Enzymatic hydrolysis of cellulose and hemicellulose can be carried out by highly specific cellulase and hemicellulase enzymes (glycosyl hydro­lases). This group includes at least 15 protein families and some sub­families [15, 27]. Enzymatic degradation of cellulose to glucose is generally accomplished by synergistic action of three distinct classes of enzymes [2]:

■ 1,4-^-D-glucan-4-glucanohydrolases or Endo-1,4-^-glucanases, which are commonly measured by detecting the reducing groups released from carboxymethylcellulose (CMC).

■ Exo-1,4-^-D-glucanases, including both 1,4-^-D-glucan hydrolases and 1,4-^-D-glucan cellobiohydrolases. 1,4-^-D-glucan hydrolases liberate D-glucose and 1,4-^-D-glucan cellobiohydrolases liberate D-cellobiose.

■ ^-D-glucoside glucohydrolases or |3-D-glucosidases, which release D — glucose from cellobiose and soluble cellodextrins, as well as an array of glycosides.

There is a synergy between exo—exo, exo—endo, and endo—endo enzymes, which has been demonstrated several times.

Substrate properties, cellulase activity, and hydrolysis conditions (e. g., temperature and pH) are the factors that affect the enzymatic hydroly­sis of cellulose. To improve the yield and rate of enzymatic hydrolysis, there has been some research focused on optimizing the hydrolysis process and enhancing cellulase activity. Substrate concentration is one of the main factors that affect the yield and initial rate of enzymatic hydrolysis of cellulose. At low substrate levels, an increase of substrate concentration normally results in an increase of the yield and reaction rate of the hydrolysis. However, high substrate concentration can cause substrate inhibition, which substantially lowers the rate of hydrolysis, and the extent of substrate inhibition depends on the ratio of total sub­strate to total enzyme [12].

Increasing the dosage of cellulases in the process to a certain extent can enhance the yield and rate of hydrolysis, but would significantly increase the cost of the process. Cellulase loading of 10 FPU/g (filter paper units per gram) of cellulose is often used in laboratory studies because it provides a hydrolysis profile with high levels of glucose yield in a reasonable time (48-72 h) at a reasonable enzyme cost. Cellulase enzyme loadings in hydrolysis vary from 5 to 33 FPU/g substrate, depend­ing on the type and concentration of substrates. ^-glucosidase acts as a limiting agent in enzymatic hydrolysis of cellulose. Adding supplemental ^-glucosidase can enhance the saccharification yield [28, 29].

Enzymatic hydrolysis of cellulose consists of three steps [12]: (1) adsorp­tion of cellulase enzymes onto the surface of cellulose, (2) biodegrada­tion of cellulose to simple sugars, and (c) desorption of cellulase. Cellulase activity decreases during hydrolysis. Irreversible adsorption of cellulase on cellulose is partially responsible for this deactivation. Addition of surfactants during hydrolysis is capable of modifying the cel­lulose surface property and minimizing the irreversible binding of cel — lulase on cellulose. Tween-20 and Tween-80 are the most efficient nonionic surfactants in this regard. Addition of Tween-20 as an additive in simultaneous saccharification and fermentation (SSF) at 2.5 g/L has several positive effects in the process. It increases the ethanol yield, increases the enzyme activity in the liquid fraction at the end of the process, reduces the amount of enzyme loading, and reduces the required time to attain maximum ethanol concentration [30].

Crop description. Linum usitatissimum L.—commonly known as lin­seed, flaxseed, lint bells, or winterlien—belongs to the family Linaceae (see Fig. 4.8). This annual herb can grow up to 60 cm in height in most temperate and tropical regions. This plant is native to West Asia and the Mediterranean [96]. The seeds contain 30-40% oil, including palmitic

acid (4.5%), stearic acid (4.4%), oleic acid (17.0%), linoleic acid (15.5%), and linolenic acid (58.6%).

Main uses. Medicinal properties of the seeds have been known since ancient Greece. It is used in pharmacology (antitussive, gentle bulk lax­ative, relaxing expectorant, antiseptic, antiinflammatory, etc.) [97]. As the source of linen fiber, it was used by the Egyptians to make cloth in which to wrap their mummies. However, today it is mainly grown for its oil [98, 99], which is used in the manufacture of paints, varnishes, and linoleum. Linseed oil is used as a purgative for sheep and horses. It is also used in cooking. There is a market for flaxseed meal as both animal feeding and human nutrition [96].

