Bradyrhizobium Bradyrhizobium
Bradyrhizobium Mesorhizobium
Escerichia• The reads with low quality were removed during the filtering
step for the classified reads (Average quality scores 7.15).
• Long reads with higher quality are more likely to be classified(min 5 bases, max 220,480 bases).

Result and discussion: Phylogenetic tree
Site CResult and discussion: Common bacteria among thedifferent sites
Escherichia spp.

Lentzea spp.

 Ubiquitous soil bacteria

 Capable of degrading high molecularweight ester [3]Bradyrhizobium spp.
Symbiotic bacteria of legume plants
N2-fixation [4]Common in South Africa and WesternAustralia soil [5]Photo from "A Comprehensive Survey of International Soybean Research - Genetics,
Physiology, Agronomy and Nitrogen Relationships"[3] Jarerat et al. 2002, [4] Zahran et al. 1999, [5] Stępkowski et al., 2005

Conclusions1. Despite the low quality scores, the MinION data
showed the key bacteria in the root zones.
2. Bradyrhizobium. spp and Lentzea. spp werecommon bacteria among the three sites.
Future research• The number of classified sequences should be
improved by modifying the protocols.
• Can Bradyrhizobium. spp and Lentzea. spp detoxify
heavy metals?

Acknowledgements
Prof. Ishizuka, Mayumi
Dr Nakayama, Shota
Ms Hirano, Nagisa
Mr Nakata, Hokuto

Mr Muntali, Kabenuka
Mr Mwansa, Mukuka
Ms Saito, Anna
Kampai project

Reference• Mendes, R., Garbeva, P., and Raaijmakers, J.M. “The rhizosphere microbiome: significance of plant
beneficial, plant pathogenic, and human pathogenic microorganisms”, FEMS Microbiology Reviews,Vol. 37, 2013, pp. 634–663.
• Jarerat, A., Pranamuda, H., and Tokiwa, Y. “Poly(L-lactide)-degrading activity in variousactinomycetes”, Macromolecular Bioscience, Vol. 2, 2002, pp. 420-428.

• Zahran, H.H. “Rhizobium-Legume Symbiosis and Nitrogen Fixation under Severe Conditions and in
an Arid Climate”, Microbiol. Mol. Biol. Rev., Vol. 63, 1999, pp. 968-989.
• Stępkowski, T., Moulin, L., Krzyżańska, A., McInnes, A., Law, I.J., and Howieson, J. “European Originof Bradyrhizobium Populations Infecting Lupins and Serradella in Soils of Western Australia andSouth Africa”, Appl. Environ. Microbiol., Vol. 71, 2005, pp. 7041-7052• Board, J.E. (Ed.) "A Comprehensive Survey of International Soybean Research - Genetics, Physiology,Agronomy and Nitrogen Relationships“, 2013