1.3. The chemical pathway

The interaction between xylose and bases, either Bransted or Lewis, is rather less reported in the literature when compared to the acid conversion of xylose to furfural indicated in the previous section. Many very different reactions have been reported as in the case of Popoff and Theander [50] that have quantified the cyclic compounds produced after a base-cata­lyzed reaction of pure D-xylose at 96 °C for 4 hours. The produced compounds are rather peculiars in comparison to other work made on the subject (see Figure 5) since most of the reported compounds are aromatics. The presence of aromatics may be a result that the reac-

tion time was long and the isomerisation that was required in order to induce such reaction was efficient. Johansson and Samuelson [51] tested the effect of alkali treatments (NaOH) on birch xylan and contrarily to the previous research; they found that the treatment led to the production of a variety of organic acids. Testing on untreated xylene showed that most of the organic acids were already obtained from xylans and the most distinctive impact was observed after a 2 day test at 40 °C where the concentrations of L-galactonic and altronic acids increased significantly which could be related to a less severe treatment of xylans that also include C6 sugars.

El Khadem et al. [52] studied the effect of xylose conversion in an alkali medium at low tem­peratures (room) and for long periods (1-4 weeks) and one of the interesting features of his work was that the process did lead to the epimerization of sugars, but furthermore, it leads to the production of C6 sugars most probably from a reverse aldol reaction. Among the sug­ars that were formed during the reaction, conversion of xylose was shown to be more effi­cient to lyxose (18 %) and arabinose (15 %) with a decrease observed for most of the compounds between 1 and 4 weeks (see Figure 6). A vast majority (more than 50 %) of xy­lose remains on its original form and the reaction leads to the production of 1 % glucose and 2.5 % of sorbose, both are C6 sugars.

Xylose, as the other carbohydrates, is converted to smaller organic acids when reacted with a strong alkali medium. As an example, Jackson et al. [53] have demonstrated that the con­version of xylose to lactic acid could reach 64 % (molar) accompanied by glyceric acid. Al­though they did not used xylose but rather ribose and arabinose, they were able to reach conversions between 35-43 % into lactic acid using potassium hydroxide as catalyst under microwave irradiation [54]. Rahubadda et al. [55] have provided a mechanism for the con­version of xylose to lactic acid under a base catalyst. The simplified pathway is depicted in Figure 7 below.

They mentioned in this report that methylglyoxal is most probably derived from glyceralde — hyde as depicted in Figure 8 below. The possible reaction leading to methylglyoxal may in­volve an E2 reaction on C2 leading to removal of the hydroxyl group on C3 then a keto-enol rearrangement to methylglyoxal.

Onda et al. [56] achieved a conversion rate of more than 20 % when using xylose as a feed­stock with a carbon-supported platinum catalyst in alkali solution. In a recent report by Ma et al. [57], it was shown that using model compounds, different carbohydrates tend to con­vert into lactic acid at different levels. Fructose was shown to be more effectively converted to lactic acid than glucose and finally than xylose. The work also showed a correlation be­tween the amount of catalyst (varying from 1-3 % wt.) of NaOH, KOH and Ca(OH)2 respec­tively. Part of the work by Aspinall et al. [58] was aimed at the non-oxidative treatment of xylans from different substrates using sodium hydroxide as solvent. The reaction was per­formed at room temperature for 25 days and amongst the products that emerged from this reaction, a majority was acidic and lactic acid as well as formic acid were the two major products. Other work by Yang et al. [59] showed that higher temperature treatments of xy­lose (200 °C) in a Ca(OH)2 solution produced about 57 % (mol.) of lactic acid with 2,4-dihy — droxybutanoic acid in second with 10 % (mol.). The same conversion patterns were observed by Raharja et al. [60] with production rates for lactic acid above 50 %.

The experimental measurements were carried out on a four-stroke, air-cooled engine. This is a one-cylinder engine with 123cm3 displacement that is connected with a phase single alter­native generator (230V/50Hz) with maximum electrical load approximately 1KW(picture 1). The engine according to the manufacturer uses as fuel gasoline. The engine functioned with­out load and under full load conditions (1KW) using different fuel mixtures: gasoline, gaso — line-10%ethanol, gasoline-20%ethanol, gasoline-30%ethanol, gasoline-40%ethanol, gasoline-50%ethanol, gasoline-60%ethanol, gasoline-70%ethanol gasoline-80%ethanol gaso- line-90%ethanol and 100% ethanol, gasoline-10% methanol, gasoline-20%methanol, gaso — line-30%methanol, gasoline-40%methanol, gasoline-50%methanol, gasoline-60%methanol, gasoline-70%methanol. During the tests, exhaust gases measurements, were also monitored for every fuel mixture and for every load conditions. Also, during the function of the engine the consumption was recorded for every fuel. There was lack of engine regulation concern-

ing the stable air/fuel ratio. For this purpose, data Acquisition cart was used with the termi­nal wiring board with on-board Cold Junction The data acquisition card was installed at a PC. This particular measuring system and software completed a scanning cycle per channel every 0.1 second approximately. This measuring speed was considered adequate for the purpose of the experiment and the sampling capabilities of the chemical sensors. For the ex­haust gas (CO and HC) measurements a analyzer was used.

The figures of CO and HC emissions, for every fuel and for every load conditions, are represented below [4—7]:

gasoline

10

9

8

7

6

5

4

time(s)

time(s)

i. The CO variation when mixture of gasoline-30%methanol is used as fuel

40%methanol

10 9 8 7 6 5 4 3 2 1 0

0 50 100 150 200 250 300 350 400 450 500

time(s)

The CO variation when mixture of gasoline-50%methanol is used as fuel.

60%methanol

70%methanol

Figure2 represents CO emissions when the fuel that is used is gasoline. The engine functions without load at first and then (after 250s) functions under full load conditions (1KW). The average value of CO emissions during the function of the engine without load is 6,41%, while at full load conditions the average value of CO emissions is 8,7%. Following, a mixture of gasoline with 10% methanol is used (fig. 3) and the same test is conducted with this mixture. From figure 3 it is being observed that the average value of CO emissions without load conditions of the engine is 4,87%, while at full load conditions the percentage of CO emissions is 6,9%. The same tests are conducted while increasing the percentage of the methanol in the fuel, using the mixtures: gasoline-20%methanol(fig. 4), gasoline-30%methanol(fig. 5), gaso- line-40%methanol(fig. 6), gasoline-50%methanol(fig. 7), gasoline-60%methanol(fig. 8), and gasoline-70%methanol(fig. 9).

