S. AL-ZUHAIR, UAE University, UAE

Abstract: This chapter discusses the enzymatic production of biodiesel using lipase enzyme as a biocatalyst. It starts by highlighting the advantages and limitations of the enzymatic approach and includes a review on the effects of the source of lipase, type and quality of feedstock, type of acyl acceptor and temperature. The chapter then discusses importance of using the lipase in immobilized form and different immobilization techniques. A kinetic model that is developed from the mechanismic steps of enzymatic transesterification of triglyceride is also presented. The chapter concludes with an exploration of the future advances in enzymatic biodiesel production.

Key words: enzymatic biodiesel production, waste oil feedstock, immobilized lipase, kinetic model.

6.1 Introduction

With the inevitable depletion of the non-renewable resources of fossil fuels, and due to its favorable environmental features, biodiesel promises to be the favorable fuel of tomorrow. Biodiesel is formed from transesterification of vegetable oils or animal fats with methanol (or ethanol) in the presence of a catalyst, as shown in Fig. 6.1. It is a renewable energy source that is non-toxic and biodegradable. Compared to petroleum-based diesel, biodiesel has lower emission levels of carbon monoxide, particulate matter and unburned hydrocarbons (Yusaf et al., 2005). In addition, using biodiesel on large scale will promote plantations of crops used to produce its feedstock, which results in more carbon dioxide recycling, minimizing its impact on the greenhouse effect (Korbitz, 1999; Agarwal and Das, 2001). Furthermore, biodiesel has a relatively high flash point (150°C) that makes it less volatile and safer to transport or handle than petroleum diesel (Krawcsyk, 1996). It provides lubricating properties, which reduce engine wear and extend engine life (Von Wedel, 1999). At the same time, biodiesel has physical properties and energetic content close to those of petroleum diesel, which allows its efficient function in conventional diesel engines without any modification.

The transesterification of triglycerides, being from vegetable oil or animal fat, is conventionally catalyzed chemically by alkaline or acid catalysts. The basic catalysts employed are sodium or potassium hydroxide because they are relatively inexpensive (Freedman et al., 1984; Akoh and Swanson, 1988). Usually, a stoichiometric excess of methanol, in a molar ratio of 6:1 (methanol:vegetable oil), is preferred to increase methyl ester yield, and the reaction can be completed in a few hours at 40-65°C. The alkali-catalyzed processes, however, are sensitive

to moisture and free fatty acids (FFA) content in feedstock. Saponification reaction of the FFA consumes the alkali catalyst and at the same time generates soaps that cause the formation of emulsions, which increase the viscosity and create difficulties in downstream recovery and purification of the biodiesel. Therefore, pre-treatment of the oil is required for commercially viable alkali- catalyzed systems. This requirement is likely to be a significant limitation to the use of low-cost feedstock, and the cost of the highly refined feedstock can account to up to 70-80% of the final cost of the biodiesel (Fukuda et al., 2001). On the other hand, the acid-catalyzed processes are insensitive towards FFA contents. However, they are rarely used because they result in much slower reactions and produce by-products, from alcohol etherification, that also results in difficulties in downstream recovery and purification. In addition, careful removal of catalyst from the biodiesel fuel is essential, since acid-catalyst residues can damage engine parts (Fukuda et al., 2001). Furthermore, acid-catalyzed reactions require higher temperatures of around 55-80°C and higher substrate molar ratios of alcohol of around 30:1 to yield approximately 99% biodiesel in 50 h (Marchetti et al., 2007). The preferred acid catalysts are sulfuric, hydrochloric and sulfonic acids (Freedman et al., 1984).

Biodiesel can also be produced in the absence of any catalyst, using supercritical methanol (Demirbas, 2002). This simple process results in high yield due to the simultaneous transesterification of triacylglycerols and esterification of fatty acids. However, this is an energy intensive process that requires operating at temperatures and pressures above the critical points for methanol, which are 512 K and 8.1 MPa, respectively. Furthermore, operating at these harsh conditions destroys the antioxidant inherently found in the feedstock, which results in reducing the oxidative stability of biodiesel.

Recently, a less energy intensive and environmental friendly procedure has been proposed by using enzymes to catalyze the transesterification of triglycerides. Enzymatic transesterification can overcome the problems facing conventional chemical methods without compromising their advantages. Biodiesel has been successfully produced in lab scale by lipase-catalyzed reactions. Conversions as high as 90% have been reached within short reaction times, provided that the reaction takes place under the appropriate conditions. Nevertheless, there are many obstacles hindering the effective use of enzymes for commercial production

of biodiesel in large scales. The most important challenges and the proposed ways to overcome them are presented in this chapter. In the following Section 6.2, a general introduction to the enzymatic approach is provided. The advantages of the enzymatic catalyzed process over conventional chemically catalyzed ones are also explained in this section. On the other hand, the limitations of enzymatic approach are presented in Section 6.3. In Section 6.4, the effectiveness of lipase, the enzyme to be used in biodiesel production, from different sources is discussed. After that the capacity of lipase to produce biodiesel from various feedstock, with special emphasis on feedstock that does not compete with food stock, is assessed in Section 6.5. This is followed by Section 6.6 that describes the effects of the type and amount of the acyl-acceptor on the enzymatic biodiesel production process and possible ways to overcome the inhibition by short-chain alcohols. In Section 6.7, the thermo-stability and optimum temperatures of lipases from different sources are presented. Section 6.8 discusses the use of immobilized lipase in biodiesel production. This is crucial since the cost of lipase remains the main obstacle facing full exploitation of its potential, the reuse of lipase is essential from the economic point of view, which can be achieved by using the lipase in immobilized form. In Section 6.9, the development of a kinetic model to describe the system, taking into consideration the inhibition effects by both substrates is presented. This chapter concludes with an explanation of the future advances in enzymatic biodiesel production and sources for further information in Sections

6.10 and 6.11, respectively.

M. ARSHADI, Swedish University of Agricultural Sciences, Sweden and H. GRUNDBERG, Processum Biorefinery

Initiative AB, Sweden

Abstract: Bioethanol can be produced from different sources of biomass including biological material from agricultural products and forest raw materials. The chapter first discusses the different biomass feedstock available for both first and second generation bioethanol production. It then discusses the various process technologies including pre-treatment, acid hydrolyses, enzymatic hydrolyses and fermentation steps to convert the various feedstock to bioethanol. The chapter includes a description of a pilot plant for production of bioethanol from lignocellulosic materials. Environmental aspects and future trends of bioethanol production have also been discussed.

