Biotechnological valorisation of agro-industrial wastes for the production of cellulases

90 Pages·2012·1.97 MB·English

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 CO released during fermentation can be
2
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.