Oxygen: The tolerance of the microorganisms in relation with oxygen classifies them as aerobic (when growth requires oxygen), facultative anaerobes (when growth may occur on oxygen when available but does not require it) and anaerobes, classified further to strictly anaerobes (when oxygen is toxic) and aerotolerant anaerobes (when growth may occur in the presence of oxygen but without utilising it). Strict anaerobes include Clostridia, methanogens, sulphate reducers and homoacetogens. The sensitivity to oxygen varies widely among the strict anaerobes. All bacteria contain enzymes to react with oxygen and produce toxic free radicals that destroy vital cellular components. However, it is the presence of other enzymes that remove the toxic oxygen radicals that determine the degree of tolerance to oxygen. In anaerobic environments the traces of oxygen are rapidly consumed by the facultative anaerobes of the consortium, decreasing the redox potential to acceptable levels (-400 mV). For this reason, the facultative anaerobes are usually found in external layers in systems where spatial distribution of the various populations is possible (e. g. lagoons, heterogeneous or hybrid bioreactors).

Ammonia: It is the degradation product of nitrogenous compounds such as proteins and amino acids. Anaerobic digestion of feedstocks such as manure results in the production of high amounts of ammonia. Non-ionised ammonia is quite inhibitory to methanogens. Since the concentration of non-ionised ammonia is a function of pH, the inhibition is less at neutral pH. There are contradictory reports on the levels of tolerance to ammonia, due to differences in substrates, inocula, environmental conditions and acclimation periods. The inhibitory total ammonia concentration causing a 50% decrease in the methane production ranges from 1.7 to 14 g/L (Chen et al., 2008). Acclimation plays a significant role in making the anaerobic consortium tolerant to high levels of ammonia (Bhattacharya and Parkin, 1989; Angelidaki and Ahring, 1993).

Long chain fatty acids (LCFAs) and other organic compounds: LCFAs tend to be adsorbed on surfaces and interfere with the molecule transfer mechanisms or the protection functions of the cell wall or membrane. Moreover, flotation of biomass can occur as a result of the adsorption of LCFA (Rinzema et al., 1989). The inhibition of LCFA in thermophilic anaerobes is more severe because of the different composition of their cell membranes (Hwu and Lettinga, 1997). Biodegradation of LCFAs, although difficult, has been observed in mesophilic and thermophilic conditions.

Other organic compounds which have been found to be toxic to anaerobic digestion are: alkyl benzenes, halogenated benzenes, nitrobenzenes, phenol and alkyl phenols, halogenated phenols, nitrophenols, alkanes, halogenated aliphatics, alcohols, halogenated alcohols, aldehydes, ethers, ketones, acrylates, carboxylic acids, amines, nitriles, amides, pyridine and its derivatives (Chen et al., 2008). The extent of toxicity depends on several factors such as the toxicant concentration, microorganism concentration, toxicant exposure time, cell age, feeding pattern, acclimation and temperature (Yang and Speece, 1986).

Metals: They can be distinguished into light and heavy metals. Light metals are present in the form of cations in solution and, in the case of anaerobic digesters, they usually include sodium, potassium, calcium and magnesium. They are usually added in the form of chemicals for pH control, but they can also arise from the breakdown of biomass. They are required for microbial growth at moderate concentrations, but they can cause severe inhibition or even toxicity at high concentrations (Soto et al., 1993).

Heavy metals (e. g. chromium, iron, cobalt, copper, zinc, cadmium and nickel) can be present in significant concentrations in municipal sewage and sewage sludge as well as in industrial wastewaters. Several metals such as iron, zinc, nickel, cobalt, molybdenum and copper are constituents of vital enzymes. Due to the non biodegradability of heavy metals, they tend to biosorb and accumulate at toxic concentrations. Apart from sorption, the heavy metals may be precipitated (reacting with sulphide, carbonate or hydroxyls) or form complexes in solution with degradation compounds produced during digestion. However, only metals in soluble free ionic form exhibit toxicity (Mosey and Hughes, 1975; Oleszkiewicz and Sharma, 1990). Therefore, immobilisation of heavy metals can take place through processes such as precipitation, sorption and chelation. The relative sensitivity of acidogenesis and methanogenesis to heavy metals is Cu > Zn > Cr > Cd > Ni > Pb and Cd > Cu > Cr > Zn > Pb > Ni, respectively (Lin, 1992, 1993).

