Sección II:

Revista peruana de biología 27(1): 067 - 078 (2020)

doi: http://dx.doi.org/10.15381/rpb.v27i1.17582
Industrial and Environmetal Biotechnology
ISSN-L 1561-0837; eISSN: 1727-9933 Article
Universidad Nacional Mayor de San Marcos
Aislamiento de Bacillus subtilis DCH4 termotolerante de la
fuente termal Chancos (Carhuaz, Perú) con potencial para
Trabajos presentados al I Congreso Internacional de degradar residuos lignocelulósicos agrícolas
Biotecnología e innovación (ICBi), 9 - 12 de julio de
2018, Universidad Nacional Agraria La Molina, Lima,
Perú. Isolation of thermotolerant Bacillus subtilis DCH4 from Chancos hot spring
Editoras: (Carhuaz, Peru) with potential to degrade lignocellulosic agriculture wastes
Ilanit Samolski Klein
Maria Lucila Hernández-Macedo
Gretty Katherina Villena Chávez
Abstract
It was isolated bacteria strains from three different types of samples: fresh water,
Autores in situ baits and ex situ enrichment. Serial dilutions were prepared and culture was
Carmen Tamariz-Angeles* 1 carried at 50 °C using a Basal-Saline medium. Isolated strains were screened for
ctamariz@unasam.edu.pe endoglucanase and xylanase activities with qualitative (Congo Red) and quantita-
https://orcid.org/0000-0001-5252-2757 tive (DNS) methods. Molecular 16S rDNA sequencing analysis was performed for
Jasmine Lázaro-Palomino 2 taxonomic identification. It was isolated 31 strains of which 14 showed hydrolytic
barinialazaro@gmail.com activities and belonged to Bacillus subtilis and Bacillus licheniformis species. More-
https://orcid.org/0000-0002-1017-0121
over, the strain B. subtilis DCH4 showed the highest endoglucanase activity at 45°C
Percy Olivera-Gonzales 1 and pH 5, and xylanase activity at 55°C and pH 6. Then, DCH4 was cultivated by
poliverag@unasam.edu.pe
https://orcid.org/0000-0002-8263-5901 submerged fermentation with two different media supplemented with sugar cane
bagasse, wheat straw, or quinoa stalk to evaluate its saccharification capability. Like-
Alberto Castañeda-Barreto 1 wise, it was screening its xylanase and cellulase genes employing specific primers;
alcastaneda24.up@gmail.com
https://orcid.org/0000-0001-9230-2177 the amplicons obtained were sequenced, and analyzed. It was found that, enzymatic
extracts of DCH4 prepared with cane bagasse or quinoa stalk media achieved the
Gretty K. Villena 3 highest endoglucanase and xylanase activities. According to molecular analysis of
gkvch@lamolina.edu.pe
https://orcid.org/0000-0001-8123-3559 genes involved in the hydrolytic process, the endoglucanase and xylanase activities
Correspondencia exhibited by DCH4 could be attributed to a bifunctional cellulase conformed by
endo-beta-1,4-glucanase (GH5) joined to cellulose binding domain 3 (CBM3), and
*Corresponding author an endo-1,4-beta-xylanase (GH11), respectively. Further transcriptomic experiments
1 Centro de Investigación y Recursos Genéticos de
Ancash (CIByRGA), Facultad de Ciencias, Universidad
would be considered to accomplish optimization strategies for biofuel production
Nacional Santiago Antúnez de Mayolo. from lignocellulosic biomass.
2 Facultad de Ciencias del Ambiente, Universidad Resumen
Nacional Santiago Antúnez de Mayolo. Se aislaron cepas de bacterias provenientes de tres tipos de muestras: agua fresca,
3 Laboratorio de Micología y Biotecnología (LMB), cebos enriquecidos in situ y ex situ. Se prepararon diluciones seriadas y el cultivo
Universidad Nacional Agraria La Molina (UNALM), fue a 50 °C usando un medio Salino-Basal. Las cepas aisladas fueron tamizadas para
Lima, Perú. las actividades endoglucanasa y xilanasa con métodos cualitativos (Rojo Congo) y
cuantitativos (DNS). Se usó el análisis molecular 16S rDNA para la identificación
taxonómica. Se aislaron 31 cepas, de las cuales 14 mostraron actividades hidrolíticas
Citación y pertenecían a Bacillus subtilis y Bacillus licheniformis. Además, B. subtilis DCH4
mostró la mayor actividad endoglucanasa a 45 °C y pH 5, y xilanasa a 55 °C y pH 6.
Tamariz-Angeles C, Lázaro-Palomino J, Olivera- Entonces, DCH4 se cultivó por fermentación sumergida con dos medios diferen-
Gonzales P, Castañeda-Barreto A, Villena GK. tes suplementado con bagazo de caña de azúcar, paja de trigo o tallo de quinua
2020. Isolation of thermotolerant Bacillus
subtilis DCH4 from Chancos hot spring (Car-
para evaluar su capacidad de sacarificación. También, se exploraron los genes de
huaz, Peru) with potential to degrade lig- xilanasa y celulasa mediante cebadores específicos; los amplicones obtenidos
nocellulosic agriculture wastes. I Congreso fueron secuenciados y analizados. Se encontró que los extractos enzimáticos de
Internacional de Biotecnología e innovación DCH4 preparados con bagazo de caña o tallos de quinua mostraron las actividades
(ICBi), Revista peruana de biología número endoglucanasa y xilanasa más elevadas. De acuerdo a los análisis moleculares de
especial 27(1): 067 - 078 (Marzo 2020). doi: los genes involucrados en el proceso hidrolítico, las actividades de endoglunacasa
http://dx.doi.org/10.15381/rpb.v27i1.17582 y xilanasa exhibidas por DCH4 podrían atribuirse a una celulasa bifuncional confor-
mada por una endo-beta-1,4-glucanasa (GH5) unida al dominio celulosa 3 (CBM3),
y una endo-1,4-beta-xilanasa (GH11), respectivamente. Posteriores experimentos
transcriptómicos podrían ser considerados para lograr estrategias de optimización
para la producción de biocombustibles a partir de biomasa lignocelulósica.
Palabras clave:
Hot spring; Bacillus; xilanasa; celulasa; lignocelulosa.
Keywords:
Hot spring; Bacillus; xylanase; cellulase; lignocellulose.

Journal home page: http://revistasinvestigacion.unmsm.edu.pe/index.php/rpb/index

© Los autores. Este artículo es publicado por la Revista Peruana de Biología de la Facultad de Ciencias Biológicas, Universidad Nacional Mayor
de San Marcos. Este es un artículo de acceso abierto, distribuido bajo los términos de la Licencia Creative Commons Atribución-NoComercial-
CompartirIgual 4.0 Internacional.(http://creativecommons.org/licenses/by-nc-sa/4.0/), que permite el uso no comercial, distribución y reproducción
en cualquier medio, siempre que la obra original sea debidamente citada. Para uso comercial, por favor póngase en contacto con revistaperuana.
biologia@unmsm.edu.pe.

