Universidad de los Andes Master Thesis

Universidad de los Andes

Master Thesis

Selection and Characterization of Active Cellulases Against Pretreated Oil Palm

Empty Fruit Bunch From a Native Endophytes Library

Luis Miguel Medina Solano

Andrés Fernando González Barrios Ph.D.

Chemical Engineering Department

Advisor

Silvia Restrepo M.Sc. Ph.D.

Microbiology Department

Co-Advisor

July 2011

CONTENTS

1. Acknowledgments __________________________________________________________ 4

2. Abstract ____________________________________________________________________ 5

3. Problem Statement _________________________________________________________ 6

4. State of the art ______________________________________________________________ 8

4.1. Lignocellulose ________________________________________________________________ 8

4.2. Enzymes _______________________________________________ ¡Error! Marcador no definido.

4.3. Bioprospecting _______________________________________________________________ 14

4.4. Endophytes ____________________________________________ ¡Error! Marcador no definido.

4.5. Extreme Environments _________________________________ ¡Error! Marcador no definido.

4.6. Bioprospection at Los Andes University _______________________________________ 14

5. Objectives ________________________________________________________________ 16

5.1. General ______________________________________________________________________ 16

5.2. Specifics _____________________________________________________________________ 16

6. Methodology _____________________________________________________________ 17

6.1. OPEFB pretreatment __________________________________________________________ 17

6.2. Culture medium ______________________________________________________________ 17

6.3. Microorganism _______________________________________________________________ 17

6.4. Screening ____________________________________________________________________ 17

6.5. Inoculum preparation and fermentation conditions _____________________________ 18

6.6. Analytical methods and bioassays_____________________________________________ 18

6.7. Partial purification of cellulase ________________________________________________ 21

6.8. SDS-PAGE ___________________________________________________________________ 22

6.9. Temperature and pH optimum _________________________________________________ 22

6.10. Kinetic assays _____________________________________________________________ 22

6.11. Saccharification assays _____________________________________________________ 23

7. Results and discussion ___________________________________________________ 24

7.1. Selection of endophytes with cellulase activity _________________________________ 24

7.2. Cellulolytic activity profiles ___________________________________________________ 24

7.3. Partial purification of cellulases from endophytes 3 and 22 ______________________ 25

7.4. Characterization of partial purified cellulase from endophyte 3 and 22 ___________ 25

8. Conclusions ______________________________________________________________ 28

9. Recommendations and future studies ______________________________________ 29

10. Security risks associated with the development of the project ______________ 30

10.1. Biological risk ______________________________________________________________ 30

10.2. Chemical risk ______________________________________________________________ 30

10.3. Mechanical risk ____________________________________________________________ 31

11. References _____________________________________________________________ 32

12. Tables __________________________________________________________________ 34

13. Figures _________________________________________________________________ 40

14. Annex __________________________________________________________________ 48

1. Acknowledgments

I want to thank with all my heart to my mother and father (Carmen Solano and Luis Medina

Young) for the unconditional support at every stage of my life, and my brothers for always

believing in me.

I want to thank especially, to Andres Gonzalez for trusting me, for all the advice and

reprimands received, which were fundamental for the development and completion of this

project and in turn, have become a core for my future, both as a person and as a scientist.

Thanks to all those involved in the realization of this project, Silvia Restrepo for his advice,

Luisa Cabezas for his support both moral and productive, also to my laboratory

companions Isabella Bahamon, Laura Palma and Camilo Beltran, without them this project

would not have been possible.

I want to thanks to Ximena Salamanca for support me; understand me and putting up with

me, for listening to my problems and for always been there for me through good and bad

times.

Thanks to Oscar Sanchez, Watson Vargas and Rocio Sierra for their advices, to my friends

and to the entire laboratory staff, especially Viviana Ferreira for helping and putting up with

me.

I want to acknowledge to the Vicerrectoria de Investigaciones and the chemical engineering

and biological science department’s at the Universidad de los Andes for the financial

support of this project.

2. Abstract

Oil palm industry generates a great number of by-products and solid wastes, which

includes the oil palm empty fruit bunch (OPEFB). The OPEFB exposes a high cellulose and

hemicelluloses content, while displaying low lignin content. This allows the substrate use, to

obtain simple sugars, as well as a carbon source for biofuels production. In this study,

cellulolytic potential for pretreated OPEFB saccharification, expressed by endophyte

microorganism was investigated. One hundred endophytes were screened on solid agar

using CMC as substrate. Those that showed cellulolytic activity were grown on cellulase

induction medium with pretreated OPEFB as carbon source. The activity of cellulase three

main components and total cellulolytic activity was measured. The screening allowed

indentifying 28 endophytes with cellulolytic activity. Based on the enzymatic activity, two

endophytes (Penicillium sp. and Aspergillus sp.) with maximum enzyme activities,

CMCase, FPase, exoglucanases and β-glucosidase, of 0.648-0.512, 0.038-0.045, 1.141-

2.989 and 0.0606-0.01 U/ml respectively, were selected and partially purified. The

cellulases produced by both endophytes were active at low pH (3) and high temperatures

(50-65 ºC) which could be a valuable tool for hydrolysis of cellulose under acidic conditions.

3. Problem Statement

Colombia, taking advantage of its location in the tropic region, has developed a important

area dedicated to cultivate oil palm, reaching values close to 300,000 hectares throughout

the national territory for the 2006 [1]. The growth of this agro-industry in Colombia has to

take into consideration the use of sustainable technology, as well as strategies that will

improve its competitiveness. A material balance of an oil palm plant yields, in terms of

mass, for every Ton of fresh fruit processed 220 kg of oil palm empty fruit bunch (OPEFB),

125-130 kg of fiber, between 50 and 60 kg of shell and 195-200 kg of oil are obtained [2].

This means that, in terms of mass flow, this industry generated approximate equals

quantities of OPEFB and oil. Considering that the average of annual increase in palm oil is

around 6% [1] it is estimated that the amount of available OPEFB will be 1.35 million tons

and 2.2 million tons by 2010 and 2015 respectively. Therefore, it is necessary to generate

alternative ideas for its use. Currently, most companies used the OPEFB as manure,

reporting different problems for composting

Studies on the lignocellulosic content of the OPEFB have shown that display a high

cellulose and hemicellulose and low lignin content [2]. This feature allows using this waste

as a substrate to produce simple sugars; these sugars can be used as carbon source in

different fermentation processes to enable the production of alcohols that can be used as

fuels. The biodegradation of cellulose is carried out by three classes of enzymes:

endoglucanase (carboxymethylcellulase), exogluconase (cellobiohydrolase) and β-

glucosidase, which together with a preliminary treatment of cellulose, manage to break the

glycosidic bonds to produce simple sugars [3]. However, these enzymes hydrolyze in

different parts of the polymer and therefore it cannot be ensure that a cellulase act in the

same way or with the same efficiency for different substrates because each substrate has a

different lignocellulosic structure [4].

Bioprospecting strategies must focus on genetic resources that generate value added

useful to the country. In the current context, extremophile microorganisms have great

biotechnological potential, because the extreme conditions in which they make its biological

activities frequently overlap with those required in industrial processes, thus providing a

source of secondary metabolites and enzymes with diverse metabolic capabilities,

operating at high and low temperatures and acidic and basic pH, which made them usable

through biotechnological processes [5].

Given the nature of the industrial pretreatment processes to produce bioethanol, in which

cellulose is hydrolyzed by acids at high temperatures, the use of enzymes active at low pH

and high temperatures could help to simplify and reduce the cost of the process.

Nevertheless, the cellulases active at low pH, reported on literature tend to be mesophilic

[6-9].

Taking all this into account, the objective of this study was to explore the cellulolytic

potential of endophytes from a Colombian extreme environment for the saccharification of

oil palm empty fruit bunch. This allowed us to obtain cellulases active at low pH and high

temperatures, capable to hydrolyze pretreated OPEFB.