Sulphide and sulphate: The presence of sulphate in the absence of oxygen causes anoxic conditions since it can be used as an electron acceptor instead of oxygen. The sulphate reducing bacteria can utilise a number of substrates (acetate, hydrogen, propionate, butyrate) in anaerobic systems and, therefore, they compete with the groups of microorganisms that degrade the same substrates. As a result the flow of electrons is diverted mostly to sulphide instead of methane production reducing the efficiency of the anaerobic systems (in terms of biogas production).

Sulphide is toxic to methanogens but also to the sulphate reducing bacteria. There is a great discrepancy in the literature concerning the mechanism of inhibition and the toxicity levels of sulphides (Chen et al., 2008). Sulphide removal can take place through stripping, coagulation, oxidation, precipitation but also through biological processes such as oxidation to sulphur (Oude Elferink et al., 1994; Song et al., 2001). Acclimation to sulphide can also be beneficiary to methanogens, increasing their tolerance levels.

Land-use change (LUC) is probably the most controversial issue associated with biofuels (Fargione et al., 2008). The main concern is related to possible additional GHG emissions when carbon stored in the soil is disturbed and released as CO2 due to the LUC. Two types of LUC are considered: direct and indirect. Direct LUC involves the conversion of existing land from a current use to the cultivation, in this instance, of biomass feedstocks for biofuel production. As shown in Table 3.2, direct LUC is considered in most international approaches and the IPCC factors are used for these purposes (IPCC, 2007). These are summarised for selected countries in Table 3.3.

Indirect LUC is associated with the displacement of existing agricultural activity (Searchinger et al, 2008). This is often difficult to assess due to the uncertainties involved, particularly at the international level. Currently, only the US approach considers indirect land-use change (US EPA, 2009).

An illustration of the influence of direct LUC for biodiesel from rapeseed is given in Table 3.4. Based on the assumptions used in this example (Fehrenbach et al., 2007), biodiesel from rapeseed can provide GHG savings of 48% compared to diesel. However, if direct LUC occurs, the saving drops to below ten per cent. Given that most countries require significantly higher GHG savings (see Table 3.2), it is important to ensure that biofuels can still meet these requirements if LUC is involved.

1Assumes worst case — conversion of land with high carbon content. Source: Fehrenbach et al. (2007).

Textbox

Therefore, 91.6% of GHG emissions are allocated to RME and 8.4% to glycerine so that:

GHG emissions allocated to RME: 0.307 kg CO2 eq. x 0.916

= 0.28 kg CO2 eq.

GHG emissions allocated to glycerine: 0.307 kg CO2 eq. x 0.084

= 0.027 kg CO2 eq.

2. Energy-content based allocation

Total energy content of RME and glycerine based on their respective LHVs:

37 MJ/kg x 1 kg RME + 17 MJ/kg x 0.092 kg glycerine = 38.76 MJ

Allocation factor:

RME: (37 x 1)/38.76 x 100 = 96%

Glycerine: (17/0.0.092)/38.76 x 100 = 4%

Therefore, 96% of GHG emissions are allocated to RME and 4% to glycerine so that:

GHG emissions allocated to RME: 0.307 kg CO2 eq./38.76 MJ x 1000 GJ x 0.96

= 7.6 kg CO2 eq./GJ RME

GHG emissions allocated to glycerine: 0.307 kg CO2 eq./38.76 MJ x 1000 GJ x 0.04

= 0.32 kg CO2 eq./GJ glycerine

Converting these back to per mass output from the process, the GHG emissions allocated to:

RME: 7.6 kg CO2 eq./GJ RME x 38.76 MJ/1000 GJ = 0.29 kg CO2 eq.

Glycerine: 0.32 kg CO2 eq./GJ RME x 38.76 MJ/1000 GJ = 0.012 kg CO2 eq.

Comparing these to the mass allocation, the results for RME are similar but by a factor of two different for glycerine. Although arguably in the example presented here, the differences in the results due to the different allocation methods are small, in many cases they can be much larger and can affect the LCA results significantly. It is therefore important that sensitivity analysis is carried out to determine the influence on the results of different allocation methods.