The HC emissions when the fuel that is used is gasoline are represented at figure 10. As it was mentioned above, the engine functioned without load at first and then (after 250s approxi­mately) functioned under full load conditions (1KW). During the function of the engine without load the average value of HC emissions is 1091ppm, while at full load conditions the average value of HC emissions is 730ppm. The mixture of gasoline with 10% methanol is illustrated at figure 11. At this figure is being observed that the average value of HC emissions without load conditions of the engine is 496ppm, while at full load conditions the HC emissions is 613ppm. When the percentage of the methanol in the fuel increases: gasoline-20%metha — nol(fig. 11), gasoline-30%methanol(fig. 13), gasoline-40%methanol(fig. 14), gasoline-50%meth — anol(fig. 15), gasoline-60%methanol(fig. 16), and gasoline-70%methanol(fig. 17).

20%methanol
50	100	150	200	250	300	350	400	450	500
0
time(s)
!. The HC variation when mixture of gasoline-20%methanol is used as fuel.
30%methanol
time(s)

0 50 100 150 200 250 300 350 400 450 500

time(s)

14. The HC variation when mixture of gasoline-40%methanol is used as fuel.

50%methanol

time(s)

0

50 100 150 200 250 300 350 400 450 500

time(s)

16. The HC variation when mixture of gasoline-60%methanol is used as fuel.

70%methanol

time(s)

In the case of HC emissions there is also a decrease of emissions when the percentage of methanol in the fuel increases at idle and under full load conditions. There is an exception at the mixture gasoline-70%methanol where the average value of HC without load is 534ppm and under full load is 367ppm. These values are higher than the values that correspond to the mixture of gasoline-60%methanol (295ppm, 298ppm). This is explained by mentioning the fact that during the use of the mixture gasoline-70%methanol there was a malfunction of the engine that was cause by the bad mixture of the air with the fuel(gasoline-70%methanol), since the engine was not regulated(ratio air/fuel) for every mixture maintaining the adjustments for gasoline. Also it must reported that the addition of methanol in the fuel led to HC decrease for the same mixture but for different load conditions. When gasoline was used HC emissions were higher at no load conditions than at full load conditions(1KW), while during the use of gasoline-methanol mixtures this was reversed. This is due to the better combustion under full load conditions because methanol has higher octane number than gasoline [4—7].

It is important to mention that when mixture gasoline-80%methanol was tested the engine could not function properly.

The CO and HC emissions are represented in the figures below, for the mixtures: gasoline, gasoline-ethanol, for every fuel and for every load conditions. For these mixtures the average values of the emissions (CO, HC) are presented at the figures below. From the average values, the variation of those emissions can be better understood.

10%ethanol

time(s)

60%ethanol

time(s)

23. TheCOvariationwhengasoline-60%ethanolmixtureusedasfuel

70%ethanol

time(s)

100%ethanol

0 50 100 150 200 250 300 350 400 450 500

time(s)

0 50 100 150 200 250 300 350 400 450 500

time(s)

29. TheHCvariationwhengasoline-20%ethanol mixtureusedasfuel

30%ethanol

0 50 100 150 200 250 300 350 400 450 500

time(s)

40%ethanol

0 50 100 150 200 250 300 350 400 450 500

time(s)

31. TheHCvariationwhen gasoline-40%ethanolmixtureusedasfuel

50%ethanol

0 50 100 150 200 250 300 350 400 450 500

time(s)

0 50 100 150 200 250 300 350 400 450 500

time(s)

Figure33. TheHCvariationwhengasoNne-60%ethanolmixtureusedasfuel

70%ethanol

0 50 100 150 200 250 300 350 400 450 500

time(s)

0 50 100 150 200 250 300 350 400 450 500

time(s)

Figures 18 — 28 present the CO variation when as fuel is used gasoline — ethanol and gasoline — methanol mixtures when the engine functioned without load and under full load condi- tions(1KW). From these figures is observed lower CO emissions when gasoline-ethanol mixtures are used compared to the mixtures gasoline-methanol, until the mixture of 70% ethanol and methanol. Over the 70% percentage of methanol the engine could not function and that is why there is no further presentation of comparative curves of CO emissions. It must also be mentioned that for the mixtures of gasoline -70% methanol, gasoline -90%ethanol and 100%ethanol the engine malfunctioned. The average values of CO emissions for the above mixtures and for both load conditions are presented in the figure 38 below [4—7]:

In the figures 28 — 37 is observed higher decrease of HC in the case were methanol is used, with exception of the use of gasoline -70%methanol mixture where the HC are higher compared to the mixture gasoline-70%ethanol. This is due to the malfunction that occurred during the use of gasoline-70%methanol mixture. There was also malfunction of the engine when the mixtures of gasoline-90%ethanol and 100%ethanol were used, which had as result the HC increase during the use of those mixtures. These observations are presented more clearly in the figure 38 below [4—7]:

Figure 39 shows the average values of HC for every mixture, when the engine functions without load and under full load conditions. It is being observed grater decrease of HC during the use of methanol in the fuel contrary to the use of ethanol.

Also is shown HC emissions decrease compared to gasoline, while the percentage of methanol and ethanol in the fuel increases without load and under full electrical load conditions (1KW). At higher percentage of ethanol in the fuel 90%ethanol and 100%ethanol it is observed HC emissions increase, which is due to incomplete combustion. Indeed, during the tests of the mixtures: gasoline-70%methanol, gasoline — 90%ethanol and 100%ethanol, there was an engine malfunction mostly at without electrical load, as it was mentioned above. This malfunction is showed from the rounds per minute recording in the figures below:

Engine rpm

time(s)

Engine rpm

During the tests the rounds per minute of the engine were recorded as it was mentioned above. The normal variation of the engine rpm appears in figure 40. The same variation that is illustrated in this figure corresponds to the mixtures gasoline until the mixtures gasoline-90% ethanol and gasoline-60%methanol, without any change. As it is presented in figure 39, the average value of the engine rpm without load (0-200s and 420-500s) is approximately 2990rpm while at full load conditions (200-420s) the average value of the engine rpm is 2880rpm. It must be noted that the engine has a round stabilizer. In figures 41, 42 and 43 the mixtures gaso- line-70%methanol, gasoline-90%ethanol and 100%ethanol are illustrated and irregular variation of the engine rpm is presented, which is caused from the engine malfunction. Higher irregular variation is observed at without load condition, and lower at full load conditions in the case of use ethanol. This malfunction is due to the smaller calorific value of methanol and ethanol than the gasoline, and to the fact that there is no adjustment of the air/fuel ratio during the use of gasoline-methanol and gasoline-ethanol mixtures. The initial adjustment that corresponds to gasoline as fuel is maintained [6,7].

