17 October 2017
Niran Roongsawang, D.Eng.
Microbial Cell Factory Laboratory,
Biodiversity and Biotechnological Resource Research Unit,
National Center for Genetic Engineering and Biotechnology (BIOTEC),
National Science and Technology Development Agency (NSTDA),
Thailand.
BIOTEC Microbial Communities : Biodiversity and Metagenomic Analysis
Thailand Bioresource Research Center (TBRC)
Culture Collection in Thailand
Microbial Communities
Little et al. 2008
�Groups of microorganisms that share a common living space.
�Play important role on both characteristic of the ecosystem and
physiology of the hosts
�Metagenomic analysis is potential approach for collect members of
community
Metagenomic Analysis
Metagenome: The genomes of total
microbiota found in nature representing the
genetic diversity of uncultured microbesthat represented of 99%
1. Activity screening and
sequencing:
2. Bacterial biodiversity by Next-gen sequencing
• Tagged amplicon
sequencing: 16S rRNA
Metagenome from
Biomass degradable
environments
Metagenomic library : the genomic DNA library representing genetic diversity of all
the microorganisms in a specific environment or biological niche
Culture-independent approach
Activity-based screening
Sequence-based screening
Pyrosequencing
Metagenomic library
Biodiversity conservation
• plasmid
• fosmid• BACs
Source Method/ Target References
Termite gut symbioint
Source: Trametes hindgut
Alkaliphilic cellulose degradation
Activity-based screening
Alkaliphilic
cellulase/hemicellulase
Nimchua et al. (2012)
J. Microbiol. Biotehnol. 22, 462-469
Cow rumen
Source: Cow rumen fluid
Anaerobic cellulose
degradation
16S rRNA
Biodiversity profiling
Cellulose degradation
Thoertkiattikul et al. (2013)
Curr. Microbiol. 67, 130-137
Peat swamp forest
Site: Narathiwas, THAOrganic carbon sink
16S rRNA
Biodiversity profilingCellulose degradation
Kanokratana et al. (2011)
Microb. Ecol. 61, 518-528
Bagasse collection site
Site: Chaiyapoom, THA
Thermophilic lignocellosedecomposition
16S rRNA
Biodiversity profiling
Acidophilic microbes
& enzymes
Kanokratana et al. (2016) Microb. Ecol. 72: 381-393
Metagenomic exploration @BIOTEC
Carnivorous plant pitcher
fluids
Source: 8 local Nepenthes sp.
Acidic hydrolytic environment
Kanokratana et al. (2013)
Microb. Ecol. 66, 322-33416S rRNA
Biodiversity profilingCellulose degradation
U.S. DOE Genomic Science Program https://microbewiki.kenyon.edu
Cellulases & Hemicellulases (Xylanases)
X0012
X1098
C0001
Cellulase (C0001)
Xylanase (X0012)
Xylanase (X1098)
Xylanase and cellulase isolated from termite gut
Effect of pH and temperature on enzyme activity
Nimchua et al. (2012)
Peat soil is originated by accumulation of
organic matters particularly plant biomass which
slowly decomposed through a very long period of
time under water logging condition.
Pru Toh Daeng,
Narathiwat
Peat swamp Metagenome
The phylogeny of bacterial 16S rRNA gene sequences from the Pru Toh Daeng peat swamp forest
The community was dominated by
aerobic, facultative and anaerobic
microbes
Acidobacteria and diverse
Proteobacteria constituted the major
group.
Planctomycetes (aquatic bacteria)
Classification of sequences encoding plant polysaccharide degrading enzymes in the
tropical peat swamp forest metagenome based on glycosyl hydrolase annotation
• Variety of cellulases, hemicellulases & amylolytic ezymes was found.
• Originate from Proteobacteria & Acidobacteria.
• Key role of these microbes in plant biomass degradation.
Sugarcane bagasse is the fibrous by-product
from sugar processing with the annual
production capacity of 20 million tones.
Currently, it is used for electricity generation
and material industry, and is considered an
rich unexplored source for lignocellulolytic
microbes working under a wide range of
ecological conditions.
surface
1 m ground level
3 m ground level
Bagasse Metagenome
Sequencing of 16SrRNA gene amplicons Annotation of genes encoding glycosyl hydrolases
surface
1 m ground level
3 m ground level
surface
ground
1 m surface
Soil contact (S1, S4, S5)
Proteobacteria,
Bacteroidetes,
Acidobacteria
Depth (S2, S3)
Spirochaetes (anaerobic)
Variety of cellulases &
hemicellulose for biomass
degradation were identified
Nepenthes pitcher fluid is highly
acidic and a rich source of un-
explored plant and microbial
hydrolytic enzymes with potential for
biotechnological application.
Close
Open
Bacterial phyla distribution profiles of microbial diversity in the pitcher fluids collected from different Nepenthes plant species
Proteobacteria is predominant
members in closed pitchers
Bacteriodetes & Actinobacteria
showed increasing abundance in
open pitchers
Acidobacteria was highly
dominant in acidic pitcher fluids
Engineering microbiomes to improve soil fertility and plant productivity
Proposed Research
! Only the bacterial communities in different environments was investigated,
microbiome of ecosystem in Thailand (especially in soil and plant) remains unexplored.
MicrobiomeMicrobiome
� The total microbial cells and their genetic materials
� Dynamic interaction effects the physiology of the hosts
� Provides novel solution for improve human and animal
health, agriculture & environment
https://www.slideshare.net/
Soil and Plant Microbiome
Soil microbiome is microbial
communities in the bulk soil
Plant microbiome is represented
as root-associated microbiome
• Imbalance of microbial communities (MC) which subsequently
increase rates of plant diseases and reduce crop productivity.• Rebalancing of MC by microbiome engineering is important.
Microbiome engineering
Franz Bender et al., 2016
Microbial strains with verified function can be combined into simple synthetic
microbiomes containing few to several dozen species and use to promote plant
health and improve soil fertility
NSTDA: driving force for Thailand’s S&T
http://www.desktopedia.com/wallpaper/Green-Hill-with-Blue-Sky-Landscape/
Acknowledgement
• National Center for Genetic Engineering and Biotechnology
• National Science and Technology Development Agency
• Enzyme Technology & Microbial Cell Factory Laboratory
members
