University of Rome “Sapienza”

Department of Chemical Engineering, Materials and Environment

Biotechnological valorisation of agro-industrial

wastes for the production of cellulases

PhD in Chemical Engineering, Environment and Safety

XXIV cycle

Tutor Candidate

Prof. Marco Bravi Dr. Giuseppe Damato

Summary

1. Biofuelling the Future

1.1. Introduction

1.2. First Generation Biofuels

1.2.1. First Generation Bioethanol

1.2.2. First Generation Biodiesel

1.3. Second Generation Biofuels

1.3.1. Cellulosic Ethanol

1.3.2. Algal biodiesel

1.4. Issues for Biofuels Commercial Success

1.4.1. Value of Biorefinery Co-products

1.4.2. Transport by Pipeline

1.4.3. Decentralized Production and Local Distribution

1.4.4. Optimized Engine Performance

2. Second Generation Bioethanol

2.1. Overview

2.2. Different Lignocellulosic Feedstocks

2.3. Production process

2.3.1. Pretreatment

2.3.1.1. Mechanical Pretreatment Processes

2.3.1.2. Chemical Pretreatment Processes

2.3.1.2.1. Alkali Methods

2.3.1.2.2. Acid Methods

2.3.1.2.3. Organosolv

2.3.1.3. Thermochemical Pretreatment Processes

2.3.1.3.1. Steam Explosion

2.3.1.3.2. Liquid Hot Water

2.3.1.3.3. Ammonia Fiber Explosion (AFEX)

2.3.1.4. Biological Pretreatment

2.3.1.5. Pretreatment Efficiency and Enzyme Loadings

2.3.2. Hydrolysis

2.3.2.1. Acid Hydrolysis

2.3.2.2. Enzymatic Hydrolysis

2.3.3. Fermentation

2.3.4. Product Recovery

2.4. Process Optimization

2.4.1. Simultaneous Saccharification and Fermentation

2.4.2. Consolidated Bioprocessing

3. Cellulase Enzymes: State of the Art and Advances in Their

Production

3.1. Cellulase Biochemistry

3.2. Cellulases from Trichoderma reesei

3.3. Industrial application of cellulases

3.4. Production of cellulolytic enzymes

3.4.1. Carbon source and inducer

3.4.2. Nitrogen source and other nutrients

3.5. Cellulase production today: issues and perspectives

3.5.1. The impact of substrate selection

3.5.2. The impact of enzymes selection: new genes versus tailored cocktails

3.5.3. The impact of process integration

4. Materials & Methods

4.1. Microorganism

4.2. Culture Media

4.3. Cellulase Production Tests

4.4. Analytical techniques

4.5. Olive Pomace

4.6. Olive Oil Mill Wastewater

5. Aim of the Work, Results and Discussion

5.1. Aim of the Work: the ETOILE Project

5.2. Results

5.2.1. Lactose-induced Cellulase Production

5.2.2. Cellulose-induced Cellulase Production

5.2.3. Olive Pomace-induced Cellulase Production

5.2.4. Comparison between Cellulose and OP as Inducers

5.2.5. OP concentration and pretreatment effects

5.2.6. Fungal Biomass Concentration

5.2.7. Effect of polyphenols on cellulase production

5.2.7.1. Effect of Gallic Acid

5.2.7.2. Effect of OOMW polyphenols

5.2.8. OOMW effect on cellulase production

5.2.9. Fungal Biomass and Olive Pomace Reuse

5.2.10. Olive Pomace as carbon source for cellulase production

5.2.10.1. OP pretreatment

5.2.10.1.1. Alkali treatment

5.2.10.1.2. Acid treatment

5.2.10.2. Biomass Growth and Cellulase Production on Hydrolyzed

Olive Pomace

5.2.11. OOMW biotreatment

5.2.11.1. Thermal-acid treatment of OOMW

5.2.11.2. Different Biotreatment Approaches

5.3. Discussion

5.4. Conclusion

6. Bibliografy

Chapter 1

Biofuelling the future

1.1 Introduction

All the energy required by our society, in terms of fuels, electricity, heat, and food,

can in principle be produced biologically, although at reduced consumption rates

pro capite compared to today’s ones. Energy - aka black - biotechnology is a multi-

disciplinary approach concerned with biological energy conversion: its main focus

is the study and the optimization of all those biological and biotechnological

processes, centered about photosynthesis, which aim at exploiting solar energy to

ultimately produce such organic energy carriers as bioethanol, biodiesel or other

renewable substitutes of fossil fuels.

The two most common types of biofuels in use today are bioethanol and biodiesel.

In the next paragraphs, the most important features of these fuels will be

summarized.

1.2 First Generation Biofuels

First-generation biofuels produced from food crops (sucrose and starch

feedstocks) and oilseeds (triglycerides feedstocks) have utilized well-known

technologies to produce liquid fuel products generally compatible with existing

mature markets, namely ethanol and biodiesel.

After much analysis, it is generally accepted that these products afford net benefits

in terms of greenhouse gas emission reduction and energy balance relative to

petroleum-based fuels. By the other hand, first generation biofuels are

characterized by a number of drawbacks, such as the "food vs fuel" debate, energy

balance and efficiency, deforestation and soil erosion, loss of biodiversity, as well

as impact on water resources.

1.2.1 First Generation Bioethanol

First generation bioethanol is produced by fermenting sugars from starch (cereal

crops, mainly corn) and sugar (such as sugar cane, bagasse and sugar beet)

biomass. It can be used in pure form in specially adapted vehicles or blended with

gasoline in any proportion up to 10%.

The principal step of bioethanol production from sugars is the fermentation

technology which involves biochemical conversion of sucrose into ethanol and

carbon dioxide in the presence of yeast. The production of ethanol from starch

requires a liquification step and a saccharification step, which are followed by the

fermentation of simple sugars.

Figure 1.1 - Bioethanol production from sugar crops (reproduced from GEA Wiegand GmbH)

A typical bioethanol production scheme is showed in Figure 1.1. During the milling

process, the starchy material is first ground into flour, which is referred to in the

industry as “meal”, which is then slurried with water to form a “mash”. Enzymes

are added to the mash to convert the starch to dextrose, a simple sugar. Ammonia

is added for pH control and as a nutrient to the yeast. The mash is then processed

in a high-temperature cooker to reduce bacteria levels ahead of fermentation and,

after cooling, it is transferred to fermenters where yeast is added and the

conversion of sugar to ethanol and carbon dioxide begins. After fermentation, the

resulting “beer” is transferred to distillation columns where the ethanol is

separated from the remaining “stillage”.

The stillage is sent through a centrifuge that separates the coarse grain from the

solubles. The solubles are then concentrated by evaporation, resulting in

Condensed Distillers Solubles (CDS) or “syrup”. The coarse grain and the syrup are

dried together to produce dried distillers grains with solubles (DDGS), a high

quality, nutritious livestock feed. The CO2 released during fermentation can be

captured and sold for use in carbonating soft drinks and the manufacture of dry

ice.

For the ethanol to be usable as a fuel, water must be removed. Most of the water is

removed by distillation, but the purity is limited to 95-96% due to the formation of

a low-boiling water-ethanol azeotrope. The 96% ethanol, 4% water mixture may

be used as a fuel, and it is called hydrated ethyl alcohol fuel. However, for blending

with gasoline, purity of 99.5 to 99.9% is required, depending on temperature, to

avoid separation. Currently, the most widely used purification method is a physical

absorption process using molecular sieves. Another method, azeotropic

distillation, is achieved by adding benzene which also denatures the ethanol.

1.2.2 First Generation Biodiesel

The first generation biodiesel is usually referred to as a mixture of fatty acid

methyl esters (FAME) produced from vegetable oils and animal fats via trans-

esterification reaction. Several production methodologies are available but the

mostly used commercial technology for biodiesel production is the trans-

esterification reaction of the triglyceride of the fatty acid with methanol under the

basic conditions.

Biodiesel is physically similar to petroleum-based diesel fuel and can be blended

with diesel fuel in any proportion. The most common blend is a mixture consisting

of 20% biodiesel and 80% petroleum diesel, called B20.

The general scheme of the transesterification reaction is shown in Figure 1.2.

Figure 1.2 – Trans-esterification reaction

The kind and quality of feedstock is a very important factor in a trans-esterification

plant as it affects corresponding material and energy flows, which are not only

indicators of technical efficiency, but also affect the economic efficiency of

biodiesel production.

An important aspect of biodiesel production is related to its main by-product, the

glycerol. It occurs in vegetable oils at a level of approximately 10% by weight.

Crude glycerol possesses very low value because of the impurities. However, as the

demand and production of biodiesel grows, the quantity of crude glycerol

generated will be considerable, and its utilization will become an urgent topic.

1.3 Second Generation Biofuels

Second generation biofuels are expected to be superior to the first generation in

terms of energy balances, greenhouse gas emission reductions and competition for

land, food and water.

The main reason why they have not yet been taken up commercially, despite their

potential advantages, is that the necessary conversion technologies are not

technically proven at a commercial scale and their costs of production are

estimated to be significantly higher than for many first generation biofuels at the

moment. Significant R&D challenges remain before wide-scale deployment is

possible, but there are now several pilot-scale plants in operation with a few larger

demonstration plants planned or under development.

The most important second generation biofuels are cellulosic ethanol and algal

biodiesel.

1.3.1 Second Generation Bioethanol

Cellulosic ethanol is an environmentally friendly and renewable transportation

fuel produced from a wide array of feedstocks, including non-food plant materials

such as agricultural wastes, dedicated energy crops such as switchgrass, sugarcane

bagasse, and wood products.

Although the technology to create cellulosic ethanol is available today, scientists

must continue to work through technical hurdles before it can be marketed at

competitive prices. Second generation bioethanol production process will be

deeply discussed in the next chapter.

1.3.2 Algal Biodiesel

Microalgae are single-cell, photosynthetic organisms known for their rapid growth

and high energy content. Some algal strains are capable of doubling their mass

several times per day. In some cases, more than half of that mass consists of lipids

or triacylglycerides - the same material found in vegetable oils.

The conversion of algae oil into biodiesel is a similar process as for vegetable oils

based on interesterification of the triglycerides after extraction, but the cost of

producing algae oil is relatively high at present.

Algae can be produced continuously in closed photo-bioreactors (Figure 1.3, left)

but oil concentration is relatively low and capital costs are high. To collect the

biodiesel feedstock more cheaply would need high volumes of algae to be

cultivated in large facilities at low cost, hence the interest in growing the algae in

open ponds (Figure 1.3, right), including sewage ponds where nutrients are in

abundance and the sewage is partly treated as a result. In practice a problem is

contamination of the desired culture by other organisms that limit algal growth.

Figure 1.3 – A photo bio reactor (left) and an open pond system (right) for micro-algae cultivation.

1.4 Issues for Biofuels Commercial Success

1.4.1 Value of Biorefinery Co-products

As second-generation biofuels emerge, so do the various types of co-products and

residuals that result from these processes. Maximum value creation from co-

products will be essential for commercial biorefinery economics.

Like crude oil, plants are composed of a huge number of different molecules. Each

constituent of the plant can be extracted and functionalized in order to produce

non-food and food fractions, agro-industrial intermediate products and synthons,

whose value is generally inversely proportional to their volume. This concept is

analogous to that of a modern oil refinery in that the biorefinery is a highly

integrated complex that will efficiently separate biomass raw materials into

individual components and convert these into marketable products such as energy,

fuels and chemicals. Figure 1.4 illustrates ax example of biorefinery scheme from

lignocellulosic biomass.

Figure 1.4 –Biorefinery scheme from lignocellulosic biomass

A biorefinery, by producing multiple products, can take advantage of the

differences in biomass components and intermediates and maximize the value

derived from the biomass feedstock. A biorefinery might, for example, produce one

or several low-volume, but high-value, chemical products and a low-value, but

high-volume liquid transportation fuel, while generating electricity and process

heat for its own use and/or for sale.

The production of biofuels in the biorefinery complex will service existing high

volume markets, providing economy-of-scale benefits and large volumes of by-

product streams at minimal cost for upgrading to valuable chemicals. A pertinent

example of this is the glycerol by-product produced in biodiesel plants. Glycerol

has high functionality and is a potential platform chemical for conversion into a

range of higher value chemicals.

An important co-product from fermentation technologies utilizing lignocellulosic

feedstocks will be the aromatic natural polymer lignin. This previously

underutilized biomass component, primarily available to date in crude form from

the pulp and paper industry, holds great promise as a feedstock for many value-

added products, rather than as a process fuel source.

1.4.2 Transport by Pipeline

A very important challenge facing biofuels - especially bioethanol - is

transportation to large markets. Ethanol production in the United States and Brazil

is dominated by decentralized plants located in rural agricultural areas and relies

on rail or truck transport to major fuel markets. Pipeline transport would be more

cost-effective but dedicated pipelines are difficult to justify for an emerging

industry and require a minimum “critical mass” of product volume for acceptable

economics.

1.4.3 Decentralized Production and Local Distribution

Globally, abundant lignocellulosic feedstocks include agricultural residues and

forest biomass, as well as wood processing residues, urban wood waste, and

perennial crops. The natural rural distribution of most biomass argues for locating

biorefineries in close proximity to suitable feedstocks in order to reduce inbound

transportation costs. However, as stated in the previous paragraph, biofuels must

also be transported to major fuel markets. An optimal future model may seek to

size the biorefinery to fit the local feedstock supply, with fuel output distributed in

closer proximity to production. This approach not only minimizes inbound and

outbound transportation costs, but also creates a truly local energy source, while

promoting local economic development.

1.4.4 Optimized Engine Performance

The biofuels ethanol, ethyl-tertiary-butyl ether (ETBE), MTBE and methanol are all

oxygenates. These kind of fuels have a high molecular oxygen content and are

either alcohols or ethers, blended with petrol leading to lower emissions of carbon

monoxide and hydrocarbons, and serving as a lead replacer. They also have higher

octane ratings than petrol.

