Merit Research Journal of Microbiology and Biological Sciences (ISSN: 2408

2408-7076) Vol. 4(1) pp. 022-038

038, January, 2016
Available online http://www.meritresearchjournals.org/mbs/index.htm
Copyright 2016 Merit Research Journals

Original Research Article

Prokaryotic diversity from the culture

culture-independent
independent
taxonomic analysis of a sugarcane bagasse
metagenome
1,2*
Ramn Alberto Batista-Garca1,
, Roco Casasanero2, Alberto Alvrez-Castillo
Castillo3, Stephen A.
4,5
4
Mallol2
Jackson , Alan D. W. Dobson and Jorge Luis Folch-Mallol
Abstract
1

Centro de Investigacin en Dinmica

Celular, Universidad Autnoma del
Estado de Morelos. Cuernavaca,
Morelos, Mexico.
2

Centro de Investigacin en
Biotecnologa, Universidad Autnoma
del Estado de Morelos. Cuernavaca,
Morelos, Mexico.
3

Instituto Tecnolgico de Zacatepec.

Zacatepec, Morelos, Mexico.
4

Marine Biotechnology Centre,

Environmental Research Institute,
University College Cork. Cork, Ireland.
5

School of Microbiology, University

College Cork. Cork, Ireland.

*Corresponding Authors E-mail:

rbatista25@yahoo.com,
rabg@uaem.mx
+(52) 777-3297057 Ext. 4550
+(52) 7773297057 Ext. 3457

A 16S rDNA metagenome library was constructed from sugar bagasse

samples and a phylogenetic analysis was conducted. This allowed us to
explore the biodiversity present in the bagasse samples and identify novel
nov
bacterial groups inhabiting this lignocellulosic rich substrate. The most
abundant phyla were Proteobacteria and Acidobacteria,
Acidobacteria while Candidate
division TM7 was the minor phyla. Overall, the prokaryotic microbial
diversity revealed the presence of 13 families and 17 genera, with
Burkholderiaceae and Burkholderia being the major family and genus
observed, respectively. Since we were also able to detect some known
bacterial genera, we could identify some specific metabolic pathways that
may be important in lignocellulose degradation. Microbial communities
inhabiting natural lignocellulosic substrates have to date received little
attention, subsequently there is little information about the main bacterial
genera present in these substrates resulting in a lack of a comprehensive
knowledge about the microbial process involved in lignocellulosic
degradation. This study provides some insight into the unique microbial
community structure in sugarcane bagasse from Meso Americ (the most
important sugar producing region in the world) and is the first such study to
characterize these bagasse inhabiting populations.
Keywords: Metagenomic approaches, 16S
S rDNA biodiversity, cellulolytic
environments, bacterial communities.

INTRODUCTION
Plant biomass is the most important, abundant and
widespread source of carbon on earth. For many years,
plant biomass was considered as a waste and as a result
its use was totally under exploited. However, more
recently there has been an increased interest in the
integrated management of biomass, and especially of
sugarcane bagasse (Li et al.,, 2009). A range of
applications, such as the production of ethanol, methanol,
biodiesel, oil, hydrogen, plastics, paper, polymers, resins,
electricity and other raw materials,
terials, have driven the use of
biomass (Demirbas, 2005). Particularly, sugarcane
bagasse obtained from sugar mills is currently widely

used in both paper and energy production (Guimares et

al., 2012).
Biomass-based
based biorefineries have gained immense
popularity
arity in recent years. Challenges still exist however,
in particularly with respect to the lack of cost-effective
cost
break-through
through technologies to allow the conversion
bagasse into alcohol or other products (Himmel, 2008).
The main problem is the low yields obtained
o
from
saccharification of the bagasse. This is a complex
process and it is often defined as a critical step,
depending on the chemical composition of the biomass
(Chen et al., 2014; Geng et al.,, 2014).

Batista-Garcia et al. 023

Sugarcane bagasse is predominantly composed of

three polysaccharides: cellulose, hemicellulose and
pectin and the heterogeneous aromatic polymer (lignin)
(Rezende et al., 2011; Liu et al., 2012). Cellulose is a
homopolymer, the major reservoir of glucose in nature;
while pectin, lignin and hemicellulose are complex
heteropolymers of organic acids, cyclic phenolic
compounds and sugars, respectively. Different molecular
interactions result in tight complexes being formed
between these polymers. Hemicellulose, for example,
has a -(14)-linked backbone made up of xyloglucans,
xylans, mannans and glucomannans, and -(13, 14)glucans (Scheller and Ulvskov, 2010). Different branching
monomers (D-galactose, D-xylose, L-arabinose, Dglucuronic acid) result in various types of hemicelluloses
being formed. Moreover several acids (ferulic and acetic,
mainly) also confer a higher level of structural complexity
because they can esterify with hemicellulose (Brink and
Vries, 2011).
Recalcitrant crystalline and amorphous structures of
these polymers limit sugarcane bagasse conversion into
fermentable sugars for further use in biorefineries (Liu et
al., 2012; Geng et al., 2014). Current understanding of
how to efficiently transform this material through
technologically scalable, replicable and sustainable
processes is as yet largely incomplete.
On average the quantitative composition of sugarcane
bagasse is generally 35 50% cellulose with 20 30%
each of hemicellulose and lignin, and usually also
contains aliphatic acids (1 3%) (acetic, formic, and
levulinic acids), furan derivatives (furfural and
hydroxymethylfurfural)
and
phenolic
compounds
(Chandel et al., 2014). Its chemical nature (qualitative
and quantitative) and structure define it as one of the
most recalcitrant wastes in the agriculture industry
(Rattanachomsri et al., 2011).
With one ton of processed sugarcane generating on
average 250 kg of bagasse (Guimares et al., 2012), and
the annual increase in demand for sugar the production
of bagasse continues to increase. Thus there is an
increased
interest
in
the
lignocellulose-based
biorefineries area with a particular focus on the use of
cultured-independent metagenomic based taxonomic
analysis of microbial communities present in the
lignocellulosic rich bagasse ecological niche. A more
comprehensive knowledge of the sugarcane bagasse
microbiome will provide further insights into the microbial
community structure and metabolic potential and help
define their potential role as lignocellulose degraders,
thereby increasing our understanding of the natural
degradation processes that occur in lignocellulolytic rich
environments such as bagasse. Moreover, it may
ultimately help facilitate a better and more optimal use of
bagasse as an industrial resource for biofuels and other
products.
Bacteria are the most abundant, diverse and
widespread microorganisms on earth. They colonize a

huge variety of natural environments, including very

specialized ecological niches such as lignocellulosic rich
habitats; which is largely possible due to the immense
metabolic diversity and genetic adaptability of microbes
(Leis et al., 2013). Given that only an estimated <1% of
microorganisms are cultivable under laboratory
conditions (Simon and Daniel, 2011), metagenomic
based tools have been successfully employed to conduct
comprehensive
ecological
studies
of
bacterial
communities in a wide variety of ecosystems. Structural
metagenomics allows both the composition and structure
of microbial communities to be assessed by describing
the major genera and species that inhabit an ecosystem
(Simon and Daniel, 2011). It can also provide information
about the role of microorganisms in the biogeochemical
cycles, propose ecological interactions between
members of different microbial communities and
postulate hypotheses about evolutionary aspects of
specific ecosystems (Simon and Daniel, 2009).
While a wide range of quite diverse metagenomes
have to date been extensively analysed (Rasheed et al.,
2013; Curson et al., 2014; Jadeja et al., 2014; Kanwar et
al., 2014; Patel et al., 2014; Tseng and Tang, 2014; Xu et
al., 2014) very few metagenomic studies have been
performed on the microbiomes of lignocellulosic based
substrates (Rattanachomsri et al., 2011; Kanokratana et
al., 2013). Sugarcane bagasse piles stored near sugar
mills are an excellent cellulolytic environment and
represent a unique habitat in which to study
lignocellulosic and metabolically versatile microbial
communities. The prokaryotic populations colonizing
sugarcane bagasse in particular and other cellulosic
materials in general have surprisingly been quite poorly
studied to date and consequently there is lack of a
comprehensive understanding of their microbial ecology.
The objective of our work was to analyse the
prokaryotic diversity of a sugarcane bagasse
metagenome,
and
subsequently,
the
structural
composition of the bacterial communities was obtained
by employing cultured-independent methods. The V3
V6 region of the 16S rDNA gene was used to analyse the
bacterial populations, and a sequence dataset was
analysed with strong algorithms using two taxonomic
classifiers. Our groups are interested in biological pre
treatments of lignocellulosic materials for biorefinery
purposes and by exploring the biodiversity profiles, this
work provides new insights to better understand the
microbial populations present in sugarcane bagasse
piles and thus may provide insights into new strategies
for more efficient lignocellulose degradation.
MATERIALS AND METHODS
Sugarcane bagasse sampling
Decaying humid sugarcane bagasse sampling was

024 Merit Res. J. Microbiol. Biol. Sci.

Figure 1. Sugarcane bagasse sampling. (A) Location of the collection site at Zacatepec municipality (Morelos State),
Mexico. Cartesian coordinates were estimated by measuring satellite instruments (GPS: global positioning system,
Model eTrex 20).(B) General scheme of sampling for one pile. Three samples in triplicates (R1, R2 and R3) of
sugarcane bagasse were taken at different levels (surface, core and bottom) of the column of eachsampled pile. A
compost sample was obtained pooling R1, R2 and R3 from each sampling level (CSS: surface compost sample, CSC:
core compost sample and CSB: bottom compost sample). A final compost sample (CSF) for further experiments was
obtained pooling CSS, CSC and CSB.

