Total contamination is defined as the quota of insoluble material retained after filtration of a fuel sample under standardized conditions. It is limited to < 24 mg/kg in the European specification for both biodiesel and fossil diesel fuels. The Brazilian and American biodiesel standards do not contain this parameter, as it is argued that fuels meeting the specifications regarding ash content will show sufficiently low values of total contamination as well. The total contamination has turned out to be an important quality criterion, as biodiesel with high concentration of insoluble impurities tend to cause blockage of fuel filters and injection pumps. High concentrations of soaps and sediments are mainly associated with these phenomena (Mittelbach, 2000).

1.7 Copper corrosion

This parameter characterizes the tendency of a fuel to cause corrosion to copper, zinc and bronze parts of the engine and the storage tank. A copper strip is heated to 50°C in a fuel bath for three hours, and then compared to standard strips to determine the degree of corrosion. This corrosion resulting from biodiesel might be induced by some sulfur compounds and by acids, so this parameter is correlated with acid number. Some experts consider that this parameter does not provide a useful description of the quality of the fuel, as the results are unlikely to give ratings higher than class 1.

Although primitive in design, open raceway ponds are still the predominant algaculture system for high-value added products and have been used for decades (Benemann and Oswald, 1996; Sheehan et al., 1998). The fact that raceway ponds are uncomplicated makes them less productive and offers little control over the culture parameters; however, the low cost of this low-tech cultivation system allows it to compete with complex photobioreactors (Gordon et al., 2007). The surface-to-volume ratio and corresponding light penetration in open ponds are not ideal. As a result, ponds can only support low culture densities; however, the ease of scaling production to industrial proportions (> 1 million liters per acre) justifies their seemingly low efficiency. The use of raceway ponds is also vindicated by their uncomplicated design, which makes them readily available for implementation and relatively simple to clean and maintain. While open ponds are cost effective, they do have a large footprint and contamination by local algal species and threat of algal grazers pose serious risks. Invasion by indigenous microorganisms may be protected against by the use of greenhouse enclosures or by growing algae that can withstand hypersaline environments, such as Dunaliella salina. For the purpose of biofuel production, however, the low areal productivities of ponds alone may not be able to provide the necessary biomass feedstock economically. Commercial scale microalgal biofuel facilities will likely rely on integrated systems of high efficiency photobioreactors to provide a dense inoculum for readily scalable raceway ponds (Huntely et al., 2007).

While genetic engineering approaches may improve the photosynthetic and biosynthetic capabilities of microalgae, many innovative methods exist for optimizing photoautotrophic culture conditions to accomplish the same goal of increased yield (Muller-Feuga, 2004). The breadth of chemical engineering knowledge being applied to photobioreactor (PBR) design in order to enhance light and nutrient availability represents an important advance in the field (Tredici and Zittelli, 1998; Miron et al., 1999), particularly for modular and scalable reactors (Janssen et al., 2003; Hu et al., 1996); however, the cost of these technologies remains the ultimate constraint on feasibility.

Photobioreactors aim to optimize many, if not all, of the culture parameters crucial to microalgal growth. One condition achieved in PBRs, but not raceway ponds, is turbulent flow to produce enhanced mixing patterns. By simply increasing the Reynolds number of these systems, the resulting fluid dynamics have a positive effect on nutrient mass transfer, light absorption, and temperature control (Ugwu et al., 2008). However, highly turbulent flow within complex geometries comes at the expense of the cells’ sensitivity to shear stress, which is one limitation of photobioreactor design. Another important consideration is the time scale over which the microalgae are transferred from the periphery of the bioreactor to the interior shaded region, as there exists a limit to the amount of light the cells can process before photoinhibiton occurs.

One of the most beneficial aspects of photobioreactors is the extended surface area achievable with tubular and flat-panel designs. This route to increased productivity takes advantage of allowing more algae to have contact with sunlight than an area of land would regularly allow with more basic cultivation systems; however, the additional cost of maintaining ideal temperatures and protecting these sometimes delicate devices from inclement weather pose some concern. Many of these nascent technologies depend entirely on the site of deployment and, as a result, require a great deal of customization (Janssen et al., 2003; Miron et al., 1999).