Lang et al. transesterified linseed oil by using different alcohols (methanol, ethanol, 2-propanol, and butanol) and catalysts (KOH and sodium alkoxides). Butyl esters showed reduced cloud points and pour points [100]. Some authors have found that biodiesel from linseed oil presents a lower cold filter plugging point (CFPP) than biodiesel from rapeseed oil, due to large amounts of linolenic acid methyl ester and their iodine value [101]. Long-term endurance tests have been carried out with methyl esters of linseed oil, showing low emission characteristics. Wear assessment has shown lower wear for a biodiesel-operated engine [102]. Experimental investigations on the effect of 20% biodiesel blended with diesel fuel on lubricating oil have shown a lubricating oil life longer while operating the engine on biodiesel [103]. Oxidation stability have shown better results compared with methyl esters of animal origin [104]. Lebedevas et al. have suggested the use of three-component mixtures (rapeseed-oil methyl esters, animal methyl esters, and linseed oil methyl esters) to fuel the engine. These three-component mixtures reduced exhaust emissions significantly, with the exception of NOx that increased them up to 13% [105].

Enzymatic transesterification of TG by lipases (3.1.1.3) is a good alter­native over a chemical process due to its eco-friendly, selective nature and low temperature requirement. Lipases break down the TAG into FFA and glycerol that exhibits maximum activity at the oil-water inter­face. Under low-water conditions, the hydrolysis reaction is reversible,

i. e., the ester bond is synthesized rather than hydrolyzed. Scientists are interested in the development of lipase applications to the inter­esterification reactions of vegetable oils for production of biodiesel.

Nag has reported [43] celite-immobilized commercial Candida rugosa lipase and its isoenzyme lipase 4 efficiently catalyzed alcoholysis (dry ethanol) of various TG and soybean oil (see Fig. 6.14). This process has many advantages over chemical processes such as (a) low reaction tem­perature, (b) no restriction on organic solvents, (c) substrate specificity on enzymatic reactions, (d) efficient reactivity requiring only the mixing of the reactants, and (e) easy separation of the product.

Kaieda et al. have developed [44] a solvent-free method for methanol — ysis of soybean oil using Rhizopus oryzae lipase in the presence of 4-30 wt%

water in the starting materials. Oda et al. [45] have reported methanol — ysis of the same oil using whole-cell biocatalyst, where R. oryzae cells were immobilized within porous biomass support particles (BSP). Kose et al. [46] have reported the lipase-catalyzed synthesis of alkyl esters of fatty acids from refined cottonseed oil using primary and secondary alcohols in the presence of an immobilized enzyme from C. antarctica, commercially called Novozym-435 in a solvent-free medium. Under the same conditions, with short-chain primary and secondary alcohols, cot­tonseed oil was converted into its corresponding esters.

Alcoholysis of soybean oil with methanol and ethanol using several lipases has been investigated. The immobilized lipase from Pseudomonas cepacia was the most efficient for synthesis of alkyl esters, where 67 and 65 mol% of methyl and ethyl esters, respectively, were obtained by Noureddini et al. [47]. Shimada et al. [48] have reported transesterifi­cation of waste oil with stepwise addition of methanol using immo­bilized C. antarctica lipase, where they have successfully converted more than 90% of the oil to fatty acid ME. They have also implemented the same technique for ethanolysis of tuna oil.

Dossat et al. [49] have found that hexane was not a good solvent as the glycerol formed after the reaction was insoluble in n-hexane and adsorbed onto the enzyme, leading to a drastic decrease in enzymatic activity. Enzymatic transesterification of cottonseed oil has been stud­ied using immobilized C. antarctica lipase as catalyst in t-butanol sol­vent by Royon et al. [50].

Sometimes, gums present in the oils used inhibit alcoholysis reactions due to interference in the interaction of the lipase molecule with sub­strates by the phospholipids present in the oil gum. Crude soybean oil cannot be transesterified by immobilized C. antarctica lipase. So, Watanabe et al. [51] have used degummed oil as a substrate for a trans­esterification reaction, in order to minimize this problem, and have effec­tively achieved conversion of 93.8% oil to biodiesel.

Methanol is insoluble in the oil, so it inhibits the lipases, thereby decreasing its catalytic activity toward the transesterification reaction. Du et al. [52] transesterified soybean oil using methyl acetate in the presence of Novozym-435 (see Fig. 6.15). Further, glycerol was also insol­uble in the oil and adsorbed easily onto the surface of the immobilized lipase, leading to a negative effect on lipase activity. They have suggested that methyl acetate was a novel acceptor for biodiesel production and no glycerol was produced in that process, as shown below:

CH,-OOC-R1 . RrCOOCH3 CH2-OOCCH4

j Lipase | “

CH-OOC-R2 + ЗСН3СООСН3 R2-COOCH3 + CH-OOCCH3

CH — OOC-R R3-COOCH3 CH2OOCCH3

They found 92% yield with a methyl acetate-oil molar ratio of 12:1, and methyl acetate showed no negative effect on enzyme activity.

