Carbon residue is defined as the amount of carbonaceous matter left after evaporation and pyrolysis of a fuel sample under specific conditions. Although this residue is not solely composed of carbon, the term carbon residue is found in all three standards because it has long been commonly used. The parameter serves as a measure for the tendency of a fuel sample to produce deposits on injector tips and inside the combustion chamber when used as automotive fuel. It is considered as one of the most important biodiesel quality criteria, as it is linked with many other parameters. So for biodiesel, carbon residue correlates with the respective amounts of glycerides, free fatty acids, soaps and remaining catalyst or contaminants (Mittelbach 1996). Moreover, the parameter is influenced by high concentrations of polyunsaturated FAME and polymers (Mittelbach and Enzelsberger 1999). For these reasons, carbon residue is limited in the biodiesel specifications.

As the name ‘microalgae’ suggests, the relative size of these organisms may seem unsuitable for generating massive quantities of biofuel on a global scale; nonetheless, microalgae offer many advantageous qualities for biofuel production, especially when compared to terrestrial bioenergy crops. The basic principle of generating biofuel from microalgae is to exploit these cells as biological factories, whose lipid output can be as much as 70% of their total dry biomass, under optimal conditions. While many species of algae exhibit the natural capacity to produce abundant amounts of oil for conversion to biofuel, a major obstacle in the commercialization of such a process lies in the scalability. Many exciting breakthroughs in algal biotechnology have occurred on the lab bench, but mass cultivation of algae is still associated with some of the most challenging problems. A few areas of intense research focus include highly productive growth systems, temperature control, photooxidative stress tolerance, light intensity regulation, harvesting, and downstream processing. Whether it is through the manipulation of culture conditions or the application of mutagenesis and genetic engineering, the biological networks of these unicellular creatures can potentially be optimized to synthesize and/or secrete biofuel metabolites, particularly in the form of lipids and other hydrocarbons, in order to overcome some of the aforementioned stumbling blocks to large-scale cultivation.

It is difficult to convey a concise list of the ideal species for biofuel production because the organism must be paired not only with the climate in which it will be cultivated, but also the specific mechanism of cultivation and desired end products. Also, the lipid content can vary considerably depending on culture conditions. For example, nitrogen and silicon deprivation has shown to augment lipid accumulation in green algae and diatoms, respectively. As investigated by NREL’s Aquatic Species Program, nutrient deprivation experiments and species collection and characterization efforts gave rise to an extensive list of microalgae with particularly high lipid contents (Sheehan et al., 1998).

There is, however, a serious caveat to high lipid accumulation in algae: the energy collected by the cell is partitioned into storage and, thus, made unavailable for immediate use. As a result, oleaginous species exhibit significantly slower growth rates than their more lean relatives. The fatty acid composition and growth characteristics of some of the more promising species are illustrated in Figure 2, where a clear balance can be seen between growth rate and lipid content. In this particular study, the algal species were cultivated first in airlift bioreactors, then in aerated polyethylene bags, and finally in outdoor raceway ponds for a period of four months. Subsequent biochemical evaluation found the neutral lipids to be predominantly C16 and C18, which are ideal chain lengths for the composition of biodiesel (Gouveia et al., 2009).

Astonishingly, of the thousands of different species of algae, a mere fifteen organisms are commonly used for commercial applications (Raja et al., 2008) and only eight species’ genomes have been sequenced (Hallmann, 2007), but future bioprospecting endeavors paired with high-throughput screening are likely to discover more exemplary candidates for algal biofuel production.

1.1 Characteristics and composition of heterogeneous wastes as feedstock

Biomass is defined as an organic material derived from plants or animals that contain potential chemical energy; for example wood, which was the first fire source used by mankind, and which is still used today by population for cooking and heating.