4. State of the art

4.1. Lignocellulose

Lignocellulose is a renewable organic material and is the major structural component of

all plants. Lignocellulose consists of three major components: cellulose, hemicellulose

and lignin. In addition, small amounts of other materials such as ash, proteins and

pectin can be found in lignocellulosic residues, in different degrees based on the source

[10].

In recent years, lignocellulosic materials have been in high demand as a carbon source

because they are abundant and inexpensive [10]. The national renewable energy

laboratory in the United States has estimated that the cost of producing ethanol from

lignocellulose projects values close to $ 0.32 per liter assuming a feedstock cost of $ 42

per Ton. The average cost of biomass corresponds to $ 0.06 per kilogram of sugar. This

means a significant cost reduction compared with the average cost of $ 1 per liter today

[11]. Nevertheless due to the complexities of lignocellulosic materials is difficult to obtain

fermentable sugars.

The different types of lignocellulosic materials can be classified in: hardwood (poplars,

aspen), softwood (pines, spruce), crop residues (cane bagasse, corn stover, rice straw,

rice hulls, sorghum bagasse, olive stones and pulp), cellulose wastes (newsprint, waste

office paper, recycled paper sludge), herbaceous biomass (alfalfa hay, switchgrass) and

municipal solid wastes [11]. The cellulose, hemicellulose and lignin proportion change

according to the lignocellulosic material, and this proportion plays a key role in the

hydrolysis process. Hardwoods have a 40-55 % of cellulose, 24-40 % of hemicellulose

and 18-25 % of lignin content, softwoods has a 40-50 % of cellulose, 25-35 % of

hemicellulose and 25-35 % of lignin content, paper has a 85-99 % of cellulose, 0 % of

hemicellulose and 0-15 % of lignin content, herbaceous biomass have a 25-40 % of

cellulose, 35-50 % of hemicellulose and 10-30 % of lignin content [3]. EOPFB has a

50.4 ± 1.2 of cellulose, 21.9 ± 1.4 of hemicellulose and 10.0 ± 1.7 of lignin content [2],

which, added with the large amount available of this agricultural waste, make the

OPEFB a viable alternative as a cheap source substrate for cellulase productions.

4.1.1. Cellulose

Cellulose, a linear biopolymer of anhydroglucopyranose molecules which are

connected by β-1,4-glycosidic bonds, the major constituent of plant materials,

and also the most abundant organic molecule on Earth, it’s found mainly in

wood and therefore in the paper. Cellulose has parallel alignment of crystal line

structures knows as microfibril because adjacent cellulose chains are coupled

by hydrogen bonds, hydrophobic interactions and Van der Waal´s forces [10].

The actions of cellulose degrading enzymes (cellulase) to produce fermentable

sugars it’s hindered due to its structure and because it’s protected by

hemicellulose and lignin.

4.1.2. Hemicellulose

Hemicellulose it’s the second most abundant component of lignocellulosic

materials, is a heterogeneous polymers of pentoses and sugar acids, and its

composition is very variable in nature which is dependent on the plant source.

Because it is a non-linear branching biopolymer, its hydrolysis is easy to

perform which produce a hetero-polymer of irregular and non-crystalline

structure.

4.1.3. Lignin

Lignin, the third main polymer in lignocellulosic biomass, it’s a heterogeneous

polymer of aromatic alcohols, which generally contains three aromatic alcohols

including coniferyl alcohol, sinapyl and p-coumaryl. Lignin acts as a barrier to

block the actions of cellulases by linking to both hemicelluloses and cellulose

and preventing the penetration of celluloses to the interior lignocellulosic

structure, hindering the productions of fermentable sugars.

4.1.4. Cellulose Hydrolysis

In order to facilitate the action of enzymes against cellulose to obtain

fermentable sugars it must be conducted a pre-hydrolysis of lignocellulosic

material to expose the cellulose. This pretreatment depends on the

lignocellulosic material of work.

4.1.5. Pretreatment types

Pretreatments are classified according to their mode of action, they vary in cost

and difficult, but can be combined in the manner most advantageous, taking

into account the type of biomass to hydrolyze. The purpose of the pretreatment

is to remove lignin and hemicellulose, reduce cellulose crystallinity, and

increase the porosity of the materials. It must improve the formation of sugars

or the ability to subsequently form sugars by enzymatic hydrolysis, avoid the

degradation or loss of carbohydrate, prevent the formation of byproducts

inhibitory to the subsequent hydrolysis and fermentation processes, and be

cost-effective [3].

4.1.5.1. Physical

4.1.5.1.1. Mechanics types

Grinding: This procedure aims to reduce the particle size,

therefore increasing the surface area which facilitates the

enzyme actions. However it requires high energy levels [3, 11].

Sieving: detach the material into different particle sizes

making it more homogenous [3, 11].

4.1.5.1.2. Thermals types

Hot liquid water: Its aims to hydrolyze the hemicellulose to

achieve a high xylose yield. Its advantage is that not requires

the addition of chemicals; however so far there are no known

costs associated to this process [3, 11].

Low-temperature pyrolysis: It breaks down all the

lignocellulosic material by heating, in the absence of oxygen

and other reagents such as water. The low-temperature

pyrolysis it has been combined with a dilute acid hydrolysis to

obtained conversions of 80 to 85% from cellulose to reducing

sugars with approximately 50% glucose [3, 11].

4.1.5.2. Physicochemical

Steam explosion: It aims to destroy the cell walls, causing

the hemicellulose auto-hydrolysis and lignin solubilization. It

doesn’t need the addition of chemicals, and it is effective and

easily implemented in batch processes. However it’s one of the

most expensive pretreatments and it may produce byproducts

inhibitory to the subsequent hydrolysis and fermentation

processes. Using this pretreatment it can be achieved

efficiencies up to 90% on enzymatic hydrolysis[3].

Catalyzed steam explosion (CSE): It’s the same as the

steam explosion pretreatment but the substrate is infused with

acid (H2SO4) or gas (CO2) to decrease the severity of the

conditions. This process reduces the productions of inhibitory

substances to the subsequent hydrolysis and fermentation

processes [3, 11].

Ammonia fiber explosion (AFEX): This pretreatment also

cause the cellulose hydrolysis and lignin solubilization, but

under temperatures and pressures more moderate than CSE.

The effectiveness of this pretreatment depends of the target

substrate [3, 12].

4.1.5.3. Chemical

Alkaline hydrolysis: this pretreatment generate the hemicellulose

hydrolysis, lignin solubilization, diminish the cellulose crystallinity

and the internal surface area [3, 13].

Acid hydrolysis: Concentrated acid hydrolysis has been used to

treat lignocellulosic material, obtaining excellent results, However

due to the toxicity, corrosive and hazardous of the concentrated

acids, requires reactors capable of sustaining this conditions, in

addition the concentrated acid must be recovered after hydrolysis to

make the process cost-feasibly. Diluted acid hydrolysis has been

successfully used for pretreatment of lignocellulosic materials, the

dilute sulfuric acid hydrolysis, one of the most studied pretreatments

due to its high yields, aims to hydrolyze the cellulose and remove

lignin. High temperatures for the diluted acid treatment are favorable

for cellulose hydrolysis [3, 11].

Ozonolysis: This pretreatment has the advantages that degrade

the hemicellulose and lignin without production of toxic residues that

may affect the downstream processes; also the reactions are

carried out at room temperatures and pressures. However, this

process is expensive due to the large amount of ozone required [3,

11].

4.1.5.4. Enzymatic

The enzymatic hydrolysis is carried out by cellulases, which are highly

specific and its efficiency depends of the lignocellulosic structure of the

target substrate. This procedure usually generate reducing sugars (e.g.

glucose), does not produce inhibitory substances to the fermentation

process, does not have corrosion problem, and its utility cost is low

compared with others hydrolysis alternatives [3, 11].