performed at the beginning of spring (April) 2008 in the

bagasse yard of a Sugar Mill (N 1839'15.9", W
9911'10.1") located in Zacatepec, a municipality in the
central-south region in the state of Morelos in Mexico
(Figure 1, A). Bagasse samples were collected from large
open-air piles (10 - 12 m height), which had been
standing in the field for approximately five months prior to
the time of collection.
Three triplicate samples (1 kg) from three different
piles were taken. These were collected from different
positions in each pile, namely from the surface (20 cm
below the surface), the core (10 m) and the bottom (20
cm above the soil) of the pile. The samples were then
mixed to homogeneity prior to DNA extraction as shown
in Figure 1, B. This strategy was employed to ensure
samples were taken from potentially different
microenvironments within the piles (oxygen, temperature,
humidity, radiation, pH). Subsequently, a composted
sample for further experiments was generated, and
consequently a representative sample was obtained
(Figure 1, B). The bagasse samples were then placed in
sterile plastic bags and stored on dry ice for transport to
the laboratory where they were subsequently frozen at o
80 C.
The sampling was performed during the dry season to
avoid ecological succession due to rain washing. The
meteorological station in the Zacatepecs locality
o
registered, an average temperature of 28 C and 0.8 mm
of accumulated rainfall in April 2008. The rainy season is
from July until September of each year, and the average

annual rainfall in this locality is typically 800 1200 mm.

The probability of rain in the dry season is very
infrequent. The temperature in the bagasse pile surface
o
o
was 30 1 C, while in the core it was 40 1 C.
Chemical sugarcane bagasse characterization
The lignin, cellulose and hemicellulose composition of the
bagasse sample was determined according to
Dominguez-Dominguez et al. (2012). The bagasse was
o
ground and dried at 70 C for 24 h. To determine the
cellulose composition, 15 mL of acetic acid (80%) and 1.5
mL of concentrated nitric acid were added to 1 g of milled
bagasse. The sample was treated at reflux for 20 min and
filtered thereafter. The residue was washed with absolute
o
ethanol, baked dried at 100 C and finally weighted
o
(material A). It was later burned at 540 C, cooled to room
temperature in a desiccator and weighted (material B).
The cellulose percentage was calculated using the
following equation:


%
=
100

To determine the lignin composition, 70 mL of sulphuric

acid (1.25%) was added to 1 g of milled bagasse. The
sample was treated at reflux for 120 min, filtered
thereafter and then washed with water. Subsequently 30
mL of sulphuric acid (72%) was added and the material

Batista-Garcia et al. 025

was agitated at 100 rpm for 4 h. The solids were then

o
recovered, filtrated, washed, dried (100 C) and weighted
o
(material C). The material was cremated at 540 C,
cooled to room temperature in a desiccator and weighted
(material D). The lignin percentage was calculated using
the following equation:


%
=
100

The percentage of hemicellulose was calculated

considering the cellulose and lignin compositions:
%
= 100% (%
+ %
)
The residual reducing sugars were calculated using the
DNS (3,5-dinitrosalicylic acid) method with a glucose
standard curve (Miller et al., 1959). The total reducing
sugars were expressed per g of bagasse.
Metagenomic
bagasse

DNA

extraction

from

sugarcane

10 g of sugarcane bagasse was suspended in 100 mL of

lysis buffer: 0.1 M Tris-HCl pH 8, 0.1 M EDTA
(ethylenediaminetetraacetic acid) pH 8, 0.1 M Na2PO4 pH
8, 1.5 M NaCl, 2% SDS (sodiumdodecyl sulfate) and 3%
CTAB (cetyltrimethylammonium bromide). The mixture
o
was frozen on dry ice and later defrosted at 80 C to
promote cell lysis. This procedure was repeated three
times. Bagasse fibres and suspended particles were
removed by centrifugation at 12000 rpm and subsequent
filtration (0.45 m) (Fermentas Ltd., Mexico). The
resulting solution was treated with phenol-chloroformisoamyl alcohol (25:24:1) to remove humic acids and
other co-extracted contaminating substances. Finally it
was treated with chloroform to remove phenol traces.
DNA was precipitated with ethanol, recovered in 50 L of
Mili-Q water, analysed by gel electrophoresis and
quantified using a spectrophotometer (Nanodrop ND1000). Finally, an additional purification step was
performed using the Fermentas DNA extraction kit
(Fermentas Ltd., Mexico). DNA was extracted from three
o
independent samples, pooled and then stored at -20 C.
PCR amplicon library preparation for sequencing
A 16S rRNA PCR amplicon gene library of the full-length
V3 V6 region (1300 bp: highly conserved region) was
prepared using metagenomic DNA from sugarcane
bagasse as a template. The PCR primers used were (i)
forward: U968-GC [5-(GC-clamp)-AAC GCG AAG AAC
CTT AC-3] and (ii) reverse: L1401 (5-CGG TGT GTA
CAA GAC CC-3] (Felske et al., 1996). Cycle conditions
o
comprised initial denaturation at 94 C for 5 min followed
o
by 30 cycles of denaturation at 92 C for 60 s, primer
o
o
annealing at 60 C for 60 s and extension at 72 C for

45 s. A final extension at 72 C for 10 min followed. Three

individual PCR reactions were performed.
Amplicons
were
purified
by
agarose
gel
electrophoresis using the Fermentas gel purification kit
(Fermentas Ltd., Mexico) as per the manufacturers
instructions and ligated to pGEM-T Easy vector
(PROMEGA Ltd., Mexico). Clones derived from
electrotransformed Escherichia coli DH5 were selected
on Luria-Bertani agar plates containing isopropyl--Dthiogalactopyranoside (IPTG)/5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal) (3 and 15 g/mL,
respectively) and ampicillin (100 g/mL). Representative
colonies were randomly selected and subsequently,
sequenced in the Langebios Genomic Services Unit
(Cinvestav-Irapuato, Mexico). Two sequences from each
clone were obtained. In this order, rounds of sequencing
using (i) PCR forward primer and vector reverse primer,
and (ii) PCR reverse primer and vector forward
primer were performed. Finally, sequences were
assembled in the DNA Baser edit program version
4.16.0.
Sequencing data analysis
All sequence reads were processed by two platforms: (i)
Ribosomal
Database
Project
(RDP)
pipeline
(http://rdp.cme.msu.edu/) and (ii) NGS analysis pipeline
of the SILVA rRNA gene database project (SILVAngs
1.1) (http://www.arb-silva.de/) (Quast et al., 2013). Both
allow analysis of large scale small- and large subunit
ribosomal (rRNA) gene tag sequencing projects.
The sequencing dataset was quality-filtered using the
RDP (Release 11.2, 2014) and SILVAngs (Release 115,
2014) pipelines. During initial processing (in both
platforms) reads with more than 2% of ambiguities or 2%
of homopolymers, shorter than 100 bases, sequences of
low alignment quality (50 alignment identity, 40 alignment
score) and identical reads were identified and excluded
from downstream analysis. Primer portions were
trimmed, and the orientation of reads and chimera
formation were checked.
Unique sequences were aligned using the secondarystructure INFERNAL aligner procedure in the RDP
pipeline (Nawrocki and Eddy, 2007) and the SILVA
Incremental Aligner (Pruesse et al., 2012). Furthermore,
(OTUs: operational taxonomic units) sequences were
clustered by complete-linkage clustering. The RDP
Classifier allowed the assignment of taxa using nave
Bayesian algorithm with a confidence threshold of 95%
(Wang et al., 2007; Cole et al., 2013); while SILVA
Classifier classified each OTU using cd-hit-est (version
3.1.2; http://bioinformatic.org/cd-hit) (Li and Godzik,
2006) running in accurate mode, ignoring overhangs, and
applying identity criteria of 1.00 and 0.98, respectively.
The classification was performed by a local nucleotide
BLAST search against the non-redundant version of the

