Category Archives: Handbook of biofuels production

Palm kernel

Elaeis guineensis is an edible oleaginous plant known as palm. The rapid increase in the production in the last 20 years has made palm oil the most important oil in the world. It is preferred for its high productivity, which explains its rapid expansion (Rupilius and Ahmad, 2007). Palm kernel oil is extremely important for the oleochemical industry because of the fatty acids profile (Ahmad, 2006). In the past decade, some researchers also found the feasibility of palm oil to produce biodiesel using either homogeneous (Darnoko and Cheryan, 2000; Crabbe et al., 2001) or heterogeneous catalysts (Jitputti et al., 2006).

Comparing the use of diesel fuel to run diesel engines, pure biodiesel exhibits an increase in BSFC up to 17% (Lin et al., 2006), while mixtures of 20% biodiesel with diesel fuel showed a lower increase of 3.3%. Altitude can play an important role, as better engine performance is achieved at high altitudes due to the influence in the duration of the premixed combustion stage (Benjumea et al., 2009). Even biodiesel from waste palm oil causes reductions in CO, HC and smoke opacity, while NOx increases (Ozsezen et al., 2009). The use of additives in 20% biodiesel blends seems to improve the previous results (Kalam and Masjuki, 2008). This oil has also been used straight and preheated, showing no negative effects on the engine, although exhaust emissions increased (Bari et al, 2002). The use of pure oil blended in low percentages with diesel fuel showed no signs of engine deterioration, while engine performance was not affected (Sapaun et al., 1996).

Influence of the feedstock and technology on biodiesel properties

The quality standards, fuel properties and performance of biodiesel are determined by the nature and quality of the feedstock, the pre-treatment and yield and the efficiency of transesterification reaction, the technology used and eventually the

Подпись: 5.18 Transesterification unit (Desmet Ballestra). image052

post-treatment. Extended reviews on the fuel properties have been recently reported (Knothe 2005; Erhan 2008).

Lipid accumulation from fermentation of sugars and related substrates used as the sole carbon source

De novo accumulation of cellular lipids is an anabolic biochemical process in which, by virtue of quasi-inverted ^-oxidation reaction series, acetyl-CoA issued by the intermediate cellular metabolism, generates the synthesis of intra-cellular fatty acids. Fatty acids will be then esterified in order to synthesize structural (phospholipids, sphingolipids, etc.) and reserve lipids (TAGs and SEs) (Moreton, 1988; Ratledge, 1988, 1994; Davies and Holdsworth, 1992; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009). In oleaginous microorganisms in which de novo lipid accumulation is conducted, acetyl-CoA that constitutes the precursor of intra-cellular fatty acids, derives from breakdown of citric acid that under some circumstances cannot be catabolized through the reactions performed in the Krebs cycle, but it is accumulated inside the mitochondria. This occurs when its concentration becomes higher than a critical value resulting in citric acid transportation into the cytosol (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006; Fakas et al., 2009b). The key-step for citric acid accumulation inside the mitochondrion matrix is the change of intra-cellular concentration of various metabolites, conducted after exhaustion of some nutrients (mainly nitrogen) in the culture medium (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006). This exhaustion provokes a rapid decrease of the concentration of intra-cellular AMP, since, by virtue of AMP — desaminase, the microorganism cleaves AMP into IMP and NH4+ ions in order to utilize nitrogen, in the form of NH4+ ions, as a complementary nitrogen source, necessary for synthesis of cell material (Evans and Ratledge, 1985).

The excessive decrease of intra-cellular AMP concentration alters the Krebs cycle function; the activity of both NAD+ and NADP+-isocitrate dehydrogenases, enzymes responsible for the transformation of iso-citric to a-ketoglutaric acid, lose their activity, since they are allosterically activated by intra-cellular AMP, and this event results in the accumulation of citric acid inside the mitochondrion (studies performed in the oleaginous microorganisms Candida sp. 107, Rhodosporidium toruloides, Y. lipolytica, Mortierella isabellina, Mortierella alpina, Mucor circinelloides and Cunningamella echinulata) (Botham and Ratledge, 1979; Evans and Ratledge, 1985; Wynn et al, 2001; Finogenova et al, 2002; Papanikolaou et al., 2004b). When the concentration of citric acid becomes higher than a critical value, it is secreted into the cytosol. Finally, in the case of lipogenous (lipid — accumulating) microorganisms, cytosolic citric acid is cleaved by ATP-citrate lyase (ACL), the key-enzyme of lipid accumulation process in the oil-bearing microorganisms, in acetyl-CoA and oxaloacetate, with acetyl-CoA being converted, by an inversion of b-oxydation process, to cellular fatty acids. In contrast, non­oleaginous microorganisms (e. g. various Y. lipolytica and Aspergillus niger strains) secrete the accumulated citric acid into the culture medium (Ratledge, 1994; Anastassiadis et al., 2002; Papanikolaou et al., 2002b) instead of accumulating significant quantities of reserve lipid. In general, production of citric acid by citrate-producing strains is a process carried out when extra — and hence intra­cellular nitrogen is depleted [overflow metabolism phenomenon (see Anastassiadis et al, 2002)], while studies of the intra-cellular enzyme activities and co-enzyme concentrations have somehow identified and clarified the biochemical events leading to citric acid biosynthesis (Finogenova et al., 2002; Morgunov et al., 2004; Makri et al, 2010) and indeed it has been demonstrated that citric acid secretion and SCO accumulation are processes indeed identical into their first steps.