067
 Tamariz-Angeles et al.

Introduction topes (Blumer-Schuette et al. 2014). In this context, the

The accelerated increasing of energy demand, deple- objective of this research was to explore the hot spring
tion of oil resources, and the need to reduce the emis- Chancos from Carhuaz (Perú) for the isolation and selec-
sion of greenhouse gases are the main reasons for devel- tion of thermotolerant native bacteria with cellulolytic
oping sustainable and renewable energy and chemical and xylanolytic activities which allow them to degrade
resources (Putro et al. 2016). In this context, current lignocellulose from agriculture wastes of the region of
research efforts are being focused on the production of Ancash, Peru.
biofuels and other useful chemicals from through lig-
nocellulosic biomass conversion. Biofuels derived from Material and methods
lignocellulosic biomass, particularly from agricultural Sampling and isolation.- Sampling and isolation
crops residues, are receiving attention around the world were performed as described by Tamariz-Angeles et al.
to attain multiple strategic objectives, such as mitigating (2014). The water samples were collected in a second-
climate change, energy security and the development of ary and rustic pool (9°19’09.39”S, 77°34’24.59”W) from
the rural economy (Nanda et al. 2015). In addition, agri- Chancos hot spring, province of Carhuaz, Ancash, Peru.
culture wastes are abundant, renewable and profitable The temperature and pH during the sampling were 47.3
resources, constituting an attractive source of carbon for ± 3.2 °C, and 6.5 ± 0.2, respectively.
the production of sugars and other chemicals (Cortes-
Tolalpa et al. 2017, Sarkar et al. 2012). Globally, the main For isolation was performed three types of samples:
crops are wheat, maize, rice, and sugarcane, which pro- fresh water for direct isolation, ex situ enrichment cul-
duce most of the lignocellulosic biomass (Carrillo-Nieves ture in the laboratory and in situ baiting-left for 14 days
et al. 2019). After rice straw, wheat straw is the second in the pool. Sugar cane bagasse, filter paper, microgranu-
largest lignocellulosic source in the world (Sarkar et al. lar cellulose or beechwood xylan were used for in situ
2012, Tian et al. 2018). In the other hand, production of and ex situ enrichment. All samples were diluted, and
quinoa is increasing due to its high nutritional value, as a each dilution was platting by duplicate on basal medium
consequence, large quantities of stalks accumulate as an salt (BMS1) supplemented with glucose (1% w/v) and
unused byproduct (Gil-Ramirez et al. 2018). Agar-agar (1.5% w/v). Incubation was at 50 °C by 15
days. Colonies with different morphology were isolated
Agricultural waste is composed of cellulose (30 – and purified. These strains were kept in the slanted tube
50%), hemicellulose (20 – 40%) and lignin (15 – 25%) with Trypticase Soy Agar (TSA) at 4 °C.
(Chen et al. 2013). Then, cellulose and hemicellulose are
fermentable polysaccharides, which can be converted Selection of cellulolytic and xylanolytic strains.-
into fermentable sugars, while lignin is a non-ferment- Each strain isolated was cultivated in 5 mL of Trypticase
able polyphenolic compound (Arora et al. 2015). Cellu- Soy Broth (TSB). Fresh culture (5µL) was inoculated on
lose is the major structural component of the plant cell Petri dishes containing BMS1 supplemented with Car-
wall, it is formed by linear chains of β-1,4-glucan that boxymethyl cellulose (1% w/v) or beechwood xylan
adhere to one another forming higher-order fibrous (1% w/v) and incubated at 50°C during 5 days (Tamariz-
crystalline structures (Kont et al. 2013). Whereas, hemi- Angeles et al. 2014) by duplicate. Qualitative cellulolytic
cellulose includes repetitive polymers of pentoses and and xylanolytic activity was tested using Congo Red meth-
hexoses, with different types of bonds, branches, and odology (Sirisena & Manamendra 1995). The clear zone
substitutions (Anwar et al. 2014, Kont et al. 2013). Cel- around the colony was measured as hydrolysis activities.
lulose fibrils are associated with hemicellulose, forming Microscopy analysis and biochemical test.- Strains
a complex network of polysaccharides, which in turn is of bacteria were grown in 5 mL of TSB (Trypticase Soy
embedded in the matrix of lignin (Ding et al. 2012). In Broth) by 16 hours, to follow according to the Gram stain
order to obtain the linked sugars, it is necessary to ef- protocol. Stained samples were observed under the im-
fectively break the blocked polysaccharides of the recal- mersion objective of light microscope (Primo star, Zeiss).
citrant lignocellulose (Bhalla et al. 2015), due that the
Biochemical characterization of strains included the
efficient conversion of lignocellulosic biomass to fer-
following tests: catalase production, urea metabolism,
mentable sugars is an essential step for its subsequent
acid production using glucose (methyl red test), citrate
conversion into value-added products such as bioetha-
metabolism, and amylase activity following universal
nol (Balat 2011, Wanmolee et al. 2014). In that sense,
protocols.
enzymes such as cellulases and xylanases have been
investigated (Blumer-Schuette et al. 2008); especially Taxonomic identification by 16S rDNA.- DNA of se-
those that are produced by thermophilic bacteria pres- lected strains was extracted with AxyPrep Bacterial Ge-
ent in hot environments - such as thermal sources- be- nomic Miniprep Kit (Axygen) following the manufacturer
cause they can be used to develop more efficient and protocol. Primers used to amplify were 27F/1492R (Re-
cost-effective processes to conversion of lignocellulosic insenbach & Longnecker 2000). PCR was performed as
biomass (Bhalla et al. 2013). These enzymes are charac- described by (Tamariz-Angeles et al. 2014) and ampli-
terized by their robustness, efficiency and conservation fication products were evaluated with agarose gel elec-
of their activity at high temperatures for long periods, trophoresis (1% w/v) with molecular marker GeneRuler
which allow them to degrade carbohydrates under harsh 1 kb DNA (Thermo Scientific). PCR products were sent
bioprocessing conditions that reflect their natural bio- for sequencing to Macrogen Korea Inc. Primers used in

068 Rev. peru. biol. 27(1): 068 - 078 (Marzo 2020)

 Isolation of thermotolerant Bacillus subtilis DCH4 from hot spring with potential to degrade lignocellulosic

sequencing reactions were 518F and 800R. Contigs of approximately 4 hours to obtain a constant weight. For
edited and assembled sequences were compared with the chemical-thermal pretreatment, 100 g of sample was
similar sequences of NCBI GenBank (The National Cen- mixed with 1 L of NaOH (24 g.L-1) and autoclaved at 121
ter for Biotechnology Information U. S. http://www.ncbi. °C, 1 atm, 20 minutes.
nlm.nih.gov/) using BLASTN. They were alignment with
Next, it was washed with tap water until pH 7 was
CLUSTALX v.2.0 and analyzed with MEGA5 using Neigh-
obtained, and it was allowed to dry at room temperature
bor-Joining methodology and Kimura 2-parameter with
until constant weight. It was ground and sieved with AST
1000 Bootstraps to make the taxonomic identification.
meshes at 2 mm diameter for submerged fermentation,
Optimum growth temperature and metabolic and 150 – 75 μm diameter for saccharification using
characterization.- Trypticase Soy Broth (5 mL) was in- crude enzymatic extract.
oculated with 250 µL of fresh culture (8 hours) diluted
Agriculture waste degradation and enzymatic ac-
to 0.1 OD620nm; and incubated in an orbital shaker at 180
tivity.- The strain with the high enzymatic activity was
rpm at 30, 35, 40, 45, 50, 55 or 60 °C. Two replicates of
evaluated in its ability to degrade pretreated substrates
each treatment were prepared. The growth was mea-
(sugar cane bagasse, quinoa stalk and wheat straw) It
sured with a UV-VIS spectrophotometer (Spectroquant®
included: (i) degradation/saccharification during sub-
Pharo 300, Merck) at 620 nm each 2.5 hours up to 12.5
merged fermentation, (ii) enzyme production with agri-
hours. For each temperature evaluated a growth curve
culture waste as carbon source, and (iii) saccharification
was obtained to calculate the growth rate during the ex-
of agriculture waste using the crude enzyme extract. For
ponential phase. A correlation between growth rate and
(i) and (ii), it was used substrates sieved at 2 mm, and for
temperature were obtained(Madigan et al. 1998) to find
(iii) substrates sieved at 75 μm of diameter.
the optimum temperature.
Submerged fermentation was performed using a bas-
Quantification of enzymatic activity.- Crude extracts
al salt medium (BMS2) with the following composition
were used for the determination of endoglucanase activity
(g·L-1): NaCl (5), MgSO4.7H2O (0.1), CaCl2.2H2O (0.6), KH-
(or CMCase), total cellulase (FPA) and xylanases.
2
PO4 (0.1), FeCl3.6H2O (0.01), nitrogen source (5) (pep-
To prepare enzymatic crude extracts, 25 mL of Luria tone or ammonium nitrate), carbon source (0.5 of agri-
broth (LB) supplemented with CMC or beechwood xylan culture waste, or 0.25 of CMC or 0.25 beechwood xylan),
(1%) was inoculated with 5% (v/v) of fresh culture (8h) and glycerol (0.5% v/v). The pH was adjusted to pH 6.5.
diluted to 0.1 OD620nm, and was incubated for 20 hours at
For saccharification during submerged fermentation
50ºC and 180 rpm. The crude extract was obtained by
(i), Flasks containing 50 mL of BMS2 were inoculated
centrifugation at 10.595 g, and filtration with bacterio-
with 2.5 mL fresh culture of 8 hours (0.08 – 0.1 OD620nm)
logical membrane (0.22 µm sterile membrane, Millipore,
and incubated at the optimum growth temperature of
Merck). All samples were prepared with three replicates.
selected strain at 180 rpm. Cultures were performed by
Enzymatic activities were quantified with the micro- duplicate for each strain.
well 3,5-dinitrosalicylic acid (DNS) method proposed
Cultures of 72, 144 or 216 hours were used to evalu-
by King et al. (2009) and modified by Tamariz-angeles
ate the degradation or saccharification ability during the
et al. (2014). Total cellulase activity and endoglucanase
submerged fermentation. Reducing sugars release in the
were calculated from a standard curve with anhydrous
submerged fermentations were measure by DNS method
glucose and with anhydrous xylose for xylanase activ-
described previously. Results were expressed as reduc-
ity. These curves were prepared at 7, 8, 9 and 10 min. of
ing sugar release per 1 mL of extract, and it was used
reaction time to get a better DNS colorimetric reaction
the glucose standard curve. Moreover, xylan and CMC
at 91.5 °C, which is the boiling temperature of water in
were considering as standard substrates, their values of
the laboratory condition (3160 m of altitude). Enzymatic
reducing sugar release were taken as 100% to compare
activity was expressed in enzyme unit (U), which is the
them with values obtained for agriculture wastes.
amount of enzyme necessary to release 1μmol of prod-
uct (reducing sugar) per minute under determined con- To evaluate the enzyme production using agriculture
ditions (temperature, pH). waste as carbon source (ii), BMS2 supplemented with ag-
riculture waste (2% w/v) and the better nitrogen source
Optimum pH and temperature, and thermal sta-
found in the previous assay was used. Culture conditions
bility.- The crude extract with the highest enzymatic ac-
were the same as above. Samples of 2 mL were collected
tivity was selected to evaluate temperature and optimum
by duplicate each 4 hours until 28 hours. Endoglucanase
pH, and its thermal stability following the methodology
and xylanase activity were quantified.
described by Tamariz-Angeles et al. (2014). For thermal
stability, the crude extract was incubated at 50, 60, 70, For saccharification assay using the diluted crude en-
and 80 °C during 1 hour previously to evaluate the re- zyme (iii), two enzymatic extracts were selected accord-
sidual enzymatic activity. ing to their enzymatic titers. 500 mL of crude enzymatic
extract was concentrated and partially purified using
Thermal-alkaline pretreatment of agriculture
centrifugal filters Ultra-15 3K (Amicon®, Merck Milli-
waste.- Agricultural waste (300 g) washed with tap wa-
pore, Ireland). Endoglucanase and xylanase activities of
ter, was dried at room temperature for 48 hours. For its
the concentrated extract were quantified and dilute with
complete drying, it was placed in an oven at 65 °C for

