The study of bioenergetics leads us into a world of novelty and greater significance and has found new encouragement in industry. The biogas generation by anaerobic fermentation has also led to new interest in research in the light of bioenergetics.

The study of energy relations for each chemical step in the living system may be an item of bioenergetics. The energy change can be calculated in terms of calories or joules per mole. This is applicable for catabolic processes, for example, the anaerobic or glycolytic paths or oxidative phosphorylation. The anabolic paths are equally fitting, e. g., the carbon fixation or the photosynthesis and nitrogen fixation by the symbiotic organisms [1].

The accounting and balancing of free energy change of certain reac­tions may lead to some fruitful conclusions. When glucose is oxidized in a bomb calorimeter (an almost one-step reaction),

C6 H12O6 + 6O2 ^ 6CO2 + 6H2O — 686,000 cal (pH 7.0)

but when equivalent CO2 is produced in a biological system (through a multistep reaction),

C6H12O6 + 6O2 + 38ADP + 38H3PO4 ^ 6CO2 + 38ATP + 44H2O

-382,000 cal (pH 7.0)

A noteworthy departure is the conservation of -304,000 cal/mol of glu­cose and gain of 38 moles of ATP, energy-rich (bond) compounds, i. e., 800 cal/mol of ATP. It also means 50,666 cal of energy are wasted if, on aver­age, 1 mole of carbon dioxide produced chemically is wasted in the form of heat, an inferior quality of energy.

A simple calculation will reveal that each nutrient has some specified energy or calorific values. This can be compared to the different energy

values of different fuels, i. e., coal, kerosene, firewood, and so forth (see Table 1.2). Taking glucose as a model carbohydrate,

C6H12O6 + 6O2 ^ 6CO2 + 6H2O — 686,000 cal
(molecular weight, MW = 180 g).

686,000 cal
180g

3800 cal/g

and taking palmitic acid as model fatty acid,

C16H32O2 + 23O2 ^ 16CO2 + 16H2O — 2,338,000 cal (MW = 256 g)

2,338,000 cal
256 g

Similarly, in amino acids, peptides show roughly the same value as that of carbohydrates. In biological systems (measurement through metabolic cage), it has been found that the biological energy values are slightly higher than those shown theoretically. This is more so by “specific dynamic action.” When mixed foods particularly protein are taken, the total calorific value is enhanced. The exact reasons are not yet clear. Let us concentrate on a few examples in the following:

In ethanol fermentation (pH 7.0),

C6H12O6 ^ 2[C2H5OH + CO2] — 56,000 cal In lactic fermentation,

C6H12O6 ^ 2[CH3CHOHCOOH] — 47,000 cal

But in lactic fermentation from polysaccharide,

(Glucosyl)n ^ 2[CH3CHOHCOOH] + (Glucosyl)n—1 — 52,000 cal
CH3CHOHCOOH + 3O2 ^ 3CO2 + 3H2O — 319,500 cal

If glucose is the starting point (as is the case of ethanol fermentation), then 2 moles of ATP are invested and finally 2 X 2 moles of ATP are regenerated and the net gain of ATP remains 2 (see Fig. 1.1). But if glyco­gen is the starting point, then only 1 mole is invested in the formation of fructose 1,6-diphosphate.

Hence, net gain in ATP is 4 — 1 = 3. Twice a mole of reduced Co I is produced by the conversion of 3 phosphoglyceraldehyde to 1,3 diphos- phoglycerate.

ATP + H2O ^ ADP + H3PO4 — 8000 cal

But AF of formation of ATP = +12,000 cal.

The energy conservation or efficiency factor can be calculated in two different ways:

1. How much potential energy-rich chemical compounds are now gained?

a. Ethanol fermentation: —16,000/—56,000, about 29%

b. Lactic fermentation: —24, 000/—52,000, about 46%

2. How much energy of reaction has been utilized as heat of formation of the energy-rich compounds?

a. Ethanol fermentation: 24,000/—56,000, about 43%

b.

Lactic fermentation: 36,000/—52,000, about 69%

1, 3-diphosphoglyceric acid ◄— 3-PhosPhoglyceraldehydes and

Dihydroxy acetone p——

2 (p) Glyceric acid—- ► ( p) Enolpyruvic acid ^ 2ATP ^ Pyruvic acid

Figure 1.1 Anaerobic part of biological oxidation.