Ethanol can be produced in two forms: hydrated and anhydrous. Hydrated ethanol

has a purity of 95-96 % v/v. As second stage refining process is required to

produce anhydrous ethanol with a purity of 99-100%. Anhydrous ethanol will

readily blend with petrol. Blends of petrol with up to till 22% anhydrous ethanol

can be readily used in unmodified cars. It is expected that factories producing

hydrated alcohol for consumption purposes will integrate the second distillation

process to produce anhydrous ethanol for blending with petrol as soon as it

attractive compared to sale to their traditional markets.

ETBE is produced by mixing ethanol and isobutylane and reacting them with heat

over a catalyst. Blended with petrol ETBE has a similar function as ethanol as an

oxygenate and anti-knock additive. However, ETBE has some logistic advantages

over ethanol, as it does not dilute with water, and therefore it is less likely that it

picks up water or other contaminants during handling, for instance in transport

lines. Another plus of ETBE is its lower vapour pressure - or evaporative

properties, which reduces the volatility of the blend, which is an environmental

advantage when air quality is considered. However, dilution of ethanol with petrol

is proven at a large scale in Brazil and the US, and the industrial stakeholders in

France and Spain had definitely a role in the choice to produce of ETBE in these

countries - a process that includes a refinery step, and thus involvement of the

traditional oil industry - instead of using blends of ethanol (Siemons et al., 2004).

Chapter 2

Second Generation Bioethanol

2.1 Overview

Lignocellulosic biomass has been the most important energy source for humans

since the discovery of fire, and today it is still the main source of energy for almost

half of the world’s population. The need to increase the use of renewable energy is

fundamental to make the world energy matrix more sustainable. Advanced

technologies are now under development to convert biomass into various forms of

secondary energy including electricity, gaseous and liquid biofuels, such as

bioethanol.

While first generation bioethanol is produced from food crops, thus generating an

economic and ethical competition between the fuels and the food markets, second

generation bioethanol can be produced from a wider range of feedstocks. The

scope of second generation biofuel processes is to extend the amount of biofuel

that can be produced sustainably by using biomass comprised of the residual non-

food parts of current crops, such as stems, leaves and husks that are left behind

once the food crop has been extracted, as well as other crops that are not used for

food purposes, such as switch grass and cereals that bear little grain, and also

industry waste such as wood chips, skins and pulp from fruit pressing etc.

2.2 Different Lignocellulosic Feedstocks

There are various forms of biomass resources in the world, which can be grouped

into four categories. Wood residues are by far the largest current source of

biomass for energy production, including paper mills and furniture manufacturing.

Municipal solid waste is the next largest, followed by agriculture residues and

dedicated energy crops. Among these biomass resources including short-rotation

woody crops and herbaceous crops, dedicated energy crops seem to be the largest,

most promising, future resource of biomass. This is because of the ability to obtain

numerous harvests from a single planting, which significantly reduces average

annual costs for establishing and managing energy crops, particularly in

comparison to conventional crops (Monique et al., 2003).

Fermentation processes from any material that contains sugar could derive

ethanol. The varied raw materials used in the manufacture of ethanol via

fermentation are conveniently classified into three main types of raw materials:

sugars, starches, and cellulose materials. While sugars can be converted into

ethanol directly, starchy materiale must first be hydrolyzed to fermentable sugars

by amylase enzymes. Also lignocellulosic biomass must be converted into sugars,

generally by the action of a thermochemical treatment followed by enzymatic

hydrolysis. Once simple sugars are formed, enzymes from microorganisms can

readily ferment them to ethanol.

Among the three main types of raw materials, cellulose materials represent the

most abundant global source of biomass and have been largely unutilized. The

global production of plant biomass, of which over 90% is lignocellulose, amounts

to about 200×109 tons per year, where about 8–20×109 tons of the primary

biomass remains potentially accessible (Polman 1994). However, the utilization of

the lignocellulosic feedstocks is characterised by a number of issues, such as their

seasonal availability, scattered stations, and the high costs of transportation and

storage.

Furthermore, lignocellulose is a more complex substrate than starch. It is

composed of a mixture of polysaccharides, namely cellulose and hemicellulose, and

lignin. These molecules are tightly bound to each other by both hydrogen and

covalent bonds.

The biochemical conversion of a lignocellulosic biomass to ethanol requires a

series of consecutive steps. After a size reduction step, the first phase of this proces

is a thermochemical pretreatment, after which cellulose and hemicellulose are

enzymatically hydrolyzed to hexose and pentose monomeric sugars, i.e. the actual

substrate of the alcoholic fermentation. Saccharification and fermentation can be

carried out separately or, more conveniently, in a single step in which the

monomeric sugars are fermented by the microorganisms as soon as they are

realeased by the enzymatic activity of cellulases and hemicellulases (Simultaneous

Saccharification and Fermentation-SSF). The final step of bioethanol production is

product recovery which is usually operated by distillation or membrane processes.

One of the most (economically and technically) critical steps in the production of

bioethanol is the enzymatic hydrolysis of the lignocellulosic biomass; the cost of

cellulase production currently accounts for a fairly large fraction of the estimated

total production costs of bioethanol.

In the next paragraphs, the production process for lignocellulosic ethanol will be

described.

2.3 Production Process

The conversion process of lignocellulosic biomass to ethanol can be described as

the integration of five unit operations: desizing, thermochemical pretreatment,

enzymatic hydrolysis, fermentation, and ethanol recovery (see figure 2.1). In the

following paragraphs this steps will be deeply described.

Figure 2.1 – Lignocellulose-to-ethanol conversion process (from Merino et al., 2007).

2.3.1 Pretreatment

The hydrolysis lignocellulose to fermentable monosaccharides is technically

problematic because the digestibility of cellulose is hindered by many physico-

chemical, structural and compositional factors. Owing to these characteristics,

pretreatment is an essential step for obtaining potentially fermentable sugars in

the hydrolysis step. In this view, the aim of the pretreatment is to break down the

lignin structure and disrupt the crystalline structure of cellulose for enhancing

enzymes accessibility to the cellulose during hydrolysis step (Mosier et al., 2005) .

Besides being considered a crucial step in the biological conversion to ethanol,

biomass pretreatment represents one of the main economic costs in the process. In

fact, it has been described as the second most expensive unit cost in the conversion

of lignocellulose to ethanol based on enzymatic hydrolysis preceded by feedstocks

cost.

Since different lignocellulosic materials have different characteristics, it is

necessary to adopt suitable pretreatments technologies based on the

lignocellulosic biomass properties of each feedstock. Furthermore, the choice of

certain pretreatment has a large impact on all subsequent steps in the overall

conversion scheme; in fact, it influences the cellulose digestibility, the generation

of toxic compounds potentially inhibitory for yeast, the stirring power

requirements, the energy demand in the downstream process and wastewater

treatment demands (Galbe and Zacchi, 2007).

There are several key properties to take into consideration for low-cost and

advanced pretreatment process (Yang and Wyman, 2008):

High yields for multiple crops. Various pretreatments have been shown to be

better suited for specific feedstocks. For example, alkaline-based

pretreatment methods can effectively reduce the lignin content of

agricultural residues but are less satisfactory for processing recalcitrant

substrate as softwoods (Chandra et al., 2007). Acid based pretreatment

processes have been shown to be effective on a wide range of lignocellulose

substrate, but are relatively expensive (Mosier et al., 2005).

Highly digestible pretreated solid. Cellulose from pretreatment should be

highly digestible with yields higher than 90% in less than three days with

enzyme loading lower than 10 FPU/g cellulose (Yang and Wyman, 2008).

No significant sugars degradation.

Minimum amount of toxic compounds. The liquid hydrolyzate from

pretreatment must be fermentable following a low-cost, high yield

conditioning step. Harsh conditions during pretreatment lead to a partial

hemicellulose degradation and generation of toxic compounds derived from

sugar decomposition that could affect the proceeding hydrolysis and

fermentation steps (Oliva et al., 2003). Toxic compounds generated and

their amounts depend on raw material and harshness of pretreatment.

Degradation products from pretreatment of lignocellulose materials can be

divided into the following classes: carboxylic acids, furan derivatives, and

phenolic compounds. Main furan derivates are furfural and 5-

hydroxymethylfurfural (HMF) derived from pentoses and hexoses

degradation, respectively; (Palmqvist and Hahn- a gerdal, ). ea

acids are mostly acetic and formic and levulinic acids Phenolic compounds

include alcohols, aldehydes, ketones and acids (Klinke et al., 2002).

Operation in reasonable size and moderate cost reactors. Pretreatment

reactors should be low in cost through minimizing their volume, employing

appropriate materials of construction for highly corrosive chemical

environments, and keeping operating pressures reasonable.

Lignin recovery. Lignin and other constituents should be recovered to

simplify downstream processing and for conversion into valuable co-

products (Yang and Wyman, 2008).

Minimum heat and power requirements. Heat and power demands for

pretreatment should be low and/or compatible with the thermally

integrated process.

Pretreatment processes can be classified into three broad categories: (1)

mechanical processes that primarily reduce the size particles of the feedstock, (2)

chemical pretreatment processes that rely on the presence of acids, bases,

solvents, or other (bio)agents to extract select components of the feedstock, or

modify its structure, and (3) thermochemical processes that rely on a combination

of heat, pressure, and mechanical energy to alter lignocellulosic feedstocks.

2.3.1.1 Mechanical Pretreatment Processes

Mechanical pretreatment methods will reduce the biomass particle sizes thereby

increasing the available surface area for enzymatic attack. Typical examples of

physical pretreatments are:

- Mechanical comminution. The reduction of particle size and cristallinity of

lignocellulosic, which also mean an increase of the specific surface and

reduction of the degree of polymerization, can be achieved by a combination of

chipping, grinding or milling depending on the final particle siza of the

material (10-30 mm after chipping, 0.2-2 mm after milling or grinding) (Sun

and Cheng, 2002). The power requirement of these pretreatments are

relatively high, depending on the final particle size and the biomass

characteristics, making them not economically feasible (Hendriks and Zeeman,

2009).

- Extrusion. This is a novel and promising method in which the materials are

subjected to heating, mixing and shearing, resulting in physical and chemical

modifications during the passage through the extruder. Screw speed and

barrel temper- ature are believed to disrupt the lignocellulose structure

causing defibrillation, fibrillation and shortening of the fibers, and, in the end,

increasing accessibility of carbohydrates to enzymatic attack (Karunanithy et

al., 2008).

2.3.1.2 Chemical Pretreatments Processes

2.3.1.2.1 Alkali methods

The effect that some bases have on lignocellulosic biomass is the basis of alkaline

pretreatments. These methods increase cellulose digestibility and they are more

effective for lignin solubilization, exhibiting minor cellulose and hemicellulose

solubilization than acid or hydrothermal processes (Carvalheiro et al., 2008).

Alkali pretreatments are described to cause less sugar degradation than acid

pretreatment and it was shown to be more effective on agricultural residues than

on wood materials (Kumar et al., 2009a).

Sodium, potassium, calcium and ammonium hydroxides are suitable alkaline

pretreatments. NaOH causes swelling, increasing the internal surface of cellulose

and decreasing the degree of polymerization and cristallinity, which provokes

lignin structure disruption (Taherzadeh and Karimi, 2008).

Ca(OH)2, also known as lime, has been widely studied. Lime pretreatment removes

amorphous substances such as lignin, which increases the crystallinity index.

Lignin removal increases enzyme effectiveness by reducing non-productive

adsorption sites for enzymes and by increasing cellulose accessibility (Kim and

Holtzapple, 2006). Pretreatment with lime has lower cost and less safety

requirements compared to NaOH or KOH pretreatments and can be easily

recovered from hydrolysate by reaction with CO2 (Mosier et al., 2005).

Addition of an oxidant agent (oxygen/H2O2) to alkaline pretreatment can improve

the performance by favoring lignin removal (Carvalheiro et al., 2008). Furthemore,

no furfural or HMF were detected in hydrolysates obtained with alkaline peroxide

pretreatment which favours the fermentation step in an ethanol production

process (Taherzadeh and Karimi, 2008).

2.3.1.2.2 Acid Methods

The main objective of the acid pretreatments is to solubilize the hemicellulosic

fraction of the biomass and to make the cellulose more accessible to enzymes. This

type of pretreatments can be performed with concentrated or diluted acid, the

former being less attractive for ethanol production due to the formation of

inhibiting compounds, equipment corrosion problems and acid recovery (Wyman,

1996).

Diluted acid pretreatment have been studied for pretreating wide range of

lignocellulosic feedstocks. Different types of reactors such as percolation, plug

flow, shrinking-bed, batch and countercurrent reactors have been applied for

pretreatment of lignocellulosic materials (Taherzadeh and Karimi, 2008). It can be

performed at high temperature (e.g. 180 °C) during a short period of time; or at

lower temperature (e.g. 120 °C) for longer retention time (30– 90 min). It presents

the advantage of solubilizing hemicellulose but also converting solubilized

hemicellulose to fermentable sugars. Nevertheless, depending on the process

temperature, some sugar degradation compounds such as furfural and HMF are

detected, and affect the microorganism metabolism in the fermentation step (Saha

et al., 2005). Anyhow, this pretreatment generates lower degradation products

than concentrated acid pretreatments.

The most studied acid for acid pretreatment is diluted H2SO4. Hydrochloric acid,

phosphoric acid and nitric acid have also been tested (Mosier et al., 2005a). Some

examples of H2SO4 utilization are:

Saccharification yield as high as 74% was shown when wheat straw was

subjected to 0.75% v/v of H2SO4 at 121 °C for 1 h (Saha et al., 2005).

Olive tree biomass was pretreated with 1.4% H2SO4 at 210 °C resulting in

76.5% of hydrolysis yields (Cara et al., 2008).

Recently, ethanol yield as high as 0.47 g/g glucose was achieved in

fermentation tests with cashew apple bagasse pretreated with diluted H2SO4 at

121 °C for 15 min (Rocha et al., 2009).

Organic acids such as fumaric or maleic acids can be efficiently utilized to pretreat

lignocellulosic biomass, with the former being more effective than than latter;

furthermore, recent studies demonstrated that less amount of furfural is formed in

the maleic and fumaric acid pretreatments than with sulfuric acid (Kootstra et al.,

2009).