Key words: biomass (lignocellulosic) feedstock, enzymatic hydrolyses, acid hydrolyses, bioethanol process tecnology, fermentation.

9.1 Introduction

For many years transport systems have relied on fossil fuels such as petrol, diesel and natural gas but these fuels are not sustainable in the long term. Petroleum prices have increased steadily over recent years which has caused much interest and investment in biofuels production. Emissions of greenhouse gases such as CO2, CH4 and N2O from the combustion of fossil fuels in the engines of motor vehicles have had negative impact on human health and also caused weather changes related to global warming. The Kyoto Protocol demands that the European Union cut CO2 emissions by 8% between 1990 and 2012. In 2007, the 27 European Union member governments approved a new target to cut their collective greenhouse gas emissions by 20% from the 1990 level by 2020.

The health and environmental problems together with increasing worldwide demands for energy and the depletion of fossil fuels in the near future call for sustainable production of fuels for the transport sector. At the same time, developing motor vehicles which increase efficiency and reduce fuel consumption has been urged. Therefore, the development of fuel systems that are based on renewable sources has been the topic of frequent international discussion.

Different transport fuels have different physical and chemical properties and they may exist as liquids or gases in many cases, e. g. biodiesel, biogas and bioethanol (ethanol derived from biological sources). The production of these from renewable resources has increased during recent decades. Liquid fuels are easily handled and possess a high energy content. The global production of bioethanol was 51 billion litres (13.5 billion gallons) in 2006 (Balat, Balat, and Oz, 2008; Sanchez and Cardona, 2008).

Bioethanol as a fuel has both advantages and disadvantages depending on the type of engine (Otto engine or diesel engine) using the fuel and there are some physical obstacles to bio-ethanol use. Bio-ethanol can be produced from different sources of biomass including biological material from agricultural products and forest raw materials, etc. The biomass feedstock can also be divided into several groups depending on the type of chemical structure of the raw material, e. g. sugar, starch or cellulosic materials. In a future bio refinery process the production of bioethanol should be integrated with the production of other value added chemical compounds and biofuels in order to be able to utilise the feedstock in an optimum way (Demirbas, 2009).

The above described bioalcohols are the most commonly discussed for fuel purposes, but they are not the only bioalcohols that can be produced. The procedure described above for producing bio-ethylene glycol from corn residue can be modified to produce erythritol and xylitol.18 These are not common alcohols, but there are more common alcohols that can be produced from biomass. One prime example is propanediol. There are two main forms of propanediol: 1,2-propanediol, which is also called propylene glycol, and 1,3-propanediol. 1,2-propanediol can
be produced from hydrogenating of biologically derived lactate or lactic acid.19

I, 2-Propanediol can also be produced from hydrogenolysis of glycerol with Raney Nickel.20 Although it has not yet been used as a fuel, it is used as a less toxic antifreeze as well as an additive for many commercially available pharmaceutical and cosmetic products. 1,3-Propanediol can enzymatically be produced from bioglycerol.21 Both 1,2- and 1,3-propanediol can be produced by hydrolysis of biomass and then fermentation of the resulting sugars.21 This can also be used to produce butanediol.21 Although it has not been used directly as a fuel, it has been reacted further to produce an octane booster for gasoline. It is important to note that these rare bioalcohols are not currently being researched for fuels, but as we get more serious about renewable energy, we will start focusing on different fuels from different biomass sources for different applications, as needed.

Branched alcohols have also been considered as potential biofuels. Biobutanol researchers have found that they can produce branched alcohols as well. Synthetic biology has allowed for the production of isobutanol in metabolically engineered bacteria with glucose as a carbon source.22 This process diverts 2-ketoacid metabolic intermediates into aldehydes via 2-ketoacid decarboxylase and then to an alcohol via alcohol dehydrogenase. This novel method for producing branched chain alcohols has also been shown to be able to produce 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol and has been licensed to Gevo (Pasadena, CA), which is focusing on commercialization of the technology.23

The present chapter was mainly aimed at presenting a discussion on several objectives of biofuels policies.

Firstly, the analysis touched on multiple effects of biofuels production and use such as the need for guaranteeing energy security and supply to an increasing number of countries currently heavily dependent on fossil fuels imports and subject to the negative effects of international fluctuations in oil prices which dramatically affect the domestic economy. Several policies and regulations are now under way in various countries to favour energy supply and safety. The European Union, for example, is moving toward a renewable energy and low carbon economy by adopting a series of directives promoting energy from renewable sources (including biofuels) or voluntary initiatives such as the 20-20-20 policy to commit to GHG emission reductions. In Brazil, the support of the electricity and heat production industries favoured the adoption of biofuels activities across country which favoured the creation of thousands of small farms. The United States is also experiencing a revision of its RFS policy allowing the country to establish biofuels targets in the future.

Secondly, bioenergy production also contributes to a number of environmental issues other than carbon (and other) emission reductions such as biodiversity, soil productivity and land-use change. A deeper analysis illustrated the effects (direct and indirect) of land conversions for biofuels feedstock production. The debate mainly concentrates on the measurement of indirect effects of land-use change and accounting practices for carbon reduction.

Thirdly, the expansion of rural areas and food safety is central to the advance in biofuels production. The nexus between rural development and bioenergy focuses on three main aspects: (1) social benefits of biofuels policies such as job and income creation having positive repercussions on rural communities; (2) public sector intervention and the progression of second-generation biofuels from non-food crops; (3) food security versus land management issues. This is at the heart of current debate on food and energy price increase. The international community through financial aid and support in technological advances plays an important role in protecting undernourished population and marginal areas in developing economies.

Fourthly, the increasing support for biofuels policies over the last years has taken place under a variety of policy tools. Subsidies to the biofuels industries have been instrumental in the international success of bioenergy practices, although the presence of distortionary effects on the society advocated by economic theory counterbalance the positive effects (on the economy and environment) arising from biofuels production. Various support policies are nevertheless being adopted across countries to promote biofuels use including capital grants, tax incentives and trade tariffs.

Finally, the agriculture and forestry conversion to bioenergy crops contributes to climate change mitigation. Currently, positive benefits of climate change mitigation from agricultural biofuels practices are not recognised within international climate change agreements such as the Kyoto Protocol. This would leave developing countries, where technology level is limited, incapable of contributing to carbon reductions and generating income from bioenergy credits. The scope for creating cap-and-trade systems for bioenergy crops and afforestation and reforestation programmes is on the way (in the United States and Brazil) for two reasons: to incentivise sugar cane industry to sell carbon emissions credits and to favour the creation of value added. This would support the international diversification of carbon markets and help distributing the benefits of carbon credits from bioenergy sources in agricultural and rural areas.

