In order to reduce the cost of enzymatic production of biodiesel, the lipase producing whole cells rather than the isolated enzyme has been used. This eliminates the need for isolation and purification steps before immobilization, which results in a considerable reduction in the cost. Air drying immobilization technique of lipase producing Rhizopus oryzae whole cells was developed by Matsumoto et al. (2001). The use of immobilized whole cells to produce biodiesel by three stepwise addition of methanol in solvent-free system was reported to achieve biodiesel yield of 71% after 165 h.

Hama et al. (2004) found that the fatty acid composition affects the activity of the whole cells by influencing their membranes. It was reported that pretreatment of the whole cells with oleic acid and linoleic acid resulted in higher enzymatic activity, whereas palmitic acid pretreated cells showed higher stability. To compensate for both activity and stability, an optimum ratio of unsaturated to total fatty acids of 0.67 was proposed. Using the pretreated whole cells, methanolysis yields were consistently above 55% even after ten repeated cycles. To explain the fatty acids composition effect, Hama et al. (2006) suggested the existence of two types of lipases: one bound to the cell wall, which plays role in stability, and the other to the cell membrane, which plays role in methanolysis activity. The increase in enzyme activity with addition of unsaturated fatty acids was expected to be due to the increase in the production of membrane-bound lipase.

The immobilized lipase producing whole cells from R. oryzae were prepared in cuboidal polyurethane foam biomass support particles in a 20 L air-lift batch cultivation bioreactor and used in a packed-bed reactor for continuous production of biodiesel by methanolysis of soybean oils (Hama et al., 2007). Compared with methanolysis reaction in a shaken bottle, the packed-bed reactor enhanced repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The flow rate of reaction mixture had to be optimized, as low flow rates resulted in a significant decrease in activity due to the covering of the immobilized whole cells with a hydrophilic layer of high methanol concentration, and high flow rates resulted in cells leaching. A highest biodiesel yield of 90% was achieved at a flow rate of 25 L h-1. The yield dropped to around 80% after the tenth cycle. To overcome the leaching problem, cross­linking treatment with 0.1% glutaraldehyhde has been proposed (Ban et al., 2002). By glutaraldehyde treatment, biodiesel yield of 83% was maintained after six batch cycles in the stepwise methanol addition process, compared to only 50% without glutaraldehyde treatment.

Recently, Tamalampudi et al. (2008) used the same immobilized whole cells prepared by Hama et al. (2007) for the production of biodiesel from relatively low
cost, inedible oil from the seeds of Jatropha curcas in a 50 ml screw-capped vessels with reciprocating shaking at 150 rpm. The activity of immobilized whole cell was compared with that of Novozym 435 and was found to be more efficient. The maximum biodiesel was 80% after 60 h using the former catalyst, whereas using the latter the maximum yield was only 76% after 90 h.

In enzymatic hydrolysis the cellulose structure is selectively converted to glucose by enzymes. The biomass has to be pre-treated, e. g. with a short, dilute acid hydrolysis step in which the structure of the cellulose is disrupted and the hemicellulose is broken down into fermentable sugars. The cellulose is then broken down by cellulases into cellobios which in turn is cleaved by b-glucosidase into glucose. The sugar losses are minimal and the amount of by-products is negligible. An optimal enzyme activity and optimal reaction conditions such as temperature (45-50°C) and pH (4.8) will increase the ethanol yield. An optimal amount of substrate has a positive effect on the reaction rate and sugar yield as well. There are two types of enzymatic hydrolysis: SHF and SSF. There are benefits and drawbacks with both methods, however, the enzymatic hydrolysis method is considered the best method to date of producing ethanol from biomass.

SHF: In the SHF method the pre-treated material is neutralised and subjected to the enzymatic activity of the cellobios and b-glucosidase. After the hydrolysis has stopped, the solid material is filtered off and the hydrolysate is fermented and then distilled. The major drawback of this method is that the enzyme activity is inhibited by the product of its work: cellobios and glucose. This means that the sugar concentration is limited to approximately 6%, giving a maximum theoretical ethanol concentration after fermentation of 3%. This is not economically viable because the cost of distillation increases dramatically when the ethanol concentration drops below 4%. The benefit is that the temperature during hydrolysis can be kept at an optimal level and that the yeast cells can be recovered after fermentation.