At world scale, biomass is now the fourth largest energy source, but it has the capacity to become the first. Photosynthesis can store up to 5-8 times more energy in biomass annually than the actual world energy consumption (Prins et al., 2005). The basic reaction of photosynthesis is as follow: carbon dioxide and water are converted to glucose and oxygen, an endothermic process for which the energy is supplied by photons. Examples of biomass are residues from agriculture or from the forest industry such as branches, straw, stalks, saw dust, etc. An important example of residual agricultural biomass is related to the ethanol production from sugar cane in Brazil which produces 280 kg of residual bagasse at 50% of dry solids. Lignocellulosic materials can be collected and recovered because this material has some energetic content (Ballerini and Alazard-Toux, 2006), however, leaving a part of this material on place is imperative since it keeps the soil fertile.

It is important for governments and citizens to realize that hydrocarbon-based waste material is another source of energy that should be taken advantage on. Waste can be solid or liquid form. It can be land filled, incinerated or converted. Municipal solid waste used electrical transmission poles and railroad ties treated with creosote, sludge from wastewater treatment and pulp and paper industries, wood from construction and demolition operations which contains paints and resins, etc., are all materials that contain carbon that can be valorized in bio-refineries such as the one Enerkem is constructing (2011) in Edmonton, AB.

Assessment of residual biomass or Municipal Solid Waste (MSW) as feedstock to produce bio-ethanol requires a basic understanding of feedstock composition and of the specific properties that dictates its performance as feed in the gasifier. The most important are: moisture content, ash content, volatile matter content, elemental composition and heating value.

The moisture content of biomass is the quantity of water in the material, expressed as percentage of material weight. This weight can be referred to on a wet basis or on a dry basis. If the moisture content is determined on a "wet" basis, the water’s weight is expressed as a percentage of the sum of the weight of the water, ash, and dry — ash free matter. It is sometimes necessary to dry the feedstock to a certain level in order to maximize the gasification reaction. Indeed, more moisture is transferred by a higher consumption of oxygen in order to keep the ideal temperature in the gasifier. Temperature of gasification is crucial on the process efficiency. An optimum exists with just the right amount of oxygen needed to perform completely the gasification reaction. This represents a temperature of about 660°C for biomass with 20 % of moisture and about 695°C for biomass with 10 % of moisture (Prins et al., 2005). If more oxygen is added, formation of carbon dioxide will increase and gasification efficiency will drop; the heating value of the synthesis gas will thus decrease (van der Drift et al., 2001). However, not enough oxygen will promote reduction of carbon leading to an increase of methane formation.

The inorganic component (ash content) can be expressed the same way as the moisture content. In general, the ash content is expressed on a dry basis. Both total ash content and chemical composition are both important in regards of the gasification process. The composition of the ash affects its behaviour under high temperatures of combustion and gasification. For example, melted ash may cause problems in both combustion and gasification reactors. These problems may vary from clogged ash-removal caused by slagging ash to severe operating issue in fluidized bed systems. Measurement of ash melting point is thus crucial.

Volatile matter refers to the part of biomass that is released when the biomass is heated beyond its dehydration temperatures. During this heating process the biomass decomposes into volatile gases and solid char. Biomass typically has a high volatile matter content (up to 80%), whereas coal has a low volatile matter content (<20%).

Elemental composition of the ash-free organic component of residual biomass is relatively uniform. The major components are carbon oxygen and hydrogen. Most biomass also contains a small proportion of nitrogen and sulfur. Table 1 presents the elementary composition of biomass as derived from ultimate analyses.

The heating value of a fuel is an indication of the energy chemically bound in the fuel with reference to a standardized environment. The standardization involves the temperature, state of water, and the combustion products. The calorific value is presented as the higher heating value and the lower heating value. The higher heating value represents the heat release per unit of mass when the material (at 25°C) is completely oxidized to carbon dioxide and water and then returned to 25°C. A calorimetric bomb is the standard instrument used to measure this value. The lower heating value is not taking into account the energy supplied by the condensation of water (latent heat of vaporization of water at 25°C which is 2440 kJ/kg). The water includes moisture from the feedstock and the product from the reaction between oxygen and hydrogen comprised in the raw material (Borman and Ragland, 1998). Basic and complementary information about biomass intended for gasification can be obtained via the proximate and ultimate analyses.

Proximate analysis measures moisture content, volatile matter, fixed carbon, ash content and calorific value.