Some additional drawbacks of photobioreactors include the chemical gradients that can develop, particularly along tubular reactors. As a result of photosynthesis, a significant amount of oxygen can accumulate in these tubes and must be purged periodically. High concentrations of oxygen can both inhibit photosynthesis and, when combined with high irradiance, result in the formation of reactive oxygen species (ROS) (Tredici and Zittelli, 1998). These added difficulties contribute to the higher complexity and cost of photobioreactor operation. Some low-cost alternatives include vertical-column or hanging — bag bioreactors, which still provide a closed system for monocultures, with less control over culture parameters. These systems often rely on sparged air to provide both CO2 and mixing force, as in airlift bioreactors. The ability to strike a balance between cost and productivity is the major challenge of microalgal cultivation, especially for applications that require closed cultivation, as is the case with genetically modified microalgae.

In this case, the main reaction leading to methanol is the hydrogenation of CO.

CO + 2H2 = CH3OH -90.64 KJ/mol (15)

CO2 + H2 = CO +H2O 41.14 KJ/mol (16)

According to equation (15) methanol is predominantly synthesised via the direct hydrogenation of CO. The second reaction is the reverse of Water Gas Shift (RWGS). Experimental data involving typical syngas mixtures that contain 3 to 9% CO2 show a decrease of its concentration in the reactor effluent stream (Sunggyu, 2007). It should be noted that the first reaction (methanol synthesis) is exothermic, whereas the second (RWGS) is endothermic. According to this depletion of carbon dioxide in the RWGS reaction, produce more reactant (CO) which overall boost the synthesis of methanol. Until 90s the role of CO2 in the methanol synthesis was not clear. Deficiency of CO2 in the feed composition can be extremely detrimental to the overall synthesis, very rapidly deactivating the catalysts and immediately lowering methanol productivity in the process. Typically, 2 to 4% of CO2 is present in the syngas mixture for the vapor-phase synthesis of methanol, whereas this value is somewhat higher, with 4 to 9%, for liquid-phase synthesis (Cybulski, 1994).

Most pre-treatments require expensive instruments or equipment that require high energy requirements, depending on the process. In particular, physical and thermo-chemical processes require ample amount of energy to change the lignocellulosic structure of biomass. Biological pre-treatment using various types of rot fungi is a process that does not require high energy for lignin removal from a lignocellulosic biomass, despite extensive lignin degradation. Biological pre-treatments are safe, environmentally friendly and less energy intensive compared to other pre-treatment methods. However, the rate of hydrolysis reaction is very slow and needs a great improvement to be commercially applicable. Biological pre-treatment comprises of using microorganisms such as brown-, white-, and soft-rot fungi for selective degradation of lignin and hemicellulose among which white-rot fungi seems to be the most effective microorganism (Fan et al., 1987). Brown rots mainly attack cellulose, while white and soft rots attack both cellulose and lignin. Lignin degradation occurs through the action of lignin-degrading enzymes such as peroxidases and laccase (Okano et al., 2005). These enzymes are regulated by carbon and nitrogen sources. The suitable fungi for biological pre-treatment should have higher affinity for lignin and degrade it faster than carbohydrate components.

Hatakka et al. (1983) studied the pre-treatment of wheat straw by 19 white-rot fungi and found that 35% of the straw was converted to reducing sugars by Pleurotus ostreatus in 5 weeks. Similar conversion was obtained in the pre-treatment by Phanerochaete sordid (Ballesteros et al., 2006) and Pycnoporus cinnabarinus (Okano et al., 2005) in 4 weeks. Akin et al. (1995) also reported the delignification of bermudagrass by white-rot fungi. The biodegradation of bermudagrass stems was improved by 29-32%, after 6 weeks, using Ceriporiopsis subvermispora and by 63-77% using Cyathus stercoreus.

In this part, a complete description of non-enzymatic catalysts which are used or potentially usable in glucose/O2 biofuel cells systems is given. The major problem in employing abiotic catalyst in such applications lies in their lack of specificity. Consequently, their application in implantable microscale devices is difficult. Nevertheless, they often lead to fast substrate
conversion kinetic characteristics and their stability is incomparably higher than enzymes one. Thus, they can be used as catalysts in biocompatible devices intended in supplying long-term high power densities.

