International Journal of Current Microbiology and Applied Science

Analysis of the cellulolytic capacity bacteria

isolated of Adonidia Merrillii rhizosphere.

Almaráz Soto Daniel Abisaí, Arellano Espino María Guadalupe, Calzada Estala Jairo Noé,
Cigarroa Huereca Karen, Escalera Villarreal Jorge Alejandro, Facio Hernández Karina,
Valenzuela Mendoza Anahí, Sánchez Muñoz Salvador.
AB STRACT

Residues rich in cellulose a complex polisacaride that can be

Key words saccharifies to obtain simples molecules,
monosaccharides. This project arises as a
Cellulose, cellulase, subject of considerable interest to reduce the
enzyme, ecological impact; unfortunately, waste and the
saccharifies, effective utilization of cellulose waste have not
degradation, been given proper importance. The production
polisacaride worldwide of nuts, it’s around millions of tons.
Melon production in Mexico passed of 543
thousand to 564 thousand tons only between
2015 and 2016, in volume. In the paper was made a research to find
optimal parameter for cellulose degradation, by Bradford and DNSA
techniques. Strain A showed the best behavior at pH 6 and CMC as
carbon source.

Introduction

Per year are produced invariable residues
rich in cellulose a complex polisacaride that
can be saccharified to obtain simples
molecules, monosaccharides. (Nandimath et
al, 2016). It is commonly degrades by an
enzyme called cellulose. This enzyme is
produced by several microorganisms, such
as bacteria and fungi (Doi, 2008; Teunissen
& Op den camp, 1993).
This project arises as a subject of
considerable interest to reduce the
ecological impact; unfortunately, waste and
the effective utilization of cellulose waste
have not been given proper importance
(Lynd et al., 2002). Cellulose-containing
waste can be of agricultural, urban or
industrial origin (Baldrian and Valaskova,
2008), such as walnut shells, which contain
between 25 and 30% cellulose (Sung and remove the indigo blue color and give a
Chen, 2002) The production worldwide of faded appearance. Another application of
nuts, it’s around millions of tons. cellulase is obtaining bioethanol which is
Nowadays, more than 80% of worldwide the great hope for a sustainable
nuts production is sold without shell development of biofuels. (Nandimath et al,
(SECOEX, 2012), which in 2007 was 2016).
produced in an amount of 8,875.90 tons
Material and methods
only in the region Lagunera de Durango and
Coahuila (SIAP 2007). Melon production in Collection of the sample
Mexico passed of 543 thousand to 564
thousand tons only between 2015 and 2016, 100 g of soil sample was collected in

in volume, Coahuila generate 119 thousand Adonidia Merrilli rhizosphere located on

187 tons; Sonora, 107 thousand 150 tons; Universidad Politécnica de Gómez Palacio

Michoacan, 93 thousand tons; Guerrero, 92 using pre-sterilized hermetic Ziploc bag and

thousand 196 tons, and Durango, 53 sterile spatula at 20 cm of deep and took to

thousand 945 tons (SAGARPA, 2017) laboratories (Illavarasi, 2014).

In addition, this is a waste that has Isolation of cellulolytic bacteria

not been given the importance as a raw
The cellulase producing bacterial
material, and also, wastewater that can be strain was serially diluted using distilled
considered as a source of cellulose. water and the diluted samples were plated
(Sternberg, 1976, Duff & Murray, 1996, on Nutrient Agar enriched with
carboxymethylcellulose (CMC) 1% by
Ding et al., 2006, Quiroz-Castañeda et al., spread plate method and were incubated for
2009). 96 hours at 37° C. The isolated colonies
were further transferred to test tubes with 5
Within the food industry, cellulase is mL of broth medium with the same
used for the extraction and filtration of fruit concentration of CMC and incubated for 72
hours at same temperature. After this, each
or vegetable juices, must filtration,
colony was plated on Nutrient Agar with
extraction of edible oils, among other CMC. (Zin et al., 2015).
applications. In the textile industry,
Experimental design
cellulases play an important role in the
fading of jeans fabrics that are used to Each one of isolated strains was
used for inoculate the reactors, the
concentrations change in ± 0.5%, in a pH of
6 and 7, using as a carbon source CMC and
Microcrystalline Cellulose. Fig. 0