The comparison of biodiesel production by acid, alkali, and enzyme is given in Table 6.2.

Cottonseed oil has been thermally decomposed at 450oC using 1% Na2CO3 as a catalyst [42]. Pyrolysis produced a yellowish-brown oil with 70OC yield. The fuel properties of original and pyrolyzed cottonseed oil are sum­marized in Table 8.7. Results of ASTM distillation compared to diesel are given in Table 8.8 showing a higher volatility for the conversion product.

Rapeseed oil was pyrolyzed in the presence of about 2% calcium oxide up to a temperature of 450OC [43]. An oil was obtained with a heating

value of 41.3 MJ/kg, a kinematic viscosity of 5.96 mm2/s, a cetane number of 53, and a flash point of 80°C. When tested on a diesel engine, the thermal efficiency (^th) and brake specific fuel consumption were improved. The concentration of nitrogen oxide in the exhaust gas was less than diesel. The absence of sulfur in the pyrolytic oil was seen as an advantage to avoid corrosion problems and the emission of polluting sulfur compounds from combustion.

Triolein, canola oil, trilaurin, and coconut oil were pyrolyzed over acti­vated alumina at 450°C and atmospheric pressure [44]. The products were characterized by IR spectrometry and decoupled 13C-NMR spec­troscopy. The hydrocarbon mixture contained both alkanes and alkenes. These results are significant for the pyrolysis of lipid fraction in sewage sludge as well as for wastes from food-processing industries [44].

Pyrolysis of rapeseeds, linseeds, and safflowers results in bio-oil con­taining oxygenated polar components. Hydropyrolysis at medium pres­sure in the presence of 1% ammonium dioxydithiomolybdenate (NH4)2MoO2S2 can remove two-thirds to nine-tenths of the oxygen pres­ent in the seeds to generate bio-oils in yields up to 75% [45]. In addi­tion, extraction with organic solvents including diesel oil gave yields up to 40%.

The potential of liquid fuels from Mesua ferrea seed oil [46], Euphorbia lathyris [47, 48], and underutilized tropical biomass [49] has been inves­tigated in the search for “energy farms” involving the purposeful culti­vation of selected plants to obtain renewable energy sources.

In photosynthesis, CO2 from the atmosphere and water from the earth combine to produce carbohydrates, which are the components of bio­mass and solar energy that drive this process. When biomass is effi­ciently utilized, the oxygen from the atmosphere combines with the carbon in plants to produce CO2 and water (see Fig. 2.1). Typically, pho­tosynthesis converts less than 1% of the available sunlight to be stored as chemical energy.

The advantages of using plants for renewable energies (fuels and chemicals) are listed follows:

■ Advances in agriculture and forestry technologies have resulted in increased utilization of land resources for cultivation of energy crops.

■ By increasing harvesting of solar energy, there is effective usage of biomass-based resources.

■ Multiple economic benefits can be derived—for example, sugar can be used as such for fermentation to alcohol—depending on the market.

■ Biomass combustion, unlike fossil fuels, does not contribute to increased CO2 levels in the atmosphere [2].

■ Increased employment opportunities resulting from the above.

While the advantages of using biomass-based energies are apparent, it is important to note that biomass cannot by itself provide complete replace­ment of fossil fuels. Hence, it is one of the solutions toward achieving energy efficiency. Further factors, such as competition for biomass between energy production and human nutritional needs, as well as the possible environ­mental effects, must be kept in mind. There are several factors that should be considered in using plants for the generation of energy; efficiency of solar energy absorption and conversion, quality of biomass produced, plant growth, growth under marginal conditions, soil characteristics, and cost — effectiveness of production of energy and conversion. We will focus on the utilization of terrestrial plants for production of renewable energies.

Process design studies of molasses fermentation have shown that the investment cost was considerably reduced when continuous rather than batch fermentation was employed, and that the productivity of ethanol could be increased by more than 200%. Continuous operations can be classified into continuous fermentation with or without feedback control. In continuous fermentation without feedback control, called a chemostat, the feed medium containing all the nutrients is continuously fed at a con­stant rate (dilution rate D) and the cultured broth is simultaneously removed from the fermentor at the same rate. The chemostat is quite useful in the optimization of media formulation and to investigate the physiological state of the microorganism [71]. Continuous fermenta­tions with feedback control are turbidostat, phauxostat, and nutristat. A turbidostat with feedback control is a continuous process to maintain the cell concentration at a constant level by controlling the medium feeding rate. A phauxostat is an extended nutristat, which maintains the pH value of the medium in the fermentor at a preset value. A nutristat with feedback control is a cultivation technique to maintain nutrient con­centration at a constant level [71].