Ultimate analysis provides information about elementary composition of the biomass in weight percentage of carbon, hydrogen, oxygen, sulphur and nitrogen. The carbon to hydrogen ratio in the feedstock has a direct impact on the syngas, more particularly on the ratio of H2/CO (Higman and van der Burgt, 2008).

Table 2 (depicted below) presents a comparison of different feedstock properties. Moisture content was provided after drying of feedstock and compositions are approximated (Ciferno and Marono, 2002).

Note that higher calorific value formulas, refering to equation 1, were developed from the composition of the feedstock. Dulong formula is one of them (Higman and van der Burgt, 2008). However, numerous correlations were developed and reported in literature. For instance, experiments on more than 200 species were performed at the Indian Institute of Technology (Bombay) and the following equation was derived from empirical data.

HHV (MJ/kg) = 0.3491C + 1.1783H — 0.1034 O — 0.0211 A +0.1005S -0.0151N (1)

Where C is the weight fraction of carbon, H of hydrogen, O of oxygen, A of ash, S of sulfur and N of nitrogen. This correlation offers an average absolute error of 1.45 % (Channiwala and Parikh, 2002).

There are other feedstock properties that are critical to determine in order to treat the material accordingly: size, distribution and shapes of the material, porous structure and bulk density. Gasification and incineration are both design for transforming hydrocarbon-based hazardous material to more simple by-products. Nonetheless, these two technologies are completely different in their operation and finalities. The goal of the combustion is to proceed to a complete oxidation of carbon in order to produce carbon dioxide and water. Thereby an excess of oxidizing agent is used. Sulfur and nitrogen comprised in the feedstock are oxidized to SOx and NOx. Whilst only gasification can lead to synthesis, both processes can lead to production of electricity. However, with the goal of generating added-value product, gasification has the possibility to convert the carbon of waste to biofuels instead of releasing the carbon as CO2. Gasification is a partial oxidation process with just enough oxygen to produce the heat to yield reduced carbon and hydrogen (Wetherold et al., 2000).

1.3.1 Steam explosion

Steam explosion is one of the most applied pre-treatment processes owing to its low use of chemicals and limited energy consumption (Harmsen et al., 2010). During steam explosion pre-treatment process, the lignocellulosic biomass is heated with high pressure saturated steam having temperatures typically in the range of 180-230oC for 2-10 minutes. Subsequently, the substrate is quickly flashed to atmospheric pressure; as a result, the water inside the substrate vaporizes and expands rapidly, disintegrating the biomass (Grous et al., 1985; Kokta and Ahmed, 1998; Zimbardi et al., 1999). This causes great reduction in the particle size of the substrate (Fig. 4). The heart of the explosion pulping process is the reactor, which allows the use of high pressure during heating and cooking. The reactor can be of either the batch (Fig. 5) (Jin and Chen, 2006) or continuous type (Fig. 6) (Kokta and Ahmed, 1998; Adapa et al., 2010a).

The extent of chemical and structural modifications from steam-explosion pre-treatment depends on residence time, temperature, particle size and moisture content (Sun and Cheng, 2002). However, the severity (Ro) of steam explosion is quantified as a function of retention time and reaction temperature (Equation 1) (Overend and Chornet, 1987; Viola et al. 2008).

r-ioo

Ro = txexpU:d

Where T is the temperature in oC and t is the time in minutes.

According to Zimbardi et al. (1999), the simplest way to carry out steam explosion is by batch procedure, hence widely reported in literature. However, the continuous reactors are of major interest for industrial applications. They have indicated that although the products obtained at the same treatment, severity in batch and continuous reactors are macroscopically different at first sight, there is still a lack of understanding to explain these differences. Consequently, they have developed experimental relationships between the two systems useful in making the data transfer straightforward (Equation 2).

log(Ro)Batch = 1.50 x (log(Ro)

Continuous 1) (2)

In addition, studies have been carried out to try to improve the results of steam explosion by addition of chemicals such as acid or alkali (Cara et al., 2008; Harmsen et al., 2010; Stenberg et al., 1998; Zimbardi et al., 2007). Limitations of steam explosion include the formation of degradation products that may inhibit downstream processes (Garcia-Aparicio et al., 2006).