2.3.1.2.3 Organosolv

Organosolvation method is a promising methodology for lignocellulosic materials

pretreatment; comparing to other chemical pretreatments, the main advantage of

this process is the recovery of relatively pure lignin as a by-product. A number of

organic or aqueous solvent mixtures can be utilized, including methanol, ethanol,

acetone, ethylene glycol and tetrahydrofurfuryl alcohol, in order to solubilize lignin

and provide treated cellulose suitable for enzymatic hydrolysis (Zhao et al.,

2009a).

In some studies these mixtures are combined with acid catalysts (HCl, H2SO4, oxalic

or salicylic) to break hemicellulose bonds; this strategy lead to high yield of xylose.

However, this acid addition can be avoided for a satisfactory delignification by

increasing process temperature (above 185 °C).

Removal of solvents from the system is necessary using appropriate extraction and

separation techniques, such as evaporation and condensation, and they should be

recycled to reduce operational costs. Solvents need to be separated because they

might be inhibitory to enzymatic hydrolysis and fermentative microorganisms

(Sun and Cheng, 2002).

2.3.1.3 Thermochemical Pretreatment Processes

2.3.1.3.1 Steam explosion

Steam explosion, today, is the most widely employed thermochemical

pretreatment for lignocellulosic biomass. It is a hydrothermal process in which the

biomass is subjected to pressurised steam for a period of time ranging from

seconds to minutes, and then suddenly depressurised. This pretreatment combines

mechanical forces and chemical effects due to the autohydrolysis of hemicellulosic

acetyl groups. The high temperature promotes the formation of acetic acid from

acetyl groups, leading to the autohydrolysis of biomass; furthermore, water can

also act as an acid at high temperatures. The mechanical effects are caused because

the pressure is suddenly reduced and fibers are separated owing to the explosive

decompression. During this process, the lignin is redistributed and to some extent

removed from the material, increases enzyme accessibility to the cellulose

microfibrils (Pan et al., 2005).

The most important factors affecting the effectiveness of steam explosion are

particle size, temperature and residence time (Alfani et al., 2000): higher

temperatures result in an increased removal of hemicelluloses from the solid

fraction and an enhanced cellulose digestibility, but they also promote higher

sugar degradation.

Steam explosion process offers several attractive features when compared to other

pretreatment technologies. These include the potential for significantly lower

environmental impact, lower capital investment, more potential for energy

efficiency and less hazardous process chemicals and conditions (Avellar and

Glasser, 1998). Among the main advantages, it is worth to mention the possibility

of using high chip size, unnecessary addition of acid catalyst (except for

softwoods), high sugar recovery, good hydrolysis yields in enzymatic hydrolysis

and its feasibility at industrial scale development.

Although acid utilization in steam explosion has been introduced with some

disadvantages, many pretreatment approaches (SO2-explosion) have included

external acid addition (H2SO4) to catalyze the solubilization of the hemicellulose,

lower the optimal pretreatment temperature and give a partial hydrolysis of

cellulose (Tengborg et al., 1998). Notwithstanding, the main drawbac s when

using acids are related to equipment requirements and higher formation of

degradation compounds ( osier et al., b almqvist and ahn- a gerdal,

2000). In general, SO2-catalyzed steam explosion is regarded as one of the most

effective pretreatment method for softwood material (Tengborg et al., 1998).

The main drawbacks of steam explosion pretreatment are the partially

hemicellulose degradation and the generation of some toxic compounds that could

affect the following hydrolysis and fermentation steps (Oliva et al., 2003). The

major inhibitors are furan derivatives, weak acids and phenolic compounds. The

main furan derivatives are furfural and 5-hydroxymethyl furfural derived from

pentoses and hexoses degradation, respectively; by the other hand, weak acids

generated during steam explosion are mostly acetic acid, formed from the acetic

groups present in the hemicellulosic fraction, and formic and levulinic acids

derived from further degradation of furfural and HMF. Wide range of phenolic

compounds are generated due to the lignin breakdown varying widely between

different raw materials.

2.3.1.3.2 Liquid hot water

Liquid hot water is an hydrothermal process which does not require rapid

decompression and does not employ any catalyst or chemicals. Pressure is applied

to maintain water in the liquid state at elevated temperatures (160–240 °C) and

provoke alterations in the structure of the lignocellulose.

The objective of the liquid hot water is to solubilize mainly the hemicellulose, to

make the cellulose more accessible and to avoid the formation of inhibitors. The

slurry generated after this kind of pretreatment can be filtered to obtain two

fractions: one solid cellulose-enriched fraction and a liquid fraction rich in

hemicellulose derived sugars. To avoid the formation of inhibitors, the pH should

be kept between 4 and 7 during the pretreatment because at this pH hemicellulosic

sugars are retained in oligomeric form and monomers formation is minimized.

Therefore the formation of degradation products is also lower (Mosier et al.,

2005a).

In general, liquid hot water pretreatments are attractive from a cost-savings

potential: no catalyst requirement and low-cost reactor construction due to low-

corrosion potential. It has also the major advantage that the solubilized

hemicellulose and lignin products are present in lower concentration, due to

higher water input, and subsequently concentration of degradation products is

reduced. In comparison to steam explosion, higher pentosan recovery and lower

formation of inhibitors are obtained, however, water demanding in the process

and energetic requirement are higher and it is not developed at commercial scale.

2.3.1.3.3 Ammonia fiber explosion (AFEX)

AFEX is an alkaline physico-chemical pretreatment methodology. In process this

the lignocellulosic biomass is exposed to liquid ammonia at relatively high

temperature (90-100 °C) for a certain period of time (usually around 30 min),

followed by immediate reduction of pressure. The effective parameters in the

AFEX process are ammonia loading, temperature, water loading, blowdown

pressure, time, and number of treatments (Holtzapple et al., 1991).

The AFEX process can either modify or effectively reduce the lignin fraction of the

lignocellulosic materials, while the hemicellulose and cellulose fractions may

remain intact. At optimum conditions, which of course depend on the selected

lignocellulosic biomass, AFEX can significantly improve the enzymatic hydrolysis.

No formation of inhibitors for the downstream biological processes is one of the

main advantages of the ammonia pretreatment, even though some phenolic

fragments of lignin and other cell wall extractives may remain on the cellulosic

surface (Chundawat et al., 2007).

However, there are some disadvantages in using the AFEX process compared to

some other processes. AFEX is more effective on the biomass that contains less

lignin, such as herbaceous crops, and it does not significantly solubilize

hemicellulose compared to other pretreatment processes such as dilute-acid

pretreatment. Furthermore, ammonia must be recycled after the pretreatment to

reduce the cost and protect the environment (Wyman, 1996; Sun and Cheng,

2002).

2.3.1.4 Biological Pretreatment

In biological pretreatment processes, microorganisms such as brown-, white- and

soft-rot fungi are used to degrade lignin and hemicellulose in waste materials

(Schurz, 1978). Brown rots mainly attack cellulose, while white and soft rots attack

both cellulose and lignin. White-rot fungi are the most effective basidiomycetes for

biological pretreatment of lignocellulosic materials (Fan et al., 1987). The white-

rot fungus P. chrysosporium produces lignin-degrading enzymes, lignin peroxidases

and manganese-dependent peroxidases; both these enzymes have been found in

the extracellular filtrates of many white-rot fungi for the degradation of wood cell

walls. Other enzymes including polyphenol-oxidases, laccases can also degrade

lignin. The advantages of biological pretreatment include low energy requirement

and mild environmental conditions. However, the rate of hydrolysis in most

biological pretreatment processes is very low (Sun et al., 2002).

2.3.1.5 Pretreatment Efficiency and Enzyme Loadings

The pretreatment process has a very important effect on enzyme loadings and

hydrolysis efficiency (see Table 2.1). High severity pretreatments, particularly

those that use acids, which tend to solubilize higher levels of hemicellulose and

lignin, usually lead to lower enzyme loadings when washed solid residues are used.

However, if the inhibitors generated during high severity pretreatments are not

removed, higher enzyme loads are required to compensate.

Table 2.1 – Pretreatments trade-offs

Pretreated solids from alkaline pretreatment processes such as AFEX generally

require a xylanase as part of the enzyme cocktail to compensate for the residual

hemicellulose and xylo-oligosaccharides that remain. However, owing to their

lower severity, lower levels of inhibitors and beneficial effects on lignin, enzyme

loadings for alkaline pretreatments tend to be lower than enzyme loadings

required for uncatalyzed pretreatment processes such as LHW and autohydrolysis,

which generally demand the highest levels of hydrolytic enzymes.

A general statement about pretreatment methodologies is that a less severe

process may give equivalent or improved results compared to an acid catalyzed

pretreatment if the downstream enzymatic hydrolysis is modified. Ultimately, the

cost of the acid catalyst and the impact of inhibitors must be weighed against the

cost of additional enzymes.

2.3.2 Hydrolysis

A number of processes for hydrolyzing cellulose into glucose have been developed

over the years. The vast majority of processing schemes utilizes either cellulolytic

enzymes or sulfuric acid of varying concentrations. Historically, enzymes have

been too expensive for economical production of fuel ethanol from biomass.

Sulfuric acid, itself, is less expensive than cellulolytic enzymes, although disposal

costs associated with the use of sulfuric acid significantly increase its cost.

However, the single largest drawback to using sulfuric acid is that it also readily

degrades glucose at the high temperatures required for cellulose hydrolysis.

Hydrolysis of lignocellulosic biomass is more complicated than that of pure

cellulose due to the presence of nonglucan components such as lignin and

hemicellulose.

2.3.2.1 Acid hydrolysis

From the research studies it was revealed acid hydrolysis of lignocellulosic

biomass mainly produced xylose from xylan with the cellulosic and lignin fractions

remaining unaltered; xylan is more susceptible to this kind of hydrolysis due to its

amorphous structure compared to cellulose, characterised by a crystalline nature

(Rahman et al., 2007). Moreover, during acid hydrolysis, xylose is degraded rapidly

to furfural and other condensation byproducts, which are inhibit the activity of

fermenting microorganisms.

2.3.2.2 Enzymatic hydrolysis

Enzymatic hydrolysis of natural lignocellulosic materials is a very slow process

because cellulose hydrolysis is hindered by structural parameters of the substrate,

such as lignin and hemicellulose content, surface area, and cellulose crystallinity

(Pan et al., 2006). Since enzymatic hydrolysis of native lignocellulose usually

results in solubilization of ≈ % of the originally present glucan, some form of

pretreatment to increase amenability to enzymatic hydrolysis is included in most

process concepts for biological conversion of lignocellulose.

The enzymatic degradation of solid cellulose is a complicated process that takes

place at a solid–liquid phase boundary, where the enzymes are the mobile

components. When cellulase enzyme systems act in vitro on insoluble cellulosic

substrates, three processes occur simultaneously (Mosier et al., 2002):

chemical and physical changes in the residual solid-phase cellulose;

primary hydrolysis, involving the release of soluble intermediates from the

surface of reacting cellulose molecules;

secondary hydrolysis, involving hydrolysis of soluble intermediates to

lower molecular weight intermediates, and ultimately to glucose.

The rate of enzymatic hydrolysis of the cellulosic materials always decreases

rather quickly. Generally, enzymatic cellulose degradation is characterized by a

rapid initial phase followed by a slow secondary phase that may last until all

substrate is consumed. This has been explained most often by the rapid hydrolysis

of the readily accessible fraction of cellulose, strong product inhibition, and slow

inactivation of absorbed enzyme molecules.

The widely accepted mechanism for enzymatic cellulose hydrolysis involves

synergistic actions by three different kind of enzymes: endoglucanses (EG),

exoglucanases or cellobiohydrolases (CB ), and β-glucosidases (BGL). Next

chapter will be entirely focused on these enzymes.

2.3.3 Fermentation

The hydrolysate resulting from the pretreatment and the biochemical hydrolysis of

lignocellulosic biomass is eventually used for bioethanol fermentation by

microorganisms. Considering that hydrolysate contains not only glucose, but also

various monosaccharides, such as xylose, mannose, galactose, arabinose, and

oligosaccharides, the fermenting microorganisms should be required to efficiently

metabolize these sugars.

According to the reactions, the theoretical maximum yield is 0.51 kg bioethanol

and 0.49 kg carbon dioxide per kg of xylose and glucose:

3 C5H10O5 5 C2H5OH + 5 CO2

C6H12O6 2 C2H5OH + 2 CO2

Fermenting microorganisms can typically use the 6-carbon sugars, one of the most

common being glucose. Therefore, cellulosic biomass materials containing high

levels of glucose are the easiest to convert to bioethanol. Microorganisms, termed

ethanologens, presently convert an inadequate portion of the sugars from biomass

to bioethanol (Demirbas et al., 2005).

Xylose-fermenting microorganisms are found among bacteria, yeast and

filamentous fungi, both native and genetically engineered ones (Hahn-Hagerdal et

al., 2006). One of the most effective bioethanol- producing yeasts, Saccharomyces

cerevisiae, has several advantages owing to its high bioethanol production from

hexoses and high tolerance to bioethanol and other inhibitory compounds in the

acid hydrolysates of lignocellulosic biomass. However, because wild-type strains of

this yeast cannot utilize pentoses, bioethanol production from a lignocellulose

hydrolysate is inadequate (Katahira et al., 2006). For xylose-using S. cerevisiae,

high bioethanol yields from xylose also require metabolic engineering strategies to

enhance the xylose flux (Hahn-Hagerdal et al., 2006).

Natural xylose-fermenting yeasts, such as Pichia stipitis, Candida shehatae, and

Candida parapsilosis, can metabolize xylose via the action of xylose reductase to

convert xylose to xylitol, and of xylitol dehydrogenase to convert xylitol to

xylulose. Therefore, bioethanol fermentation from xylose can be successfully

performed by recombinant S. cerevisiae carrying heterologous XR and XDH from P.

stipitis, and xylulokinase from S. cerevisiae (Katahira et al., 2006). The

ethanologenic bacteria that currently show the most promise for industrial

exploitation are Escherichia coli, Klebsiella oxytoca and Zymomonas mobilis (Dien et

al., 2003).

