Butanol can be produced by chemical synthesis. One process is Oxo-synthesis, which in­volves the reaction of propylene with carbon monoxide and hydrogen in the presence of co­balt or rhodium as the catalyst. The mixture of n-butyraldehyde and isobutyraldehyde are obtained and then the mixture can be hydrogenated to the corresponding n-butanol and iso­butyl alcohols (Park, 1996).The reactions are as following:

CH3CH2CH2CHO + H2 ® CH3CH2CH2CH2OH (a)

(CH3 )2 CHCHO+H2 ® (CH3 )2 CHCH2OH (b) (2)

When using cobalt as the catalyst, the reaction processes at 10~20MPa and 130~160°°C, the products ratio of n-butyraldehyde and isobutyraldehyde is 3. Rhodium as the catalyst used in industry from 1976 and the reaction processes at 0.7-3MPa and 80-120°°C. The products ratio of n-butyraldehyde and isobutyraldehyde can reach 8-16. Hydrogenaration processes by using the catalyst of nickel or copper in gaseous phase or nickel in liquid phase. Some by­products can be transferred into butanol at high temperature and high pressure that will en­hance the product purity.

Another route is aldol condensation, which involves the reaction of condensation and dehy­dration from two molecules of acetic aldehyde. And then, the product crotonaldehyde was transformed into n-butanol by hydrogenation at 180°°C and 0.2MPa. The reaction is as fol­lowing: ch3ch=chcho+2h2—> ch3ch2ch2ch2oh

Comparing the two processes, Oxo-synthesis route has the advantages of materials easily obtained, comparable moderate reaction conditions, enhanced ratio of n-butanol to isobutyl alcohol. So, Oxo-synthesis process is the main industrial route for n-butanol production. There are also some other fossil oil derived raw materials such as ethylene, propylene and triethylaluminium or carbon monoxide and hydrogen are used in butanol production (Zver- lov, et al., 2006).

As shown in Figure 3, fatty acid biosynthesis interfaces with the primary metabolism at the acetyl-CoA node. Fatty acid biosynthesis is initiated by the formation of acetoacetyl-ACP, the substrate for fatty acid chain elongation. The conversion of acetyl-CoA to acetoacetyl-ACP includes two key enzymatic steps: (1) the conversion of acetyl-CoA to malonyl-CoA, catalyzed by acetyl-CoA carboxylase (ACC) and (2) the conversion of malonyl-ACP to acetoacetyl-ACP via p-ketoacyl-ACP synthase III (KASHI). These two enzymes are common metabolic engi­neering targets for improving fatty acid biosynthesis. In fact, ACC has been shown to be a rate — limiting step of fatty acid synthesis in Escherichia coli, and overexpression of ACC has been shown to yield more than a 5-fold increase in FFA production [5]. Overexpression of KASIII in E. coli also improved FFA synthesis, increasing lipid production by 20-60% [6]. After acetoacetyl-ACP formation, fatty acid chain elongation proceeds by an iterative process, whereby the hydrocarbon chain is elongated in increments of 2 carbons. Once the elongation process terminates, the final acyl-ACP is divided among three possible paths: one leading to membrane biosynthesis, an essential pathway for cell growth, and the other two yielding hydrocarbon fuels or fuel precursors (Figure 3).

To produce biofuels with an even-numbered carbon chain, the acyl-ACP is cleaved by a thioesterase (TE), releasing the FFA. The TE is yet another key target for metabolic engineering. The final fuel properties, including viscosity, cloud point, flash point, oxidative stability, ignition delay, and combustion quality, are largely determined by the hydrocarbon chain length and degree of saturation [7]. Accordingly, numerous TEs have been cloned and characterized, predominantly from plant sources, to control the carbon chain length of the FFAs. Engineering strategies often exploit this collection of TEs to tailor the biofuel product. Favored TEs include a truncated TE (‘tesA) from E. coli and acyl-ACP TEs from Umbellularia californica and Cuphea hookeriana, producing FFAs with carbon lengths of 16:0, 12:0, and 10:0 and 8:0, respectively [8—10]. The FFAs themselves can be extracted as fuel precursors and converted into biodiesel (FAMEs or FAEEs) using acid-catalyzed chemical processes [11]. To allow for FFA accumulation, the p-oxidation pathway and free fatty acid recycling are often eliminated by gene knockout of acyl-CoA synthetase (acs) and acyl-ACP synthetase (aas) [12]. An alternative strategy was recently demonstrated, whereby FFAs were synthesized through an engineered reversal of the p-oxidation cycle [13]. In this strategy, acetyl-CoA is used directly for fatty acid chain elongation, allowing for improved carbon and energy efficiency compared to the fatty acid biosynthesis pathway which requires activation of acetyl-CoA to malonyl — CoA. Engineering a reversed p-oxidation cycle required modification of multiple regulatory mechanisms, knockout of other fermentative pathways, expression of a TE or other fuel producing enzyme, and overexpression of key enzymes in the p-oxidation pathway [13]. While this strategy yielded the highest reported concentration of FFAs in E. coli (7 g/L), its application to other host organisms may be restricted by inadequate knowledge of the native regulatory mechanisms.