Rev. peru. biol. 27(1): 069 - 078 (March 2020) 069

 Tamariz-Angeles et al.

phosphate buffer (50 mM pH 6) to obtain 120 U/L-1 of situ enrichment bait of sugar cane bagasse yield more
endoglucanase activity (DE). Immediately, 35 mL of di- isolated and selected strains (Figure 1A). Tamariz-Ange-
luted DE was mixed with 1.4 g of substrate (agriculture les et al. (2014) found similar results for in situ enrich-
waste, CMC or beechwood xylan) and incubated in agita- ment with sugar cane bagasse, but they did not isolate
tion at 180 rpm and optimal temperature for 12, 24, 36, any strains from direct isolation from Huancarhuaz hot
48, 60 and 72 hours. Negative control without the crude spring water samples (near 70°C). Sugar cane bagasse
enzyme extracts were prepared. Reducing sugar release is known as a suitable substrate for production of cellu-
(RSR) was quantified using the DNS method Results lases and xylanases (Adsul et al. 2004, Camassola & Dil-
were expressed as RSR (mg) per gram of substrate (g). lon 2007, Ajijolakewu et al. 2013, Saha 2003).
Amplification of endoglucanase and xylanase
genes.- DNA was used for the amplification of endogluca- A
nase and xylanase genes. Primers CelF/CelR were used 10
for endoglucanases of Bacillus (Nurachman et al. 2010), Isolated
CbgF/CbglR for endoglucanase bgl C of B. licheniformis Selected

Number of strains
8
(Aftab et al. 2012), xynAoli1/xynAoli2 for xylanase xynA
gene of B. subtilis (Wolf et al. 1995), and XtF/XtR for cel- 6
lulase free alkaline xylanase gene of Bacillus sp. (Kumar
et al. 2011). The master mix of 50 µL was prepared in 1X 4
Dream Taq reaction buffer with 0.2 mM dNTP, 0.1 mM of
each primer, 0.5 U Dream Taq polymerase (Thermo Sci- 2
entific), and 5 ng of template DNA. PCR protocol for CelF/
CelR, xynAoli1/xynAoli2, and XtF/XtR includes 2 min of 0
None SCB FP MC BX SCB FP MC+FP BX
initial denaturation step at 94 °C, followed by 30 cycles Direct Ex situ In situ
of 1 min at 94 °C for denaturation, 1 min at 53 °C for an-
nealing, and 2 min at 72 °C for extension; and 8 min at B 30
72 °C for final extension. PCR protocol for CbglF/CbglR Endoglucanase (mm)
consists of 5 min of initial denaturation at 94 °C, followed 25 Xylanase (mm)
by 30 cycles of 30 s at 94 °C for denaturation, 30s at 54 °C
Diameter of hydrolysis halo (mm)

for annealing, and 90s at 72 °C for extension; and 10 min 20

at 72 °C for final extension.

15
PCR products were evaluated by agarose gel electro-
phoresis (1% w/v), and sequenced in Macrogen Korea Inc. 10

Both, consensus DNA sequence and translated amino acid

5
residues chain were prepared with Codoncode aligner
v.8.0.2. The consensus DNA sequences were analyzed with 0
BlastN; adding similar sequences of Genbank, sequences
ICHB1

ICHB2

ICHB4

ICHB6

ICHB7

ICHP1
ECHC4
DCH2

DCH3

DCH5

ICHB3

ICHB5
DCH1

DCH4

were aligned with ClustralX v.2.0, and the phylogenetic

tree was prepared with MEGA7 using Neighbor-Joining,
Kimura 2-parameter, and 1000 Bootstraps methodology. Figure 1: Isolation and selection of thermotolerant bacterial strains
The translated amino acid residues chains were analyzing of Chancos hot spring. A. Isolated and selected bacterial strains (SCB,
in Protein Blast and Protein SmartBlast online platform of sugar cane bagasse; FP, filter paper; MC, microgranular cellulose; BX,
NCBI (http://www.ncbi.nlm.nih.gov/). According to Yang birchwood xylan); B. Diameter of the clear zone around selected
et al. (2009), the model of protein structure was predic- strains. Values represent the mean of four replicates ± SD.
tive using Swiss-Model Server online [http://swissmodel.
(https://swissmodel.expasy.org). Molecular identification and biochemical charac-
terization test.- Edited ribosomal 16S sequences were
Statistical analyses.- All qualitative and quantitative
assembled until 1537 bp size corresponding to the com-
data were prepared with four or six replicates, means
plete gen. From BlastN analyses, 99.14 – 99.87% range
and standard deviation were calculated. ANOVA to Lin-
of identity with sequences of type collection of Genbank
ear Models, T-student, and Duncan’s analyses were used
was found. According to the phylogenetic tree (data not
to evaluate significant differences between treatments (p
showed), 12 strains were identified as B. licheniformis and
< 0.05 or p < 0.01).
only two strains were B. subtilis (Table. 1). DNA sequences
were submitted to Genbank to get the accession numbers
Results and discussion MK524697 – MK564710 (Table. 1). In addition, micro-
Isolation and selection.- Water samples from Chan- scopic analysis showed that all selected strains were rod
cos hot springs were used for isolation cellulase produc- shape and spore-forming gram-positive bacteria. Similar
ing bacteria. From 31 bacterial strains isolated from three studies found that some species of Bacillus are natural
different types of samples, 14 strains were screened and inhabitants of hot springs around the word (Acharya &
selected as endoglucanase and xylanase producers (Fig- Chaudhary 2011, Tamariz-Angeles et al. 2014) with useful
ure 1). Direct culture from freshwater samples and in applications in industry (Mehta & Tulasi 2013).