Furthermore, during the tests the consumption of the fuel was recorded for every mixture separately and for every load conditions. The results of the consumption recording are illustrated in the figure below:

Figure 44 shows an increase of fuel consumption when the percentage of methanol and ethanol in the fuel increases than gasoline. Also, between the use of the mixtures of methanol and ethanol is observed small increase during the use of methanol because of the smaller calorific value that methanol has compared to ethanol. The smaller calorific value of methanol and ethanol compared to gasoline and also the lack of regulation (ratio air/fuel) of the engine, results to the consumption increase contrary to the use gasoline. This increase of consumption happens automatically for the rounds regulator that the engine has, for the maintaining of the rounds constant.

3. Conclusion

From the observations above is appeared that methanol and ethanol as mixture with gasoline results in an emissions (CO and HC) decrease when the engine functions without load and under full load conditions. There is an exception in the use of the mixtures: gasoline-70%meth — anol, gasoline -90% ethanol and 100%ethanol where there is observed an HC emissions increase because of the incomplete combustion and consequently due to engine malfunction. Also, it must be mentioned that the adjustment of the engine (air/fuel ratio) was that which referred to the use of gasoline as fuel. From the aspect of consumption, there was a consump-

tion compared to gasoline increase when the percentage of the methanol and ethanol in the fuel was increased in both load conditions. Between the use of methanol and ethanol mixtures is observed higher increase of consumption when the mixtures of methanol are used due to the fact that methanol has lower calorific value compared to ethanol. From the aspect of emissions, when the mixtures of gasoline with methanol and ethanol are compared, there is grater reduction of emissions in the case where methanol is used. It can be said that this is caused because of the smaller carbon chain of the methanol molecule, which results to the better combustion of methanol. It is also observed that the engine functions with the mixtures of methanol until the use of 70%methanol mixture with gasoline, while with ethanol mixtures until 100% ethanol as fuel (with the initial adjustment of the air/fuel ratio that is made for gasoline). This is due to the fact that ethanol has higher octane number compared to methanol. Finally, it is important the fact that methanol and ethanol are a renewable fuels, which present emissions decrease compared to gasoline, when they are used, in a time period where petroleum reservations are depleted and the environmental pollution is one of the most important problems that humanity faces [4—7].

Author details

Charalampos Arapatsakos*

Department of Production and Management Engineering, Democritus University of Thrace, Xanthi, Greece

Entrained flow reactor is the preferred route for large scale gasification of coal, petcoke and refinery residues because of high carbon conversion efficiencies and low tar production [22]. This mode of gasifier does not require inert bed material but relies on feeding the feedstocks co-currently with oxidizing agent at high velocity to achieve a pneumatic transport regime

[18]. At operating temperature of 1200-1500oC, this method is able to convert tars and meth­ane, resulting in better syngas quality [18]. Importantly this technology requires the feed­stocks to be pulverised into fine particles of ~50 |om before feeding, which is not a major issue for coal but very difficult and costly for biomass sources [18, 22].

Amongst the different options for the conversion of xylose reported in the previous chap­ter, production of lactic acid via the microbial route is a vastly studied field [61—63] since currently, all of the production of lactic acid at an industrial scale in the world is biologi­cally based. Traditionally, the concept evolves around fermenting carbohydrate-based syr­up by homolactic organisms, mostly lactic acid bacteria (LAB). The most common carbohydrate-based substrates used to this purpose may be molasses, corn syrup, whey, sugarcane or even beet bagasse. Highly efficient LAB includes Lactobacillus delbrueckii, L.

amylophilus, L. bulgaricus and L. leichmanii. Mutant Aspergillus niger has also been reported to be effective at an industrial scale [64]. LAB have the particularity to possess an homo­fermentative metabolism producing only lactic acid as extracellular waste product, instead of the heterofermentative pathway yielding by-products such as aldehydes, organic acids and ketones. The catabolic pathway yielding lactic acid is essentially the same across all organisms; the pyruvate intermediate is converted to lactic acid by a lactate dehydrogen­ase (LDH). Thus for hexose sugars, the theoretical yield is 2 moles of lactate per mole of sugar (or 1g sugar for 1g lactate). This enzymatic catalysis has the advantage over its chemical counterpart to be stereospecific: both L-lactate-dehydrogenase (L-LDH) and D — lactate-dehydrogenase (D-LDH) exist, generating either L-lactate or D-lactate respectively [65]. Both are NAD-dependant (nicotinamide adenine dinucleotide) and may be found alone or together in wild lactate-producing microbial strains. Since optical purity of lac­tate is a major requirement for the lactate industry, research focuses on stereospecificity as much as yields and productivity [61,66—70].

An efficient lactate producer has to display specific attributes, mainly the adaptability to low-cost substrates, high selectivity of desired enantiomer (L, D or both), high optimal tem­perature for decreased contamination risks, low pH tolerance and high performances (yield and productivity). LAB display appreciable performances, but lack a low pH tolerance, which implies uses of a pH control apparatus during the fermentation process. LAB optimal pH is near neutral, but the pKa of lactic acid being 3.8, an alkali agent, usually Ca(OH)2 must be used thus generating calcium lactate. After typical batch fermentation, the medium is acidified with H2SO4 therefore regenerating and purifying the lactic acid [64]. Another drawback of LAB is their requirement for a complex growth medium, since they are auxo — troph for certain amino acids and vitamins [71]. In order to overcome this problem, many fungi were also investigated for lactate production. Strains of Rhizopus, Mucor and Monilla sp. have shown potential whilst other fungi even displayed amylolytic activity, which could lead to a direct starch-to-lactate conversion [72—74].

Most researches still focuses on hexose conversion, and research group have optimized strains and process strategies in order to obtain high lactate titers, yields and productivities. Ding and Tan [75] developed a glucose fed-batch strategy using L. casei and generating up to 210 g/L of lactic acid with a 97 % yield. Chang et al. [76] proposed a continuous high cell density reactor strategy yielding a titer of 212.9 g/L and productivity of 10.6 g/L/h with Lb. rhamnosus. Dumbrepatil et al. [77] created a Lb. delbrueckii mutant by ultraviolet (UV) muta­genesis producing 166 g/L with productivity of 4.15 g/L/h in batch fermentation. Genetically engineered non-LAB biocatalysts yet have to match the performances of highly efficient wild LAB. In fact, C. glutamicum, S. cerevisiae and E. coli recombinant have been developed, but with limited success [61].