With an intact acs, FFAs can be converted into acyl-CoA, a precursor for other fuel products including the biodiesel precursor, TAG, and fuels such as FAEEs and fatty alcohols (Figure 3). The conversion of acyl-CoA to TAG requires the provision of 1,2-diacylglycerol and a diacylglycerol acyltransferase (DGAT) to catalyze transfer of the acyl chain. While DGAT has been overexpressed to improve TAG production in plants [14], the utility of this strategy still remains to be tested in microorganisms. Most metabolic engineering strategies for microbial TAG synthesis focus on improving the supply of the precursors: FFA and glycerol-3-phosphate (G3P) [15, 16]. Microbial production of FAEEs typically involves heterologous expression of both the pathway for ethanol production and an acyltransferase (AT) [17—19]. Selection of the two genes required for ethanol synthesis, pyruvate decarboxylase (pdc) and alcohol dehydro­genase (adh), will largely depend on the host organism, but generally, efforts involving prokaryotic hosts such as E. coli and cyanobacteria will use pdc and adh from Zymomonas mobilis due to their capacity for high ethanol production [20]. To date, only one AT has been heterologously expressed for FAEE production: the wax synthase gene (aftA) from Acineto- bacter baylyi ADP1 [17—19]. A third biofuel product derived from acyl-CoA is fatty alcohols. The enzymatic conversion of acyl-CoA to a fatty alcohol is dependent upon whether the fatty acyl-CoA reductase (far) is of prokaryotic or eukaryotic origin. Most prokaryotic FARs reduce acyl-CoA to a fatty aldehyde, requiring another enzyme, fatty aldehyde reductase (ALR), for conversion to the fatty alcohol product. On the other hand, eukaryotic FARs catalyze the direct conversion of acyl-CoA to fatty alcohol without release of an aldehyde intermediate [21]. Metabolic engineering strategies for fatty alcohol production include: expression of a pro­karyotic FAR, acr1 from Acinetobacter calcoaceticus BD413, with reliance on native fatty alde­hyde reductases for fatty alcohol synthesis [19]; expression of 5 different eukaryotic FAR homologs from the model plant organism Arabidopsis thaliana [22]; and expression of a eukaryotic FAR, far1 from mouse [23]. The recent discovery of a prokaryotic FAR from Marinobacter aquaeolei VT8, capable of catalyzing the direct conversion of acyl-CoA to fatty alcohol, may be a beneficial alternative to the use of eukaryotic FARs for fatty alcohol pro­duction in prokaryotic hosts such as E. coli and cyanobacteria [24]. An alternative strategy used by Dellomonaco and colleagues identifies surrogates for far and adh in the native E. coli genome based on sequence homology [13]. With the numerous biofuel products derived from acyl — CoA and the natural enzymatic diversity for these conversions, we have only just begun to explore and develop the metabolic engineering tools essential to enable large-scale synthesis.

