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Received: 9 December 2017 Revised: 29 January 2018 Accepted: 12 February 2018 DOI: 10.1002/mbo3.615
ORIGINAL RESEARCH
Microbial diversity of thermophiles with biomass deconstruction potential in a foliage-rich hot spring Li Sin Lee1
| Kian Mau Goh2
| Chia Sing Chan2 | Geok Yuan Annie Tan1 |
Wai-Fong Yin1 | Chun Shiong Chong2 | Kok-Gan Chan1,3 1 ISB (Genetics), Faculty of Science, University of Malaysia, Kuala Lumpur, Malaysia 2
Abstract The ability of thermophilic microorganisms and their enzymes to decompose biomass
Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia
have attracted attention due to their quick reaction time, thermostability, and de-
3
drolases (GHs) could accelerate the industrialization of biofuels and biochemicals.
Jiangsu University, Zhenjiang, China
Correspondence Kok-Gan Chan, International Genome Centre, Jiangsu University, Zhenjiang, China. Email:
[email protected] Funding information Postgraduate Research Fund grant, Grant/ Award Number: PG124-2016A; University of Malaya, Grant/Award Number: GA0012016 and GA002-2016; Universiti Teknologi Malaysia GUP, Grant/Award Number: 09H98, 16H89 and 4B297; JBK; NRE
creased risk of contamination. Exploitation of efficient thermostable glycoside hyHowever, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus,
Desulfurobacterium,
Fervidobacterium,
Geobacillus,
Meiothermus,
Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes. KEYWORDS
Biofilm, biofuel, biomass degradation, cellulase, hot spring, thermophile
1 | I NTRO D U C TI O N
production (Xing, Zhang, & Huang, 2012). Fungi and bacteria are major decomposers of organic matter, with bacteria being more in-
Lignocellulolytic biomass is a sustainable resource for second-
fluential due to their metabolic versatility (Liu et al., 2011; López-
generation biofuel production (Xia, Ju, Fang, & Zhang, 2013).
González et al., 2015). Although fungi have been widely used for
Global demand for biofuel, combined with the depletion of nonre-
lignocellulolytic biomass degradation, bacteria have an equally im-
newable fossil fuels, has resulted in the rapid expansion of biofuel
portant role in the degradation process (Takaku, Kodaira, Kimoto,
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. MicrobiologyOpen. 2018;e615. https://doi.org/10.1002/mbo3.615
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Nashimoto, & Takagi, 2006; Watanabe, Nagao, Toda, & Kurosawa,
flora in biomass- enriched samples and GBS indigenous samples
2009; Partanen, Hultman, Paulin, Auvinen, & Romantschuk, 2010;
were different. Therefore, Peacock et al. (2013) suggested that the
Karadag et al., 2013). Bacteria also have a remarkable ability to
additional lignocellulosic biomass stimulated the growth of the po-
tolerate changes in environmental conditions compared to fungi,
tent biomass degraders in a natural environment.
including extreme pH and temperature (Li et al., 2013). Research
One of the quickest approaches for examining microbial pop-
has been focusing on finding new thermostable enzymes or isolate
ulations is 16S rRNA amplicon sequencing. The genera involved in
thermophilic cells for lignocellulolytic degradation. The bacteria
high-temperature biomass degradation have been studied in labo-
class Clostridia and its order Thermoanaerobacterales have been
ratory setups with a predefined medium or type of biomass (Park
extensively studied (Chang & Yao, 2011). Additionally, Deinoccocus-
et al., 2012; Eichorst et al., 2013; Peacock et al., 2013; Xia et al.,
Thermus spp. (Wu et al., 2015), Geobacillus spp. (Zambare, Bhalla,
2014; Vishnivetskaya et al., 2015; Yu et al., 2015). In this study, we
Muthukumarappan, Sani, & Christopher, 2011; Brumm, et al.,
analyzed the microbial diversity in a Malaysian hot spring (60–90°C,
2015; Brumm, Land, & Mead, 2015), Melioribacter spp. (Rakitin,
mean 68°C, pH 8.6) using 16S rRNA amplicon-based sequencing.