026 Merit Res. J. Microbiol. Biol. Sci.

SILVA SSU Ref dataset (release 115; http://www.arbsilva.de) using blastn (version 2.2.28+; http://blast.ncbi.
nlm.nih.gov/Blast.cgi) with default settings (Camacho et
al., 2009). According with the function [(% sequence
identity + % alignment coverage)/2], reads without any
BLAST hits or reads with weak BLAST hits showing
values lower than 93, remain unclassified (Ondov et al.,
2011). Subsequently, phylum and full-taxonomic
fingerprints were constructed (Ionescu et al., 2012;
Klindworth et al., 2013). The classification and taxonomic
nomenclature considered in this analysis are in
accordance with the last edition of Bergeys Manual of
Systematic Bacteriology (Garrity and Hold, 2012).
Rarefaction curves, Shannon Weaver index, the Chao1
richness estimator and the abundance-base coverage
estimator (ACE) were obtained using RDP tools.
Phylogenetic trees were prepared using the server
Phylogeny.fr (http://www.phylogeny.fr/). MUSCLE and
ClustalW for the multiple alignments, Gblocks for the
alignment curation and neighbour-joining method were
used in the analysis. Phylogeny.fr considers various
bioinformatics algorithms to construct a robust
phylogenetic tree from a set of sequences (Deereper et
al., 2008).
An OTUs network visualization showing OTUs
interaction was generated to compare the prokaryotic
diversity derived of our bagasse sample with a dataset
obtained from the Thai bagasse deposited in GenBank
under
accession
numbers
HM362440-HM362617
(Rattanachomsri et al., 2011). The network map was
prepared in Qiime (Quantitative Insights Into Microbial
Ecology) version 1.8.0 (Caporaso et al., 2010) and was
visualised in Cytoscape program version 3.1.1 (Shannon
et al., 2013). Silva and RDP classifiers were used to
prepare the network files in Qiime.
The 16S rRNA gene dataset was deposited in
GenBank-NCBI (National Center for Biotechnology
Information) under accession numbers: KM882649KM882821.
RESULTS AND DISCUSSION
It is clear that lignocellulosic rich environments contain
microbial diversity that remains as yet largely
understudied. The increasing demand of bagasse-based
biorefineries to use sugarcane bagasse as a raw material
to obtain products of associated high-value supports our
approach of attempting to gain a better understanding of
the overall structures of the microbial communities
present in the bagasse (Lima et al., 2014; Silva et al.,
2014). Thus a more comprehensive knowledge of the
microorganisms present within sugarcane bagasse may
allow greater potential access to their metabolic
capabilities which are important in allowing them inhabit
this particular lignocellulose rich ecosystem. With this in
mind this study focused on analysing the prokaryotic

diversity of a sugarcane bagasse metagenome, using

cultured-independent methods.
Chemical sugarcane bagasse composition
The chemical composition revealed that the bagasse
from the Zacatepecs Sugar Mill used in this work was
composed of 53.73% cellulose, 26.37% lignin and 19.9%
hemicellulose. The residual reducing sugar analysis
showed 0.18 mg/g bagasse. This composition is similar
to levels previously reported in other studies
(Rattanachomsri et al., 2011; Rezende et al., 2011;
Siqueira et al., 2013) and confirms the recalcitrant nature
of this agricultural waste. The polymeric composition both
qualitative and quantitative of the bagasse, suggest that
quite unique microbial diversity is likely to be present in
this lignocellulosic rich environment. Analysis of these
bacterial population should provide valuable insights into
not only overall population structures but also potentially
of methods or approaches to control the microbial
processes within these populations for optimal utilization
of the bagasse for bio-fuel applications.
Metagenomic DNA isolated from sugarcane bagasse
For metagenomic analysis, the quantity of DNA isolated
is important to ensure adequate representation of the
microbial communities in the sample. Isolation and
purification of metagenomic DNA from lignocellulosic rich
substrates has a number of drawbacks including the fact
that there are no standardized methods available for
nucleic acid extraction and no commercially available
extraction kits are available. In this study an average of
0.5 g metagenomic DNA per 1 g of bagasse was
isolated from each sample. This yield of metagenomic
DNA is low however when compared with previously
described yields from soils; where DNA yields around 15
- 50 g of DNA per 1 g of soil have been reported (Henne
et al., 1999; Yun et al., 2004; Amorim et al., 2008). As
previously stated sugarcane bagasse is a very
recalcitrant lignocellulosic waste, primarily due to its
chemical structure and composition (Chandel et al.,
2014). This recalcitrance is likely not to allow the
development of a wide variety of microbial communities
(Silva et al., 2009); which may explain the low yields of
DNA obtained from the sugarcane bagasse samples.
Furthermore, the fibrous nature of the bagasse also
constitutes a limitation for DNA extraction, while the
granularity of a sample is also known to have a marked
effect on the yield of DNA (Brady et al., 2007).
Crude DNA solutions prepared from the sugarcane
bagasse were extremely dark and very viscous, and
additionally showed a brownish colour indicating the
presence of contaminants (Brady et al., 2007).
Sugarcane bagasses samples are known to contain

Batista-Garcia et al. 027

Figure 2. Isolation and purification ofsugarcane bagasse metagenomic DNA. (A) Extraction of metagenomic DNA
from three independent samples of bagasse. Lane 1: 1 kb ladder. Lanes 2, 3 and 4: Crude metagenomic DNA. (B)
Purification of metagenomic DNA. Lane 1: 1 kb ladder. Lane 2: Bagasse metagenomic DNA before additional
purification step. Lane 3: Bagasse metagenomic DNA after additional purification step. (C) PCR reaction to amplify
approximately 1300 bp of the gene coding for 16S rRNA from sugarcane bagasse metagenomic DNA. Lane 1: 1 kb
ladder. Lane 2 and 3: Bands corresponding to gene fragments coding for 16S rRNA from metagenomic DNA and
Rhizobium meliloti (PCR control) respectively.

humic and aliphatic acids (acetic, formic, and levulinic

acid),
furan
derivatives
(furfural
and
hydroxymethylfurfural)
and
phenolic
compounds
(Chandel et al., 2014). These common compounds are
derived from the production of molasses, which are
concomitantly extracted with metagenomic DNA and can
be observed as a large smear following agarose gel
electrophoresis (Figure 2, A). They are considered as
strong pollutants in DNA solutions because they
contribute to the denaturation of nucleic acids, can affect
their stability, inhibit numerous enzymes such as DNA
polymerases and moreover, negatively interfere with
PCR reactions (Tebbe and Vajhen, 1993; Lim et al.,
2005). The isolation of DNA from bagasse resulted in coextraction of other undesirable compounds, thus this
aforementioned dark colour is most likely explained by
the fact that co-extracted substances are known to cause
a blackish colour in crude DNA solutions (Tebbe and
Vajhen, 1993) and the observed physical properties of
the DNA suggest that co-extracted contaminants were
not completely removed during the purification step (Lim
et al., 2005). Repeated DNA purification steps were
therefore undertaken to ensure the removal of these
contaminants, with the DNA being purified until complete
decolouration was observed. The final gel electrophoresis
analyses showed a defined DNA band of high molecular
weight (> 10000 bp) (Figure 2, B).
Overall DNA concentration and quality was assessed
by reading absorbance (A) of the bagasse
samples at 260 and 280 nm using Nanodrop, with a ratio

value of 1.79 being obtained, confirming that DNA purity

was suitable for further metagenomic analyses.
Prokaryotic microbial diversity
A library of 500 clones with amplicons derived from 16S
rDNA PCR (approximately 1300 bp; Fig. 2, C) was
obtained, and 200 of these clones were subsequently
analysed. Following quality filtering, 177 reads with an
average length of 1305 bp were analysed. The minimal
and maximum sequence length was 513 and 1355
respectively. During the quality control we identified and
discarded: five sequences that showed more than 2% of
ambiguities, nine that were shorter than 100 bases, four
that revealed low alignment quality and five that were
classified as duplicated reads. A histogram from the
aligned reads displaying the number of sequences vs. the
position (start and end positions) was generated during
the alignment process (Supplementary Figure 1).
Three bacterial phyla (Acidobacteria, Candidate
division TM7 and Proteobacteria) were observed in
bagasse-derived reads. Comparison of OTUs against
RDP and SILVA shows that the Proteobacteria and
Acidobacteria were the largely predominant bacterial
lineages constituting 60 and 34% of the gene amplicon
sequences respectively, while Candidate division TM7
was the minor phylum (Figure 3, A). These three phyla
have previously been reported to be dominant in
lignocellulosic rich environments such as native forest

028 Merit Res. J. Microbiol. Biol. Sci.

Supplementary Figure 1

Figure 3. Taxonomic fingerprint. (A) Phylum fingerprint. (B) Full-taxonomic fingerprint. Fingerprints were derived from
SILVA pipeline analysis.

soil enriched with cellulose (Ghio et al., 2012); the gut of

Holotrichia parallela larvae (Huang et al., 2012) and
cellulose rich soil from the Chaco region of South
America (Talia et al., 2012). According to the SILVA
classifier the taxonomic classification resulted in 100% of
the sequences being classified (phylum level) in more

than 40 OTUs, and subsequently the full-taxonomic

fingerprint was obtained (Figure 3, B). Overall, the
prokaryotic microbial diversity revealed the presence of
13 families and 17 genera, with Burkholderiaceae and
Burkholderia being the major family and genus
respectively. On the other hand, the taxonomic analysis

Batista-Garcia et al. 029

Figure 4. Relativetaxonomic representation. (A) Classified genera in the phylum

Acidobacteria and its relative abundance. (B) Relative abundance of Proteobacterialclasses.
(C, D, E and F) Classified genera and relative abundance of Alphaproteobacteria,
Betaproteobacteria, Deltaproteobacteria and Gammaproteobacteria respectively. All values
are shown according to the total sequences analysed. This classification was obtained from
SILVA.

based on the RDP classifier also resulted in 100% of the

sequences being classified at the phylum level. However,
this classifier (with similar confidence threshold as that of
the SILVA classifier) identified 48 hits as unclassified
sequenced at the division-class-genera-species levels;
with a large number of acidobacterial sequences (78.7%)
not being classified below the phylum level. Similarly one
sequence from the phylum Proteobacteria could not be
classified below the phylum level. This information
strongly suggests that sugarcane bagasse is an
interesting lignocellulosic rich habitat in which to identify
new strains, species and even genera of bacteria.
Although all sequences were assigned to phyla, 27.1% of
bacterial reads were not classified at genus level. A
notable proportion of sequence reads without
classification to phylum level or below the phylum level
has previously been found in structural metagenomic
studies from cellulolytic and non-cellulolytic ecosystems
(Engelbrektson et al., 2010; Kanokratana et al., 2013; Xu
et al., 2014).
Five genera (Acidobacteriaceae family) belonging to

the phylum Acidobacteria phylum were identified:

Granulicella, Telmatobacter, Terriglobus, Edaphobacter
and Acidobacterium (Figure 4, A). The phylum
Proteobacteria was also highly represented. A relative
abundance analysis with the full-taxonomic fingerprint
shows the following distribution: Betaproteobacteria
(35%),
Alphaproteobacteria
(18%),
Gammaproteobacteria (5%) and Deltaproteobacteria
(2%) (Figure 4, B). Taxonomic classification revealed that
the
best
represented
genus
in
the
class
Alphaproteobacteria is Hyphomicrobium, with an
additional four genera being identified (Methylovirgula,
Bradyrhizobium, Rhizomicrobium and Pseudolabrys)
(Figure 4, C). Nine reads which classified in the
Caulobactereaceae and Beijerinckiaceae families were
related to uncultured bacteria. Two (Achromobacter and
Burkholderia) and one (Haliangium) genera respectively
represent Betaproteobacteria and Deltaproteobacteria
classes (Figure 4, D and E). Based on their 16S rDNA,
62 sequences were grouped in the genus Bulkholderia
(Figure 3, B and Figure 4, D). Nine reads were classified

030 Merit Res. J. Microbiol. Biol. Sci.

Figure 5. Culture-independent taxonomic analysis reveals significant differences in the microbial composition derived
from cellulolytic (box enclosed with solid lines) and non-cellulolytic (box enclosed with dashed lines)environmental
metagenomic sequence sets.

as Gammaproteobacteria and related to different genera

(Escherichia-Shigella, Pseudomonas and Dyella). Three
sequences belonging to the Xanthomonadaceae family
also showed similarity with uncultured bacteria (Figure 4,
F).
A large number of environmental metagenomes
(rumen, rivers, seas, oceans, lakes, soils, foods, faecal
materials, sediments, gut insects and sludge) have to
date been structurally and functionally analysed
(Rasheed et al., 2013; Curson et al., 2014; Jadeja et al.,
2014; Kanwar et al., 2014; Patel et al., 2014; Tseng and
Tang, 2014; Xu et al., 2014). However, the composition
of microbial communities inhabiting lignocellulosic
environments and in particular lignocellulosic rich wastes
remains largely under-studied. Only two other studies
have to date undertaken a phylogenetic analysis of the
complex microbial community structure in industrial
bagasse feedstock piles. Both were undertaken by the
Champreda group and reported much higher levels of
diversity than had previously been anticipated
(Rattanachomsri et al., 2011; Kanokratana et al., 2013).
Those studies identified between ten and seven bacterial
phyla associated with sugarcane bagasse in Thailand.
Rattanachomsri et al. (2011) also report the prevalence
of Proteobacteria in their taxonomic classification, with
Proteobacteria, Firmicutes, Bacteroides, Acidobacteria,
Actinobacteria and TM7 as the major represented phyla.

On other hand, Kanokratana et al. (2013) found a

differential microbial distribution among different samples
of sugarcane bagasse, depending on the location of the
sample within the bagasse; but again Proteobacteria
represent the major phylum in many of these samples. It
should also be noted that a widespread bacterial diversity
was not observed in these samples with Firmicutes,
Proteobacteria and Bacteroides primarily predominating.
However, structural metagenomic analyses from other
environmental metagenomes (soils, sediments, marine
sponge) show a vast microbial biodiversity (Webster et
al., 2010; Lee et al., 2011; Valverde and Mellado, 2013;
Xu et al., 2014), with some studies describing more than
40 phyla (Valverde and Mellado, 2013; Xu et al., 2014).
The low microbial diversity present in the samples is
more than likely related to the structural complexity and
recalcitrant nature of bagasse, resulting in a somewhat
limited number of bacterial genera being capable of
colonizing this ecosystem. Notwithstanding this, clear
differences were observed in the structural composition
of microbial communities in our bagasse samples when
comparing community structures reported for other
lignocellulosic and non-lignocellulosic environments
(Figure 5). Talia et al. (2012) found 10 phyla of cellulolytic
bacteria inhabiting soil samples; while the rumen
microbiome analysis of both ruminants, Odocoites
virginianus and Rangifer tarandus, revealed seven and

Batista-Garcia et al. 031

Figure 6. Sample-basedrarefaction curves of 16S rDNA sequences amplified from sugarcane bagasse metagenome.
Rarefaction curves were calculated in RDP pipeline. Assignments of OTUs at different genetic distance levels were
obtained: 0.01 (strain), 0.03 (species), 0.05 (genus), 0.15 (class) and 0.28 (phylum).

four different phyla (Pope et al., 2012; Gruninger et al.,

2014). Only four phyla were described from the
metagenome analysis of the gut of Holotrichia parallela
(larve) (Huang et al., 2012). The previous studies are
consistent that Proteobacteria and Bacteroides are the
main
phyla
identified
in
these
lignocellulosic
environments. Proteobacteria have also been identified
as the main phylum in non-lignocellulosic metagenomes
isolated from Amazon River water (Ghai et al., 2011),
mangrove sediment, desert and artic soil (Xu et al.,
2014). Only the sediment sample collected from
mangrove showed a low bacterial diversity with four
phyla, while the Amazon River water revealed high
microbial diversity levels representing more than ten
phyla.
Rarefaction curves were obtained at different genetic
distance levels in order to compare the richness of the
genetic diversity as a function of number of sequences
(Figure 6). A typical trend was observed at higher genetic
distance level (0.01, 0.03, 0.05, 0.15 and 0.28). The
curve at a genetic distance level of 0.28 (phylum level)
showed a rapid asymptotic behaviour according to the
low phylum diversity found in the bagasse metagenome.
Curves representing the genus and class levels (0.05 and
0.15) demonstrated a sigmoid trend up to saturation.
Gradual increases observed in the rarefaction curves at
different levels (strain, species, genus) support the
taxonomic analysis proposed by the RDP and SILVAs

classifier algorithms. Rarefaction curves for the

sugarcane bagasse samples showed some levelling off
indicating that the library was representative and that the
estimations of microbial diversity were likely to be
accurate (Jackson et al., 2012).
The number of OTUs in the sample was determined
with Shannon and Chao1 diversity indexes being
calculated (Table 1). Identification of unique phylotypes
using RDP and SILVA pipelines showed 42 OTUs from
the metagenomic dataset analysis. Chao1 species
richness estimates predict 59.21 OTUs at 98%
sequence identity for the sugarcane bagasse sample
suggesting that 29% of the diversity was not sampled,
while on the other hand, Chao1 phylum and class
richness show that 100% of the phylum and class level
diversities were sampled. Notwithstanding this, our
results are comparable with other metagenomic studies
which report effective sampling values close to 70%,
while contrasting with those published results by authors
that only recovered sequences representing between 5060% of the metagenomic biodiversity (Rattanachomsri et
al., 2011; Jackson et al., 2012; Kanokratana et al., 2013).
Finally, we compared our results with those obtained
by the Thai group since these are the only ones currently
available. OTUs-level comparisons using a network map
was prepared in Qiime and its visualization showed that
there is a close relationships between the identified OTUs
in both sets of samples (our bagasse sample and the

032 Merit Res. J. Microbiol. Biol. Sci.

Table 1. Analysis of 16S rRNA sequences from sugarcane bagasse.

Chao1 species richness and Shannon diversity indexes were calculated at
different genetic distance levels.

Genetic distance
0.02
0.03
0.05
0.15
0.3

Richness
42
35
17
10
3

Chao1 richness
61.37
50.00
36.00
10.00
3.00

Shannon index
2.88
2.70
2.47
0.77
0.61

Figure 7. OTU network map showing OTU interaction between a sugarcane bagasse of this study (red network) and
Thai sugarcane bagasse (green network) (Rattanachomsri et al., 2011).

Thai bagasse sample) (Figure 7). The network map did

not reveal a limited degree of shared OTUs between both
bagasse samples. This suggests that the microbiome of
the bagasses was very similar. In addition while a high
convergence in network interaction was apparent, it
should be noted that no relationships between some
branches of the networks were observed.This
visualization is in accordance with the 16S rDNA-based
taxonomic classification obtained for the two sugarcane
bagasse samples.
Linking phylogenetic
capability

taxonomy

to

functional

The composition of microbial communities within a given

ecosystem is known to reflect the metabolic roles of
these bacteria (Kassen & Rainey 2004; Achtman &
Wagner, 2008; Jackson et al., 2012; Prosser, 2012). All
the genera identified in the bagasse samples contain at
least one species with the potential to degrade
lignocellulosic materials. Sugarcane bagasse is known to

contain very low levels of available nitrogen and

accessible organic matter. Its enriched composition of
lignin, cellulose and hemicellulose together with the low
free water content and other factors such as its acidic pH,
largely limits the ability of many bacteria to colonize the
bagasse resulting in somewhat limited microbial diversity
coupled with quite long natural degradation processes
(Goldbeck et al., 2014).
There was a predominance of aerobic, microaerophilic
and facultative anaerobic genera, while 16S rDNA gene
sequences related to strict anaerobic genera were not
observed. Bacterial genera which are involved in key
steps of biogeochemical nitrogen cyclling: N2 fixation
(Pseudomonas,
Burkholderia,
Rhizomicrobium),
amomonia oxidation (Burkholderia, Achromobacter),
nitrite and nitrate reduction (Bradyrhizobium, Dyella,
Acidobacterium) were also identified in this study (Torres
et al., 2011; Kundu et al., 2012; Kim et al., 2014; Zuleta
et al., 2014). Moreover, some of the species were related
to genera that have been shown to possess
thermotolerant properties (Faoro et al., 2012; Tajima et
al., 2012).