In a third category of microorganisms, the accumulated (inside the cytosol) citric acid provokes inhibition of the enzyme 6-phospho-fructokinase, and the above fact results in the intra-cellular accumulation of polysaccharides based on the 6-phospho-glucose (Evans and Ratledge, 1985). Schematically, the intermediate cellular metabolism resulting in the synthesis of either citric acid or storage lipid is presented in Figure 8.3 (Ratledge, 1994; Ratledge and Wynn, 2002; Papanikolaou and Aggelis, 2009).

After the biosynthesis of intra-cellular fatty-CoA esters, an esterification with glycerol takes place in order for the reserve lipids to be stocked in the form of TAGs (Ratledge, 1988, 1994). This synthesis in the oleaginous microorganisms is conducted by virtue of the so-called pathway of a-glycerol phosphate acylation (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Mullner and Daum, 2004; Fakas et al., 2009b). In this metabolic pathway, free fatty acids are activated by coenzyme A and are subsequently used for the acylation of the glycerol backbone to synthesize TAGs. In the first step of TAGs assembly, glycerol-3-phosphate (G-3-P) is acylated by G-3-P acyltranferase (GAT) at the sn-1 position to yield 1-acyl-G-3-P (lysophospatidic acid-LPA), which is then


Citrate Extra-cellular medium

8.3 Pathways involved in the breakdown of glucose by microbial strains capable of producing SCO and/or citric acid in nitrogen-limited conditions. FFA: free-fatty acids; TRSP: citric acid transporting system; a, b, c: systems transporting pyruvic acid from cytosol to mitochondrion and inversely; d: system transporting citric and malic acid from cytosol to mitochondrion and inversely; ACL: ATP-citrate lyase; FAS: fatty acid synthetase; ICDH: iso-citrate dehydrogenase; MDc: malate dehydrogenase (cytoplasmic); MDm: malate dehydrogenase (mitochondrial); ME: NADPH+-malic enzyme; PD: pyruvate dehydrogenase; CS: citrate synthase; ICL: iso-citrate lyase; EMP: Embden-Mayerhoff-Parnas pathway. Pathways described by Ratledge (1994), Ratledge and Wynn (2002), Papanikolaou and Aggelis (2009).

further acylated by lysophosphatidic acid acyltransferase (also named 1-acyl-G — 3-P acyltransferase-AGAT) in the sn-2 position to yield phosphatidic acid (PA). This is followed by dephosphorylation of PA by phosphatidic acid phosphohydrolase (PAP) to release diacylglycerol (DAG). In the final step DAG is acylated either by diacylglycerol acyltransferase or phospholipid diacylglycerol acyltransferase to produce TAGs (Ratledge, 1988; Davies and Holdsworth, 1992; Athenstaedt and Daum, 1999; Mullner and Daum, 2004; Fakas et al, 2009b).

As far as the structure of the microbial TAGs produced is concerned, although their final composition could theoretically be a random substitution of acyl-CoA

groups into glycerol, in the case of the oleaginous microorganisms that have been examined, the glycerol sn-2 position is almost always occupied by unsaturated fatty acids [production of vegetable-type TAGs (see Ratledge, 1988; 1994; Guo and Ota, 2000)]. Therefore, various oleaginous microorganisms (principally yeasts belonging to the species Rhodosporidium toruloides, Apiotrichum curvatum and Y. lipolytica) have long been considered as promising candidates for the production of equivalents of exotic fats (fats that are principally saturated but containing unsaturated fatty acids esterified in the sn-2 glycerol position) (Moreton 1985, 1988; Moreton and Clode 1985; Ykema et al, 1989, 1990; Davies et al., 1990; Lipp and Anklam, 1998; Papanikolaou et al., 2001, 2003; Papanikolaou and Aggelis 2003b; Papanikolaou and Aggelis, 2010).