It has been proven that certain microorganism, fungi and bacteria,

thermophilic or mesophilic, anaerobic or aerobic has the ability to produce

cellulose for the hydrolysis of lignocellulosic materials. In fungi, the

hydrolysis is made primary by 3 enzymes, endoglucanase,

cellobiohydrolase, and β-glucosidase, which work efficiently on

lignocellulosic materials in a synergistic manner. The most used fungi to

produce cellulases are Trichoderma reesei, Aspergillus niger, and

Chaetomiun globosum [4, 14-17].

4.1.6. Cellulase

Cellulases are mixtures of hydrolytic enzymes capable of degrading cellulose to

fermentable sugars. There are 3 groups of fungal cellulose-degrading enzymes

which act in synergistic manner: endoglucanase, which hydrolyze the internal

glycosidic bonds in cellulose chains, cellobiohydrolase that acts on chain ends.

The enzymatic reactions of these two mostly generate a disaccharide known as

cellobiose and, to a lesser extent, cello-oligosaccharides. The third group of

fungal cellulose-degrading enzymes, known as β-glucosidase, hydrolyzes the

cellobiose to fermentable sugars (Table 1, Fig. 1) [3, 10-11, 18].

Endoglucanase, also referred to as carboxymethylcellulase due to the artificial

substrate (carboxymethylcellulose) used to measure its enzyme activity, initiate

breakdown by cleaving the internal 1,4-β-glicosidic bonds of cellulose, making it

more accessible to cellobiohydrolases due to the new free chains ends added.

The endoglucanases are generally monomerics, with pH optima between 4.0-

5.0 and temperature optima from 50º to 70º C, have an exposed active site,

besides having the ability to bind to the substrate at any point on the surface.

Some fungi produce multiples endoglucanases (e. g. T. reesei, Phanerochaete

chrysosporium) [3, 10-11].

Cellobiohydrolases hydrolyze 1,4-β-glicosidic bonds at chain ends, producing

cellobiose as the main product, to a lesser extent, cello-oligosaccharides.

Cellobiohydrolases are monomers with pH optima between 4.0 and 5.0,

temperature optima from 37 to 60ºC and create a substrate binding-tunnel with

their extended loops which surround the cellulose. Some cellobiohydrolases

has the ability to attack the cellulose on non-reducing ends, increasing the

synergy with endoglucanases. The end product of cellobiohydrolases

(cellobiose) acts as a competitive inhibitor, limiting the ability of the enzymes to

degrade all of cellulose molecules in a system [3, 10-11].

β-glucosidase are responsible to hydrolyze soluble cellobiose and cellodextrins

to glucose, therefore are competitively inhibited by glucose. β-glucosidase

posses the most variability among the cellulolytic enzymes do to their structure

and localization. Some β-glucosidase have a simple monomeric structure with

around 35 kDa molecular mass (e.g. from Pleurotus ostreatus), some others

have dimeric structures with around 146 kDa (e.g. Sporobolomyces singularis)

or even trimeric structures with more than 450 kDa (e.g. pisolithus tinctorius),

also some monomeric β-glucosidase are 300 kDa (e.g. Trametes versicolor).

Regarding location, β-glucosidase can be intracellular, extracellular and wall-

associated, because of this, the pH optima of β-glucosidase is very variable,

nevertheless the temperature optima range from 45 to 70ºC [3, 10-11].

Commercially, these enzymes are mainly used in the textile industry, cereals

processing, grain alcohol fermentation, in the recovery of color in worn cotton

clothes, starch processing, animal food production, malting and brewing, fruit

extraction and vegetable juices, paper industry and in detergents [19]. Its most

significant potential use resides in the saccharification of cellulose to produce

fermentable sugar, which coupled to different fermentation processes enable

the production of alcohols that can be used as fuels [20].

The main problem with the application of cellulose in the industry is the high

cost of enzyme production. Therefore it is important to search or/and modify

microorganism with a high rate of cellulase production and use a inexpensive

lignocellulosic material (e.g. agricultural waste) on cultures in order to reduce

the costs of cellulase production [19].

Another problem it’s that few celluloses have optimum activities at the extreme

conditions used in the processes to produce bioethanol (High temperatures and

low or high pH) (Table 1); therefore there is a need to find celluloses with high

activities at this conditions in order to reduce the cost of the process.

4.2. Bioprospecting

Bioprospecting broadly defined as the systematic search for genes, compounds,

designs and organisms that may lead to the development of products with economic

potential. Bioprospecting strategies must focus on genetic resources that generate

value added useful to the country. The organisms that have evolved over millions of

years of natural selection in different environments, represent a wide range of

metabolic capabilities that may be of interest to industrial, pharmaceutical,

agricultural, health and environment sectors [21].

4.3. Studies at Universidad de los Andes

The Mycology and Plant Pathology Laboratory from Los Andes University (LAMFU)

has isolated 609 endophytes from two fraylejon species (Espelieta grandiflora and

E. corymbosa) on the Páramo Cruz Verde. From 2006 to 2008, with Colciencias’s

financing, it was evaluated in vitro the antagonist activity of 100 endophytes towards

phytopathogens disease-causing on crops with economic relevance in Colombia

[22]. During the characterization of the endophytes, some of them showed

cellulolytic activity, revealing its great biotechnological potential.

The Grupo de Diseño de Productos y Procesos (GDPP) from Los Andes University

has lead studies with a focus on harnessing the cellulose and hemicellulose content

in the waste generated from the extraction of palm oil and its potential use to

produce simple sugars [23-24]. The first was an exploratory work carried out in

which it was characterized the lignocellulosic content of the 3 major wastes: The

OPEFB, the fiber and the shell. It was found that the OPRFB has the highest

percentage of cellulose and hemicellulose and lower lignin percentage, the shell

has the highest percentage of lignin and lower of cellulose and hemicellulose, and

the fiber has intermediate values between the other two residues. It also was

carried out a pre-hydrolysis with sulfuric acid for the three residues, in which the

time, temperature and acid concentration were varied, and it was found that the

OPEFB and the fiber produced more reducing sugars than the shell, due to theirs

high content of hemicellulose and cellulose and low lignin content [23].

Furthermore, studies have also been made from a combination of dilute acid

pretreatment followed by enzymatic hydrolysis using commercial enzymes. The two

types of enzymes studied were commercial enzymes recorded under the name of

Celluclast® and Viscozyme® [24]. It was tested loads of 0.5 and 1.0 ml of each

enzyme for each 5 g of OEPFB fed to the process. It was found that the enzyme

Celluclast® had higher yields than the Viscozyme® in both cases. For the

Celluclast®, the yield of fermentable sugar production was around 27% when used

1 ml of enzyme and 23% with 0.5 ml of enzyme, meanwhile the Viscozyme®

achieved yields of 9% with 1 ml of enzyme and 6% with 0.5 ml of enzyme.

5. Objectives

5.1. General

To explore the cellulolytic potential of extremophiles microorganisms from an

established library for the saccharification of oil palm empty fruit bunch.

5.2. Specifics

To determine the cellulolytic activity (FPA, CMCase, β-glucosidase and

Cellobiohydrolase) in vitro of 100 endophytes from LAMFU collection using

the pretreated oil palm empty fruit bunch as substrate.

To determine the pH and temperature optimum of the best partially

purified cellulases obtained.

To obtain the kinetics constant of Michaelis-Menten of the best partially

purified cellulases obtained.

To study the saccharification of pretreated oil palm empty fruit bunch

with the best partially purified cellulases obtained.

