Merit Research Journal of Microbiology and Bi Available online http://www.meritresearchjourn Copyright © 2016 Merit Research Journals
Original Research Article
Prokaryotic dive taxonomic an
Ramón Alberto Batista-García
1,
Jackson
4
, Alan
1
Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos. Cuernavaca, Morelos, Mexico.
2
Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos. Cuernavaca, Morelos, Mexico.
3
Instituto Tecnológico 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 Author’s E-mail: rbatista25@yahoo.com, rabg@uaem.mx +(52) 777-3297057 Ext. 4550 +(52) 7773297057 Ext. 3457
A 1 sa exp bac abu divi div
Bur
obs bac ma inh atte gen kno deg co imp cha Key
envi
INTRODUCTION
Plant biomass is the most important, widespread source of carbon on earth. plant biomass was considered as a waste its use was totally under exploited. recently there has been an increased integrated management of biomass, an sugarcane bagasse (Li
et al.
, 2009) applications, such as the production of eth biodiesel, oil, hydrogen, plastics, paper, p electricity and other raw materials, have d biomass (Demirbas, 2005). Particula bagasse obtained from sugar mills is
logical Sciences (ISSN: 2408-7076) Vol. 4(1) pp. 022-03 als.org/mbs/index.htm
sity from the culture-in alysis of a sugarcane b metagenome
2*
, Rocío Casasanero
2
, Alberto Alvárez- . W. Dobson
4,5
and Jorge Luis Folch-M
Abstract
6S rDNA metagenome library was construct ples and a phylogenetic analysis was condu lore the biodiversity present in the bagasse sa erial groups inhabiting this lignocellulosic ri ndant phyla were
Proteobacteria
and
Acidob
ion TM7 was the minor phyla. Overall, th rsity revealed the presence of 13 familie
kholderiaceae
and
Burkholderia
being the erved, respectively. Since we were also able erial genera, we could identify some specific be important in lignocellulose degradation. biting natural lignocellulosic substrates hav ntion, subsequently there is little information era present in these substrates resulting in a l wledge about the microbial process invo radation. This study provides some insight imunity structure in sugarcane bagasse from ortant sugar producing region in the world) and racterize these bagasse inhabiting populations.
words:
Metagenomic approaches, 16S rDNA ronments, bacterial communities. abundant and or many years, and as a result owever, more interest in the d especially of . A range of anol, methanol, olymers, resins, riven the use of rly, sugarcane urrently widely used in both paper and energ
al.
, 2012). Biomass-based biorefineri popularity in recent years. Cha in particularly with respect to break-through technologies bagasse into alcohol or other The main problem is the l saccharification of the baga process and it is often de depending on the chemical c (Chen
et al.
, 2014; Geng
et al.
,
, January, 2016
ependent gasse
astillo
3
, Stephen A. llol
2
d from sugar bagasse ted. This allowed us to ples and identify novel ch substrate. The most
cteria
, while Candidate e prokaryotic microbial and 17 genera, with ajor family and genus to detect some known metabolic pathways that Microbial communities to date received little bout the main bacterial ack of a comprehensive lved in lignocellulosic to the unique microbial Meso Americ (the most is the first such study to
biodiversity, cellulolytic production (Guimarães
et
s have gained immense
llenges still exist however, the lack of cost-effective o allow the conversion products (Himmel, 2008). ow yields obtained from sse. This is a complex ined as a critical step, mposition of the biomass 2014).
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
β
-(1
→
4)-linked backbone made up of xyloglucans, xylans, mannans and glucomannans, and
β
-(1
→
3, 1
→
4)-glucans (Scheller and Ulvskov, 2010). Different branching monomers (D-galactose, D-xylose, L-arabinose, D-glucuronic 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 (Guimarães
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
Batista-Garcia et al. 023
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 18°39'15.9", W 99°11'10.1") 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 -80
o
C. The sampling was performed during the dry season to avoid ecological succession due to rain washing. The meteorological station in the Zacatepec´s locality registered, an average temperature of 28
o
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 was 30 ± 1
o
C, while in the core it was 40 ± 1
o
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 ground and dried at 70
o
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 ethanol, baked dried at 100
o
C and finally weighted (material A). It was later burned at 540
o
C, cooled to room temperature in a desiccator and weighted (material B). The cellulose percentage was calculated using the following equation:
% = − ℎ ∗ 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
Premia la tua curiosità
Tutto ciò che desideri leggere.
Sempre. Ovunque. Su qualsiasi dispositivo.
Nessun impegno. Annulla in qualsiasi momento.