When lignocellulosic hydrolyzates are added at a low feed rate in con­tinuous fermentation, low concentration of bioconvertible inhibitors in the fermentor is assured. In spite of a number of potential advantages in terms of productivity, this method has not developed much yet in fermentation of the acid hydrolyzates. One should consider the following points in continuous cultivation of acid hydrolyzates of lig — nocelluloses:

■ Cell growth is necessary at a rate equal to the dilution rate in order to avoid washout of the cells in continuous cultivation.

■ Growth rate is low in fermentation of hydrolyzates because of the presence of inhibitors.

■ The cells should keep their viability and vitality for a long time.

The major drawback of the continuous fermentation is that, in contrast to the situation in fed-batch fermentation, cell growth is necessary at a rate equal to the dilution rate, in order to avoid washout of the cells in continuous cultivation [21]. The productivity is a function of the dilu­tion rate, and since the growth rate is decreased by the inhibitors, the productivity in continuous fermentation of lignocellulosic hydrolyzates is low. Furthermore, at a very low dilution rate, the conversion rate of the inhibitors can be expected to decrease due to the decreased specific growth rate of the biomass. Thus, washout may occur even at very low dilution rates [18]. On the other hand, one of the major advantages of continuous cultivation is the possibility to run the process for a long time (e. g., several months), whereas the microorganisms usually lose their activity after facing the inhibitory conditions of the hydrolyzate. By employing cell-retention systems, the cell-mass concentration in the fermentor, the maximum dilution rate, and thus the maximum ethanol productivity increase. Different cell-retention systems have been inves­tigated by cell immobilization and encapsulation, and cell recirculation by filtration, settling, and centrifugation. A relatively old study [72] shows that the investment cost for a continuous process with cell recir­culation has been found to be less than that for continuous fermentation without cell recirculation.

Biostil® is the trade name of a continuous industrial process for ethanol production with partial recirculation of both yeast and waste­water. The fermentor works continuously; the cells are separated by using a centrifuge, and a part of the separated cells is returned to the fermentor. Most of the ethanol-depleted beer including residual sugars is then recycled to the fermentor. In this process, besides providing enough cell concentration in the fermentor, less water is consumed and a more concentrated stillage is produced. Therefore, the process has a lower wastewater problem. However, the process needs a special type of centrifuge (which is expensive) in order to avoid deactivation of the cells [47, 73].

Application of an encapsulated cell system in continuous cultivation has several advantages, compared to either a free-cell or traditionally entrapped cell system, e. g., in alginate matrix. Encapsulation provides higher cell concentrations than free-cell systems in the medium, which leads to higher productivity per volume of the bioreactor in continuous cultivation. Furthermore, the biomass can easily be separated from the medium without centrifugation or filtration. The advantages of encap­sulation, compared to cell entrapment, are less resistance to diffusion through beads/capsules, some degree of freedom in movement of the encapsulated cells, no cell leakage from the capsules, and higher cell con­centration [74].

Bovine spongiform encephalopathy (BSE), commonly known as mad cow disease, is a fatal neurodegenerative disease of cattle. BSE has attracted wide attention because it can be transmitted to humans. Pathogenic prions are responsible for transmissible spongiform encephalopathies (TSE), and especially for the occurrence of a new variant of Creutzfeldt-Jakob disease (nvCJD), a human brain-wasting disease. Due to this problem, the specified risk material is burned under high temperatures to avoid any hazards for humans and animals. However, another possibility could be to consider this material as a source for producing biodiesel by transesterification. In fact, production of biodiesel from the risk material could represent a more economic usage than its combustion. Siedel et al. have found that almost every single step of the process leads to a significant reduction in the concen­tration of the pathogenic prion protein (PrPSc) in the main product and by-products. They concluded that biodiesel from materials with a high concentration of pathogenic prions can be considered safe [163]. Animal fats, such as tallow or lard, have been widely investigated as a source of biodiesel [164-169]. Muniyappa et al. have found that transesterifi­cation of beef tallow produced a mixture of esters with a high concen­tration in saturated fatty acids, but with physical properties similar to esters of soybean oil [37]. Ma et al. found that 0.3% NaOH completed methanolysis of beef tallow in 15 min [170]. Some authors have found that absolute ethanol produced higher conversion and less viscosity than absolute methanol at 50°C, after 2 h [171]. Nebel and Mittelbach have found n-hexane was the most suitable solvent for extraction of fat from meat and bone meal. The extracted material was converted into fatty acid methyl esters through a two-step process [172]. Lee et al. have performed a three-step transesterification to produce biodiesel from lard and restaurant grease. They found that a porous substance, such as silica gel, improved the conversion when more than 1 M methanol was used as reaction substrate [173]. Mbaraka et al. also synthesized propylsulfonic acid-functionalized mesoporous silica materials for methanol esterification of the FFA in beef tallow, as a pretreatment step for alkyl ester production [174].