In addition to LUC, different crop management practices can also influence emissions of carbon from soil. However, there is still considerable uncertainty and lack of knowledge regarding the loss from or sequestration of carbon in soils due to this. It is also unclear how temperature increase due to climate change might alter farm management practices (and other activities) and what effect that will have on the change of carbon in soils (Baker et al, 2007; Bellamy et al., 2005; Davidson and Janssens, 2006).

A recent study in the UK found that the Soil Organic Carbon (SOC) stocks are being depleted at an alarming rate due to a combined effect of these factors. The measurements of SOC on 6000 sites across all types of land use over the past 25 years have shown that the estimated annual losses of carbon are equal to 13 million tonnes. This is equivalent to eight per cent of the UK emissions of CO2 in 1990 and as much as the entire UK reduction in CO2 emissions achieved between 1990 and 2002 (Bellamy et al, 2005).

It is widely believed that soil disturbance by tillage was a primary cause of the historical loss of SOC in North America. It is also believed that substantial SOC sequestration can be accomplished by changing from conventional ploughing to less intensive methods known as conservation tillage (Baker et al., 2007). However, some studies have demonstrated that conservation tillage leads to higher concentrations of S OC near the surface while conventional tillage accrues more S OC in deeper soil layers. Long-term measurements have also been unable to detect carbon gain in soil due to reduced tillage. Overall, although there are other good reasons to use conservation tillage, there is no proven evidence that it promotes carbon sequestration in soil (Baker et al., 2007).

Similarly, adding fresh organic matter (e. g. crop residues, compost, livestock manure and green manure) is widely practised as a way of increasing the nutrient levels in soils. However, there is also evidence that this may stimulate microbiological activity in the soil and can lead to the decomposition of ancient carbon buried in deep soil layers (Davidson and Janssens, 2006).

Fruit and vegetable wastes present low total solids and high volatile solids and are easily degraded in anaerobic digesters. The rapid hydrolysis of these feedstocks may lead to acidification of the digester and the consequent inhibition of methanogenesis. Many carbohydrate-rich feedstocks require either co-digestion with other feedstocks or addition of alkaline buffer to ensure stable performance (Wieger et al., 1978). A solution may be provided by two-stage reactors that use the first stage as a buffer against the high organic loading rate, which offers some protection to the methanogens. Separation of the acidification process from methanogenesis by the use of sequencing batch reactors has shown to give high stability, a significant increase in biogas production and an improvement in the effluent quality when used with fruit and vegetable waste (Bouallagui et al, 2004).

To minimize substrate diffusion limitations, enzymes are usually attached on non­porous materials. However, the non-porous supports exhibit low enzyme loading capabilities (Chen and Su, 2001). On the other hand, porous materials have high enzyme loading capabilities, but suffer from a high limitation of substrate (Hayashi et al., 1993). In order to minimize the substrate diffusion limitation and enhance the enzyme loading at the same time, nano-size particles have been receiving great attention in recent years due to their large interfacial area and unique physical properties. Nanoparticle materials have been used in various bioprocesses including enzyme immobilization. For example, Tang et al. (2007) immobilized lipase onto nano-sized biopolymer Chitosan particles.

On the other hand, due to their high mechanical strength and thermal resistance, polyacrylonitrile (PAN) were used to generate electrospun nanofibrous membranes, which were used as the support for immobilizing C. rugosa lipase (Li and Wu, 2009). Lipase was bound covalently to PAN nanofibers ranged from 150 to 300 nm by amidination and used in a membrane reactor. The reactor was used for hydrolysis of oil and could also be used for biodiesel production.