In a microfluidic channel, the relationship between the fluid velocity and the absolute pressure for an incompressible viscous liquid is given by the classical fluid dynamics theory and the well-known Navier-Stokes equation:

9V — — (P’] —

—— НІ»-V) v = — VI — I +u Av

9t { ’ (1)

Where V stands for the fluid velocity vector with components (u, v, w), each expressed for a set of Euler components (x, y, z, t), P is the absolute pressure, p is the relative density, and |i is the kinematic viscosity.

In the case of a microfluidic horizontal straight channel (x-direction), the flow is always laminar under low pressure drop (typically a few bar), leading thus to a unidirectional flow and a uniform absolute pressure in the cross-section. For a fixed pressure drop AP between the inlet and the outlet of the channel, Eq. 1 simplifies to:

9u 1 AP (9 2u 92u

91 p L ^ 9y 2 9z 2

Where L is the length of the microchannel. When the permanent flow is reached, the time derivative term becomes zero and Eq. 2 simplifies to:

-AP 92u 92u pL 9y2 9z2

Where p is the dynamic viscosity (10-3 Pa. s for water at 20°C), defined as the product of the dynamic viscosity and the relative density p. Due to the very large aspect ratio of the rectangular cross-section of the microchannel, a 2D approach is usually considered that leads to a pseudo infinite-plate flow (except in the borders). The directions along the length and height of the microchannel are indicated as x and y coordinates, respectively (see fig. 2). A typical parabolic rate profile is obtained for pressure driven flow:

AP h2

— у 2)

2.p. L 4

Where h is the height of the microchannel and y is defined as y=0 at the middle of the microchannel and y= ± h/2 at the upper and under walls. By considering a rectangular microchannel (with l the width of the microchannel) in Eq. 4, the flow rate, Q, in laminar regime, is deduced and is proportional to the applied pressure (Eq. 5):

AP. l.h3

12. p. L

In electrochemical laminar flow systems, the mass transport is achieved by both diffusion and convection transport. In the case of Y-shaped microchannel, the mixing between the two laminar streams occurs by transverse diffusion. Microscale devices are generally characterized by high Peclet number, Pe, (Pe = Uavh/ D, with Uav the average velocity of the flow, h the height of the microchannel and D the diffusion coefficient of the molecule). In

this condition, the transverse diffusion is much lower than the convection, and the diffusive mixing of the co-laminar streams is restricted to a thin interfacial width, Smix, in the center of the channel (Fig. 3) that grows as a function of the downstream position (x) and the flow rate, determined from Eq. 6 (Ismagilov et al., 2000):

<6>

Where D is the diffusion coefficient for ions of type i and Uav is the average flow velocity defined as:

AP. h2

12iyL

For fast electron transfer and in excess of supporting electrolyte, the kinetics of a simple electrochemical redox reaction is controlled by diffusion and convection. The concentration profiles of the chemical species involved in the reaction are determined by solving the convective diffusion equation (Eq. 8):

-c — + V(-DVc) + vVc + R = 0 dt ‘ ‘

Where ci is the concentration of species i, Di its diffusion coefficient, t the time, v the fluid velocity vector (given by Eq. 1) and Ri a term describing the rate of net generation or consumption of species i formed by homogeneous chemical reaction.

In the case of a microfluidic biofuel cell as described in this work, Eq. 8 can be simplified into a 2-dimensionnal cartesian steady state (Eq. 9):

I C2c d2c і dc

-D I + Cc — 1 + u(y)^ = 0

‘ y dx dy2 ) dx

The boundary conditions associated are usually: (i) c = c° (bulk concentration) at the inlet of the microchannel, (ii) c = 0 at the electrode surface and (iii) no flux at the other walls (no electrochemical reaction).

Those simulations were exploited in order to calculate the diffusive flux at the electrode, defined as:

(x) = D

electrode

And, therefore, the total current is expressed as:

Where n is the number of electron exchanged, and F is the Faraday constant.

The pumping power Wpumpmg, required to sustain steady laminar flow in the microchannel by the syringe pump, is estimated on laminar flow theory (Bazylak et al., 2005) as the pressure drop multiplied by the flow rate:

One can note that the contributions from inlet and outlet feed tubes are not included, because they are negligible.