Engine tests also showed a reduction in emission, except oxides of nitrogen that increased up to 11% for the yellow grease methyl ester [157]. Cold-flow properties of the fat-based fuels were found to be less desirable than those of soy-based biodiesel, with comparable lubricity and oxidative stability [175]. To solve this problem, Kazancev et al. blended up to 25% of pork lard methyl esters with other oil methyl esters and fossil diesel fuels. In this case, the CFPP showed a value of — 5°C. In winter, only up to 5% of esters can be added to the fuel. Depressant Viscoplex 10-35 with an optimal dose of 5000 mg/kg was found to be the most effective additive to improve the cold properties [101].

Although the physical and thermodynamic characteristics of alcohols do not make them particularly suitable for compression ignition (CI) engines, with certain modifications, however, they can also be used in CI engines. In heavy vehicles powered by CI engines, ethanol carbure — tion can be employed for bi-fuel operation of the engine with proportional savings in diesel oil. The various methods for using alcohols with diesel are fumigation, dual injection, and alcohol-diesel emulsions.

In a fumigation system the engine is fitted with a suitable carbure­tor and auxiliary ethanol tank. An ethanol-air mixture is carbureted during the induction stroke to provide 50% of the total energy of the cycle and the remaining energy is provided by diesel oil being injected in the conventional manner near the end of the compression stroke. The mate­rials of a fuel tank and fuel system must be compatible with alcohol. The entire system can be used as a retrofit kit, as shown in Fig. 7.6.

Ghosh et al. [4] carried out an investigation on the performance of a tractor diesel engine with ethanol fumigation (see Figs. 7.7 and 7.8). The following observations were recorded:

1. The brake thermal efficiency decreases with an increase in ethanol fumigation rate at a constant engine speed.

2. The BSFC decreases with an increase in ethanol fumigation rate at a constant engine speed

Figure 7.7 Experimental setup of ethanol fumigation.

3. The diesel substitution and the energy replacement increases with an increase in an ethanol fumigation rate at a constant engine speed.

4. The NOx emission level and the exhaust gas temperature decreases with an increase in a ethanol fumigation rate at a constant engine speed.

5. The CO emission level increases with an increase in an ethanol fumi­gation rate at a constant engine speed.

6. The smoke level decreases with an increase in an ethanol fumigation rate at a constant engine speed.

7.

The fumigation rate of 1.06 kg/h (40% diesel substitution) is optimal for good engine performance.

Ethanol fumigation in diesel engines can play a major role in envi­ronmental air pollution control, and ethanol is a viable alternative fuel for diesel engines.

Ethanol is a very good SI engine fuel and a rather poor CI engine fuel. Ethanol has a high octane rating of 90 and a low cetane rating of 8, and will not self-ignite reasonably in most CI engines. Dehydrated ethanol is fumigated into the air stream in the intake manifold of a 42-hp trac­tor diesel engine to improve its self-ignition quality. The performance of the engine under dual-fuel (diesel and fumigated ethanol) operation is compared with diesel fuel operation at various speeds (800, 900, 1000, 1100 rpm), loads (0, 4, 8, 12, 16 kgf), and fumigation rates (0.00, 1.06, 1.45, 2.06 kg/h). Analysis of the results shows that ethanol fumigation has the advantages of reduction in BSFC, NOx emission, and smoke level and the disadvantage of slight reduction in brake thermal efficiency. The fumigation rate of 1.06 kg/h (40% diesel substitution) is optimal for good engine performance.

It has been concluded that ethanol is a viable alternative fuel for diesel engines. A dramatic reduction in the NOx and the smoke level sug­gests that fumigation, as an emission control technique in diesel engines, can play a vital role in environmental air pollution control on a farm.