The percentage efficiency figures raise doubt about the interpreta­tion. Such efficiency is never achieved by a man-made machine but biological systems can. If we accept the lower figures with a margin, we are conserving no less than 25% of our expenditure in the form of provident fund energy, even under sudden stress, i. e., anaerobic conditions.

Let us look at the situation when a reduced coenzyme is regenerated or oxidized (brief and simplified):

-ATP — ATP — ATP + і O2

NADH (H+)——- > FAD——- > Cytochrome——- ► Cytochrome—— ► H2O

Stoichiometrically,

CoIH(H+) + 1 O2 + 3ADP + 3H2PO4 ^ CoI++ 3ATP + 4H2O

Similarly in the oxidative part, through the tricarboxylic acid cycle, the major aspects may be represented as in Fig. 1.2.

From alpha ketogluterate to succinate, 1 mole of energy-rich phos­phate in the form of guanosine triphosphate (GTP) is gained. Succinate to fumarate mediated by FAD coenzymes generates two equivalents of ATP. In the rest of the events, 4 sets of reduced Co I, when regenerated, give rise to 4 X 3 = 12 equivalents of ATP. In the entire sequence of events, from pyruvate plus oxaloacetate into citrate/isocitrate and finally back to oxaloacetate, a total of 15 equivalents of energy-rich phosphate bonds (ATP) are gained.

In combining the anaerobic part, 2 additional moles of reduced Co I will be reoxidized and 6 ATP equivalents will be regenerated. Starting from glucose-6-P all the way to CO2 and H2O, we see that 2 + 6 + (2 X 15) = 38 equivalents of ATP are gained. The balance of the equation has been

Oxaloactate————— ► Citrate/Isocitrate

Co I

‘ —CO2, Co I

Fumarate -4————————— Succinate GTP

FAD (=2ATP)

Figure 1.2 Tricarboxylic acid cycle (oxidative pathway).

cited earlier. An oxidative pathway is considered to be more effective from a biochemical energetic viewpoint.

One anabolic example of photosynthesis is briefly discussed. Theoretically, reversal of this known reaction should fit well for photo­synthesis:

C6H12O6 + 6O2 > 6(CO2 + H2O) — 686,000 cal

But in fact, we find a slightly different figure. The entire reaction may be symbolically represented as

2H2O + 2NADP+ -—-> 2NADPH (H+) + O2

2 Chloroplast 4 ‘ 2

3CO2 + 9ATP + 5H2O Triosephosphate + 9ADP

+ 6NADPH (H+) + 8H3PO4 + 6NADP+

But the actual stoichiometric presentation shows

n(CO2 + H2O) > ( CH2O)n + nO2 + n(113,000 cal)

almost 22,000 cal higher than expected; fortunately, however, the ender- gonic reaction derives its energy from light energy. These figures are jus­tified because the part of the reaction occurring in the absence of light needs a large excess of energy-rich compounds (ATP). The deficiency of ATP is, however, taken care of by two linked reactions:

Cyclic photophosphorylation:

nADP + nH3PO4 hv > nATP + nH2O

Noncyclic photophosphorylation:

4Feox + 2ADP + 2H3PO4 + 4H2O — h> 4Fered + 2ATP + O2 + 2H2O + 4H+

or 2Co IIred + 2ATP + O2 + 2H2O + 2H+

The deficiency of 1 mole of ATP per mole of CO2 fixed is provided by cyclic photophosphorylation. The other anabolic process is the nitrogen fixa­tion, which is also highly energy consuming.

The heat of formation of NH3 by a chemical pathway can only be determined indirectly. By the Haber process, high pressure and tem­perature is needed and the yield remains very low. So the input in energy in the technological process remains in large excess than the the­oretical heat of formation of NH3.

Nitrogen fixation can take place in nature in two major ways. Molecular nitrogen is converted to oxides of nitrogen in the atmosphere

by electrical discharge and gets into soil by rainwater in the form of nitrites and nitrates. These are reduced to ammonia by the biological nitrogen fixation of symbiotic organisms or by blue-green algae.