Ermakova, & Ravin, 2015), and Thermoanaerobacterium spp. (Currie
The Y-shaped Sungai Klah (SK-Y ) hot spring was studied because it
et al., 2014) are known for decomposing biomass at high tempera-
is a natural “biomass degrading bioreactor” due to the presence of a
tures (Bhalla, Bansal, Kumar, Bischoff, & Sani, 2013). Furthermore,
submerged foliage bed. The data and results obtained add to the list
members of the genera Thermotoga (Yu et al., 2016), Rhodothermus
of important thermophiles for biomass degradation at high tempera-
(Keshk, 2016), Anoxybacillus (Chan et al., 2016), Rhodothermaceae
tures, suggesting that the microbial populations involved in biomass
strain RA (Goh et al., 2016), and Caldicellulosiruptor (Peng et al.,
degradation in natural environments are far more complicated than
2015) were found to produce thermostable enzymes for biomass
in laboratory setups.
saccharification. Thermostable biomass- acting enzymes are promising due to their suitability for industrial applications (Duan & Feng, 2010). Other advantages of thermophiles and their enzymes have been reported (Taylor et al., 2009; Vishnivetskaya et al., 2015). Heated
2 | M ATE R I A L S A N D M E TH O DS 2.1 | Sample collection and water analysis
environments such as hot springs are potential sources of thermo-
The Y-shaped Sungai Klah hot spring (SK-Y ) (3°59′50.50″N,
philes and thermozymes (Urbieta et al., 2015; Zhao et al., 2017).
101°23′35.51″E) is located in Perak, Malaysia. Previously, we con-
Due to high temperatures, most known hot springs lack vegeta-
ducted microbial diversity analysis of the main water source of the
tion sources. In one report, the microbial community in heated
Sungai Klah (SK) hot spring (Chan, Chan, Tay, Chua, & Goh, 2015). In
(68°C) sediments surrounding vegetated (Juncus tweedyi) wetland
this work, samples of the trapped heated spring water were taken
in Obsidian Pool (site OBP10) in Yellowstone National Park (YNP)
from the SK-Y. The SK-Y is located approximately 10 meters from the
was found to mainly include Firmicutes, Proteobacteria, Aquificae,
SK hot spring, as reported by Chan et al. (2015).
Deinococcus-Thermus, Spirochaetes, and Verrucomicrobia phyla,
Sampling was performed on March 24, 2016. A clean stainless
and a huge proportion of unclassified bacteria. The majority of
water sampling dipper was used to collect water samples without
Firmicutes members including lignocellulolytic degraders were
any foliage at four different spots with approximately 5 m between
Clostridium, Anaerobacter, Caloramator, Caldicellulosiruptor, and
sampling locations. Water was stored in sterile glass Schott bot-
Thermoanaerobacter (Vishnivetskaya et al., 2015). When OBP10
tles and immediately transported to the laboratory within 2.5 hr
samples were inoculated with various lignocellulolytic materials,
and stored at 4°C overnight. On the following day, water analysis
including Avicel, switchgrass, Populus, and xylan, and incubated
was conducted by MyTest Lab Sdn Bhd (Malaysia) using American
at 55–85°C in anaerobic laboratory conditions, the main bacteria
Public Health Association standard protocols. At least 20 pieces of
after three culturing rounds were Thermoanaerobacter, Caloramator,
submerged foliage with no apparent biofilm were collected with a
Caldicellulosiruptor, Clostridium, Dictyoglomus, and Fervidobacterium;
sampling dipper and transferred to polypropylene Ziploc bags using
their distributions in these experiments varied with experi-
a tweezer. Submerged foliage with green biofilm was collected and
mental parameters such as temperature and type of substrate
stored separately in 1 L Schott bottles. Degraded foliage was col-
(Vishnivetskaya et al., 2015).
lected at the base of SK-Y with green biofilm, and nondegraded plant
Another site that lacks lignocellulosic plant material is the Great
litters were carefully removed in situ.