In addition to oxygen-containing biofuels, acyl-ACP can also be converted into pure hydro­carbon fuels in the form of alkanes and alkenes (Figure 3). In 2010, the discovery of an alkane synthesis pathway in cyanobacteria provided the genetic knowledge necessary for engineering microbial alkane production [25]. The pathway consists of two enzymatic steps: (1) reduction of acyl-ACP to a fatty aldehyde by means of an acyl-ACP reductase (AAR) and (2) decarbon — ylation of the aldehyde to an alkane or alkene, catalyzed by an aldehyde decarbonylase (ADC). Due to the recent discovery of this pathway, few metabolic engineering strategies have been applied for alkane production. Some strategies focus on improving supply of the acyl-ACP precursor, relying on the native cyanobacterial pathway for alkane synthesis [23], while others have simply transferred the alkane pathway (AAR and ADC) into another host organism [25—27]. With the rapidly growing database of genome sequence information, numerous homologs of AAR and ADC have been identified [26, 27], representing a diverse range of targets for metabolic engineering. Future optimization of the alkane biosynthesis pathway may result in the high alkane yields needed for biofuel production.

Glycerol hydrogenolysis to PDOs consists of hydrogen addition and removal of one oxygen atom in the form of H2O. In order to design efficient catalysts, it is fundamental to under­stand the mechanism of this reaction. Three main reaction mechanisms have been proposed in the literature, depending on whether the reaction runs on acid or basic catalytic sites and with or without the formation of intermediate compounds:

i. dehydrogenation — dehydration — hydrogenation (glyceraldehyde route),

ii. dehydration — hydrogenation,

iii. direct glycerol hydrogenolysis.

Below, the main features of each mechanism will be discussed

1.3.2. First stage

First and second phase: The quantity and percentage of wastes generated at the Central Wholesaler of Antioquia during the year 2011 are show at the Table 3 and Figure 6. The highest production of wastes was associated to cabbage and lettuce leaves then wastes of citrics (orange and lemon) and finally wastes of mango, guava and others tropical fruits. The Figure 7 shows some pictures of wastes in the storage containers at the Central Wholesaler of Antioquia. With the information of production were selected wastes of cabbage and lettuce leaves, orange, mango, papaya and guava to be employed at the bioprocess.

Third phase: The elemental analysis of wastes selected is show at the Table 4. To orange wastes, the relation C/N obtained was less than values reported in others research. In the others cases the results were close to values reported for wastes with similar characteristics. A relation C/N close to 30 is considered appropriate to growth of anaerobic bacteria [22].

Organics waste

■ Lettuce and cabbage leaves

■ Orange and lemon Pimento, cucumber

■ Mango

■ Tomato

The chemical composition analysis of wastes showed that the highest values of volatile solids were found in the tropical fruits (mango, orange, guava and papaya). The volatile solids are the proportion of the raw material that bacteria using to generate biogas and have an out­standing role during the anaerobic fermentation process.

In order to determinate the quantity of wastes to be used was obtained the density of each wastes, to this were taken samples and then were triturated, weighed and finally was calcu­lated the volume to employ. The bioreactor was loaded with 422 kilograms of wastes and 1110 kilograms of water, this provided an average relation (wastes: water) of 1:2,5.

Each waste was triturated and mixed with water during three minutes until to reach an average size of 2 centimeters. In order to reduce the quantity of methanogenic bacteria, the wastes were submitted to acidic conditions during three months with a value of pH close to 3,5. Afterwards was added during three days agricultural lime until to reach a pH of 6,2; in that moment the production of biohydrogen started. The quantity of agricultural lime added was 7 kilograms (Figure 9).

The organics load showed an important reduction during the process, the total suspend solids were reduced in 83%, the chemical oxygen demand was reduced in 65% and the biochemical

oxygen demand was reduced in 63,6%. The environment temperature was between 21,8 у 31 °C, this meanthat the biohydrogen productionwas developed under mesophilic conditions. The averagereiativehumiditywas between 38y 73%.

Table 7. Organic load of wastes at the first stage (Laboratory of Sanitary Engineering, National University of Colombia)

80 70 60 E 50

40

30

І 20 10

6 11 16 21 26 31 36 41 46 51 56 61 66 71 16 80 8l 00 9h day

Humedad relativa

Figure 10. Behavioroftemperatureandrelativehumidityaverage

The gas production started three days after application of agricultural lime and continued for 22 days more. The hydrogen (biohydrogen) percentage found in gas ranged between 6,37 y 17,26; with a percentage of hydrogen less than 13,3; there was carbon dioxide and nitrogen in the biogas, however when the percentage of hydrogen was greater than 13,3; the gas compo­sition was only hydrogen and carbon dioxide. The greater value of methane was 1,25% and less was 0%, this mean that the pretreatment to reduce the methanogenic bacteria was satisfactory.