Batista-Garcia et al. 033

The phylum Acidobacteria comprises two classes:

Acidobacteria and Holophagae, ofwhich, Acidobacteria
comprises 19 species in seven genera. In this study we
only found sequences related with the Acidobacteria
class. Acidobacteria is one of the most widespread
bacterial phyla found in nature with members being
detected in soil, freshwater, marine, extreme and polluted
environments (Ward et al., 2009). Species of the
Subdivision 1 (Acidobacteria class) grow very slowly and
can often take weeks to form visible colonies. They also
can grow under conditions of nutrient deprivation and
over quite a broad pH range (Koch et al., 2008;
Pankratov & Dedysh, 2010; Mnnist et al., 2012).
Sugarcane bagasse is a moderately acidic and lownutrient environment and therefore not surprisingly,
perhaps around 34% of the analysed sequences were
identified as Acidobacterium, Terroglobus, Edaphobacter,
Granulicella and Telmatobacter (all genera belonging to
the Acidobacteria class). Representatives of the phylum
Acidobacteria have previously been reported to possess
a high potential to degrade plant biomass materials,
being able to metabolise complex carbon sources such
as xylan, crystalline and amorphous cellulose,
hemicellulose, pectin, starch and chitin (Ward et al.,
2009). Polymeric carbon is in fact a useful substrate for
the cultivation of Acidobacteria (Joseph et al., 2003;
Schoenborn et al., 2004). This explains the high
percentage of Acidobacteria, which we observed in the
sugarcane bagasse samples.
The phylum Acidobacteria is a non-autotrophic phylum
since enzymes related to autotrophic metabolism have
not been found in their members. Acidobacteria have
been reported to oxidize carbon monoxide (CO) and are
believed to significantly contribute to the removal of CO
present in the atmosphere (King et al., 2007; Ward et al.,
2009). This capability together with their capacity to
degrade complex lignocellulosic materials, suggests a
potential active role in carbon cycling by Acidobacteria
within the sugarcane bagasse. Indeed Acidobacteria are
known to be adapted to growth in environments with low
substrate availability and that as slow growing oligotrophs
their overall abundance within a microbial community is
strongly regulated by pH (Naethern et al., 2014).
Similarly, Acidobacteria are known to play a significant
role in nitrogen metabolism, with genes encoding
nitrogen transporters and regulatory proteins involved in
nitrogen metabolism, enzymes such as nitrate and nitrite
reductases, ammonium oxidase together with enzymes
involved in denitrification and enzymes involved in
nitrogen metabolism have been reported (Ward et al.,
2009; Lin et al., 2014). Thus, the metabolic plasticity of
members of the Acidobacteria phylum helps to ensure
their survival in low-nitrogen environments such as
sugarcane bagasse.
Acidobacteria also possess the capacity to produce
extracellular cellulose, in both microfibril and filamentous
forms; with cellulose synthesis being implicated in the

molecular mechanisms involved in survival under

stressful conditions, such as those
likely to be encountered in lignocellulosic rich
ecosystems. In addition the ability to synthesise
extracellular cellulose suggests that at least some
members of the Acidobacteria possess the ability to
survive repeated cycles of rehydration and drying (Ward
et al., 2009). The production of extracellular cellulose in
bacteria is also known to enhance biofilm production,
thereby promoting aeration and hydrations of
microenvironments by retaining water under dry
conditions (Ude et al., 2006; White et al., 2006). In
addition cellulose derived from acidobacterial metabolism
(cellulose synthesis and/or bagasse degradation) is
believed to promote colonization due to enhanced
adherence to the substrate, again through biofilmformation; thereby providing a loose network for nutrient
and water retention. Thus these physiological aspects of
extracellular cellulose production are likely to be
important for survival of the Acidobacteria in the
sugarcane bagasse piles.
A phylogenetic reconstruction was conducted
involving 60 sugarcane bagasse derived sequences from
the phylum Acidobacteria (class Acidobacteria) (Figure
8). A 16S rDNA sequence subset of members of all
species of this class was collected from NCBI. Overall,
the phylogenic analysis reveals that the majority of the
16S rDNA reads are not grouped with previously
described acidobacteria. The RR168 sequence shows no
phylogenetic relationship with the rest of the analysed
sequences (Figure 8). Only three 16S rDNA sequences
were directly grouped with species of the genera
Telmatobacter, Acidobacterium and Edaphobacter.
These results indicate the existence of a substantial
number of new ribotypes from the 16S rDNA sequences
obtained in this study. They may represent novel genera,
species and even strains given that the aforementioned
phylogenetic reconstruction shows a distant relationship
with the previously reported genera of the Acidobacteria
class.
Betaproteobacteria was the most represented class in
the phylum Proteobacteria, with the genus Burkholderia
(62 sequences) being the most representative
Betaproteobacteria present in the 16S rDNA library from
the bagasse. This genus comprises more than 70
described species (Mathew et al., 2014) with a wide and
intriguing biological versatility displayed across a broad
range of lifestyles (Compant et al., 2008; Via et al., 2011).
The genus has a worldwide distribution, it has been
described from diverse environments and has been
implicated in the degradation of xenobiotics, in plant
growth promotion, in biocontrol strategies in agriculture
and in symbiotic and non-symbiotic biological nitrogen
fixation (Suarez-Moreno et al., 2012; Zuleta et al., 2014).
Several species have also been described which possess
lignocellulolytic potential (Maki et al., 2011; Fujii et al.,
2012).

034 Merit Res. J. Microbiol. Biol. Sci.

Figure 8. Radial phylogenetic reconstruction for the phylum Acidobacteria. The relationship between 16s rDNA gene
dataset derived from sugarcane bagasse (only reads classified in Acidobacteria phylum) and 19 sequences of 16S
rDNA belonging at 19 species of the class are showed.

One of the most distinctive physiological characteristics

of Burkholderia is their ability to produce siderophores
2+
with the primary role of iron (Fe ) acquisition/chelation
(Vandamme et al., 2007). Siderophore-mediated nutrient
acquisition has been identified as an important survival
factor in members of the genus Burkholderia particularly
in metal-poor environments
(Thomas et al., 2007).
While siderophore production is typically induced under
iron-limiting conditions, siderophores can form complexes
2+
2+
2+
2+
with other essential metals (e.g. Mo , Mn , Co , Ni )
(Ahmen and Holmstrom, 2014). Moreover in various
bacteria, they have also been reported to be involved in
metal homeostasis and transport of other biologically
2+
2+
2+
2+
2+
relevant metals such as Cd ,Cu ,Ni ,Pb ,Zn (Schalk
et al., 2011). Thus siderophore production is a likely
strategy employed by Burkholderia to successfully
colonize low-metal environments such as sugarcane
bagasse, to not only allow metal acquisition but to also
promote mineral dissolution from fibrous materials within
the bagasse (Shirvani and Nourbakhsh, 2010). Thus
siderophore production by Burkholderia could play an
important role in bagasse degradation by facilitating Fe
and other metal uptake by the degrader microbial
populations (e.g. Acidobacteria and Burkholderia
2+
species) under Fe -limiting conditions.
In addition there are other important links between
microbial siderophore production and lignocelloluse.
Different species of Burkholderia have been reported to

produce catecholate and hydroxamate (Barelmann et al.,

1996; Kvitko et al., 2012; de los Santos-Villalobos et al.,
2012). Some of these siderophores have been implicated
2+
in the redox speciation of Fe , by facilitating the
3+
reduction of Fe . The reaction between hydrogen
peroxide and reduced Fe generates oxygen radicals,
which are known to play an important role in the
depolymerization of lignin, hemicellulose and cellulose
(Xu and Goodell, 2001; Arantes and Milagres, 2007).
While Burkholderia are known to protect themselves from
H2O2 through the production of catalases/peroxidases
(Lefebre et al., 2005).
It has been observed that siderophores (with the same
chemical nature that catecholate and hydroxamate) are
capable of decreasing lignocellulosic viscosity and
promote its transformation (Milagres et al., 2012). Thus
siderophore production by Burkholderia sp. within the
sugarcane bagasse would clearly facilitate an
important survival strategy both from a nutrient
uptake
and
a
lignocellulose
biotransformation
perspective.
Other minority taxonomic classes and subdivisions
were found to represent approximately 29% of the
analysed sequences. Cellulolytic activities have
previously been described in some of the identified
genera within this population (Lednick et al., 2000;
Wadell and Bang, 2008; Huang et al., 2012; Song et al.,
2013). For example a read classified as Methylovirgula