Biopropanol is a rarely discussed biofuel. Propanol is an alcohol with a three carbon chain (C3H7OH). Propanol is less toxic and less volatile than methanol, so it has some interesting properties as a fuel, although it is rare to consider it a fuel,



Crushing and drying carbohydrate


Gasification and partial oxidation of carbohydrate with oxygen and water


Hydrogen, carbon monoxide, carbon dioxide, and water mixture


Catalytic production: CO + 2Н,—► CH3OH





11.1 Schematic of biomethanol production of biomass carbohydrates.

since most propanol produced is used as a chemical solvent. There are two main types of propanol, n-propanol and isopropanol. Biopropanol is n-propanol that is produced from biomass. The University of British Columbia has developed technology for producing biopropanol (as well as biobutanol and bioethanol) from syn-gas using novel catalysts. Syntec Biofuels is commercializing this technology.9 The other method for producing biopropanol is from microbial fermentation of biomass (cellulose), but that is extremely inefficient, because very little propanol is traditionally produced and propanol is toxic to the cell in any significant concentration, so it is impractical at this stage of biotechnology. The issues with microbial production of biopropanol as analogous to the issues with microbial production of biobutanol, so if biobutanol becomes a more practical biofuel to produce, then biopropanol will also become more feasible.

Food safety and development of rural areas

At the heart of current debate on biofuels markets, the development of rural areas and food safety issues are of great concern. When considering the nexus between biofuels and rural development, four main aspects are specified in current literature: (1) social benefits of biofuels policy; (2) food security versus land management; (3) public sector intervention; and (4) enhancement of second — generation biofuels from non-food crops. Dufey (2006) offers a comprehensible review of social benefits of biofuels production accruing in developing as well as developed countries. In general, increase in employment generation in rural areas is mostly dependent on the type of crop used for biofuels production (e. g. sugar cane), although this should be seen according to market structure and income distribution. Given that agricultural production in rural areas is mostly labour­intensive, extra demand for agricultural products is likely to increase wages and employment. There are significant effects on job creation by either employing feedstock conversion practices or acquiring feedstock locally. Small-sized farmers could accelerate multiple income effects (Hazell and Pachauri, 2006). As a consequence, increased liquidity in local markets would have positive repercussions on the economy of rural areas. In Brazil or United States, for example, large firms control the bioenergy industry, whereas in developing countries small-sized growers organised in cooperatives represent an important link between large corporations and independent farmers.

The second aspect of biofuels policy is the question of food safety versus land management. Rosengrant et al. (2006), with the use of the IMPACT model (developed by the International Food Policy Research Institute at the Consultative Group on International Agricultural Research), examine the interactions between the demand of land for biofuels feedstock and the demand of land for food purposes and analyse how these interdependences affect food commodities and prices. The authors consider three main scenarios: (a) a massive growth in biofuels and no changes in productivity; (b) use of second-generation biofuels in current agricultural practices; and (c) considerable biofuels growth with changes in agricultural productivity and switch to production of second-generation biofuels. Results suggest in case (a) a remarkable increase in food prices causing sizeable losses in rural areas in developing countries. The need of subsidising biofuels would then arise with consequent distorting mechanisms due to unproductive agriculture and bioenergy sectors. In the second scenario (b), a change in technology would increase food price but at a lower rate compared to the first scenario. Finally, the last scenario (c) shows that a combination of technology improvements and productivity increases would alleviate shocks in food prices and favour the growth of small-sized farmers devoted to the supply and development of local markets.

The International Centre for Trade and Sustainable Development (2008) argues on competition of land for food versus land for biofuels feedstock. In principle, higher food prices would not automatically affect poor people. Rather, increases in food prices could be seen as an income generator for farmers working in poor rural communities. This vision is, however, not totally shared by a number of researchers (Naylor et al., 2007; Goldemberg, 2008) and institutions (World Bank, 2008). In particular, Goldemberg (2008) recalls that the problem of land competition over food and biofuels production should be seen as a problem of food safety versus climate issues. The entire ‘food question’ is the consequence of a renewed interest in the agricultural sector because of the ease of profits in biofuels energy production. Extended agricultural practices affect increases of indirect emissions of carbon as well as other dangerous GHGs (e. g. NOx) and contribute to deforestation and biodiversity losses.

