It is of great interest to develop new non-damaging immobilization techniques of enzyme for the development of stable biofuel cells. In fact, it is very difficult to propose a technique that does not affect the stability of biomolecules. Enzymes are proteins which possess tridimensional structures in which active centers are insulated. To keep the stability of the molecule and to preserve its catalytic efficiency it is necessary not to modify this tridimensional structure and particularly not to affect the environment of the active center. Different combining techniques can be used to immobilize enzymes onto the surface of solid electrodes:

— immobilization into a polymer network

— adsorption on an electrode material

— covalent grafting to an electrode

— immobilization within a membrane.

The first technique consists in immobilizing enzyme in an electropolymerized thin film. It is a very simple technique since it only needs to dip the electrode into a solution containing both monomers and biomolecules. Then the growth of polymer film can be realized by different ways: chronoamperometry (Brunel et al., 2007), chronopotentiometry or cyclic voltammetry (Fei et al., 2007). Different monomers such as pyrrole (Habrioux et al., 2008), aniline (Timur et al., 2004) or phenol (Bartlett et al., 1992) can be electropolymerized. This kind of films can be either conductive or not. The main advantage in using tridimensional conductive films lies in their ability to transfer electrons. Moreover, to increase the number of enzymatic molecules immobilized close to the electrode surface, a first adsorption step of enzymatic molecules can be performed (Merle et al., 2009). Other non-electropolymerized films can be used for enzyme holding. Currently, both chitosan and Nafion® are commonly used (Habrioux et al., 2010; Klotzbach et al., 2008). These two polymers possess surfactant properties interesting to immobilize enzymes in micellar structures (Moore et al., 2004). Moreover, the hydrophobic/hydrophilic property of the polymers can be tuned by modifying the chemical structure of these molecules (Klotzbach et al., 2008; Thomas et al., 2003). It is also possible to simply use retention properties of the Nafion® film for buffering its sulfonic groups (Habrioux et al., 2010). The main problem associated with the use of these polymers lies in the non-control of the film thickness. One of the most promising immobilization techniques has been proposed by Heller’s group. This approach consists in immobilizing enzymes in an osmium-based redox polymer (Mao et al., 2003) which is able to swell in contact with water. It acts both as an immobilizing network and an electrochemical mediator. The whole structure of the film leads to very fast electron transfer between the active centers of enzymes and the electrode surface. Another smart technique consists in covalent grafting of enzyme to the electrode surface. Thus Merle et al. (Merle et al., 2008) realized the grafting of amino groups on a carbon electrode before coupling these functions with amino-groups of enzymes using glutaraldehyde. This seems to confer a remarkable stability to the resulting electrode. Another well-known approach has been proposed by Willner et al. (Willner et al., 1996) that consisted in the reconstitution of the enzyme after the grafting of its active center on a gold electrode.

Ethanol: In compliance with the provisions of Act 693/01, the country began to implement initiatives for alcohol fuel from sugar cane. At the moment 5 ethanol plants are running: Incauca, Providencia, Manuelita, Mayaguez and Risaralda refineries that produce about 1,050,000 liters of alcohol fuel a day and this production is mainly to supply the domestic market. It is estimated a domestic demand close to 1,500,000 liters per day to cover the 10% of blending needs.

Likewise, in the country several alcohol production projects are being implemented in several departments: Antioquia, Boyaca, Santander and the coast, derived from different raw materials such as sugar cane, sugar beet, banana and cassava.

Unfortunately, due to the economic crisis there is absence of new plants. Projects are standstill, and Ecopetrol plant would only come into operation in 2011, starting with a production of 385,000 liters a day. At the moment there is another project being developed in Magdalena, where an international company sowed a very large sugar cane area for producing an average of 300,000 liters a day. With this, the 20% blending could be reached by in 2012 without any problem.

Biodiesel: At the moment there are five projects under construction for producing biodiesel from oil palm (Oleoflores — already in production, Odin Energy, Biocombustibles Sostenibles del Caribe) and two in the eastern region (Biocastilla, Bio D. SA). In addition, they are other projects under development, one in the central region (ECOPETROL), one in the eastern region (Manuelita), one in the west region and another in the north region. In 2008 it is expected they shall enter into production, with a total amount of 400,000 t/year (19).

How are investments for biodiesel production doing? Construction of the Ecopetrol plant in Barrancabermeja is almost over. With this in total there will be seven plants in the country. A total installed capacity of 526,000 biodiesel tonnes a year may be achieved.

2. Conclusions

It must be accepted that the so-called modern man now has the same challenge our ancestors solved centuries ago, that life is not over. Availability of natural resources and the way we use them, force us to shape a scenario of technological innovations and social coexistence, in which the ethics of life prevails over money; this becomes more valid in this global world that requires new economic, lifestyle, consumption and value models.