Nano-sized magnetite (NSM) particles have been used as support for immobilization of enzymes. In addition to the larger surface area, due to the nano­size used, immobilization on magnetite materials allows easy enzyme recovery from the medium under the magnetic force, due to the magnetic response of the support material. Hence, there is no need for expensive liquid chromatography systems, centrifuges or filters. However, efficient loading of enzymes onto nano-sized magnetite (NSM) particles requires the surface functionalization by polymerization or sol-gel entrapment, which reduces the magnetic response of NSM particles (Lee et al., 2009). To avoid this limitation, Huang et al. (2003) immobilized lipase covalently to NSM particles. However, the covalent binding results in structural changes that can greatly reduce the activity of the enzyme. Therefore, coordinating NSM particles with a low molecular weight ligand has been proposed to overcome the abovementioned problem as the attachment would be via physical adsorption in this case, rather than by covalent bonding (Lee et al., 2009). At the same time, the particle sizes do not increase, as when the NSM particles are wrapped with polymers (Ma et al., 2003). In addition, the ligand acts as a spacer between NSM and the immobilized enzyme to prevent direct contact of lipase to the surface of the magnetites that may hinder the flexible enzyme structure. The immobilized lipase on the NSM particles showed higher specific activity and thermal stability than the free one and the activity of the immobilized lipase remained almost constant over five uses and recoveries (Bastida et al., 1998). The stable reuse as well as the convenience in the recovery offered by magnetic separation ensures that a surface-modified NSM particle is a good support material for lipase immobilization.

Model organism for the solventogenic clostridia is C. acetobutylicum. The metabolism of this organism is well understood, and the genes and enzymes needed for butanol production are already identified and characterized (see below and Fig. 10.2). C. acetobutylicum typically performs a biphasic fermentation, often referred to as ABE (for acetone/butanol/ethanol) fermentation (Durre, 2005a; Jones and Woods, 1986).

During exponential growth (in the so-called acidogenic phase or acidogenesis),

C. acetobutylicum follows the standard butyric acid pathway producing acetate, butyrate, CO2 and H2 (see Fig. 10.2). In addition, small amounts of ethanol and

Table 10.1 Butanol-producing microorganisms

Genus Species Reference

Clostridium septicum Clostridium chavoei — Clostridium aurantibutyricum Clostridium tetanomorphum

Clostridium sporogenes Clostridium pasteurianum Clostridium carboxidivorans — Butyribacterium methyotrophicum — T. thermosaccharolyticum Hyperthermus butylicus

10.1 Relationship of butanol-producing microorganisms. Tree was created with Ribosomal Database Project (Cole et al., 2007) on the basis of 16s rRNA gene sequences.

Acetaldehyde —

Thiolase (Th1A)

Acetoacetyl-CoA

I 3-Hydroxybutyryl-CoA dehydrogenase (Hbd) 3-Hydroxybutyryl-CoA

I Crotonase (Crt)

Crotonyl-CoA

I Butyryl-CoA dehydrogenase (Bed)

► Butyraldehyde —

Butyraldehyde dehydrogenase Butanol dehydrogenase (AdhE) (AdhE, BdhA/B)

10.2 Catabolic pathways of acid and solvent formation in Clostridium acetobutylicum. The single reactions shown do not represent stoichiometric fermentation balances.

acetoin are formed successively, and under certain conditions, lactate is produced as well. Typically, about twice as much butyrate is formed compared to acetate. Accumulation of the excreted acids causes a rapid decrease in pH of the surrounding medium. This poses a serious threat to C. acetobutylicum, since anaerobic bacteria are unable to maintain a constant internal pH, which is generally 1 unit higher than the external pH (Durre et al., 1988; Gottwald and Gottschalk, 1985; Huang et al., 1985). When the external pH drops to the critical point of 4.5, considerable levels of undissociated acetic and butyric acid are present (pKa of acetic acid = 4.75 and pKa of butyric acid = 4.82), which can then pass the cytoplasmatic membrane via diffusion. Due to the higher internal pH, these acids dissociate into salts and protons again and thus destroy the essential proton gradient across the membrane needed for energy conservation and several transport mechanisms.

To avoid this deleterious effect, a major metabolic shift takes place in C. acetobutylicum at the end of exponential growth. The organism takes up acetate and butyrate and converts these organic acids into the solvents acetone and butanol, respectively (solventogenic phase or solventogenesis; see Fig 10.2). A butanol/acetone ratio of 2:1 is typical for C. acetobutylicum, whereas some strains of C. beijerinckii form isopropanol instead of acetone. While the reassimilation of
the excreted acids leads to an increased pH, the solvents acetone, isopropanol and, especially, butanol are also toxic for the cell (see Section 10.5). However, the cell gains enough time to initiate the formation of endospores and thus secures long-time survival.