Naylor et al. (2007) argue on the increasing rate, over the last years, in demand for energy commodities as incomes rise. This scenario would determine increases in energy as well as food commodities prices reversing, in the latter case, what was once the long-term declining trend in agricultural prices. The volatility of food prices causes strong impacts on undernourished population which typically spends almost all its income on food commodities. Linkages between food and energy prices are inevitable. While these were once seen in terms of agricultural energy inputs, nowadays these could be determined by the revenue prices of feedstock for biofuels production required to cover production costs. At international level, these relationships would be most difficult to determine given a number of determinants affecting food and energy prices such as the demand elasticity of agricultural commodities, national policies over land management for biofuels and food crops and the presence of institutional support to incentivise biofuels production. There are only few quantitative models which explain international transmission of price volatility for biofuels and agricultural commodities (Abdulai, 2000; Conforti, 2004; Schmidhuber, 2006; Peri and Baldi, 2008; Hertel and Beckman, 2010), and these focus either on national case studies (i. e. Ghana, Iran, Italy, United States) or selected agricultural crop and biofuels commodities. A further implication on food security and undernourished population is food aid. There is an inverse relationship between shipment aids (from richer countries) and food prices (Falcon, 1991; del Ninno et al, 2007). Countries relying on food aid (i. e. Sub-Saharan African or Southern Asian countries) are subject to substantial domestic critical effects (i. e. production and land availability, internal market prices instability, government responses) in the presence of global food price increases. The general trend in food and energy prices and the consequences on world food safety is also recognised by the World Bank in its recent document submitted for and approved by the G8 meeting in 2008 (World Bank, 2008). The rise in food and energy prices (Fig. 2.1) causes important macroeconomic effects mostly on domestic economies.

Inflation, for example, is hitting developing economies that are fighting to keep the percentage between five and seven per cent (Fig. 2.2). The same countries are now experiencing fluctuations in inflation rates because of price increase in oil, food and other basic commodities.


2.1 Commodity price indexes in nominal terms (author’s elaboration on World Economic Outlook Database [International Monetary Fund, 2009]).


2.2 Inflation rates for selected economies (author’s elaboration on World Economic Outlook Database [International Monetary Fund,


Worsening of balance of payment also causes a reduced capacity of developing countries to sustain (by reducing official reserves) import exposure in the immediate future. Most of the emerging economies show in fact a negative trend in changes of official reserves over the last decade (Table 2.1).

Furthermore, when emerging economies are also energy-intensive importers, a damaging effect in terms of trade contributes to exacerbate their institutional and economic vulnerability. Pressures on wages and other costs become inevitable for

Table 2.1 Changes in official reserves in billion US dollars






Commonwealth of Independent States







































































Source: World Economic Outlook Database (International Monetary Fund, 2009).

such countries where fiscal and monetary policies are too vulnerable to food and energy price fluctuations. This and the rise of income inequality (including the aggravation of poverty) in developing countries asks for immediate implementation of adequate policies.

G8 as well as United Nations countries agreed on a number of initiatives. First, a continuous support to fund the World Food Programme in addition to the provision of financial and technical assistance for the supply of agricultural commodities. Second, in a longer term perspective, investments in agricultural and rural infrastructures to guarantee market access especially in African, Southern Asian and small island countries. Third, enhancing technological investments in developing as well as developed countries for second — and third-generation biofuels from cellulose-based ethanol products. And fourthly to promote the reduction in trade tariffs for biofuels commodities and improve the functioning and implementation of international agreements (e. g. the Doha Round) affecting agricultural markets (World Bank, 2008).

The public sector plays a substantial role in the development of rural (and also industrialised) areas and the mitigation of competing food markets when enhancing biofuels activities. The use of land for biofuels feedstock could have negative impacts on the demand for food commodities causing food prices to increase due to scarcity of productive land for food production. Lack of sufficient natural resource endowments for biofuels crops causes consistent losses especially in poor areas. A price increase in food commodities would in fact be detrimental to those farmers experiencing a net deficit of food production. Unjustified repercussions on consumer prices would then occur (in rural/poor areas) where demand elasticity of agricultural products is high. To avoid the occurrence of vast social costs, public intervention becomes a necessary tool which helps reduce market failures and rebalance trade-offs between food and bioenergy through adequate supporting policies (Hazell, 2006). These can be in the form of incentives: to increase the productivity of food production such that additional land and water can be used for biofuels crops; to convert infertile lands to second-generation biofuels; to use by-products from food production to boost bioenergy commodities; and to remove barriers to trade and promulgate the benefits of competitive markets for biofuels commodities at any scale of technology. Supporting policies would also guarantee independent and small-sized farmers in less developed countries the opportunity to process bioenergy commodities at local level. In addition, the identification of all stakeholders in the biofuels chain becomes fundamental when setting policy targets in the food sector at national level. The Brazilian example is a success. First for the recognition of new demand in environmentally friendly automobile industry through the use of ethanol fuels; second for setting subsidies to enhance economies of scale in the agricultural as well as the automobile sector; third for integrating the private sector in the public management for electricity supply from bioenergy products; and fourth for creating new stimulus to rural activities employed in biofuels production.