070 Rev. peru. biol. 27(1): 070 - 078 (Marzo 2020)

 Isolation of thermotolerant Bacillus subtilis DCH4 from hot spring with potential to degrade lignocellulosic

Table 1. Molecular and biochemical test characterization of selected strains from Chancos hot spring.
16S DNAr identification GenBank Accession Growth temperature (C°) Metabolic characteristics
Strain
Specie Identity (%)* number 16S DNAr Optimum Interval Citrate Urea Catalase RM Amylase
ICHB1 B. subtilis 99.33 MK564697 45 30-55 + + + + +
ICHB2 B. licheniformis 99.8 MK564698 50 30-55 + + - - -
ICHB3 B. licheniformis 99.46 MK564699 50 30-55 - + + - -
ICHB4 B. licheniformis 99.53 MK564700 50 30-55 - + + - -
ICHB5 B. licheniformis 99.87 MK564701 50 30-55 - + + - -
ICHB6 B. licheniformis 99.66 MK564702 50 30-55 - + + - -
ICHB7 B. licheniformis 99.06 MK564703 50 30-55 - + + - -
ICHP1 B. licheniformis 99.87 MK564704 50 30-55 - + - - -
DCH1 B. licheniformis 99.87 MK564705 50 30-55 - + + + -
DCH2 B. licheniformis 99.66 MK564706 50 30-55 - + + - +
DCH3 B. licheniformis 99.87 MK564707 50 30-55 - + + - -
DCH4 B. subtilis 99.19 MK564708 45 30-50 + + + + +
DCH5 B. licheniformis 99.53 MK564709 45 30-55 - + + - -
ECHC4 B. licheniformis 99.14 MK564710 45 30-50 - + - - -
*, it shows the highest percentage obtained using BlastN platform and type culture collection of Genbank (www.ncbi.nlm.nih.gov). RM, red methyl test.

The optimum growth temperatures found of selected was higher with xylan in most of the strains (Fig 2B). Ac-
strains (Table 1) suggest that most of B. licheniformis cording to Jonnadula et al. (2018), xylan or intermediates
strains are moderate or facultative thermophiles accord- produced during xylan degradation might be involved in
ing to the classification proposed by Mehta and Tulasi cross-induction of endoglucanase, and some of the heter-
(2013). Likewise, B. subtilis strains exhibited optimum ologous oligosaccharides structurally related may cause
growth temperature at 45 °C, showing their thermotol- cross-induction of unrelated carbohydrolases.
erant condition. A similar result has been reported for
In submerged fermentation, enzymatic production
a strain of B. subtilis isolated from YangLing hot springs
could be influenced by nutritional (especially carbon
in China (Li et al. 2008). According to biochemical tests
and nitrogen sources) and environmental culture factors
performed, all strains were urease producers and most
(Acharya & Chaudhary 2011). In some species of Bacil-
of them were catalase positive. A reduced number of
lus cultured with organic source of nitrogen increased
strains were citrate positive, and acid producers. Finally,
the production of xylanases, while (NH4)2SO4 could in-
only two strains showed amylolytic activity.
hibit secretion and synthesis of xylanases (Sepahy et al.
Determination of enzymatic activity.- Both endo- 2011). According to the quantitative method, B. subti-
glucanase and xylanase activities of selected strains were lis DCH4 showed better results for both endoglucanase
tested using DNS method (Figure 2A-B). From 31 isolates, and xylanase activities with tryptone and yeast extract
only six strains showed endoglucanase activity, and 14 se- as a nitrogen source. Additionally, the extract LB-CMC of
lected strains displayed xylanase activity. It was observed B. subtilis DCH4 showed the best total cellulase activity
that endoglucanase activity was significantly induced by reaching 108 ±14 UL-1. This strain was selected for agri-
xylan (Fig 2A) while xylanase activity promoted by CMC culture waste degradation assays.

A NS
LB-X
B 2500
NS

LB-X
1250
LB-CMC LB-CMC
2000

1000
Endoglucanase activity (U.l-1)

1500
Xylanase activity (U.l-1)

750

1000
500
NS
NS NS NS

500
250

0 0
ECHC4

ICHB1

ICHB2

ICHB3

ICHB4

ICHB5

ICHB7
DCH1

DCH2

DCH3

DCH4

DCH5

DCH2 DCH4 ECHC4 ICHB1 ICHB2 ICHB3

ICHB6

ICHP1

Figure 2: Hydrolytic activities of selected strains. A. Endoglucanase activity of crude extract; and B. Xylanase activity of the crude extract.
LB-X, Luria broth-xylan; LB-CMC, Luria broth-Carboxymethyl cellulose; NS, none significant t-student with 99% of confidence. Values repre-
sent the mean of six replicates ± SD.

Rev. peru. biol. 27(1): 071 - 078 (March 2020) 071

 Tamariz-Angeles et al.

Optimum pH and temperature; and thermal sta- the neutralization step after acid or alkali pre-treatment
bility.- According to quantitative enzymatic assay, the of biomass (Bhalla et al. 2013).
strain DCH4 was selected to evaluated hydrolytic activi-
In the other hand, thermal stability is the capacity of
ties using agriculture waste. Due that enzymes require
an enzyme to retain its active structural conformation
optimum environmental conditions such as temperature
at a selected high temperature for a prolonged period
and pH (Anwar et al. 2014), these parameters were de-
(Bhalla et al. 2013). According to figure 3C, crude enzy-
termined for endoglucanase and xylanase of B. subtilis
matic extract of DCH4 retained 37 %, 25, 25% and 25%
DCH4 as well as thermal stability (Fig. 3). Both, tempera-
of endoglucanase activity after 1 h of incubation to 60,
ture fluctuation and pH changes can affect the integrity
70 and 80°C respectively. However, CelDR −cataloged
of the secondary, tertiary and quaternary structure of
with high thermal stability retained 70% of its cellulase
the enzyme protein, denaturing and inactivating it, thus
activity after incubation at 75°C for only 30 min. (Li et
affecting enzymatic activity (Chen et al. 2016). Accord-
al. 2008). Our results suggest that endoglucanase DCH4
ing to the results, the optimal temperatures for both en-
could be exhibited moderate thermal stability.
zymes were around 55 oC while optimal pH was in the
range 5-6 (Fig. 3A and 3B). But endoglucanase activity of About xylanase activity, thermostable xylanases
DCH4 showed more thermal stability (Fig. 3C). which can tolerate extreme conditions pH, and high salt
concentrations are of great interest for bioprocessing
Specifically, endoglucanase activity of crude extract
(Bhalla et al. 2013). The optimum xylanase activity was
of B. subtilis DCH4 showed optimum endoglucanase ac-
55°C, retaining 87% and 74% of its activity at 50°C and
tivity between 45 – 55°C and retaining more than 70% of
60 °C, respectively (Fig. 3A). The optimum pH was 6, re-
activity when the hydrolysis was performed at 70°C (Fig.
taining 51, 72 and 40 % of activity at pH of 5, 7and 8 re-
3A). This feature shows that DCH4 has endoglucanase
spectively (Fig. 3B). But the crude extract of DCH4 lost its
activity useful for some industrial bioprocess, especially
xylanase activity after 1 hour of incubation at 60°C. Simi-
in lignocellulose conversions that has several limitations
lar results were described for xylanase of a mesophyll
when being carried out at ≤50 °C (Bhalla et al., 2013).
B. subtilis R5 (Jalal et al., 2009). However, thermostable
Li et al. (2008) purified endoglucanase of family 5 (Cel-
xylanases from thermo-alkaliphilic, thermo-acidophil-
DR) from B. subtilis DR isolated from a hot spring, which
ic, and thermo-halophilic bacteria have been reported
showed 40°C optimum temperature and retained more
(Bhalla et al. 2013).
than 80% of its maximum activity at temperatures be-
tween 45°C and 70°C. Another condition to get optimum Agriculture waste degradation and enzymatic
enzymatic activity is the pH. Adaptability of thermophilic activity.- Biochemical conversion of lignocellulosic sub-
microbes and thermostable enzymes to a wide range of strates through saccharification and fermentation is a
pH also makes them suitable candidates bioprocess- major pathway for bioethanol production (Balat 2011).
ing (Bhalla et al. 2013). Hence, DCH4 showed optimum Then, three lignocellulosic agriculture wastes (sugar-
endoglucanase activity at pH 5, but retained more than cane bagasse, wheat straw, quinoa stalks) were selected
52% activity between 5 and 7.8 of pH (Fig. 3B) DCH4 and to evaluate the degradation ability of B. subtilis DCH4.
certain stability at pH range 4 – 7. For lignocellulose bio- Then, saccharification and enzyme production assays
conversion, acidophilic and alkalophilic thermotolerant were performed to evaluate the potential of B. subtilis
enzymes have several advantages because could avoid DCH4.