In Escherichia coli and Bacillus subtilis, NO 3 is reduced to NH3

[NO—3 ^ NO2—1 ^ N2O2—2 ^ NH2OH ^ NH3]

and an oxidation reduction potential of 0.96 V (pH 7.0) is utilized by these systems to convert other materials to a more oxidized state.

3

NH3 + 2"O2 ^ NO2 + H2O + H+ — 36,500 cal
NO— + 2-O2 ^ NO— — 17,500 cal

2e— 2e— 2e—

N = N———- » HN = NH———- » H2NNH2———- » 2NH3

Via Mo-protein complex

Hydrogen is made available from reduced coenzymes, and the energy
is available from ATP produced by the oxidation of general metabolites.

In some systems, H2 becomes the by-product, and this could be an ideal fuel or it can be used in a suitable chemical cell for the production of energy.

Sugar substances (such as sugarcane juice and molasses), starchy mate­rials (such as wheat, corn, barley, potato, and cassava), and lignocellu — losic materials (such as forest residuals, straws, and other agricultural by-products) are being considered as the raw materials for ethanol pro­duction. The dominating sugars available or produced from these pop­ular raw materials are

■ Glucose, fructose, and sucrose in sugar substances

■ Glucose in starchy materials

■ Glucose from cellulose and either mannose or xylose from hemicel — lulose of lignocellulosic materials

Most ethanol-producing microorganisms can utilize a variety of hex — oses such as glucose, fructose, galactose, and mannose, and a limited number of disaccharides such as sucrose, lactose, cellobiose, and maltose, and rarely their polymers. Therefore, it is necessary to convert the com­plex polysaccharides, such as cellulose and starch, to simple sugars or disaccharides. Different types of substrates that need treatment are presented in Table 3.1, prior to fermentation.

In this section, sugar production from starchy materials is discussed; lignocellulosic materials are discussed in Sec. 3.5.

Ethanol has been well established in the fuel market, where its share from less than 1 GL in 1975 is expected to increase to 100 GL in 2015. Grains, sugarcane juice, and molasses are the dominant raw materials for the time being, while lignocellulosic materials are expected to have a significant share in this market in the future. There have been great achievements in the development of ethanol production from lignocel — lulosic materials, and large-scale facilities are expected to be built within a few years. However, several challenges will still persist in this process in the future, until the process is fully established.

One of the major problems associated with the use of biodiesel is poor low-temperature flow properties, documented by relatively high cloud points (CPs) and pour points (PPs) [1, 2]. The CP, which usually occurs at a higher temperature than the PP, is the temperature at which a fatty material becomes cloudy due to the formation of crystals and solidifi­cation of saturates. Solids and crystals rapidly grow and agglomerate, clogging fuel lines and filters and causing major operability problems. With decreasing temperature, more solids form and the material approaches the PP, the lowest temperature at which the material will still flow. Saturated fatty compounds have significantly higher melting points than unsaturated fatty compounds (Table 5.1), and in a mixture, they crystallize at higher temperatures than the unsaturates. Thus, biodiesel fuels derived from fats or oils with significant amounts of sat­urated fatty compounds will display higher CPs and PPs.

Besides the CP (ASTM D2500) and PP (ASTM D97) tests, two test methods for the low-temperature flow properties of petrodiesel exist, namely, the low-temperature flow test (LTFT) (used in North America; e. g., ASTM D4539) and cold filter plugging point (CFPP) (used outside North America; e. g., the European standard EN 116). These methods have also been used to evaluate biodiesel and its blends with No. 1 and 2 petrodiesel. Low-temperature filterability tests were stated to be nec­essary because of better correlation with operability tests than the CP or PP test [31]. However, for fuel formulations containing at least 10 vol% methyl esters, both LTFT and CFPP are linear functions of the CP [32]. Additional statistical analysis have shown a strong 1:1 correlation between LTFT and CP [32].

Several approaches to low-temperature problems of esters have been investigated, including blending with petrodiesel, winterization, additives, branched-chain esters, and bulky substituents in the chain. The latter approach may be considered a variation of the additive approach, as the corresponding compounds have been investigated in biodiesel at additive levels. Blending of esters with petrodiesel will not be discussed here.