There exists, undoubtedly, a connection between developed and rural areas for biofuels production. Large-scale biofuels activities in developed countries may reduce the export of food products pushing the prices of these goods up. This would in turn positively affect rural areas in developing countries benefitting from higher net surpluses in food commodities. Contrarily, higher world food prices would also mean scarcity of food products for poor households living in rural areas. When this negative effect is counterbalanced by higher employment and income perspectives in the biofuels industry, the net impact at aggregate social level generates economic growth led by the agricultural system. From this angle, biofuels chain can make a substantial contribution to combat poverty and improve food safety. The production of energy from bioenergy crops, together with the sustainable use of local resources, could result in higher standards of living for the rural society as a whole. Additional energy resources to the local community would finally contribute to the local development of rural economic activities including agricultural enhancements and food security.

A final aspect to discuss concerning the link between biofuels/bioenergy and rural development is the enhancement of second-generation biofuels. Studies on jatropha production in African countries (Venturini Del Greco and Rademakers, 2006) indicate several benefits at community level. These benefits derive from an integrated approach run by public enterprises (and managed by private firms) to jatropha production such as electricity consumption, milling services, additional oil for sale purposes, by-products for use in soap manufacturing and fertilisers use. Van der Plas and Abdel-Hamid (2005) argue in favour of biofuels from wood production in rural areas in Sub-Saharan African countries. Of relevant interest is the demand from urban centres and the transparency of relationships (contractors, distribution of rents, etc.) between these urban centres and rural areas supplying biofuels. The intricate but efficient legal network thus running in these areas contributes either to the enrichment of small farmers’ wealth or to the sustainable resource use.

Starchy materials

Starch is a biopolymer, defined as a homopolymer, consisting of only one monomer, D-glucose (Pongsawatmanit et al., 2007). To produce bioethanol from starch, it is necessary to break down by hydrolysis the chains of this carbohydrate to glucose syrup or fermentable sugar that can be converted into bioethanol by yeasts (Balat et al., 2008). This type of feedstock, mainly corn and wheat, is the most utilised for bioethanol production. The starch-based bioethanol industry has been commercially viable for about 30 years (Barretts de Menezes, 1982). In that time, tremendous improvements have been made in enzyme efficiency, reducing process costs and time and increasing bioethanol yields (Kim and Dale, 2004). However, there are two main reasons for the present high cost: on the one hand, the usual yeast S. cerevisiae cannot utilise starchy materials, so large amounts of amylolytic enzymes, namely glucoamylase and a-amylase, need to be added (Apar and Ozbek, 2004); on the other hand, the starchy materials need to be cooked at a high temperature (413-453 K) to obtain a high bioethanol yield. In the last years, the possibility of hydrolysing starch at low temperatures (liquefaction) for achieving energy savings has been investigated (Shigechi et al., 2004; Mojovic et al., 2006; Robertson et al., 2006).

Effect of temperature

The increase in temperature of a reaction mixture usually results in an increase in the reaction rate. This is mainly due to the increase in rate constants with temperature and partly due to the reduction in viscosity and mass transfer resistances. However, in enzymatic catalyzed reaction, this increase in reaction rate with temperature persists up to a certain optimum temperature, after which the rate decreases sharply. This sharp drop takes place at the onset of the denaturation of the enzyme that occurs at elevated temperatures. In addition to the deactivation of the enzyme, the presence of the inactive enzyme at the interface blocks the active enzyme from penetrating the interface, which would further decrease the reaction rate. This trend has been consistently observed in all studies that investigated the effect of temperature on the production of biodiesel by lipase. The critical temperature, at which the enzyme starts to deactivate, was different as shown in Table 6.1. Generally, lipases from bacterial sources, such as those from Pseudomonas species, have relatively higher thermo-stability than lipases from yeast source, such as those from Candida species that include Novozym 435. For example, the optimum operating temperature of lipase from P. fluorescens has been reported to be 65°C (Fukuda et al., 2001), whereas that of lipase from Novozym 435 has been reported to be 35-40°C (Chang et al., 2005). Immobilization provides a more rigid external backbone for lipase molecule, allowing it to maintain its activity at higher temperatures than if it is in free-form. Hence, the reaction optimum temperature is expected to increase, which results in faster rate of reaction.


After the pre-treatment and the degradation stage that releases the sugar units it is possible to convert the carbohydrates to ethanol by the technique of fermentation. Baker’s yeast (Saccharomyces) is most commonly used and is able to convert glucose to ethanol under both aerobic and anaerobic conditions. During the fermentation process, two moles of carbon dioxide and two moles of ethanol are produced from one mole of a sugar unit. In order to achieve optimal fermentation there are several characteristics of the fermenting microorganisms that should be considered, e. g. temperature range, pH range (3.5-5.0 for yeast, 6.5-7.0 for bacteria), alcohol tolerance, growth rate, genetic stability, inhibitor tolerance, yield, etc. (Bai, Anderson, and Moo-Young, 2008). The fermentation process can occur either in separate batches or as a continuous process which is often more preferable economically (Sanchez and Cardona, 2008).