Batista-Garcia et al. 035

Figure 9. Radial phylogenetic reconstruction for the phylum Proteobacteria. The relationship between 16S
rDNA gene dataset derived from sugarcane bagasse (only reads classified inthe phylumProteobacteria) and 43
sequences of the 16S rDNA belonging to 43 type species and/or genera are shown.

sp. was found in the 16S rDNA library. This genus

contains only one described species to date namely
Methylovirgula ligni, an obligate acidophile with a pH
optimum between 4.5 and 5 (Vorober et al., 2009),
consistent with the moderate pH of bagasse.
A radial phylogenetic visualization shows the
relationships
of
the
sequences
classified
as
Proteobacteria (60% of the total) derived from sugarcane
bagasse metagenomic DNA (Figure 9). 16S rDNA
sequences (37 in total) belonging to type species of the
genera and classes identified in the full-taxonomic
fingerprint were collected in the NCBI. The phylogenic
analyses revealed that only four 16S rDNA bagasse
sequences were directly grouped with species,
Burkholderia caribiensis, Pseudomonas aeruginosa,

Methylovirgula ligni and Beijerinckia derxii. The

phylogenetic visualization for the bagasse sequences
identified as Proteobacteria demonstrates once again
that there is a clear predominance of bacteria showing a
truly distant relationship with the type species.
To the best of our knowledge, this is the first study in
Mexico and indeed in Central or South America
describing the complex composition of the microbial
populations inhabiting sugarcane bagasse feedstock
piles from sugar mills. Sugar mills in Mexico and in
Central and South America, represent an important
economic activity and bagasse generation is around 350
million metric tons per year on a worldwide basis
(http://www.fas.usda.gov). These production levels
generate serious pollution problems and demand the

036 Merit Res. J. Microbiol. Biol. Sci.

need for proper waste management strategies

(Kiatkittipong et al., 2009). These reasons support this
type of study, whereby exploration of microbial diversity
can shed further light on not only the likely metabolic
activities of the microbial populations present, but of their
potential in facilitating further degradation of this quite
recalacitrant biomass.
ACKNOWLEDGMENTS
This work was funded by grant CB-153789-Q from the
National Council for Science and Technology
(CONACyT)
from
the
Mexican
government:
http://www.conacyt.gob.mx, and Proyecto Biomasa
(Acuerdo 13/2007) of UAM-Cuajimalpa. RB-G and R-C
received a scholarship from CONACyT. Additionally RBG received a short-term fellowship from the Europe
Molecular Biology Organization (EMBO): ASTF503-2013
to perform part of this work at University College Cork, in
Ireland. We are grateful to Dr. Verawat Champreda
(Enzyme
Technology
Laboratory,
Bioresources
Technology Unit, National Center for Genetic Engineering
and Biotechnology, Thailand) who shared with us his
dataset derived from the structural metagenome analysis
from sugarcane bagasse and Jamie Fitzgerald for his
help with the network visualization.
REFERENCES
Achtman M, Wagner M (2008). Microbial diversity and the genetic
nature of microbial species. Nat. Rev. Microbiol. 6, 431-440.
Ahmed E, Holmstrm SJ (2014). Siderophores in environmental
research: roles and applications. Microb. Biotech.7, 196208.
Amorim JH, Macena TN, Lacerda GV, Rezende RP, Dias JC, Brendel
M, Cascardo JC (2008). An improved extraction protocol for
metagenomic DNA from a soil of the Brazilian Atlantic Rainforest.
Genet. Mol. Res.7, 12261232.
Arantes V, Milagres AM (2007). The effect of a catecholate chelator as
a redox agent in Fenton-based reactions on degradation of ligninmodel substrates and on COD removal from effluent of an ECF kraft
pulp mill. J. Hazard Mater.141, 273279.
Barelmann I, Meyer J, Taraz K, Budzikiewicz H (1996). Cepaciachelin,
a new catecholate siderophore from Burkholderia cepacia. J.
Biosc.51, 627630.
Brady SF (2007). Construction of soil environmental DNA cosmid
libraries and screening for clones that produce biologically active
small molecules. Nat. Protoc.2, 12971305.
Brink J, Vries P (2011). Fungal enzyme sets for plant polysaccharide
degradation. Appl. Microb. Biotech.91, 14771492.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K,
Madden TL (2009). BLAST+: architecture and applications. BMC
Bioinformatics10, 421-430.
Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman F,
Costello E, Fierer N, Pea A, Goodrich J, Huttley G, Kelley S,
Knights D, Koenig J, Ley R, Lozupone C, McDonald D, Muegge B,
Pirrung M, Reeder J, Sevinsky J, Turbaunh P, Walters W, Widmann
J, Yatsunenko T, Zaneveld Jm Knight R (2010). QIIME allows
analysis of high-throughput community sequencing data. Nat.
Methods7, 335336.
Chandel AK, Antunes FA, Anjos V, Bell M, Rodrigues L, Polikarpov I, de
Azevedo E, Bernardinello O, Rosa C, Pagnocca F, da Silva S
(2014). Multi-scale structural and chemical analysis of sugarcane
bagasse in the process of sequential acidbase pretreatment and

ethanol
production
by
Scheffersomyces
shehatae
and
Saccharomyces cerevisiae. Biotech. Biofuels7, 63-70.
Chen C, Ding S, Wang D, Li Z, Ye Q (2014). Simultaneous
saccharification and fermentation of cassava to succinic
acid by Escherichia coli NZN111. Bioresour. Technol.163, 100105.
Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, Brown CT,
Porras-Alfaro A, Kuske CR, Tiedje JM (2014). Ribosomal Database
Project: data and tools for high throughput rRNA analysis. Nucl.
Acids Res. 42, 633-642.
Compant S, Nowak J, Coenye T, Clment C, Ait Barka E (2008).
Diversity and occurrence of Burkholderia spp. in the natural
environment. FEMS Microb. Rev.32, 607626.
Curson AR, Burns OJ, Voget S, Daniel R, Todd JD, McInnis K, Wexler
M, Johnston AW (2014). Screening of metagenomic and genomic
libraries reveals three classes of bacterial enzymes that overcome
the toxicity of acrylate. PLoS ONE9, doi:10.1371.
De los Santos-Villalobos S, Barrera-Galicia GC, Miranda-Salcedo MA,
Pea-Cabriales JJ (2012).Burkholderia cepacia XXVI siderophore
with biocontrol capacity against Colletotrichum gloeosporioides.
World J. Microb. Biotech. 28, 26152623.
Demirbas A (2005). Bioethanol from Cellulosic Materials: A Renewable
Motor Fuel from Biomass. Energy Sources 27, 327337.
Dereeper A, Guignon V, Blanc G, Audic S, Buffet S, Chevenet F,
Dufayard J, Guindon S, Lefort V, Lescot M, Claveire J, Gascuel O.
(2008). Phylogeny.fr: robust phylogenetic analysis for the nonspecialist. Nucleic. Acids Res.36, 465469.
Engelbrektson A, Kunin V, Wrighton KC, Zvenigorodsky N, Chen F,
Ochman H, Hugenholtz P (2010). Experimental factors affecting
PCR-based estimates of microbial species richness and evenness.
ISME J.4, 642647.
Faoro H, Glogauer A, Couto GH, de Souza EM, Rigo LU, Cruz LM,
Monteiro RA, Pedrosa F (2012). Characterization of a new
Acidobacteria-derived moderately thermostable lipase from a
Brazilian Atlantic Forest soil metagenome. FEMS Microbiol. Ecol.81,
386394.
Felske A, Engelen B, Nbel U, Backhaus H (1996). Direct ribosome
isolation from soil to extract bacterial rRNA for community analysis.
Appl. Environ. Microb.62, 41624167.
Fujii K, Ikeda K, Yoshida S (2012). Isolation and characterization of
aerobic microorganisms with cellulolytic activity in the gut of
endogeic earthworms. Int. Microb.15, 121130.
Garrity GM, Holt GJ (2012). Bergeys Manual of Systematic
Bacteriology. (Williams ST, Sharpe ME & Holt JG, eds) New York,
USA.
Geng W, Huang T, Jin Y, Song J, Chang HM, Jameel H (2014).
Comparison of sodium carbonate-oxygen and sodium hydroxideoxygen pretreatments on the chemical composition and
enzymatic saccharification of wheat straw. Bioresour. Technol.161,
6368.
Ghai R, Rodriguez-Valera F, McMahon KD, Toyama D, Rinke R,
Cristina Souza de Oliveira T, Wagner Garcia J, Pellon de Miranda
F, Henrique-Silva F (2011). Metagenomics of the Water Column in
the Pristine Upper Course of the Amazon River. PLoS ONE 6,
e23785.
Ghio S, Lorenzo GS, Lia V, Talia P, Cataldi A, Grasso D, Campos E
(2012). Isolation of Paenibacillus sp. and Variovorax sp. strains from
decaying woods and characterization of their potential for cellulose
deconstruction. Int. J. Biochem. Mol. Biol.3, 352364.
Goldbeck R, Damasio AR, Gonalves TA, Machado CB, Paixo DA,
Wolf D, Mandelli F, Rocha GJ, Ruller R, Squina F M (2014).
Development of hemicellulolytic enzyme mixtures for plant biomass
deconstruction on target biotechnological applications. Appl. Microb.
Biotech.98, 8513-25.
Gruninger RJ, Sensen CW, McAllister TA, Forster RJ (2014).
Diversity of Rumen Bacteria in Canadian Cervids. PLoS ONE9,
e89682.
Guimares KA, Alves L, Sacramento TM, Frdric L (2012). Application
of succinylated sugarcane bagasse as adsorbent to remove
methylene blue and gentian violet from aqueous solutions Kinetic
and equilibrium studies. Dyes Pigments92, 967974.
Himmel ME (2008). Biomass Recalcitrance - Deconstructing the plant
cell wall for bioenergy. (Oxford: Blackwell Publishing).