Society needs energy for its development, but development does not necessarily imply a waste of energy. In any productive process, materials and water may or may not be wasted, but it is certain that it will consume energy and that energy consumption will be associated with a real environmental impact. If energy production takes on all costs, it would be much more expensive.

New energy sources are the new economic, political and even environmental strategy. Their importance is such that currently over 30 raw materials are being tested worldwide. Despite this big boost, they do not yet provide a solution to the global energy problems.

Biofuels should not be taken as the solution to the energy and environmental problem, but as part of a complex human and energy project where leading countries still disagree on a solution. If Bioenergy is properly used, it provides a historic opportunity to contribute to the growth of many of the world’s poorest countries.

A reality must be emphasized; alcohol fuel is more expensive than gasoline and biodiesel. It is not good business that a market economy develops isolated and organically; the market must be intervened so these alternatives are viable, because rival fuel is cheaper. Oil is in the reservoir, while cassava, sugar cane, oil palm or other crops used as raw material must be planted, and in expensive lands. Then, by definition, we talk about a project that is viable only if the State intervenes so it can be operated outside the framework of the market.

The world faces complex challenges and life’s survival on the planet can not be supported on the solution to the renewable energy alternative based on biofuels, as it would grow the replacement of food crops with monocultures, deforestation for energy crops, while it would boost the diversity extinction, fertile lands and water reduction, and the social consequences population displacement causes.

In that sense the FAO has declared: Biofuel policies and subsidies should be urgently reviewed in order to preserve the goal of world food security, protect poor farmers, promote broad-based rural development and ensure environmental sustainability. But also states: Growing demand for biofuels and the resulting higher agricultural commodity prices offer important opportunities for some developing countries. Agriculture could become the growth engine for hunger reduction and poverty alleviation, production of biofuel feedstocks may create income and employment, if particularly poor small farmers receive support to expand their production and gain access to markets.

It also requires a certification system that ensures that biofuels will be marketed only if they have the necessary environmental requirements.

Colombia is not and cannot be indifferent to the global market trend for crude oil and its derivatives. This fact gives the opportunity for goods production, such as biofuels, that allow diversity in the energy basket available in the domestic market and that can be exported to international markets. However, a necessary condition for competing in the international market is efficient conditions for the production of these goods.

Colombia has enough available land for growing biofuels, from 14 million hectares for agriculture business and 20 for livestock, only 5 million are currently in use and the remaining is for extensive cattle ranching; a better use could be biofuels which would provide plant cover and rural income opportunities. It also holds high productivity in sugar production from sugar cane, but such activity has been focused on agribusiness models, where production is held in few companies from renowned economic groups.

Although in Colombia ethanol has been a biofuel pioneer, biodiesel projects are gaining strength and this fuel can have a greater impact and national coverage.

In the country there is a poor use of natural resources and a high dependence on them; there is not full agreement between vocation or fair and the use of resources. Productivity paradigm boosts to predatory models and the economic efficiency and profitability fallacy as sole indicator, productive projects that do not consider social and environmental benefits are presented.

Then, in the previous horizon, it is required to develop a long-term sustainable agriculture that is compatible with the environment. The aim of this is a critical reassessment of the current modernizing model, taking into account that different technological offers, articulated to a diverse set of socio-economic and environmental factors, require different technological solutions. Consequently, decisions about biofuels should consider the food safety situation but also land and water availability.

Energy has deep and broad relations with the three sustainability dimensions (economic, social and environmental); i. e., it must go into the integration, harmonization and optimization. The services energy provides help to meet several basic needs such as: water supply, lighting, health, ability for producing, transporting and processing food, mobility and information access so that access to a certain amount of advanced forms of energy such as electricity or liquid fuels and gaseous fuels, should be included among the inalienable human rights in the XXIst century. Energy supply safety and energy prices are crucial for economic development. On the other hand, it is clear that many ways of producing and consuming can reduce environmental sustainability. We must ask: is the current energy production and consumption sustainable? One of the most important challenges humanity faces is to find the way to produce and use energy so that in the long term human development is promoted, in all its dimensions: social, economic and environmental.

Finally, to balance the enthusiasm with objectivity: it is necessary to carefully study the economic, social and environmental bioenergy impact before deciding how fast it is desired to be developed, and what technologies, policies, investment and research strategies to follow.

Ethyl tertiary butyl ether (ETBE) is a high-octane bioethanol product obtained mainly by making the ethanol react with isobutylene (a byproduct of oil refining) under the effect of heat and various catalysts. It is consequently considered as being partially renewable.

ETBE has technological and functional features that are very like and distinctly better than those of the alcohol it is obtained from. Moreover, it lacks the latter’s problems of volatility or miscibility with petrol and it features a high octane number.

