Gasification process is an extension of the pyrolysis process except that it is conducted at elevated temperature range of 800-1300 °C so that it is more favourable for gas production [20]. The gas stream is mainly composed of methane, hydrogen, carbon monoxide, and car­bon dioxide. Biomass gasification offers several advantages, such as reduced CO2 emissions,

compact equipment requirements with a relatively small footprint, accurate combustion control, and high thermal efficiency. The main challenge in gasification is enabling the py­rolysis and gas reforming reactions to take place using the minimum amount of energy and gasifier design is therefore important [21].

Ogi et al. used an entrained-flow gasifier for OPEFB gasification at 900°C [22]. During gasifi­cation with H2O alone, the carbon conversion rate was greater than 95% (C-equivalent), and hydrogen-rich gas with a composition suitable for liquid fuel synthesis ([H2]/[CO] = 1.8-3.9) was obtained. The gasification rate was improved to be greater than 99% when O2 was add­ed to H2O; however, under these conditions, the gas composition was less suitable for liquid fuel synthesis due to the increase of CO2 amount. Thermogravimetric (TG) analysis suggest­ed that OPEFB decomposed easily, especially in the presence of H2O and/or O2 suggesting that OPEFB is an ideal candidate for biomass gasification. Lahijani and Zainal investigated OPEFB gasification in a pilot-scale air-blown fluidized bed reactor [23]. The effect of bed temperature (650-1050°C) on gasification performance was studied and the gasification re­sults were compared to that of sawdust. Results showed that at 1050°C, OPEFB had almost equivalent gas yield and cold gas efficiency compared with saw dust, however, with low maximal heating values and higher carbon conversion. In addition, it was realized that ag­glomeration was the major issue in OPEFB gasification at high temperatures. This can be overcome by lowering the temperature to 770 ± 20 °C. Mohammed et al. studied OPEFB gas­ification in a bench scale fluidized-bed reactor for hydrogen-rich gas production [24]. The total gas yield was enhanced greatly with the increase of temperature and it reached the maximum value (~92 wt.%) at 1000 °C with big portions of H2 (38.02 vol.%) and CO (36.36 vol.%). The feedstock particle size of 0.3-0.5 mm, was found to obtain a higher H2 yield (33.93 vol.%), and higher LHV of gas product (15.26 MJ/m3). The optimum equivalence ratio (ER) (0.25) was found to attain a higher H2 yield (27.31 vol.%) at 850 °C. Due to the low effi­ciency of bench scale gasification unit the system needs to be scaling-up. The cost analysis for scale-up EFB gasification unit showed that the hydrogen supply cost is $2.11/kg OPEFB. Recently, a characterization and kinetic analysis was done by Mohammed et al. and it was found that a high content of volatiles (>82%) increased the reactivity of OPEFB, and more than 90% decomposed at 700 °C; however, a high content of moisture (>50%) and oxygen (>45%) resulted in a low calorific value [25]. The fuel characteristics of OPEFB are compara­ble to those of other biomasses and it can be considered a good candidate for gasification.

Soybean meal (SM) is still finding its way within scientific researches towards biobased material (biomaterial) applications. The number of publications on this topic is limited compared to corn gluten meal. SM has been characterized for its chemical composition, moisture content, thermal behavior and infrared spectrum and its potential as a particulate filler in value-added biocomposites was evaluated by compounding with polycaprolactone (PCL) [235]. The composite of PCL/SM (70/30) was prepared by extrusion and injection molding and then tested for mechanical properties such as tensile, flexural and impact. It has been observed that PCL/SM composite exhibited higher tensile/flexural modulus, but lower strength, elongation and impact strength compared to PCL. At the same time, the resulted biocomposite had relatively less cost than PCL itself. Thus, addition of SM to PCL increases the rigidity, but the particle-matrix adhesion needs to be improved.

SM has been plasticized and blended with other polymers such as polycaprolactone (PCL), poly(butylenes succinate) (PBS), poly(butylene adipate terephthalate) (PBAT) [236—237], and natural rubber [238—239]. SM was plasticized and destructurized successfully using glycerol and urea in a twin screw extruder and then blended with biodegradable polyesters, PCL and PBS [236]. As a result of destructurization phenomenon, improvement occurred in mechanical properties of the protein-based blends. In another work, SM was plasticized using glycerol in presence of two different denaturants (destructurizers), and the resulted thermoplastic SM was blended with different polyesters, PBS, PCL and PBAT [237]. Taguchi experimental design was adopted to investigate the effect of each constituent on the tensile properties of the final blend. Wu et al. [238] produced vulcanized blend of natural rubber and 50 wt% SM. The rubber phase was embedded by the SM matrix suggesting the interaction between phases, also approved by the increase in the glass transition temperature of the rubber phase. The produced blend exhibited good elasticity and water resistance.

Another area of research conducted on SM is producing edible films from defatted SM for food packaging applications [240—241]. For this purpose, SM was fermented in a soybean meal solution (15 g/100 ml of water) by inoculation with Bacillus subtilis bacteria fermentated under optimum conditions of 33°C and pH 7.0-7.5 for 33 h. Then, the fermented soybean solution was heated for 20 min at 75°C with 2-3 ml glycerol added to the solution to overcome film brittleness. The filtered solution was finally casted in a petri dish to produce films. Increasing amount of plasticizer in the fermented film led to a decrease in tensile strength and an increase in % elongation of the film compared to the ordinary soybean film. Moreover, the SM-based film exhibited higher water vapor permeability. On the other hand, experiments showed that growth inhibition of the produced SM-based film in the agar media containing E. coli was much higher than the ordinary soy protein film. These results indicated that the fermented SM-based films can be used as a new packaging material to extend the shelf-life of foods; however mechanical and physical properties need to be improved for more industrial applications.