SSF: To solve the problem of the low concentration of ethanol in SHF, SSF mixes the pre-treated material with both enzymes and yeast. This means that as soon as glucose is formed, the yeast will consume it and produce ethanol. The result is that the enzymes never ‘sense’ a high glucose or cellobiose concentration giving a higher ethanol concentration. The major drawback is that during hydrolysis the temperature must be held at 35°C due to the presence of the yeast cells, slowing the hydrolysis down and the yeast cells cannot be recovered. Since there is solid material together with the yeast cells neither centrifugation nor filtration is an option for separating the yeast for recovery. Both processes have common drawbacks also: first enzymatic hydrolysis is relatively slow compared to a thermochemical process. This means that the reaction vessels in a large scale production unit will be very large with challenges with agitation and temperature control. Secondly the enzymes are presently too expensive to make the process economically viable. However, it is generally agreed that the cost of the enzymes will drop drastically when large scale production has started. Cellulase can be produced by fungi and bacteria under aerobic and anaerobic conditions and the microorganisms can be both mesophilic or thermophilic. The cellulase can be recovered after the reaction which will improve the yield of the hydrolysis and reduce the enzyme cost (Balat, Balat, and Oz, 2008; Sun and Cheng, 2002).

Biogas consists primarily of methane and carbon dioxide, but also smaller amounts of hydrogen sulphide, ammonia and traces of hydrogen, nitrogen, carbon monoxide, saturated or halogenated carbohydrates and oxygen may be present. The biogas is usually saturated with water vapour and may also contain particles and siloxanes. The energy content is determined by the methane content (1 kWh per m3 of biogas with 10% of methane).

The biogas can be used in as many applications as the natural gas (heating, combined heat and power systems, fuel cells).There may be different specifications for biogas to be used in different applications, especially, when biogas is to be used in stationary appliances or to be fed to a pipeline grid. The biogas needs purification to improve its quality in most cases.

Hydrogen sulphide and its oxidation products are the major ‘contaminants’ of the biogas (corrosive) with a maximum permitted concentration of 5 ppm.

Hydrogen sulphide reacts with most metals. Conditions of high pressure and temperature (prevailing during storage or usage of biogas) favour the reactivity of this contaminant. Sulphur dioxide also lowers the dew point (temperature to which a given volume of gas must be cooled, at constant barometric pressure, for water vapour to condense into water) in the stack gas. There are biological methods for hydrogen sulphide removal that can be applied in the anaerobic digester as well as other physicochemical methods applicable after biogas has been collected. The biological methods include the supply of small air amounts to activate the sulphide oxidising microorganisms (Thiobacillus) grown in a micro-aerophilic environment on CO2 (autotrophic). The hydrogen sulphide is converted to elemental sulphur but also to sulphate. A combination of biological filter (containing sulphide oxidising microorganisms) and a water scrubbing step can be used alternatively. Physicochemical methods include usage of iron containing compounds (iron chloride, iron oxide), activated carbon, water scrubbing, dimethylether of polyethylene glycol (or selexol) scrubbing and NaOH scrubbing. Iron chloride can be supplied into the digester which forms iron sulphide (insoluble) and is applied when hydrogen sulphide is produced at high concentrations.

Humidity should also be removed because the presence of water favours the formation of sulphur oxidation products. Water is condensed and frozen under conditions of high pressure during biogas storage.

Carbon dioxide must also be removed if the biogas has to meet the natural gas specifications. Especially if biogas has to be used a vehicle fuel, it must be enriched in methane. Suitable methods for carbon dioxide removal include water absorption, polyethylene glycol absorption (the carbon dioxide is better dissolved in selexol), carbon molecular sieves (a series of carbon columns is used to save energy required for pressure application) and membrane separation (with gas phase in both sides of the membrane — high pressure or with a liquid phase in the one side for absorption of the carbon dioxide while diffusing through the membrane — low pressure). Halogenated compounds (present in landfill biogas) and oxygen (due to air entrance when landfill biogas is collected) must be removed too. The requirements for removal of these constituents are reported in Table 12.3 depending on the biogas usage.

Estimates of biofuel prices are given in Table 3.8. Although these are uncertain, several general points can be drawn from these estimates (The Royal Society, 2008):

• higher oil prices are beginning to make current biofuels commercially more attractive;

• cost reductions through economies of scale are expected for all biofuels, with lignocellulose technologies anticipated to be in the same range as food-crop technologies; and

• the post-tax prices of petrol and diesel fuels in Europe (less so in the USA) are often much higher than the pre-tax costs of biofuels; hence tax credits or other incentives, for example in the form of reductions in excise taxes on biofuels, would have a large effect on substitution.

However, these estimates do not take into account changes in prices and land values that may arise from competing demands from agriculture.

The economic prospects of biofuels will depend on improvements in yields both in the growth of the crops and in the efficiency of the conversion processes. Feedstock costs will also influence biofuel prices.