Fig. 0 Experimental Design

Growth assay
Reactors were prepared using
Enzyme assay
Bushnell Haas broth with CMC and
Microcrystalline cellulose at .5%, 1%, and Concentration of cellulose was
1.5%. Initial pH of the production medium determined by the DNS method. In
was adjusted to 6 and 7. Reactors with 50 Eppendorf tubes was added 500 μL of
mL of work volume were autoclaved and sample, 200 μL of DNS reactive and the
then inoculated with 2.5 mL of tubes were placed in a water bath at 100° C
preinoculum. Biomass increase was for 5 minutes. The tubes get cool at room
measured by optical density at 600 nm in temperature, and then 300 μL of distilled
spectrophotometer Genesys 10 UV Thermo water was added to the tubes and mixed by
Scientific collecting samples each 24 hours inversion. The concentration was measured
for 7 days (Nandhini et al, 2014) in spectrophotometer at 545 nm (Miller,
1959)
Extracellular protein
DNA extraction protocol
Extracellular protein was measured
by Bradford technique at 595 nm with a DNA extraction was made by the
calibration curve made with egg albumin Moore, E 2004. Adapted protocol.
and an optical density of 595 nm (Bradford,
1976)
Results

Was achieved the growth of 40% of

the isolated strains, with which the reactors Fig.2 Cellular Growth (A-CMC 1.5%-pH7)
was inoculated. The strain A in pH 6,
demonstrated be constant as soon as it show
greater growth in OD Fig. 1 (0.501 CC
1.5%), in almost all the concentrations in
both carbon sources, being, except in CMC
1.5% Fig. 2 and CC 0.5% Fig. 3 in which
the greater growth (0.316, 0.342
respectively) it had the same strain but in
pH 7, the maximum point was at the fifth Fig.3 Cellular Growth (A-CC 0.5%-pH7)
day.
In the extracellular protein
production, the strain A in pH 6 showed
greater growth Fig. 4 (442.87 ppm) in CMC
0.5% and the strain B in pH 6 Fig. 5 (580.71
ppm) in CC 1.5%. The increases in the
proteins concentration were higher between
the sixth and seventh day. A significate
difference in pH is not appreciable.

Fig.1 Cellular Growth (A- CC1.5%-pH6)

Fig. 4 Proteins Production (A-CMC 0.5%- Fig.6 Cellulose consumption with higher
pH6) yield (A-CMC 1.5%-pH6)

Fig. 5 Proteins Production (B-CC 1.5%- Fig. 7 Cellulose consumption with higher
pH6) yield (A-CC 1.5%-pH7)

The results in reductor sugars and

the protein production match with the strain
A in pH 6 and CMC 1.5%, had a better
consumption Fig. 6 (81 a 352.05 ppm),
meanwhile in CC the best consumption Fig.
7 (71.95 a 123.104 ppm) was in pH 7 in
concentration of 1.5%. Fig. 8 DNA extraction strain B (last
channel)

Discussion
 In this research, was made an strains is the carbon source and the
analysis to found cellulolytic bacterias. concentration. At the same, the degradation
Bacteria show an attractive potential for the of cellulose was better with CMC as carbon
exploitation of cellulases and source.
hemicellulases due to their rapid growth
Future visions
rate, enzyme complexity and extreme
habitat variability (Maki et al., 2009). It will realize an isolation of
Cellulase can also be isolated from Cattle genomic DNA and then make an
waste (Sharma et al., 1985), woody biomass identification of the bacteria by sequencing
(Sleat et al, 1984), Cow manure (Palop et genomic DNA, for now the strain that we
al., 1989) and compost (Lee et al., 1975). morphology mind identified, it will give an
Cellulase producing Bacteria were found optimization using different carbon source
commonly in all environments which such like nut shell, melon shell, waste
enables them to degrade the cellulose found wood, paper, etc.
prevalent in waste materials (Bai et al.,
2012).
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