6. Methodology

6.1. OPEFB pretreatment

The OPEFB was shredded by grinding in a hammer mill. Then a diluted acid pre-

hydrolysis was carried out, by soaking the OPEFB in 1% (w/v) H2SO4 (100 ml for

each 5 g of OPEFB) for 1 h, followed by autoclaving the solution at 121 ºC for 15

min. The pretreated OPEFB was filtered, washed with distilled water until no traces

of acid could be detected and then dried in an oven at 100 ºC for 3 days[16].

6.2. Culture medium

The endophytes were growth on potato/dextrose/agar medium (PDA).

The screening medium was a solid CMC medium [25], and the induction medium for

enzymatic activity determination was a modified Fries’ basal medium [26] with the

following composition: 1% w/v NH4NO2, 1% w/v K2HPO4, 0.5% w/v MgSO4, 0.1%

w/v NaCl, 0.13% w/v CaCl2, 0.01% w/v MnSO4, 0.01% w/v boric acid, 0.001% w/v

CuSO4, 0.2% w/v FeSO4 y 0.001% w/v ZnSO4, 10 g/L OPEFB and 6 g/L peptone.

6.3. Microorganism

The fungi under study were 100 isolated endophytes maintained in vitro on the

mycological collection of LAMFU. The endophytes were isolated from young leaves

presenting herbivory symptoms, and from leaves with no visible attacks, from

Espeletia grandiflora and E. corymbosa plants. The fungi were grown on PDA at

room temperature for 14 then stored at 4 ºC for subsequent use in inoculum

preparations. Fungal strains that showed significant cellulolytic activity were

identified by morphological characterizations at LAMFU. Morphological

characterizations were performed with microscopic observation of preparations in

water, lactophenol blue and lactophenol. Identifications were made following the

traditional taxonomy keys [27], [28] and [29], and by macroscopic descriptions of

the colonies.

6.4. Screening

The microorganisms were grown on solid CMC medium. The growth time depended

on the growth rate of the microorganism and enzyme activity. The Petri dish was

stained by adding 20 ml of Congo red solution at room temperature for 30 min. The

residual dye on the dish was rinsed using distilled water. The Congo red distain with

~20 ml of 1 M NaCl for 30 min [30]. Positive endophytes were selected based on

yellow halo formation (Figure 2).

6.5. Inoculums preparation and fermentation conditions

In all fermentations, one fungus’s plug was inoculated for each 30 ml of modified

Fries’ basal medium. The cultures were incubated at room temperature for 14-20

days with agitation speed of 150 rpm. Each experiment was carried out in duplicate.

Samples were withdrawn at regular time intervals and centrifuged at 13000 rpm at 4

ºC for 20 minutes. The supernatant was filtered using a Whatman Nº 1 filter paper.

The filtrated was used on the enzymatic assays.

6.6. Analytical methods and bioassays

The activities of the cellulase complex: endoglucanase, β-glucosidase and

cellobiohydrolase, as well as the total cellulolytic activity (FPase) were measured,

using the procedures described by Zhang et. al (2009). The results were expressed

as µmol reducing sugar released * ml enzyme-1 * min-1. Reducing sugars

concentrations were determined according to dinitrosalicylic acid (DNS) method

[31].The protein content was measured using the Folin-Lowry method, with bovine

serum albumin as standard [32-33], and by spectroscopy (absorbance at 280 nm) in

order to obtain an estimate of the protein levels at the fraction obtained with the

DEAE-Sephadex A-50 column. Sodium dodecyl sulfate–polyacylamide gel

electrophoresis (SDS–PAGE) was used to verify the partial purification process of

the enzymes, under denaturing conditions, as described by Laemmli [34] using a

broad range molecular weight standard (BioRad®).

6.6.1. Protein quantification

i) To 10 ul of the sample were added 0.1 ml of SDS (0.1%) and 0.1 ml of cupper

reagent ii) The solution stand at room temperature for 10 min. iii) It was added

0.4 ml of Folin reagent, using a vortex mixer, and the mixture stand at 55 ºC for

5 min and then incubated at 4 ºC for 5 min iv) The absorbance is read at 610

nm. To determine the protein concentration a standard curve of absorbance as

a function of initial protein concentration is made using the same procedure

described with different concentrations of bovine serum albumin (0, 10, 20, 50,

100, 200, 500, 1000 y 2000 µg/ml in NaCl (80%) [32].

6.6.2. Quantification of reducing sugars

The quantification of reducing sugars is performed by three different methods

depending on the enzyme activity to be performed.

i) Dinitrosalicylic acid (DNS) Method: 3 ml of DNS reagent were added to

1.5 ml of sample. The mix was put on boil water for 15 min and the let it

cool to room temperature. 0.5 ml of the mix were centrifuged at 10000

rpm for 3 min. 0.2 ml of the supernatant were mixed with 2.5 ml of water

and the absorbance was read at 540 nm [31, 35]. To determine the

reducing sugar concentration a standard curve of absorbance as a

function of reducing sugar concentration was made using the same

procedure described with different concentrations of glucose depending

on the assay. For the CMCase assay: 0.25, 0.50, 0.66, 1.00, mg/ml in

0.05 M sodium acetate buffer at pH 5 (buffer A). For the FPA assay

1.00, 1.65, 2.5, 3.35 mg/ 0.5 ml in buffer A.

ii) Phenol-sulfuric acid assay: 0.7 ml of 5% phenol solution was mixed with

0.7 ml of the sample. Then 3.5 ml of 96% sulfuric acid were added with

vigorous mixing. The absorbance was read at 490 nm after cooling the

mix to room temperature. The absorbance values (after subtraction of

regent blank) were then translated into glucose equivalent using a

standard graph plotting glucose (5-150 µg/mL) against absorbance [31,

35].

iii) Commercial glucose oxidase kit: This procedure was used according to

manufacturer (Sigma®).

6.6.3. Total cellulase assay

The filter paper activity (FPA) assay is one of the most common total cellulose

activity assay used, which is based on a fixed degree of conversion of

substrate, e.g. a fixed amount (2 mg) of glucose (based on reducing sugars

measured by the DNS assay) released from 50 mg of filter paper within a fixed

time (e.g., 60 min).

One milliliter of buffer A is added to a test tube followed by a 0.5 ml enzyme

suitable diluted in the same buffer and 50 mg of Whatman No 1 filter paper. At

least to dilutions of enzyme most be used, whit one dilution releasing slightly

more than 2.0 mg of glucose and one slightly less than 2.0 mg of glucose. The

reaction mixture was incubated at 50 ºC for 60 min and 3 ml of DNS was added

to stop the reaction. Then the amount of reducing sugar was measured by the

DNS method. Enzyme blanks, reagent blanks and glucose standard solution

are treated in exactly the same way [31, 35]. Using the standard curve

(subheading 6.6.2.i), the absorbance values of the diluted enzymes, after

subtraction of enzyme blank and reagent blank, were translated into milligrams

of glucose produce. FPase activity was calculated as:

6.6.4. Endoglucanase assay

The CMCase assay is a fixed conversion method, which requires 0.5 mg of

glucose released under the reaction [31, 35]. The reducing sugar concentration

is measured by the DNS method.