The search for lignocellulose-to-lactate biocatalysts have led to the discovery of many strains of pentose-utilizing LAB. Lb. pentosus ATCC8041 [78, 79], Lb. bifermentans DSM20003 [80], Lb. brevis [81], Lb. Plantarum [82], Leuconostoc lactis [83, 84], and E. mundtii QU 25 [85, 86]. Lactic acid produced from xylose per say has been investigated by few [84,85, 87, 88], but with mitigated results, mainly due to the fact that the pentose-utilizing

LAB do not perform as well in pentoses as in hexoses-rich metabolism. This phenomenon is most likely due to the fact that pentoses are metabolized in the PK pathway (phospho — ketolase), thus for a given strain, even if hexoses are fermented through an homofermen­tative route, pentose will yield heterofermentative products (i. e. acetic and lactic acid) [78, 89]. Nevertheless, Tanaka et al. [84] have shown that in addition to the PK, L. lactis could metabolize xylulose-5-phosphate (X5P), an intermediate pentose catabolite, through the pentose phosphate pathway (PPP). The theoretical yield through the PPP is 5 moles of lac­tate for 2 moles of pentoses, but through the PK it decreases to 1:1 [61], thus, the conver­sion advantage of the PPP is obvious. Okano et al. [87,89] demonstrated this approach by creating a pentoses-utilizing Lb. plantarium recombinant in which the native L-lactate de­hydrogenase (L-LDH) gene was disrupted, leaving only the homologous D-lactate dehy­drogenase (D-LDH) active. However, this strain produced both acetic and D-lactic acid; hence the PK gene (xpk1) was substituted by a heterologous transketolase (tkt) from L. lac — tis, thereby shifting heterolactic fermentation to a homolactic one.

Modification of yeast strains in order to achieve xylose-to-lactate conversion has also been investigated, as an example Ilmen et al. [90] expressed the L-LDH gene from L. helveticus in P. stipitis and was able to reach a titer of 58 g/L of lactate with a yield of 58 %. These results were obtained despite the fact that no effort had been made to silence the native PDC/ADH (pyruvate decarboxylase/alcohol dehydrogenase) ethylic pathway, consequent­ly 4.5 g/L of ethanol was simultaneously produced as the endogenous PDC rivalled against the recombinant L-LDH for pyruvate. Tamakawa et al. [88] went further by trans­forming C. utilis, disrupting the native pdc1 gene, and expressing heterologous LDH, XR (xylose reductase), XDH (xylitol dehydrogenase) and XK (xylulokinase) enzymes. Further­more, to prevent the redox imbalance, they increased the XR’s NADH (reduced nicotina­mide adenine dinucleotide) affinity by site-directed mutagenesis. In batch culture this recombinant was able to yield titers up to 93.9 g/L of lactate at a yield of 91 %. Table 3 shows the most recent and most efficient strains developed for lactic acid production, both from hexoses and pentoses.

of bovine L-LDH.

Disruption of
PDCand PDH
genes.

K. lactis Glucose Semi-Batch 60 500 0.85 0.12 [99]

Expression of

bovine L-LDH

gene.

Disruption of endogenous PDC gene. Expression of heterologous LDH, XR, XDH

C. utilis and XK. XR gene site-specific mutation for preferential NADH cofactor utilization

Expression of

P. stipitis LDH from L. Xylose Batch 58 147 0.58 0.39 [90]

helveticus.

* No xylose consumption occurred

**SSF = simultaneous saccharification and fermentation

Table 3. Lactic acid concentration (LA), time of fermentation (Tf), yield and production rate for the most common microorganisms used for the biological conversion of xylose to lactic acid

Lactic acid seems to be, on the biological as well as on the chemical point of view the best possible compound that could be derived from a based-catalysed reaction of xylose. Race­mic mixtures of lactic acid (most probably derived from chemical synthesis) can be evaluat­ed to 1150 USD/tonne [100] whilst the pure isomer was reported to have a price market around 1750 USD/tonne [101]. As in many cases, the price will vary proportionally with pu­rity of the compound. Utilisation of lactic acid on the market is mostly related to polymers, food, pharmaceutical and detergents. The annual world demand for the compound should reach a little more than 367 Ktonnes/year by 2017 [102].

Fabiana Aparecida Lobo, Fernanda Pollo,

Ana Cristina Villafranca and Mercedes de Moraes

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/52514

1. Introduction

Many private and governmental initiatives have been established worldwide to identify viable alternatives to petroleum derivatives [1,2].The goals are to reduce dependence on imported en­ergy from non-renewable sources, while mitigating environmental problems caused by petro­leum products, and to develop national technologies in the alternative energy field.

Ethyl alcohol (ethanol) is considered to be a highly viable alternative fuel. Its production from biomass means that it can provide a source of energy that is both clean and renewable. The in­clusion of ethanol as a component of gasoline can help to reduce problems of pollution in many regions, since it eliminates the needto use tetraethyl lead (historically notorious as a highly tox­ic trace component of the atmosphere in major cities) as an anti-knock additive.

The quantitative monitoring of metal elements in fuels (including gasoline, alcohol, and die­sel) is important from an economic perspective in the fuel industry as well as in the areas of transport and environment. The presence of metalspecies (ions or organometallic com­pounds) in automotive fuels can cause engine corrosion, reduce performance, and contrib­ute to environmental contamination [2—5].

The low concentrations of metals in fuels typically require the use of sensitive spectrometric an­alytical techniques for the purposes of quality control. Atomic absorption spectrometry (AAS) can be applied for the quantitative determination of many elements (metals and semi-metals) in a wide variety of media including fuels, foodstuffs, and biological, environmental, and geo­logical materials, amongst others. The principle of the technique is based on measurement of the absorption of optical radiation, emitted from a source, by ground-state atoms in the gas phase. Atomization can be achieved using a flame, electrothermal heating, or specific chemical

reaction (such as the generation of Hg cold vapor). Electrothermalatomizers include graphite tubes, tungsten filaments, and quartz tubes (for atomization of hydrides), as well as metal or ce­ramic tubes. Flame atomic absorption spectrometry (FAAS) is mostly used for elemental analy­sis at higher concentration levels, of the order of mg L-1[3—5]. Table 1 lists some of the published studies concerning the application of AAS for determination of metals in fuels.

