Different, so-called solventogenic species of the genus Clostridium, like Clostridium acetobu — tylicum, Clostridium beijerinckii or Clostridium saccharoperbutylacetonicum, can be used for 1- butanol production by ABE fermentation. The fermentation usually proceeds in two steps; at first butyric and acetic acids, along with hydrogen and carbon dioxide, are formed and then metabolic switching leads to the formation of solvents (mainly 1-buta­nol and acetone) and the cessation/slowdown of acid and gas production (for recent re­views see [111—114]). Industrial fermentative ABE (butanol) production, which has quite a long and impressive history connected with both World Wars, is nowadays carried out only in China and Brazil (estimated annual production of 100 000 t and 8 000 t from corn starch and sugar cane juice, respectively) [10]. However, many corporations such as BP, DuPont, Gevo, Green Biologics, Cobalt Technologies and others have declared their inter­est in this field. A unique example of the use of lignocellulosic hydrolysate on an indus­trial scale is the former Dukshukino plant (operated in the Soviet Union up to 1980s) producing acetone and butanol by fermentation. The plant was based on current "very modern" biorefinery concepts which assumed the conversion of complex feedstocks (hy­drolysates of agricultural waste + molasses or corn) into many valuable products i. e. in addition to solvents (acetone, butanol and ethanol), it was possible to produce liquid CO2, dry ice, H2 fodder yeast, vitamin B12 and biogas [115].

The most interesting approach to fermentation of any lignocellulosic substrate is probably consolidated bioprocessing (CBP) i. e. a method in which a single microorganism is used for both substrate decomposition and fermentation to produce the required metabolites. Although some clostridial species such as Clostridium thermocellum can utilise cellulosic substrates and produce ethanol [116, 117], the ABE fermentation pattern unfortunately cannot be produced using clostridia. However, C. acetobutylicum ATCC 824 possesses genes for various cellullases and a complete cellulosome [118—120]. But even if production of some cellulases by C. acetobutylicum ATCC 824 was induced by xylose or lichenan [118], cellulose utilization was not achieved, possibly because of insufficient or deficient synthesis of an unknown specific chaperone that could be responsible for correct secre­tion of cellulases [119]. Nevertheless as solventogenic Clostridium species are soil bacteria that differ significantly in fermentative abilities and genome sizes, it is not excluded that in the future, some solventogenic species with cellulolytic activity will be isolated from an appropriate environment. Recently, a new strain of Clostridium saccharobutylicum with hemicellulolytic activity and ABE fermentation pattern was found amongst 50 soil-borne, anaerobic, sporulating isolates [121].

Until now, lignocellulosic substrates must be prehydrolysed for the ABE process. In the case of fermentation of lignocellulosic hydrolysate, usually containing low concentrations of fer­mentable sugars, one of the main bottlenecks in the ABE process, the low final titre of buta­nol (caused by severe butanol toxicity towards bacterial cells), is of minor importance. In fact hydrolysates are very good substrates for clostridia that express extensive fermentative abilities [122, 123]and can utilise not only cellulose-derived glucose but also hemicellulose monomers (xylose, arabinose, galactose, mannose). Co-fermentation of various sugar mix­tures was described for Clostridium beijerinckii SA-1 (ATCC 35702) [124], Clostridium acetobu — tylicum DSM 792 [125], C. acetobutylicum ATCC 824 [126] and C. beijerinckii P260 [127] however, at the same time, catabolic repression of xylose utilization in the presence of glu­cose was demonstrated in C. acetobutylicum ATCC 824 [128, 129].

An overview of fermentation parameters achieved in batch ABE fermentations of different hydrolysates is presented in Table 3. The most promising results were obtained by Lu et al. [130] using cassava bagasse and a mutant strain, C. acetobutylicum JB200; the results of Marchal et al. [131] were unique at the scale used (48 m3) as shown in Table 3. A frequent problem of lignocellulosic hydrolysates is a low final concentration of fermentable sugars caused by low density of the original substrate. This can be overcome by evaporation of the hydrolysate [132](see Table 3) or by addition of glucose and/or other carbohydrates present in the hydrolysate (this is only possible in laboratory scale experiments) [85,133—135]. In the case of glucose supplemented corn stover and switchgrass hydrolysates, final ABE concen­trations of 26 and 15 g/l were achieved [135]. With C. beijerinckii P260, use of diluted and Ca(OH)2 treated barley straw hydrolysate supplemented with glucose resulted in a solvent concentration of 27 g/l, a yield of 43% and productivity of 0.39 g/l/h [133]. In addition to ma­terials presented in Table 3, other substrates like diluted sulfite spent liquor supplemented with glucose [134], palm empty fruit bunches [136, 137] or hardwood [138] were used in the ABE process but in these cases, additional optimizations were necessary.