Being an ether, it contains oxygen in the molecule, and this enables it to help improve the vehicle’s emissions of pollutants. A recent paper (Da Silva et al., 2005) conducted a study on the effects of the anti-detonating properties and Reid vapor pressure (RVP) of petrols mixed with various additives, concluding that adding ETBE improves the mixture’s anti­detonating properties and reduces the vapor pressure without interfering with the volatility needed to start a cold engine.

ETBE obtained from bioethanol (also called bioETBE) offers the same benefits as bioethanol, i. e. a lower emission of pollutants, a higher octane number and a reduction in crude oil imports, without the technical and logistic problems posed by the alcoholic nature of bioethanol. BioETBE also contributes to the diffusion of biofuels in the transport sector.

One of the early efforts on miniature microbial biofuel cells reported a surface power output of 0.023 mW / m2 and current density of 150 mA/ m2 based on the 10pm diameter circular anodic electrode (Chiao et al., 2006). The miniature microbial biofuel cells were limited by relatively low volumetric power density and coulombic efficiency due to their high internal resistances. Compared with macro scale microbial biofuel cells using the same microbes (Ringeisen et al., 2006), miniature microbial biofuel cells generated similar volumetric current density but significantly lower volumetric power density, which is insufficient for the anticipated applications. It was pointed out that the internal resistances of miniature microbial biofuel cells were around 40 fold higher than that in the macro scale microbial biofuel cells. The ohmic loss was higher in the micropillar devices with the same catholyte and anolyte; however, they generated higher volumetric power density (32 A/ m3) than the serpentine-channel devices (0.5 A/m3) (Ringeisen et al., 2006). The high surface area-to — volume ratio and good microbe adaptivity of the micropillar electrodes decreased the anode resistance and resulted in higher volumetric power output. Carbon based anodes are known for high surface area-to-volume ratio and easy adaptation of microorganism and they are widely used in macro scale microbial biofuel cells. The recent investigations using carbon nanotubes (CNT) as electrodes (Qiao et al., 2007 and Timur et al., 2007) provide promising solutions for constructing carbon-based anodes in miniature microbial biofuel cells. The CNT based electrodes showed great improvement in the electricity generation and biocompatibility. Its maximum power density was 42 mW/m2 using E. coli as the microbial catalyst.

In the pursuit to improve the miniature microbial biofuel cell performance, different strategies were employed such as increasing the anode surface area, improving coupling of microorganism to anode surface, developing electrochemically active microbes and decreasing proton diffusion resistance. In summary, the enhancement strategies resulted in enhanced mass transport, improved reaction kinetics, and reduced ohmic resistance. Based on these developments, the ability to generate sufficient current and power from miniature devices was realized, thus breaking the conventional concept that small scale microbial biofuel cells would perform unsatisfactorily due to limited amount of substrates and microorganism. Since the development of the first miniature microbial biofuel cells in 2006, the volumetric power density and coulombic efficiency have been increased over 5 times. Although the output potential from the miniature MFCs is still insufficient for powering conventional equipment, they are promising options for on-chip power sources, especially for medical implants, which only require several millivolts to operate. Given the evidence that volumetric current density of the miniature MFC was achieved to be 2400 mA/m3 and required power from the cell was therefore 960 mW / m3, which is sufficient for existing devices (Wang & Lu, 2008). However, higher current density can result in excessive ohmic heating and electrolysis during the operation. Therefore, study in optimizing current density, overall output voltage and stability of the miniature MFCs as well as electrode design and device configuration for implantation rejection, microbe leakage, and analysis of the composition and distribution of internal resistances is necessary before further implementation in practical applications.

1.1 History of industrial biobutanol production

The initiation of the industrial acetone-butanol-ethanol (ABE) production by Clostridium fermentation is connected with the chemist Chaim Weizmann, working at the University of Manchester UK, who wished to make synthetic rubber containing butadiene or isoprene units from butanol or isoamyl alcohol and concentrated his effort on the isolation of microbial producers of butanol. Further, the development of acetone-butanol process was accelerated by World War I when acetone produced by ABE fermentation from corn in Dorset, UK was used for cordite production. However in 1916, the German blockade hampered the supply of grain and the production was transferred to Canada and later with the entry of the United States to the war, two distilleries in Terre Haute were adapted to