With regard to future trends, several scenarios can be delineated for multiple objective policy approaches for biofuels production. Advances in technological research and development and learning processes from past and current experiences (i. e. international food and oil price increases, land management competition for food and biofuels feedstock debate) indicate that one of the main pathways toward a long-term sustainability of the human and natural environment is a bio-based economy. The European Union, United States and a number of other countries have recognised, through recent regulation, that a substantial reduction in oil and petroleum products should be adopted in order to face increasing demand for energy and mitigate climate changes at the same time.

The European Union, for example, is aiming at achieving a reduction of 20% of carbon emissions by 2020 with increasing use of renewable sources by 20%. It is an ambitious policy given the current economic crisis and unemployment pressures and restructuring of the economy in new Member States. Nonetheless, the European Union is moving toward an energy-efficient market with ample space for the implementation and diffusion of biofuels technologies and products to renovate the agricultural sector and promote bio-refineries installations. The United States, on the other hand, is currently experiencing a revision of its RFS policy. The adoption of a strategic approach at all levels of the biofuels production chain would ensure coordinated measures: across governmental departments and agencies in view of economic, environmental and social concerns; and between research and commercialisation phases to converge a multitude of stakeholders’ needs. Also, monitoring the implementation of biofuels projects would result in further advantages for the entire biofuels supply industry.

Efficiency in strategic planning is also claimed to improve the quantification of indirect effects of biofuels production and use. These may come in the form of displacement effects of fossil fuels emissions gained over new lands for bio-crop production which are not taken into account in current carbon reduction inventories. Furthermore, intergenerational issues (such as discounting rates and time management) are also relevant for valuing life-time effects of biofuels plants over different generations and natural resource use.

Efficient management of biofuels production also aims at rural development in developing countries. The Brazilian experience is a unique case where strong market integration (across the sugar cane industry, electricity supply and transport sector, for example) and transparent institutional framework have favoured the launch of biofuels production. Replication of this mechanism, including the lessons from Brazil’s learning-by-doing experiences, elsewhere becomes essential to promote agricultural growth, income generation and biodiversity protection in developing economies.

It is essential at this stage of the biofuels chain development to sustain technology advances for second — and third-generation biofuels (i. e. lignocellulosic). This would aim at reducing current land competition between food and non-food crops. Current support for research is therefore a strategic element toward worldwide reduction in food and energy prices. The European Union, the World Bank and the United States agree that enhancing continuous support to research and development for next-generation biofuels would serve as a key factor to favour the improvement of current international food crisis, energy dependence and carbon emission reductions. From a developing countries’ perspective (granted that new forms of biofuels technologies are being implemented locally through international financial support) this would also help in reducing the dependence on foreign markets in food and energy. A number of macroeconomic positive impacts would follow such as improving balance of payment accounts, boosting employment and income generation and reducing the gap in poverty conditions. However, enhancing agricultural activities and new forms of biofuels locally would not have expected positive effects if the international community does not apply reductions in trade tariffs on biofuels commodities. Efforts in this direction become essential in particular for improving the functioning of international agreements (e. g. the Doha Round) affecting agricultural markets (World Bank, 2008).

Support policies for the biofuels industry are crucial for the development of new markets for bio-commodities. Though governmental subsidies are playing an important role in supporting bio-crops production, these nonetheless generate distortionary effects when used for unproductive reasons. Government aid is therefore called for implementing alternative incentivising mechanisms to ensure adequate measures for land conversion. Long-term forms of investment grant (either from public or private sources) subject to continuous monitoring of land management practices would guarantee the efficiency of bio-based projects and avoid waste of financial resources.

Land-use practices for bioenergy production are vital to mitigate climate change. Land conversions for fuel feedstock would produce net benefits to the society (in terms of carbon emission reductions) which are not fully internalised in social well-being. Post-Kyoto negotiations should address land-use changes to compensate countries for the credits gained from carbon emission reductions. There exists a possibility to develop a carbon trading system for bioenergy commodities. Brazil is moving toward a cap-and-trade mechanism for ethanol, and voluntary agreements are under way with the United States to adopt a bilateral trade market for carbon credits from bioenergy sources and afforestation activities. This would not only guarantee the creation of value added for the domestic economy, but also serve as attraction to foreign investors to invest in agricultural activities in support of a bio-based economy.

Lignocellulosic biomass, including agricultural residues, wood and energy crops, is an attractive material for bioethanol production. Lignocellulosic biomass could produce up to 442 billion litres per year of bioethanol (Bohlmann, 2006), which is about 16 times higher than the current world bioethanol production (Kim and Dale, 2004). Furthermore, about 3.6% of the world’s electricity production and 2.6 x 1012 MJ of steam are also generated from burning lignin-rich fermentation residues, a co-product of bioethanol made from crop residues and sugar cane bagasse. Most potential electricity and steam production could be provided by burning fermentation residues in the utilisation of wheat straw (Sun and Cheng, 2002).

Conversion of cellulosic biomass is a future alternative of biofuel production. However, bioconversion of cellulosics and lignocellulosics to bioethanol is difficult due to the resistant nature of biomass to breakdown, the variety of sugars that are released when the hemicellulose and cellulose polymers are broken, the need to find or genetically engineer organisms to efficiently ferment these sugars and costs for collection and storage of low density lignocellulosic feedstocks (Balat et al, 2008). Provided that cellulases and pretreatment processes are expensive, genetically modified crops to reduce the needs for pretreatment processes are promising paths to solve this problem, together with other strategies, such as increasing plant polysaccharide content and overall biomass (Sticklen, 2008).

Forest and agricultural residues may be used to produce bioethanol. As an advantage, there would not be a strong competition between the use of land for food and for energy. Sorghum seeds can be used for food, while the stems could be optimised for different chemical platforms. Recent studies concluded that sweet sorghum is a very useful plant, whereby the complete plant can be used without leaving any waste (Smith and Buxton, 1993; DSD, 2005). Lignocellulosic biomass of cardoon can be used as a solid biofuel, while seed oil can be derived for biodiesel production (Fernandez et al., 2006). Lately, the production of ethanol fuel from cardoon stems and leaves has been proposed (Martinez et al, 1990).