PRE-STEAMING BIN

— ▼ STEAM " 4 INJECTION

FEEDING SCREW

Г DIRECTIONAL * VALVE

WAX INJECTION

REFINER

160 kW VARIABLE SPEED MOTOR

MDF/HDF

Fig. 6. The Andritz (ANDRITZ AG, Graz, Austria) continuous biomass steam explosion facility for manufacturing of Medium Density and High Density Fiberboards (MDF/HDF), Forintek pilot plant at the FPInnovations, Quebec City, Quebec (Adapa et al., 2010a).

The development of efficient enzymatic electrodes to oxidize glucose is of relevance for the development of implantable glucose/O2 biofuel cells. Nowadays, enzymes classically used to perform efficient oxidation of glucose to gluconic acid are either glucose oxidase (GOD) or glucose dehydrogenase (GDH). In the next part the properties of these two enzymes will be explained in details.

1.1.1 Glucose oxidation catalyzed by glucose dehydrogenase

Contrary to GOD, GDH is non-sensitive towards oxygen (Zhang et al., 2007). This is an attractive property for its use in glucose oxygen biofuel cells. However, GDH is an NAD — dependant enzyme. It is well-known that the oxidation of glucose catalyzed by GDH is rather limited by oxidation kinetics of NADH into NAD+. Even if the use of modified electrodes allows to reduce the overpotential associated to the oxidation of NADH into NAD+ (Delecouls-Servat et al., 2001), the stability of the electrodes remains poor. Another solution based on the use of a pyrroloquinoline quinone (PQQ) cofactor proposes to suppress the NAD dependence. Nevertheless, the PQQ cofactor has a limited stability (Wang, 2007).

1.1.2 Glucose oxidation catalyzed by glucose oxidase

1.1.2.1 Properties of the enzyme

GOD is by far the most used anode catalyst in glucose/O2 biofuel cells. Its molecular weight (155 kDa) and molecular size (60 A x 52 A x 77 A) are high (Alvarez-Icaza et al., 1995). This constitutes a limitation for current densities obtained with a solid electrode since the footprint of the enzyme is great. This enzyme possesses two identical linked FAD (Flavine Adenine Dinucleotide) subunits which are responsible for |3-D-glucose oxidation (Zhu et al., 2006) to gluconic acid (two electrons reaction product). The redox potential of FAD-FADH2 cofactor is ca. -0.36 V vs. Ag/AgCl/KCl(sat.) at a pH value of 7.2 (Stankovich et al., 1978), which is of particular interest for biofuel cells applications since it allows low-potential glucose oxidation. In this reaction, dioxygen is the natural electrons acceptor. Therefore, during the oxidation process, dioxygen is reduced towards hydrogen peroxide. The formation of H2O2 leads to inhibition of the enzyme because it modifies the amino groups in the vicinity of the active center (Kleppe, 1966). The pH value and the temperature have also
an effect on GOD performances. Temperatures higher than 40 °C lead to a drastic decrease of activity (Kenausis et al., 1997). The pH value which optimizes GOD activity greatly depends on the electron acceptor. This value is equal to 5.5 and 7.5 when oxygen (Kenausis et al., 1997) methylene blue (Wilson & Turner, 1992) are used, respectively.

1.1.2.2 Performances of GOD electrodes towards P-D-glucose oxidation

In the case of MET, the use of suitable electrochemical mediators is of importance to increase the rate of electron transfer between the enzyme and the electrode surface since it allows to raise current densities. The second interest lies in the possibility to inhibit the formation of peroxide. Actually, it is just necessary to use a mediator which is able to realize faster electron transfer with GOD than oxygen can do. One of the most efficient systems has been developed by Heller’s group (Mano et al., 2005; Mao et al., 2003). It consists of a tridimensional matrix of an osmium based redox polymer containing GOD. The formal potential of the polymer is -195 mV vs. Ag/AgCl at pH 7.2. The covalent chain composed of thirteen atoms long allows the increase of the electron diffusion coefficient (Mao et al., 2003) by increasing the collision probability between reduced and oxidized forms of the osmium centers. The reticulation with PEGDGE (polyethyleneglycoldiglycydilether) allows the formation of a redox hydrogel capable of swelling in contact with water. It is probable that the matrix structure is responsible for a weak deformation of the protein structure. Such electrodes are able to deliver a catalytic current at potentials as low as -360 mV vs. Ag/AgCl in a physiologic medium containing 15 mM glucose (Mano et al., 2004).