The total production of hydrogen was 177 liters in 22 days, with a maximum value of 14,5 liters, an average of 7,4 liters of H2/day and maximum yield of 83 liters of H^/m3 of bioreactor. The maximum value of generation of hydrogen was registered 7 days after from started the gas production and the maximum rate of hydrogen generation was obtained between first and seventh days. The Figure 12 shows a several pictures of biohydrogen generated, the color blue is from silica gel used to remove the wet of the gas. The quantity of organic load removed was 26.400 mg/liter of O2 (COD).

210 "vT a>

180

150

0 ~o

<D

120 ГО

3

1 90

о (D

60 о

о

30

О

0

Figure 13. Productionaccumulatedofhydrogen

The ABE fermentation is a complicated multistage process with a series of consecutive and parallel reactions influenced by a series of technological factors. The presence or absence of natural constituents or contaminations in the used raw materials has important influence on the productivity and product distribution. The ABE fermentation is controlled by intracellular redox processes which is influenced by a variety of technological conditions.

2.1. General mechanism of ABE fermentation

The ABE fermentation is a two-stage process: first, an acid-producing and then a solvent producing process takes place, but the solvent producing metabolic pathway could be observed only above 20 g/L starting sugar concentration [8]. Key factors in starting of solven — togenesis are the undissociated intracellular butyric acid concentration and the summarized amount of the undissociated butyric and acetic acids within the cells. These are in relationships with the pH and the concentration of butyric and acetic acids in the ferment mash of course, and a boundary condition is that glucose concentration should be above 15 g/L at the moment of the final consumption of butyric acid, because a high glucose flux is required to generate as much amount of ATP as is enough to supply the energy demand of the butyric acid-butanol transformation [9]. Hartmanis et al. studied the pathway for uptake of acids during the solvent formation phase of ABE fermentation by C. acetobutylicum using 13C NMR [10]. Actively metabolizing cells showed that butyrate can be taken up from the medium and quantitatively converted to butanol without accumulation of intermediates. The activities of acetate phos — photransacetylase, acetate kinase, and phosphate butyryltransferase rapidly decreased to very low levels when the organism began to form solvents. This indicates that the uptake of acids does not occur via a reversal of these acid-forming enzymes. No short-chain acyl-CoA synthetase activity could be detected. Apparently, an acetoacetyl-CoA:acetate (butyrate) CoA — transferase is solely responsible for uptake and activation of acetate and butyrate in C. acetobutylicum. The transferase exhibits broad carboxylic acid specificity. The key enzyme in the uptake is acetoacetate decarboxylase which is induced late in the fermentation and pulls the transfer reaction towards formation of acetoacetate. The major implication is that it is not feasible to obtain a batch-wise BuOH fermentation without acetone formation and retention of a good yield of BuOH [10]. Ferredoxin enzymes also play important role in the ABE processes, thus the presence of iron in the appropriate form and concentration is essential factor in the appropriate solvent production. When Clostridium acetobutylicum was grown in batch culture under Fe limitation (0.2 mg/L) at pH 4.8, glucose was fermented to BuOH as the major fermentation end product, and small quantities of HOAc were produced. The final conversion yield of glucose into BuOH could be increased from 20% to 30% by Fe limitation. The BuOH- acetone ratio was changed from 3.7 (control) to 11.8. Hydrogenase specific activity was decreased by 40% and acetoacetate decarboxylase specific activity by 25% under Fe limitation. Thus, Fe limitation affects C and electron flow in addition to hydrogenase [11].

Terracciano and Kashket investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undis­sociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM [12]. C. acetobutylicum ATCC 824 cells harvested from a phosphate-limited chemostat culture maintained at pH 4.5 had intracellular concentrations of acetate, butyrate and butanol which were 13-, 7- and 1.3- fold higher, respectively, than the corresponding extracellular concentrations. Cells from a culture grown at pH 6.5 had intracellular concentrations of acetate and butyrate, which were only 2.2-fold higher than the respective external concentrations. The highest intracellular concentrations of these acids were attained at pH 5.5. When cells were suspended in anaerobic citrate-phosphate buffer at pH 4.5, exogenous acetate and butyrate caused a concentration — dependent decrease in the intracellular pH, while butanol had relatively little effect until the external concentration reached 150 mM. Acetone had no effect at concentrations <200 mM. These data demonstrate that acetate and butyrate are concentrated within the cell under acidic
conditions and thus tend to lower the intracellular pH. The high intracellular butyrate concentration presumably leads to induction of solvent production thereby circumventing a decrease in the intracellular pH great enough to be deleterious to