Boiling Spring (GBS), located in Nevada (77–85°C) (Peacock et al.,
Freshly picked foliage samples from trees growing along the SK-Y
2013). Microbial diversity analysis was conducted to compare the
were collected and stored in sterile polyethylene bags. The lignin,
microbial diversity in GBS water- sediments with man- made in
cellulose, and hemicellulose content of these foliage samples were
situ enrichment using ammonia fiber explosion-treated corn sto-
analyzed at the Malaysian Agricultural Research and Development
ver and aspen shavings. The microbial community attached to the
Institute (MARDI), a local service provider of acid detergent lignin
supplemented biomass consisting of potential biomass degraders,
(ADL), acid detergent fiber (ADF), and neutral detergent fiber (NDF)
sugar fermenters, and hydrogenotrophs that included Thermotoga,
analysis using a modified protocol based on Van Soest and Wine
Dictyoglomus, Desulfurococcales, and Archaeoglobales. The microbial
(1967).
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2.2 | Total community DNA extraction
spectrophotometer (Thermo Scientific, Waltham, MA, U.S.), and a Qubit® 2.0 Fluorometer (Invitrogen, Merelbeke, Belgium).
Unless specified, all samples were kept at 4°C. DNA extraction was completed within 2 days after sample collection. To study the microbial diversity of the SK-Y, total community DNA extraction of the following samples was performed: (1) pooled water of four sites with an
2.3 | Library construction and 16S rRNA amplicon- based sequencing
equal volume ratio, (2) submerged foliage with no apparent biofilm,
The Illumina 16S rRNA Metagenomic Sequencing Library Preparation
(3) submerged foliage with green biofilm, and (4) degraded foliage
Guide was followed for the preparation of the libraries. Two sets
collected at the base of the SK-Y.
of primers were used to target bacterial and archaeal hypervariable
Four liters of pooled water were filtered through a filter mem-
16S rRNA conserved regions (Klindworth et al., 2012): (1) 16S rRNA
brane with a 0.22-μm pore size (Sartorius, Göettingen, Germany).
V3 and V4 bacterial amplicon polymerase chain reaction (PCR) for-
Then, the membrane was placed in 10 ml of autoclaved 1 × con-
ward primer 5′-TCG TCG GCA GCG TCA GAT GTG TAT AAG AGA
centration phosphate- buffered saline (PBS; 137 mmol/L sodium
CAG CCT ACG GGN GGC WGC AG-3′; (2) 16S rRNA V3 and V4 bac-
chloride, 2.7 mmol/L potassium chloride, and 10 mmol/L phos-
terial amplicon PCR reverse primer 5′-GTC TCG TGG GCT CGG AGA
phate buffer) containing sterile glass beads and shaken vigorously
TGT GTA TAA GAG ACA GGA CTA CHV GGG TAT CTA ATC C-3′; (3)
for 5 min. Next, the membrane was removed and the leftover liquid
targeted archaeal amplicon PCR forward primer 5′-TCG TCG GCA
was centrifuged at 17,000g for 2 min at 4°C. The supernatant was
GCG TCA GAT GTG TAT AAG AGA CAG CMG CCG CGG TAA-3′;
discarded and the pellet was resuspended using Tris-EDTA buffer
and (4) targeted archaeal amplicon PCR reverse primer 5′-GTC TCG
(10 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA) and modified cetyl-
TGG GCT CGG AGA TGT GTA TAA GAG ACA GTA CNV GGG TAT
trimethylammonium bromide (CTAB) lysis buffer (100 mmol/L Tris-
CTA ATC C-3′. The underlined and nonunderlined sequences refer
HCl, 100 mmol/L EDTA, 100 mmol/L K 2HPO 4, 1.5 mol/L NaCl, and
to the Illumina adapter overhang nucleotide sequences and locus-
1% w/v CTAB) (Murray & Thompson, 1980; Zhou, Bruns, & Tiedje,
specific primers for the regions to be targeted, respectively. The
1996). Subsequently, enzymatic lysis was performed with overnight
amplicons were then subjected to a series of library quantification
incubation at 37°C in the presence of 10 mg/ml of lysozyme (Sigma-