A. Zebda1, C. Innocent1, L. Renaud2, M. Cretin1, F. Pichot3, R. Ferrigno2 and S. Tingry1

1Institut Europeen des Membranes 2Institut des Nanotechnologies de Lyon 3Centrale de Technologies en Micro et Nanoelectronique, Universite de Montpellier 2

France

1. Introduction

Enzymatic biofuel cells (BFCs) employ enzymes to catalyse chemical reactions, thereby replacing traditional electrocatalysts present in conventional fuel cells. These systems generate electricity under mild conditions through the oxidation of renewable energy sources (Calabrese Barton et al., 2004). At the anode side the fuel is oxidized and the electrons, which are released by the oxidation reaction, are used to reduce the oxidant at the cathode side (Fig. 1).

Efficient connection is achieved by the use of appropriate redox mediators that are typically dyes or organometallic complexes, responsible for transferring the electrons from the enzymes to the electrode surface. The advantages of biocatalysts are reactant selectivity, activity in physiological conditions at room temperature, and manufacturability, compared to precious metal catalysts. Abundant organic raw materials such as sugars, low aliphatic alcohols, and organic acids can be used as substrates for the oxidation process, and mainly molecular dissolved oxygen acts as the substrate being reduced. The concept of biochemical fuel cell appeared in 1964 with the works of Yahiro and co-workers (Yahiro et al., 1964), which described the construction of a methanol/O2 cell. In the nineties, BFCs have come in to prominence with the recent advancements in novel electrode chemistries developed by Katz and Willner (Wilson, 2002), and Heller (Degani et al., 1987). The most studied biofuel

cell operates with glucose as fuel and oxygen as oxidant (Service, 2002). At the anode, glucose is oxidized to gluconolactone by the enzymes glucose oxidase (GOx) or glucose dehydrogenase, and at the cathode, dioxygen is reduced to water by the enzymes laccase or Bilirubin oxidase (BOD), which are multicopper oxidases.

Typical enzymatic fuel cells demonstrate powers in the range of microwatt to milliwatt. However, the tests are often performed under quite different conditions (concentration, temperature, pH, mass transport conditions, etc.), which complicates the comparison between different configurations in literature. Compared to conventional fuel cells, BFCs show relatively low power densities and short lifetime related to enzyme stability and electron transfer rate (Bullen et al., 2006). The improvement of the performance requires optimization of the components and solutions are described in literature in terms of catalysts, enzymatic electrode assemblies and design (Davis et al., 2007; Ivanov et al., 2010). Nowadays, the explosive growth of portable, wireless consumer electronics and biomedical devices has boosted the development of new micro power sources able to supply power over long periods of time. Miniature BFCs are considered as promising alternative (Gellett et al., 2010) to power supply in wireless sensor networks (WSN). However, the miniaturization of these devices imposes significant technical challenges based on fabrication techniques, cost, design of the device, and nature of the materials. These devices must provide similar performances to larger biofuel cells in terms of efficiency and power density while using less reagents, space and time consumption.

The number of miniaturized biofuel cells mentioned in literature is mainly restricted to a few devices. These devices include conventional devices that have been miniaturized, as well as micro-devices that use completely novel methods of energy conversion. Therefore, the present chapter presents the recent advances in the miniaturization of BFCs. Miniature conventional BFCs will be first presented but, here, we will focus the discussion on the development of microfluidic enzymatic BFCs, where microfluidics plays a direct and essential role. These micro-devices operate within the framework of a microfluidic chip. They exploit the laminar flow of fluids that limits the convective mixing of fuel and oxidant within a microchannel, eliminating the need for a membrane. As a result, the reaction kinetics can be optimized for both the cathode and anode independently by adjusting the composition of the fuel and oxidant stream. A discussion on the parameters affecting the performances of the microfluidic BFCs is proposed and directed towards interesting theoretical and experimental works. Finally, issues that need to be considered are presented to improve microfluidic device performances for desirable solution in the energy conversion process.