In the dual-injection method, two injection systems are used, one for diesel and the other for alcohol. This method can replace a large per­centage of diesel fuel. In this method, air is sucked and compressed, and then methanol is injected through a primary injector. To ignite this, a small amount of diesel is injected through a pilot injector. The relative injection timing of alcohol and diesel is an important aspect of the system.

As two injection systems are required, two injectors are required on the cylinder head, which limits the application of this method to large-bore engines. An additional pump, fuel tank, and fuel line are also required, making the system more complicated. But this method replaces 60% of diesel at a partial load and 90% at a full load, and provides higher ther­mal efficiency.

A biofuel cell operation is very similar to a conventional fuel cell, except that it uses biocatalysts such as enzymes, or even whole organisms instead of inorganic catalysts like platinum, to catalyze the conversion of chemical energy into electricity. They can use available substrates from renewable sources and convert them into benign by-products with the generation of electricity. As mentioned earlier, in recent years, med­ical science is increasingly relying on implantable electronic devices for treating a number of conditions. These devices demand a very reliable and maintenance-free (any maintenance that might require surgery) power source. Biofuel cells can provide solutions to most of these prob­lems. A biofuel cell can use fuel that is readily available in the body, for example, glucose in the bloodstream, and it would ideally draw on this power for as long as the patient lives. Since they use concentrated sources of chemical energy, they can be small and light.

A biofuel cell can operate in two ways: It can utilize the chemical pathways of living cells (microbial fuel cells), or, alternatively, it can use isolated enzymes [7, 28]. Microbial fuel cells have high efficiency in terms of conversion of chemical energy into electrical energy; however, they suffer from the low volumetric catalytic activity of the whole organ­ism and low power densities due to slow mass transport of the fuel across the cell wall. Isolated enzymes extracted from biological systems can be used as catalysts to oxidize fuel molecules at the anode and to enhance oxygen reduction at the cathode of the biofuel cell. Isolated enzymes are attractive catalysts for biofuel cells due to their high cat­alytic activity and selectivity. The theoretical value of the current that can be generated by an enzymatic catalyst with an activity of 103 U/mg is 1.6 A, a catalytic rate greater than platinum! However, practical observed currents are much lower due to the loss of catalytic activity from immobilization of the enzymes at the electrode surface and energy losses of the overall system. A major challenge in the biofuel cell design
is the electrical coupling of the biological components of the system with the fuel cell electrodes. Molecules known as electron-transfer mediators are needed to provide efficient transport of electrons between the bio­logical components (enzymes or microbial cells) and the electrodes of the biofuel cell. Integrated biocatalytic systems that include biocatalysts, electron-transfer mediators, and electrodes are under research and development. Biofuel cells have much wider fuel options; enzymatic bio­fuel cells can operate on a wide variety of available fuels such as ethanol, sugars, or even waste materials.

A basic microbial biofuel cell consists of two compartments, an anode compartment and a cathode compartment, separated by a PEM as shown in Fig. 9.11. Usually, Nafion-117 film (an expensive material) is used as the PEM; it allows hydrogen ions generated in the anode compartment to be transferred across the membrane into the cathode compartment [8].

Previously, graphite electrodes were used as the anode and cathode, but they are now replaced by woven graphite felt as it provides a larger surface area than a regular graphite electrode of similar dimensions. This facilitates an increased electron transfer from the microorganisms. A microorganism (e. g., Escherichia coli) is used to breakdown glucose in order to generate adenosine triphosphate (ATP), which is utilized by cells for energy storage. Methylene blue (MB) or neutral red (NR) is used as an electron mediator to efficiently facilitate the transfer of electrons from the microorganism to the electrode. Electron mediators tap into the electron transport chain, chemically reducing nicotinamide adenine din­ucleotide (NAD+) to its protonated form NADH. The exact mechanism by which the transfer of electrons takes place through these electron mediators is not fully known [29]; however, it is known that they insert themselves into the bacterial membrane and essentially “hijack” the electron transport process of glucose metabolism of the bio-electrodes in a biofuel cell. Their activity is very dependent on pH, and a potassium phosphate buffer (pH 7.0) is used to maintain the pH value in the anode compartment. The cathode compartment contains potassium ferricyanide,

a potassium phosphate buffer (pH 7.0), and a woven graphite felt elec­trode. Potassium ferricyanide reaction helps in rapid electron uptake. Hydrogen ions (H+) migrate across the PEM and combine with oxygen from air and the electrons to produce water at the cathode. The cath­ode compartment has to be oxygenated by constant bubbling with air to promote the cathode reactions. It may be worth mentioning that the electron transport chain occurs in the cell membrane of prokaryotes (a unicellular organism having cells lacking membrane-bound nuclei, such as bacteria), while this process occurs in the mitochondrial mem­brane of eukaryotes (animal cells). Therefore, attempts to substitute eukaryotic cells for bacterial cells in a biofuel cell may present a sig­nificant challenge.