steps to accurately quantify the NGS (next-generation sequencing)
Aldrich, Saint Louis, MO, U.S.) and gently swirled at 20-min inter-
sample libraries. The amplicons were quantified with Qubit dsDNA
vals. The solution was then incubated at 90°C for 1 hr (Murray &
HS Assay Kit (Invitrogen) on a Qubit® 2.0 Fluorometer, and the li-
Thompson, 1980). Subsequently, sodium dodecyl sulfate was added
brary size was selected based on Agilent Technologies 2100 Expert
to the final concentration of 1% (w/v), followed by the introduction
Bioanalyzer using Agilent High Sensitivity Kit (Agilent Technologies,
of 20 mg/ml of proteinase K (Qiagen, Valencia, CA, U.S.), incubated
Santa Clara, CA, U.S.). The number of amplifiable molecules in a li-
at 60°C for 2 hr with gentle shaking at 15-min intervals. RNA was
brary were quantified absolutely using Eco Real-Time PCR System
broken down using 100 mg/ml RNase A (Qiagen) and incubated at
with KAPA Library Quantification Kit (KAPA BioSystems, Boston,
37°C for 30 min. Proteins were removed by washing the DNA pel-
MA, U.S.) prior to sequencing. Next, libraries were sequenced using
let with phenol/chloroform/isoamyl alcohol (25:24:1). The resulting
paired-end sequencing on an Illumina MiSeq sequencer (Illumina,
DNA pellet was precipitated with 0.6 volume of isopropanol, fol-
San Diego, CA, U.S.) with MiSeq Reagent Kit V2 (2 × 250 base pairs)
lowed by 70% (v/v) ethanol, and was rehydrated with 60 μl of elution
and V3 (2 × 300 base pairs) for the archaeal primers amplicons and
buffer (Qiagen) (Manjula, Sathyavathi, Gunasekaran, & Rajendhran,
bacterial primers amplicons, respectively.
2011). Approximately 100 g of foliage samples were separately placed according to sample type (foliage with no apparent bio-
2.4 | Sequence analysis
film, foliage with green biofilm, or degraded foliage) inside a
The raw sequence reads generated by the Illumina sequencer
500 ml autoclaved glass bottle containing sterile PBS with 0.05%
were processed in CLC Genomic Workbench 7.0 (CLC Bio, Aarhus,
Tween 20 (4 ml PBS per 1 g leaf, pH 7.4), and sonicated (Branson
Denmark). Adapter sequences were trimmed and reads were fil-
Ultrasonics, Danbury, CT, U.S.) for 1 min at 25°C. The prepara-
tered to ensure an average Phred score of 20. Paired-end reads were
tions were then hand-s haken vigorously for 30 s, and leaf debris
merged (mismatch cost = 2; gap cost = 3; maximum unaligned end
was discarded, and the remaining liquid was aliquoted into a 50 ml
mismatches = 0; minimum score = 8) in CLC Genomics Workbench
tube and centrifuged at 14,800g for 10 min at 4°C. The pellet was
7.0. The assembled reads were then subjected to chimera filter-
subjected to the aforementioned conventional total community
ing and microbial taxonomic classification using the Quantitative
DNA extraction.
Insights Into Microbial Ecology (QIIME) pipeline (Caporaso et al.,
To improve the purity of the extracted total community DNA,
2010). Analyses performed using the QIIME (version 1.9.1) pipeline
inhibitors such as humic acids were removed using the Agencourt
were based on default parameters, unless otherwise stated. Briefly,
AMPure XP System (Beckman Coulter, Brea, CA, U.S.). Quality
the steps included removing chimera sequences, picking operational
and yield of the purified total community DNA were examined
taxonomic units (OTUs) based on an open reference clustering ap-
using 1% w/v agarose gel electrophoresis, a Nanodrop™ 1000
proach using the UCLUST tool, and taxonomic assignment using
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BLAST with the National Center for Biotechnology Information
database (CAZy) to determine the number and types of GH families
(NCBI) 16S Microbial database with an e-value of 0.001. The NCBI
for these taxa.