The thermospray (TS) technique was originally developed by Vestal et al. in 1978 [23]as an interface between liquid chromatography and mass spectrometry. In atomic absorption spectrometry, the tube was heated electrically in order to maintain a constant temperature, which restricted use of the method to only a few elements. However, Gasparand Berndt (2000) proposed the TS-FF-AAS procedure, in which a metal tube is positioned above the flame of the atomic absorption spectrometer, as a reactor. The sample solution is transported through a metal capillary, connected to the tube, and heated simultaneously by the flame. On reaching the hot tip of the capillary, the liquid partially vaporizes, forming an aerosol. In turn, the aerosol is vaporized within the tube, producing an atomic cloud that absorbs the radiation emitted by the lamp.

The TS-FF-AAS method was used as an interface between high performance liquid chroma­tography (HPLC) and FAAS, employing a flow injection system [25-60].

The objective of this work is to describe the analysis of Cu present in hydrated ethyl alcohol fuel (HEAF) using the technique of atomic absorption spectrometry with thermal nebuliza — tion in a tube heated in a flame (TS-FF-AAS). The atomizers used were a metal tube (Ni-Cr alloy) and a ceramic tube (Al2O3).

1.4. The chemical pathway

Xylose, as all the other carbohydrates that can be isolated from lignocellulosic biomass, has a carbonyl function that is susceptible to transformations, including reduction. One of the most common compounds that can be derived from xylose is xylitol, a pentahydroxy chiral compound as depicted in Figure 9.

Amongst the most reported catalysts in the literature are nickel and Raney nickel. According to Wisniak et al. [103] they are good catalysts for the production of xylitol from xylose with total conversion at 125 °C and 515 psi. In the same year, the authors published the use of ruthenium, rhodium and palladium for the reduction of xylose [104] concluding that the ef­ficiency of those metals was declining in the order Ru>Rh>Pd at temperatures around 100-125 °C under pressure. Mikkola et al. [105, 106] also used nickel as a catalyst by ultrason­ic process that generated close to 50 % conversion of xylose to xylitol. From this process was reported that an important problem was the deactivation of the catalyst. Utilisation of nickel also led to the publication of two patents, one in 2003 [107] and another in 2007 [108]. In the case of the first, the concept relied on the isomerization of D-xylose to L-xylose prior to cata­lytic reduction under a nickel catalyst.

Ruthenium as well as ruthenium-based compounds has also been reported as catalysts for the reduction of xylose to xylitol. Ruthenium has been operated at temperatures between 90 °C and 110 °C under pressure using ruthenium supported either on silica [109] or on carbon [110]. Conversion rates for the latter have been reported to reach 35 % to xylitol for the latter with coproduction of glycerol and ethylene glycol. Ruthenium chloride (RuCl3) has also been reported as a catalyst for the reduction of xylose to xylitol [111, 112].

Treatment of carbohydrates at a higher severity leads to the hydrogenolysis, implying not only the carbonyl compounds being reduce to alcohol but a breakage of the carbon-carbon bonds in the original carbohydrate. Recent work [113] shows that temperature above 250 °C and pressure between 600-1000 psi, can lead to conversion of xylose to ethylene glycol, pro­pylene glycol and glycerol, as depicted in Figure 10 below.

Production of ethylene glycol and glycerol has also been reported by Guha et al. [110] as a side product of their xylitol production. Hydrogenolysis of xylitol is a logical suite for re­duction of xylose and specific work has been reported using different catalytic systems and experimental setups. As an example, it was recently reported [114] that xylitol could be con­verted into a mixture of polyols and different other products as formic acid and lactic acid as well as xylitol, which, according to the previously mentioned work in this chapter, is giv­en when xylose is submitted to a noble metal catalyst under hydrogen. In this specific case, the catalyst was platinum supported on carbon under a base-catalyzed matrix. Chopade et al. [115] also presented a patent reporting the conversion of carbohydrates (including xylose) into polyols using a ruthenium catalyst as did Dubeck and Knapp in 1984 [116].

In 2010 it was reported the use of nickel as a catalyst for hydrogenolysis of xylose [117] whilst Kasehagen [118] reported hydrogenolysis of carbohydrates under a nickel-iron-cop­per catalyst using a matrix of alkali salts with glycerol as the main product. The effects of nickel was studied by Wright [119] but this time using tungsten as a co-catalyst. Finally, there is a report about hydrogenolysis of carbohydrates under a rhenium catalyst [120].

1.1. Instruments and accessories

The instrumentation consisted of an atomic absorption spectrometer fitted with a flame atomizer (Perkin-Elmer, model AAnalyst 100), a hollow cathode Cu lamp (Л = 324.8 nm, slit width = 0.7 nm, i = 15 mA), with an air/acetylene (4:2 ratio) flame gas mixture, and back­ground correction using a deuterium lamp. Other equipment comprised an analytical bal­ance (Sartorius BL 2105) and a peristaltic pump (Ismatec, model ICP 8).

The TS-FF-AAS assembly employed a Rheodyne RE9725 injection valve, PEEK tubing, and a ceramic thermocouple insulator capillary (OMEGATITE450, OMEGA, USA). The capillary wascomposed of Al2O3(>99.8%), resistant to temperatures up to 1900 °C, with 0ext= 1.6 mm and two orifices with 0int = 0.4 mm (this capillary provided better results than a stainless steel HPLC capillary, with less noise in the absorbance signal). The atomization tubes were a metal tube composed of Ni-Cr super-alloy (Inconel, length 100 mm, 0int = 10.0 mm, 0ext = 12.0 mm, 6 orifices with 0 = 2.5 mm, perpendicular to an orifice with 0 = 2.0 mm), and a ceramic tube (99.9% Al2O3, length 100 mm, 0int = 10.0 mm, 0ext = 12.0 mm, 6 orifices with 0 = 2.5 mm, perpendicular to an orifice with 0 = 2.0 mm).

Data acquisition employed the software MQDOS (Microqrnmica), and the absorbance val­ues were proportional to the height of the transient signals.

The temperature in the interior of the atomization tube was measured in two ways. The first method employed a thermocouple with an earthed connection, positioned adjacent to the metal tube, oriented towards the orifice where the ceramic capillary used to introduce the sample into the atomizer was located. The temperature measured for the metal tube was 983 ± 1°C. Secondly, the thermocouple with connection exposed was positioned adjacent to the ceramic capillary within the tube, where a temperature of between 1030 °C and 1060°C was measured, at which the tube glowed ruby-red above the flame [16,40,45,54].