Electrochemistry of microbial fuel cells. In a microbial fuel cell, two redox couples are required in order to generate a current: (a) coupling of the reduction of an electron mediator to a bacterial oxidative metabolism and (b) coupling of the oxidation of the electron mediator to the reduction of the electron acceptor on the cathode surface. The electron acceptor is subsequently regenerated by the presence of O2 at the cathode surface. The electrochemical reactions in a biofuel cell using glucose as a fuel are

At the anode:

C6H12O6 + 6H2O S 6CO2 + 24e + 24H+

At the cathode:

4Fe(CN)63~ + 4e2 s 4Fe(CN)642
4Fe(CN)642 + 4H+ + O2 s 4Fe(CN)632 + 2H2O

Complete oxidation of glucose does not always occur. One might often get additional products besides CO2 and water. For example, E. coli forms acetate, being unable to completely breakdown glucose, thereby limit­ing electricity production. Recently, an elegant approach to address this long-standing problem of limited enzyme stability has been reported [30]. It is suggested that the immobilization of enzymes in Nation layers to create a bio-anode results in stable performance over months.

Another way of using a microorganism’s ability to produce electro­chemically active substances for energy generation is to combine a biore­actor with a biofuel cell or a hydrogen fuel cell. The fuel can be produced in a bioreactor at one place and transported to a (H2 or bio-) fuel cell to be used as a fuel. In this case, the biocatalytic microbial reactor produces the fuel, and the biological part of the device is not directly integrated with the electrochemical part (see Fig. 9.12).

The advantage of this scheme is that it allows the electrochemical part to operate under conditions that are not compatible with the biological part of the device. The two parts can even be separated in time, oper­ating completely independently. The most widely used fuel in this scheme is hydrogen gas, allowing well-developed and highly efficient H2/O2 fuel cells to be conjugated with a bioreactor.

In recent years, ethanol has been developed as an alternative to the traditional methanol-powered biofuel cell due to the widespread avail­ability of ethanol for consumer use, its nontoxicity, and increased selec­tivity by alcohol. Ethanol fuel cells with immobilized enzymes have provided higher power densities than the latest state-of-the-art methanol biofuel cells. Open-circuit potentials ranging from 0.61 to 0.82 V and power densities of 1.00-2.04 mW/cm2 have been produced.

Mediatorless microbial fuel cells. Most biofuel cells need a mediator mol­ecule to speed up the electron transfer from the enzyme to the electrode. Recently, mediatorless microbial fuel cells have been developed. These use metal-reducing bacteria, such as members of the families Geobacteraceae or Shewanellaceae, which exhibit special cytochromes bound to their membranes. These are capable of transferring electrons to the elec­trodes directly. Rhodoferax ferrireducens, an iron-reducing microor­ganism, has the ability to directly transfer electrons to the surface of electrodes and does not require the addition of toxic electron-shuttling mediator compounds employed in other microbial fuel cells. Also, this metal-reducing bacterium is able to oxidize glucose at 80% electron effi­ciency (other organisms, such as Clostridium strains, oxidize glucose at only 0.04% efficiency). In other fuel cells that use immobilized enzymes, glucose is oxidized to gluconic acid and generates only two electrons, whereas in microbial fuel cells (MFCs) using R. ferrireducens, glucose is completely oxidized to CO2 releasing 24 electrons. These MFCs have a remarkable long-term stability, providing a steady electron flow over
extended periods. Current density of 31 mA/m2 over a period of more than 600 h has been reported [31]. MFCs using R. ferrireducens have the abil­ity to be recharged, and have a reasonable cycle life and low capacity loss under open-circuit conditions. They allow the harvest of electricity from many types of organic waste matter or renewable biomass. This is an advantage over other microorganisms in the family Geobacteraceae, which cannot metabolize sugars.