New crops that have been evaluated as bioenergy crops over the last years include switchgrass and elephant grass. Provided they cannot be used for feeding purposes, they seem to successfully substitute cereals, such as corn, to produce bioenergy. Lignocellulosic perennial crops (e. g. short rotation coppices and inedible grasses), especially warm-season (plants with C4 carbon fixation) perennial grasses, are promising feedstocks because of high yields, low costs, good suitability for low quality land (which is more easily available for energy crops) and low environmental impacts (Cherney et al, 1991).

Another group of dedicated bioenergy feedstocks is woody plants, including hybrid poplar, willow and pines. Hybrid poplar is considered a model woody biomass feedstock because of its broad adaptation, available genome sequence and fast growth. The biomass accumulation of hybrid poplar is reported to be between 7 and 20 mg/ha/year depending on the nutrition and environmental conditions (Christersson, 2006; Yuan et al., 2008).

Beside the adsorption technique, lipase can be immobilized on support surfaces by covalent anchorage, electrostatic binding and entrapment within inorganic or organic
inert matrices. Adsorption techniques are simple, but the binding forces between the enzyme and the support are weak and enzyme leaching often occurs. A higher degree of stability can be achieved by covalent bonding between the enzyme and the solid surface (Shamel et al., 2005; Shamel et al., 2007); however this requires several chemical steps that are accompanied by loss in enzyme activity. High stability can also be achieved by electrostatic interaction, but this technique is limited to be used at pH values compatible with the electrostatic point, which also may affect the activity of the enzyme, since the enzyme conformation changes as function of pH (Macario et al., 2007). On the other hand, the immobilized lipase by entrapment within a polymer matrix is much more stable than physically adsorbed lipase (Hartmeier, 1985), and unlike the covalent bonding this method uses a relatively simple procedure. Enzyme entrapment in a silica matrix by sol-gel offers a good compromise between stability of the heterogeneous biocatalyst and activity loss, and hence this technique has received considerable attention in recent years (Frings et al., 1999). Entrapment of lipase in an inorganic polymer matrix, which is based on sol-gel process, is well documented (Reetz, 1997). The method involves an aqueous solution of the enzyme, an acid or base (NaOH, NaF or HCl) as catalyst and an alkoxysilanes as inorganic — organic matrix precursor. The sol-gel material is then obtained by hydrolysis and condensation of the precursor to result in an amorphous silica matrix that entraps the enzyme. The lipase entrapped in sol-gel has been used for biodiesel production (Orcaire et al., 2006; Al-Zuhair et al., 2008) and was easily recovered from reaction media. However, under the same operating conditions, it was found that immobilized lipases, from P. cepacia, on ceramic beads were more capable of transesterifying WO of high water contents to biodiesel than lipase, from the same course, entrapped in sol-gel matrix (Al-Zuhair et al., 2008), which is mainly due to diffusional limitations.

Covalently immobilized lipases are usually prepared in almost anhydrous media. This usually results in a problem, especially in porous structures, which is mainly used to enhance the interfacial area. At the oil water interface, lipases are in open active form, where a flap (or lid) that would seclude the active cites is moved to allow substrate accessibility to the active sites (Verger et al., 1973; Brady et al., 1990). When inside a porous structure, lipase molecules become inaccessible to external surfaces, which prevent their activation. Therefore, it has been proposed to use hydrophobic support that resembles the surface of drops of the natural substrates to immobilize lipase on. In this case, the adsorbed lipases are in open form, with the active sites accessible for substrate and the immobilized enzyme in this case exhibit significantly enhanced activity (Bastida et al., 1998). Based on that Palomo et al. (2002) used an epoxy acrylic matrix, Sepabeads, with the surface covered by octadecyl groups, yielding a very hydrophobic surface that has large pores to allow intensive protein interaction. The support permits in one step immobilization, purification, hyper-activation and stabilization of surface in a very simple protocol: the mere addition of support to the lipase solution at very low ionic strength. In addition, the support is rigid enough to be used in packed — bed reactor and does not swell in any reaction media. The stability and activity of lipases from C. antarctica, C. rugusa and M. miehei immobilized on this support were found to be superior to other covalently attached derivatives.

During hydrolysis, water molecules react with the glycosidic bonds in the structure of cellulose and hemicellulose and degrade them to sugar units such as glucose, xylose, etc. Free sugar units can be obtained from lignocellulosic material by either thermochemical processes or a combination of thermochemical and biochemical processes.

The chemical processes are divided into two general types, one using high acid concentration in the hydrolysis step, one example is called the Concentrated Hydrochloride Acid Process (CHAP), and one using dilute acid in the hydrolysis step, one example was developed in cooperation between Canada, America and Sweden (called CASH).

CHAP process

The Concentrated Hydrochloride Acid Process (CHAP) is based on the hydrolysis of lignocellulosic feedstock by concentrated hydrochloric acid at low temperature. The process was developed for cellulose rich raw material since a high concentration of the acid may cause the degradation of pentose in hemicellulose to furfural derivatives. The ethanol yield is typically about 35%. The concentrated acid is corrosive and the process needs higher capital investment due to more expensive materials. The dangers associated with the recovery of the concentrated acid make this method less attractive. In addition, during combustion of lignin which is contaminated with hydrochloric acid there is some risk for dioxin emissions. Due to the corrosive problems with hydrochloric acid, focus has moved to concentrated sulphuric acid however, the major problem of recovering the acid remains unsolved so far.

CASH process

The Canada, America and Sweden Hydrolysis (CASH) process was developed in cooperation between Canada, America and Sweden. In this method, hydrolysis occurs with dilute sulphuric acid at a temperature of around 200°C (pressure 8-25 bar). Previous studies have shown that by using SO2 and dilute sulphuric acid in two steps, this increases the sugar and also ethanol yield since the amounts of inhibitors such as furfural are decreased. The process was developed for woody biomass. The ethanol yield is around 20% of the energy content in the raw material, however, up to 40% of the energy content of the biomass is bound in the hydrolysis residue, mostly the insoluble and condensed lignin, but also a large portion of unreacted cellulose. The reason why there is cellulose left in the hydrolysis residue is due to the reaction kinetics of the hydrolysis compared to the kinetics of the sugar break down reactions. At the end of the reaction, only the very stable form of the cellulose is left, making the hydrolysis reaction very slow, but at the same time, the sugar concentration has increased, making the breakdown reactions faster. At one point, the breakdown of the carbohydrates is faster than their formation and thereby the sugar concentration declines. The hydrolysis residue can be used as a solid biofuel in boilers or directly in powder fuel turbines or pelletised.