Numerous, usually polymeric, additives were synthesized and reported mainly in the patent literature to have the effect of lowering the PP or sometimes even the CP. A brief overview of such additives has been presented [33]. Similarly, the use of fatty compound-derived mate­rials with bulky moieties in the chain [34] at additive levels has been investigated. The idea associated with these materials is that the bulky moieties in these additives would destroy the harmony of the crystal­lizing solids. The effect of some additives appears to be limited because they more strongly affect the PP than the CP or they have only a slight influence on the CP. The CP, however, is more important than the PP for improving low-temperature flow properties [35].

The use of branched esters such as isopropyl, isobutyl, and 2-butyl esters instead of methyl esters [36, 37] is another approach for improv­ing the low-temperature properties of biodiesel. Branched esters have lower melting points in the neat form (Table 5.1). These esters showed a lower TCO (crystallization onset temperature), as determined by dif­ferential scanning calorimetry (DSC) for the isopropyl esters of soybean oil (SBO) by 7-11°C and for the 2-butyl esters of SBO by 12-14°C [36]. The CPs and PPs were also lowered by the branched-chain esters. For example, the CP of isopropyl soyate was given as —9°C [7] and that of 2-butyl soyate as — 12°C [36]. In comparison, the CP of methyl soyate is around 0°C [32]. However, in terms of economics, only isopropyl esters appear attractive as branched-chain esters, although even they are more expensive than methyl esters. Branching in the ester chain does not have any negative effect on the CN of these compounds, as dis­cussed above.

Winterization [35, 38, 39] is based on the lower melting points of unsaturated fatty compounds than saturated compounds (Table 5.1). This method removes by filtration the solids formed during the cooling of the vegetable oil esters, leaving a mixture with a higher content of unsaturated fatty esters and thus with lower CP and PP. This procedure can be repeated to further reduce the CPs and PPs. Saturated fatty compounds, which have higher CNs (Table 5.1) than unsaturated fatty compounds, are among the major compounds removed by winterization. Thus the CN of biodiesel decreases during winterization. Loss of mate­rial was reduced when winterization was carried out in presence of cold — flow improvers or solvents such as hexane and isopropanol [39].

In other work [40], tertiary fatty amines and amides have been reported to be effective in enhancing the ignition quality of biodiesel without negatively affecting the low-temperature properties. Also, sat­urated fatty alcohols of chain lengths >C12 increased the PP substan­tially. Ethyl laurate weakly decreased the PP.

7.10.1 Aquatic system impacts

The biological consequences of alcohol spills or leaks into marine water are sensitive to many factors such as scale and duration of the spill, tidal patterns, water currents, flow rate, temperature, and available oxygen. Marine life can tolerate low concentrations of alcohol.

In general, methanol and ethanol are significantly less toxic than gasoline or crude oil. Because alcohols are miscible, volatile, and degrad­able, they are dispersed readily, and diluted and neutralized in aquatic environments. The aquatic environment recovers more rapidly and com­pletely from an alcohol spill than from a gasoline or crude oil spill of the same volume.

While a significant number of scientists are assessing the future of renewable and nonrenewable sources of energy, and their potential use­fulness and costs, a few of them are busy exploring existing storehouses of nature and modifying the renewable resources into direct conventional fuels. Prof. Melvin Calvin and his group at the University of California at Berkeley emphasize the importance of a group of plants which, in addi­tion to producing polysaccharide, also produce polyisoprenes (rubber) and similar associated products [4]. While the Hevea produces rubber, different euphorbiacea produce polyhydrocarbons that have molecular weights lower than 10% of that of average natural rubber. It is likely that chemical manipulation may yield liquid fuels similar to that of conventional gasoline or diesel out of these products.

The interesting aspect of these plants is that rubber plants demand good insolation and high moisture content in soil as well as in the atmos­phere. But many subspecies of Euphorbia can grow comfortably in sunny semiarid lands, where standard cultivations are not economically viable [5]. This leads us to two major considerations: (1) soil conservation, eco­logic improvement, and increase in P/R (productivity/respiratory) ratio; (2) production of hydrocarbon and biomass, both of which have energy value.

Avalois is the North Brazil variety, and Euphorbia tirucalli is the Southern Californian equivalent of the plant. Both of them usually con­tain 30% hydrocarbon in their latex. Similar or parallel plants in the Indian Subcontinent are not yet well known. But like rubber plantation, which successfully migrated from Brazil to Malaysia, one may try a few

species of Euphorbia—particularly on the rocky, arid, or laterite belts, which are rather unproductive for forestry or cultivation. It is worth­while to take a glance at some information already available on these products [6].