The fuel utilization (FU) is estimated by the following Eq. 13, defined as the current output divided by the flux of reactant entering the channel (Bazylak et al., 2005):

I

’.F. C.Q

The fuel utilization is maximized for the lowest flow rate and decreases with flow rate. Typical fuel utilization for microfluidic fuel cell is ~ 1% (Hayes et al., 2008).

This tuber is of considerable interest not only for ethanol production but also to produce glucose syrup, and it is available in tropical countries. The ethanol yield from the whole manioc is equivalent to the ethanol produced from cereals using dry milling methods. The only known lies in that the manioc has to be processed 3-4 days after it was harvested. To avoid such lengthy processing times, the manioc is first sliced and then left to dry in the sun. The waste water produced in the process can be treated by means of anaerobic digestion to produce bio gas.

1.4 Spruce (picea abies)

This tree has attracted a great deal of attention as a raw material for ethanol production because it is a lignocellulose material mainly composed of hexose sugars, which are more readily convertible than pentose sugars.

1.5 Willow (salix)

This is a member of the Angiosperm family and is consequently characterized by a hard wood. In this species, a fraction of the xylose units is acetylated. Some of the OH groups of the xylose carbons C2 and C3 are replaced by O-acetyl groups. With pretreatment, these groups release acetic acid that, in high enough concentrations, inhibits the yeasts involved in the fermentation process, according to some studies (Sassner et al., 2008a). It was recently demonstrated (Sassner et al., 2008b) that, by pretreating willow with sulfuric acid before the enzymatic hydrolysis process, and then simultaneously performing saccharification and fermentation, they succeeded in obtaining a global ethanol yield of 79%.

Diazonium grafting is a promising alternative to conventional electrode functionalization method. In this approach the electrochemical reduction of diazonium forms an aryl centered radical. The resulting aryl radical can then form a covalent bond with conducting and semiconducting surfaces. The CV results shown in Fig. 4. indicate the first cycle of electro­reduction process from NO2 to NH2 at different diazonium concentrations. The reduction of NO2 to NH2 occurs on the first negative-going sweep in a range of potential from -1.0 V to —

1.1 V forming a clear irreversible anodic peak. From the results, it was confirmed that the amino groups are successfully grafted on the carbon surface for further immobilization.

Optimum values of reduced bulk density Or for soils are around 1.2 g. cm-3, but more important is the minimum value of bulk density for the restriction of root growth which is about 1.7 — 1.8 g. cm-3 for light soils and only 1.40 — 1.45 g. cm-3 for heavy-textured clay soils. Bulk density Or is a crucial parameter for the assessment of the soil compaction rate as an important negative factor of soil productivity. Bulk density of topsoil in the range of 0.95 — 1.15 g. cm-3 shows loose topsoil while the value > 1.25 g. cm-3 indicates heavily compacted topsoil.

Another important value of soil is soil aeration VZ. It is expressed in volume % as the difference between porosity Po and momentous soil moisture Wobj.

vz = Po — Wobj. (16)

Optimum aeration e. g. for grasslands is 10% by volume, for soils for barley growing it is already as much as 24% by volume. Soil porosity Po is the sum of all pores in volume per cent, in topsoils it is around 55%, in subsoil it decreases to 45 — 35%. Sandy soils have on average P = 42% by vol., out of this 30% are large pores and 5% are fine pores while heavy — textured clay soils have the average porosity of 48% by vol., out of this only 8% are large pores and 30% are fine pores. Fine pores are capillary and large pores are non-capillary ones. Cereals should be grown in soils with 60 — 70% of capillary pores out of total porosity and 30 — 40% of non-capillary pores. Forage crops and vegetables require the soils with 75 — 85% of capillary pores and only 15 — 25% of non-capillary pores out of total porosity. Ploughing resistance P is also significant. It is a specific resistance that must be overcome during cutting into and turning over the soil layer. It is expressed by the drawbar pull measured dynamometrically on the coupling hook of a tractor. It is related to the texture and moisture of soil, to its content of organic substances and ploughing depth. Ploughing resistance for light soils is 2 — 4 t. m-2, for heavy-textured soils it is 6 — 8 t. m-2. The units kp. dm-2 are also used. For sandy soils the ploughing resistance of 25 — 28 kp. dm-2 is usual, for clay soils it is 70 kp. dm-2.