Microorganisms for bioethanol fermentation can best be described in terms of

their performance parameters, which are: temperature range, pH range, alcohol

tolerance, growth rate, productivity, osmotic tolerance, specificity, yield, genetic

stability, and inhibitor tolerance (Demirbas et al., 2004). All the recombinant

strains are mesophilic organisms and function best between 303 and 311 K

(Hettenhaus, 1998). An organism must maintain a fairly constant balance of pH to

survive: most bacteria grow best in a narrow range of pH from 6.5 to 7.5

(Aminifarshidmehr et al., 1996), while yeast and fungi tolerate a range of pH 3.5–

5.0. The ability to lower pH below 4.0 offers a method for present operators using

yeast in less than aseptic equipment to minimize loss due to bacterial

contaminants. The majority of organisms cannot tolerate bioethanol

concentrations above 10–15% (w/v) (Hettenhaus, 1998).

2.3.4 Product recovery

As biomass hydrolysis and fermentation technologies approach commercial

viability, advancements in product recovery technologies will be required. For

cases in which fermentation products are more volatile than water, recovery by

distillation is often the technology of choice. A distillation system separates the

bioethanol from water in the liquid mixture. Water content of virgin bioethanol is

generally higher than 80%. Large quantities of energy are required to concentrate

the ethanol to 95.6% (azeotrope mixture of ethanol with water). The beer column

separates most of the bioethanol from water and produces a top stream rich in

bioethanol, and a bottom stream rich in water [145]. In this flow, bioethanol from

cellulosic biomass has likely lower product concentrations (<5 wt%) than in

bioethanol from corn. The maximum concentration of bioethanol tolerated by the

microorganisms is about 10 wt% at 303 K but decreases with increasing

temperature. To maximize cellulase activity, the operation is rather at maximum

temperature (310K), since the cost impact of cellulase production is high relative

to distillation [49,77,146].

2.4 Process Optimization

Reducing process complexity remains a major challenge for the commercialization

of LCB to ethanol. Current research is focused on eliminating the need for

detoxification of hydrolysates, developing robust biocatalysts capable of

fermenting pentose and hexose sugars simultaneously, reducing water usage,

increasing ethanol yield and titer, and decreasing cellulase usage.

Various process configurations are shown in Figure 2.2. These decrease in

complexity from separate hydrolysis and fermentation (SHF) to consolidated

bioprocessing (CBP).

Figure 2.2 - Lignocellulose to ethanol process configurations. The cellulose could be hydrolyzed

alone before fermentation (SHF) or with the hemicellulose (SHcF) followed by fermentation

process. Cellulose hydrolysis could also occur simultaneously with fermentation in the presence

(SScF) or absence (SSF) of hemicellulose. CBP involves a biocatalyst that is capable of producing all

the hydrolytic enzymes and is also capable of fermenting all the resulting sugars (from Geddes et

al., 2011).

2.4.1 Simultaneous Saccharification and Fermentation

Enzymatic hydrolysis and fermentation can be performed separately in a process

named SHF (Separate Hydrolysis and Fermentation) or, more conveniently, in a

combined step - the so-called simultaneous SSF.

SSF gives higher reported bioethanol yields and requires lower amounts of enzyme

because end-product inhibition from cellobiose and glucose formed during

enzymatic hydrolysis is relieved by the yeast fermentation (Dien et al., 2003;

Chandel et al., 2007).

Major advantages of SSF as described by Sun (Sun et al., 2002), include: (i) increase

of hydrolysis rate by conversion of sugars that inhibit the cellulase activity, (ii)

lower enzyme requirement, (iii) higher product yields, (iv) lower requirements for

sterile conditions since glucose is removed immediately and bioethanol is

produced, (v) shorter process time; and (vi) less reactor volume. SSF process has

also some disadvantages. The main disadvantage of SSF lies in different

temperature optima for saccharification and fermentation (Krishna et al., 2001). In

many cases, the low pH (e.g. < 5), and high temperature (e.g. >313 K), may be

favorable for enzymatic hydrolysis, whereas the low pH can surely inhibit the lactic

acid production and the high temperature may affect adversely the fungal cell

growth (Huang et al., 2005). Trichoderma reesei cellulases, which constitute the

most active preparations, have optimal activity at pH 4.5 and 328K. For

Saccharomyces cultures SSF are typically controlled at pH 4.5 and 310 K (Dien et

al., 2003).

2.4.2 Consolidated Bioprocessing

Consolidated bioprocessing (CBP) is a highly integrated process configuration in

which the main steps in lignocellulosic ethanol production (hydrolytic enzymes

production, hydrolysis of carbohydrate, fermentation of both hexose and pentose

sugars) take place in a single reactor.

CBP has the potential to provide the lowest cost route for biological conversion of

cellulosic biomass to fuels and other products in processes featuring hydrolysis by

enzymes and/or microorganisms. To realize this potential, the first step to be

overcome is the development of a microorganism capable to produce hydrolytic

enzymes and efficiently metabolise all the components of the lignocellulosic

biomass; this microorganism should also produce a desired product at high yield

and titer. Both of these capabilities are possessed by known microorganisms, but

to date have not been combined in a single microorganism or microbial system.

Several lines of evidence support the feasibility of such combinations using

biotechnology, which could proceed through two distinct strategies each with

several potential host organisms: a native cellulolytic strategy, which involves

engineering naturally occurring cellulolytic microorganisms to improve product-

related properties; and a recombinant cellulolytic strategy, which involves

engineering non-cellulolytic organisms that exhibit high product yields so that they

express a heterologous cellulase system that enables cellulose utilization (Lynd et

al., 2005).

Chapter 3

Cellulase Enzymes:

State of the Art and Advances

in Their Production

3.1 Cellulase Biochemistry

Cellulose, the primary product of photosynthesis in terrestrial environments,

represents an important source of carbon and energy for many bacterial and

fungal microrganisms. The hydrolysis of this polysaccharide is catalyzed by a

number of enzymes which are collectively known as cellulase.

Cellulases are members of the glycoside hydrolase family of enzymes (Henrissat et

al., 1997).

The widely accepted mechanism for enzymatic cellulose hydrolysis in fungal

species involves synergistic actions by three kind of enzymes: endoglucanase (EC

3. .1.4), exoglucanase or cellobiohydrolase (EC 3. .1.91), and β-glucosidase (EC

3.2.1.21) (Henrissat, 1994; Zhang and Lynd, 2004). Endoglucanases hydrolyze

intramolecular β-1,4 glucosidic bonds of cellulose chains randomly to produce new

chain ends; exoglucanases processively cleave cellulose chains at the reducing and

non-reducing ends to release soluble cellobiose (a glucose dimer) or glucose β-

glucosidases eventually hydrolyze cellobiose to glucose. These three hydrolysis

steps occur simultaneously (see figure 3.1). Primary cellulose hydrolysis occurs on

the surface of solid substrates and releases small soluble oligosaccharides into the

liquid phase upon hydrolysis by endoglucanases and exoglucanases. The enzymatic

depolymerization step performed by endoglucanases and exoglucanases is the

rate-limiting step for the whole cellulose hydrolysis process; this is mainly due to

the highly crystalline structure of the cellulose polymer. Secondary hydrolysis,

which occurs in the liquid phase, involves primarily the hydrolysis of cellobiose to

glucose by β-glucosidases (Zhang and Lynd, 2004). During cellulose hydrolysis, the

solid substrate characteristics vary, including changes in the cellulose chain end

number resulting from generation by endoglucanases and consumption by

exoglucanases and changes in cellulose accessibility resulting from substrate

consumption and cellulose fragmentation.

Figure 3.1 – Mechanistic scheme of enzymatic cellulose hydrolysis by Trichoderma non-complexed

cellulase system (from Zhang et al., 2006)

Industrial cellulases are produced by fungi. The primary interest in fungal

cellulases stems from the fact that several fungi produce significant amount of

extracellular cellulases. Typical examples of fungal mesophilic strains known to

produce cellulases are Trichoderma viride, T. reesei, Aspergillus niger, A. fumigatus,

Fusarium oxysporium, Piptoporus betulinus, Penicillium echinulatum and P.

purpurogenum. Thermophillic fungi such as Sporotrichum thermophile, Scytalidium

thermophillum, Clostridium straminisolvens and Thermonospora curvata are also

known to produce cellulase enzymes, particularly importants for their

thermostable features (Kumar et al., 2008).

Bacterial cellulases, differently from fungal ones, exist as discrete multi-enzyme

complexes, named cellulosomes. This complex is composed by multiple subunits

that interact with each other synergistically and degrade cellulosic substrates

efficiently (Bayer et al., 2004). The most important components and its structure

are illustrated in Figure 3.2.

The main advantage of cellulosome is that it allows concerted enzyme activity in

close proximity to the bacterial cell, concomitantly minimizing the distance over

which cellulose hydrolysis products must diffuse; this allow efficient uptake of

these oligosaccharides by the bacteria.

Figure 3.2 – Bacterial cellulosome structure and main components (from Kumar et al., 2008)

3.2 Cellulases from Trichoderma reesei

Trichoderma reesei, also known as Hypocrea jecorina, is a mesophilic filamentous

which produce and excrete efficiently cellulase and xylanase enzymes. Industrial

strains of Trichoderma reesei can produce extracellular protein level up to 100 g/L

(Cherry et al., 2003). This ability together with its cheap cultivation make it a

useful organism for the large-scale production of enzymes for a variety of

industrial applications (Hui et al., 2001). Trichoderma cellulases and

hemicellulases are currently used in several kinds of industries. For examples, they

have been used for animal food processing (Henk et al., 1992) and textile

treatment (Lange, 1993); in addition, the potential applications for the pulp and

paper industry have been developed (Viikari, 1996).

Among the many mutants of T. reesei, Rut C-30 is a widely studied strain

(Montenecourt et al., 1979). It can grow on a single carbon source, such as

cellulose and xylan, and expresses both cellulases and xylanases. The repression by

glucose to the cellulase expression is less sensitive in Rut C-30 than in some other

strains (Verdoes et al., 1995; Paloheimo et al., 2003).

Cellulase production in T. reesei is regulated at the transcriptional level (Abrahao-

neto et al., 1995). The main genes cbh1, cbh2, egl1 and egl2 for cellulase

expression are assumed to be regulated coordinately and their relative expression

levels have been shown to be similar in different culture conditions; in addition,

cbh1 gene always has the highest expression level (Ilmen, 1997; Fowler et al.,

1999). Cellulase genes expression is induced by oligosaccharides directly or

indirectly derived from cellulose such as cellobiose (two β-1, 4-linked glucose

units) or sophorose (two β-1,2 linked glucose units) (Fritscher et al., 1990; Ilmen,

1997). Furthermore, cellulase genes are found to be induced when T. reesei grows

in the presence of several disaccharides, namely, laminaribiose, gentiobiose,

lactose and xylobiose (Vaheri et al., 1979; Durand et al., 1988). T. reesei is also able

to metabolize sorbitol and glycerol to grow; however, these carbon sources

hydrates don’t induce cellulase production (El-Gogary et al., 1989).

The most powerful inducer of cellulase expression in Trichoderma reeesei is

sophorose; nevertheless, this compound is specific to T. reesei since it does not

induce cellulase expression in other fungal species, such as Aspergilus niger,

Phanerochaete janthinellum and Phanerochaete chrysosporium (Hrmova et al.,

1991; Gielkens et al., 1999).

Considering that cellulose is too large to be transported into cells, an inducer

capable of passing through the cell wall needs to be formed when cellulose is used

as the inducer for cellulase production. One of the most important enzymes

implicated in cellulase expression is the β-glucosidase, which has two functions:

the first one is the cleavage of cellobiose into glucose, which leads to repression to

cellulase expression; the second function is the transglycosylation of cellobiose to

sophorose (Fowler et al., 1999). This molecule is considered to be a poor substrate

for β-glucosidase, while it is readily transported by a permease into the mycelium,

where it induces the expression of cellulase genes.

The presence of glucose, which is easy to metabolize and energetically favorable to

the microbe, leads to the repression of other genes expression needed for the use

of other carbon sources. The controlling mechanism is called glucose (carbon

catabolite) repression. The cellulase production by T. reesei is under glucose

repression. Trichoderma reesei Rut-C30 is a glucose repression less sensitive

strain, which contains a truncated cre1 gene. Transformation of a full-length cre1

gene into this strain can restore the glucose repression of cellulase genes, which

demonstrated the glucose repression is regulated by CREI (Ilmen et al., 1996;

Margolles-Clark et al., 1997).

3.3 Industrial application on cellulases

Commercial production of cellulase enzymes by culture fermentation began in the

early 1970s, with cellulase made by Trichoderma mainly sold for research studies.

The mid 1980s saw the first large industrial uses of cellulase for stonewashing

denim and as an additive for animal feeds. This was accompanied by the

introduction of commercial cellulases made by fungi of the genera Aspergillus,

Penicillium and Humicola (Nielsen et al., 1995). Growth in cellulase use has

continued into the late 1990s with other textile applications such as biopolishing,

animal feed applications in increased digestibility of barley and wheat- based

feeds, clarification and yield improvement for fruit juice, and laundry detergent.

Today the main fields of application of cellulase enzymes are (Tolan and Foody,

1999):

Stonewashing denim - Denim stonewashing originated in the 1970s as a way to

deliver pre-softened blue jeans to the public. The sewn denim was washed in the

presence of pumice stones for roughly 60 minutes to shear and abrade the

garments. The resulting jeans were softened by the stonewashing and therefore

“ready to wear” at the time of purchase. The use of stones had some inconvenients,

such as the damage of the washing machines, the dust provided in the plant and

the process effluent, and worker injuries. Cotton is pure cellulose, and cellulase

attacks cellulose, breaking it down, and thereby weakening the surface of the fabric

in the same way that stoning does. Cellulase is now used to treat virtually every

pair of stone-washed jeans sold in the world.