Hydrogen production by cyanobacteria and microalge through photosynthesis can be represented by the following reactions:

In indirect biophotolysis, the electrons are derived from water by photoautotrophic cells. As presented in reactions [13.3] and [13.4], the process consists of two stages in series: the first one is photosynthesis for carbohydrate accumulation and the second one, is dark fermentation of the endogenous carbohydrates for hydrogen production. In this way, the oxygen and hydrogen evolutions may be temporally and/or spatially separated (Benemann, 1996). This separation not only avoids the incompatibility of oxygen and hydrogen evolution (e. g. enzyme deactivation and the explosive property of the gas mixture), which are key barriers to direct biophotolysis, but also makes hydrogen purification relatively easy, because CO2 can be conveniently removed from the generated H2/CO2 mixture (Belafi-Bako et al., 2006).

Cyanobacteria have attracted more research interest for hydrogen production via indirect biophotolysis than microalgae. Such cyanobacteria species include Anabaena sp., Spirulina sp., marine cyanobacteria such as Calothrix sp., Synechococcus sp. and Geobacter sp. Anabaena cylindrical is a well-known hydrogen producing cyanobacterium, but Anabaena variabilis has received more attention in the recent years, because of higher hydrogen yields compared to the other species (Masukawa et al., 2001). Emphasis has been given to increase the activity of hydrogen producing enzymes and to develop mutants of Anabaena sp. to increase the rate of hydrogen production. However, at the present time, the hydrogen production rate by Anabaena sp. is considerably lower than that obtained by dark or photo­fermentations which are described below (Pinto et al., 2002; Liu et al., 2006a).

Nowadays, indirect biophotolysis, just like direct biophotolysis, is an immature technology, applied only at laboratory scale. It should be noted that indirect water photolysis, is under active research and development, since several factors are still crucial for further improvement in technology. Environmental conditions, such as light, temperature, salinity, nutrient availability and gas atmosphere (the presence of oxygen, nitrogen or methane) play an important role in the hydrogen production efficiency (Dutta et al., 2005). In addition, in order to improve hydrogen production rates and yields using cyanobacteria, methods such as screening of wild-type strains possessing highly active hydrogen evolving enzymes (nitogenases and/or hydrogenases) (Pinto et al., 2002), or genetic modification of strains to increase the hydrogenase activity, are under investigation. Finally, optimization of cultivation conditions such as light intensity, pH, temperature, and nutrient content, will contribute to increased H2 production.

Sunflower (Helianthus annuus L.), a member of the Compositea family, is an important oilseed crop worldwide (Fig. 4.2), yielding approximately 45-50% oil and the quality depending on the region (Pereyra-Irujo et al., 2009). The feasibility of sunflower oil used as a raw material for biodiesel production has been extensively researched in Spain, including homogeneous and heterogeneous catalysts (Vicente et al., 1998, 2004, 2005; Antolin et al., 2002; Arzamendi et al., 2006, 2008; Ramos et al., 2008). Auxiliary energies, such as low frequency ultrasonication, have been proposed to enhance the reaction yield in transesterification reactions using ethanol (Georgogianni et al., 2008).

Diesel engine tests have also been performed showing a power loss up to 10% when the engine was run on biodiesel (Kaplan et al., 2006). However, the use of blends with diesel fuel up to 30% biodiesel reported no significant changes in BSFC (Neto da Silva et al., 2003). CO2, CO and NOx emissions seem to be lower than those of diesel fuel (Ilkilic, 2008). The use of straight sunflower oil in

an indirect injection diesel engine also exhibits exhaust emissions reduction and no negative effects on the engine performance (Canakci et al, 2009).

Biodiesel can be produced on industrial scale using a batch or continuous process. The most suitable oils are produced from soybean (USA, Latin America), rapeseed (EU) and palm (Southeast Asia) oils. Refined vegetable oils are the best resource due to the high conversion of triglycerides into esters in a short time of reaction. Nearly only methanol is used to the lowest price and the easiest production process is employed. The most commonly used catalysts are NaOH, KOH, NaOCH3 with a loading in the range between 0.3% and 1%. The operating temperature is 60-70°C and molar ratio of CH3OH:oil is 6:1. In a batch process, the oil is charged in the reactor followed by the catalyst and methanol addition. After stirring, the reaction mixture is settled, centrifuged or pumped to another vessel in order to separate the glycerol layer from the biodiesel. The methanol is recovered from the ester layer and glycerol by flash evaporation.