In a second generation bioethanol plant, lignocellulosic polysaccharides (cellulose and hemicellulose) are broken down into monosaccharides (hexose and pentose) to be further fermented into ethanol. This includes a sequence of processes such as pretreatment, hydrolysis (enzymatically or chemically), fermentation and purification. At the end, the residual from the original lignocellulosic biomass is the coproduct mainly in the form of lignin [83]. The amount and quality of the produced lignin depends on the original lignocellulosic matter and the process. Typically, lignocelluloses contain 10-30 % lignin, which depends on various factors such as nature of biomasses, growth as well as isolation process [84]. As we know, lignin is a polymer that exists in the cell walls of plants, which is available in nature next to cellulose. The role of lignin in plant is to save them from compression, impact and bending. In addition to that, the major role of lignin extends to prevent the plant tissues from various kinds of naturally occurring microorganisms [85].

Chemically the polymeric structure of lignin is highly complicated and consists of three different monomer units (Figure 6) and they are called as p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol [86—87]. In addition to plant varieties, the lignin extraction process (known to be delignification) play a major role in the determination of local structure. Thus, the chemical structure of lignin extracted from plant biomass is never similar as exist in plant. There is a possibility for the alteration of monomer arrangement in the lignin structure. Hence, the lignin in plants, called as "natural lignin", is termed as "technical lignin" after isolation. These technical lignins can be further classified into three classes based on the domination of monomer units and they are [88]: (i) Softwood lignin: dominated with coniferyl monomer units, (ii) Hard wood lignin: combinations of equal quantities of guaiacyl and syringyl monomer units and (iii) Grass lignin: equally formulated with all three monomers of coniferyl, sinapyl and p-coumaryl.

The sources for lignin production can be divided into two major categories and they are (i) paper and (ii) bioethanol industries. They use different types of de-lignification process thus the lignin from paper industries and the lignin from bioethanol industries are not similar. Paper industries adopt Kraft, Sulphite, and Soda pulping processes to remove lignin from biomass, where as lignocellulosic ethanol industries, prefer to go with organosolv, steam explosion, dilute acid as well as ammonia fibre explosion for the removal of lignin [89]. Due to the tough food-versus-fuel concerns, non-food crops such as switchgrass, miscanthus and sugarcane bagasse become an effective feedstock for ethanol production and those related biofuel are named as second generation biofuels [90]. As the global demand for biofuel continues to grow, there will be emerging opportunities for lignocellulosic ethanol industries, which is expected to create a huge amount of lignin and it is predicted to be ~225 million tons by 2030 [85]. The challenge is to dispose them effectively that includes using them as a feedstock for energy products as well as for the fabrication of various chemicals and materials [91]. In common practice, lignin has been used for energy fuel, which is also the easiest way of disposal. In other hand, value-added uses of lignin can give economic return to the lignocellulosic ethanol industries and can improve their sustainability.

Only 2% of the lignin, which is produced from various sources, has been used as the feedstock for various chemicals including phenol, terephthalic acid, benzene, xylene, toluene, etc. [89] In addition to these, lignin has also been used in fertilizer, wood adhesives, surfactants, and also some kind of coloring agents [89]. Recently, lignin has been included as filler/reinforcing agent for blends and composites in both thermoplastic as well as thermoset platforms [85, 92—94]. In addition to that, lignin is found to be a suitable renewable carbon source for the synthesis of carbon materials [95]. Lignin has been widely exploited for the fabrication of activated carbon for various purposes including hydrogen storage, waste water removal and energy storage/conversion. Especially synthesizing nanostructured carbon materials from renewable resource-based lignin receives a great scientific interest due to the unique mor­phology as well as their physicochemical properties. As the global demand grows for the carbon fibre composites, there is a huge demand for the low cost carbon fibres. Lignin-based carbon fibres can substitute polyacrylonitrile (PAN)-based carbon fibre, hence the opportunity for lignin as a successful feedstock for carbon fibre is in near future [96]. This will be possible by understanding the basics of lignin chemistry and their application for fibre fabrication.

The increasing genetic knowledge provides feasible technique for the strain modification. Many efforts have been made to construct the strain with high butanol tolerance, superior butanol yield, productivity and less byproduct. The process can be classified into pathway — based construction and regulation-based construction.

Except butanol, acetone and ethanol are main products in ABE fermentation. The byprod­uct, especially acetone is low valuable and undesirable. Blocking the expression key enzyme gene for acetone is thought perfect to decrease the split flux and enhance butanol yield. However, the results were not ideal as expected. Knocking out the C. acetobutylicum EA 2018 adc gene, the acetone is still produced in low level (Jiang et al., 2009). In C. beijerinckii 8052, the strain with adc gene disruption produced similar acetone with the original wild type strain (Han et al., 2011). To block acetate and acetone pathway by knocking out gene
adc and ctfA reduced solvent production (Lehmann et al., 2012). These results demonstrated that the butanol metabolic mechanism is more complicated than expected.

Acetate and butyrate are produced during acidogenesis, and then they are transformed into acetyl-CoA and butyl-CoA to participate the solvent formation during solventogenesis phase. It seems an ineffective loop. In fact, the "inefficiency" loop is necessary for acid accu­mulation and switching to solventogenesis, at the same time, energy and reduction force were reserved. Disruption of acetate and butyrate pathway didn’t enhance butanol produc­tion. Knocking out acetate biosynthetic pathway gene by Clos Tron had no significant influ­ence on the metabolite distribution (Lehmann et al., 2012). Disruption of ptb gene blocked the butyrate synthesis and led to acetate and lactate accumulation. Some mutant strain with­out bk gene even can’t survival (Sillers et al., 2008). It indicated that the pathways seeming useless were necessary for butanol synthesis. What’s more, it is not possible to improve per­formance by decrease acid formation.