Control of anaerobic digestion is crucial in order to secure or even to maximise the performance of the process. In order to develop a control scheme the following steps should be considered:

• Definition of the control objective: The objective could be as simple as the pH stabilisation or more complicated involving stabilisation and optimisation of the bioreactor operation in terms of biogas production or chemical oxygen demand removal. Since optimisation and stabilisation are conflicting objectives, the control law should be sophisticated enough to meet these targets in the best way.

• Selection of the suitable measurements: The properties of a suitable measurement to be used in a control scheme are the ability to reflect the process state and its changes due to disturbances (sensitivity), as well as the time response and the simplicity of the measurement method. The most common measurements in anaerobic digesters (Table 12.2) are:

— Biogas flow: The biogas production rate and especially the performance in methane is the most commonly used measurement to detect the process stability. A reduction in the biogas production rate usually suggests that the volatile fatty acids have been accumulated as a result of overloading or presence of a toxicant. However, any change in this parameter is caused by process instability and cannot be an early warning, that is, it is not sensitive enough.

— Biogas composition: The principle gases in the headspace of an anaerobic digester are CO2 and CH4. When CO2 increases relatively in proportion to CH4, process imbalance has already evolved and, consequently, this index cannot be used as an early indicator. On the other hand, CO2 in the gas phase is influenced by changes in alkalinity and pH in the bioreactor, and as a result when pH control is applied in low buffered systems, changes in its value do not reflect process instability (Ryhiner et al., 1992). Another important gas found at very low concentrations is hydrogen. Hydrogen has been suggested as an early reliable measurement for early detection of an imminent imbalance (Archer et al., 1986, Molina et al., 2009). Hydrogen is a significant intermediate compound regulating the performance of the acetogens. Accumulation of hydrogen entails accumulation of volatile fatty acids due to thermodynamic limitations of acetogenesis. It should be kept lower than 40 nM (which corresponds to a partial pressure less than 6 Pa at 35°C). Archer et al., (1986) monitoring hydrogen partial pressure in the headspace predicted an accumulation of volatile fatty acids 3-6 h before it happened. However, the changes in the hydrogen concentration cannot be correlated necessarily with imbalance (Guwy et al., 1997). Measuring hydrogen in the headspace does not correspond to the actual concentration sensed by the microorganisms which are in the aqueous phase. This is why measurement of dissolved hydrogen is suggested as a more reliable index (Pauss et al., 1990; Frigon and Guiot, 1995). Hydrogen sulphide and carbon monoxide can also be detected but they are not important for control purposes.

— Volatile fatty acids: They are the most important intermediate compounds in anaerobic digestion since their accumulation leads to pH decrease, stressing the methanogens further. The increase in acetate concentration under overload conditions does not indicate necessarily process imbalance if the biogas production rate has also increased. In this case, the system may operate at a higher acetate concentration at a new steady state, without rejecting the possibility of process failure. However, propionate and butyrate accumulation denote signs of imbalance since it usually happens when hydrogen concentration increases. Propionate is accumulated first, since its conversion requires six times lower concentration of hydrogen than butyrate (Ozturk, 1991). Therefore propionate has been suggested as a suitable indicator for process imbalance (Pullammanappallil et al., 1998; Boe et al., 2008), along with butyrate (Renard et al., 1991), the ratio of propionate to butyrate (Hill, 1982), and the iso forms of butyrate and valerate (Cobb and Hill, 1991; Ahring et al., 1995). Depending on the metabolic pathways prevailing in an anerobic bioreactor, volatile fatty acids may be formed at various concentrations and there cannot be a rule of thumb for a ‘safe’ level of volatile fatty acids securing stable operation. For example, Pullammanappallil et al., (2001) found that operation of a controlled, glucose fed bioreactor in the presence of phenol remained stable at a high propionate concentration (2750 mg/L). Moreover, the inhibition of volatile fatty acids is pH dependant and their inhibitory effect increases at pH values ranging from 6 to 7.5.

— pH: Monitoring pH is very important since it affects the microorganisms activity and can be correlated with changes in acids and bases as well as anions and cations produced or consumed as a result of the metabolic activity. However, it cannot be used to evaluate the state of the system since it is affected by the buffer capacity of the liquid (determined mostly by the bicarbonate, ammonia, volatile fatty acids).

— Alkalinity: It is distinguished in total and bicarbonate alkalinity. Total alkalinity is measured through titration to pH 3.7 (Powel and Archer, 1989) and expresses the capacity of an anaerobic system to maintain the pH under acidification. However, total alkalinity increases as the volatile fatty acid concentration increases. Therefore, the bicarbonate alkalinity, measured through titration to 5.75, can reflect the effective buffer capacity of the system. Various methods have been developed for the on-line measurement of the bicarbonate alkalinity (Table 12.2).

— Organic matter: Common parameters such as the total and volatile solids, chemical oxygen demand, total organic matter and biochemical methane potential (preferable to biological oxygen demand in the case of anaerobic systems) express the aggregate organic matter present in a digester and, correlated with the organic matter of the influent, give an accurate estimate of the organic matter removal. However, these are time consuming, off­line measurements, except from the total organic carbon method which can be applied on-line in the case of anaerobic systems with low

solid content (Table 12.2). Therefore, they are not suitable for on-line controllers.

— Metabolic activity: The physicochemical parameters available for measurement respond to changes in the metabolic activity of the anaerobic microorganisms, but the correlation is not always direct. Since the success of a control scheme applied on anaerobic systems is based on directing the microbial activity to the desired performance, its assessment is very important. The microbial activity can be evaluated through measurement of the specific methanogenic activity (Ince et al., 1995; Garcia-Morales et al., 1996; Fountoulakis et al., 2004; Dong et al., 2009; Montero et al., 2009), application of molecular techniques (for the qualitative and quantitative detection of specific microorganisms based on the DNA and RNA probing (Macario and de Macario, 1993; Macario et al., 1989; Raskin et al., 1994; Montero et al., 2009) and detection of changes in cellular components such as enzymes (NADH and coenzyme F420) (Perk and Chynoweth, 1991; Amann et al., 1998), ATP (Chung and Neethling, 1988) and phospholipid fatty acids (Nordberg et al., 2000). Moreover measurement of the activity of certain enzymes and application of microcalorimetry (heat released in an anaerobic ecosystem which can be correlated to the size of the microbial population, the metabolic state and activity) have also been used for monitoring (Switzenbaum et al., 1990). Since most of the analytical procedures required for assessing the metabolic activity are elaborate and time consuming or require samples of low solid content, the utilisation of these measurements is limited for on-line control, but can be used off-line to give a better insight of the system status.