Harris et al. suggested [14] that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum. Determination of BuP and acetyl phosphate (AcP) levels in various C. acetobutylicum strains (wild(WT), M5, a butyrate kinase (buk) and a phosphotran — sacetylase (pta) mutant) showed that the buk mutant had higher levels of BuP and AcP than the wild strain; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production [15]. Consistently with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains. For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry wt.), and higher and sustained levels corresponded to higher butanol formation fluxes. The BuP levels never exceeded 40 to 50 pmol/g (dry wt.) in strain M5, which produces no solvents. The BuP profiles were bimodal, and there was a second
peak midway through solventogenesis that corresponded to carboxylic acid reutilization. AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutiliza­tion. As expected, in the pta mutant AcP levels were very low, yet this strain exhibited strong butanol prodn. These data suggest that BuP is a regulatory mol. that may act as a phospho — donor of transcriptional factors. DNA array-based transcriptional anal. of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress

The toxicity of accumulated butanol and the intermediates is a very important feature of the ABE fermentation. Costa studied [16] the growth rates of Clostridium acetobutylicum in presence of BuOH, EtOH, Me2CO, acetate and butyrate. Acetate and butyrate were the most toxic compounds, with concentrations of 5 and 8.5 g/L, respectively, stopped the cell growth. An EtOH concentration of 51 g/L or 11 g BuOH/L reduced cell growth by
50%. Acetone did not inhibit cell growth at 29 g/L, thus ethanol and acetone were non­toxic at a normal fermentation. Some mutant strains, however, more tolerant towards bu­tanol, for example Lin and Bladchek [17] obtained a derivative of C. acetobutylicum ATCC 824 which grew at concentrations of BuOH that prevented growth of the wild — type strain at a rate which was 66% of the uninhibited control. This strain produced con­sistently higher concentrations of BuOH (5-14%) and lower concentrations of acetone (12.5-40%) than the wild-type strain in 4-20% extruded corn broth. Characterization of the wild-type and the mutant strain demonstrated the superiority of the latter in terms of growth rate, time of onset of BuOH production, carbohydrate utilization, pH resist­ance, and final BuOH concentration in the fermentation broth [17]. Moreira et al. [18] ini­tiated a fundamental study attempting to elucidate the mechanism for BuOH toxicity in the acetone-BuOH fermentation by Clostridium acetobutylicum. Butanol as a hydropho­bic compound inserted into the membrane increases the passive proton flux, forms a "hole" for proton on the membrane. This eliminates hydrogen ions form the cell and the intracellular pH increases. The strains which are able to decrease the membrane fluidity are more resistant towards butanol. The cells have deacidifying mechanism to keep the intracellular pH value at 6 when the pH value of the ferment liquor is located between 4 and 5 can reduce acids into alcohols, which increases their butanol producing ability. Lepage et al [19] studied the changes in membrane lipid composition of C. acetobutyli­cum during ABE fermentation. Large changes were found in phospholipid composition and in fatty acid composition, the latter characterized mainly by a decrease in the unsa- turated/saturated fatty acid (U/S) ratio.

Effects of the addition of alcohols (EtOH, BuOH, hexanol, and octanol) and of acetone were also studied. In all cases, large changes were observed in the U/S ratio, but with differences which were related to the chain length of the alcohols. The effect of solvents appears to account for a large part of changes in lipid composition observed during the fermentation. The pH was also important, a decrease in pH resulting in a decrease in the U/S ratio and in an increase in cyclopropane fatty acids. The effect of increasing temperature was mainly to increase fatty acid chain lengths [19].

3. General conditions of the ABE fermentation

Optimal conditions of ABE fermentation strongly depend on many factors such as the selected raw materials or their composition, and are essentially influenced by the selected strain as well. Furthermore, a series of important factors can decrease or increase the yield and changes the distribution of the ABE solvents even with the same raw material or bacterium strain. Some selected pieces of information are summarized below.