When 50 |jL of HNO3 (~0.1 mol L-1) was injected at a rate of approximately 1.5 mL min-1,there was a temperature reduction of around 50°C, due to cooling of the tube by the solution, followed by a rapid return to the maximum temperature range.

5.1. The chemical pathway

Oxidation of xylose has been numerously reported in the literature although focus interest, both on the biological as well as chemical point of view has been focused toward a simple oxidation of xylose to xylonic acid (see Figure 12).

Oxidation of xylose has been reported for a variety of different metallic catalyst including gold for high conversion rates [171]. Using a process performed a little higher than room temperature in a basic pH for 1 hour, they were able to reach a 78 % conversion of xylose to xylonic acid. Using comparable catalyst, Pruesse et al. [172] were able to reach 99 % selectivi­ty with a conversion rate of 21 mmol/min/g (Au) in a continuous reactor. Nevertheless, con­trarily to Bonrath, Pruesse and co-worker used a mixture of gold and palladium to perform this oxidation and temperature slightly higher (60 °C as compared to 40 °C).

Copper has also been indirectly investigated for the conversion of xylose to xylonic acid in that sense that Van der Weijden et al. [173] used C5 sugars (including xylose) for the reduc­tion of copper sulfate in wastewater with very promising results. Although emphasis was not put on the carbohydrate itself, results showed that the reduction of copper from (II) to elemental was possible yet economical at larger scale. Xylonic acid was also observed as by­product of xylose oxidation using chlorine, as a side reaction of lignin oxidation. In this work [174], the concentration of xylonic acid increased by a factor of 40 after the chlorination process. Interesting enough, the xylitol concentration also increased, which might lead to the conclusion that oxidation, was probably not the sole factor here and that side reactions as the Cannizarro reaction between two xylose molecules could have been occurring. Jokic et al. [175] showed that it was possible up to an efficiency of 80 % to convert xylose simultane­ously to xylonic acid and xylitol using electrotechnologies. Such process could be to a cer­tain extent compared to the Cannizarro reaction where the original aldehyde is acting as redox reagent.

Further oxidation of xylose leads to a trihydroxydiacid, more specifically xylaric acid as de­picted in Figure 13 below.

Conversion of C5 sugars and to a smaller extent of xylose into aldaric acids has been descri­bed in literature in a few reports. Kiely et al. [176] reported that a conversion up to 83 % xy­lose into 2,3,4-trihydroxyglutaric acid was achievable in a reaction mixture composed of nitric acid and NaNO2. The side product of this reaction was reported to be disodium tetra — hydroxysuccinate. Conversion of xylose to xylaric adic was also reported [177] using oxygen under a platinum catalyst all of this in an alkali promoted medium. Comparable conversion process [178] was obtained without any alkali, though still performed the reaction in water at 90 °C under 75 psi of oxygen. The conversion for this process was 29 %. Fleche et al. [179] reported a maximum conversion of 58% once again using platinum supported on alumina.

Severer oxidizing conditions leads to a breakage of the carbon-carbon bonds in the carbohy­drate molecule leading to the production, mostly, of small organic acids as formic and acetic acid on glucose [180]. A simplified scheme of such a reaction is presented in Figure 14 below:

An example of sever oxidation of xylose in a mixture of hydrogen peroxide and ammonium hy­droxide have been recently reported [181] with a conversion of 96 % at room temperature for 1 h. Similar conversion of xylose was reported [182] for a process using oxygen and a molybde­num and vanadium catalyst. The reaction was done for 26 h at 353 K and 30 bar for a conversion of up to 54 % into formic acid with carbon dioxide as by-product.

5.2. The biological pathway

Xylonic acid synthesis from xylose has been reported for Acetobacter sp. [183], Enterobacter cloacea [184], Erwinia sp. [185, 186], Fusarium lini [187], Micrococcus sp. [188], Penidllium cory- lophilum, Pichiaquer cuum [185], Pseudomonas sp. [189, 190], Pullularia pullulans [191], Glucono — bacter and Caulobacter [192, 193].

In metabolic pathways, xylose is converted to xylonate via 2 key enzymes. First, a xylose de­hydrogenase (XD) oxidizes xylose to D-xylono-1,4-lactone (xylonolactone) using either NAD + or NADP+ as cofactor. This reaction is followed by the hydrolysis of xylonolactone to xylo­nate either spontaneously or by an enzyme with lactonase activity [194, 195]. It is hypothe­sized that Pseudomonas and Gluconobacter sp. both carry a membrane-bound pyrroloquinoline quinine (PQQ)-dependent XD and a cytoplasmic one [195, 196]. Stephens et al. [193] recently proposed a full xylose catabolic pathway for C. crescentus. Note that a similar pathway was proposed for arabinose yielding L-arabonate [197]. As shown in Figure 15, the proposed metabolic pathway for C. crescentus shows that xylonate is an intermediate in catabolic reactions that is quite different from the XI or XR/XDH previously discussed which were more intensively studied.

Researches on highly efficient microbial xylonic acid production are scarce compared to bio­fuels or xylitol. Even if the identification of xylonate producing species began as early as 1938 [187], the first attempt to isolate a possible industrial biocatalyst was done by Buchert et al. [185], who identified P. fragi ATCC4973 as a potentially high efficiency xylonate producer (92 % of initial sugar converted to xylonic acid with initial xylose concentration of 100 g/L). In further work, P. fragi and G. oxydans showed yields of over 95 % but the low tolerance of those native strains to inhibitors tends to be problematic for industrial uses [192]. As dis­cussed above, the metabolic pathways implied by xylonate have been investigated in the re­cent years [193,196]. The first recombinant microorganism engineered for the industrial production of xylonate was done by Toivari et al. [198]. By introducing the heterologous Tri — choderma reesei xydl gene (coding for the NADP+ dependant XD) in S. cerevisiae, they were able to obtain up to 3.8 g/L xylonate with 0.036 g/L/h productivity and 40 % yield. Nygard et al. [195] engineered K. lactis by introducing T. reesei xydl and deleting the putative xyll gene coding for the XR. Up to 19 g/L xylonate where produced when grown on a xylose (40 g/L) and galactose (10.5 g/L) medium. The native ability of fast xylose uptake was an advantage, but high intracellular xylonate concentration was observed, which may indicate difficulties with product export. Liu et al. [199] used similar approach engineering E. coli by disrupting the native xylose metabolic pathways of XI and XK (as shown in Figure 16). The native path­way of xylonate was also blocked by disrupting xylonic acid dehydratase genes. The XD from C. crescentus was introduced and 39.2 g/L of xylonate from 40 g/L of xylose in minimal medium was obtained at high productivity 1.09 g/L/h. From these results it is clear that re­search is at its genesis and significant efforts will be required for the creation of a highly pro­ductive and effective xylonate production biocatalyst.