Distillation and purification

During the fermentation process it is important to separate the produced ethanol from the original liquid since several microorganisms are not able to survive the high concentration of ethanol (more than 15-20%). The remaining liquid contains ethanol and water (about 80%) and other soluble compounds and ethanol can be separated by distillation or supercritical fluid technology (Schacht, Zetzl, and Brunner, 2008). Unfortunately the distillation requires a lot of energy to obtain 95.6% ethanol (azeotrope mixture of ethanol and water). In the next step the ethanol is further purified (99%) by adding a drying agent to the solution or by molecular sieve adsorption. But the 99% ig (industrial grade) ethanol is hygroscopic and may absorb water again from the surrounding air during storage. The purification (i. e. dehydration) steps are necessary since the ethanol/gasoline blend will separate in the presence of water and is difficult to remix (Szulczyk, McCarl, and Cornforth, 2010).

Anaerobic digesters

The anaerobic continuous stirred tank reactor (CSTR) is the most basic bioreactor configuration. The major advantage of the CSTR is its simplicity in construction and operation. However, large bioreactor volumes are required to provide the high retention time necessary to sustain the slow growing anaerobic microbial mass inside the bioreactor, which raises the cost of the process. Therefore, for an efficient anaerobic system with relatively small bioreactor volume, the design of anaerobic digesters should aim at providing an optimum environment for the growth of the anaerobic microorganisms given the complexity of their physiology and the syntrophic and/or antagonistic interactions among them. Lettinga (1995) specified certain criteria:

• High retention of the active biomass (microorganisms) inside the bioreactor.

• Sufficient contact between the biomass and the substrate.

• High reaction rates and elimination of the limiting transport phenomena.

• Suitable environment for the adaptation of the biomass to various types of feedstocks.

• Suitable environment for all organisms under the operating conditions.

Depending on the solid content of the feedstocks, different bioreactor configurations can be used:

• Low solid content feedstocks (e. g. secondary wastewater treatment, wastewater from food industry, hydraulic flush manure systems; swine)

— Anaerobic lagoons — fixed, floating, or submerged covers

— Completely mixed reactors

— Anaerobic filter reactors

— Fluidised bed reactors

— Upflow anaerobic sludge blanket reactors (U ASBR)

— Anaerobic baffled reactors (ABRs).

• Medium solid content feedstocks (e. g. dairy manure, ‘scraped’ swine manure, municipal or food industries sludge)

— Plug flow reactors

— Completely mixed reactors

— Contact reactors.

• High solid content feedstocks (e. g. organic fraction of municipal solid wastes, agricultural residues, food processing waste; food residuals; pulp — paper sludge)

— Plug flow

— Completely mixed

— Leach-bed.

A brief description of the main bioreactor types follows:

• Fixed-bed anaerobic reactor (anaerobic filter): The wastewater is introduced from the bottom or the top of a column which is filled with inert material (rocks, cinder, plastic or gravel). The filling material provides the surface upon which microorganisms are attached forming a biofilm. The microorganisms can also be retained through entrapment in the micro-porous structure of the filling material. Clogging is a typical problem with this type of digester. The organic load of the wastewater must be low to medium. Recirculation must be applied so that the organic load in the entrance is maintained between 8 and 12 g/L. Wastewaters containing significant amounts of suspended solids or constituents that cause precipitation of organic and inorganic compounds are not suitable for this bioreactor type. The filling material must provide large void space to avoid clogging (95%) and have large specific surface (100-200 m2/m3).

• Expanded and fluidised bed anaerobic digester: This type of configuration allows a more effective mass transfer from the liquid phase to the membrane, because fine filling material is used (0.2-0.5 mm). The upflow velocity must be high enough (through recirculation) to maintain the expansion of the bed between 15% and 30%, while if the expansion raises up to 300%, the bed is characterised as fluidised (Hall, 1992). Energy consumption required to provide recirculation is the main disadvantage of this bioreactor. The wastewater must contain low suspended solids as in the case of the fixed bed bioreactors.