The catalytic hydrotreatment of liquid biomass converts the contained triglycerides/lipids into hydrocarbons at high temperatures and pressures over catalytic material under excess hydrogen atmosphere. The catalytic hydrotreatment of liquid biomass process is quite simi­lar to the typical process applied to petroleum streams, as shown in Figure 3. A typical cata­lytic hydrotreatment unit consists of four basic sections: a) feed preparation, b) reaction, c) product separation and d) fractionation.

In the feed preparation section the liquid biomass feedstock is mixed with the high pressure hydrogen (mainly from gas recycle with some additional fresh make-up hydrogen) and is preheated before it enters the reactor section. The reactor section consists normally of two hydrotreating reactors, a first guard mild hydrotreating reactor and a second one where the main hydrotreating reactions take place. Each reactor contains two or more catalytic beds in order to maintain constant temperature profile throughout the reactor length. Within the re­actor section all associated reactions take place, which will be presented in more detail at a later paragraph.

The reactor product then enters the separator section where, after it is cooled down, it enters the high pressure separator (HPS) flash drum in which the largest portion of the gas and liq­uid product molecules are separated. The gas product of the HPS includes the excess hydro­gen that has not reacted within the reactor section as well as the side products of the reactions including CO, CO2 H2S, NH3 and H2O. The liquid product of the HPS is lead to a second flash drum, the low pressure separator (LPS), for removing any residual gas con­tained in the liquid product, and subsequently is fed to a fractionator section. The fractiona­tor section provides the final product separation into the different boiling point fractions that yield the desired products including off-gas, naphtha, kerosene and diesel. The heaviest molecules return from the bottom of the fractionator into the reactor section as a liquid recy­cle stream.

In order to improve the overall efficiency, a liquid recycle stream is also incorporated, which in essence consists of the heavy molecules that were not converted. The gas product from the HPS and LPS, after being treated to remove the excess NH3, H2S, CO and CO2, is com­pressed and fed back to the reactor section as a gas recycle stream in order to maintain a high pressure hydrogen atmosphere within the reactor section.

Gas

-►Naphtha

-►Kerosene

-►Diesel

Figure 3. A typical process diagram of catalytic hydrotreatment of liquid biomass

1.1. Reaction mechanisms

Several types of reactions take place during catalytic hydrotreatment of liquid biomass, based on the type of biomass processed, operating conditions and catalyst employed. The types of reactions that liquid biomass undergoes during catalytic hydroprocessing include: a) cracking, b) saturation, c) heteroatom removal and d) isomerization, which are described in more detail in the following section.

1.1.1. Cracking

As the molecules included in the various types of liquid biomass can be relatively large and complicated, cracking reactions are desired to convert them into molecules of the size and boiling point range of conventional fuels, mainly gasoline, kerosene and diesel. A character­istic reaction that occurs during catalytic hydrotreating of oils / fats is the cracking of trigly­cerides into its consisting fatty acids (carboxylic acids) and propane as shown in Scheme 1 [5][6]. This reaction is critical as it converts the initial large triglycerides molecules of boiling point over 600°C into mid-distillate range molecules (naphtha, kerosene and diesel).

Other cracking reactions may take place however such as those described in Schemes 2 and 3, depending on the type of molecules present in the feedstock. For example Scheme 2 is a cracking reaction which may occur during catalytic hydrotreatment of pyrolysis oil which includes polyaromatic and aromatic compounds. Alternatively Scheme 3 may follow deoxy­genation of carboxylic acids on the produced long chain paraffinic molecules, leading to smaller chain paraffins, during the upgrading of Fischer-Tropsch wax.

O

ch2-o-c-r

I

Ch-o-co-r + 2h2 —► 3r-ch2cooh + ch3-ch2-ch3

CH2-O-C-R

O

Triglyceride carboxylic acid propane

Scheme 1.

Scheme 2.

R — R’ + H2

Scheme 3.

1.1.2. Saturation

Saturation reactions are strongly associated with catalytic hydrotreating as the introduction of excess hydrogen allows the breakage of double C-C bonds and their conversion to single
bonds, as shown in the following reactions. In particular the saturation of unsaturated car­boxylic acids into saturated ones depicted in Scheme 4, is a key reaction occurring in lipid feedstocks. Furthermore other saturation reactions lead to the formation of naphthenes by converting unsaturated cyclic compounds and aromatic compounds as in Scheme 5 and 6, which are likely to occur during upgrading of pyrolysis oils.