The genes participate in butanol synthesis including of thl, BCS operon, and add, bdh. Over­expression these genes are thought useful to increase the butanol yield. Overexpression of aad gene alone could enhance butanol production (Nair and Papoutsakis, 1994; Tummala et al., 2003). Transformed strain M5 (sol operon deficient because of lose of plasmid pSOL) with a plasmid carrying aad gene restored butanol-producing capability (Nair and Papoutsa­kis, 1994). Overexpression of aad gene and down-regulated ctf gene increased the butanol and ethanol production. To boost the butyryl-CoA pool, the strain with both thl and aad overexpression was constructed. However, butyrate and acetone concentration were in­creased, not butanol. The thl overexpression with ctf knock down didn’t change the product significantly (Sillers et al., 2009). So, the metabolic is more complicated than it seems. Theo­retical analyses also suggested alteration single solvent-associated gene is not sufficient to increase butanol yield (Haus et al., 2011).

Low butanol tolerance of the strains is another problem of butanol production. Although butanol synthesis is spontaneous in clostridium, the wild type strains can’t endure high bu­tanol concentration upper than 2%. Butanol stress influence gene expression of amino acid, nucleotide, glycerolipid biosynthesis and the cytoplasmic membrane composition (Janssen et al., 2012). Cells have heat shock response system will protect it from heat or other stress (Bahl, Muller et al. 1995). Overexpression of grosESL improved the strain tolerance and bu­tanol titer (Tomas et al., 2003).

The utilization of xylose and other carbon sources was inhibited by glucose is a phenomen­on called as Carbon catabolite repression (CCR). CCR limited the efficiency of butanol fer­mentation with lignocellulosic material as substrate. The utilization rate of pentose was improved efficiently by knocking out pleiotropic regulator gene ccpA, glcG (responsibility for phosphoenoopyruvate-dependent phophotransferase system, PTS) and overexpressing the genes of xylose utilization (Ren et al., 2010; Xiao et al., 2012). By heterogonous expres­sion transaldolase gene talA in ATCC 824, the xylose utilization was improved significantly (Gu et al., 2009). Knocking out xylose repressor gene XylR also increased the fermentation efficiency (Xiao et al., 2012).

There also some strategies aim at the upstream regulation. Global transcription machinery engineering (gTME) is thought to be a promising method to improve the butanol-producing performance (Alper et al., 2006; Papoutsakis, 2008). By regulating the transcription factor, the gTME strategy is thought to be able to change the metabolic strength and direction. gTME has been shown an efficient solution to improve substrate utilization, product toler­ance, and production in yeast (Alper et al. 2006) and E. coli (Chen et al., 2011). In butanol — producing Clostridium, the metabolic pathway have been described clearly, however, the mechanism of metabolism regulation is still not fully understood. This situation keeps the gTME strategy away from butanol-producing strains. Much effort should be devoted on the proteomics and transcriptomics etc. that will increase more details behind the appearance of ABE fermentation. A true gTME strategy will bring fresh and effective innovation to the bu­tanol fermentation.

The concept of metabolic engineering is to develop strains as "cell factory" which is efficient for desired products production from renewable sources (Na et al., 2010). Some microbes at­tracted interests because they are more tolerant to butanol than Clostridium, although these bacteria haven’t natural solvent-producing ability. Some kinds of Lactic acid bacteria can grow in 3-4% butanol (Liu et al., 2012) after long term adaption, that makes them promising host for butanol producing. The synthetic biology strategy has been implemented by con­structing the whole butanol-producing pathway in Escherichia coli, Bacillus subitilis, Saccharo — myces cerevisiae and Pseudomonas putida (Shen and Liao, 2008; Nielsen et al., 2009). This strategy deserves further attempts in spite of the poor final butanol concentration.

The direct conversion of CO2 into hydrocarbon-based fuels could greatly simplify the overall production process and reduce the cost of biofuel production (Figure 1). The search for autotrophic microorganisms capable of performing this CO2-to-fuel conversion started in the late 1970’s with the U. S. Department of Energy’s Aquatic Species Program (ASP) [96]. The ASP isolated and screened over 3,000 species of microalgae from a diverse range of environmental habitats. The program focused mainly on eukaryotic algae, as they naturally produce signifi­cant amounts of TAG. During the course of the program, the recombinant DNA technology used in metabolic engineering was developed, yet due to the infancy of this technology, it was not applied to microalgae for fuel applications until near the end of the ASP [15]. With the development of recombinant DNA technology, prokaryotic microalgae (i. e. cyanobacteria, previously known as blue-green algae) were recognized as potential hosts for fuel production, and the successful engineering of cyanobacteria for ethanol production confirmed their potential [97]. Unfortunately, research funding for microalgal fuel production waned as crude oil prices fell in the 1990’s. However, in the late 2000’s, the cost of crude oil soared, spurring a resurgence of interest in microalgae for fuel production and in the application of metabolic engineering to enhance fuel yields. In general, both eukaryotic microalgae (referred to as algae in the subsequent text) and prokaryotic microalgae (referred to as cyanobacteria in the subsequent text) utilize photosynthesis for energy generation and the Calvin-Benson-Bassham cycle for CO2 fixation (Figure 4). However, due to the cellular differences between algae and cyanobacteria, the strategies for engineering autotrophic fuel production will be discussed based on this host division.