A 3500 B C A Xyl
4000 a 100 a
A EG
3000 3500
B
80
Enzymatic activity (UL-1)

Enzymatic activity (UL-1)

2500 3000 b
Residual activity (%)

C
2500
2000 60
D c
2000
1500 a d B
ab ab 40
b E 1500
d C C
1000 A
e c 1000 B B
F 20
500 D
500 C b b b
G
0 0 0
40 45 50 55 60 65 70 4 5 6 7 8 50 60 70 80
Temperature (°C) pH Temperature (°C)

Figure 3. Temperature and pH condition for enzymatic activity of crude extract of B. subtilis DCH4. A. optimum temperature, B. op-
timum pH, and C. thermal stability (Residual activity after incubation for 1 hour at 50, 60, 70°C). Values represent mean of six replicates ±
SD, and means with different letters show significance difference (p<0.05).

072 Rev. peru. biol. 27(1): 072 - 078 (Marzo 2020)

 Isolation of thermotolerant Bacillus subtilis DCH4 from hot spring with potential to degrade lignocellulosic

A 4500 a
B 70 α α
SCB
b
WS β
4000 60
QS

3500 X
β
50 α
CMC
a
Reducing sugar (µg.mL-1) 3000 a a
c µ
40 b µ
2500
β β

Percentage (%)
b b b
d
2000 30
e c c
1500 g g f
20
h
h
1000 h
h,i
j j i j
j 10
500 k l
l
m m m mm mmm m
0 0
P AN P AN P AN X (100%) CMC (100%) X (100%) CMC (100%) X (100%) CMC (100%)

72 h 144h 216h 72 h 144 h 216 h

C 2000
D 2000 a
SCB
SCB b
WS WS
a*
QS c QS

d
b* e
1500 1500
c*
Endoglucanase activity (U.L-1)

d* e* g f
f*

Xylanase activity (U.L-1)

h
i* h*
j* k*
1000 l* 1000
m* i
j k
n* n*

l
l
o* m
m
500 500
12 16 20 24 28 32 36 40 44 48 12 16 20 24 28 32 36 40 44 48

hours hours
E a F a
G *
b *
140 SCB b 140 SCB16
d c
QS 35 QS16
e
WS c
120 120 *
X d
30
CMC
100 100
e 25 *
*
Reducing sugar (mg.g-1)

Reducing sugar (mg.g-1)

f
f
80 80
20
Percentage (%)

60 60 a
a b 15
b a*
d* c* c
40 e* b* 40
c d 10
f* e
d a
e c b a
20 f c b 20 f e dD a* 5
d Ff E C
c* Bb* A
e A d*
f B f* e*
E D C
F
0 0 0
12 24 36 48 60 72 12 24 36 48 60 72 12 24 36 48 60 72
Time (hours) Time (hours) Time (hours)

Figure 4: Agriculture waste degradation and enzymatic activity of a selected strain. Submerged fermentation: A. Reducing sugars release;
B. Percentage of reducing sugars released from agriculture (considering 100% RSR of CMC or beechwood xylan). Enzyme production by
submerged fermentation in medium with agriculture wastes: C. Endoglunacase activity; D. Xylanase activity. Saccharification using diluted
crude enzyme: E. Reducing sugar released using crude enzyme of sugar cane bagasse fermentation; F. Reducing sugar released using crude
enzyme of quinoa stalk fermentation; and G. Percentage of saccharification of wheat straw related to xylan (100%). P, peptone; AN, ammo-
nium nitrate; SCB, sugar cane bagasse; QS, quinoa stalks; WS, wheat straw; X, xylan of beechwood; CMC, carboxymethyl cellulose. Values
represent mean of six replicates ± SD, and means with different letters show significance difference (p<0.01). *, significance difference (p<0.01)

First, saccharification during submerged cultures was found that sugar cane bagasse released 44% of reducing
measure as reducing sugars release of different lignocel- sugar compared with xylan during the first 72 hours, and
luloses substrates (sugarcane bagasse, wheat straw, qui- the other substrates attained percentages at around to
noa stalks, CMC and xylan). It was found that B. subtilis 30%. These results showed that B. subtilis DCH4 not only
DCH4 hydrolyzed all carbon sources substrates assayed have the ability to hydrolyze commercial substrates, fur-
(Fig.4A). Due that xylan and CMC are common substrates thermore, it can degrade natural pre-treated substrates
for cellulase and xylanase assays (Acharya & Chaudhary such as sugar cane bagasse, quinoa stalks, and wheat
2011, Lynd et al. 2002, Sharma & Kumar 2013) and are straw. Additionally, the influence of nitrogen source in
less recalcitrant than agriculture wastes, they have been the saccharification yield was evaluated using organic
used as a reference substrate to evaluate the level of deg- and inorganic source. Peptone allows the higher yield of
radation ability calculating an index expressed yield of reducing sugars while ammonium nitrate produced the
reducing sugar released as percentage (Fig. 4B). It was lower release of reducing sugars. Nitrogen source used

Rev. peru. biol. 27(1): 073 - 078 (March 2020) 073

 Tamariz-Angeles et al.

in the production medium is one of the major factors cal step for bioethanol production because various fac-
affecting the level of enzyme production (Kumar et al. tors influence yields of monomer sugars from lignocel-
2016). Organic nitrogen influences the formation of ex- lulose such as temperature, pH, enzyme doses, substrate
tracellular enzymes as a precursor for protein synthesis concentration (Sarkar et al. 2012).
(Bajaj et al. 2011). Furthermore, peptone has been re-
Endoglucanase and xylanase genes.- For improv-
ported as a good supplement for microbial growth and
ing enzymatic lignocellulose hydrolysis it is necessary
xylanase production (Raj et al. 2013, Shanthi & Roymon
to know which enzymes are involved in the bioprocess.
2018). The largest amount of reducing sugars released
Some researches of hydrolytic B. subtilis have been fo-
occurred at 72 hours and decreased after that time prob-
cused on fermentation optimization to obtain higher
ably by the depletion of the macro and micronutrient in
yields, and increase recovery rates employing genetic
the growth medium (Bibi et al. 2014). Moreover, hydro-
engineering (Sarkar et al. 2012). In this sense, there are
lysis of carbon substrates could provide an easily acces-
some reports of characterization of complete genomes,
sible source of simple sugars for bacterial growth, which
genes and proteins and cloning to get more efficient en-
decreased in the culture medium in the resting 144 and
zymes (Yang et al. 2009, Nurachman et al. 2010, Vallenet
216 hours. Bibi et al. (2014), suggest that high yield of
et al., 2017, Chang et al. 2018).
the final product (xylose) produced after the degrada-
tion of xylan during fermentation could be responsible Then, some genes involved in the enzymatic activity of
of enzyme inhibition due to feedback regulation. DCH4 were explored. Four sets of primers proposed for
Bacillus genus were used but only two of them allowed
The use of inexpensive agriculture residues as sub-
the amplification at performed PCR conditions. Primer
strates for the production of industrial enzymes is a
set that amplified endoglucanase gene was CelF/CelR,
significant way to reduce the cost of the overall process
which was designed using conserved sequences from
and literature shows that several microbial species have
GenBank of endoglucanase of genus Bacillus (Nurachman
been reported to use lignocellulose for xylanase produc-
et al. 2010). Primer set that amplified xylanase gene was
tion (Bhalla et al. 2015). Related with that, it was evalu-
xynAoli1/xynAoli2 designed for xynA gene from Bacillus
ated the production of hydrolytic enzymes (cellulases
subtilis (Wolf et al. 1995). CbgF/CbglR and XtF/XtR prim-
and xylanases) using a culture medium supplemented
er sets did not produce any amplification products.
with agricultural waste as the sole carbon source, find-
ing that sugar cane bagasse, wheat straw and quinoa The DNA sequence of the cellulase gene was edited
stalk induced the production of endoglucanase and xy- to 1401 pb, deposited to Genbank as AN MK644599, and
lanase (Fig. 4C, 4D). The highest production in all cases compared with GenBank data using BlastN. It was found
was attained at 16 hours of culture, and quinoa stalk 99.57% of identity with CDS of completed genome of B.
produces the highest endoglucanase and xylanase activi- subtilis (AN: CP0237555, CP011534), 98.86% with cellu-
ties (1865.67 U·L-1 and 1986.43 U·L-1, respectively) and lase gene of B. subtilis (EF070194, JQ346089), 98.79 and
sugar cane bagasse reached similar xylanase activity 98.72% with endo-beta-1,4-glucanase gene of B. subtilis
(1963.82 U·L-1). Enzyme production is dependent on the (MH923517, KC477685, respectively), and 98.50% with
nature of carbon source, favorable degradability, chemi- endo-beta-1,4-glucanase gene of B. amyloliquefaciens
cal composition, physical associations, accessibility of strain IARI-SP-2 (KF240848). Phylogenetic tree obtained
substrate, and presence of some nutrients (Kumar et al. is shown in figure 5A. The translated chain had 463 ami-
2016). Thus, when microorganisms degrade different no acid residues and was compared with other proteins
polysaccharides, they released small, medium or large using Standard Blast Protein. It was found that 35 - 281
sized oligosaccharides that enter the cell and induce the fragment showed high identity with the cellulase - gly-
expression of enzymes responsible for respective poly- cosyl hydrolase domain family 5 (AN pfam0150); while
saccharide degradation (Jonnadula et al. 2018). In conse- the interval of 341 to 422 has a high identity with cel-
quence, the chemical composition of each type of agricul- lulose binding domain family 3 (CBM-3) (AN pfam0042).
ture waste evaluated may influence on the induction and This information was confirmed with protein SmartBlast
production of hydrolytic enzymes; but others micro and analysis (Fig 5B.), where DCH4 cellulase chain earned
macronutrients could be necessary to evaluate for gain 98% of identity with an endo-beta-1,4-glucanase of B.
better cellulase and xylanase production. subtilis subsp. subtilis 168 (NP_389695). According to
protein structural analysis, translated chain contains
In addition, enzymatic crude extracts of quinoa stalks
two monomers, a catalytic domain which has 99.34%
and sugar cane bagasse cultures by 16 hours were used
identity with endo-1,4-beta-glucanase with manganese
to saccharification of SCB, QS, and WS. Beechwood xy-
(II) ion from Bacillus subtilis 168, and other non-catalytic
lan and CMC was used as standard substrates. After 72
domain with 98.45% of identity to CBM3 lacking the cal-
hours of saccharification, low yield of reducing sugar
cium-binding site from Bacillus subtilis 168. Both struc-
were obtained for all substrates including standard ones
tures were used for the model of cellulase of DCH4 (Fig.
(Fig. 4E, 4F). Wheat straw yields 35 and 38% of reducing
5C-E). The endoglucanase of DCH4 model comprised 14-
sugars with quinoa or bagasse extract, respectively (Fig.
326 residual amino acids, it includes a ligand for manga-
4G). Miyazaki et al. (2005) found that during enzymatic
nese (Mn+2) shaped by four residues (142, 180, 182, 183)
hydrolysis, wheat straw releases more sugars than other
where 180 and 182 residues are metal complexes. The
lignocellulosic substrates probably by its higher content
cellulose binding CBM3 model used 339-467 residues.
of cellulose and xylan. Enzymatic hydrolysis is the criti-