• Manipulated variables: The manipulated variables are operating parameters through which the process state can be affected and led to the satisfaction of the control objective according to the applied control law.

The most common manipulated variable is the dilution rate, or equivalently, the hydraulic retention time (inverse of the dilution rate). The dilution rate should generally be lower than the maximum specific growth rate constant of the slowest growing microorganisms group to avoid wash out in a continuously stirred tank reactor. In such type of bioreactor, the sludge (solids) retention time coincides with the hydraulic retention time. In order to increase the conversion rate, recirculation of the sludge is often applied to increase the biomass concentration. In systems fed with waste of high solid content, the liquid effluent stream is recirculated to provide it with nutrients and microorganisms. In both cases, the hydraulic and sludge retention times are separated and can be manipulated independently. The extent of manipulation of the hydraulic retention time is restricted in practice given the waste storage capacity of the treatment plants (a few hours to a few days). The hydraulic retention time in thermophilic conditions can be as low as 4-6 d, while in mesophilic conditions it is 10-15 d, although higher values of the hydraulic retention time result in more stable operation (Pind et al, 2001).

The organic loading rate, influenced by the organic content of the waste at a given hydraulic retention time, is another manipulated parameter, but since the organic content of the waste does not vary, its use is rather restricted.

In the case of more than one waste stream being commonly digested (codigestion), the composition of the waste mixture is another manipulated variable. In codigestion, wastes can be combined to make up for nutrient deficiencies, dilute the inhibitory compounds of waste stream, enhance the process yield of low potential waste (Angelidaki and Ahring 1997; Gavala et al., 1999; Angelidaki and Ellegaard 2003; Alatriste-Mondragon et al., 2006; Nielsen and Angelidaki 2008; Dareioti et al, 2009; Li et al., 2009; Shanmugam and Horan, 2009).

Other manipulated variables are the acid, base or bicarbonate addition rates to control the pH or alkalinity in the bioreactor or the feed (Pind et al., 2001). pH and alkalinity control require the addition of chemicals, which raise the cost of the process. An alternative is to recycle the CO2 produced in order to increase the alkalinity, but this is not effective in case the bioreactor pH is lower than 6.5 (Romli et al., 1994).

• The control law: It is the information flow structure through which the manipulated variables are handled based on the measurements. The complexity of the control law is determined by the diversity of the control objective. As a result, the controller can be simple (on-off, proportional, proportional-integrated — differential), more complicated adaptive model-based, empirical (expert systems), fuzzy or neural network-based. Detailed references on the various control systems having applied on anaerobic digesters can be found in Pind et al. (2003), Liu (2003) and Boe (2006).

Estimates of capital costs for biofuel plants (or any other developing technology) are uncertain due to the many influencing factors. An example is the 18 million litres/yr CHOREN bioethanol plant whose costs escalated from €500 million in early 2007 to €1000 million in early 2008 (Bridgwater, 2009). Nevertheless, several estimates are available for different biofuel technologies. One of the most comprehensive and consistent studies currently available, carried out by DENA (2006), puts the cost of thermo-chemical plants between €525 and €650 million for plants treating 1 million tonnes of wet biomass and producing 105 000-120 000 tonnes of biofuel per year. In addition to the economic benefits, this option provides operational and organisational synergies and significantly lowers the plant availability risk. Integration into an existing refinery or chemical plant can also accelerate the planning procedure and can lower investment costs by around 25% (DENA, 2006).

Table 3.6 shows the process options considered, and Table 3.7 shows the breakdown of costs. Processing route 1 appears to be economically the most sustainable option.

It is interesting to note that integration into an existing refinery or chemical plant is the most cost-effective option across the different processing routes. In addition to the economic benefits, this option provides operational and organisational synergies and significantly lowers the plant availability risk. Integration into an existing refinery or chemical plant can also accelerate the planning procedure and can lower investment costs by around 25% (DENA, 2006).

Even fewer estimates are available for the capital costs of bio-chemical plants. A recent study by the U S EPA (2009) estimates the costs for a bio-chemical plant producing 56 million gallons/yr of ethanol from 849 385 dry tonnes/yr of corn stover at $133 million/yr (for the year 2010). With other costs added (including site development, project contingency, etc.), the total project investment costs are estimated at $232 million/yr (US EPA, 2009). For the years 2015 and 2020, the annual costs are predicted to go down to $220 million and $198 million, respectively.

Numerous publications and patents are describing various routes for the production of biodiesel from different feedstocks. An overview is given in Mittelbach and Remschmidt (2004) and Mittelbach (2009).

Alkaline transesterification is the traditional process. Acid catalyzed transesterification is not industrially applied. However, it is seldom used in

combination with alkaline transesterification and the catalyst can be homogeneous and heterogeneous. The new developments are non-catalyzed interesterification but not industrially operational. 5.4.1 Homogeneously catalyzed production of biodiesel Today the most commercially available biodiesel production plants are using homogeneous alkaline catalyst. The reaction is a nucleophilic addition of an alkoxide anion to the carbonyl function followed by an expulsion of the glyceroxide anion (Fig. 5.4). The catalysts used are sodium and potassium methoxides or hydroxides. The advantage of using sodium and potassium methoxides is that no water is formed
ROH + В > SOR + BH® Nucleophile
CH2—Oe
CH2—OH HC—— O—C——- R2 + в I II о CH2—О—C——— R3 II о
Reverse reaction: removal of glycerine Rate limiting step: first step leading to DG
CH2—OH
CH2—O-
“OR
HC—— О—C—— R + OR	HC—— О—C—— R + RCOOR’
CH2—OH
5.4 Reaction scheme for the base-catalyzed reaction.

and no saponification is occurring. The use of hydroxide involves the formation of water resulting in hydrolysis of the acylglycerides or alkyl esters with formation of soaps (Fig. 5.5).

Potassium catalysts are favorable compared to sodium catalyst due to the acceleration of the phase separation (higher density of the glycerol layer) and lower soap formation. Furthermore, salts can be used as fertilizer after neutralization with an acid. However, the price of the potassium-derivatives is higher.

In addition, due to the fact that during the reaction glycerol is separated out (glycerol has a limited solubility in lipids and biodiesel), water is also removed, shifting the equilibrium of the reaction towards alkyl ester formation. Kinetic studies of this multiple phase reaction show that the formation of the diglyceride is the slowest, whereas the next steps are much faster (Mittelbach and Trathnigg, 1990). The standard conditions for the alkaline transesterification are 6:1 molar ratio of methanol to oil, concentration of catalyst in the range of 0.5-1.5% (depending on the FFA content of the feedstocks) and temperature of 60°C.