0.5 ml enzyme suitable diluted in the buffer A was added to a test tube of

volume at least 25 ml. At least two dilutions of enzyme most be used, with one

dilution releasing slightly more than 0.5 mg of glucose and one slightly less than

0.5 mg of glucose. The reaction mixture was incubated at 50 ºC for 5 min, 0.5

ml of 2 % (w/v) CMC in buffer A was added, and then incubated for 30 min at

50 ºC. 3 ml of DNS was added to stop the reaction, and boiled for 5 min in

vigorously boiling water bath. Finally 20 ml distillated water was added and

mixed by inverting the tubes several times, and then the absorbance was

measured at 540 nm. Using the standard curve (subheading 6.6.2.i), the

absorbance values of the diluted enzymes, after subtraction of enzyme blank

and reagent blank, were translated into milligrams of glucose produce. The

dilutions were the converted into enzyme concentration. Enzyme blanks,

reagent blanks and glucose standard solution were treated in exactly the same

way. CMCase activity was calculated as:

6.6.5. Cellobiohydrolase assay

The reaction mixture, 1.6 ml of Avicel solution (1.25 % w/v Avicel FMC PH101

on sodium acetate buffer 0.1 M pH 5 (buffer B)), 0.4 ml of enzyme diluted on

buffer B, were incubated at 50 ºC for 2 h. the reaction was stopped by

submerging it in ice-cooled water bath. 1 ml of the reaction was withdrawn and

centrifuged at 13000 rpm for 3 min. the total soluble sugars in the supernatant

was determined by Phenol-sulfuric acid assay (subheading 6.6.2.ii). One unit of

cellobiohydrolase activity is defined as the amount of enzyme that release a

micromole of glucose equivalent per minute from Avicel [31, 35].

6.6.6. β-glucosidase assay

1.0 ml enzyme suitable diluted in the buffer A was added to a test tube. At least

two dilutions of enzyme most be used, with one dilution releasing slightly more

than 1.0 mg of glucose and one slightly less than 1.0 mg of glucose. 1 ml of 1.5

mM cellobiose in buffer A was added, and then incubated for 30 min at 50 ºC.

The reaction was terminated by immersing the tubes in boiling water for 5 min.

The tubes were transferred to a cold water bath and the glucose released

determined by a glucose oxidase reaction kit [31, 35]. Enzyme blanks and

reagent blanks were treated in exactly the same way. β-glucosidase activity

was calculated as:

6.7. Partial purification of cellulase

Cellulases were partially purified when the activity pick was present.

Methodology

1. Raw extract clarification: the raw enzyme extract was precipitated with

ammonium sulfate (90%) [36]. The preparation was kept overnight at

4°C to allow precipitated protein to sediment and then was recovered

by centrifugation at 10000 rpm for 30 min at 4 ºC and dissolved in

buffer A (two-fold of the volume of the precipitate) .

2. Desalting: The enzyme solution was desalted by overnight dialysis

with three changes of buffer A.

3. Sample purification: Desalted enzyme solution was placed on a DAE-

shepadex A-50 (Sigma) column previously equilibrated with buffer A.

Fractions were withdrawn with a linear NaCl gradient.

4. Fraction concentration: Fractions were withdrawn with a linear NaCl

gradient (1M). The ones, that presented cellulolytic activity were

pooled and then concentrated by ultrafiltration with a centriprep

(Amicon®) having a 10 kDa molecular mass cut-off.

6.8. SDS-PAGE

Sodium dodecyl sulfate–polyacylamide gel electrophoresis (SDS–PAGE) was used

to verify the partial purification process of the enzymes, under denaturing

conditions, as described by Laemmli [34] using a broad range molecular weight

standard (BioRad®).

6.9. Temperature and pH optimum

CMCase and the FPA activities of the partially purified cellulase was assessed at

pH ranging from 1 to 10, and temperatures ranging from 30 to 70 °C in order to find

the optimum conditions for the cellulases.

6.10. Kinetic assays

Kinetics studies were performed at the optimum temperatures and pH of the

endoglucanases under study, containing amounts of CMC ranging from 1% to 2.5%

w/v for the determination of maximum velocity (Vmax) and Michaelis constant (Km).

The reaction is started by addition of 1 U of CMCase to the CMC solution (final

volume 200 µl), then stopped by adding up DNS solution (600 µl) at the requested

times (60, 120, 180 and 240 seconds) and boiled for 5 min in vigorously boiling

water bath. Finally 1 ml of distillated water was added and mixed by inverting the

tubes several times, and then the absorbance was measured at 540 nm. Using the

standard curve (subheading 6.6.2.i), the absorbance values obtained, after

subtraction of reagent blank, were translated into micromoles of glucose produce.

Each assay was carried out by duplicate. The data was plotted and analyzed in a

Lineweaver-Burk plot.

6.11. Saccharification assays

Enzymatic saccharification of OPEFB fibers were carried out in a shake flask at the

optimum temperature of the cellulase and 125 rpm for 8 days. Saccharification

started by the addition of 0.418 FPA units of cellulase preparation into 2 mL of an

appropriated buffer, containing 0.1 g of pretreated or un-pretreated (without diluted

acid pre-hydrolysis) OPEFB fiber. Sodium azide (0.02% w/v) was added to the

reaction mixture to avoid bacterial contamination. Each experiment was carried out

in duplicate. 0.1 ml samples were withdrawn at regular time intervals for analysis,

centrifuged for 3 min at 13000 rpm, and the supernatant was used for the

determination of reducing sugar hydrolysis (%) was calculated qualitatively using

the equation [37]:

Hydrolysis (%) = [reducing sugars (mg/ml) x 0.9 x 100]/[OPEFB (mg/ml) x 0.77].

The results were compared by one-way analysis of variance (one-way ANOVA) to

find the differences between saccharification assays with pretreated and un-

pretreated OPEFB means at 5% (0.05) significance level.

.

7. Results and discussion

7.1. Selection of endophytes with cellulase activity

One hundred endophytes were screened to determine which of them presented

cellulolytic capacity on solid agar with CMC as substrate followed by Congo red

staining. This methodology allowed us to identify 28 endophytes with cellulolytic

activities, which were grown in the induction medium (30 ml) plus 5 g/L of glucose, and

the total activity (FPase) was determined. Out of them, sixteen showed total activity and

the endophytes 3, 22, 18 and 56 showed the highest activities (Table 2).

7.2. Cellulolytic activity profiles

The endophytes with higher total activities were grown on the induction medium (150

ml), and the activity of the three main components of cellulase (endoglucanase

(CMCase), β-glucosidase and cellobiohydrolase (exoglucanase)), and total cellulolytic

activity (FPase) were determined (Fig. 3). The fungus 3 and 22 showed the highest

enzyme activities, CMCase, FPase, exoglucanase and β-glucosidase, of 0.648-0.512,

0.038-0.045, 1.141-2.989 and 0.0606-0.01 U/ml respectively (Fig. 3a-c).

It can be seen that for all fungi there was a trend to increase the FPA activity with time,

and these days also correspond with high exoglucanase and/or β-glucosidase activities.

The activity profiles of the cellulases obtained in all fungi shows some erratic behavior

for all the cellulases, primary due to experimental errors: (i) small changes in the

absorbance measured to quantify the reducing sugar and glucose concentration has a

significant impact in the activity determined, due to the low enzymatic activity of the

cellulases and (ii) due to the medium is heterogeneous it cannot be ensured that

samples withdrawn represent the whole system, at any time.

One of the most important requisites for the cellulose saccharification is the ability of the

endoglucanases and exoglucanases to act synergistically to hydrolyze crystalline

cellulose, since neither is effective on its own. These reactions produce cellobiose,

which in high concentrations is inhibitory to both cellulases; Hence, high levels of β-

glucosidases activities are required to hydrolyzes cellobiose to relieve the inhibition [4,

35]. We believe that the low total activity obtained on the studied fungus is mostly due to

the low production of β-glucosidase.

7.3. Partial purification of cellulases from endophytes 3 and 22

Endophytes 3 and 22 which displayed the highest activities, identified as Penicillium sp.,

and Aspergillus sp respectively, were grown on the induction medium and the enzyme

extract was used to partially purify the cellulases.

There is a significant loss of total activity and proteins in each step of the purification,

which is normal in any purification process (Table 3). Nevertheless, the low yields

obtained for both fungus is due to the loss of total activity at the ammonium sulphate

precipitation step (< 70 %), mainly caused by the use of a dialysis membrane made of

cellulose, which could be degraded by the cellulases, allowing the lost of proteins.