database was selected because it is large and diverse, with the capability to provide a greater depth of information during taxonomic profiling compared to RDP, GreenGenes, or SILVA databases (Chan et al., 2015). All samples were randomly subsampled to the same sequencing depth prior to analysis. Microbial diversity was as-
3 | R E S U LT S 3.1 | General site descriptions
sessed using rarefraction analysis, the number of observed OTUs
More than a dozen hot spring sites are present in the Sungai Klah
per sample, Shannon–Wiener, and Simpson using QIIME. Beta di-
hot spring park. Previously, we performed a 16S rRNA amplicon and
versity measurements between all the samples were calculated
shotgun sequencing for samples obtained from one of the SK hot
using Unifrac distance (Lozupone & Knight, 2005), implemented
springs (Chan et al., 2015). Although the site is located in a wooded
in QIIME. Principal coordinates analysis (PCoA) was performed on
area, plant litter does not accumulate in the hot spring, as the stream
the weighted UniFrac distance matrix, which accounts for commu-
flows rapidly. Approximately 10 m away from the previously stud-
nities’ membership and relative abundance of OTUs. The resulting
ied location (Chan et al., 2015), man-made drainage, 30 m long and
sequencing data were submitted to NCBI SRA under Bioproject
0.5 m deep, was built to trap heated spring water (Figure 1a). As the
PRJNA353967.
shape of the drainage is Y-shaped, we therefore named the samples obtained from this site as SK-Y, to differentiate current work from
2.5 | Carbohydrate-active gene prediction
our earlier study that was identified as SK hot spring (Chan et al., 2015). The temperature at the SK-Y spring head was approximately
After taxonomic assignment using QIIME, taxa with a relative abun-
90°C, but was lower (60–70°C; mean 68°C) adjacent to the water’s
dance of ≥0.85% were individually checked against the complete
surface and further away from the spring head. The pH for SK-Y
genome information available in the Carbohydrate-Active EnZymes
ranged between 7.5 and 8.6. The most interesting feature of SK-Y is
(a)
(c)
(b)
(d)
(e)
F I G U R E 1 Y-shaped Sungai Klah hot spring (SK-Y ) and types of samples. (a) Illustration of sampling site for water and foliage, (b) SK-Y, (c) foliage with green biofilm, (d) nondegraded foliage with no apparent biofilm, and (e) degraded foliage
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the presence of fallen plant litter that is mainly foliage, accumulated
from 3.0% to 16.7%, whereas hemicellulose content ranged be-
in the heated water, resulting in the formation of a bed of foliage
tween 2.9 and 4.5%. Vitex and Stenochlaena samples were relatively
with a thickness of approximately 20 cm. The average size of the
higher in cellulose content than the Ficus and Adenanthera samples.
fresh fallen foliage is about 15 cm long by 6 cm wide.
The lignocellulosic content of the plant genera (Ficus, Stenochlaena, and Adenanthera) were not found in the literatures to date, except as reported by Codron, Lee-Thorp, Sponheimer, and Codron (2007)
3.2 | Physicochemical analysis of water
for Vitex sp. foliage, which stated that foliage contained 6.0% lignin,
Water analysis was completed to determine the physicochemical
7.2% cellulose, and 8.1% hemicellulose.
condition of SK-Y (Table S1). The temperature and pH during sampling were 68°C and 8.6, respectively. The color of the SK-Y water was 68 true color units (TCU). Aluminum (0.96 mg/L) and iron (0.65 mg/L)
3.4 | 16S rRNA gene sequencing data analysis
were detected in the SK hot spring (Chan et al., 2015), but these
Total community DNA were extracted for four different types of
metal ions were not detected in the SK-Y water sample. SK-Y has
samples. These sample types were (1) SK-Y water, (2) submerged fo-
higher fluoride (6 mg/L), nitrate (0.29 mg/L), and zinc (0.17 mg/L)
liage with no apparent biofilm (labeled as nondecay), (3) submerged
content compared to the SK hot spring (1.1 mg/L,