• UASBR: The UASBR was designed as an alternative to wastewater treatment without the operating problems of bioreactors with filling materials but incorporating the concept of biomass immobilisation (Lettinga et al., 1980). In this bioreactor type, the microorganisms are agglomerated to form a dense structure (granule) with excellent settling properties and strength under adverse conditions. The granular sludge blanket remains in the bottom of the bioreactor. The feed is introduced from the bottom and the motion of the flow is upwards. The upflow velocity is very important since it influences the formation of the granules. Typical upflow velocities range between 0.5 and 3 m/h (Annachhatre, 1996). The biogas produced is often entrapped in the granules making them lighter and buoyant with their potential wash out. An effective three phase separator on the top of the bioreactor results in the retention of the granule and their return to the sludge blanket. UASBR is a reliably tested technology for the treatment of a wide range of wastewaters (from municipal wastewater to high strength agro industrial wastewater) with low solid content. It has low installation, operation and maintenance costs. More than 900 full-scale units are currently being operated all over the world (Garcia et al., 2008). Hybrid systems have been developed to combine the characteristics of a UASBR and an anaerobic filter, expanded or fluidised bed reactor. Hybrid UASBR have been used to treat a variety of industrial wastewaters over the years (Banu and Kaliappan, 2008; Sunil Kumar et al., 2007; Ramakrishnan and Gupta, 2006; Sandhya and Swaminathan, 2006).

• ABR: It is a rectangular tank with baffles. The wastewater flows above and below a series of baffles successively coming into contact with the biomass which is accumulated in the bottom of the bioreactor (McCarty and Bachmann, 1992). This bioreactor type is simple in structure, with no moving parts or mixers. The biomass is not necessary to have good settling properties as in the UASB, in order to be retained in the bioreactor. It is an efficient system at low retention times and its operation is stable under sudden changes in the organic loading rate (Barber and Stuckey, 1999). A modification of this bioreactor type led to the periodic anaerobic baffled reactor (PABR) which is based on the periodic feeding mode to all compartments. In PABR, the switching frequency of the feed allows flexibility in operation; the PABR can be operated as a simple ABR, if the switching frequency is set to zero, and, in the extreme case of very high switching frequency, as a single-compartment upflow bioreactor (Skiadas and Lyberatos, 1998; Stamatelatou et al., 2009).

• Plug flow: It is a long narrow insulated and heated tank. The digested material flows from one end of the tank to the other as fresh feedstock enters the bioreactor. The bioreactor can be placed horizontally or vertically. It is used in the case of solid feedstocks. In order to provide mixing, various practices are applied (de Baere, 2008). In the Dranco process (vertical, downflow plug flow digester), the fresh feedstock is mixed with a portion of the digested material and is introduced from the top of the bioreactor. The same concept can be applied while the plug flow reactor is placed horizontally (Kompogas process). In this case slowly rotating impellers inside the reactor can aid the horizontal movement of the mixture, also serving for mixing, degassing and suspension of the heavier particles. In another plug-flow type configuration (Valorga process), the horizontal flow is circular and biogas injection at intervals under pressure through a network of nozzles provides mixing.

• Leach bed: The feedstock is loaded in a vertical bioreactor to form a bed through which a liquid stream percolates as a leachate and is recirculated to the top of the same reactor where it is produced (Biocel process). This process is implemented in Lelystad, The Netherlands (ten Brummeler, 1999).

• Complete mixed anaerobic digester — anaerobic contact process. It usually consists of a round insulated tank, above or below ground. Heating is provided through coils with hot water inside the tank or an external heat exchanger. Mixing is achieved through a motor driven mixer, recirculation of the mixed liquor or biogas. The cover can be floating or fixed. In the case of low solid content feedstocks and in order to enhance the biomass concentration in the bioreactor, a modification of the complete mixed anaerobic digester led to the anaerobic contact process. In this configuration, the bioreactor is followed by a settling tank (or inclined parallel plates, membranes, etc.; Defour et al., 1994) to separate the sludge from the supernatant. The sludge is recycled to the bioreactor increasing the biomass concentration.

• Covered anaerobic lagoon: It is a large earthen impoundment, lined with appropriate geomembranes and covered with a flexible or floating gas tight cover. They are used mostly for manure treatment. No heat and mixing are provided, therefore the ambient temperature is prevailed making this type of digester unsuitable in cold climatic conditions.

There are more parameters according to which an anaerobic system can be


1 Temperature of operation: All digesters usually operate within two temperature ranges, either at 35-40°C (mesophilic) or 50-60°C (thermophilic). Mesophilic anaerobic digestion is applied for digesting rumens of animals and feedstock from industrial and farm activities, while thermophilic anaerobic digestion is more suitable for sanitation of pathogen-bearing feedstocks. Another advantage of thermophilic anaerobic digestion is the fast conversion rates of the feedstock (induced by the fast metabolism of the microorganisms due to the high temperature) and, consequently, the lower retention time (and reactor volume) required. However, the psychrophilic range of temperatures (<20°C) have also been studied, especially in lagoons and swamps. Thorough studies on reactor design and in-depth parametric analysis for psychrophilic consortia are lacking. It has been acknowledged, however, that, in psychrophilic conditions, systems favouring biomass accumulation are required to secure high efficiency (Kotsyurbenko et al, 1993; Lettinga et al., 2001). Another possibility has been to apply genetic engineering in the attempt to introduce stable enzymes, active in cold temperatures to give improved catalysts for the biomethanation process (Kashyap et al., 2003).