Household laundry detergent - Cellulase in laundry detergent removes the hairs,

known as pills, that occur on cotton clothes after repeated wearing and machine

washing. The cellulase removes the existing pills, and conditions the surface of new

or unpilled clothes. The result is an appearance that more closely resembles a new

garment in sharpness of color and smoothness of appearance. Cellulase also

enhances the softness and removal of soil from the garment.

Animal feed - The primary use of cellulase in the feed industry has been in barley-

and wheat-based feeds for broiler chickens and pigs. The barley and wheat contain

soluble beta-glucans that increase the viscosity of the feed in the gut of the ani-

mal. This, in turn, causes an uptake of water, which decreases the amount of car-

bohydrate and vitamins that the animal obtains from the feed, as well as causing

sticky stool and related problems of disease and effluent disposal. Inclusion of

cellulase in the feed, as well as xylanase and other enzymes, helps to overcome

these problems.

Deinking and dewatering paper - Deinking is the process by which the ink is

removed from paper to allow it to be recycled. Cellulase enzymes increase the

amount of ink removed from the fibers, thereby increasing the cleanliness of the

sheet. This results in a brighter, cleaner sheet, or alternatively a reduction in the

use of surfactants and bleaching chemicals. Paper dewatering is most important on

the paper machine, where an aqueous slurry of pulp and additives are pressed into

paper sheets: cellulase enzymes increase the rate of drainage of pulp, thereby

offering the potential to increase the speed of the paper machine.

Beverage processing - In the production of fruit juice, wine, beer, and other

beverages, the raw juice is in a slurry with solid fruit. Cellulase enzymes break

down cellulose and beta-glucan associated with the plant cell walls, thereby

decreasing the viscosity of the slurry and increasing the ease of the juice recovery.

The enzyme treatment can also increase the clarity of the juice by solubilizing

small particles and enhance the flavor of the juice by increasing the extractability

of flavor compounds.

Baking - Cellulase is used to break down gums in the dough structure, so as to

allow a more even dough rise and flavor distribution. However, too much action

can damage the dough structure and degrade the baked goods.

3.4 Production of cellulase by Trichoderma reesei

Trichoderma reesei is one of the most important fungal strain for cellulase

production. In the last decades, several studies focused their attention to find the

best cultural conditions to improve enzyme production with this strain.

The most widely used culture medium for the cellulase production was developed

by Mandels and Weber (Mandels, 1969), the composition of which is showed

below in Table 3.1. In this medium, there are carbon source, nitrogen source,

sulfur, phosphate, mineral nutrients and the antifoam tween 80. In the next

paragraphs are summarized the main aspects of submerged fermentation for

cellulase production.

Nutrient Concentration

(NH4)2SO4 1,4 g/l

KH2PO4 2 g/l

MgSO4 – 7 H2O 0,3 g/l

CaCl2 – 2 H2O 0,4 g/l

Proteose Peptone 1 g/l

Tween 80 0,2 g/l

FeSO4 5 mg/l

MnSO4 1,6 mg/l

ZnSO4 1,4 mg/l

CoCl2 2 mg/l

Table 3.1 – Composition of Mandels culture medium for Trichoderma growth

3.4.1 Carbon source and inducer

Solid cellulosic materials have been used as the carbon source and inducer for

fungus growth and cellulase production in different studies (Suto et al, 2001);

however, the high level of solid content in the liquid burdens the agitation, lowers

the availability of oxygen and adsorbs some of the enzymes in the bioreactor

(Oashima et al., 1990). Soluble substrates and inducers, by the other hand, have the

above advantages compared to the solid substrates and, in addition, the process

conditions can be optimized better and run as a fed-batch or a continuous mode to

maximize the productivity (Ju et al., 1999). The utilization of soluble substrates

may hamper the cellulase synthesis due to the accumulation of free small

oligosaccharydes and the consequent catabolite repression.

Whether cellulase production is mostly growth or non-growth-associated is still a

subject of some debate (Singhania et al., 2010; Velkovska et al., 1997; Lo et al.,

2010) attached, however, with a significant process economics relevance. In the

former case, the outgrown fungal biomass would be a continuous net process loss,

while in the latter case the fungal biomass could (in principle) be recycled and

reused for multiple cellulase production cycles. Indeed, if cellulase production

were fully non-growth associated, its production cost would be the sum of: the cost

of the substrate supporting (1) the maintenance of a steady state fungal biomass

concentration (i.e., in the absence of decay), (2) the growth compensating the

fungal biomass decay, (3) the energy supply for cellulase synthesis, (4) the

component supply supporting cellulase synthesis. If cellulase production were

growth associated, to following would add up to the previous items: (5) the

component and energy supply for the fungal biomass growth associated to the

planned cellulase production.

Cellulase process design and media formulation aim at maximizing induction of

cellulase production while minimizing catabolite repression arising from

accumulated breakdown products. Batch processes achieve simplicity and

maximal theoretical terminal enzyme activity but may incur in catabolite

repression if the instantaneous production of substrate/inducer breakdown

products from substrates is not balanced by their instantaneous consumption; if

pre-hydrolysed material is used as the feed, a mismatch in time between

increasing cell concentration and inducers concentration (oligosaccharides being

hydrolysed) may cause these latter may fail to exert their full potential (Lo et al.,

2010); fed-batch processes may be used to adjust the rate of production of

breakdown products from substrates to their rate of consumption by suitably

dosing the former and continuous processes may be used to diminish the

accumulation of reducing sugars; however, continuous culture production of

secondary metabolites may lead to internal contaminations by mutations and

replacement of the initial culture by faster-growing but less productive species.

Finally, multiple-stage processes may be used to match process conditions (e.g. pH

and temperature) to physiological stage.

Cellulase production from pure cellulose, or from soluble sugars such as lactose,

cellobiose, and sophorose, was deeply investigated in the past years. However, the

production of the enzyme using high-value substrates is not economically feasible

for the large-scale bioethanol production process. To overcome this problem, in

the past decade, a number of low-cost lignocellulosic substrates have been

investigated as a feedstock for cellulase production. They include wastepaper

materials (Shin et al., 2000; Wang et al., 2010), sawdust (Lo et al., 2005), wheat

straw (Chahal, 1996) and mixed agricultural wastes (Shiahmorteza et al., 2003; Xin

et al., 2010). Frequently, researchers working in a specific country focus their

attention on local significant lignocellulosic wastes coming from relevant

agricoltural crops and related industrial transformations thereof: it is the case of

orange wastes in Nigeria (Omojasola et al., 2008), banana wastes in India (Baig et

al., 2003) and tequila industry wastes in Mexico (Huitron et al., 2008).

3.4.2 Nitrogen source and other nutrients

The effect on cellulase production of different nitrogen sources such as ammonium

sulfate, ammonium nitrate, ammonium ferrous sulfate, ammonium chloride and

sodium nitrate have been studied Among these, ammonium sulfate led to

maximum production of cellulases while nitrate is generally considered not

suitable for T. reesei cultivations due to increase of medium pH during

fermentation.

Typical inorganic nitrogen sources in T. reesei cultivation are ammonium sulfate or

ammonia water solution. Nitrate is generally considered not suitable for T. reesei

cultivations due to increase of medium pH during fermentation (Olsson et al.,

1994). The organic nitrogen sources, such as peptone, yeast extract and corn steep

liquor, are better with an increase in cellulase production; however, the utilization

of these nitrogen sources for the culture medium scale up the cost of the process

(Kumar et al., 2008).

Besides carbon and nitrogen sources, several other factors have also been reported

to be important in optimization of cultivation conditions. The morphological and

physiological changes of T. reesei have an effect on cellulase production (Mcintyre

et al., 1998). In the cultivation medium, potassium phosphate serves as a

phosphate source to form part of the cell and buffer in the cultivation, which

concentration could be adjusted to certain extent and will not have an effect on

cellulase expression. Magnesium sulfate and calcium chloride are very important

to the enzyme function inside the cell. All of the trace-elements are essential to the

cell growth and enzyme functions, however, the amounts are very small, otherwise

it will cause cell death.

Another important element of the culture medium is the surfactant, the most

widely used of which is Tween-80. This substance is beneficial for the cellulase

production with its optimal concentration is 0,2 mL/L, while higher concentration

is harmful (Olsson et al., 1994). The rational for the enhanced cellulase production

by Tween-80 may be due to the increased permeability of the cell membrane,

contributing a more rapid secretion of the enzymes, and as a result, which leads to

a greater synthesis.

3.5 Cellulase production today: issues and perspectives

The hydrolysis of lignocellulosic biomass by cellulase enzymes accounts for the

40% of current bioethanol cost input (Zhang and Lynd, 2004). To turn the prospect

of replacing a significant proportion of fossil fuels into reality, the lignocellulose-

to-ethanol conversion process has to become less expensive. Current estimates

suggest that the cost of producing cellulosic ethanol is $1.80/gallon or higher,

which is almost twice as high as the cost of first generation bioethanol (Gallagher,

2001).

In the last decades, much effort has been focused on understanding the factors that

mainly contribute to the production cost of cellulase enzymes; the next paragraphs

are focused on the theoretical ways to address these challenges.

3.5.1 The impact of substrate selection

Lignocellulisic biomasses are characterized by an intrinsic biochemical variability

between different plant species. The principal components of biomass are cellulose

(30–50%), hemicellulose (20–30%) and lignin (20–30%), with minor percentages

of starch, proteins and oils. Table 3.2 shows the biochemical variety of different

typical substrates for lignocellulosic ethanol production.

Table 3.2 – Chemical composition of different plant species (from Merino and Cherry, 2007)

The biochemical composition of the lignocellulosic substrate has a direct impact on

enzymatic digestibility: the main characteristics that have been shown to influence

the hydrolysis include accessibility, degree of cellulose crystallinity, and the type

and distribution of lignin (Mansfield et al., 1999).

Considering the great influence of substrate composition on enzymatic hydrolysis

effectiveness, one of the most promising strategy in this field consists in the

genetic alteration of the most abundant substrates, to obtain an increased

susceptibility to enzyme digestion. Modifying lignin biosynthetic enzymes to lower

lignin in cell walls is an obvious way to reduce biomass recalcitrance, and sugar

yields from modified alfalfa lines with lower lignin-forming enzymes were nearly

double over wild type (Chen and Dixon, 2007). A decrease in lignin content was

also achieved in aspen by down-regulation of the coumarate-coenzyme A ligase

(Pt4CL1), which led to a 45% decrease in lignin and a compensatory 15% increase

in cellulose content of the modified plant. This altered ratio favors bioethanol

production (Li et al., 2003).

Another way to increase enzymatic digestibility of plants consists in reducing the

levels of synthetic enzymes and/or increasing the levels of degradative enzymes.

Reducing the expression of poplar glycosyltransferase using RNA interference led

to a reduction in the glucuronoxylan content of poplar and consequently increased

its digestibility by cellulase (Lee et al., 2009). Arabidopsis plants expressing a

repressor derived from a secondary cell wall thickening-promoting factor (NST1)

were twice as susceptible to enzymatic hydrolysis as control plants (Iwase et al.,

2009).

3.5.2 The impact of enzymes selection: new genes versus tailored cocktails

Three categories of enzymes in the glycosyl-hydrolase superfamily are required for

deconstruction of cellulose after the biomass has undergone pretreatment. These

hydrolases are endoglucanase, exoglucanase (also named cellobiohydrolase), and

β-glucosidase. These enzymes work synergically, which means, their combined

effect on cellulose hydrolysis is greater than the individual effects added together.

Since the 1970s, the search for new genes has led to the discovery of many sources

of cellulase from fungi, termites, aerobic and anaerobic bacteria.

While in the past selection and screening was performed in order to isolate a pure

culture, today the metagenomic approach permits to analyze abundant and

biodiverse environments, such as soil, sea and ocean water, to revealed the

presence of many new microorganisms; the discovery of new microbes, in turn,

leads to the characterization of new genes and then proteins. Figure 3.3 illustrates

the typical approach of metagenomic gene discovery. After biotope selection and

sample or culture enrichment, nucleic acid is extracted from the environmental

sample. The approach might involve metagenomics (environmental genomic DNA)

or metatranscriptomics (environmental mRNA reversed transcribed to comple-

mentary DNA, cDNA) and an enrichment or selection can be applied. Gene

enrichment selects for differentially expressed genes using techniques such as

differential expression analysis (DEA) and gene targeting. Genome enrichment

uses techniques such as stable isotope probing (SIP), 50Bromo-2-deoxyuridine

(BrdU)-labelling and suppressive subtractive hybridization (SSH) to enrich or

select for genomes of interest. Downstream screening approaches can be activity-

based through the screening of expression libraries, sequence-dependent by using

gene targeting or can be sequence-independent through the direct sequencing of

the metagenome. The final expression requires a full-length open reading frame

(ORF) expressed in a suitable host to generate a functional gene product (Cowan et

al., 2005).

Figure 3.3 – Metagenomic approach for new genes discovery (from Cowan et al., 2005)

Instead of looking for new microbial species and new genes in nature, scientists

today have the possibility to use genetic engineering to improve the the specific

activity of cellulolytic enzymes. There are two main strategies: directed evolution

and rational design. In the first approach, DNA shuffling using PCR is used as a

powerful way to randomly modify the structure of enzymes, which are later

screened for activity (Rabinovich et al., 2002). By the other hand, rational design

uses targeted approaches to modifying enzymes: the availability of

crystallographic and site-directed mutagenesis data allows understanding of the

structure of the catalytic site of cellulases and provides the basis for a rational

design approach to optimize the interaction of the enzyme with the substrate

(Zhang et al., 2006).

Although it could guarantee the best results in long term perspectives, the search

of new cellulase genes and the optimization of the existing ones through genetic

engineering techniques are very slow activities. A very important and effective

strategy used today consists in creating cocktails of known cellulolytic enzymes

tailored to specific biomass substrates. The optimization of enzyme mixture may

lead to improved hydrolysis performance and, more importantly, to a substantial

decrease of enzyme load, which means a reduction of costs.

3.5.3 The impact of process integration

The production process of lignocellulosic ethanol is made of different steps, from

biomass pretreatment to ethanol fermentation and recovery. In the last decades,

researchers focused their attention to the single steps, for example improving

sugar yield from pretreatment, biochemical hydrolysis rate and ethanol yield from

yeasts.