The ester is neutralized by diluted acid, washed with water and dried under vacuum. The glycerol is neutralized, the FFAs separated and eventually refined for further use. In many cases the batch process is carried out in a two-step reactor.

An example of this is the Lurgi technology, in which most of the glycerin is separated at the first reactor supplied with esterification column for the separation of the excess of methanol and glycerin. Biodiesel produced after the second reactor is treated in a wash column to remove the glycerin and methanol.

Biodiesel production can also be carried out in a continuous process using tubular system as in the Desmet Ballestra biodiesel technology. This technology is characterized by its integrated feedstock pre-treatment and transesterification. Crude oils and fats are first pretreated to meet certain preset quality standards and are then processed in the standard transesterification process. This approach allows the processing of a whole range of feedstocks, including traditional biodiesel feedstocks (rapeseed, soybean, palm oil and sunflower) but also alternative and/or lower quality feedstocks (animal fat, used cooking oil, jatropha oil, etc.). A typical Desmet Ballestra biodiesel plant configuration (including pre-treatment section) is outlined in Fig. 5.17.

The Desmet Ballestra biodiesel process technology uses three reactors in series which operate under mild conditions (temperature of 55°C and atmospheric pressure) (Fig. 5.18). Pre-treated feedstock is continuously fed to the first loop reactor 1 together with methanol and catalyst (NaOCH3). Methanol is added in a proper excess compared to the required stoichiometric amount in order to maximize the degree of transesterification and to minimize soap formation. Loop reactor 1 has a settling zone in the bottom part from which spent glycerin is continuously discharged. The reacted light phase overflows to the second loop reactor 2 where fresh methanol and catalyst are added. Loop reactor 1 and 2 are identical and operate under the same conditions. The light phase leaving the

5.17 Desmet Ballestra biodiesel process.

second loop reactor consists of almost fully converted biodiesel. It is transferred to a third stirred-tank (safety) reactor in which the final conversion takes place.

8.3.1 General remarks

When various sugars or similarly metabolized compounds (e. g. glycerol, polysaccharides, etc.) are utilized for the production of SCO, accumulation of lipid in the microbial cells or mycelia (the so-called ‘de novo’ lipid accumulation process) is triggered by exhaustion of nitrogen from the growth medium, which allows the conversion of sugar to storage lipid (Ratledge, 1988, 1994; Ratledge and Wynn, 2002; Wynn and Ratledge, 2006; Papanikolaou and Aggelis, 2009;

Fakas et al, 2009b). In contrast, when growth is conducted on hydrophobic carbon sources (e. g. fats, oils), accumulation of storage lipids (the so-called ‘ex novo’ lipid accumulation process) is a primary anabolic process occurring simultaneously with the production of lipid-free material, being independent from the nitrogen exhaustion in the medium (Fickers et al., 2005; Papanikolaou and Aggelis, 2010).

In the case of SCO utilization for biodiesel production, research interest is focused only upon the process of de novo lipid accumulation. In this case, there is continuously increasing interest upon the potentiality of transforming abundant renewable materials (like waste glycerol, flour-rich waste streams, cellulose and hemicellulose hydrolysates, etc.) into SCO that will be further transformed into biodiesel. The process of ex novo lipid accumulation aims at adding value to low-cost fatty materials so that speciality high-value lipids (e. g. cocoa-butter or other exotic fats substitutes) will be produced (Papanikolaou et al., 2001; 2003; Papanikolaou and Aggelis 2003a, 2003b, 2010).

The lipids produced by oleaginous microorganisms are mainly composed of neutral fractions [principally triacylglycerols (TAGs) and to lesser extent steryl — esters (SEs)] (Ratledge, 1994; Ratledge and Wynn, 2002). As a general remark it must be stressed that when growth is carried out on various hydrophobic substances, the microbial lipid produced contains lower quantities of accumulated TAGs compared with growth elaborated on sugar-based substrates (Koritala et al., 1987; Guo et al., 1999; Kinoshita and Ota, 2001; Papanikolaou et al., 2001, 2002a; Fakas et al. 2006, 2007, 2008a). In any case, accumulation of storage lipids is accompanied by morphological changes in the oleaginous microorganisms, since ‘obese’ cells with large lipid globules can generally appear during the lipid-accumulating phase (Figure 8.1). Storage lipids, unable to integrate into

phospholipid bi-layers, cluster to form the hydrophobic core of the so-called ‘lipid bodies’ or ‘oil bodies’ (Mlickova et al., 2004a, 2004b). Lipid bodies of the oleaginous Y. lipolytica yeast are illustrated in Figure 8.2. As previously stressed, the biochemical pathways of de novo and ex novo lipid accumulation process present fundamental differences. These differences will be presented, explained, clarified and comprehensively discussed in the following sections.