2 Solid content of digesting mixture: When the solid content of the digesting mixture is less than 3-4% (little or no suspended solids), then the digesters are usually a single phase liquid system. Digesters treating solids are characterised as wet or dry depending on whether the solid content is up to 12-15% or more. Wet anaerobic systems are in a slurry form and can still be mixed through agitation, while for the dry anaerobic systems, the plug-flow type digesters are most suitable.

3 Number of bioreactors: The anaerobic systems may consist of a single bioreactor or a combination of bioreactors of different or the same design. Especially, in the case of anaerobic digestion of solid or slurry feedstocks, the use of more than one bioreactors is a common practice. Typically, two stages are used, with the first one being the hydrolytic-acidogenic step and the second one being the methanogenic step. In a two-stage process, it is possible to optimise the operational conditions of both steps since they take place in different bioreactors. The application of this concept has resulted in a great variety of two-stage configurations. The main advantage of the two-stage systems is the process stability in the case of feedstocks that would cause an unstable performance in single stage systems.

Two or three bioreactors of leach-bed type may be used in series, as in the sequential batch anaerobic composting (SEBAC) process (Chynoweth et al., 1992, 2006). Leachate is transferred from a ‘mature’ bioreactor to a bioreactor filled with the fresh feedstock and recycled to the top of the ‘mature’ bioreactor until methanogenic conditions in the first stage prevail. Then, the bioreactor is switched to internal leachate recirculation until methanogenesis is completed. The volatile fatty acids from the first stage are transferred through the leachate into the ‘mature’ bioreactor with active methanogenic populations, while microbes from the second ‘mature’ bioreactor are recycled to the first one, enhancing its microbial activity.

In a similar concept, another configuration also uses batch loading to stimulate rapid volatile fatty acid production in a two-stage system. It combines one or more high solid bioreactors of leach-bed type in the first stage with a high rate and low solids bioreactor (such as an anaerobic filter or a UASBR) in the second stage (Zhang and Zhang, 1999; Lehtomaki et al., 2008). The high-solids reactors are loaded and the leachate from the batch reactors is continuously circulated through a single low-solids digester. The effluent of the second bioreactor, with reduced organic load and high alkalinity, is pumped back to the first stage bioreactor(s).

Temperature phased systems is another case of multistage configuration with each stage operating at a different temperature. This process has been implemented most often using thermophilic digestion (with a temperature range of 45°C to 65°C) as the first phase, followed by mesophilic digestion (with a temperature range of 25°C to 42°C). This is designed to produce a final product with a minimal odour level, better dewatering properties and low content in pathogens.

4 Continuous or batch mode of operation: There are systems that operate in a continuous mode, while others are loaded in batches and, upon completion of the waste degradation up to a degree, are emptied and left with a 10-15% of the digested content as a seed for the next cycle of batch (sequencing batch reactors).

5 Small or large scale systems: Anaerobic digestion has been extensively applied in agriculture at small scale in the form of on-farm digesters, and the produced biogas is utilised for heat as well as electricity production. The solid and liquid residues from the anaerobic digesters can be recycled in the farm. The digesters are constructed as simple as possible in order to be economic. They are heated containers, shaped like silos, troughs, basins or ponds and may be placed underground or on the surface. They may be batch type (much simpler to construct and maintain) or continuous type. On-farm digesters usually operate at a mesophilic range of temperatures at a typical retention time of 10-30 d.

However, operation of large scale systems have the advantage of being more economically profitable; integrated farm waste management takes all factors into account (feedstock, products) with the aim of maximising the economies of scale and of eliminating the impacts to the environment. Moreover, very large scale anaerobic digestion plants, the so-called ‘centralised anaerobic digestion plants’, have been developed to use feedstock from a variety of sources. The primary source of feedstock is farm wastes, but also other non-toxic types of wastes, such as those coming from food processing industries or the organic fraction of the source-sorted municipal solid wastes, can be introduced in a centralised anaerobic digestion (CAD) facility. The anaerobic digesters may be either mesophilic or thermophilic and operate at typical retention times of 12-20 d. Process control schemes are usually applied in this scale since it is affordable to employ trained staff. Farmers may have an additional income through tipping fees by providing the feedstock to a CAD, but they may also benefit more through applying the digestate on their farms as a fertiliser. The location of CAD plants is also crucial and they usually serve either a single large farm or several farms within a radius of about 10 km.