Batista-Garcia et al. 037

Huang S, Sheng P, Zhang H (2012). Isolation and Identification of

Cellulolytic Bacteria from the Gut of Holotrichia parallela Larvae
(Coleoptera: Scarabaeidae). Int. J. Mol. Sci.13, 25632577.
Ionescu D, Siebert C, Polerecky L, Munwes Y, Lott C, Bizic-Ionescu M,
Quast C, Peplies J, Olivier F, Ramette A, Rodiger T, Dittmar T, Oren
A, Geyer S, Stark H, Sauter M, Licha T, Laronne J, de Beer D
(2012). Microbial and chemical characterization of underwater fresh
water springs in the Dead Sea. PLoS ONE7, e38319.
Jackson SA, Kennedy J, Morrissey JP, OGara F, Dobson AD (2012).
Pyrosequencing reveals diverse and distinct sponge-specific
microbial communities in sponges from a single geographical
location in Irish waters. Microb. Ecol.64, 105116.
Jadeja NB, More RP, Purohi HJ, Kapley A (2014). Metagenomic
analysis of oxygenases from activated sludge. Bioresour. Technol.
46, 799-809.
Johnson DB, Yajie L, Okibe N (2008). Bioshrouding: a novel approach
for securing reactive mineral tailings. Biotech. Lett.30, 445449.
Joseph SJ, Hugenholtz P, Sangwan P, Osborne CA, Janssen H (2003).
Laboratory cultivation of widespread and previously uncultured soil
bacteria. Appl. Environ. Microb.69, 72107215.
Kanokratana P, Mhuantong W, Laothanachareon T, Tangphatsornruang
S, Eurwilaichitr L, Pootanakit K, Champreda V (2013). Phylogenetic
Analysis and Metabolic Potential of Microbial Communities in an
Industrial Bagasse Collection Site. Microb. Ecol.66, 322334.
Kanwar N, Scott HM, Norby B, Loneragan GH, Vinasco J, Cottell JL,
Chalmers G, Chengappa MM, Bai J, Boerlin P (2014). Impact of
treatment strategies on cephalosporin and tetracycline resistance
gene quantities in the bovine fecal metagenome. Sci. Rep.4, 5100.
Kassen R, Rainey PB (2001). The ecology and genetics of microbial
diversity. Ann. Rev. Microb. 58, 207-231.
Kiatkittipong W, Wongsuchoto P, Pavasant P (2009). Life cycle
assessment of bagasse waste management options. Waste
Manag.29, 16281633.
Kim M, Hyun DW, Kim JY, Kim S, Bae JW, Park EJ (2014). Dyella
jejuensis sp. nov., isolated from soil of Hallasan Mountain in Jeju
Island. J. Microb.52, 373377.
King GM, Weber CF (2007). Distribution, diversity and ecology of
aerobic CO-oxidizing bacteria. Nat. Rev. Microb.5: 107118.
Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M,
Glckner FO (2013). Evaluation of general 16S ribosomal RNA
gene PCR primers for classical and next-generation sequencingbased diversity studies. Nucleic. Acids Res. 41, e1.
Koch IH, Gich F, Dunfield PF, Overmann J (2008). Edaphobacter
modestus gen. nov., sp. nov., and Edaphobacter aggregans sp.
nov., acidobacteria isolated from alpine and forest soils. Int. J. Syst.
Evol. Microb.58, 11141122.
Kundu P, Pramanik A, Mitra S, Choudhury JD, Mukherjee J, Mukherjee
S (2012). Heterotrophic nitrification by Achromobacter xylosoxidans
S18 isolated from a small-scale slaughterhouse wastewater. Biop.
Biosyst. Eng. 35, 721728.
Kvitko BH, Goodyear A, Propst KL, Dow SW, Schweizer HP (2012).
Burkholderia pseudomallei known siderophores and hemin uptake
are dispensable for lethal murine melioidosis. PLoS Negl. Trop. Dis.
6, e1715.
Lednick D, Mergaert J, Cnockaert MC, Swings J (2000). Isolation and
identification of cellulolytic bacteria involved in the degradation of
natural cellulosic fibres. Syst. Appl. Microb. 23, 292299.
Lee OO, Wang Y, Yang J, Lafi FF, Al-Suwailem A, Qian PY (2011).
Pyrosequencing reveals highly diverse and species-specific
microbial communities in sponges from the Red Sea. ISME J. 5,
650664.
Lefebre MD, Flannagan RS, Valvano MA (2005). A minor
catalase/peroxidase from Burkholderia cenocepacia is required for
normal aconitase activity. Microb. 151, 19751985.
Leis B, Angelov A, Liebl W (2013). Screening and expression of genes
from metagenomes. Adv. Appl. Microb. 83, 168.
Li LL, McCorkle SR, Monchy S, Taghavi S, van der Lelie D (2009).
Bioprospecting metagenomes: glycosyl hydrolases for converting
biomass. Biotech. Biof. 2, 10-17.
Li W, Godzik A (2006). Cd-hit: a fast program for clustering and
comparing large sets of protein or nucleotide sequences. Bioinf. 22,
16581659.

Lim HK, Chung EJ, Kim JC, Choi GJ, Jang KS, Chung YR, Cho KY, Lee
SW (2005). Characterization of a Forest Soil Metagenome Clone
That Confers Indirubin and Indigo Production on Escherichia coli.
Appl. Environ. Microb. 71, 77687777.
Lima MA, Gomez LD, Steele-King CG, et al. (Provide names of other
authors) (2014). Evaluating the composition and processing
potential of novel sources of Brazilian biomass for sustainable
biorenewables production. Biotech. Biof. 7, 10-19.
Lin X, Tfaily MM, Steinweg JM, Chanton P, Esson K, Yang ZK, Chanton
JP, Cooper W, Schadt CW, Kostka JE (2014). Microbial
community stratification linked to utilization of carbohydrates and
phosphorus limitation in a boreal peatland at Marcell
Experimental Forest, Minnesota, USA. Appl. Environ. Microb. 80,
35183530.
Liu S, Lu H, Hu R, Shupe A, Lin L, Liang B (2012). A sustainable woody
biomass biorefinery. Biotech. Adv. 30, 785810.
Maki M, Broere M, Leung KT, Qin W (2011). Characterization of some
efficient cellulase producing bacteria isolated from paper mill
sludges and organic fertilizers. Int. J. Biochem. Mol. Biol. 2, 146
154.
Mnnist MK, Rawat S, Starovoytov V, Hggblom MM (2012).
Granulicella arctica sp. nov., Granulicella mallensis sp. nov.,
Granulicella tundricola sp. nov. and Granulicella sapmiensis sp.
nov., novel acidobacteria from tundra soil. Int. J. Syst. Evol. Microb.
62, 20972106.
Mathew A, Eberl L, Carlier AL (2014). A novel siderophore-independent
strategy of iron uptake in the genus Burkholderia. Mol. Microb. 91,
805820.
Milagres A, Arantes V, Medeiros C, Machuca A (2002). Production of
metal chelating compounds by white and brown-rot fungi and their
comparative abilities for pulp bleaching. Enz. Microb. Techn. 30,
562565.
Miller GL (1959). Use of Dinitrosalicylic Acid Reagent for Determination
of Reducing Sugar. Anal Chem. 31, 426428.
Naether A, Foesel BU, Naegele V, Wust P, Weinert J, Bonkowski M, Atf
F, Oelmann Y, Polle A, Lohaus G, Gockel S, Hemp A, Kalko E,
Eduard K, Pfeiffer S, Renner S, Schoning I, Weisser W, Wells K,
Fischer M, Overmann J, Friedrich M (2012). Environmental Factors
Affect Acidobacterial Communities below the Subgroup Level in
Grassland and Forest Soils. Appl. Environ. Microb.78, 7398- 7406.
Nawrocki EP, Eddy SR (2007). Query-dependent banding (QDB) for
faster RNA similarity searches. PLoS Comput. Biol.3, e56.
Ondov BD, Bergman NH, Phillippy AM (2011). Interactive metagenomic
visualization in a Web browser. BMC Bioinf. 12, 385.
Pankratov TA, Dedysh SN (2010). Granulicella paludicola gen. nov., sp.
nov., Granulicella pectinivorans sp. nov., Granulicella aggregans sp.
nov. and Granulicella rosea sp. nov., acidophilic, polymer-degrading
acidobacteria from Sphagnum peat bogs. Int. J. Syst. Evol. Microb.
60, 29512959.
Patel DD, Patel AK, Parmar NR, Shah TM, Patel JB, Pandya PR, Joshi
CG (2014). Microbial and Carbohydrate Active Enzyme profile of
buffalo rumen metagenome and their alteration in response to
variation in the diet. Gene 545, 8894.
Pope PB, Mackenzie AK, Gregor I, Smith W, Sundset MA, McHardy
AC, Morrison M, Eijsink VG (2012). Metagenomics of the Svalbard
reindeer rumen microbiome reveals abundance of polysaccharide
utilization loci. PLoS ONE 7, e38571.
Prosser J (2012). Ecosystem processes and interactions in a morass of
diversity. FEMS Microb. Ecol. 81, 507-519.
Pruesse E, Peplies J, Glckner FO (2012). SINA: accurate highthroughput multiple sequence alignment of ribosomal RNA genes.
Bioinf. 28, 18231829.
Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies
J, Glckner FO (2013). The SILVA ribosomal RNA gene database
project: improved data processing and web-based tools. Nucleic.
Acids Res. 41, 590596.
Rasheed Z, Rangwala H, Barbar D (2013). 16S rRNA metagenome
clustering and diversity estimation using locality sensitive hashing.
BMC Syst. Biol. 7, 4-11.
Rattanachomsri U, Kanokratana P, Eurwilaichitr L, Igarashi Y,
Champreda V (2011). Culture-independent phylogenetic analysis of
the microbial community in industrial sugarcane bagasse feedstock

038 Merit Res. J. Microbiol. Biol. Sci.

piles. Biosc. Biotech. Biochem. 75, 232239.