i. Noble metals

Hydrogenolysis reactions involve the addition of hydrogen to an organic molecule. There­fore, hydrogenolysis catalysts must be able to activate hydrogen molecules. Noble metals are known to be active for the dissociation of hydrogen molecules and are widely used in hydrogenation reactions. The first studies on glycerol hydrogenolysis were carried out using Ru based catalysts [56]. Feng et al. [57] studied the effect of different supports (TiO2 SiO2 NaY, y-Al2O3) on Ru based catalysts. The TiO2 supported catalyst exhibited the highest ac­tivity giving a glycerol conversion of 90.1%; however, it also favored the production of eth­ylene glycol over 1,2-PDO. In contrast, Ru/SiO2 showed the lowest activity, but resulted in much higher selectivity to 1,2-PDO. They also performed blank reactions with the supports, achieving no significant conversions; which indicated that the supports cannot catalyze the reaction independently. Ru particle size was affected by the type of support, and a correla­tion was established between the size of the Ru particle and the activity of the catalyst, being higher with decreasing Ru particle size.

Apart from Ru, other noble metals have also been studied. For instance, Furikado et al. [58] compared the activity of various supported noble-metal catalysts (Rh, Ru, Pt and Pd over C, SiO2 and Al2O3). Among all the catalysts, the best results in terms of 1,2-PDO selectivity were achieved with Rh/SiO2 at low reacting temperature and low glycerol conversions (7.2). Nevertheless, the selectivities to 1,2-PDO obtained were rather low, due to the over-hydro — genolysis of 1,2- and 1,3-PDO to 1 and 2-PO.

The use of noble metal-base bifunctional catalytic systems has also been reported. As it was previously described in the glyceraldehyde based mechanism, the dehydration of glycerol to glyceraldehyde, and further dehydration of glyceraldehyde to pyruvaldehyde are both thought to be catalyzed by adsorbed hydroxyls. The effect of different base additives on the performance of Ru/TiO2was reported [45]. The addition of Li or Na hydroxides dramatically increased the glycerol hydrogenolysis activity of Ru/TiO2and the selectivity to 1,2-PDO. The highest conversion of glycerol (89.6%) and the highest selectivity to 1,2-PDO (86.8%) were observed with LiOH. The selectivity to 1,2-PDO was similar with all the bases added, which showed that the selectivity to 1,2-PDO is independent of base concentration within a certain range. However, the selectivity to ethylene glycol decreased no matter which base was add­ed. Almost no reaction was observed in the absence of Ru/TiO2, indicating that the presence of metal is required in order to take place glycerol hydrogenolysis. The lower selectivity to ethylene glycol with increasing base addition to the reacting solution was explained by the fact that ethylene glycol presented higher affinity to adsorb in the surface of the catalyst and to suffer the attack of hydroxyl groups, whose concentration was higher at elevated pH val­ues [59].

Noble metal-acid catalytic systems have also been used. According to the mechanism in Fig­ure 10, glycerol is firstly dehydrated to acetol, which is then hydrogenated to 1,2-PDO. The first dehydration step is supposed to be catalyzed by acid sites while the second one by met­al sites. Therefore, one interesting option to increase the selectivity to target product, 1,2-

PDO, is the use of bifunctional noble metal-acid catalysts. Different Bronsted acids like sulfonated zirconia, zeolites, homogeneous H2SO4 and Amberlyst 15 were tested together with Ru/C [60,61]. Acid-type cation-exchange resin Amberlyst 15 was the most effective co­catalyst. Nevertheless, a weak point in the system of Ru/C with Amberlyst 15 is that the re­action temperature is limited to 393 K. At higher temperatures sulfur compounds such as SO2 and H2S, which are formed by the thermal decomposition of the sulphonic groups of the resins, poison the catalyst. Using Amberlyst 70 the reacting temperature can be increased to 453 K before observing thermal decomposition [62].

Table 2. Selected examples of hydrogenolysis of aqueous glycerol over heterogeneous catalysts. PDO: Propanediol,

PO: Propanol, EG: Ethylene Glycol.

The use of more stable inorganic salts can avoid the temperature problems related to ion — exchange resins. Balaraju et al. [67] used the combination of Ru/C catalyst with different in­organic salts such as niobia, zirconia-supported 12-tungstophosphoric acid or acid caesium 12-tungstophosphate in glycerol hydrogenolysis at 453 K. The best results were achieved with those co-catalysts presenting a high number of medium strength acid sites. Particular­ly, with niobia as co-catalyst 62.8% glycerol conversion and 66.5% 1,2-PDO selectivity were reported. Another option is the use of a noble metal on acid supports. Vasiliadou et al. [68] investigated glycerol hydrogenolysis on Ru-based (y-Al2O3, SiO2 ZrO2) catalysts at 513 K and 80 bar. The nature of the oxidic support was found to influence the ability of the catalyst to both activate the glycerol substrate and selectively convert it to propanediol. The charac­terization of the catalytic materials revealed a correlation between catalytic activity for the hydrogenolysis reaction and total acidity, as the yield to hydrogenolysis products increased with the concentration of the acid sites. However, increased acidity was also responsible for the promotion of the excessive hydrogenolysis of the desired 1,2-propanediol to propanols.

1.1. Experimental set up

The experimental setting is shown in Fig. 1. Its base is a cylindrical quartz chamber (1) with diameter of 90 mm and height of 50 mm. Top (2) and bottom (3) it is hermetically closed with metal flanges. Camera is filled with fluid (4), the level of which has been maintained by the injection pump through the hole (5). Bottom flange is made of stainless steel. The stainless steel T-shaped cylindrical electrode (6), cooled with water, immerses in the liquid through the central hole in the bottom flange. There is a 5 mm thick metal washer on its surface (7) in the middle of which there is a hole in diameter of 10 mm. Sharp corners are rounded. This washer is used for reducing the waves (which have been moving to the quartz wall) amplitude on the liquid surface.

