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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