074 Rev. peru. biol. 27(1): 074 - 078 (Marzo 2020)

 Isolation of thermotolerant Bacillus subtilis DCH4 from hot spring with potential to degrade lignocellulosic

Bhalla et al. (2013) refer that endoglucanases contain- tilis (AN CP007173), 99.07% with xylanase gene of B.
ing carbohydrate-binding modules (CBM) − also called subtilis (Z34519, KJ540228), 99.38% endo-1,4-beta-
primary cellulases − are the most crucial in enabling ef- xylanase of B. subtilis MW10 (DQ100307), and 98.92%
ficient utilization of crystalline cellulose. This type of cel- with xlnA gene of xylanase EC 3.2.1.8 from B. circulans
lulase is bifunctional, where both two domains interact strain (XO7723) were obtained. Figure 6A shows the
together in cellulose recognition increasing the enzyme corresponding phylogenetic tree. Its translated chain
efficiency. In this case, DCH4 endoglucanase is supposed containing 217 amino acids was trimmed to 214 and
to have a ligand to Mn+2. Studies of endoglucanase of B. compared with other protein chains of NCBI database
subtilis 168 (BsCel5A) found that Mn+2 improved drasti- using Standard Blast Protein tool. It was found 100%
cally the thermal stability and lifetime at high temper- of identity with a secreted endo-1,4-beta-xylanase of B.
ature, did not change of substrate preference affinity subtilis 168 (NP389765) and 99.53% with an endo-1,4-
(Santos et al. 2012). In the same way, studies of the role beta-xylanase of B. pumilus (AAZ17390). Concordantly,
of CMB3 of Bacillus sp. KD1014 suggest that it contribute Protein SmartBlast analysis found 100% of identity with
in thermostability, and affinity and substrate specificity a multispecies endo-1,4-beta-xylanase A for Bacillales
for small substrates (Lee et al. 2018). (WP003231377), and endo-1,4-beta-xylanase of B. sub-
tilis subsp. subtilis str. 168 (Fig. 6B). Structural protein
Endo-β-1,4-glucanases are the major enzymes re-
analysis showed that the protein is a monomer similar
sponsible for the breakdown of internal glycosidic bonds
to endo-1,4-beta-xylanase from Bacillus subtilis 168
of cellulose chains producing oligosaccharides, cellobi-
(1A1) (98.36%), which was used as a template to pre-
ose, and glucose (Bhalla et al. 2013, Santos et al. 2012).
dict a structural model for xylanase DCH4 (Fig. 6C) with
Xylanase consense sequence was edited to 651 30-212 residues. This model included a ligand to meso-
pb and deposited in Genbank with MK644600 acces- tartaric acid shaped by six residues (131, 132, 133, 161,
sion number. From BlastN analysis, a 99.69 % of iden- 162, 163) where 133, 162 and 163 residues form hydro-
tity with CDS sequences of complete genome B. sub- gen bonds, and 161 form salt bridges.

A C

Figure 5. Analysis of endoglucanase gene of B. subtilis DCH4. A. Phylogenetic tree of endoglucanase gene using Neighbor-Joinning,
Kimura 2-parameter, and 1000 bootstraps; B. Protein SmartBlast of the translated endoglucanase gene (NCBI, Standard protocol); C. Struc-
tural model of endoglucanase with cellulose binding molecule (CMB3); D. Alignment of DCH4 translated chain with endoglucanase of B.
subtilis 168; and E. Alignment of DCH4 translated chain with CMB3 of B. subtilis 168. C, D, E were prepared with (https://swissmodel.expasy.
org).

Rev. peru. biol. 27(1): 075 - 078 (March 2020) 075

 Tamariz-Angeles et al.

Enzymatic hydrolysis of xylan involves a multi-enzyme Finally, it was found the presence of interesting re-
system where the endoxylanase attack the main chain of sults obtained for isolation of cellulolytic and xylanolytic
xylan (Balat 2011). Xylanase of DCH4 showed adequate microorganisms from Chancos hot spring, indicating that
optimum temperature (55 °C), but after incubation at 60 the strain DCH4, which showed the better performance
°C by 1 hour, its activity has lost completely. Protein struc- is a B. subtilis thermotolerant strain. Its enzymatic activi-
ture studies of endoxylanase rXynA from Bacillus subtilis ties could be related with bifunctional thermostable en-
168 (1A1) ‒ similar to xylanase DCH4 ‒ revels that the doglucanase of family 5, and an endoxylanase of family
transition from the native to the denatured enzyme starts 11 identified by molecular analysis from genomic DNA.
at a temperature of 55 ° C and for higher temperatures, This results, suggest that the strain B. subtilis DCH4 is a
both the catalytic activity and β-sheet secondary structure suitable candidate to perform biochemical and genetic
were lost (Murakami et al. 2005). Then, some studies to studies in order to improve its enzymatic activity for bio-
improve thermal stability have been performed by other technological applications related to lignocellulose con-
researchers (Miyazaki et al. 2006). version process.

A C

Figure 6. Analysis of xylanase gene of B. subtilis DCH4. A. Phylogenetic tree of xylanase gene using Neighbor-Joining, Kimura 2-parameter,
and 1000 bootstraps; B. Protein SmartBlast of the translated xylanase gene (NCBI, Standard protocol); C. Model of xylanase of DCH4; D.
Alignment of DCH4 translated gene with the closer template (xylanase of B. subtilis 168 (1A1). SRT, meso-tartaric acid. C and D were pre-
pared with (https://swissmodel.expasy.org).