Methanol is the simplest alcohol. It has the chemical formula (CH3OH) and is more volatile and more toxic than ethanol. Traditionally, methanol has been considered wood alcohol, because it was produced by pyrolysis of wood. In theory, pyrolysis of wood could be considered biomethanol, because it is producing the alcohol-based fuel from a biological source (wood). However, the term biomethanol is typically used to describe methanol produced from one of two methods: Fischer Tropsch reaction of syn gas or biomethane. A common source of biomethane is landfills. The process of making biomethanol from landfill gas and syn gas is a cost-intensive chemical process. Although in theory this is a large source of energy, because current residues/waste by-products from agricultural and forest products amount to approximately one-third of the total commercial energy use.6

In general, we would break biomethanol production into three methods: syn­gas, bio-gas/bio-methane, and carbohydrates. Syn-gas contains carbon monoxide (~30vol%), hydrogen (25-30vol%), carbon dioxide (20-30vol%), methane (~10vol.%), and ethane (~3vol.%). Typically, purification and/or gas conditioning are needed before syn-gas can be catalytically converted to biomethanol. The catalyst used in the reactor for methanol synthesis is typically copper oxide, zinc oxide, or chromium oxide.7 The two standard chemical reactions for methanol synthesis at these catalysts are shown below.

CO + 2H2 « CH3OH

CO2 + 3H2 « CH3OH + H2O

Both of these reactions are exothermic and result in a loss of moles of gas, so Le Chatelier’s principle would dictate that the reaction is favored by high pressure and low temperature. Side products can be produced and need to be considered if depending on the purification needed of the methanol product. Side products could include dimethyl ether, formaldehyde, or more complex alcohols.7 These side products may decrease the energy density of the fuel as well as the toxicity.

Biogas or biomethane that is captured from landfills is often times called landfill gas. Landfill gas is typically considered to be the same as natural gas, but it is not. Natural gas is more than 80% methane and landfill gas is typically 40-60% methanol with the remaining gas being mostly carbon dioxide. The EPA predicts that each pound of biodegradeable waste in the landfill will produce 10-12 standard cubic feet of gas over a 25-year period.8 This landfill gas accounts for 34% of the methane emissions,8 so it is clearly a large source of methane that could be tapped for fuel purposes. The overall reaction for methanol production from landfill gas is:

CH4 + H2O « CH2OH + H2O

However, this process is not direct. The landfill gas is reformed to syn gas after a pretreatment to remove sulfur compounds and a compression to 400 psi and then the syn gas is reacted to methanol and purified.8

The general method for biomethanol production from carbohydrates is shown in Fig. 11.1 where carbohydrates are gasified and partially oxidized to hydrogen and carbon monoxide which is used to catalytically produce methanol via the same catalytic methanol synthesis method described above for syn gas.7 Clearly, biomass is more complex than syn gas, so more pretreatment, gas cleaning, and gas conditioning is needed. In general, pretreatment involves chipping to a size below 5 cm and drying, whereas gas cleaning involves removing tars, soot, alkali metals, BTX (benzene, toluene, and xylenes), and inorganic impurities (HCl, ammonia, HCN, H2S, and COS).7 Gas conditioning is involved in getting rid of the methane and other hydrocarbons in the gas as well as altering the ratios of CO:CO2:H2 if necessary via the water gas shift reaction, amine stripping of carbon dioxide, or other CO2 scrubbing methods. Steam reforming of methane (and other light hydrocarbons) over nickel catalysts will form carbon monoxide and hydrogen,7 which then can be used directly for producing methanol. Overall, this is an area of research that appears to be the future of considering methanol as a biofuel, but currently researchers in the United States are more focused on ethanol as a fuel due to volatility and toxicity issues. Methanol also has lower volumetric and gravimetric energy density, which limits its usefulness for portable or transportation power.