Reaction times can be shortened using a two-step procedure or a continuous reaction with simultaneous separation of the glycerol. A typical reaction scheme involving a two-step reaction is depicted in Fig. 5.6 using potassium hydroxide as catalyst.

In the alkaline catalyzed process, it is important that the feedstock is as much as possible water-free as well as with low FFA content in order to prevent

RCOOH + M+OH

M+=Na+, K+

5.5 Reaction scheme for soap formation.

hydrolysis. FFAs are not converted into esters but are transformed into soaps which cause problems in separating the glycerol layer and the water washing due to the formation of emulsions. In addition, FFAs are deactivating the catalyst with the soap formation. A feedstock with a high FFA content needs a higher concentration of catalyst. Preferably the FFA content should be less than 0.5% to ensure a complete conversion and efficient post-treatment. The glycerol layer separated from the biodiesel contains methanol, catalyst and soaps. After acidifying, the FFAs can be separated, the methanol evaporated and the sodium or potassium salts separated, purification steps which result in crude glycerol.

Biodiesel is then dried and used as such without post-treatment (if the feedstock is a refined lipid) after recovering of the excess of methanol and water washing.

Biodiesel can also be produced by transesterification of the oil in situ. In this procedure there is no need for the extraction of the oil from seeds. The liquid phase and solid oil containing feedstock are mixed and stirred. The disadvantage of this process is that large quantities of methanol and high concentration of catalyst are required. Furthermore, soaps are formed and additional solvent is needed to wash the seeds in order to ensure the complete separation of the oil and the transesterification reaction (Haas et al., 2007; Georgogianni et al., 2008; Qian et al., 2008).

At 60°C, the highest yield of esters is obtained using a molar ratio 226:1:1.6 of methanol:oil:NaOH. The FFA content in the biodiesel was lower than one per cent and contains no acylglycerols. By applying a drying step of the flakes prior to the reaction, the amount of methanol and catalyst can be reduced by 50% (Haas and Scott, 2007). If the feedstock contains more than three per cent FFAs, a combination of esterification and transesterification can be used (see Section 5.4.3).

In the ‘Alcohol Refining’ process (Fig. 5.7) developed by Westfalia Separator (Harten, 2006), feedstocks with high FFA content can be transformed into

biodiesel by extraction of the FFA in the glycerol layer (still containing alkaline catalyst) separated from the alkaline transesterification step. The extracted FFAs are converted into soaps. The glycerol layer is acidified and the FFAs separated. The advantages of alcohol refining are that it provides degumming and that during the transesterification less fouling and emulsion formation is observed. The disadvantages are that the FFAs are not converted into esters (loss of yield) and that the glycerol layer (which cannot be purified economically) has to be discarded.

The addition of solvents such as tetrahydrofuran and methyl t-butyl ether can accelerate the transesterification due to an increased solubility of methanol in the oil (Boocock et al.,1998). Another alternative to prepare biodiesel involves the application of microwave irradiation (Breccia et al, 1999) and ultrasonic irradiation (Stavarache et al, 2003).

Microwave heating looks very promising for a continuous flow preparation using a commercially available scientific microwave apparatus. The methodology allows for the reaction to be run under atmospheric conditions with flow rates up to 7.2 L/min, using a 4 L vessel. It can be utilized with methanol 1:6 molar ratio of oil:alcohol (Barnard et al., 2007).

The apparatus can be also used with butanol and sulphuric acid or potasium hydroxide as catalyst (Leadbeater et al., 2008).

A novel laminar flow biodiesel reactor/separator has been developed achieving high conversion rate while simultaneously allowing glycerol to separate and to settle from the reaction flow. At 40-50°C, a feed of 1.2 L/min (1:6 oil:methanol molar ratio) and 1.3% potassium hydride, a 99% conversion of waste canola oil was achieved with the removal of 70-99% of produced glycerol (Boucher et al., 2009).

Transesterification can also be performed in the presence of strong acid catalysts such as sulfuric acid and p-toluene sulfonic acid, normally for feedstocks with high FFA (>0.5%) (Fig. 5.8).

Methanesulfonic acid has been introduced by BASF (Lutropur®) as an efficient catalyst for esterification of low quality oils. This acid is less corrosive, non­oxidizing and more environmentally friendly than sulfuric or phosphoric acid. It is also used as a neutralizing agent in this base-catalyzed transesterification.

The advantage of this process is that FFAs are simultaneously converted into esters. Therefore, acid-catalyzed transesterification can be used for feedstocks which are containing high amounts of FFAs such as crude palm oil (up to 8%), used frying oils (3-7%), animal fats (up to 30%), grease and side-streams from oil refining (10-90%).

The mechanism involves protonation of the carbonyl function giving rise to a carbonium ion which is attached by the nucheophilic alcohol followed by splitting of the diglyceride and the aliphatic ester. The reaction is repeated with the diglyceride and monoglyceride. Acid transesterification has a number of disadvantages:

• Acid catalyzed transesterification is much slower than alkali catalysis (Canada 1999).

• Due to the slow reaction rate higher temperatures and pressure have to be applied (100°C/5 bar) which can also result in formation of by-products (formaldehydes and glycerol ethers).

• Feedstocks containing 0.5% water give rise to a yield decrease of one to five per cent (Canacki 1999).

• During the esterification water is formed which causes hydrolysis of the triglycerides

• The most employed acid catalyst (sulfuric acid) is very corrosive and causes dark colouring of the produced biodiesel.

The most economical conditions involve the use of molar ratio of 20:1 of methanol:oil, three per cent of sulfuric acid at 65°C for 48 hours. A comparison of base and acid catalyzed transesterification with methanol (with KOH and H2SO4 as catalysts) has also been reported (Nye et al., 1983).

The reaction kinetics of acid catalyzed transesterification of used frying and cooking oils has been studied by Zheng et al, (2006). The oil:methanol molar ratio and the temperature were the most significant factors influencing the conversion. Using a large excess of methanol and oil:methanol:acid ratio of 1:245:38 at 70°C and respectively a ratio of 1:75:1.9 at 80°C, gave a conversion of 99% in ca. 4 hours (pseudo-first-order kinetics). However, large scale production may not be economically feasible due to the large excess of methanol employed. It was reported that waste palm oil has also been transesterified in acid conditions (Al-Widyan and Al-Shyoukh, 2002).

Other homogeneous catalysts which can be used are phosphoric acid, hydrogen chloride, sulfonic acids and Lewis acids (BF3, SnCl2, AlCl3, FeCl3, CaCl2) (Mittelbach and Trathnigg, 2004).