Hence heavy-textured soils are more responsive to the higher reduced bulk density of soil when roots develop poorly, they need more non-capillary pores to allow for the better infiltration of precipitation water, they also need higher aeration because they are mostly too moist and many aerobic processes including the microbial activity take place with difficulty. Of course, the high ploughing resistance is not desirable either for the economics of soil cultivation or for the production process of any crop. Therefore it is necessary to improve heavy-textured soils and the question is how. Organic fertilisers are not sufficient; peat was used previously but now it is banned to use it for the reason of the peat bog conservation, and synthetic soil amendments (Krilium, Flotal etc.) are currently too costly for the agriculture sector. An excellent material for the improvement of heavy-textured soils is the solid phase of digestate if ploughed down at higher doses than those used for the application of farmyard manure or compost, i. e. 100 — 150 t. ha-1. Even though we cannot expect any great release of mineral nutrients from organic matter of the solid phase of digestate due to high stability of this material, the improvement and aeration of heavy — textured soil with better conditions for the microbial activity of soil and undisturbed root growth often bring about a higher yield effect than is the yield effect of nutrients from high — quality organic fertilisers as shown by the results of this field trial:

When we still believed that the solid phase of digestate was an organic fertiliser, we laid out an exact field trial on a heavier-textured, loamy-clay soil with medium to good reserve of available nutrients. The trial had two treatments: the one treatment was fertilisation with the solid phase of digestate only (after fugate centrifugation) and the other treatment was the application of only mineral fertilisers in the form of pure salts at such a dose that the level of these easily available nutrients to plants was the same as the amount of unavailable or little available nutrients in the treatment fertilised with digestate. We wanted to find out from the yield of the grown crop what amount of mineral nutrients would be released from the digestate in comparison with completely available nutrients in the first year and in subsequent years of the crop rotation: early potatoes — winter barley — red clover — oats. We intended to compare the digestate with other organic fertilisers, e. g. farmyard manure which in the first year mineralises about a half of its nutrients bound in organic matter. But the result we obtained was surprising: in the first year the yield of early potatoes was higher by 12% in the digestate treatment although nobody could doubt that this treatment had a lower amount of nutrients than the variant fertilised with pure salts. The only explanation is that the higher yield effect in the digestate treatment was not caused by the higher input of nutrients but by the improvement in physical properties of heavy-textured soil that surely occurred as seen in Tab. 7. The favourable effect of the heavy-textured soil improvement on yield was positively reflected in subsequent years also in other crops of the crop rotation that were fertilised in both treatments in the same way, i. e. mineral fertilisers were applied. We drew a conclusion that in practice the yield effect is often ascribed to digestate nutrients although it is caused by better soil aeration and better root growth due to soil loosening after the application of digestate.

Most of work was performed with the strain Clostridium pasteurianum NRRL B-592 which differed from usually employed solvent producing clostridia significantly, especially in sooner onset of solvents production i. e. during exponential growth phase. The strain was also chosen because of its properties i. e. stable growth and solvents production, robustness regarding minor changes in cultivation conditions and resistance toward so-called strain degeneration. Nevertheless in some cases, other, more typical solventogenic strains, C. acetobutylicum DSM 1731 and C. beijerinckii CCM 6182 were used, too.

Compositions of cultivation media, strains maintenance, description of cultivation, used analytical methods and expressions describing calculation of fermentation parameters i. e. yield and productivity for batch, fed-batch and continuous fermentations are given in Patakova et al., (2009 and 2011a).

3.1 Methods of ABE study

Despite complex process character, fermentation control, which is of key importance, relies only on few on-line measurable values like pH or redox potential of the medium and off-line determined concentrations of substrate(s), biomass and metabolites. In order to understand the process better and to improve fermentation control, fluorescence labelling of selected traits together with microscopy and flow cytometry was applied. Flow cytometry, as high — throughput, multi-parametric technique capable of analysis of heterogenic populations at the level of individual cells, has recently been used for description of clostridial butanol fermentations for the first time, but in totally different context (Tracy et al., 2008).