The development and increasing use of diesel and bioethanol mixtures in diesel engines has been driven mainly by the European countries needing to comply with the European Union directive 2003/30/CE (which establishes that at least 5.75% of the fuels market must consist of biofuels by the year 2010), as well as the need to dispose of a petrol surplus in the refineries due to the greater demand for diesel vehicles. The drawbacks of the so-called e — diesel mainly concern a reduced viscosity and lubrication issues, a lower cetane number and injection capacity, a greater volatility (which can lead to an increase in the emissions of uncombusted hydrocarbons) and a lesser miscibility (Marek & Evanoff, 2001; Hansen et al., 2005; Lapuerta et al., 2007). In particular, Lapuerta et al. studied different diesel-bioethanol mixtures in different conditions of temperature, water content and additives, developing level maps that give a precise idea of the mixtures’ areas of stability and of kinetic separation, that prompt the following conclusions:

• the presence of water in the mixture facilitates the separation of the ethanol phase;

• when its temperature increases, the mixture becomes more stable and the solubility of the ethanol in the diesel also increases;

• the mixture’s sensitivity to the effects of water content and additives is higher, the higher the temperature of the mixture;

• mixtures with a bioethanol content up to 10% (v/ v) can be used in diesel engines in regions where temperatures in winter rarely drop below -5°C;

• using stability-improving additives can increase the range of ethanol proportions in the mixtures, or the geographical extension of their applicability, enabling any phase separation to be avoided.

1.1 Research projects and bioethanol promotion

To succeed in demonstrating the feasibility of replacing petrol and diesel oil with bioethanol, the European Union developed the BEST project (BioEthanol for Sustainable Transportation) (European Union, 2011), involving six European countries (Sweden, the Netherlands, the United Kingdom, Ireland, Spain and Italy), and also Brazil and China: the global aims of the project are to introduce at least 10,500 FFV and 160 bioethanol-fueled buses, as well as to build 148 service stations, 135 to provide E85 and 13 to provide E95.

The NILE project (New Improvements for Lignocellulosic Ethanol) (Eurec, 2011) focuses instead on proposing the best processes for an economically effective production of bioethanol from lignocellulose biomass, suitable for use in internal combustion engines. The main goal of the NILE project is to reduce the cost of producing bioethanol from this type of raw material so as to make the technology commercially competitive. The NILE project brings together 21 industrial and research organizations from 11 member states, with complementary professional backgrounds and expertise so as to cover the whole cycle of bioethanol production and usage. On a technical level, the problems that remain to be solved concern reducing the cost of the enzymatic hydrolysis process by developing new artificial enzymatic systems, eliminating the current drawbacks intrinsic in converting fermentable sugars into ethanol, and validating the artificial enzymatic systems and yeast strains in a fully-integrated pilot plant.

Finally, there is the European LAMNET research program (Latin America Thematic Network on Bioenergy) (LAMNET, 2011), the main aim of which is to establish a trans­national forum to promote the sustainable use of biomass in Latin America and other emerging countries.