Conversion of simple hexose sugars, such as glucose and mannose, in fermentation into ethanol can take place anaerobically as follows:

C6H12O6 (Hexoses) Micr°°rganisms> 2C2H5OH (ethanol) + 2CO2

If the entire sugar is converted into ethanol according to the above reac­tion, the yield of ethanol will be 0.51 g/g of the consumed sugars, mean­ing that from 1.0 g of glucose, 0.51 g of ethanol can be produced. This is the theoretical yield of ethanol from hexoses. However, the ethanol yield obtained in fermentation does not usually exceed 90-95% of the theoretical yield, since part of the carbon source in sugars is converted to biomass of the microorganisms and other by-products such as glycerol and acetic acid [9, 31].

A similar reaction for anaerobic conversion of pentoses, such as xylose to ethanol, might be considered. Xylose is generally converted first to xylulose by a one-step reaction catalyzed by xylose isomerase (XI) in many bacteria, or by a two-step reaction through xylitol in yeasts and fungi. It can then be converted to ethanol anaerobically through a pentose phosphate pathway (PPP) and glycolysis. The general reaction can be written as

3C5H10O5 (Pentoses)———- ii——— * 5C2H5OH (Ethanol) + 5CO2

In this case, we can expect a theoretical ethanol yield of 0.51 g/g from xylose, as we had from glucose. However, the redox imbalance and slow rate of ATP formation are two major factors that make anaerobic ethanol production from xylose very difficult [32, 33]. A few anaerobic ethanol — producing strains have been developed from xylose in research labora­tories, but no strain is so far available for industrial-scale processes. Attempts have been made to overcome this problem of xylose assimila­tion by cometabolization or working with microaerobic conditions, where oxygen is available at low concentrations. A number of microorganisms can produce ethanol aerobically from xylose, where the practical yield of ethanol from xylose and other pentoses is usually lower than its the­oretical yield. The challenges in ethanol production from xylose have been reviewed by van Maris et al. [34].

Crop description. Calophyllum inophyllum—commonly known as nagchampa, ballnut, ati tree, kamani, ndamanu, fetau, Alexandrian laurel, nambagura, Indian laurel, and tamanu oil—belongs to the family Guttiferae and is native to the Indo-Pacific region, particularly Malaysia [112]. This evergreen tree is commonly found in the coastal regions of South India and Madagascar (see Fig. 4.10). It usually reaches up to 25 m high [113]. It tolerates varied kinds of soil, coastal sand, clay, and degraded soil. The average kernel oil content is about 60.1% [114]. The fatty acids present in crude oils are stearic (14.3%), palmitic (13.7%), oleic (39.1%), and linoleic (31.1%) acids [115].

Main uses. It is known best as an ornamental plant. Besides this, its wood is hard and strong and has been used in construction. The seeds yield oil for medicinal use and cosmetics. A number of medicinal and therapeutic properties of various parts of Calophyllum have been described, including the treatment of rheumatism, varicose veins, hem­orrhoids, and chronic ulcers [116]. Fatty acid methyl esters of C. ino­phyllum oil have been found suitable for use as biodiesel that meets biodiesel standards of the United States and European Standards Organization [78].

Figure 4.10 Calophyllum ino­phyllum. (Photo by Forest Starr and Kim Starr, courtesy of the U. S. Geological Society [www. hear. org/ starr/hiplants/images/hires/ html/starr_040711_0232_ calophyllum_inophyllum. htm].)

The estimated amount of good quality and nutritive-value oils and fats used for frying around the world is around 20 million metric tons (MT). In frying, the hot oil serves as a heat exchange medium by which heat is transferred to the material being fried. As a result of frying, the oil darkens from the formation of polar materials such as minor phenolic components; elevated FFA; high total polar materials; compounds having high foaming property, low smoke point, low iodine value, and increased viscosity; and color compounds.

Sims [62] reported has conversion of tallow, a by-product of the meat industry, into esters. The fuel properties of methyl, ethyl, and butyl esters of tallow were similar to diesel fuel, particularly ME, which were remarkably similar except for the higher liquidification temperature of tallow esters. Short-term engine performance tests with methyl, ethyl, and butyl esters gave comparable results as diesel fuel, but at higher BSFC. Blends with diesel in 50-50% proportion by volume gave inter­mediate results between esters and neat diesel fuel.