In addition to a batch fermentation arrangement, semi-continuous fermentation of enzymat­ically hydrolyzed SO2 pretreated pine wood using C. acetobutylicum P262 resulted in 18 g/l of solvents, a yield of 36% and solvent productivity of 0.73 g/l/h [142]. Further, fed-batch fer­mentation of wheat straw hydrolysate supplemented with varying concentrations of hydro­lysate sugars (glucose, xylose, arabinose and mannose) using C. beijerinckii P260 yielded a solvent productivity of 0.36 g/l/h if gas stripping was used [127]. In the cases shown in Table 3, enzyme hydrolysis preceeded fermentation, however simultaneous saccharification and fermentation (SSF) was also tested. In SSF of acid pre-hydrolyzed wheat straw using C. beijer — inckii P262 and solvent removal by gas stripping, 21 g/l of ABE was produced with a pro­ductivity of 0.31 g/l/h [127] Nevertheless, the solvent yield from hardwood using SSF was rather low, at 15% [138].

5. Conclusion

Intensive research over the last decades on lignocellulose-derived ethanol have focused mainly on intensification of biomass pretreatment, production of cellulolytic enzymes, and strain and process improvements, and have eliminated some of the main technologi­cal bottlenecks. Although a number of projects on 2nd generation bioethanol ended with the opening of pilot and demonstration plants around the world (production capacity in millions of gallons for the year 2012 given in brackets) e. g. the POET demonstration plant in Iowa (0.02 from corn stover and cobs), Abengoa in Kansas (0.01 from corn stover), Blue Sugarsin Wyoming (1.3 from stover and cobs), Chempolis in Finland (3.7 from paper waste), Fiberight in Iowa (6.0 MSW), Iogen in Canada (0.48 from stover), Praj MATRIX in India (0.01 from cellulose), UPM-Kymemene/Mesto in Finland (0.68 from mixed cellulose) and in spite of several proclamations, none of them is operating at the industrial scale [9]. To make this possible, further reductions in processing costs will be necessary to achieve a product that is competitive with 1st generation bioethanol. Further process integration is required, including decreased energy demand during pretreatment, increased sugar concentration, higher enzyme activity and strain recycling. By-products, e. g. lignin separated after pretreatment procedure can be used to generate energy for ethanol plant operations (lignin has higher caloric value (25.4 MJ/kg) then the biomass it­self [8]) or used as a dispersant and binder in concrete admixtures, as an alternative to phenolic and epoxy resins, or as the principal component in thermoplastic blends, poly­urethane foams or surfactants [143]. A combination of 1st and 2nd generation feedstocks (e. g. corn cobs together with stover ) can eliminate bottlenecks and lead to product com­petitiveness. Higher bioethanol production costs can also be compensated for by political and economic instruments such as tax incentives (e. g. tax exemption on biofuels and higher excise taxes for fossil fuels) and legislation (mandatory blends) to enable ready ac­cess of 2nd generation biofuels to the market [30]. Butanol, as a second generation biofuel, might be produced via fermentation and used as an excellent fuel extender in addition to ethanol if the technological bottleneck of a low final concentration, yield and productivity could be overcome, and the assumption that suitable cheap waste pretreatments were possible.

Acknowledgement

The. review was performed thanks to financial support of the projects Kontakt ME10146 of Ministry of Education, Youth and Sport of Czech Republic and BIORAF No. TE01020080 of the Technological Agency of the Czech Republic.

Author details

Leona Paulova, Petra Patakova, Mojmn Rychtera and Karel Melzoch *Address all correspondence to: Leona. paulova@vscht. cz

Department of Biotechnology, Institute of Chemical Technology Prague, Prague, Czech Re­public