Although homogeneous catalysis is the traditional and very efficient process to convert lipids into alkyl esters, it has a number of disadvantages. The catalyst cannot be reused and has to be discarded after the reaction. In addition, catalyst residues have to be removed from crude biodiesel using several water washing steps that increases the production cost and complicates the purification of the glycerol. Recently an excellent review was published dealing with heterogeneous
catalyst for biodiesel production (Di Serio et al, 2008). Various processes are available using heterogeneous catalysts which are simplifying the purification costs of the biodiesel and the glycerol. The advantage of heterogeneous catalysis is that the catalyst can be either recovered by filtration and/or decantation or applied in a fixed bed reactor and the post-treatment of the biodiesel and glycerol is easier.

Gliperol® is another biofuel integrating glycerol recently patented by the Industrial Chemistry Research Institute of Warsaw (Poland).59 It is composed of a mixture of three molecules of FAMEs and a molecule of glycerol triacetate (triacetin). It can be obtained after the transesterification of a mole of TG with three moles of methyl acetate using lipases or an ion-exchange acidic resin as catalysts.59-61 When ethyl acetate is used, the corresponding FAEEs with triacetin are obtained,62 following the enzymatic process summarized in Fig. 7.4.

In both processes, enzymatic and acidic, glycerol is not isolated as a by-product but utilized in the form of esters with low-molecular weight carboxylic acids as biofuel components. The methodology to prepare this novel biofuel employing heterogeneous catalyst allows the reduction of biofuel production costs by running the reaction without having to remove the catalyst. This allows to run the process in a continuous manner.59 The process patented by the Industrial Chemistry Research Institute of Warsaw also consists of a post-treatment of the reaction

mixture in order to remove, by distillation, excess of ester acidic alcohol used (ethyl acetate). Removal of the reactant from the mixture after reaction allows the reutilization of the reactant and, consequently, reduces the process costs.

In the case of an enzymatic process, immobilized lipases have been normally employed. Methyl or ethyl acetates can be used as acyl acceptors in the interesterification reaction, and the deactivation of enzyme by glycerol is minimized as no glycerol is produced in the reaction. Moreover, the use of ethyl acetate could be interesting because of the production of ethyl esters (an extra carbon atom) that increases the heat content and the cetane number of the final biofuel. Using ethyl esters instead of methyl esters also decreases the cold and pour points as well as increases the flash and combustion points, which improves cold starts and safety in handling the biofuel.63 Modi et al. have obtained over 90% yield in ethyl esters by using 10% Novozyme 435 as lipase (wt/wt to sunflower oil) at 50°C after 12 hours, using an ethyl acetate/oil molar ratio of 11:1.62 The reusability of the heterogeneous enzymatic catalyst (Novozyme 435) was also investigated in the same study, both in ethyl acetate and in ethanol. The stability of lipases after 12 reaction cycles was found to be constant: 91.3% and 93.7% as relative activity for interesterification and ethanolysis, respectively. Under these optimized conditions, Glyperol® production by enzymatic interesterification of vegetable oils could be technically and industrially feasible, nearly as much as the acidic process proposed by the Industrial Chemistry Research Institute of Warsaw.

A closing favourable point is also the good market of triacetin as a by-product. Triacetin has widespread applications in food, feed, printing, tanning, cigarettes, cosmetics, pesticides and pharmaceutical industries as well as in medical field.

10.5.1 Butanol versus ethanol

Alcoholic fuels are a perfect replacement for gasoline. Most commonly used is bioethanol with an annual production of 17 335.2 million gallons in 2008 (Renewable Fuels Association, 2009). However, biobutanol offers a number of major advantages over bioethanol (Table 10.7). First and foremost, butanol has a

significantly higher energy content and air-fuel ratio (similar to those of gasoline) and thus an increased mileage. Butanol is well suited to existing car engines without any modifications (ButylFuel, 2009), and it can also be mixed with gasoline in any concentration, while ethanol can only be blended up to 85% with gasoline. Another problem of ethanol is its hygroscopic and corrosive nature, which requires transportation in special tanks and blending shortly before use. Butanol, in contrast, can be blended at the refinery and distributed using the existing infrastructure (pipelines, tanks, pumps, filling stations, etc.). Due to the significantly lower vapor pressure, butanol is safer to handle as well.