The top flange, made from duralumin, contains copper sleeve (13) with a diameter of 20 mm is placed in the center (2), and plays the role of the second electrode. The nozzle with diameter of 4 mm and length of 6 mm is located in the center of the copper sleeve (8). Gas is introduced into the flange (2) through the aperture (9). Gas flow changes the direction at 90 degrees inside the flange and injects tangentially into the channel (10). (10) The gas is rotated in the circular channel. Rotating gas (11) lands on the surface liquid and moves to the central axis of the system, where fells into the quartz cell through the nozzle (14), forming a plasma torch (12). Camera (14), in its turn, plays a role of pyrolytic chamber. Flow rate reaches the maximum value near the nozzle. Due to this, the zone of lower pressure is formed in the center of the gas layer, compared to the periphery. The conical structure appears over the liquid’s surface near the system axis (Fig. 1). External static pressure is 1 atm. and internal — 1.2 atm (during discharge burning). Gas from quartz chamber (14) gets into the refrigerator (15), which is cooled with water at room temperature.

Condensed matter (16) together with the gas from the refrigerator gets to the chamber (17). At the chamber exit (17) there’s a flask (18), where gas is gathered for its composition diagnostics by means of mass spectrometry and gas chromatography. Study of plasma parameters is performed by emission spectrometry. The emission spectra registration procedure uses the system which consists of optical fiber, the spectral unit S-150-2-3648 USB, and the computer.

Fiber is focusing on the sight line in the middle between the top flange (2) and the surface of the liquid (4).

The spectrometer works in the wavelength range from 200 to 1100 nm. The computer is used in both control measurements process and data processing, received from the spectrometer.

The voltage between the top flange and electrode, immersed in the liquid, is supplied by the power unit "PU". DC voltage provided is up to 7 kV. Two modes of operation have been considered:

1. "liquid" cathode (LC) — electrode immersed in the liquid has "minus" and the top flange has "plus";

2. solid" cathode (SC) — with the opposite polarity.

Electrode which has "plus" is grounded. Breakdown conditions are controlled by three parameters: the fluid level, the gas flow value and the voltage magnitude between the electrodes. The several modes of operation have been studied:

1. Various air flow and CO2 ratio;

2. Discharge voltage varied within Ud = 2.2 4 2.4 kV;

3. Discharge current varied within Id = 220 4 340 mA (ballast resistance hasn’t been used).

At first, for the analysis of the plasma-chemical processes kinetics the distilled water (working fluid), and ethanol (ethyl alcohol solution in distilled water with a molar ratio C2H5OH/H2O = 1/9.5), as a hydrocarbon model have been used. As the working gas mixture of air with CO2, in a wide range of air flow and CO2 ratios has been used. The ratio between air and CO2 in the working gas changes in the ranges: CO2/Air = 1/20 4 1/3 for the working fluid C2H5OH/H2O (1/9.5) and CO2/Air = 0/1 4 1/0 (by pure air to pure CO2) — for distilled water.

Plasma component composition and population temperature of the excited electron (Te’), vibration (Tv’) and rotational (Tr’) levels of plasma components and relative concentrations of these components have been determined by the emission spectra. For the temperature population determination of the excited oxygen atoms electron levels the Boltzmann diagram method has been used [5]. Te’ of oxygen atoms has been determined for the three most intense lines (777.2 nm, 844 nm, 926 nm). Temperature population of excited hydrogen atoms electron levels has been determined by the two lines of 656.3 nm and 486 nm relative intensities.

The effect of the presence of CO2 in the system on the initial gas products has been investigated by means of "TORNADO-LE" current-voltage characteristics with changes in the working gas composition. Tv’ and Tr’ have been determined by comparing the experimentally measured emission spectra with the molecules spectra simulated in the SPECAIR program [9]. With help of this program and measured spectra, relative component concentrations in plasma have been determined. Also, the concentration of atomic components has been obtained by calculating the amount of oxygen that fell into a working system with the working gas flow. The hydrogen amount has been received from the electrolysis calculations. The output gas in reforming ethanol has been analyzed by gas chromatography and infrared absorption.

Acetogens are defined as obligate anaerobes that utilize the reductive acetyl-CoA pathway for the reduction of CO2 to the acetyl moiety of acetyl-coenzyme A (CoA), for the conserva­tion of energy, and for the assimilation of CO2 into cell carbon [33]. In addition to the reduc­tive acetyl-CoA pathway, four other biological pathways are known for complete autotrophic CO2 fixation: the Calvin cycle, the reductive tricarboxylic acid (TCA) cycle, the 3-hydroxypropionate/malyl-CoA cycle and the 3-hydroxypropionate/4-hydroxybutyrate cy­cle [34]. Since the earlier atmosphere of earth was anoxic and the acetyl-CoA pathway is bio­chemically the simplest among the autotrophic pathways (the only linear pathway, whereas the other four pathways are cyclic), it has been postulated to be the first autotrophic process on earth [35, 36]. The reductive acetyl-CoA pathway is also known as the ‘Wood-Ljungdahl’ pathway, in recognition of the two pioneers, Lars G. Ljungdahl and Harland G. Wood, who elucidated the chemical and enzymology of the pathway using Moorella thermoacetica (for­merly: Clostridium thermoaceticum) [35] or CODH/ACS pathway after the key enzyme of the pathway Carbon Monoxide dehydrogenase/Acetyl-CoA synthase. This ancient pathway is diversely distributed among at least 23 different bacterial genera: Acetitomaculum, Acetoa — naerobium, Acetobacterium, Acetohalobium, Acetonema, Alkalibaculum, "Bryantella", "Butyribacte- rium",Caloramator, Clostridium, Eubacterium, Holophaga, Moorella, Natroniella, Natronincola, Oxobacter, Ruminococcus, Sporomusa, Syntrophococcus, Tindallia, Thermoacetogenium, Thermoa — naerobacter, and Treponema [33]. A selection of mesophilic and thermophilic acetogens are presented in Table 2. Acetogens are able to utilize gases CO2 + H2, and/or CO to produce acetic acid and ethanol according to the following stoichiometries:

The Acetyl-CoA pathway is essentially a terminal electron-accepting process that assimilates CO2 into biomass [35]. It constitutes an Eastern (or Carbonyl) branch and a Western (or Meth-

yl) branch (Figure 2.). The Western branch employs a series of enzymes to carry out a six — electron reduction of CO2 to the methyl group of acetyl-CoA, starting from the conversion of CO2 to formate by formate dehydrogenase. Formyl-H4folate synthase then condenses for­mate with H4folate to form 10-formyl-H4folate, which is then converted to 5,10-methenyl — H4folate by a cyclohydrolase. This is followed by a dehydrogenase that reducesmethenyl- to 5,10-methylene-H4hydrofolate, before (6S)-5-CH3-H4folate is formed by methylene-H4folate reductase [37]. A B12-depedent methyltransferase (MeTr) then transfer the methyl group of (6S)-5-CH3-H4folate to corrinoid iron-sulphur protein (CoFeSP) of the bi-functional carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex [37]. The bi-function­al CODH/ACS enzyme complex is formed by two autonomous proteins, an a2p2 tetramer (CODH/ACS) and a y& heterodimer (CoFeSP), and the genes are often arranged in an oper — on, together with MeTr [37, 38]. In the Eastern branch, the CODH component catalyzes the reduction of CO2 to CO. The central molecule, acetyl-CoA, is finally generated when CO, methyl group (bound to CoFeSP) and CoASH are condensed by ACS. Given the pivotal role of CODH/ACS, it is unsurprising that this complex was found to be the most highly ex­pressed transcripts under autotrophic conditions in C. autoethanogenum [27], and can repre­sent up to 2% of the soluble cell protein of an acetogen [39]. CODH/ACS is not unique to acetogenic bacteria, as it is also present in sulphate-reducing bacteria, desulfitobacteria, and Archaea (methanogens and Archaeoglobus) [38, 40].

The reducing equivalents required for fixation of CO2 carbon into acetyl-CoA come from the oxidation of molecular hydrogen under chemolithoautotrophic growth, or NADH and re­duced ferredoxin under heterotrophic growth [75]. An extensive review by Calusinska et al. (2010) highlighted the diversity of ubiquitous hydrogenases that Clostridia possess although only one acetogen C. carboxidivorans was included in this study [76], which catalyze the re­versible oxidation of hydrogen:

H2 « 2 H + + 2 e — (5)

The direction of the hydrogenase reaction is directed by the redox potential of the compo­nents able to interact with the enzyme. Hydrogen evolution occurs when electron donor is available, whereas the presence of electron acceptor results in hydrogen oxidation [77]. Hy — drogenases can be classified into three phylogenetically distinct classes of metalloenzymes: [NiFe]-, [FeFe]-, and [Fe]-hydrogenases [76]. In Methanosarcina barkeri, the Ech hydrogenase, a [NiFe]-hydrogenase, was demonstrated to oxidize H2 to reduce ferredoxin [78]. During acetoclastic methanogenesis, Ech hydrogenase oxidize ferredoxin to generate H2 [78]. Al­though genome analysis revealed the presence of Ech-like hydrogenase in C. thermocellum, C. phytofermentans, C. papyrosolvens, and C. cellulolyticum, their physiological roles remained unknown [76]. Clostridia harbour multiple distinct [FeFe]-hydrogenases, perhaps reflecting their ability to respond swiftly to changing environmental conditions [76]. The monomeric, soluble [FeFe]-hydrogenase of C. pasteurianum is one of the best studied. It transfer electrons from reduced ferredoxins or flavodoxins to protons, forming H2 [79]. A trimeric [FeFe]-hy — drogenase found in C. difficile, C. beijerinckii, and C. carboxidivorans were hypothesized to
couple formate oxidation to reduce protons into H2 [76]. In Thermotoga maritima, an electron bifurcating, trimeric [FeFe]-hydrogenase was identified, that was shown to simultaneously oxidize reduced ferredoxin and NADH to evolve hydrogen under low H2 partial pressure [80]. Under high H2 partial pressure, the authors hypothesized that the NADH is oxidized to produce ethanol. In silico analysis revealed homologs of this bifurcating hydrogenase in a few Clostridia including C. beijerinckii and C. thermocellum [80]. In addition to classical hy — drogenases, CODH/ACS and pyruvate:ferredoxin oxidoreductase (PFOR) from M. thermoa — cetica were shown to have hydrogen evolving capability, possibly as a mean of disposing excess reducing equivalents when electron carriers are limited and/or CO concentration is sufficient to inhibit conventional hydrogenases [81].