RCH = CH-COOH + H2 —► rch2ch2cooh

Scheme 4.

Scheme 5.

Scheme 6.

As a result of this reaction the produced saturated molecules are less active and less prone to polymerization and oxidation reactions, mitigating the sediment formation and corrosion phenomena appearing in engines.

Armando T. Quitain, Shunsaku Katoh and Motonobu Goto

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/52539

1. Introduction

Recent concerns about climate change and problems associated with the use of fossil-de­rived fuels and nuclear energy have inspired researchers to seriously explore environmen­tally benign and economically viable renewable energy and fuels. As potential solution to reduce fossil-derived carbon dioxide (CO2) emissions from gasoline-run automobiles, addi­tion of biomass-derived oxygenates was proposed. Bioethanol has been considered, howev­er, ether oxygenates such as ethyl tert-butyl ether (hereby referred to as ETBE), has gained popularity over ethanol (EtOH) due to its superior properties which blend well with gaso­line [1]. ETBE also outranks MTBE as an octane enhancer due to its low blending Reid vapor pressure. Moreover, ETBE is a better option because it is derived from EtOH which can be obtained from biomass. ETBE is produced from the reaction of isobutene (IB) and EtOH, however, the current supply of IB, which is mostly derived from non-renewable crude oil, may not be sufficient to cope up with the expected high demand in the future. For this rea­son, alternative routes for its synthesis are also currently being explored. tert-Butyl alcohol (TBA), which can also be derived from biomass can be employed instead of IB [2]. Research for the development of efficient and energy-saving methods for the production of these gas­oline oxygenates had gained significant momentum over the past few years. The application of microwave technology was proposed for the synthesis of the above mentioned gasoline oxygenate.

Microwave technology relies on the use of electromagnetic waves to generate heat by the os­cillation of molecules upon microwave absorption. Unlike the conventional heating, the heat is generated within the material, thus rapid heating occurs. Other than the advantages of rapid heating, microwave effects on reaction likely occur, thus obtaining dramatic increase

in the yield even at low temperatures. The benefits of using microwave irradiation to the synthesis of the abovementioned biofuels include energy efficiency, development of a com­pact process, rapid heating and instant on-off process (instant heating-cooling process), among others.

In this chapter, syntheses of ether oxygenates by microwave irradiation will be discussed and summarized, focusing on our recent studies on microwave-assisted heterogeneously catalyzed processes.

Lipids are a general set of cellular components that are grouped together by the common trait that they are soluble in non-polar solvents. Throughout living organisms, there are sev­eral sources of lipids that play various roles in biochemical processes including energy stor­age and water insoluble nutrient transport across cell membranes that include neutral lipids, phospholipids, steroids, waxes, and carotenoids. Since lipids have a generally low oxygen and high carbon and hydrogen content, they are very energy dense molecules. This charac­teristic, along with their natural abundance and similarities with petroleum based fuels, make them ready targets for processing and use as a blend or replacement to traditional fuels.

Neutral lipids (commonly referred to as "fats"), which are widely regarded as one of the most common sources of lipids, and which has the highest potential for use as an alternative fuel, can be found in various forms throughout different organisms, and will be the primary topic of focus for this discussion. Most marine and aquatic biomass can store lipids within the cell that can range from a small fraction to upwards of 80% of the cellular weight. Due to this trait, research and production scale operations have been centered on utilizing aquatic biomass for lipid production and conversion to fuel with the remaining cellular components being recycled for mineral content or discarded.

Scheme 1. Transesterification reaction schematic

Figure 2 shows an example of a marine diatom Niztchia sp. stained with Nile Red fluores­cence stain (red color shows chlorophyll and yellow shows lipid fluorescence).

Neutral lipids consist of a glycerol molecule (a three carbon alcohol) and one to three fatty acids (referred to as mono-, di-, or tri — acylglycerols depending on number of fatty acids present) with the fatty acids being various carbon chain lengths and having various levels of unsaturation (unsaturated, mono-unsaturated, poly-unsaturated, etc.). Fatty tissues in ani­mals serve as both an energy storage mechanism as well as a means of insulation against temperature extremes. Algae primarily store fats in the cell membrane to serve as an energy storage medium as well as a nutrient transport system to shuttle metabolites into and out of the cell. Several studies have been conducted to attempt to identify the distribution of fatty acids in algae and other aquatic biomass [13—15].