At this point it is rather hard to verify the potential or the economic value of oxidation prod­ucts from xylose. Complete oxidation to formic acid could be the most suitable approach at this point since the market for xylonic and xylaric acid is not as well defined as for the sim­ple methanoic acid with its actual market value between 750-950 USD/tonne [200] and an annual world demand suspected to reach 573 Ktonnes in 2012 [201]. Conversion of xylaric acid into glutaric acid (pentanedioic acid) would lead to a very interesting market as a plas­ticizer but dehydration or reduction of the three central hydroxyl groups may be a challenge that could be winning at lab scale although a multiple synthesis pathway would be very dif­ficult to reach economic at an industrial level.

D-xylonic acid

Figure 16. D-xylose and D-xylonic acid metabolic pathways in E. coli. The symbol X denotes that the gene is disrupted.

6. Conclusion

Second-generation ethanol or "cellulosic ethanol" relies on the utilisation of lignocellulosic biomass as a source of carbohydrates via the "bio" conversion route (keeping in mind that other pathway, as thermocatalytic pathways, may also lead to cellulosic ethanol). Produc­tion of ethanol thus requires isolation of cellulose from lignocellulosic matrix, then hydroly­sis of cellulose to glucose prior to fermentation. Both of the previously mentioned steps represent challenges for industry, but the whole economic of the process is perhaps the most challenging part of cellulosic ethanol production. Cellulose is usually available in lignocellu­losic biomass in the 45-60 % range which, assuming a perfect conversion implies production of 300-400 L/tonne of lignocellulosic biomass processed. At an actual price of 0.48 USD/L, each ton of biomass has a potential value of about 150-200 USD/tonne of biomass processed.

The conversion of lignocellulosic biomass is rather more complex and to a certain extent more expensive than starch-based feedstock as corn and therefore, one can assume that the
conversion price is going to be higher than classical or first generation ethanol production. Keeping that fact in mind, the conversion of cellulose to glucose itself is a major technologi­cal challenge since it either requires enzymes, ionic liquids or strong acids that are rather ex­pensive to buy or expensive to recycle and since it is of outmost importance for the production of the ethanol, technology is to a certain extent limited by this reality.

The remaining carbon content of lignocellulosic biomass is also an important factor to be considered. Since the maximum production of ethanol from the total feedstock could vary around 300-400 L per tonne, there is at this point a necessity to generate co-products from the biomass in order to make this whole process economic at the end thus coping for techno­logical problem as conversion of cellulose to glucose. Lignin is one of the most abundant macromolecule on earth bested only by cellulose. The aromatic nature of lignin is a chal­lenge for ethanol production but not for added value compounds as aromatic monomers that could displace actual monomers used in the polymer industry that are usually obtained from non-renewable materials.

Hemicelluloses are also an important part of the lignocellulosic biomass. Hemicelluloses, contrarily to cellulose that is characterized by an amorphous and a crystalline part, are high­ly ramified and easy to hydrolyse. Usually, a simple diluted alkali solution, acidic solution or even hot water can allow conversion of hemicellulose to simple sugars. The major prob­lem with hemicellulose is the heterogeneous composition including but not limited to small acids and a variety of C6 and C5 sugars. Whilst the C6 sugars could be easily fermented to ethanol, pending reduction of the organic acids and other inhibitors, the C5 sugars require speciality yeasts for fermentation.

Other than the classical fermentative pathway, C5 sugars can as well be converted, biologi­cally as well as chemically into a wide variety of added value products and "green" com­pounds. In this paper, we have identified 4 pathways for the conversion of C5 sugars but more specifically xylose, a common carbohydrate in biomass hemicelluloses.

Reaction of xylose under an acid catalyst is probably one of the most investigated fields in this domain. The target for this conversion being furfural, a well-known chemical as well as precursor for other compound as furan, Me-THF, THF and furfuryl alcohol, a reactant used in the polymer industry. The best approach for the conversion of xylose furfural, to the best of our knowledge, is chemical as no microorganism allowing conversion of C5 sugars to fur­fural has been identified so far. The conversion of xylose to furfural was reported to reach more than 95 % for both heterogeneous and homogeneous catalyst. On the other hand, the selectivity toward furfural is not always as efficient since the latter undergoes polymerisa­tion in acidic medium, which often also leads to deactivation of the catalyst.

A basic catalyst leads to a conversion of C5 sugars to lactic acid although this pathway as not been deeply investigated in the literature. Lactic acid is a compound well in demand on the market but the limitations for the chemical transformation is the lack of stereospecificity of the products. Conversion of xylose under a base catalyst leads to the production of a race­mic mixture of D — and L-lactic acid and thus reducing the market value of the product, par­ticularly if the polymer industry is targeted. On the other hand, the biological conversion of xylose to lactic acid is a well-known and extensively reported process for which the produc­tion was reported to reach 6.7 g/L/d for genetically modified organisms as, in this specific case, Lactobacillus sp. RKY2. According to the reports, the production of lactic acid would be more efficient by the biological approach since it can lead to a stereospecific and a higher market value.

Reduction of xylose can lead to many different products including xylitol for lower severi­ty up to diols as ethylene glycol and propylene glycol at higher severity. It is ambiguous to determine at this point if either the chemical or the biological pathway is more efficient for the production of xylitol since reports on both pathways have shown promising re­sults. The main problem with the xylitol market is that although it is increasing, it is fair­ly small and therefore it is harder to fit in a new production of xylitol. On the other hand, a more severe reduction of xylose, leading to diols, could be a very interesting opportuni­ty for the production of ethylene glycol and propylene glycol, two very important prod­ucts in the chemical industry. The downside of this approach would be the production of glycerol as a side-product.

Finally, oxidation of xylose is, at this point, the approach with the lower potential for a rapid commercialisation since the market for xylonic acid and xylaric acid is hard to size at present. The conversion process, both chemical and biological seems to have significant po­tential in terms of scalability but the end usage is not well defined at this point. The best option would be to produce glucaric acid from xylaric acid, which could be used as a plasti­cizer. On the other hand, such a process, overall rather complicated, would add a significant cost for a product that would land in the commodity range.