It is clear today that to further improve the process, the different steps of

pretreatment, hydrolysis, and fermentation need to be viewed holistically. As

discussed previously, the choice of a particular pretreatment impacts on the

following hydrolysis step, both for the enzyme cocktail and for the enzyme load.

Similarly, the selection of the fermenting microorganism determines optimal

process parameters, such as pH and temperature, which in turn can affect enzyme

performance and loading since hydrolysis and fermentation are often combined

hydrolysis in a single reactor.

The enzymatic hydrolysis can either be done separately from the fermentation

(SHF, separate hydrolysis and fermentation) or in combination with the

fermentation (SSF, simultaneous saccharification and fermentation).

In SHF, hydrolysis is allowed to proceed to a point of completion at reaction

conditions optimal for enzyme action, (50° C and pH 5 for T. reesei cellulases), then

the process parameters are adjusted to allow survival of the fermenting organism

(≈3 ◦ C and pH 5,5-7). The primary drawback of this process configuration is the

low hydrolysis rate due to end-product inhibition of enzymes. On the contrary, SSF

process is capable of improved hydrolysis rates, yields, and product concentrations

compared to SHF because of the continuous removal of the reaction end products

by the yeast, provided the parameters required for fermentation does not

drastically slow enzyme action. Ideally we will see organisms and enzymes

developed that have similar growth and reaction optima, allowing optimal growth

and enzyme action to occur in a single vessel. In hybrid hydrolysis and

fermentation (HHF), the biochemical hydrolysis and fermentation take place in the

same reactor but they are temporally separated to optimize the two single

processes: firstly, enzymatic hydrolysis is allowed to proceed to a point at which

glucose release is almost completed, then the temperature is dropped, the pH

increased, and fermentation is started by addition of the organism.

In recent years, the concept of consolidated bioprocessing (CBP), has been

garnering a lot of interest because of the potential for drastically reducing

production cost. This process is characterized by the presence of a single

microorganism capable of both cellulolytic enzymes production (so to hydrolyze

lignocellulosic biomass) and ethanol fermentation. It is evident that the main

requirement to develop this process is the creation of a microorganism with the

selected abilities. Although no natural microbe exhibits all the features desired for

CBP, a number of microorganisms, both bacteria and fungi, possess some of the

desirable properties. These microorganisms can broadly be divided into two

groups: first, native cellulolytic microorganisms that possess superior

saccharolytic capabilities, but not necessarily product formation; second,

recombinant cellulolytic microorganisms that naturally give high product yields,

but into which saccharolytic systems need to be engineered. Examples of native

cellulolytic microorganisms under consideration include anaerobic bacteria with

highly efficient complexed saccharolytic systems, such as mesophilic and

thermophilic Clostridium species, and fungi that naturally produce a large

repertoire of saccharolytic enzymes, such as Trichoderma species and Fusarium

oxysporum. However, the anaerobic bacteria produce a variety of fermentation

products, limiting the ethanol yield, whereas the filamentous fungi are slow

cellulose degraders and give low yields of ethanol. Candidates considered as

potential recombinant cellulolytic microorganisms into which saccharolytic

systems have been engineered include the bacteria Zymomonas mobilis, Escherichia

coli and Klebsiella oxytoca, and the yeast Saccharomyces cerevisiae and xylose-

fermenting yeasts Pachysolen tannophilus, Pichia stipitis, and Candida shehatae

(van Zyl et al., 2007).

Chapter 4

Materials and Methods

4.1 Microorganism

The mutant cellulase-producing strain Trichoderma reesei Rut-C30 (NRRL 11460)

used in this work was obtained from United States Department of Agriculture

(Agricultural Research Service Patent Culture Collection, Peoria, Illinois). The

microorganism was maintained at 4 °C on Petri plates of Potato Dextrose Agar,

with regular subculturing every 4–6 weeks.

4.2 Culture media

Trichoderma reesei Rut-C30 pre-culture was carried out on two types of

propagation medium: glucose-based and pomace-based.

Glucose-based medium was based on the Mandels one (Mandels and Weber, 1969)

with the exception that urea was omitted while the peptone content was elevated

by 25%: glucose (10 g/l) KH2PO4 (2 g/l); (NH4)2SO4 (1.4 g/l), MgSO4•7 2O (0.3

g/l); FeSO4•7 2O (5 mg/l); MnSO4• 2O (1.6 mg/l); ZnSO4• 2O (1.4 mg/l);

CoCl2•6 2O (2 mg/l); CaCl2• 2O (0.4 g/l); Proteose Peptone (1 g/l); Tween 80

(0.2 g/l). The composition of the culture medium used for the cellulase production

test was the same as that of the corresponding pre-culture medium, but for the

supplementation with 10 g/l of a specific inducer (lactose, Avicell cellulose, OP)

and the absence of glucose (except in some runs where this has been explicitly

stated). Pomace-based propagation media were obtained by enzymatically

hydrolysing (15 FPU/g biomass) finely ground olive pomace previously subjected

to acidic-thermal pretreatment (50 g/L olive pomace, 45 min at 120 °C in 1.5%

H2SO4). After hydrolysis, the suspension was centrifuged and the supernatant,

added with the remaining compounds, adjusted to the growth medium final

volume, and thermally sterilised.

4.3 Cellulase Production Tests

All the performed tests were carried out in 300-ml Erlenmeyer flasks with a

working volume of 100 ml; the flasks were incubated at room temperature (24° C)

and agitated on a rotary shaker (200 rpm). A 10% or 50% (v/v) inoculum

concentration (resulting in a biomass concentration of 0.3 or 1.5 g/l, respectively),

from a 3-day old pre-culture, was used to initiate the cellulase production tests.

The production flasks were periodically sampled and reducing sugars

concentration and enzymatic activity (Filter Paper Activity, FPA), were measured.

All filter paper tests were run in duplicate.

OP was sterilised by autoclaving (120 °C, 20 min) in its culture medium before

inoculation. Sugar release during autoclaving, determined by the Miller method,

was less than 0.1 g/l.

4.4 Analytical Techniques

Reducing sugars were estimated by their glucose equivalents generated during the

assay, as determined by the 3,5-dinitrosalicylic acid method (Miller, 1959) with

glucose as standard. The enzymatic activity was measured according to the filter

paper activity (FPA) method (Ghose et al., 1987) and expressed as international

Filter Paper Units (FPUs); one FPU is defined as the amount of enzyme that

releases 1 μmol of glucose/min under the assay conditions. Activities were

reported as FPU/ml. Polyphenols were measured by the Folin-Ciocalteau

(Singleton, 1965) method using gallic acid as standard.

4.5 Olive Pomace

The olive mill solid byproducts used in this study were collected from an olive oil

production plant located in Southern Italy (Monopoli plant of Casa Olearia Italiana,

Marseglia Group; Bari, Italy). The size distribution of the solid residue, in the form

of dried pellets, was determined by sieving and the results are shown in Fig. 1. For

cellulase production induction, only the 710-1 μm fraction of the supplied raw

material was used; for fungal biomass inoculum growth, on the other hand, the

whole olive pomace was taken, finely ground and then treated as described in

Subsection “Culture media”.

4.6 Olive Oil Mill Wastewater

OOMW was obtained from an olive oil mill located in Central Italy (Oleificio

Fraterna Seconda; Breccelle, Isernia, Italy) after ~5 months of local storage in an

underground tank. Its characteristics were measured right before its use and were:

COD (measured by Hach-Lange kit): 22 g/l; polyphenols content (measured by the

Folin-Ciocalteau method): 1.9 g/l.

Chapter 5

Aim of the Work,

Results and Discussion

5.1 Aim of the Work: the ETOILE Project

This experimental work is embedded in a wider European-funded project named

Etoile (FP7/2007-2013, Project n° 222331).

The aim of Etoile project was to develop a new integrated process where the two

main wastes coming from olive oil traditional three-phase production process, the

solid lignocellulosic olive pomace (OP) and the liquid olive oil mill waste water

(OOMW), are exploited for the production of cellulolytic enzymes and bioethanol.

Figure 5.1 illustrates the process with its various steps. This project was carried on

through the cooperation of different international research partners, both public

and private:

1. Università degli Studi di Roma Sapienza

2. Copenhagen Institute of Technology (Aalborg University)

3. Labor S.r.l.

4. Explora Biotech S.r.l.

5. Foundation for Research and Technology Hellas (FORTH)

6. ARGUS Umweltbiotechnologie GmbH

7. PRISMA DOMI ATE

Figure 5.1 – The Etoile project: bioethanol production via lignocellulosic fermentation of olive oil

residues.

More specifically, this experimental work was focused on the exploitation of the

two selected wastes for the production of cellulase enzymes. In a first stage, olive

pomace has been tested as a new low-cost inducer for the production of enzymes

by the myceliar fungus Trichoderma reesei Rut-C30, in different cultural and

metabolic conditions. Considering that OOMW may be present (as an entrainment)

together with OP in the case of 3-phase processing or of drained alperujo, we also

investigated the effect of that liquid residue and the effect some of the most

representative OOMW polyphenols (vanillic acid, caffeic acid and tyrosol) on

cellulase production. Olive pomace ahs also been tested as carbon source (after a

thermochemical pretreatment) for the growth of the mold.

Finally, the ability of T. reesei to grow on and biotreat the olive oil mill waste water

has been unsuccessfully tested.

5.2 Results

5.2.1 Lactose-induced cellulase production

One essential culture medium component in a process of enzyme production is the

inducer: it is a compound, usually organic, that stimulates the production of the

desired enzyme in a particular microorganism. Typical inducers of cellulase

production in Trichoderma strains are cellulose (the enzyme target), lactose and

sophorose.

Shake-flask experiments, inoculated with a 10% v/v inoculum (i.e around 0.3 g/l of

biomass) from a pre-colture grown for 3 days, were initially performed to evaluate

the production of cellulase by Trichoderma reesei RUT-C30 using the carbohydrate

lactose as classical inducer. As showed in Figure 5.2, after a one-day lag phase, the

production of cellulase start to increase and reach a peak of 1.43 FPU/mL at 3rd

fermentation day.

Figure 5.2 - Fermentation profiles for T. reesei RUT-C30 cellulase activity (red curve) while

growing in a lactose-based medium; the blue curve shows the decrease of the reducing sugars.

5.2.2 Cellulose-induced cellulase production

Shake-flask experiments, inoculated with a 10% v/v inoculum (i.e around 0.3 g/L

of biomass) from a pre-colture grown for 3 days, were performed to evaluate the

induction power of cellulose (Avicel) in two different nutritional situations, i.e. in

the presence and in the absence of glucose as carbon and energy source. During

fermentations, samples were withdrawn every 24 hours and analyzed for enzyme

activity levels. The production of cellulase in the studied conditions is illustrated in

Figures 5.3 and 5.4.

When glucose is added (Fig. 5.3) to the colture medium, the measured FPA shows a

fast increase with a peak of 0.90 FPU/mL at 2nd fermentation day, followed by a

significant drop in the last two days; this is probably due to a catabolite repression

system, as confirmed by the observed decrease in glucose consumption rate. By

the other hand, in absence of glucose (Fig. 5.4), cellulase activity show a completely

different profile, characterized by a slow but constant increase up to the value of

0.68 FPU/mL at 4rd fermentation day.

Figure 5.3 - Fermentation profiles for T. reesei RUT-C30 cellulase activity and reducing sugars

concentration while growing in presence of cellulose.

Figure 5.4 - Fermentation profiles for T. reesei RUT-C30 cellulase activity while growing in

presence (red curve) or absence (blue curve) of glucose, with cellulose as inducer of enzyme

production.

5.2.3 Olive Pomace-induced cellulase production

Shake-flask experiments were conducted, as described in the previous paragraph,

to evaluate the induction power of lignocellulosic Olive Pomace in two different

nutritional situations, i.e. in the presence and in the absence of glucose as carbon

and energy source.

Figure 5.4 clearly indicates that OP can be effectively used as inducer of cellulase

production by T. reesei RUT-C30: when OP is added in the colture medium and

glucose is absent, the profile of the enzymatic activity show a constant increase,

reaching the maximum value of 1.16 FPU/mL at 4rd fermentation day. By the other

hand, when in a OP plus glucose colture medium (Fig. 5.6), the measured FPA

shows a trend similar to the cellulose-induced one, with a fast increase with a peak

at 2nd fermentation day (of 0.79 FPU/mL), followed by a significant drop in the

last two days

Figure 5.5 - Fermentation profiles for T. reesei RUT-C30 cellulase activity while growing in

presence (red curve) or absence (blue curve) of glucose, with Olive Pomace as inducer of enzyme

production.

Figure 5.6 - Fermentation profiles for T. reesei RUT-C30 cellulase activity and reducing sugars

concentration while growing in presence of Olive Pomace as inducer.

5.2.4 Comparison between cellulose and OP as inducers

Shake-flask experiments, inoculated with a 10% v/v inoculum (i.e around 0.3 g/L

of biomass) from a pre-colture grown for 3 days, were performed to evaluate the

induction power of OP in two different nutritional situations, i.e. in the presence

and in the absence of glucose as carbon and energy source. During fermentations,

samples were withdrawn every 24 hours and analyzed for enzyme activity levels.

The production of cellulase in the studied conditions is illustrated in Figure 5.7.

The first notable result, here, is that OP is actually usable as an inducer for

cellulase production in Trichoderma reesei RUT-C30. As it can be seen in Figure 5.7

(left), when glucose is added to the colture medium, the enzymatic activity profiles

are almost identical for the two tested inducers, cellulose and OP: after a 24-hour

lag phase, cellulase production started to increase, reaching a maximum value of

0.8–0.9 FPU/ml after 48 hours and slightly decreasing afterwards.