acetone production. After the war, the group of American businessmen bought Terre Haute plant and restored the production in 1920; at that time butanol was appreciated as solvent for automobile lacquers. Subsequently, with decreasing price of molasses new solventogenic strains were isolated and first plant using this feedstock was built at Bromborough in England near the port, in 1935. In 1936 the Weizmann patent expired and new acetone — butanol plants were erected in U. S.A., Japan, India, Australia and South Africa using usually molasses as the substrate. The Second World War again accelerated the process development and acetone became the most required product; the plant at Bromborough was expanded and semi continuous way of fermentation which cut the fermentation time to 30- 32h was accomplished here together with continuous distillation. At the end of the war, two thirds of butanol in U. S.A. was gained by fermentation but rise of petrochemical industry together with increasing price of molasses that started to be used for cattle feeding caused gradual decline of industrial acetone-butanol fermentation. Most of the plants in Western countries were closed by 1960 with the exception of Germiston factory in South Africa where cheap molasses and coal enabled to keep the process till 1983 (Jones & Woods, 1986). In addition to Western countries, the production of acetone and butanol was also supported in the Soviet Union. Here, in Dukshukino plant, in 1980s, the process was operated as semi continuous in multi-stage arrangement with possibility to combine both saccharidic and starchy substrates together with small portion (up to 10%) of lignocellulosic hydrolyzate and continuous distillation (Zverlov et al., 2006). In China, industrial fermentative acetone and butanol production began around 1960 and in 1980s there was the great expansion of the process. Originally, batch fermentation was changed to semi continuous 4-stage process in which the fermentation cycle was reduced to 20 h, the yield was about 35-37% from starch and the productivity was 2.3 times higher in comparison with batch process (Chiao & Sun, 2007). At the end of 20th century the most of Chinese plants were probably closed (Chiao & Sun, 2007) but now hundred thousands of tons of acetone and butanol per year are produced by fermentation in China (Ni & Sun, 2009).

Industrial production of ABE in the former Czechoslovakia started with a slight delay comparing with other already mentioned countries. Bacterial cultures were isolated, selected and tested for many years by professor J. Dyr, head of the Department of Fermentation Technology of the Institute of Chemical Technology in Prague who lead a small research team and preparatory works for the plant design (Dyr & Protiva, 1958). Acetone — butanol plant was fully in operation from 1952 till 1965. The main raw materials were firstly potatoes which were later changed for rye. Various bacteria cultures (all were classified as Clostridium acetobutylicum) were prepared for several main crops (potatoes, rye, molasses) which increased flexibility of the production. Annual production of solvents increased from year to year but did not exceed 1000 tons. Concentration of total solvents in the broth varied around 17-18 g. LA Process itself was run as batch, pH was never controlled, propagation ratio in large fermentation section was 1 : 35. The whole fermentation time was on average 36-38 h. Critical point for each fermentation was "break" in acidity after which started a strong evolution of gases and solvents. In case of potatoes and rye there were no nutrients supplied to the fermentation broth. The only process necessary for the pre-treatment of the raw materials of starch origin was their steaming under pressure in Henze cooker. Initial concentration of starch ranges from 4.5 to 5% wt. In spite of keeping all sanitary precaution (similarly today’s GMP) two types of unexpected failures occurred. Firstly it was contamination by bacteriophage (not possible to analyze it in those times) which appeared approx. three times during the lifetime and always was followed by a total sanitation and complete change of the producing strain. Secondly there appeared another unexpected event, i. e. a final turn to a complete acidification without initiation of solvent production indicated by a spore creation. This situation appeared in the range from 1 to 4% of the total number of batches.

All plasmids were prepared and isolated from bacterial hosts using the QIAPrep Kit (Qiagen) following the supplier’s protocol. E. coli DH5a cells (Invitrogen) were grown axenically either on LB agar or in LB medium containing pertinent antibiotic selective pressure (ampicillin or kanamycin) at 37° C. Genomic DNA was extracted from D. salina using standard CTAB protocol adapted from Volvox carteri. PCR was performed using the MJ Mini (Bio-Rad) to amplify the actin (GenBank: AF541875) and rubisco small subunit (GenBank: AY960592) promoters and nitA 3′-UTR (GenBank: EF156403) from D. salina UTEX 1644 genomic DNA. Likewise, the genes ble and bar were amplified from the plasmids pSP124 and pGR117, respectively. All primers employed in this study are listed below in Table 2. Each PCR product was subsequently ligated into the subcloning plasmid pGEM®-T Easy (Promega) using T4 DNA Ligase and its corresponding buffer (Invitrogen). Sequencing of the genetic fragments derived from PCR was performed at the UMBC Biological Sciences Dept. DNA sequencing facility using BigDye® (Applied Biosystems).

Didem Ozgimen and Sevil Yucel

Yildiz Technical University, Bioengineering Department, Istanbul

Turkey

1. Introduction

The depletion of fossil fuels and their effects on environmental pollution necessitate the usage of alternative renewable energy sources in recent years. In this context, biodiesel is an important one of the alternative renewable energy sources which has been mostly used nowadays. Biodiesel is a renewable and energy-efficient fuel that is non-toxic, biodegradable in water and has lesser exhaust emissions. It can also reduce greenhouse gas effect and does not contribute to global warming due to lesser emissions. Because it does not contain carcinogens and its sulphur content is also lower than the mineral diesel (Sharma & Singh, 2009; Suppalakpanya et al., 2010). Biodiesel can be used, storaged safely and easily as a fuel besides its environmental benefits. Also it is cheaper than the fossil fuels which affect the environment in a negative way. It requires no engine conversion or fuel system modification to run biodiesel on conventional diesel engines.