Acknowledgement

We would like to acknowledge Enerkem, Greenfield Ethanol, CRB Innovations and the Min­istry of Natural Resources of Quebec for financial support of the Industrial Chair in Cellulo — sic Ethanol.

Author details

Ariadna Fuente-Hernandez, Pierre-Olivier Corcos, Romain Beauchet and Jean-Michel Lavoie*

*Address all correspondence to: jean-michel. lavoie2@usherbrooke. ca

Industrial Research Chair on Cellulosic Ethanol (CRIEC), Departement de Genie Chimique et de Genie Biotechnologique, Universite de Sherbrooke, Sherbrooke, Quebec, Canada

Working standard solutions were prepared from a stock 1000 mg L-1 copper standard solu­tion (spectroscopic grade), by dilution in 0.14 mol L-1 HNO3 (Synth).

The HEAF samples were prepared by mixing the fuel with an equal volume of 0.14 mol L-1 HNO3,with final volumes of 50 mL [3—5]. Subsequent quantification employed the standard additions procedure.

1.2. Assembly of the TS-FF-AAS system

A schematic diagram of the TS-FF-AAS system is shown in Figure 1.

It is recommended that the Inconel tube should only be positioned above the burner head after lighting the flame, to avoid the possibility of an explosion within the tube due to gas accumulation. The TS-FF-AAS system was therefore first assembled, after which the spec­trometer flame was ignited immediately after opening the gas valves to avoid any explosion risk. This procedure facilitated the positioning of the tube above the burner head, which was performed while the flame was extinguished. All analyses employed a fixed volume of sam­ple, injected into the flow of air as the carrier, since previous work has shown that injection using carrier solutions results in greater sample dilution and dispersion [40,45,54,58].

The sample was introduced into the system using a manual Rheodyne valve (Figure 1), after which it was transported to the ceramic capillary in the flow of air. Since the capillary was heated simultaneously with the metal or ceramic reactor tube, the liquid was partially va­porized, forming a thermospray, and atomization occurred on arrival in the tube, generating a transient signal that was captured and stored by the software. The determination em­ployed the height of the transient signal peak.

1.3. Optimization of carrier flow rate and sample volume

The influences of the carrier flow rate (in the range 9.0-18.0 mL min-1) and the sample vol­ume (50, 100, and 200|jL) were evaluated using a standard of 200|jg Cu L-1.

1.4. Construction of analytical curve

After optimization of the system, analytical curves were constructed in the concentration range 0.1-0.4 mg Cu L-1 in 0.14mol L-1 HNO3. Additions of analyte were made to the sample mixed with an equal volume of 0.14 mol L-1 HNO3. The detection limit (DL) was calculated from 12 blank readings for each type of tube (metal or ceramic).

Leona Paulova, Petra Patakova, Mojiw Rychtera and Karel Melzoch

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/53492

1. Introduction

Fluctuations in the price of oil and projections on depletion of accessible oil deposits have led to national and international efforts to enhance the proportion of energy derived from renewable sources (bioenergy) with special emphasis on the transport sector (e. g. according to Directive 2009/28 EC, by 2020, 20% of energy in EU-27 should be met from renewable sources and 10% should be used in transportation). To fulfil the legal requirements, wider exploitation of biofuels made from renewable feedstocks, as a substitute for traditional liq­uid fuels, will be inevitable; e. g. the demand for bioethanol in the EU is expected to reach 28.5 billion litres by 2020 [1], while in America 36 billion gallons of ethanol must be pro­duced by 2022 [2]. Bioethanol, which has a higher octane level then petrol but only contains 66% of the energy yield of petrol, can be used as blend or burned in its pure form in modi­fied spark-ignition engines [2]. This will improve fuel combustion, and will contribute to a reduction in atmospheric carbon monoxide, unburned hydrocarbons, carcinogenic emis­sions and reduce emissions of oxides of nitrogen and sulphur, the main cause of acid rain [2]. Butanol-gasoline blends might outcompete ethanol-gasoline ones because they have bet­ter phase stability in the presence of water, better low-temperature properties, higher oxida­tion stability during long term storage, more favourable distillation characteristics and lower volatility with respect to possible air pollution. Recently performed ECE 83.03 emis­sion tests [3] have shown negligible or no adverse effects on air pollution by burning buta­nol-gasoline blends (containing up to 30% v/v of butanol) in spark ignition engines of Skoda passenger cars.

Although most of the world’s bioethanol is currently produced from starch or sugar raw materials, attention is increasingly turning to 2nd generation biofuels made from lignocellu — lose, e. g. agriculture and forest wastes, fast growing trees, herbaceous plants, industrial

wastes or wastes from wood and paper processing. The concept of ethanol production from lignocellulose sugars is not new. Probably the first technical attempt to degrade polysac­charides in wood was carried out by the French scientist Henri Braconnot in 1819 using 90% sulfuric acid [4]. His findings were exploited much later, in 1898, with the opening of the first cellulosic ethanol plant in Germany, followed by another one in 1910 in the US [5, 6]. During World War II, several industrial plants were built to produce fuel ethanol from cellu­lose (e. g. in Germany, Russia, China, Korea, Switzerland, US), but since the end of the war, most of these have been closed due to their non-competitiveness with synthetically pro­duced ethanol [7]. In spite of all the advantages of lignocellulosic as a raw material (e. g. low and stable price, renewability, versatility, local availability, high sugar content, noncompeti­tiveness with food chain, waste revaluation) and extensive efforts of many research groups to reduce bottlenecks in technology of lignocellulosic ethanol production (e. g. energy inten­sive pretreatment, costly enzymatic treatment, need for utilization of pentose/hexose mix­tures, low sugar concentration, low ethanol concentration), large scale commercial production of 2nd generation bioethanol has not been reopened yet [8], although many pilot and demonstration plants operate worldwide [9]. Identically, only first generation biobuta­nol is produced in China (approx. annual amount 100 000 t) and Brazil (approx. annual amount 8 000 t) [10]. At the 2012 London Olympic Games, British Petrol introduced its three most advanced biofuels i. e. cellulosic ethanol, renewable diesel and biobutanol. At a demon­stration plant at Hull UK, biobutanol, produced by Butamax (joint venture of BP and Du­Pont) was blended at 24 % v/v with standard gasoline and used in BMW-5 series hybrids without engine modifications [11]. As the final price of both ethanol and 1-butanol produced by fermentation is influenced mostly by the price of feedstock, the future success of industri­al ABE fermentation is tightly linked with the cost of pre-treatment of lignocellulosic materi­al into a fermentable substrate.