Richardson et al. [63] have tested an engine with ME of tallow. Preliminary engine tests indicated that the use of 10% and 20% blends (volume basis) performed similar to diesel fuel. However, lubricant qual­ity aspects were not studied and an endurance test was not conducted. The ignition quality of the blend was significantly better than that of diesel. Overall, it was concluded that tallow ME on 10% (volume basis) can be successfully used as diesel fuel where large amounts of tallow are produced and temperatures below 10°C are not encountered. The fuel consumption of ME of used frying oil has been measured by Mittelbach and Tritthard [64]. The ester fuel showed slightly lower hydrocarbon and carbon monoxide emissions but increased oxides of nitrogen, compared with that of diesel fuel. The particulate emissions, however, were sig­nificantly lower for used frying oil. But, they suggest long-term engine testing to prove the quality of this fuel.

The results discussed contribute to a better understanding of the structure—physical property relationships in different fatty acid esters from different vegetable oils which give the desired biodiesel quality and optimal performance of engines.

A. K. Sinha

9.1 Introduction

Global primary energy consumption (i. e., energy used for space heating, transportation, generating electricity, etc.) is expected to triple from about 400 exajoules (EJ = 1018 joules) per year in 2000 to about 1200 EJ/yr in 2050 at the present rate of increase in consumption. However, due to increased energy efficiency of the devices, the actual increase is expected to be about 800-1000 EJ.

More than 80% of the present primary energy requirements are met by fossil fuels. The consequences of burning hydrocarbons at such a large scale for our energy needs are already evident in the form of global warming and its disastrous environmental effects. In order to permit sta­bilization of anthropogenic greenhouse gases, fossil fuel consumption will have to be limited to about 300 EJ/yr by 2050. Hopefully, the con­cern about global warming, limit on fossil fuel supplies, and rise in their prices will force us to gradually decrease the use of fossil fuels in the future. Reducing hydrocarbon consumption to 300 EJ requires carbon — free energy sources to supply the difference ~700 EJ/yr. This shortfall is a problem that requires immediate attention and proactive action for sustainable development.

The need for an efficient, nonpolluting energy source for transportation, large-scale generation, and portable devices has spurred the develop­ment of alternative energy sources. Fuel cells are a promising alternative energy source that fits the above requirements [1-6]. A fuel cell is an electrochemical device that converts the chemical energy of a fuel (hydro­gen, natural gas, methanol, gasoline, etc.) and an oxidant (air or oxygen) into electricity, with water and heat as by-products. Since no combustion

251

is involved in the hydrogen fuel cell process, no NOx are generated. Since sulfur is a poison to fuel cells, it has to be removed from fuel before feed­ing it to a fuel cell; therefore, no SO2 is generated in the fuel cell.

The trend toward portability and miniaturization of computing and communication devices has created a requirement for very small and lightweight power sources that can operate for long periods of time with­out any refill or replacement. Also, advances in the medical sciences are leading to an increasing number of electrically operated implantable devices like pacemakers, which need power supplies to operate for an extremely long duration (years) without maintenance, as any mainte­nance would necessitate surgery. Ideally, implanted devices would be able to take advantage of the natural fuel substances found in the body [7-8]. The idea of a biofuel cell that can generate electricity based on var­ious metabolic processes occurring in our own cells is very appealing. A biofuel cell converts chemical energy to electrical energy by the catalytic reaction of microorganisms. Most microbial cells are electrochemically inactive, and electron transfer from microbial cells to the electrode requires mediators such as thionine, methyl viologen, methylene blue, humic acid, and neutral red. In recent years, mediatorless microbial fuel cells have also been developed; these cells use electrochemically active bacteria (Shewanella putrefaciens, Aeromonas hydrophila, etc.) to transfer elec­trons to the electrode. A major advantage of the biofuel cell over the hydro­gen fuel cell is the replacement of expensive and precious platinum (Pt) as a catalyst by much cheaper hydrogenase enzymes. A brief description of the development and state of the art of hydrogen and biofuel cells is presented in this chapter.