13.3.1 Feedstocks for fermentative hydrogen production

It is well founded that carbohydrates are the main source of hydrogen during fermentative processes and therefore wastes/wastewater or agricultural residues, rich in carbohydrates, can be considered as potential hydrogen feedstocks (Kapdan and Kargi, 2006). The main criteria for substrate selection are: availability, cost, carbohydrate content and biodegradability. Glucose, sucrose and to a lesser extent starch and cellulose are the fermentation substrates mostly studied in the laboratory (Mizuno et al., 2000; Ueno et al., 2001; Fang et al., 2004). They have been used as model substrates for research purposes due to their easy biodegradability and because they can be present in different carbohydrate-rich wastewaters and agricultural wastes. However, synthetic carbohydrates are expensive raw materials for a pilot or full-scale hydrogen production process and therefore the use of zero-cost, rich in carbohydrates wastes, seems to be ideal in real hydrogen production applications.

Rice, winery, noodle, sugar, and molasses manufacturing, olive pulp and cheese whey are among actual wastewaters that have been studied for hydrogen production at a laboratory scale (Table 13.4). In addition, hydrogen could be produced using as feedstocks complex solid wastes, such as wastes from kitchen, food processing, mixed wastes, and municipal wastes containing along with carbohydrates, proteins and fats. In the later case, the hydrogen conversion efficiencies are low, due to the complex structure of the wastes. In general the hydrogen yield from wastes rich in carbohydrates is higher than those rich in proteins and fats.

Moreover, the rich in sugars energy crops, sugar beet, sugar cane and sweet sorghum, as well as the rich in starch energy grains, corn and wheat are among the most suitable substrates for hydrogen production (Table 13.5). However, the potential to produce hydrogen from the residues remaining after harvesting and processing of these starch or sugar crops, that cannot be further exploited in the food industry chain, is more likely to yield a solution with far better overall prospects for economic and environmental sustainability (Lynd et al., 2005). Hydrogen generated from such feedstocks can be characterized as ‘second — generation hydrogen’ since its production is not competitive to food production, but rather a side product of the food production industry. The agricultural residues contain carbohydrate polymers such as cellulose, hemicellulose and lignin, and thus a pre-treatment process (mechanical, chemical or enzymatic) is always necessary, for solubilization of cellulose and hemicellulose to simple sugars, which could easily be degraded by hydrogen-producing bacteria. Rice and wheat straws, corn stover, wheat bran are some of the lignocellulosic feedstocks used for hydrogen generation (Table 13.5). Figure 13.3 presents different potential feedstocks for hydrogen production.

Thermochemical conversion processes remained largely unexplored until relatively recent despite their important involvement in catalysis.14,15 Catalytic cracking and/or pyrolysis of vegetable oils and biomass were, until very recently, the most common thermochemical processes for the production of fuels and high — added-value chemicals. Lately, other key thermochemical processes have joined these promising technologies, in particular for the production of biofuels. These mostly include a variety of technologies such as biomass gasification to bio­syngas and other biofuels via hydrothermal upgrading (HTU), reforming and/or synthetic pathways (Fischer-Tropsch synthesis [FTS]), production of bioalcohols from biomass gasification, and so on.7

Several feedstocks can be employed in these processes, from virgin vegetable oils (e. g. palm, canola, soybean, etc.) for catalytic cracking to waste oils and fats as well as all different types of biomass, including residual oils, sewage sludge, organic and/or agricultural waste, black liquor and many others.7 The use of the feedstock is highly dependent on the process and the biofuel to be obtained (e. g. steam reforming and HTU biofuels can be prepared from either dry or wet biomass, irrespectively).

Thermochemical conversion processes and technologies are largely complex and varied. They will be fully described in Part IV of the book (Chapters 12-20), so we refer the readers to these chapters for more information.

The Moringaceae is a single-genus family of oilseed trees with 14 known species. Of these, the fast growing, drought-tolerant Moringa oleifera, which ranges in height from 5 to 10 m, is the most widely known and utilised (Morton, 1991). M. oleifera thrives best in a tropical insular climate and is plentiful near the sandy beds of rivers and streams. M. oleifera can tolerate wide rainfall range (25-300 cm per year) and soil pH from 5.0 to 9.0 (Rashid et al, 2008). M. oleifera seeds contain between 33% and 41% w/w of vegetable oil (Somali et al., 1984; Anwar and Bhanger, 2003; Anwar et al, 2005) and are rich in oleic acid (> 70%). M.

oleifera is commercially known as ‘ben oil’ or ‘behen oil’, due to its content of behenic (docosanoic) acid, and possesses significant resistance to oxidative degradation (Lalas and Tsaknis, 2002).

M. oleifera has many medicinal uses and significant nutritional value (Anwar, 2007). A survey conducted on 75 indigenous (Indian) plant-derived non-traditional oils concluded that M. oleifera oil has a good potential for biodiesel production (Azam et al., 2005). Acid pretreatment is needed to reduce the acid value, but the resulting biodiesel exhibits one of the highest cetane number (around 67) found for biodiesel (Rashid et al., 2008).