Today, biodiesel is commonly produced in many countries of the world such as Malaysia, Germany, USA, France, Italy and also in Australia, Brazil, and Argentina. Biodiesel production of EU in 2009 was presented in Table 1 (European Biodiesel Board, July 2010). As can be seen from Table 1, 9 million tons biodiesel were produced in European Union countries in 2009. Germany and France are the leaders in biodiesel production. EU represents about 65% of worldwide biodiesel output. Biodiesel is also main biofuel produced and marketed in Europe. In 2009, biodiesel represented is about 75% of biofuels produced in Europe.

The world production of biodiesel between 1991 and 2009 was presented in Figure 1. From Figure 1, biodiesel production increased sharply after 2000s in the world.

Firstly in 1900, Rudolph Diesel showed that diesel engines could work with peanut oil. And then, the different kinds of methods such as pyrolysis, catalytic cracking, blending and microemulsification were used to produce biodiesel from vegetable oil for diesel engines (Sharma & Singh, 2009; Varma & Madras, 2007). Finally, transesterification process was developed as the most suitable method to overcome problems due to direct use of oil in diesel engines (Varma & Madras, 2007).

Biodiesel is generally produced from different sources such as plant oils: soybean oil (Kaieda et al., 1999; Samukawa et al., 2000; Silva et al., 2010; Cao et al., 2005; Lee et al., 2009; Yu et al., 2010), cottonseed oil (Kose et al., 2002; He et al., 2007; Royon et al., 2007; Hoda, 2010; Azcan & Danisman, 2007; Rashid et al., 2009), canola oil (Dube et al., 2007; Issariyakul et al., 2008), sunflower oil (Madras et al., 2004), linseed oil (Kaieda et al., 1999), olive oil (Lee et al., 2009), peanut seed oil (Kaya et al., 2009), tobacco oil (Veljkovic et al., 2006), palm oil (Melero et al., 2009), recycled cooking oils (Issariyakul et al., 2008; Rahmanlar, 2010; Zhang et al. 2003; Demirba§, 2009) and animal fats (Da Cunha et al., 2009; Oner & Altun, 2009; Guru et al., 2009; Guru et al., 2010; Tashtoush et al., 2004; Teixeira et al., 2009; Chung et al., 2009).

The major economic factor to consider for input costs of biodiesel production is the feedstock. 90 % of the total cost of the biodiesel production is the resource of the feedstock. Studies to solve this economic problem especially focused on biodiesel production from cheaper raw material. Using agricultural wastes, high acid oils, soapstock, waste frying oil and alg oil as raw materials for biodiesel production are being reported in literature (Haas & Scott, 1996;Ozgul & Turkay, 1993; Ozgul & Turkay, 2002; Leung & Guo, 2006; Yucel et al., 2010; Ozgimen & Yucel, 2010).

Table 1. Biodiesel production of EU in 2009 (EBB 2010)

Transesterification process, as showed in Figure 2 (Barnard et al., 2007) is a conventional and the most common method for biodiesel production. In transesterification reaction homogeneous catalysts (alkali or acid) or heterogeneous catalysts can be used. The catalysts split the oil into glycerin and biodiesel and they could make production easier and faster.

(FAME)

Fig. 2. Biodiesel production via transesterification reaction (Barnard et al., 2007)

In this method, fatty acid alkyl esters are produced by the reaction of triglycerides with an alcohol, especially ethanol or methanol, in the presence of alkali, acid or enzyme catalyst etc. The sodium hydroxide or potassium hydroxide, which is dissolved in alcohol, is generally used as catalyst in transesterification reaction (Dube et al., 2007). The products of the reaction are fatty acid methyl esters (FAMEs), which is the biodiesel, and glycerin (Vicente et al., 2004). Ethanol can be also used as alcohol instead of methanol. If ethanol is used, fatty acid ethyl ester (FAEE) is produced as product (Hanh et al., 2009b). Methyl ester rather than ethyl ester production was preferred, because methyl esters are the predominant product of commerce, and methanol is considerably cheaper than ethanol (Zhou & Boocock, 2003). However, methanol usage has an important disadvantage, it is petroleum based produced. Whereas ethanol can be produced from agricultural renewable resources, thereby attaining total independence from petroleum-based alcohols (Saifuddin & Chua, 2004; Encinar et al. 2007). Ethanol is also preferred mostly in ethanol producing countries. Propanol and butanol have been also used as alcohols in biodiesel production.