The first micro-sized enzymatic biofuel cells reported in 2001 (Chen et al., 2001). A glucose/O2 biofuel cell consisted of two 7 pm diameter, 2 cm long electrocatalyst-coated carbon fibers operating at ambient temperature in a pH 5 aqueous solution. The areas of the anode and the cathode of the cell were about 60 times smaller than those of the smallest reported fuel cell and 180 times smaller than those of the previously reported smallest biofuel cell. The power density of the cell is 64 pW/cm2 at 23 °C and 137 pW/cm2 at 37 °C, and its power output is 280 nW at 23 °C and 600 nW at 37 °C. The results revealed that the miniature enzymatic biofuel cells could generate sufficient power for small power­consuming CMOS circuit. Later, a miniature enzymatic biofuel cell with the same carbon fibers operating in a physiological buffer was reported (Mano et al., 2002). In a week operation the cell generated 0.9 J of electrical energy while passing 1.7 C charge. Based on this result, Mano developed a miniature compartment-less glucose/O2 biofuel cell operating in a living plant. Implantation of the fibers in the grape leads to an operating biofuel cell producing 2.4 pW at 0.52 V, which is adequate for operation of low-voltage CMOS/SIMOX integrated circuits. The performance of the miniature enzymatic biofuel cell was upgraded to 0.78 V operating at 37 °C in ph 5 buffer later on (Mano et al., 2003). In 2004, a miniature single-compartment glucose/O2 biofuel cell made with the novel cathode operated optimally at 0.88 V, the highest operating voltage for a compartmentless miniature fuel cell (Soukharev et al., 2004). The enzyme was formed by "wiring" laccase to carbon through an electron conducting redox hydrogel, its redox functions tethered through long and flexible spacers to its cross-linked and hydrated polymer, which led to the apparently increased electron diffusion coefficient. The latest report on miniature glucose/O2 biofuel cells demonstrated a new kind of carbon fiber microelectrodes modified with single-wall carbon nanotubes (CNTs) (Li et al., 2008). The power density of this assembled miniature compartment-less glucose/O2 BFC reaches 58l Wcm-1 at 0.40 V. When the cell operated continuously with an external loading of 1 M resistance, it lost 25% of its initial power in the first 24 h and the power output dropped by 50% after a 48 h continuous work. Although from the practical application point of view, the performance and the stability of the current emzymatic biofuel cells remain to be improved, the miniature feature and the compartment­less property as well as the tissue-implantable biocapability of enzymatic biofuel cell essentially enable the future studies on in vivo evaluation of the cell performance and stability in real implantable systems.

In an effort to miniaturize the EBFCs, we have developed a versatile technique based on C — MEMS process for the miniaturization of electrodes. Our research focuses on the fabrication of 3D microelectrodes for miniature enzymatic biofuel cells. First, the functionalization methods for EBFCs enzyme immobilization were studied. Then we apply finite element approach to simulate the miniature EBFCs to attain the design rule such as electrode aspect ratio, configuration as well as orientation of the chip. Building an EBFC based on the design rule we obtained is on-going.

The butanol production through acetone-butanol-ethanol (ABE) fermentation is an unique feature of some species of the genus Clostridium; the most famous of them are strains of

C. acetobutylicum, C. beijerinckii and C. saccharoperbutylacetonicum but others with the same ability exist, too. Together with all Clostridium bacteria, solvent producers share some common characteristics like rod-shaped morphology, anaerobic metabolism, formation of heat resistant endospores, incapability of reduction of sulphate as a final electron acceptor and G+ type of bacterial cell wall (Rainey et al., 2009).

ABE fermentation consists of two distinct phases, acidogenesis and solventogenesis. While the first one is coupled with growth of cells and production of butyric and acetic acids as main products the second one, started by medium acidification, can be characterized by initiation of sporulation and metabolic switch when usually part of formed acids together with sugar carbon source are metabolized to 1-butanol and acetone. The biphasic character of ABE fermentation coupled with alternation of symmetric and asymmetric cell division, first mentioned by Clarke et al., (1988), is shown in Fig. 1. In the batch cultivation, first acidogenic phase is connected with internal energy generation and accumulation and also cells growth while second solventogenic phase is bound with energy consumption and sporulation. The tight connection of sporulation and solvents production was proved by finding a gene spoOA responsible for both sporulation and solvent production initiation (Ravagnani et al., 2000).

Metabolic pathway leading to solvents production and originating in Embden-Mayerhof — Parnas (EMP) glycolysis is shown in Fig.1, too. Pentoses unlike hexoses are converted to fructose-6-phosphate and glyceraldehyde-3-phosphate prior to their entrance to EMP metabolic pathway. Major products of the acidogenic phase — acetate, butyrate, CO2 and H2 are usually accompanied by small amounts of acetoin and lactate (not shown in Fig.1). The onset of solvents production is stimulated by accumulation of acids in cultivation medium together with pH drop. Butanol and acetone are formed partially from sugar source and partially by reutilization of the formed acids; and simultaneously a hydrogen production is reduced to a half in comparison with the acidogenic phase (Jones & Woods, 1986; Lipovsky et al., 2009). Functioning of all enzymes involved in the butanol formation has been reviewed, recently (Gheslaghi et al., 2009). Unfortunately, butanol is highly toxic to the clostridia and its stress effect causes complex response of the bacteria in which more than 200 genes regulating membrane composition, cell transport, sugar metabolism, ATP formation and other functionalities are involved and complicate any effort to increase butanol resistance (Tomas et al., 2004).