Most acetogens are also able to utilize another gas carbon monoxide (CO). In contrast to CO2, CO can serve as both a source of carbon btut also as source of electrons such that hy­drogen is not necessarily required. With a CO2/CO reduction potential of -524 to -558mV, CO is approximately 1000-fold more capable of generating extremely low potential electrons than NADH, capable of reducing cellular electron carriers such as ferredoxin and flavodoxin [38, 82]. The reducing equivalents generated from CO oxidation can be coupled to reduction of CO2 into acetate, butyrate and/or methane, evolution of molecular hydrogen from pro­tons, reduction of nitrate/nitrite, reduction of sulfur species and reduction of aldehydes into alcohols [35, 83]. However, relatively few microorganisms are able to utilize CO as sole car­bon and energy source, probably due to growth inhibition from sensitivity of their metallo — proteins and hydrogenases towards CO [38, 83]. During exponential growth of Pseudomonas carboxydovorans (an aerobic carboxydotroph), it was demonstrated via immunological locali­zation studies that 87% of the key enzyme CODH is associated with the inner cytoplasmic membrane, but this association was lost at the end of the exponential growth phase and a reduction in CO-dependent respiration rate was observed [84, 85]. It should be mentioned that aerobic and anaerobic CODH enzymes are structurally very different. CODH has been reported to be a very rapid and efficient CO oxidizer at rates between 4,000 and 40,000 s-1, and reduces CO2 at 11s-1 [86, 87]. Other electron donors commonly used by acetogens in­clude formate, CH3Cl, lactate, pyruvate, alcohols, betaine, carbohydrate, acetoin, oxalate and citrate [88]. CODH is able to split water in a biological water-gas shift reaction into hydro­gen and electron according to the stoichiometry:

The operation of this water gas shift reaction is the biochemical basis for the tremendous flexibility that acetogens have in terms of input gas composition. Via this reaction these or­ganisms can flexibly use CO or H2 as a source of electrons. Recently, some acetogens such as C. Ijungdahlii, C. aceticum, M. thermoacetica, Sporomusa ovata, and S. sphaeroides have addition­ally been shown to utilize electrons derived from electrodes to reduce CO2 into organic com­pounds such as acetate, formate, fumarate, caffeine, and 2-oxo-butyrate [89]. Termed microbial electrosynthesis, this nascent concept offers another route for acetogens to harvest the electrons generated from sustainable sources (e. g. solar and wind) to reduce CO2 into useful multi-carbon products such as biofuels [90].

Under chemolithoautotrophic conditions, acetogenesis must not only fix carbon but also conserve energy. Approximately 0.1 mol of ATP is required for generation of 1g of dry bio­mass in anaerobes [82]. Acetyl-CoA is an energy rich molecule that through the combined actions of Pta (phosphotransacetylase) and Ack (acetate kinase), one ATP can be generated via substrate level phosphorylation (SLP). However, the activation of formate to 10-formyl — H4folate in the methyl-branch of Acetyl-CoA pathway consumes one ATP so no net gain in ATP is achieved via this mechanism [35, 75]. Furthermore, the reduction of CO2 to the car­bonyl group also requires energy, estimated at one third of ATP equivalent [35]. Recent ad­vances indicated that other modes of energy conservation such as electron transport phosphorylation (ETP) or chemiosmotic processes that are coupled to the translocation of protons or sodium ions are implicated in acetogens. Acetogens such as M. thermoacetica har­bour membrane-associated electron transport system containing cytochrome, menaqui — nones, and oxidoreductases that translocate H+ out of the cell [33]. For acetogens that lack such membranous electron transport system, such as Acetobacterium woodii and C. Ijungdahlii, a membrane-bound corrinoid protein is hypothesized to facilitate extrusion of Na+ or pro­tons during the transfer of methyl group from methyl-H4F to CODH/ACS [75]. However, all enzymes involved are predicted to be soluble rather than membrane bound. Recent evi­dence suggested coupling to an Rnf complex in A. woodii, and C. ljungdahlii (Figure 3) which acts as ferredoxin:NAD+-oxidoreductase [62, 91—93]. The Rnf complex is also found in other Clostridia (but not in ABE model organism C. acetobutylicum) and bacteria, and was original­ly discovered in Rhodobacter capsulatus where it is involved in nitrogen fixation [93]. Using reduced ferredoxin (Fd2-) generated from CO oxidation, carbohydrate utilization and/or hy — drogenase reactions, this membrane-bound electron transfer complex is predicted to reduce NAD+ with concomitant translocation of Na+/ H+. The ion gradient generated from the above processes is harvested by H+- or Na+- ATP synthase to generate ATP [33, 93]. The recent ge­nome sequencing of A. woodii revealed that Rnf complex is likely to be the only ion-pump­ing enzyme active during autotrophic growth and the organism’s entire catabolic metabolism is optimized to maximize the Fd2-/NAD+ ratio [42]. Recently, a third mechanism of energy conservation which involves bifurcation of electrons by hydrogenases was pro­posed for anaerobes [94] and demonstrated for enzymes hydrogenase (see above; [80]), bu — tyryl-CoA dehydrogenase [94, 95], or an iron-sulfur flavoprotein Nfn [96]. A similar mechanism has also been proposed for the methylene-THF reductase of the reductive acetyl — CoA pathway, which would enable this highly exergonic reduction step (AG0′ = -22 kJ/mol) to be coupled with the Rnf complex for additional energy conservation [62]. However, no experimental proof to support this hypothesis has been published to date.

In an attempt to generate an autotrophic E. coli, the genes encoding MeTr, the two subunits of CODH/ACS, and the two subunits of CoFeSP from M. thermoacetica were cloned and het­erologously expressed in E. coli [97]. Although the MeTr was found to be active, the other subunits misassembled hence no active enzymes were found [97]. Autotrophic capability is clearly a very complex process that involves many genes other than the CODH/ACS com­plex and tetrahydrofolate pathway, including compatible cofactors, electron carriers, specif­ic chaperones and energy conservation mechanisms. For instance, more than 200 genes are predicted to be involved in methanogenesis and energy conservation from CO2 and H2 in methanogens [98]. A recent patent application described the introduction of three Wood — Ljungdahl pathway genes encoding MeTr, CoFeSP subunit a and p from C. difficile into C. acetobutylicum [99]. The recombinant strain was shown to incorporate more CO2 into extrac­ellular products than wild-type [99].