Alkali-catalyzed transesterification proceeds much time faster than that catalyzed by an acid and it is the one most used commercially (Dube et al., 2007; Freedman et al., 1984). The most commonly used alkali catalysts are NaOH, CHeONa, and KOH (Vicente et al., 2004). Potassium hydroxide (KOH) and sodium hydroxide (NaOH) flakes are inexpensive, easy to handle in transportation and storage, and are preferred by small producers. Alkyl oxide solutions of sodium methoxide or potassium methoxide in methanol, which are now commercially available, are the preferred catalysts for large continuous-flow production processes (Singh et al., 2006).

For acid-catalyzed systems, sulfuric acid has been the most investigated catalyst, but other acids, such as HCl, BF3, H3PO4, and organic sulfonic acids, have also been used by different researchers (Lotero et al, 2005). But in alkali catalyzed method, glycerides and alcohol must be substantially anhydrous, otherwise it leads to saponification (Helwani et al., 2009). Due to saponification the catalytic efficiency decreases, the separation of glycerol becomes difficult and it also causes gel formation (Helwani et al., 2009). In homogeneous catalyzed reactions, separation of catalyst from the reaction mixture is hard and expensive. With this purpose, large amount of water is used to separate catalyst and product (Vyas et al., 2010). On the other hand, undesired by-product formation such as glycerin can be seen, the reaction lasts very long and energy consumption may be very high. Thus, researchers have focused on development of new biodiesel production methods and the optimization of the processes (Sharma et al., 2008). So, various processes such as supercritical process,
microwave assisted method and ultrasound assisted method have recently developed. Alternative energy stimulants or non-classical energies have been used for many years to increase the reaction rate and to enhance the yield of particular reaction products. Novel methods or combining innovative methods and techniques are a challenge that can lead to unexpected advances in biodiesel production techniques (Nuechter et al., 2000). In this study, biodiesel production in supercritical conditions, in microwave and ultrasound techniques as novel methods through the years (2000-2011) was reviewed and presented in detail.

The moisture in biomass both acts as a facilitator of natural binding agents and a lubricant (Kaliyan and Morey, 2006a). Many studies have indicated that the production of high quality pellets is possible only if the moisture content of the feed is between 8 and 12% (wb). Moisture contents above or below this range would lead to lower quality pellets (Hill and Pulkinen, 1988; Kashaninejad et al., 2011; Li and Liu, 2000; Obernberger and Thek, 2004; Shaw and Tabil, 2007). In general, an increase in moisture content from 10 to 44% could result in up to 30-40% decrease in pellet densities of biomass (Chancellor, 1962; Grover and Mishra, 1996; Gustafson and Kjelgaard, 1963; Kaliyan and Morey, 2006a; Mani et al., 2002 and 2006b; Smith et al., 1977). However, the percentage decrease in density depends on the type of biomass. Therefore, a moisture content of 10% (w. b.) is considered as optimal moisture content to obtain high density and durability pellets.

Gas Chromatography/ Mass Spectrometry (GC/MS) is a very popular technique used to separate and identify each organic compound from mixtures. It provides both qualitative and quantitative information about the sample. Gas Chromatography (GC) separates the molecules based on their molecular weight and their volatility. Gas chromatography uses a conventional oven with a column of very small internal diameter. The temperature of the oven is changed and based on the volatility of the compounds they travel through the column at different rates. Hence they separate and reach the mass spectrometer at different times. The time a compound takes to reach the mass spectrometer from the time it is injected into the column is called retention time. The retention time of a compound depends on its molecular weight and structure (thus its volatility). The retention time of the compounds is dependent on the initial temperature and temperature ramp rate of the GC. The compounds injected into the GC reach the MS at different times based on their volatility and complex mixtures are thus separated, but this separation is not enough to understand their composition. Two compounds of different molecular weight and structure may reach the MS at the same time if they have similar volatility. A mass spectrometer is used to identify these compounds eluting from the GC column. The mass spectrometer charges the compounds, accelerates them under high magnetic fields and then breaks them into ions. Based on their mass and charge, the ions hit the detector at various positions and are thus detected. Unlike it is the case for TGA and DSC mass spectrometry analysis does not require a a thorough understanding of the mixture composition of the mixture is not necessary before analysis. However confirming the identity of the predicted compound, after the analysis of the unknown sample, with a known standard is necessary. The mass spectrometer is usually run in scan mode where it detects all the ions that hit the detector. However, scans of samples containing known compounds can be made very sensitive by forcing the mass spectrometer to detect the ions specific to the compounds of interest (selective ion mode). Based on the fragmented ions and the retention time, the chemical structure of the compound can be determined.

The mass spectrometer is very sensitive and detects even a small concentration of impurities. Hence the sample needs to be extremely pure and free of any oxygen or moisture. This implies the need for tedious sample preparation and purification step before the introduction of the sample into the instrument. It is also important to maintain the instrument itself and the column used in the GC. There is good chance of introduction of moisture and column degradation products into the sample without proper care. The quadruple of the MS itself needs to be cleaned and tested for its sensitivity frequently.