The observed experimental results are quite different, and more interesting, in the

absence of glucose, when cellulose or OP are the unique carbon and energy sources

in the colture medium. In these conditions, compared to cellulose, OP seems to be a

better inducer of cellulase production. As showed in Figure 5.7 (right), cellulase

production starts after a 24-h lag phase in both fermentations but the maximum

activity reached is higher in the OP-induced system (~1.2 FPU/mL) than in the

cellulose-induced one (about 0.7 FPU/ml). Furthermore, apparently, production

does not appear to reach a plateau over the test time.

Figure 5.7 - Fermentation profiles for T. reesei RUT-C30 cellulase activity while growing in

presence (left) or absence (right) of glucose; the inducers tested are Olive Pomace (red curves) and

cellulose (blue curves).

Cellulase production by T. reesei RUT-C30 was also studied in 9-day-long shake-

flask experiments in which the inoculum size was increased to 50% v/v of the pre-

colture (i.e around 1.5 g/L of biomass, centrifugated before inoculation in a fresh

medium). The increased fermentation time permitted us to better evaluate the

enzyme production, which usually reached a plateau.

The induction power of OP in 9-day-long fermentations was compared with the

effect of cellulose, the classical inducer of cellulolytic enzymes; the basal

expression level of cellulase in the studied conditions was evaluated by analyzing

the enzyme activity levels in inducer-lacking flasks. As it can be observed in Fig.

5.8, the enzyme activity reached in the studied condition are higher if compared

with the values reached in 4-day- long fermentations; this is obviously due to the

higher concentration of biomass but it also indicates that the chosen

microorganism does not fully express its productive potential over a four-day

bioreaction time. 9-day-long fermentations confirmed that OP represents a better

inducer of cellulase production compared to cellulose, the classical cellulase

production inducer used by most experimenters. Again, the maximum activity was

reached at the end of the fermentation run and a plateau was not evident in the

activity trend. The filter paper activity measured in OP- based medium was nearly

20% higher than in cellulose-based medium (3.0 vs 2.4 FPU/ml).

Figure 5.8 - Enzymatic activity profiles for 9-day fermentations with the two tested inducers: Olive

Pomace (green curve) and cellulose (red curve). The basal enzymatic activity (i.e. no inducer

present in culture media) measured in the studied conditions is also shown (blue curve).

5.2.5 OP concentration and pretreatment effects

The influence of Olive Pomace concentration and thermal pretreatment on

cellulase production was investigated.

The induction power of different concentrations of OP, ranging from 2.5 to 20

grams per liter, was tested in 9-day long fermentations as previously described. As

it can be seen in Figure 5.9, the maximum cellulase activity reached at 9th

fermentation day increase in a linear manner up to a 10 g/L OP concentration

while at 15 and 20 grams per liter the measured increments are more modest.

Figure 5.9 – Correlation between OP concentration and maximum enzymatic activities reached at

the end of 9-day fermentation.

In all the experiments, the OP was added to the colture media before its thermal

sterilization in an autoclave (at the temperature of 120° C for 20 minutes). In this

process, the lignocellulosic biomass can undergo a light hydrolysis with a

consequent release of small sized oligosaccharides (soluble residue). For this

reason, we decided to evaluate if the induction power of the Olive Pomace was

exclusively due to that soluble residue or if the pellet-sized biomass was also

important in the studied process.

Shake-flask fermentations, inoculated with a 50% v/v inoculum (i.e around 1.5 g/L

of biomass) from a pre-colture grown for 3 days, were performed; in these

experiments, after the thermal sterilization, the solid OP was separated from the

liquid medium.

As showed in Figure 5.10, the soluble residue has an inductive effect on cellulase

production; the maximum FPA reached in the studied conditions is 1.15 FPU/mL,

which is significantly higher compared with tha basal activity (0.79 FPU/mL).

Nevertheless, the induction power of the soluble residue is definitely lower

compared with that of the solid OP (2.98 FPU/mL).

Figure 5.10 – Enzymatic activity profiles for 9-day fermentations with: solid OP (red curve), liquid

residue (green curve) and basal enzymatic activity (i.e. no inducer, blue curve).

5.2.6 Fungal Biomass Concentration

Shake flask experiments were carried out to investigate the dependence of

cellulase productivity on fungal biomass concentration (ranging from 0.3 to 6 g/L

of dry fungal biomass). The experimental results show a monotonically growing

activity in all the performed runs within the allotted 9-day fermentation time and a

maximum in the attained activity value over the tested fungal concentration range

placing at 3 g/L (Figure 5.11).

Figure 5.11 – Effect of biomass concentration on cellulase production.

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

0.3 0.8 1.5 3.0 6.0

Enzy

mat

ic a

ctiv

ity

(FP

U/m

L)

Biomass concentration (g/L)

5.2.7 Effect of polyphenols on cellulase production

5.2.7.1 Effect of Gallic Acid

To investigate the effect of this class of molecules on cellulase production, gallic

acid was first chosen as model molecule. The effect of gallic acid, used at the

concentration of 3 g/L, on cellulase production has been tested in two different

nutritional situations: the first is in a glucose-based medium and in absence of any

inducer, the second is in the presence of lactose, a classical inducer of cellulase

production.

Figure 5.12 - Fermentation profiles for T. reesei RUT-C30 cellulase activity while growing on

culture media containg glucose (blue curve), glucose plus gallic acid (red curve), lactose (gray

curve) and lactose plus gallic acid (green curve).

The experimental results are illustrated in Figure 5.12. As it can be seen, in the

presence of glucose, gallic acid seems to slightly improve the enzyme production in

the studied microorganism: the maximum activity reached in the gallic acid based

medium was ≈ . F U/ml, compared to the value of ≈ . F U/ml of the negative

control. In the presence of lactose, on the other hand, gallic acid does not seem to

have any effect (neither positive nor negative) on cellulase production: in both

cases, the enzyme activity profiles are almost identical.

5.2.7.2 Effect of OOMW polyphenols

The effect of some of the most representative OOMW polyphenols (vanillic acid,

caffeic acid and tyrosol) on cellulase production by T. reesei RUT-C30 were

invastigated in 9-day long experiments.

The induction power of the selected polyphenols were studied at the concentration

of 0.5 grams per liter, and the results, illustrated in Figure 5.13, were also

compared to the basal cellulase activity (i.e. no inducer present in the culture

media).

As it can be seen in Figure 5.13, caffeic acid is the only phenolic molecule that

stimulate the cellulase productivity in the studied conditions: when this chemical

is added to the culture medium, the filter paper activity is ≈7 % higher compared

with the basal activity (1.35 vs 0.79 FPU/mL). Vanillic acid, when added to the

culture media, does not seem to significantly interphere with cellulase production;

compared with the basal activity curve, the production of enzyme seem to be

slower in the first days but, at the end of the fermentation, the reached FPA is very

similar. Finally, only tyrosol has a negative effect on cellulose production: in the

studied condition, tyrosol added fermentation have a 35% lower enzymatic

activity compared to the basal curve (0.50 vs 0.79 FPU/mL).

Figure 5.13- Fermentation profiles for T. reesei RUT-C30 cellulase activity while growing on

culture media containg caffeic acid (violet curve), vanillic acid (red curve) and tyrosol (green

curve); the basal enzymatic activity (i.e. no inducer present in culture media) measured in the

studied conditions is also shown (blue curve).

5.2.8 OOMW effect on cellulase production

Main aim of this work was studying the effect the lignocellulosic OP as inducer of

cellulase production and as organic substrate for the mold T. reesei RUT-C30.

However, considering that OOMW may be present (as an entrainment) together

with OP in the case of 3-phase processing or of drained alperujo, we also

investigated the effect of that liquid residue on cellulase production in T. reesei

RUT-C30.

Shake-flask experiments were conducted by using OP as inducer and OOMW was

added at different concentrations (2.5, 5 and 10% of the total colture medium

volume); the flasks were inoculated as previously described for the 9-days

fermentation runs. The experimental results obtained are shown in Figure 5.14.

The trends illustrated in the graph clearly show that OOMW does not interphere

with cellulase production of T. reesei RUT-C30 below the critical concentration of

10% v/v; at this concentration value, enzyme production is observed after a 24-

hour lag phase and, on the 9th fermentation day, the filter paper activity measured

was ~20% lower compared to that measured in the OOMW-free production

medium.

Figure 5.14 – Effect of different concentration of OOMW on cellulase production.

5.2.9 Fungal Biomass and Olive Pomace Reuse

Given that biomass should be developed before inoculation in production cultures

and both development time and raw substrate costs add up to the final cellulase

production cost, a series of test runs was carried out with the objective of testing

the capability of the same inoculum of fungal biomass to support multiple

production cycles. The experimental setup and process configuration are

illustrated in figure 5.15.

Figure 5.15 – Process configuration for fungal biomass and OP reuse study.

These repeated runs were carried out in two different ways: by recycling both the

biomass and the admixed OP (i.e., by using olive pomace over multiple cellulase

production runs) or refreshing this latter. However, given that it is not possible to

separate the mycelium from any solid residue of OP after their mixing, the second

series of runs was actually carried out by adding a fresh batch of OP at the

beginning of any production phase, meaning that the fermentation is actually

taking place with an increasing solids load (fresh olive pomace plus olive pomace

residues from previous production runs). Therefore, the first production phase of

the two test series took place in the same way and the two run series differentiated

at the beginning of the following production phase.

A 7-day fermentation time was allotted in order to align the required sampling,

unloading, centrifugation and resuspending work to week boundaries.

As it can be observed in Figure 5.16, the first sample of the second production runs

exhibits a non null enzyme activity. The non-supplemented run exhibits a linear

enzyme activity buildup to a final value slightly exceeding the value reached during

the first phase. The OP-supplemented run shows a steeper initial response and a

later slow-down in activity buildup to a final value approximately equal to that

reached in the non-OP-supplemented run.

At the beginning of the third production phase, the initial activity is, again, non

null. In the non-OP-supplemented run, this zero-time activity value is lower than

what was measured at the beginning of the second phase. Then, during the run,

activity buildup during the run is progressive but very slow, up to a final value

which is about one third of the value attained during the first and second phase.

The OP-supplemented run shows a zero-time activity value comparable to that

measured at the beginning of the second production run, and then also shows an

activity buildup profile comparable to that measured in the previous production

runs.

Figure 5.16 - Fungal Biomass and Olive Pomace Reuse: the red curve shows the fermentation

profile of the OP-supplemented cultures while the blue curve refers the non-supplemented ones.

5.2.10 OP as carbon source for cellulase production

Olive pomace is an abundant lignocellulosic waste coming from olive oil three-

phase production process. In this experimental work, OP has been successfully

tested as a powerful inducer for cellulase production in Trichoderma reesei Rut-

C30. We also investigated the possibility to exploit this biomass as source of

carbon and energy for cellulase production process.

5.2.10.1 OP pretreatment

The pretreatment of lignocellulosic biomass is a fundamental step and it’s

necessary to release the monomeric sugars necessary for the fermentation

processes.

On the basis of specific pretreatment studied conducted by Labor S.r.l. in the

framework of the ETOILE Project, a few experimental runs have been performed to

investigate the effect of alkaline and acid pretreatment on OP, followed by

biochemical hydrolysis.

For these pretreatment studies, the selected OP load was 50 grams per liter; this

concentration was chosen to avoid an excessive density of the resulting solution,

which would constitute the culture medium for fungal growth. For the biochemical

hydrolysis, the selected enzyme load was 15 FPU for each grams of lignocellulosic

biomass (enzyme used: Novozymes Cellulase NS-50013).

5.2.10.1.1 Alkali pretreatment

Shake-flask run were conducted to evaluate the effectiveness of 2% NaOH

thermochemical pretreatment on OP. After the addition of the base and its

complete dissolution, the flasks were incubated in autoclave at 120° C for 45

minutes, the precipitate was left to settle one hour. The liquid phase was then

separated, the pH was adjusted to 5 and, after the addition of the cellulolytic

enzymes, the solution was incubated 20 hours at 47° C. The resulting solution was

eventually analyzed for reducing sugars and total polyphenols concentration. As

shown in figure 5.17, this alkaline treatment release 5,1 g/L of reducing sugars and

2.7 g/L of polyphenols.

The hydrolyzed resulting from this kind of pretreatment, after the addition of all

the salts normally presents in the Mandel medium, was tested as culture medium

for T. reesei growth; unfortunately, the fungus was not able to grow in the studied

conditions, probably due to the high concentration of polyphenols.

Figure 5.17 – Reducing sugars and total polyphenols concentration measured after acid (blue bar)

and alkaline (red bar) treatment.

0

2

4

6

8

10

12

14

Reducing sugars Polyphenols

Co

nce

ntr

atio

n (

g/L)

5.2.10.1.2 Acid pretreatment

Shake-flask run were conducted to evaluate the effectiveness of 2% NaOH

thermochemical pretreatment on OP. Three concentration of H2SO4 were tested:

0.5, 1 and 1.5%. The experimental conditions and the general procedure were the

same of the alkaline pretreatment and the results for reducing sugars release are

illustrated in figure 5.18. The biggest concentration of reducing sugars was

released when the concentration of H2SO4 was 1.5% (13.2 g/L versus 7.5 g/L at

0.5% and 10.2 g/L at 1%). As shown in figure X, also the concentration of total

polyphenol found in the final solution was lower after the acid treatment

compared to alkaline one (0.59 versus 2.7 g/L).

Figure 5.18 – Reducing sugars concentration after thermochemical treatment with H2SO4 and

enzymatic hydrolysis.

5.2.10.2 Biomass growth and cellulase production on hydrolyzed olive

pomace

Shake-flask experiments, inoculated with a 10% v/v inoculum (i.e around 0.3 g/L

of biomass) from a pre-culture grown for 3 days, were performed to evaluate

fungal biomass growth and cellulase production on hydrolyzed olive pomace. The

0

2

4

6

8

10

12

14

0.5% 1% 1.5%

Re

du

cin

g su

gar

rele

ase

d (

g/L)

Acid concentration

lignocellulosic substrate for the growth was treated with 1.5% H2SO4 and high

temperature, as previously described, and eventually hydrolyzed by commercial

cellulases; before inoculation, the solution was diluted 1:2 (to lower the toxic

polyphenol concentration) and the andel’s medium salts were added.