Other promising catalysts are acetates and stearates of calcium, barium, magnesium, mangane, cadmium, bad, zinc, cobalt and nickel. A ratio of oil:alcohol of 1:12 at 200°C for 200 minutes is used. Stearates gave better yields due to higher solubility in the lipophilic phase. The advantages of these catalyst in comparison with Brpnsted acids are the lower alcohol used and a less sensitivity towards the content of water in the feedstock (Di Serio et al., 2005).

In addition in situ acid catalyzed transesterification were successfully performed. Using sulfuric acid in situ transesterification of homogenized sunflower seeds, an esters yield up to 20%, greater than with extracted oil, was reported due to the transesterification of the seed with lipids (Harrington and D’Arcy-Evans, 1985; Siler-Marinkovic and Tomasevic, 1998).

Similar processes have been used for rice bran oil (Ozgul-Yucel and Turkay, 2002). Recently, an acid catalyzed in situ transesterification of soybean oil in carbon dioxide expanded methanol was published (Wyatt and Haas, 2009). A 1.2 N sulfuric acid solution in methanol containing 50% mol fraction CO2 resulted in a 90% conversion of the triacylglycerol within 10 hours. Introduction of CO2 into the system increases the rate of reaction 2.5-fold. Alkaline transesterification in gas expanded methanol was unsuccessful.

Tin(Sn2+) complexes using the liganol 3-hydroxy-2-methyl-4-pyronate (maltolate) have been used to convert various vegetable oils into FAME at 80°C using a molar ratio of 400:100:1 of methanol:oil:catalyst. Yields up to 90% can be obtained but the methanolysis is dependent on the nature of acid chain favoring the presence of unsaturation and chain length. Technological potential is rather low as the complexes remain dissolved in the reaction medium. Attempts have been made to immobilize the complexes (Suarez et al., 2008).

A single acid or alkaline catalysis is not efficient to produce alkyl esters meeting the EU and US biodiesel standards if crude oils, fats and waste oils are being used. Therefore, a combined process with both acidic and alkaline catalyst in a two step reaction is required in which the acid treatment convert soaps and FFA into esters while the alkaline catalyst converts the acylglycerides into esters.

A dual process (Fig. 5.9) has been developed by Canacki and Van Gerpen (2003). A batch reactor was used to produce biodiesel from crude soybean oil, yellow grease (9% FFA) and brown grease (40% FFA). The high FFA feedstocks are firstly esterified with H2SO4 to reduce the FFA content to 1%, followed by transesterification with methanol and KOH.

The reaction rate is dependent upon the concentration of methanol and is increased if higher concentrations of H2SO4 are used. A ratio of methanol:oil (40:1) is used for feedstocks with high FFA content in comparison with the 6:1 ratio in alkaline catalysis. Replacing methanol by ethanol shows faster acidic esterification. Similar processes have been developed by Issariyakul et al. (2007) using a mixture of methanol/ethanol.

A more efficient procedure to convert high acidic feedstocks using short chain alcohols and a combination of acidic esterification and an alkaline process in the presence of ethylene glycerol and glycerol (temperature lower than 120°C and pressure lower than 5 bar) was described by Lepper and Friesenhagen (1986). Due to the immiscibility with the lipophilic layer, the water formed is entrained.

The acid treated oil containing fatty acid esters and mono — and diesters of ethylene glycol and glycerol is treated with alkaline catalyst for the transesterification. The advantage of the process is a short reaction time due to the continuous removal of the water and no soap formation is taking place. The disadvantage is the removal of the catalyst which requires two separations if the acidic catalyst has not been neutralized by an additional amount of alkaline catalyst. Canacki and Van Gerpen (1999) stated that using in feedstocks like crude and waste oil can result in a 25% reduction in cost relative to fully refined edible oils. However, Zhang et al. (2003) explained that it seems not to be clear. The traditional alkaline process requires the lowest fixed capital cost, but a much higher feedstock cost. The acid-alkaline process has a lower manufacturing cost, an attractive tax return (green certificates) and a lower biodiesel breakeven price. On the contrary, the corrosive nature of the acid catalysts should be included in the economic evaluation.

Biodiesel from low grade animal fat mixed with soybean oil has been synthesized in a combined esterification and transesterification process. A mixture of 50% of both raw materials has been selected and a computer simulation of the production process using Aspen Plus software has been carried out to evaluate the industrial feasibility. The acid esterification was performed with p-toluene sulfonic acid instead of sulfuric acid. The use of the latter one can cause a high concentration of sulfur in the biodiesel. However, the reaction rate is much faster than the reaction rate with p-toluene sulfonic acid (Canoira et al., 2008).

A variant of the acidic alkaline process is an alkaline transesterification followed by an acidic esterification on condition that the feedstock is containing maximum ten per cent FFAs (Verhe et al., 2009). Higher acid concentrations deactivate the alkaline catalyst in a too large extent. Both reactions are carried out in one reactor without intermediate separation of the layers (Fig. 5.10).

Reaction steps:

1. Neutralization of FFA and alkaline transesterification

2. Acidic esterification

3. Evaporation of methanol

4. Separation of glycerol layer

5. Washing of biodiesel with water.

Reaction conditions:

• Alkaline transesterification:

— catalyst 0.6% KOCH3 (33% in CH3OH) + calculated amount of KOCH3 in order to neutralize the FFA

— methanol: 20%

— temperature: 65°C

— reaction time: 90 minutes

• Acidic esterification:

— catalyst: 2% H2SO4 + calculated amount to neutralize the excess of KOCH3 and split the soaps

— temperature: 65°C

— reaction time: 180 minutes

The oil is heated at 65°C and mixed with the alkaline catalyst and methanol. Without separation of the glycerol that calculated amount of H2SO4 is added. After 3 hours, the methanol is stripped off and the crude biodiesel is transferred to a separator for glycerol removal. Water is added and the glycerol/water layer is separated. The biodiesel is washed with water until neutral and dried at 65°C under vacuum.

The main advantage of this process is that the feedstock does not require a pre­refining for the removal of the phospholipids, gums, traces of proteins and carbohydrates. During the esterification and work-up in acidic conditions at 65°C, a degumming is occurring, pigments are decomposed and are discarded with the aqueous layers. Separation of the glycerol and aqueous layers is much easier in acidic medium as no emulsions are formed. During the acidic esterification the residual acylglycerols are also transesterified into FAMEs resulting in higher conversion rates.