The partial purification process was effective, because allowed us to concentrate the

enzymes and to increase the specific activity of the cellulases by at least 2 fold (Table 3,

Fig. 4). But, it does not allow us to determine the molecular weight of the cellulases, due

to the presence of several proteins besides them (Fig. 4).

Both endophytes showed similar specific activities at the beginning of the purification

process. But the specific activity, at the end of the process, of the fungus 22 was better

than the fungus 3, primary due to different losses of protein at the ammonium sulphate

precipitation step (Table 3), mainly caused by the use of a more efficient centrifuge,

which could reach 10.000 rpm instead of 4.000 rpm, therefore allowing a higher

recovery of protein. With this into consideration, and the possibility of using different

desalting processes (e.g. desalting columns), it could increase the final yield and

specific activity at the purification process.

The presence of several peaks of protein, at the fractions eluted with different salt

concentrations in the DEAE-sepheadex A-50 column, with FPA activity for the fungus

22 (Fig. 5) possibly indicates that this fungus is capable of producing different forms of

cellulases.

7.4. Characterization of partial purified cellulase from endophyte 3 and 22

It was assessed the CMCase and the FPA activities of the partially purified cellulase at

pHs and temperatures ranging from 1 to 10 and 35 to 75 °C, respectively, in order to

find the best conditions for saccharification and kinetics assays.

The highest CMCase activities for both endophytes were equal, at 65 ºC and pH 3 (Fig.

6 a-b); meanwhile the highest FPA activities for fungi 3 and 22 were different, at 50 and

65ºC and pH 3 and 5 respectively (Fig. 6 c-d). The CMCase activity for the both

endophytes were closed to the optimum (<70%) at a wide range of pH and

temperatures. In contrast, the FPA activities of both fungi were greatly reduced at pH

and temperature near the optimum (Fig. 6), which could be due to the fact that the

FPase activity depends on the performance of all cellulases (exoglucanases,

endoglucanases and β-glucosidase). This assay allowed to increase the FPase and

CMCase activities of both fungi by more than 1.7 and 2.5 fold, respectively (Table 4);

and also gave some insight about how to improve the cellulases induction medium.

It has been reported on literature, cellulases active at low pH, but most of them tend to

be mesophilic [6-9]; Given the nature of the industrial pretreatment process to produce

bioethanol, in which cellulose is hydrolyzed by acids at high temperatures, in the

subsequent step of enzymatic hydrolysis the use of enzymes, such as the produced by

the endophytes under study, could help to simplify and reduce the cost of the process.

Kinetics studies and saccharification assays were performed with the partial purified

cellulases from endophytes 3 and 22, and a commercial preparation on cellulase from

A. niger (MP Biomedicals) diluted to have enzymatic activities near to that from fungus 3

and 22 (Table 5).

The Lineweaver-Burk plot of enzyme activities versus [CMC] allowed to obtain the

Michaelis-Menten constants and Vmax of the endoglucanases under study (Fig. 7,

Table 6). The enzyme with highest Michaelis constant (Km) is produced by fungus 3,

and the one with the lowest Km is the produced by the fungus 22, indicating that

endoglucanases produced by fungus 22 has a higher affinity for the substrate (CMC)

than the produced by fungus 3 and the commercial enzyme. The highest Vmax

correspond to the commercial cellulase and the lowest one from fungus 3 (Table 6).

These results show that the commercial cellulase has a higher reaction rate for the

substrate than the produced by the endophyte 22 and 3.

The endoglucanases under study have higher affinity for the substrate CMC, than

several endoglucanases reported on literature, but lower reaction rates. CMCases of

Thermomonospora Curcata hydrolyze CMC substrate with Vmax of 833 µmol glucose

min-1, it’s Km for CMC was 0.733 %w/v [38]. CMCases isolated by Lucas et al. from

Chalara paradoxa has Vmax of 1.1 µmol min-1 whereas, it’s Km was 0.83 % w/v [39].

Nevertheless, due to differences in experimental conditions, between this study and

those reported on literature, such as enzymatic load and the use of a cellulase cocktail

instead of a purified cellulase, one must be careful when comparing these results

The qualitatively hydrolysis (%) of pretreated OPEFB obtained with cellulases from

fungal strains 3 and 22 were closed to 6% and 8% respectively; meanwhile the

commercial preparation of cellulases from A. niger reaches hydrolysis (%) close to

7%(Fig. 8). For all the tested cellulases, the hydrolysis obtained with untreated OPEFB

(without diluted acid pre-hydrolysis) were significantly (P < 0.05) lower than the obtained

with pretreated OPEFB (Fig. 8).

The hydrolysis obtained for all the cellulases tested were lower than those reported on

literature [15-16], nevertheless, this is due to the low enzymatic load (4.18 FPA units/g

of OPEFB) used in this assays. Umikalson et al. (1998) reported hydrolysis (%) of

pretreated OPEFB near 85%, but using a higher enzymatic load (20 FPA units/g of

OPEFB) [15].

Díaz (2007) tested loads of 0.5 and 1 ml of commercial enzymes (Celluclast® and

Viscozyme®) for each 5 g of pretreated OPEFB (same pretreatment of this study),

reporting hydrolysis near 27 % [24]. Taking into account the low hydrolysis obtained

with such a high enzymatic load and that the differences in hydrolysis obtained with the

tested cellulases for pretreated and un-treated OPEFB were ~2.5 % (Fig. 8), could

mean that the pretreatment used in this study does not increase the expose of cellulose

in an efficient way.

It has been reported that alkali pretreatment of wood materials produced the most

suitable substrate for enzymatic saccharification compared to acid treatment [15, 37,

40], due to alkali treatment dissolve the hemicelluloses and causes the opening of the

crystalline structure of cellulose and lignin; also, lignin is decomposed to CO2, H2O, and

carboxylic acids [40]. With this into consideration and the use of a higher enzymatic

load, the enzymatic saccharification of OPEFB with the cellulases produced by

endophytes 3 and 22 could be improved, therefore reducing the cost of the process.

8. Conclusions

Endoglucanases produced by endophytes of study, are a valuable tool for cellulose

hydrolysis under acidic conditions, due to been active at low pH and high temperatures.

Endoglucanases produced by the endophyte Aspergillus sp. has higher affinity for the

substrate CMC than the commercial endoglucanase of Aspergillus niger.

Cellulases produced by Aspergillus. has the capacity to hydrolyze the OPEFB

analogous the commercial cellulase of Aspergillus niger.

Bioprospections studies on Colombia to generate added value are promising, due to in

this study it was found cellulases active at low pH and high temperatures with the

capacity to hydrolyze OPEFB, from endophytes isolated of an extreme environment.

9. Recommendations and future studies

Although the best endophytes obtained has lower cellulolytic activity than those

reported on literature, using pretreated OPEFB; a best culture medium (ie. changing the

nitrogen source, improving the OPEFB pre-treatment and adding cellulases inducers),

and changes in the fermentation process, may be used to improve the production of

cellulases by the Aspergillus sp. and Penicillium sp.

The saccharification yields of OPEFB obtained by the endophytes under study are lower

than those reported on literature. Nevertheless, by using a higher enzymatic load, and a

better pretreatment process coupled with the properties of the cellulases found (active

at low pH and high temperatures), kept the possibility to improve the hydrolysis of

OPEFB.

With the use of a more efficient centrifuge (10000 rpm) and using a different desalting

process (e.g. desalting columns) the final yield and specific activity at the purification

process could be increased.

10. Security risks associated with the development of the project

To reduce the possibility of damage to health during the project, it was necessary to

know the risks associated with this activity, among which are biological, mechanical and

chemical.

10.1. Biological risk

The microorganisms of study, were endophytic fungal and according to ATCC this

microorganisms have a biosafety level 1.