Solventogenic clostridia are known for their capabilities to utilize various mono-, di-, oligo — and polysaccharides like glucose, fructose, xylose, arabinose, lactose, saccharose, starch, pectin, inulin and others but usually the specific strain is not able to utilize efficiently all of named substrates. Although all genes of cellulosome were identified in C. acetobutylicum ATCC 824 genome, the whole cellulosome is not functional what results in incapability of cellulose utilization (Lopez-Contreras et al., 2004). At first, starchy substrates like corn and potatoes were used for ABE fermentation but later blackstrap molasses became the preferential feedstock. Nowadays, a lot of researchers aim to use lignocellulosic hydrolyzates which, if available at a reasonable price and quality (no inhibitors), would be ideal feedstock for this process because clostridia can utilize diluted solutions of various hexoses, pentoses, disaccharides and oligosacharides efficiently.

A population of D. salina CCAP 19/18 cells was harvested from a 250-ml culture in its exponential growth phase (approximately 1 x 106 cells ml-1), washed repeatedly with pre­electroporation buffer (0.2 M mannitol, 0.2 M sorbitol) to remove residual salts, and resuspended in electroporation buffer (0.08 M KCl, 0.005 M CaCl2, 0.01 M HEPES, 0.2 M mannitol, 0.2 M sorbitol) to achieve a final density of 8 x 107 cells ml-1 (Sun et al., 2005). Next, 500 pl electroporation samples were prepared in 0.4 cm electrode gap cuvettes (Bio-Rad), which contained either 4 x 107 or 1 x 106 cells ml-1, 20 mg ml-1 of pSP124, and 2 mg ml-1 of herring or salmon sperm DNA (Sigma). After a 10 minute incubation on ice, electroporation was executed using the Gene Pulser® II with Capacitance Extender Plus (Bio-Rad). Two electroporation conditions were tested, which correspond to the following respective parameters: [1] capacitance of 500 pF and voltage of 400 V (1 kV cm-1) and [2] capacitance of 25 pF and voltage of 1.6 kV (4 kV cm-1); the resistance of each sample was 50 0. Following the electric pulse, the cells were immediately supplemented with DM and allowed to recover in the dark for 12 hours at room temperature. For subsequent selection of potential transformants, the cells were plated on 1.5% agar DM plates containing 4 mg bleocin L-1 and monitored for a one week period.

Microparticle Bombardment

Spherical gold particles of less than 10 pm in diameter (Aldrich) were prepared by repeatedly washing with and resuspending in sterile dH2O to achieve a concentration of 50 mg ml-1. For twenty shots from the microparticle gun, approximately 20 pg of DNA was ethanol-precipitated onto 12.5 mg of gold particles (250 pl) for one hour at -80° C. After briefly spinning the gold solution at 14,000 RPM, the pellet was washed with 70% ethanol, spun again, and finally resuspended in 78% ethanol and kept on ice for use with the transformation gun.

Just before transformation, a 1-L D. salina CCAP 19/18 culture in exponential phase (approximately 1 x 106 cells ml-1) was collected by centrifugation (Sorvall® RC-5B Refrigerated Superspeed Centrifuge, Du Pont Instruments) at 5,000 RPM for 10 minutes at 25° C (± 5) and resuspended in 5 ml of fresh DM. Cell bombardment and recovery were as described previously for transformation of Volvox (Schiedlmeier et al., 1994). Each filter paper was then submerged in 25 ml of DM and allowed to recover without antibiotic selective pressure for two days using the culture conditions mentioned previously, with the exception of aeration, and transformed colonies were subsequently selected for using the minimum inhibitory concentration (M. I.C.) of the appropriate agent on solid medium.

3.2 Results