In liquid GC/MS the samples are usually extracted into volatile organic solvents and are then injected into the GC. The compounds thus detected are biased towards non-polar organic molecules of low molecular weight. However, it is also possible to detect polar organic molecules like organic acids after derivatization thus rendering them non polar. For quantitative analysis one injects a know quantity of a compound called internal standard (IS). An internal standard is a compound, which is similar in all respects to the compounds of interest but can be separated from the original compounds in the column. Deuterated counterparts of the original compounds are usually used as internal standards for the GC/MS analysis. Internal standards are injected into the original solid or liquid prior to extraction. The compounds of interest and the original solids are then extracted into a volatile organic solvent through soxhlet extraction or liquid-liquid extraction. Specific ASTM (American Society for Testing Materials) guidelines exist for the extraction procedures for quantitative analysis of various volatile organic pollutants. A standard mixture of known quantities of the internal standard and the compound of interest is made in similar solvent. This standard mixture is then analyzed in GC/MS using the same protocol used for the sample and the standard.

Differences in efficiency of the extraction of both the compounds and the internal standard can lead to erroneous results such as incorrect concentration predictions. Only samples which contain extractible substances of lower molecular weight can be analyzed using this technique.

These shortcomings can be overcome by using pyrolysis GC-MS. In this technique, the sample is pyrolyzed at high temperatures for very short amount of time and the products thus produced are directly injected into GC/MS. This technique is usually used to find the structures of heteropolymers (polystyrene and PVC blends, lignin, etc.). The primary advantage of this technique is that it requires a small sample volume and no sample preparation, but due to measurement errors of such very small volumes pyrolysis GC-MS is difficult to quantify and inaccurate. Hence the ratio of breakdown products is often a better measurement estimate to describe the original sample.

Both GC/MS and pyrolysis GC/MS are used extensively in biofuel research. Lignin as a heteropolymer present in the biomass is often analyzed for its chemical composition using pyrolysis GC/MS (Galletti et al. 1997; Mills et al. 2009). Lignin typically contains three major constituents called p-hydroxyphenyl lignin (H-lignin), guaiacyl lignin (G-lignin) and syringyl-lignin (S-lignin). The structure and strength of lignin is dependent on the ratio of these three kinds of lignin (Boudet et al. 1998). Lignin rich in G-lignin is supposed to be highly condensed when compared to lignin rich in S — lignin (Chiang and Funaoka 1990). Ralph and Hatfield (1991) were one of the first ones to analyze the composition of lignin using pyrolysis GC/MS. They show that the pyrolysis products of lignin can be a variety of products derived from H, G and S-lignin. They suggest using a ratio of the total amount of H to the total amount of G to the total amount of S products to find the H:G:S ratio. The S: G ratio of a feedstock has been shown to affect the efficiency of both the pretreatment technology and the enzymatic saccharification. Pyro-GC/MS has been used to find the changes in S:G ratio due to different pretreatments (Samuel et al. 2010; Huyen et al. 2010; Ibarra et al. 2007; Jung et al. 2010; Papatheofanous et al. 1995). The S:G ratio decreases as a result of kraft pulping (Ibarra et al. 2007) and thus hardwoods (S:G ratio >1) have always been a choice for the paper and pulp industry. Ammonia treated Miscanthus showed a decrease in the S:G ratio as result of pretreatment (Huyen et al. 2010). Samuel et al. (2010) have shown that the S:G ratio of switchgrass decreased due to dilute acid pretreatment of biomass, showing it is easier to break S-lignin when compared to G-lignin. Based on this hypothesis genes have been identified which increase the amount S-lignin present in the plant biomass (Marita et al. 1999; Ralph 2007). The mechanism of how lignin is formed and how each gene effects the formation of these individual components of lignin has been found by both NMR and pyro-GC/MS. Pyro-GC/MS is used to differentiate between various plant mutants with different S:G ratios. The changes to S:G ratio as function of harvesting time has also been identified using pyro-GC/MS (Huyen et al. 2010). Feedstocks with similar lignin content have been shown to have different saccharification yields based on their S:G ratio (Davison et al. 2006). Feedstocks with higher amount S-lignin were easier to pretreat but had lower xylose yields when compared to feedstocks with higher G-lignin (Davison et al. 2006). The differences in S:G ratio has also been correlated to in-rumen digestibility of plant biomass (Guo et al. 2001; Baucher et al. 1999; Vailhe et al. 1998). As the S:G ratio decreased the in-rumen digestibility increased, showing that feedstocks such as grass are ideal for animal feed. The lignin degradation products produced during the pretreatment process are also dependent on the S:G ratio of the original lignin polymer (Chiang and Funaoka 1990). S:G ratio, as measured from pyro-GC/MS, offers a platform to correlate its effects on different biofuel production steps and thus helps us design better feedstocks.