Literature cited Ajijolakewu KA, Sani A, Oyeyiola GP, et al. 2013. Cellulase pro-
duction potentials of the microbial profile of some
Acharya S, Chaudhary A. 2011. Effect of nutritional and envi- sugarcane bagasse dumping sites in Ilorin , Nigeria.
ronmental factors on cellulases activity by thermo- Notulae Scientia Biologicae 5: 445–449. https://doi.
philic bacteria isolated from hot spring. Journal of Sci- org/10.15835/nsb549176
entific and Industrial Research. 70, 142–148. http://
nopr.niscair.res.in/handle/123456789/10976 Anwar Z, Gulfraz M, Irshad M. 2014. Agro-industrial lignocel-
lulosic biomass a key to unlock the future bio-energy:
Adsul MG, Ghule JE, Singh R, et al. 2004. Polysaccharides from A brief review. Journal of Radiation Research and Ap-
bagasse: applications in cellulase and xylanase pro- plied Sciences 7: 163–173. https://doi.org/10.1016/j.
duction. Carbohydrate Polymers 57: 67–72. https:// jrras.2014.02.003
doi.org/10.1016/j.carbpol.2004.04.001
Arora R, Behera S, Kumar S. 2015. Bioprospecting thermo-
Aftab S, Aftab MN, Ikram U-H, et al. 2012. Cloning and expres- philic/thermotolerant microbes for production of
sion of endo-1,4–β-glucanase gene from Bacillus li- lignocellulosic ethanol: A future perspective. Renew-
cheniformis ATCC 14580 into Escherichia coli BL21 able and Sustainable Energy Reviews 51: 699–717.
(DE 3). African Journal of Biotechnology 11: 2846– https://doi.org/10.1016/j.rser.2015.06.050
2854. https://doi.org/10.5897/ajb11.2902

076 Rev. peru. biol. 27(1): 076 - 078 (Marzo 2020)

 Isolation of thermotolerant Bacillus subtilis DCH4 from hot spring with potential to degrade lignocellulosic

Bajaj BK, Sharma M, Sharma S. 2011. Alkalistable endo-β-1,4- Ding SY, Liu YS, Zeng Y, et al. 2012. How does plant cell wall
xylanase production from a newly isolated alkali- nanoscale architecture correlate with enzymatic di-
tolerant Penicillium sp. SS1 using agro-residues. 3 gestibility? Science 338: 1055–1060. https://doi.
Biotechnology 1: 83–90. https://doi.org/10.1007/ org/10.1126/science.1227491
s13205-011-0009-5
Jalal A, Rashid N, Rasool N, et al. 2009. Gene cloning and char-
Balat M. 2011. Production of bioethanol from lignocellulosic acterization of a xylanase from a newly isolated Bacil-
materials via the biochemical pathway: A review. lus subtilis strain R5. Journal Bioscience and Bioen-
Energy Conversion and Management 52: 858–875. gineering 107: 360–365. https://doi.org/10.1016/j.
https://doi.org/10.1016/j.enconman.2010.08.013 jbiosc.2008.12.005
Bhalla A, Bansal N, Kumar S, et al. 2013. Improved lignocel- Jonnadula RC, Imran M, Poduval PB, et al. 2018. Effect of poly-
lulose conversion to biofuels with thermophilic bac- saccharide admixtures on expression of multiple poly-
teria and thermostable enzymes. Bioresource Tech- saccharide-degrading enzymes in Microbulbifer strain
nology 128: 751–759. https://doi.org/10.1016/j. CMC-5. Biotechnology Reports 17: 93–96. https://doi.
biortech.2012.10.145 org/10.1016/j.btre.2017.12.008
Bhalla A, Bischoff KM, Sani RK. 2015. Highly Thermostable Xy- King BC, Donnelly MK, Bergstrom GC, et al. 2009. An optimized
lanase Production from A Thermophilic Geobacillus microplate assay system for quantitative evaluation
sp. Strain WSUCF1 Utilizing Lignocellulosic Biomass. of plant cell wall-degrading enzyme activity of fungal
Frontier in Bioengineering and Biotechnology 3: 1–8. culture extracts. Biotechnology and Bioenginery 102:
https://doi.org/10.3389/fbioe.2015.00084 1033–1044. https://doi.org/10.1002/bit.22151
Bibi Z, Ansari A, Zohra RR, et al. 2014. Production of xylan Kont R, Kurašin M, Teugjas H, et al. 2013. Strong cellulase inhib-
degrading endo-1 , 4- b -xylanase from thermophilic itors from the hydrothermal pretreatment of wheat
Geobacillus. Journal of Radiation Research and Ap- straw. Biotechnology for Biofuels 6: 1–14. https://doi.
plied Sciences 7: 478–485. https://doi.org/10.1016/j. org/10.1186/1754-6834-6-135
jrras.2014.08.001
Kumar A, Dutt D, Gautam A. 2016. Production of crude enzyme
Blumer-Schuette SE, Kataeva I, Westpheling J, et al. 2008. from Aspergillus nidulans AKB-25 using black gram
Extremely thermophilic microorganisms for bio- residue as the substrate and its industrial applications.
mass conversion: status and prospects. Currient Journal Genetic Engineering and Biotechnology 14:
Opinion in Biotechnology 19: 210–217. https://doi. 107–118. https://doi.org/10.1016/j.jgeb.2016.06.004
org/10.1016/j.copbio.2008.04.007
Kumar SS, Panday DD, Naik GR. 2011. Purification and molecu-
Blumer-Schuette SE, Brown SD, Sander KB, et al. 2014. Ther- lar characterization of low molecular weight cellulase-
mophilic lignocellulose deconstruction. FEMS Mi- free xylanase from thermoalkalophilic Bacillus spp. jb
crobiology Reviews 38: 393–448. https://doi. 99. World Journal of Science Technology 1: 9–16.
org/10.1111/1574-6976.12044
Lee JP, Shin E-S, Cho MY, et al. 2018. Roles of Carbohydrate-
Camassola M, Dillon AJP. 2007. Production of cellulases and Binding Module (CBM) of an Endo- β -1,4- Glucanase
hemicellulases by Penicillium echinulatum grown on (Cel5L) from Bacillus sp. KD1014 in Thermostabil-
pretreated sugar cane bagasse and wheat bran in sol- ity and Small-Substrate Hydrolyzing Activity. Journal
id-state fermentation. Journal of Applied Microbiology of Microbiology and Biotechnology 28: 2036–2045.
103: 2196–2204. https://doi.org/10.1111/j.1365- https://doi.org/10.4014/jmb.1802.10001
2672.2007.03458.x
Li W, Zhang WW, Yang MM, et al. 2008. Cloning of the ther-
Carrillo-Nieves D, Rostro MJ, de la Cruz Quiroz R, et al. 2019. mostable cellulase gene from newly isolated Bacil-
Current status and future trends of bioethanol pro- lus subtilis and its expression in Escherichia coli.
duction from agro-industrial wastes in Mexico. Re- Molecular Biotechnology 40: 195–201. https://doi.
newable and Sustainable Energy Reviews 102: 63–74. org/10.1007/s12033-008-9079-y
https://doi.org/10.1016/j.rser.2018.11.031
Lynd LR, Weimer PJ, Zyl WH, et al. 2002. Microbial Cellulose
Chang S, Wu B, Schenk G, Pedroso MM et al. 2018. Processivity Utilization: Fundamentals and Biotechnology. Micro-
and enzymatic mechanism of a multifunctional fam- biology Molecular Biolology Reviews 66: 506–577.
ily 5 endoglucanase from Bacillus subtilis BS-5 with https://doi.org/10.1128/MMBR.66.3.506
potential applications in the saccharification of cellu-
losic substrates. Biotechnology for Biofuels 11: 1–15. Madigan MT, Martinka JM, Parker J. 1998. Brock: Biología de
https://doi.org/10.1186/s13068-018-1022-2 los Microorganismos, Octava. ed. Prentice Hall Iberia,
Madrid.
Chen SS, Tung KL, Abdolali A, et al. 2013. Typical lignocellu-
losic wastes and by-products for biosorption process Mehta D, Tulasi S. 2013. Diversity of Hot Environments and
in water and wastewater treatment: A critical review. Thermophilic Microbes, in: Tulasi, S., Littlechild, J.,
Bioresource Technology 160: 57–66. https://doi. Yutaka, K. (Eds.), Thermophilic Microbes in Envi-
org/10.1016/j.biortech.2013.12.037 ronmental and Industrial Biotechnology. Spring-
er Dordrecht Heidelberg, London. https://doi.
Chen Q, Li M, Wang X. 2016. Enzymology properties of two org/10.1007/978-94-007-5899-5
different xylanases and their impacts on growth per-
formance and intestinal microflora of weaned piglets. Miyazaki K, Hirase T, Kojima Y, et al. 2005. Medium to large-sized
Animal Nutrition 2: 18-23. https://doi.org/dx.doi. xylo-oligosaccharides are responsible for xylanase in-
org/10.1016/j.aninu.2016.02.003 duction in Prevotella bryantii B14. Microbiology 151:
4121–4125. https://doi.org/10.1099/mic.0.28270-0
Cortes-Tolalpa L, Salles JF, van Elsas JD. 2017. Bacterial syner-
gism in lignocellulose biomass degradation - comple- Miyazaki K, Takenouchi M, Kondo H, et al. 2006. Thermal sta-
mentary roles of degraders as influenced by complex- bilization of Bacillus subtilis family-11 xylanase by
ity of the carbon source. Frontier in Microbiology 8: directed evolution. Journal of Biological Chemistry
1–14. https://doi.org/10.3389/fmicb.2017.01628 281: 10236–10242. https://doi.org/10.1074/jbc.
M511948200