Since both acetic and butyric acids are intermediate products of ABE fermentation, and butyrate is almost completely consumed during the solventogenic phase, addition of these intermediates to increase the yield of butanol has already been studied in detail. Beneficial effects were observed with addition of AcOH (completely reduced to Me2CO), butyric acid (50-80% recovery as BuOH), and sodium acetate NaOAc (60% recovery as Me2CO), while bad results were obtained with addition of formic acid and calcium acetate [61,62]. Nakhmanocivh and Shcheblikina [63] used a 4% glucose medium, with corn gluten or flour mash, and additon of 0.1N Ca(OAc)2 raised acetone yield by 20-24% and 0.1 N calcium butyrate raised BuOH yield by 45-60% in C. acetobutylicum fermentations. Though Ca(OAc)2 accelerated the fermentation, it was only 40-50% fermented itself. Utilization of Ca(OOCPr)2 goes further (above 70%), mostly by conversion to Ca(OAc)2.

Tang concluded [64] that addition of 1.5 g/L acetic acid increased the cell growth and enhanced acetone production in ABE fermentation. The final concentration of acetone was 21.05%, and the butanol production was not improved. Similarly, addition of 1.0 g/L butyric acid increased the cell growth and enhanced butanol production, the final concentration of butanol was 24.32% while the acetone production was not improved. Additon of acetic acid and butyric acid together (10 mM each) to C. acetobutylicum grown on glucose (2%) in a pH-controlled minimal medium caused rapid induction of acetone and butanol synthesis (within 2 h) [65]. The specific growth rate of the culture and the rate of H2 production decreased gradually from the onset of the experiment, whereas the rate of CO2 production remained unchanged. No correlation was found between solvent production and sporulation of the culture [65]. A 32 % conversion rate of the glucose into solvents took place when the same fermentation was carried out on a synthetic medium (BuOH:acetone:EtOH was 0.6:1.9:6). This was changed to 34 and 35 % (BuOH:ace — tone:EtOH was 5:3:6 or 0.8:2.4:6) by adding HOAc or butyric acid, respectively [66].

Fond et al. [67] studied the effect of HOAc and butyric acid additon in the fermentation of vari­ous kinds of carbohydrates using fed-batch fermentations. Different specific rates of carbohy­drate utilisation were obtained by variations in feeding rates of sugar. At low catabolic rates of sugar addition of acetic acid or butyric acid, alone or together, increased the rate of metabolic transition by a factor 10 to 20, the amount of solvents by a factor 6 and the percentage of ferment­ed glucose to solvents by a factor 3. The same results were obtained with both glucose and xy-

lose fermentations. Depending on the rates of growth, butanol production began at acid levels of 3-4 g L-1 for fast metabolism and at acid levels of 8-10 g L-1 for slow metabolism. Associated with slow metabolism, reassimilation of acids required values as high as 6.5 g L-1 of acetic acid and 7.5 g L-1 of butyric acid. At a high rate of metabolism, acetic and butyric acids were reassimi­lated at concentrations of 4.5 g L-1 [67]. Significant increases in acetone and BuOH production could be observed by Yu and Saddler [68] by growing C. acetobutylicum on xylose in presence of added HOAc or butyric acid. Increased yields could not be accounted for by conversion of the low amounts of acetic or butyric acid added. The effect was greater when the acid was added be­fore, rather than during fermentation, so pH change alone is probably not responsible and en­zyme induction may be involved in this process. Addition of acetate or butyrate ensures fermentation at neutral pH conditions as well. Holt et al. used C. acetobutylicum NCIB 8052 (ATCC 824) and monitored a batch culture at 35 °C in a glucose (2%) minimal medium. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the un­supplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt./vol.]). Substantial solvent concentra­tions obtained at pH 7 in a 2% glucose medium supplemented with high concentrations of ace­tate plus butyrate (100 mM each, supplied as their K salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, are able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to pro­duction of some n-propanol as well as acetone and n-butanol; the final culture medium was vir­tually acid free. At pH 7, supplementation with propionate (150 mM) again led to formation of n-propanol but also provoked production of some acetone and n-butanol, although in consider­ably smaller amounts than those obtained when the same basal medium had been fortified with acetate and butyrate at pH 7 [69].

Liquid hourly space velocity (LHSV) is defined as the ratio of the liquid mass feed-rate (gr/h) over the catalyst mass (gr) and as a result is expressed in hr-1. In fact the inverse of LHSV is proportional to the residence time of the liquid feed in the reactor. In essence the higher the liquid hourly space velocity, the less time is available for the contact of the feed molecules of the reaction mixture with the catalyst, thus the less the conversion. However, maintaining large LHSV imposes a faster degradation of the catalyst therefore in industrial applications the LHSV is maintained in as high values as it is practically possible.

1.1.6. Hydrogen feed-rate

The hydrogen feed-rate is another important parameter as it also defines hydrogen partial pressure depending on the hydrogen consumption of each application. It actually favours both heteroatom removal and saturation reaction rates. However, as hydrogen cost defines the overall unit operating cost, hydrogen feed-rate is normally optimized depending on the system requirements. Furthermore the use of renewable energy sources for hydrogen pro­duction is also envisioned as a potential cost improvement option.