Lignin and some of its monomers have been shown to inhibit both cellulases during enzymatic saccharification (Sewalt et al. 1997; Ximenes et al. 2010; Ximenes et al. 2011) and microbes during fuel production (Mills et al. 2009). These lignin degradation products produced during the pretreatment may have an inhibitory role on the downstream processes. These products can be identified and quantified using GC/MS (Pecina et al. 1986). Identifying these lignin degradation products is also important for the biofuel industry as it gives us an idea about the byproducts produced during the pretreatment process itself (Ehara et al. 2005). As pyro-GC/Ms can give a ratio of the amount of G-lignin and S-lignin present in the lignin, derivatization followed by the reductive cleavage (DFRC) method has been developed by Ralph et al. to measure the quantitatively the various constituents of lignin in the biomass (Lu and Ralph 1997; Lu and Ralph 1998; Lu and Ralph 1999; Peng et al.1998; Ralph and Lu 1998). This method uses GC/MS to quantify various derivatized and reduced products. Genetic pathway engineering, also known as synthetic biology, is gaining popularity as it aims to produce microbes which may in turn act as biofuel factories to produce the desired kind of fuel from the glucose units. Qualitative analysis of the composition of the fuel allows the identification of the right modification of the microbes to produce fuel. Quantification of the products is also necessary to understand the most efficient mechanism and the toxicity limit of the fuel until which the microbes can sustain. GC/MS is also used to qualitatively and quantitatively measure the fatty acids and fuels produced from the microbes ((Akiyama et al. 2008; Atsumi et al. 2008; Dai et al. 2007; Lu et al. 2008; Tang et al. 2007).

In general there are numerous combinations of enzymes and mediators that have been employed in biofuel cells but the respective studies typically involve monoenzymatic systems, which are capable of only partial oxidation of the fuel. Improvement of fuel
utilization can be achieved by complete oxidation, which can be realized by introduction of enzyme cascades to increase the overall efficiency of the fuel cell (Nick et al., 2005; Sokic — Lazic et al., 2010; Addo et al., 2010). By this way, the overall performance of the BFC is increased as well.

In a theoretical work described by Kjeang and co-workers, a concept of an enzymatic fuel cell is proofed, without presenting experimental system (Kjeang et al., 2006). This work describes how to optimize the structure of a microfluidic enzymatic fuel cell, involving three-step-catalyzed methanol oxidation (Fig. 9). Different enzyme patterning strategies are tested, e. g., spatially distributed — or evenly-mixed enzymes along the electrode surface. The model predicts high fuel utilization at low flow rates, i. e., in the diffusion dominated and mixed mass, transfer conditions. According to the model, the investigated theoretical concept is reaction limited, which means that the system performance can be improved by improving enzyme turnover numbers. This work also demonstrates that the power required for pumping of the fuel is negligible in comparison to predicted power of the fuel cell.

Based on this work, we built a methanol/O2 microfluidic BFC able to completely oxidize methanol, according to the electron steps described for each electrode in Fig. 10. The enzymes and the mediators were immobilized in poly-L-lysine.

These enzymes are NADH-dependant. The electrochemical connection and regeneration of NADH at the anode is achieved using the enzyme diaphorase and the redox mediator benzylviologen as already described (Palmore et al., 1998). At the anode, a mixture of the three dehydrogenase enzymes was deposited along a gold electrode surface according two configurations (Fig. 11). When enzymes are mixed along the anode, optimal power density is achieved as observed in Fig. 12. The Y-channel device delivers a power density of 70 pW cm-2 at a cell voltage 0.25 V.

(B)

2. Conclusion and perspectives

Microfluidic BFCs could be an effective solution for small power sources applications such as biological sensors, implantable medical devices or portable electronics. However significant research efforts must be made for practical applications. Researches must be
aimed at identifying most robust and active enzymes, more efficient immobilization environment for enzymes and mediators in microfluidic environment, and at increasing enzyme lifetimes.

To deliver higher power densities, the challenges are in the area of energy density and fuel utilization. Microfluidic BFCs designs should integrate advanced immobilization configurations to improve enzyme performance and high surface area electrodes to enhance rates of convective-diffusive reactant transport. More nanofluidic research and development will be needed to demonstrate the real potential of this form of energy conversion system. Besides, the power output of single microfluidic BFC is inadequate for most practical applications. The enlargement of a single cell by increasing the geometrical electrodes area and the microchannel is constrained by fuel/oxidant crossover and higher ohmic losses. In order to produce adequate power, multiple independent cells could be stacked as in typical fuel cells.

3. Acknowledgment

This work was supported by a CNRS postdoctoral fellowship and by the Project PIE CNRS "Energie" 2010-2013. Nanolyon clean room facilities are acknowledged for the fabrication of the microfluidic devices.