Rev. peru. biol. 27(1): 077 - 078 (March 2020) 077

 Tamariz-Angeles et al.

Murakami MT, Arni RK, Vieira DS, et al. 2005. Correlation of Tamariz-Angeles C, Olivera-Gonzales P, Villena GK, et al. 2014.
temperature induced conformation change with opti- Isolation and Identification of Cellulolytic and Xylano-
mum catalytic activity in the recombinant G/11 xyla- lytic Bacteria from Huancarhuaz Hot Spring. Annual
nase A from Bacillus subtilis strain 168 (1A1). FEBS Research & Review in Biology 4, 2920–2930. https://
Letters 579: 6505–6510. https://doi.org/10.1016/j. doi.org/10.9734/ARRB/2014/10699
febslet.2005.10.039
Tian SQ, Zhao RY, Chen ZC. 2018. Review of the pretreat-
Nanda S, Azargohar R, Dalai AK, et al. 2015. An assessment ment and bioconversion of lignocellulosic biomass
on the sustainability of lignocellulosic biomass for from wheat straw materials. Renewable and Sus-
biorefining. Renewable and Sustainable Energy Re- tainable Energy Reviews 91: 483–489. https://doi.
views 50: 925–941. https://doi.org/10.1016/j. org/10.1016/j.rser.2018.03.113
rser.2015.05.058
Vallenet D, Danchin A, Médigue C, et al. 2017. Bacillus subtilis,
Nurachman Z, Kurniasih SD, Puspitawati F, et al. 2010. Cloning the model Gram-positive bacterium: 20 years of anno-
of the endoglucanase gene from a Bacillus amylolique- tation refinement. Microbiology and Biotechnololgy
faciens PSM 3.1 in Escherichia coli revealed catalytic 11: 3–17. https://doi.org/10.1111/1751-7915.13043
triad residues Thr-His-Glu. American Journal of Bio-
chemistry and Biotechnology 6: 268–274. https://doi. Wanmolee W, Sornlake W, Laosirippojana N, et al. 2014. Devel-
org/10.3844/ajbbsp.2010.268.274 opment of efficient fungal biomass-Degrading enzyme
mixtures for saccharification of local lignocellulosic
Putro JN, Soetaredjo FE, Lin SY, et al. 2016. Pretreatment and feedstock. International Journal of Environmental,
conversion of lignocellulose biomass into valuable Chemical, Ecological, Geological and Geophysical En-
chemicals. Royal Society Chemistry Advances 6: gineering 8: 69–73. http://doi.org/10.5281/zeno-
46834–46852. https://doi.org/10.1039/c6ra09851g do.1337001
Raj A, Kumar S, Singh SK. 2013. A Highly Thermostable Xyla- Wolf M, Geczi A, Simon O, et al. 1995. Genes encoding xy-
nase from Stenotrophomonas maltophilia : Purifica- lan and β-glucan hydrolysing enzymes in Bacillus
tion and Partial Characterization. Enzyme Research subtilis: Characterization, mapping and construc-
2013: 1–8. https://doi.org/10.1155/2013/429305 tion of strains deficient in lichenase, cellulase and
xylanase. Microbiology 141: 281–290. https://doi.
Reinsenbach AL, Longnecker K. 2000. Novel bacterial and ar- org/10.1099/13500872-141-2-281
chaeal lineages from an in situ growth chamber de-
ployed at a Mid-Atlantic Ridge hydrothermal vent. Yang D, Xu W, Yang H, et al. 2009. Cloning and expression of
Applied Environment Microbiolology 66: 3798–3806. a novel thermostable cellulase from newly isolated
https://doi.org/10.1128/AEM.66.9.3798-3806.2000 Bacillus subtilis strain I15. Molecular Biology Reports
37: 1923–1929. https://doi.org/10.1007/s11033-
Gil-Ramirez A, Salas-Veizagab DM, Greyb C, et al. 2018. Inte- 009-9635-y
grated process for sequential extraction of saponins,
xylan and cellulose from quinoa stalks (Chenopodium
quinoa Willd.). Industrial Crops & Products 121: 54–
65. https://doi.org/10.1016/j.indcrop.2018.04.074
Saha BC. 2003. Hemicellulose bioconversion. Journal of Indus-
trial Microbiolology & Biotechnology 30, 279–291.
https://doi.org/10.1007/s10295-003-0049-x
Santos CR, Paiva JH, Sforça ML, et al. 2012. Dissecting struc-
ture–function–stability relationships of a thermo-
stable GH5-CBM3 cellulase from Bacillus subtilis
168. Biochemistry Journal 441: 95–104. https://doi.
org/10.1042/bj20110869
Sarkar N, Ghosh SK, Bannerjee S, et al. 2012. Bioethanol produc-
tion from agricultural wastes: An overview. Renew- Agradecimientos / Acknowledgments:
able Energy 37: 19–27. https://doi.org/10.1016/j. CTA, POG, GKV want to express their special gratitude and honor
renene.2011.06.045 the memory of their mentor, Dr. Marcel Gutiérrez-Correa (RIP)
who actively participated in the conception and design of this
Sepahy AA, Ghazi S, Sepahy MA. 2011. Cost-effective produc- research.
tion and optimization of alkaline xylanase by indig-
enous Bacillus mojavensis AG137 fermented on ag- Conflicto de intereses / Competing interests:
ricultural waste. Enzyme Research 2011. https://doi. The authors have declared that no competing interests exist.
org/10.4061/2011/593624
Shanthi V, Roymon M. 2018. Isolation, Identification and Partial Rol de los autores / Authors Roles:
Optimization of Novel Xylanolytic Bacterial Isolates CTA, POG, GKV conceptualized the research and designed the
from Bhilai-Durg Region, Chhattisgarh, India. Iran experiments. CTA, JLP, POG, ACB carried out the assays. CTA, JLP
analyzed the data. CTA, ACB, GKV wrote the first draft. CTA, POG,
Journal of Biotechnology 16: 200–212. https://doi. GKV, JLP, ACB read and approved the manuscript.
org/10.21859/ijb.1333
Sharma M, Kumar A. 2013. Xylanases : An Overview. Brithis Bio- Fuentes de financiamiento / Funding:
technology Journal 3: 1–28. https://doi.org/10.9734/ Components of this research were financially supported by a
BBJ/2013/1784 doctoral scholarship to CTA of the CONCYTEC, and an undergra-
duate thesis scholarship to BLP of the Dirección del Instituto de
Sirisena DM, Manamendra TP. 1995. Isolation and character- Investigación - Vicerrectorado de Investigación de la UNASAM.
ization of cellulolytic bacteria from decomposing rice
straw. Journal of the National Science Foundation of Aspectos éticos / legales; Ethics / legals:
Sri Lanka 23: 25–30. This investigation did not falling into any legal or ethical pro-
blems.

078 Rev. peru. biol. 27(1): 078 - 078 (Marzo 2020)