A biosafety level 1, is one in which the microorganisms are fully characterized and

do not cause any disease or risk to human health on a consistent and repetitive way

[41]. Taking into account that many microorganisms were opportunistic pathogens it

was important to follow these security measures [41]:

To have a minimal personal protection (goggles, gloves, and lab coat).

After and before handling the microorganism was necessary to wash hands

properly.

Do not eat, drink, or store them inside the laboratory.

Procedures should minimize aerosol formation and spread, the use of a

laminar flow chamber is recommended.

Disinfect the workspace after and before using the microorganisms.

Waste must be sterilized by approved methods before being discarded, it is

recommended to autoclave the waste.

The biological samples should be kept inside the laboratory.

10.2. Chemical risk

Risks associated with the implementation of chemicals necessary for the

development of this project are shown in annex table 1; the most common risk and

the security needs to use them are shown in table 2 and 3 respectively (Annex).

10.3. Mechanical risk

Risks associated with the implementation of equipments necessary for the

development of this project are shown in annex table 4.

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12. Tables

Table 1. Overview of cellulases and their main features (adapted from [10])

Optimum

Substrate

Molecular

Mass (kDa)

Optimum

Temperature (ºC)

pH optimum References

Endoglucanases Cellulose

(amorphous

regions)

Monomeric

(22-45)

50-70 Mostly 4-5 [3, 6-11, 42-

44]

Exoglucanases Cellulose

(crystalline

regions)

Monomeric

(50-65)

37-60 Mostly 4-5 [3, 6-11, 42-

44]

β-glucosidases Cellobiose Monomeric,

dimeric, trimeric

(35-450)

45-75 Vary [3, 6-11, 42-

44]

Table 2. Total cellulolytic activity of positive endophytes obtained on the screening.

Strain FPA at day 7

(U/ml)

FPA at day 14

(U/ml)

18 0 0.084

62 0 0.011

3 0.010 0.091

6 0.034 0

72 0.048 0

14 0.011 0

22 0.058 0.042

65 0.034 0

74 0.034 0.006

37 0.068 0.018

73 0.053 0.005

56 0.073 0.021

5 0. 0.043

32 0.026 0.047

2p 0 0.030

17p 0.016 0

*Each assay was made by duplicate

Table 3. Mass balance and recovery percentage during partial purification

Volume

(ml)

Total protein

(mg)

Total activity

(U)

Specific activity

(U/mg)

Yield

(%)

Endophyte 3 22 3 22 3 22 3 22 3 22

Raw extract 980 500 1852 841 39.25 16.54 0.021 0.020 - -

90% (NH4)2SO4 24.0 15.0 148.87 58.25 3.99 4.61 0.027 0.079 10.17 27.88

DEAE-sephadex A-50 10.0 30.0 85.40 42.26 3.00 3.39 0.035 0.080 7.64 27.88

Ultrafiltration 1.5 7.0 40.95 35.80 1.81 3.08 0.044 0.086 4.61 18.61

Table 4. Improvement of FPase and CMCase activities partially

purified cellulases at optimum ph and temperature

FPase (U/ml) CMCase (U/ml)

Initial Optimum Fold Initial Optimum Fold

Endophyte 3 1.21 2.09 1.74 44.52 211.82 4.76

Endophyte 22 0.44 0.84 1.90 6.84 17.07 2.50

Table 5. Activities of partial purified cellulases and commercial enzyme

PC FPase β-glucosidase Exoglucanase CMCase

[mg/ml] (U/ml) (U/mg) (U/ml) (U/mg) (U/ml) (U/mg) (U/ml) (U/mg)

Endophyte 3 27.30 1.21 0.04 0.45 0.02 48.36 1.77 44.52 1.63

Endophyte 22 5.11 0.44 0.09 0.33 0.06 81.31 15.90 6.84 1.44

CE 10.00 0.90 0.09 2.33 0.23 34.71 3.47 49.50 4.95

PC, denote: protein concentration; CE denote: Commercial enzyme.

Table 6. Kinetics constant of Michaelis-Menten on CMC as substrate

Endophyte 3 Endophyte 22 Commercial enzyme

Vmax (µmol*min-1) 3.38 ± 0.368 7.39 ± 0.0781 12.17 ± 3.805

Km (%w/v) 0.52 ± 0.013 0.078 ± 0.054 0.079 ± 0.021

13. Figures

Figure 1. Schematic representation of the hydrolysis of amorphous and microcrystalline cellulose by

cellulases. The solid squares represent reducing ends, and the open squares represent nonreducing

ends. Amorphous and crystalline regions are indicated. Cellulose, enzymes, and hydrolytic products

are not shown to scale [18].

Figure 2. Detection of cellulolytic activity by Red Congo staining method. A) example of a fungal

strain with cellulolytic activity; B) example of a fungal strain without cellulolytic activity.

Time (days)

0 2 4 6 8 10 12 14 16 18

FP

A (

U/m

l)

-g

luco

sid

ase

(U/m

l)

0.00

0.01

0.02

0.03

0.04

Exo

glu

conase (

U/m

l) C

MC

ase(U

/ml)

0.0

0.2

0.4

0.6

0.8

1.0

Time (days)

0 2 4 6 8 10 12 14 16 18

FP

A (

U/m

l)

-g

luco

sid

ase

(U/m

l)

0.00

0.02

0.04

0.06

0.08

Exo

glu

conase (

U/m

l) C

MC

ase(U

/ml)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

FPA

-glucosidase

Exogluconase

CMCase

Time (days)

0 2 4 6 8 10 12 14 16 18

FP

A (

U/m

l)

-g

luco

sid

ase

(U/m

l)

0.00

0.01

0.02

0.03

0.04

0.05

Exo

glu

conase (

U/m

l)

CM

Case(U

/ml)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Time (days)

0 2 4 6 8 10 12 14 16 18

FP

A (

U/m

l)

-g

luco

sid

ase

(U/m

l)

0.00

0.02

0.04

0.06

0.08

Exo

glu

conase (

U/m

l)

CM

Case(U

/ml)

0.0

0.2

0.4

0.6

0.8

1.0

A B

C D

Figure 3. Cellulolytic activities profiles of the fungus 3, 22, 18 and 56, growth on pretreated OPEFB

and peptone as carbon and nitrogen sources. A) Endophyte 3; B) endophyte 18; C) endophyte 22; D)

endophyte 56.

Figure 4. Coomasie-blue stained 10 % sodium dodecyl sulphate-polyacrylamide gel electrophoresis

of each purification step of cellulases from fungi 3 (A) and fungi 22 (B). Lane 1, raw extract; lane 2,

sulphate amonium precipitation (90%); lane 3, DEAE-shephadex column pool; lane 4, partially

purified sample; lane 5, molecular weigth markers.

B A

Figure 5. Distribution of proteins and total cellulolytic activity after chromatography on DEAE-

Sephadex A-50 column. A salt (NaCl) gradient in acetate buffer (1 M, pH 5) was used to elute the

proteins. The pooled fractions are designated as bars at the top.

pH

0 1 2 3 4 5 6 7 8 9 10 11

Ac

tivid

ad

re

lati

va

(%

)

0

20

40

60

80

100

120

Temperatura (ºC)

30 35 40 45 50 55 60 65 70 75 80

Ac

tivid

ad

re

lati

va

(%

)

0

20

40

60

80

100

120

pH

0 1 2 3 4 5 6 7 8 9 10 11

Ac

tivid

ad

re

lati

va

(%

)

-20

0

20

40

60

80

100

120

Temperatura (ºC)

30 35 40 45 50 55 60 65 70 75 80

Ac

tivid

ad

re

lati

va

(%

)

0

20

40

60

80

100

120

Hongo 3

Hongo 22

A B

C D

Figure 6. Effects of pH and temperature on the enzyme activities. A) Effect of temperature on CMCase

activity. B) Effect of pH on CMCase activity. C) Effect of temperatures on FPase activity. D) Effect of pH on

FPase activity. The effect of pH was performed on the indicated buffer at optimum temperature for 30 min

and 60 min for CMCase and FPase respectively; () 0.05 M sodium phosphate buffer pH 1-3; () 0.05 M

citrate buffer pH 3-4; () 0.05 M sodium acetate buffer pH 4-6; () 0.05 M sodium phosphate buffer pH 6-

8; () 0.05 M glycine-NaOH buffer pH 8-10. The effect of temperature was performed on sodium acetate

buffer (0.05 M, pH 5) for 30 min and 60 min for CMCase and FPase respectively.