Rezende CA, de Lima MA, Maziero P, Deazevedo ER, Garcia W,
Polikarpov I (2011). Chemical and morphological characterization of
sugarcane bagasse submitted to a delignification process for
enhanced enzymatic digestibility. Biotech. Biof. 4, 54-60.
Schalk IJ, Hannauer M, Braud A (2011). New roles for bacterial
siderophores in metal transport and tolerance. Environ. Microb. 13,
28442854.
Scheller HV, Ulvskov P (2010). Hemicelluloses. Ann. Rev. Plant Biol.
61, 263289.
Schoenborn L, Yates PS, Grinton BE, Hugenholtz P, Janssen PH
(2004). Liquid serial dilution is inferior to solid media for isolation of
cultures representative of the phylum-level diversity of soil bacteria.
Appl. Environ. Microb. 70, 43634366.
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, Amin
N, Schwikowski B, Ideker T (2003). Cytoscape: a software
environment for integrated models of biomolecular interaction
networks. Genome Res. 13, 24982504.
Shirvani M, Nourbakhsh F (2010). Desferrioxamine-B adsorption to and
iron dissolution from palygorskite and sepiolite. Appl. Clay Science
48, 393397.
Silva LF, Taciro MK, Raicher G, Piccoli RA, Mendona TT, Lopes M,
Gomez JG (2014). Perspectives on the production of
polyhydroxyalkanoates in biorefineries associated with the
production of sugar and ethanol. Int. J. Biol. Macromol. 14, 497-508.
Simon C, Daniel R (2009). Achievements and new knowledge
unraveled by metagenomic approaches. Appl. Microb. Biotech.85,
265276.
Simon C, Daniel R (2011). Metagenomic analyses: past and future
trends. Appl. Environ. Microb. 77, 11531161.
Siqueira G, Vrnai A, Ferraz A, Milagres A (2013). Enhancement of
cellulose hydrolysis in sugarcane bagasse by the selective removal
of lignin with sodium chlorite. Appl. Energy 102, 399402.
Song N, Cai HY, Yan ZS, Jiang HL (2013). Cellulose degradation by
one mesophilic strain Caulobacter sp. FMC1 under both aerobic and
anaerobic conditions. Bioresour. Tech. 131, 281287.
Surez-Moreno ZR, Caballero-Mellado J, Coutinho BG, MendonaPreviato L, James EK, Venturi V (2012). Common features of
environmental
and
potentially
beneficial
plant-associated
Burkholderia. Microb. Ecol. 63, 249266.
Tajima K, Han X, Satoh Y, Ishii A, Araki Y, Munekata M, Taguchi S
(2012). In vitro synthesis of polyhydroxyalkanoate (PHA)
incorporating lactate (LA) with a block sequence by using a newly
engineered thermostable PHA synthase from Pseudomonas sp.
SG4502 with acquired LA-polymerizing activity. Appl. Microb.
Biotech. 94, 365376.
Talia P, Sede SM, Campos E, Rorig M, Principi D, Tosto D, Hopp HE,
Grasso D, Cataldi A (2012). Biodiversity characterization of
cellulolytic bacteria present on native Chaco soil by comparison of
ribosomal RNA genes. Res. Microb. 163, 221232.
Tebbe CC, Vahjen W (1993). Interference of humic acids and DNA
extracted directly from soil in detection and transformation of
recombinant DNA from bacteria and a yeast. Appl. Environ. Microb.
59, 26572665.
Thomas MS (2007). Iron acquisition mechanisms of the Burkholderia
cepacia complex. Biometals 20, 431452.
Torres MJ, Bueno E, Mesa S, Bedmar EJ, Delgado MJ (2011).
Emerging complexity in the denitrification regulatory network of
Bradyrhizobium japonicum. Biochem. Soc. Trans. 39, 284288.
Tseng CH, Tang SL (2014). Marine microbial metagenomics: from
individual to the environment. Int J Mol Sci 15: 88788892.

Ude S, Arnold DL, Moon CD, Timms-Wilson T, Spiers AJ (2006). Biofilm

formation and cellulose expression among diverse environmental
Pseudomonas isolates. Environ. Microb. 8, 19972011.
Valverde JR, Mellado RP (2013). Analysis of metagenomic data
containing high biodiversity levels. PLoS ONE 8, e58118.
Vandamme P, Opelt K, Knchel N, Berg C, Schnmann S, De Brandt E,
Eberl L, Falsen E, Berg G (2007). Burkholderia bryophila sp. nov.
and Burkholderia megapolitana sp. nov., moss-associated species
with antifungal and plant-growth-promoting properties. Int. J. Syst.
Evol. Microb. 57, 22282235.
Vial L, Chapalain A, Groleau MC, Dziel E (2011). The various lifestyles
of the Burkholderia cepacia complex species: a tribute to
adaptation. Environ. Microb. 13, 112.
Vorobev AV, de Boer W, Folman L, Bodelier PL, Doronina NV, Suzina
NE, Trotsenko YA, Dedysh SN (2009). Methylovirgula ligni gen.
nov., sp. nov., an obligately acidophilic, facultatively methylotrophic
bacterium with a highly divergent mxaF gene. Int. J. Syst. Evol.
Microb. 59, 25382545.
Wadell E, Bang S (2008). Exploring the phylogenetic diversity of the
homestake gold mine, lead, SD. J. Ind. Microb. Biotech.36, 585
598.
Wang Q, Garrity GM, Tiedje JM, Cole JR (2007). Naive Bayesian
classifier for rapid assignment of rRNA sequences into the new
bacterial taxonomy. Appl. Environ. Microb. 73, 52615267.
Ward NL, Challacombe JF, Janssen PH, Henrissat B, Coutinho P, Wu
M, Xie G, Haft D, Sait M, Badger J, Barabote R, Bradley R, Brettin
T, Brinkac L, Bruce D, Creasy T, Daugherty S, Davidsen T, DeBoy
R, Detter J, Durkin A, Ganapathy A, Gwinn-Giglio M, Han C, Khouri
H, Kiss H, Kothari S, Madupu R, Nelson K, Paulsen I, Penn K, Rem
Q, Rosovitz M, Selengut J, Shrivastava S, Sulivan S, Tapia R,
Thompson L, Watkins K, Yang Q, Yu C, Zafar N, Zhou L, Kuske C
(2009). Three genomes from the phylum Acidobacteria provide
insight into the lifestyles of these microorganisms in soils. Appl.
Environ. Microb. 75, 20462056.
Webster NS, Taylor MW, Behnam F, Lcker S, Rattei T, Whalan S,
Horn M, Wagner M (2010). Deep sequencing reveals exceptional
diversity and modes of transmission for bacterial sponge symbionts.
Environ. Microb. 12, 20702082.
White AP, Gibson DL, Kim W, Kay WW, Surette MG (2006). Thin
aggregative fimbriae and cellulose enhance long-term survival and
persistence of Salmonella. J. Bacteriol. 188, 32193227.
Xu G, Goodell B (2001). Mechanisms of wood degradation by brown-rot
fungi: chelator-mediated cellulose degradation and binding of iron
by cellulose. J. Biotech. 87, 4357.
Xu Z, Hansen MA, Hansen LH, Jacquiod S, Srensen SJ (2014).
Bioinformatic approaches reveal metagenomic characterization of
soil microbial community. PLoS ONE 9, e93445.
Yun J, Kang S, Park S, Yoon H, Kim MJ, Heu S, Ryu S (2004).
Characterization of a novel amylolytic enzyme encoded by a gene
from a soil-derived metagenomic library. Appl. Environ. Microb. 70,
72297235.
Zuleta LF, Cunha CO, de Carvalho FM, Prioli L, Celso R, Martins F,
Miana S, Ivo J, Straliotto R, Hungria M, Ribeiro A (2014). The
complete genome of Burkholderia phenoliruptrix strain BR3459a, a
symbiont of Mimosa flocculosa: highlighting the coexistence of
symbiotic and pathogenic genes. BMC Genomics 15, 535.