The partial pressure of syngas components have a major influence on microbial growth and product profiles because the enzymes involved are sensitive to substrate exposure [194]. Due to the low solubility of CO and H2 in water, the growth of dense bacterial cell cultures can face mass transfer limitations, so increasing the partial pressure of gaseous substrates can help alle­viate this problem. For instance, studies in which the CO partial pressure (PCo) increased from 0.35 to 2.0 atm showed that this resulted in a 440% increase in maximum cell density, a signifi­cant increase in ethanol productivity and a decrease in acetate production in C. carboxidivorans strain P7 [195]. In another study involving C. ljungdahlii, the increase of PCO from 0.8 to 1.8 atm had a positive effect on ethanol production, and the microbe did not exhibit any substrate in­hibition at high PCO [196].In less CO-tolerant microorganisms, the effect of increasing PCO parti­al pressure range from non-appreciable in the case of Rhodospirillum rubrum [197], to negative impact on doubling time of Peptostreptococcus productus (now: Blautia product) [194] and Eubac — terium limosum [198]. Similar to CO, the increase in partial pressure of H2 (pH2) to 1700 mbar en­hanced acetate productivity of A. woodii to 7.4g acetate/ l/day [199].

Qureshi reviewed the ABE fermentation in various types of reactor systems and recovery by gas stripping with 13 references. Gas stripping is a simple technique which does not require expensive apparatus, does not harm the culture, does not remove nutrients and reaction intermediates and reduces butanol toxicity (inhibition). As a result of butanol removal by gas stripping, concentrated sugar solutions can be used to produce ABE solvents. Compared to sugar utilization of 30 g L-1 in a control batch reactor, sugar utilization of 199 g L-1 has been reported with 69.7 g L-1 solvent production. In fed-batch reactors concentrated sugar solutions (350 g L-1) have been used. Additionally, the process of ABE production results in concentrated product streams containing 9.1-120 g L-1 ABE solvent. In the integrated ABE production and recovery systems, selectivity figures of 4-30.5 have been reported [118]. The effect of factors such as gas recycle rate, bubble size, presence of acetone, and ethanol in the solutions or broth were investigated in order to remove butanol from model solution or fermentation broth. Butanol stripping rate was found to be proportional to the gas recycle rate. In the bubble size range attempted (< 0.5 and 0.5-5.0 mm), the bubble size did not have any effect on butanol removal rate. In C. beijerinckii fermentation, ABE productivity was reduced from 0.47 g L-1 h-1 to 0.25 g L-1 h-1 when smaller (< 0.5 mm) bubble size and an excessive amount of antifoam (to inhibit production of foam caused by smaller bubbles) were used. This suggested that fermentation was negatively affected by antifoam [119].

Gas stripping can be performed by using fermentation gases (H2 and CO2) formed during fermentation. Concentrated sugar solutions (250-500 g/L) were used in continuous fermenta­tion of Clostridium beijerinckii BA101, which operated for 21 d (505 h), producing 460 g acetone-BuOH/L [120]. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose L-1 and produced 17.6 g total solvents L-1 (yield 0.39 g g-1, productivity 0.29 g L-1 h-1). Using integrated fermentation-gas stripping product recovery system with CO2 and H2 as carrier gases, the fed-batch reactor was operated for 201 h. At the end of fermentation, an unusually high concentration of total acids (8.5 g L-1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 L culture broth. The average solvent yield and productivity were 0.47 g g-1 and 1.16 g L-1 h-1, respectively [121]. Using a potential industrial substrate (liquefied corn starch, 60 g L-1) in a batch process integrated with gas stripping resulted in the production of 18.4 g L-1 ABE solvents, with 92% utilization of sugars present in the feed. In a fed-batch reactor fed with saccharified liquefied corn starch, 81.3 g L-1 ABE was produced as compared to 18.6 g L-1 in the control. In this integrated system, 225.8 g L-1 corn starch sugar (487% of control) was consumed. In absence of product removal, it is not possible for C. beijerinckii BA101 to utilize more than 46 g L-1 glucose [122].