1 / ( S [ % w / v ] )

- 0 , 2 0 , 0 0 , 2 0 , 4 0 , 6 0 , 8 1 , 0

1/V

o

0 , 0

0 , 5

1 , 0

1 , 5

2 , 0

2 , 5

Figure 7. Lineweaver-Burk plot of enzyme activities versus [CMC] of CMCases. Initial velocity (Vo) =

µmol*min-1; S = [CMC %(w/v)].The enzymatic load was 1U of CMCase for all experiments. Kinetics

assays were performed at different conditions depending of cellulases: () Endophyte 3 at pH 3 and 65

ºC; () Endophyte 22 at pH 3 and 65 ºC; () Commercial Enzyme at pH 5 and 50 ºC

Time (days)

0 1 2 3 4 5 6 7 8 9

% H

yd

roly

sis

0

1

2

3

4

5

6

7

8

9

Figure 8. Time courses of saccharification of oil palm empty fruit bunch fibers. Saccharification was

performed at different conditions depending of the cellulases: () Endophyte 3 at pH 3 and 50 ºC;

() Endophyte 22 at pH 5 and 65 ºC; () Commercial Enzyme at pH 5 and 50 ºC. Black symbols

denoted pretreated OPEFB; white symbols denoted untreated OPEFB (without diluted acid pre-

hydrolysis).

14. Annex

Table 1. Risks associated with the implementation of chemicals necessary for the development of this project.

Reagent Hazard category Human health risk security measures

Health Rating: 2 May cause a reaction. Minimal personal protection.

Flammbility Rating: 1 Causes irritation to skin. do not throw into sewer.

Reactivity Rating: 3 Harmful if swallowed. Stored in a cool, dry, ventilated area.

Health Rating: 1 May cause a reaction. Minimal personal protection.

Flammbility Rating: 0 Causes irritation to skin.

Reactivity Rating: 0 Harmful if swallowed. the use of a ventilation system is needed

Health Rating: 0

Flammbility Rating: 0 Minimal personal protection.

Reactivity Rating: 0

Health Rating: 1 Causes irritation to skin.

Flammbility Rating: 0 Vapors can affect the eyes. Minimal personal protection.

Reactivity Rating: 3 Harmful if swallowed.

Health Rating: 2 Inhalation may cause irritation

Flammbility Rating: 0 in airways. Minimal personal protection.


Health Rating: 1 Inhalation may cause irritation

Flammbility Rating: 0 in airways. Minimal personal protection.

Reactivity Rating: 0 Harmful if swallowed.

Health Rating: 3 Inhalation may cause irritation in airways.

Flammbility Rating: 0 Harmful if swallowed. Minimal personal protection.

Reactivity Rating: 0 My cause alergics.

Health Rating: 0



Health Rating: 2 Highly exothermic reaction.

Flammbility Rating: 0 Contact with eyes may produce permanent Minimal personal protection.

Reactivity Rating: 2 injuries.


Flammbility Rating: 1 Contact with eyes may produce permanent Minimal personal protection.

Reactivity Rating: 2 injuries.




Health Rating: 0






Health Rating: 0



Health Rating: 0






Health Rating: 0



Health Rating: 0



Health Rating: 3 may cause cancer in long periods

Flammbility Rating: 0 of exposure. Minimal personal protection.


Health Rating: 3 Highly corrosive. Minimal personal protection.

Flammbility Rating: 0 May cause severe skin burns. nitrile gloves.

Reactivity Rating: 2 extraction cabin.

Carboxymethylcellulose

sephadex G-25

DEAE-sephadex A-50

acrylamide 30%

sulphuric acid 96%

Folin reagent

glucose

dinitrosalicylic acid (DNS)

sodium acetate buffer 0,05 M pH 5

sodium acetate buffer 0,1 M pH 4.8

NH4NO3

K2HPO4

MgSO4

CaCl2

CuSO4

FeSO4

ZnSO4

peptone

NaOH 2N

Na2CO3

Table 2. The most common risk associated with the implementation of chemicals necessary for the development

of this project.

Table 3. The security needs to use the chemicals necessary for the development of this project.

Risk Description

CaCl2 NaOH 2N

R15 Reacts with water releasing inflammable gases CaCl2

CaCl2 ZnSO4

Na2CO2

R21 Causes irritation to skin. NH4NO3 K2HPO4

NH4NO3 K2HPO4

FeSO4 Foling reagent

R33 Danger of cumulatives effects

R34 Cause burns

R35 Cause serious burns

MnSO4 CuSO4

FeSO4 ZnSO4

Sulphuric acid 96 %

Foling reagent

Acrilamyde 30%

Irritates the respiratory systemR37

NaOH 2N

Harmful if sallowedR22

Na2CO2

Carboxymethylcellulose

Reagents

R10 Inflammable

Sulphuric acid 96 %R14 React violently with water

R20 Harmful by inhalation

Security Description

do not breathe the gases/smokes CaCl2 ZnSO4

/vapors/aerosols

S24 Avoid contact with skin NH4NO3 K2HPO4

NH4NO3 K2HPO4

NaOH 2N sulphuric acid 96%

S29 do not throw wastes into sewer

in case of insufficient MnSO4 CuSO4

ventilation, wear suitable FeSO4 ZnSO4

respiratory equipment

in case of ingestion, seek medical advice NH4NO3 K2HPO4

immediately and show the container or label FeSO4 Foling reagent

in case of accident by inhalation: MnSO4 CuSO4

FeSO4 ZnSO4

S38

S46

remove casualty to fresh air and keep at rest.S63

Foling reagent

Foling reagent

NH4NO3

Reagent

S23

S25 Avoid contact with eyes

Na2CO3

Table 4. Risks associated with the implementation of equipments necessary for the development of this project

Equipment Mechanical risk Electrical risk Thermal risk Others

Phase constrast

microscopyNo

There is a electrical

risk when

connecting the

equipment to the

power source

No No

Microcentrifuge

there is a mechanical

risk when the

equipment is in

operation


risk when

connecting the

equipment to the

power source

No No

Bain-marie No


risk when

connecting the

equipment to the

power source

depending on

operating

conditions, it can

produce skin

burns

No

Oven No


risk when

connecting the

equipment to the

power source

has a high risk

and can cause

severe burns if

not used

properly

No

Sonicator No


risk when

connecting the

equipment to the

power source

No No

Laminar flow chamber No


risk when

connecting the

equipment to the

power source

When working

with lighter

inside the

chamber, this

can be a thermal

risk

when using the

ultra violet light

is at risk of being

exposed to UV

light, making it

essential to use

the filters

Auto clave Sanyo No


risk when

connecting the

equipment to the

power source

If the work cycle

is interrupted

improperly,

there is a high

risk of direct

contact with

water vapor

No

Analytical balance No


risk when

connecting the

equipment to the

power source

No No

Peristaltic pump No


risk when

connecting the

equipment to the

power source

No No

Shaker

there is a mechanical

risk when the

equipment is in

operation. Samples can

not be manipulated

inside while in

operation


risk when

connecting the

equipment to the

power source

No No

Universidad de los Andes Master Thesis

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