Thrichoderma reesei Rut-C30 showed a modest grow on the studied hydrolyzed:

the measure biomass yield of 0.257 g biomass/g OP.

Cellulase production by T. reesei Rut-C30 growing on hydrolyzed olive pomace was

also studied in two different conditions: in presence and in absence of solid OP to

the previously described culture medium. As shown in figure 5.19, cellulase

production in the two cases is very similar, with a slightly advantage for the OP-

added culture (2.2 versus 1.9 FPU/mL).

Figure 5.19 – Enzymatic activity profiles for 18-day fermentations on hydrolyzed Olive Pomace in

presence (blue curve) and in absence (red curve) of solid OP.

5.2.11 OOMW biotreatment

5.2.11.1 Thermal-acid treatment of OOMW

The first aim of this work was to find a way to couple two very different biological

processes: the “dirty” biotreatment of an agricultural liquid residue, the OO s,

and the “clean” cellulase production process. The main difference between the two

0.0

0.5

1.0

1.5

2.0

2.5

0 5 10 15 20

Enzy

mat

ic A

ctiv

ity

/FP

U/m

L)

Days

processes is that the former is usually conducted by using a consortium of

uncharacterised microorganisms while the viability of the latter process relies on

the manteinance of axenic conditions. In this view, our first goal was to devise a

low-cost pretreatment carrying about the microbiological stabilisation of OOMW to

be performed prior to the biotreatment and the enzyme production processes.

Considering that the autoclave sterilisation of huge volumes of a waste is not

economically feasible, we devised an acid-pasteurisation pretreatment process

nicely fitting the needs and the features of the overall biotreatment and enzyme

production process. The devised pretreatment process consists of two phases. In

the first phase, OOMW, whose natural pH is usually about 5, is acidified down to

pH=3, its temperature is increased to and held at 65°C for 30'. The target pH was

chosen because lethal/inhibitory to most bacteria and fungi but harmless for

Trichoderma reesei RUT-C30. Moreover, the low pH is responsible of the

coagulation of some components present in the liquid waste and the formed flocs

can be separated by settling in 24 hours, thereby also reducing the COD content of

OOMW by about 30%. The reason of the adopted temperature value is that it can

be obtained by using solar heat exchangers, thereby almost entirely offsetting the

treatment costs.

The acid-pasteurization process was successfully experimentally tested to evaluate

its effectiveness in lowering the microbial load; the OOMWs that undergo the

thermal-acid treatment, if plated on Petri plates, exhibit a total absence of colony-

forming microorganisms growth.

5.2.11.2 Shake-flasks experiments

OOMWs represent a serious environmental problem in the Mediterranean area:

they are characterized by a high COD, a low pH, a significant suspended solids

fraction and feature the presence of biorecalcitrant and inhibiting compounds,

mainly polyphenols, which make traditional biological processes poorly effectives.

Previous studies (D'Urso et al., 2007 and 2008) have demonstrated that mold

strains belonging to the Trichoderma genus are able to withstand the critical

characteristics (pH and composition) of OOMWs and successfully operate their

biotreatment. Considering this, we investigated the possibility to grow the hyper-

producing mold Trichoderma reesei Rut-C30 on OOMW. To reach this goal,

different approaches have been unsuccessfully followed:

OOMW was used as culture medium as such and diluted with water (1:2,

1:5 and 1:10);

an external carbon source (glucose) was added to OOMW (as such and/or

diluted) to support the biomass growth;

an external source of salts (from andel’s medium) was added added to

OOMW (as such and/or diluted) to support the biomass growth;

OOMW was pretreated with aluminium sulphate, as suggested by Labor

S.r.l. research group.

5.3 Discussion

Cellulases are currently the third largest industrial enzyme worldwide because of

their use in cotton processing, paper recycling, as detergent enzymes, in juice

extraction, and as animal feed additives. However, cellulases will become the

largest volume industrial enzyme, if ethanol, butanol, or some other fermentation

product of sugars, produced from biomass by enzymes, becomes a major

transportation fuel. In this work we presented a strategy to lower the production

costs of cellulase enzymes by exploiting olive pomace, an abundant agricultural

waste, as carbon source and inducer for this process.

The comparison between the induction power of OP and that of cellulose appears

to depend upon the presence or absence of glucose. In the presence of glucose, OP

inducing power is equivalent to that of cellulose. In glucose-lacking media, OP

induces a higher productivity (+25% to +67%) than cellulose.

OP higher inducing power than cellulose does not represent an incompletely

unespected result; the lignocellulosic substrate object of this study, in fact, is made

of a complex matrix of lignin, cellulose and hemicellulose, representing thus the

real substrate which is found in nature by the fungus. Moreover, similar findings

have been made on many other lignocellulosic byproducts (Mathew et al., 2008).

Even though 4-day, low fungal biomass (0.3 gFBl-1) fermentations resulted in about

threefold specific cellulase productivity than 9-day, high fungal biomass (1.5 gFBl-1 )

runs, these latter attained a higher (about threefold) maximum enzymatic activity

(see Table 5.1). This finding has a practical relevance because, as hinted by Merino

and Cherry (Merino and Cherry, 2007), if the enzyme activity of culture media is

high enough, they can be directly added to the target lignocellulosic biomass to

hydrolyze, thus avoiding the step (and the involved costs) of enzyme separation

and formulation. The utilization of the whole culture medium as source of enzymes

for biomass hydrolysis could be an important strategy to lower the bioethanol cost

by producing this biofuel locally.

Parameter Inducer

Cellulose OP Cellulose OP

Biomass inoculum

concentration (g l-1) 0.3 0.3 1.5 1.5

Enzymatic Activity

(FPU ml-1) 0.68 1.16 2.37 2.98

Fermentation time

(h) 96 96 216 216

Cellulase

Productivity

(FPU h-1 g-1)

23.6 40.3 7.3 9.2

Table 5.1 – Chemical composition of different plant species (from Merino and Cherry, 2007)

Varying the amount of suspended OP brings about a continuous increase in

maximum attained cellulase activity, which reaches 3.50 FPU ml-1 at 20 g l-1 of olive

pomace load. Specific productivity is mathematically infinite under zero inducer

load and basal expression, but under no induction maximum activity is too low for

practical application.

Specific productivity decreases at increasing olive pomace loads. At 10 g l-1 of olive

pomace load, specific productivity attains an intermediate value while enzyme

activity is close to the absolute maximum value achieved at the maximum OP load.

Szengyel et al. (1997) made the same observation, and argued that the increased

mass transfer resistance in the shake flask shown at higher solids concentrations is

responsible for this. We checked for possible oxygen limitation conditions at the

central test point of our experimental design (a 9-day long production culture was

carried out at 1.5 gMB l-1 and 10 gOP l-1) and found that oxygen was above 75% of

the saturation value during more than 90% of the fermentation time. The lowest

dissolved oxygen level (15% saturation) was reached shortly after the beginning of

the run likely due to the fast consumption of oligosaccharides released during the

thermal sterilisation of OP. Once this initial small stock was exhausted the release

rate of sugars from the large-grained OP was sufficiently slow to maintain their

concentration at a very low level, thus preventing both the onset of a fast oxydative

metabolism response and the repression of enzyme production.

Olive pomace is the result of centrifugal separation from oil and OOMW in 3-phase

processing, or from drained alperujo in 2-phase processing and entrainments of

OOMW may be present; therefore, the effect of OOMW on cellulase production by

T. reesei Rut-C30 was also investigated. OOMW shows minimal effects on cellulase

production up to 5%: in these conditions, an activity drop less than 10% is

measured. Above 10%, an initial production lag and a final activity drop by more

than 20% is recorded.

The possible advantage of adopting a higher fungal concentration under OP

induction was also investigated. As observed in Table 1, a maximum is observed in

maximum attained enzyme activity over the tested fungal biomass concentration

range (3.55 FPU ml-1 at 3 gFBl-1). However, this is a modest activity gain over the

value reached at half the fungal biomass load (+20%), hence it actually entails a

significant specific productivity loss (-60%).

Inducer Type

(FT=Fermentation Time;

FB=Fungal Biomass)

MCC +Glc

(FT=4 d

FB=0.3 g/L)

OP +Glc

(FT=4 d

FB=0.3 g/L)

MCC

(FT=4 d

FB=0.3 g/L)

OP

(FT=4 d

FB=0.3 g/L)

No Ind

(FT=9 d

FB=1.5 g/L)

MCC

(FT=9 d

FB=1.5 g/L)

OP

(FT=9 d

FB=1.5 g/L)

Maximum Enzyme Activity

0.90

(@48 h)

0.79

(@48 h)

0.68 1.16 0.79 2.37 2.98

Enzyme Productivity +63 +55 +24

2361

+40

4028

+2.4 +7.3

731

+9.2

920

Inducer Concentration

0 2.5 5 10 15 20

(g/L)

Maximum Enzyme Activity

0.79 1.30 1.63 2.98 2.94 3.50

Enzyme Productivity (∞) 1605 1006 920 605 540

OOMW Concentration

(%)

0 2.5 5.0 10.0

Maximum Enzyme Activity

2.98 2.87 2.75 2.32

Enzyme Productivity 920 886 849 716

Fungal Biomass Concentration

(g/l)

0.3 0.75 1.5 3 6

Maximum Enzyme Activity

2.10 2.07 2.98 3.55 2.88

Enzyme Productivity 3241 1278 920 548 222

Biomass and Inducer

Recyclability

1st PP

+FPM

+FB

+OP

2nd PP

+FPM

--

--

3rd PP

+FPM

--

--

1st PP

+FPM

+FB

+OP

2nd PP

+FPM

--

+OP

3rd PP

+FPM

--

+OP

Maximum Enzyme Activity

2.37 2.62 0.81 2.37 2.74 2.85

Enzyme Productivity *940

**940

***940

*1040

**(∞)

***1040

*321

**(∞)

***(∞)

*940

**940

***940

*544

**1087

***544

*377

**1131

***565

Table 2. Compared maximum cellulase activity and specific productivity as a function of: inducer

type and concentration; under olive pomace induction: OOMW concentration and fungal biomass

concentration; under olive pomace induction and fungal biomass and inducer recycle: subsequent

fermentation rank and fresh inducer supplementation. Maximum Enzyme Activity is measured in

FPU/ml; Enzyme Productivity is reported as FPU ml-1 h-1 gMB-1 ml gIND

-1 ml. In inducer-less runs,

specific productivity is calculated as: FPU ml-1 h-1 gMB-1 ml (+) and, where both productivity

definitions are applicable, both productivity values have been calculated for the relevant run series.

(*): Specific productivity referred to total suspended olive pomace; (**): specific productivity

referred to fresh olive pomace only; (***): specific productivity referred to olive pomace which has

been used in not more than two production phases; MCC: Microcrystalline celulose; No Ind: No

inducer; FPM: Fresh Production Medium; FB: Fungal Biomass.

Cellulase production, like any other biological production process, requires the

previous production of the microbial biomass, typically in a series of subsequent

steps from small shake-flask scale up to big reactor; this process (i) is time

consuming, (ii) involves reactor capacity engagement, and (iii) is chatacterized by

significant costs for substrate and nutrients supply. In this work, we demonstrate

the feasibility to recycle the fungal biomass and reuse it over multiple production

cycles, leading to a net increase of the specific bioreactor production capacity.

The lignocellulosic olive pomace has also been successfully tested as carbon source

for T. reesei growth and cellulase production. Based on previous experimental

work performed in Labor S.r.l. laboratories, alkaline and acid pretreatment have

been investigated. NaOH 2% pretreatment resulted in an hydrolysed with low

monomeric sugar and high polyphenol concentrations (5.1 and 2.7 g/L

respectively): this solution, even with the addition of other Mandel’s salts, was not

able to support the fungal growth, probably due to the inhibitory effect of phenolic

compounds. H2SO4 treatment, by the other hand, resulted in an higher free sugars

concentration (7.5, 10,2 and 13,2 g/L at 0.5%, 1% and 1.5% respectively) and in a

low polyphenols concentration (0.59 g/L at 1.5% of H2SO4); this hydrolysate was

successfully exploited, with the addition of other classic nutrients, as culture

medium. The fungal biomass yield on this substrate was 0.257 g biomass/g OP. In

the studied conditions on OP hydrolysate, cellulase production by T. reesei Rut-C30

is lower if compared with a synthetic medium case but still significative: at the end

of a 18-day fermentation, the measured FPA is equal to 2.2 FPU/mL when solid OP

is added to the medium (as a supplement inducer), while the enzymatic activity is

1.9 FPU/mL in absence of the solid inducer.

5.4 Conclusions

Despite recent advances, the availability of low-cost cellulase is still recognized as

an hindrance to the deployment of lignocellulosic bioethanol. Production of

cellulase at a low cost is an outcome of process optimization, including the choice

of a low-cost carbon source and a suitable inducer.

In the present experimental thesis we successfully demonstrated that the

lignocellulosic agricultural waste Olive Pomace is effectively usable as a new, low-

cost inducer for the production of cellulase, in turn devoted to the production of

lignocellulosic bioethanol, with the mesophilic filamentous fungus Trichoderma

reesei Rut-C30.

Shake flask runs demonstrated that OP is a more effective inducer of cellulase

expression than cellulose, the classical inducer.

It was found that OOMW presence in culture media does not affect cellulase

production by T. reesei RUT-C30 below the critical concentration of 10% v/v, and

slightly (-20%) depressed above. Moreover, the effect of phenolic compounds on

cellulase production, in the absence of other inducers, appears slightly promotive.

It was also investigated the possibility of the microbial biomass to support

multiple production cycles; in this sense, we demonstrated the feasibility to reuse

the fungal mycelium for three consecutive production batches.

Olive pomace has also been successfully tested, after a thermochemical and

biochemical pretreatment, as carbon and energy source for T. reesei growth and

cellulase production.

Finally, different approaches have been investigated to develop an OOMW

biotreatment process with the mold T. reesei: a new thermal-acid treatment for the

liquid waste has been developed (which could be exploited in other processes) but

the selected fungus didn’t show the ability to grow and biotreat the OOMW.

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