Another recent development has been the use of microfibers rather than flat electrodes and the enzyme-based electroactive coatings. The anode coating used is glucose oxidase, which is covalently bound to a reducing-potential copolymer and has osmium complexes attached to its backbone. The cathode coating contains the enzyme laccase and an oxidizing-potential copolymer. The osmium redox centers in the coatings electrically “wire” the reaction centers of the enzymes to the carbon fibers. This electrode design avoids glucose oxidation at the cathode and O2 reduction at the anode, eliminating the need for an electrode — separating membrane. This has led to miniature “one-compartment bio­fuel cells” for implantable devices within humans, such as pacemakers, insulin pumps, sensors, and prosthetic units. Biofuel cells with two 7-^m-diameter, electrocatalyst-coated carbon fiber electrodes placed in 1-mm grooves machined into a polycarbonate support with a power output of 600 nW at 37°C (enough to power small silicon-based micro­electronics) have been reported [32].

Microbial fuel cells have a long way to go before they compete with more established hydrogen fuel cells or electrical batteries. However, a number of factors provide motivation for research into microbial fuel cells for electricity production.

1. Bacteria are adapted to feeding on virtually all available carbon sources (carbohydrates or more complex organic matter present in sewage, sludge, or even marine sediments). This makes them poten­tial catalysts for electricity generation from organic waste.

2. Bacteria are omnipresent in the environment and are self — reproducing, self-renewing catalysts; thus a simple initial inoculation of a suitable strain could be cultured continuously in an MFC for long­term operation.

3. The catalytic core of conventional fuel cells uses very expensive pre­cious metals such as platinum, and biocatalysts like bacteria may become a serious cost-reducing alternative.

Although biofuel cells are still in an early stage of development and work toward optimizing the performance of a biofuel cell system is needed, the utilization of white blood cells as a source of electrons for a biofuel cell could mark an important step in developing a perpetual power source for implantable devices. There is still a lot of work to be done as there are many unanswered questions; however, the feasibility of con­structing commercially viable biofuel cell power supplies for a number of applications is very promising.

Energy can be derived from living systems in restricted forms only. Lignocelluloses are burned to get heat, and vegetable oils are often used for illumination. These may also serve as nutrients for different biotic species in various forms, i. e., cellulose, starch, and sugars. In other words, chemically stored energy may be reused in the form of fuel (fire­wood) or nutrients (food, feed, fodder, etc.). Animals can be employed to do different mechanical work. Animals directly (fish, meat) or indirectly (egg, milk) may provide nutrients for others. Use of dried cow dung as

cooking fuel in rural areas is also a well-known example of animal prod­ucts indirectly contributing to this field. But examples of direct energy flow from living systems are still in the conceptual state. Scientists dream that, one day, light emitted by fireflies or high voltage generated by electric eels may be of great use in the near future.

The production of alcohol or methane by microbial fermentation of common plant wastes are well-known phenomena. Recently however, scientists have started looking into these phenomena with greater inter­est, so that, in either gas or liquid form, their production and use can be optimized and made efficient. Plant bodies have been used as anten­nas, and plant leaves have been demonstrated to work as batteries. The survival of all biotic species depends directly or indirectly on solar energy. Studying the energy-based ecosystem raises awareness of this fact. Obviously, the most common question becomes: If the sun happens to be the source of all energy, why then is the solar energy not har­nessed by different devices? There are inherent limitations of most of the physical devices by (a) way of efficiency, (b) critical cost, (c) mainte­nance, (d) reliability, and (e) other factors.

In photosynthetic systems operating in green vegetations of the above points, (b), (c), and (d) are enormously better. Its characteristic limita­tions are [for point (a)] the incident insolation, the ability to use only a narrow spectrum [for point (e)], and requiring the proportionate amount of soil surface area for insolation, optimal nutrients, temperature, and moisture in the microenvironment. Here nature provides several mutants from which we can take, pick, screen, or select the most toler­ant variety. We may resort to genetic engineering for tissue cultures or selective hybridization.

What is our objective? Along with the effort to harness the solar energy by different physical methods, parallel efforts of optimal use of solar energy through biotic fixation should be attempted. This involves under­standing the following:

1. The living world in its entirety, i. e., ecology.

2. The photosynthetic systems in different species: terrestrial, aquatic, or mixed.

3. Application of the above to develop science and technology for:

a. Better management of the biotic systems useful for our purpose

b. Conversion of biological raw materials into energy rich products

4. Coordination for quality of life, pollution abatement, and sparing of nonrenewable resources for future generations.

A few examples that may not be out of place include potato, tomato, eucalyptus, and so forth. Though of wild origin, they have been appreci­ated and have been cultivated for this use after studying and admiring

their productivity and receptivity. Later by scientific manipulation, new strains have been developed for cultivation.

It is justified to discuss certain established facts for making suffi­cient conceptual clarity for special topics. Some aspects of energy rela­tions in living systems will be discussed in detail. Some other aspects will not be discussed in detail because existing “know-how” is rather limited.