4.1. Ethanol fermentation

Fermentation of lignocellulose hydrolysates is more complicated compared to fermentation of 1st generation feedstock (sugar cane juice, molasses, grains) for several reasons: a) pentose sugars (predominantly xylose) are present along with hexoses (mainly glucose, mannose, galactose) in the hydrolysate, b) toxic compounds released during pretreatment can influ­ence metabolic activity of the fermentation strain, c) low concentrations of fermentable sug­ars hamper the attainment of a high ethanol concentration. Because lignocellulose hydrolysates are poor in some nutrients (phosphorus, trace elements, and vitamins) they are usually supplemented, e. g. by addition of corn steep or yeast extract before being used as a substrate for fermentation. For an efficient process it is necessary to identify a strain that uti­lizes both pentose and hexose sugars, produces ethanol with a high yield and productivity and is tolerant to both inhibitors and ethanol. One of the main challenges is to simultaneous­ly co-ferment pentose and hexose sugars, but neither yeast S. cerevisiae nor the bacterium Z. mobilis, which are usually used for ethanol production, contain genes for expression of xy­lose reductase and xylitol dehydrogenase [89]. In order to enhance process effectiveness, co­fermentation or sequential fermentation of hexoses and pentoses has been examined by combining good ethanol producers with strains naturally utilizing pentoses e. g. Pichia stipi — tis, Candida shehatae, Pachysolen tannophillus, Klebsiella oxytoca. However, xylose utilization is the rate limiting step due to catabolite repression by hexoses and the low availability of oxy­gen, and inhibition of pentose-utilizing strains by ethanol [90, 91]. Moreover, the yield of ethanol by co-fermentation is usually lower than with separate processes, e. g. yields of 0.5 g ethanol per g glucose (98% of theoretical) and 0.15 g/g xylose (29% theoretical) were ach­ieved by separate cultivation of Z. mobilis and P. tannophillus respectively, but in optimized co-fermentation, the yield was just 0.33 g ethanol/g sugar. The same yield was obtained in a 5-reactor process combining P. stipitis and S. cerevisiae [92], but it was enhanced to 0.49 g/g sugars (96% theoretical) by cultivation of an adapted co-culture of S. cerevisiae, P. tannophilis and recombinant E. coli in dilute-acid softwood hydrolysate [93]. In a subsequent process employing P. stipilis and S. cerevisiae, which was inactivated before Pichia inoculation to avoid oxygen competition, 75% of theoretical ethanol yield was achieved [94]. A different approach is represented by the use of a recombinant strain prepared either by cloning genes encoding xylose utilization into good ethanol producers or to construct synthetic pathways for ethanol production in pentose-utilizing hosts. Wild type yeasts can be genetically modi­fied to utilize xylose by introducing fungal genes encoding xylose reductase and xylitol de­hydrogenase or bacterial/fungal genes for xylose isomerase [95]. Yeast S. cerevisiae was transformed with the xylA gene from Thermus thermophiles and Piromyces sp. to produce xy­lose isomerase, but unfortunately, this enzyme was inhibited by xylitol, favouring instead, its formation. Recently a recombinant strain of S. cerevisiae expressing a heterologous xylA gene produced 0.42 g/g of ethanol from xylose [96]. A strategy using xyl1 and xyl2 genes from P. stipitis introduced into S. cerevisiae produced transformants that exclusively con­sumed xylose, but produced significant amounts of xylitol [97]. On the other hand, with re­combinant Z. mobilis, which carried E. coli genes encoding for xylose isomerase, xylulokinase, transketolase and transaldolase, 86% ethanol yield from xylose was achieved. Another strain of Z. mobilis, expressing genes araABD from E. coli, encoding L-arabinose iso­merase, L-ribulokinase, L-ribulose-5-P-4 epimerase together with genes for transketolase and transaldolase, was able to grow on arabinose with 98% ethanol yield. E. coli, which nat­urally utilizes a wide range of substrates including pentoses, was transformed by genes en­coding pyruvate decarboxylase and alcohol dehydrodenase, resulting in enhanced ethanol production [96]. Adaptation of recombinant strains to inhibitors can further increase the yield of ethanol, e. g. the ethanol yield achieved with a genetically engineered strain of S. cer­evisiae grown on bagasse hydrolysate was increased from 0.18 g/g to 0.38 g/g after adapta­tion [98]. Recombinant strains that not only consume pentoses but also hydrolyse hemicelluloses by co-expressing endoxylanase, p-xylosidase and p-glucosidase activities has recently been constructed [95] and yields of 0.41 g/g of ethanol were obtained from total sugars in a rice straw hydrolysate.

In addition to the wide range of sugars, their low concentration in hydrolysates is problem­atic. Since ethanol recovery by distillation is only economically viable on the industrial scale for yields greater than 4% (w/w), which for most hydrolysates requires a dry mass concen­tration greater than 20% [45], the use of high substrate loading is needed. Effect of substrate concentration (unbleached hardwood pulp and organosolve pretreated poplar) on glucose concentration resulting from the enzyme hydrolysis was studied in [52] and [99]. In labora­tory scale after 48 h of enzymatic hydrolysis 158 g/l glucose in the hydrolyzate was reached, ethanol concentration after fermentation ranged between 50.4 and 63.1 g/l. The general problem for this kind of conversions is that high load of the pulp or pretreated lignocelulo — sic material gives rise to high viscosity and thus also to mixing and transport problems. These extremely high yields of glucose can be attributed to a very efficient peg mixer. Prob­lems connected with use of such high viscosity slurries can be overcome by various strat­egies, e. g. maximizing dry matter by removing most hemicellulose and lignin, utilizing alternative bioreactors with novel mixing modes (e. g. peg mixer, shaking, gravitational tum­bling, hand stirring) or gradual dosing of substrate into the bioreactor (fed-batch), which en­ables the use of more substrate and thus increases the yield of ethanol above values achievable in batch mode. Moreover, the actual concentration of toxic substrates is reduced and yield and/or productivity is enhanced by controlled dosing of substrate and prolonged cultivation time, thus shortening unprofitable periods between batches [45, 89]. Feed rates should reflect the type of hydrolysate and strain. Continuous cultures usually using immo­bilized cells (to prevent their wash out from the bioreactor at high dilution rates) is another strategy to increase process productivity [89].

Integration strategies, which replace classical separate hydrolysis and fermentation process­es (SHF) by combining several process steps in one vessel represents another approach for lignocellulosic ethanol production. Simultaneous saccharification and fermentation (SSF), which combines enzymatic hydrolysis and fermentation in one step, permits an increased rate of cellulose hydrolysis by elimination of product inhibition (the released glucose is con­sumed by the microbial strain), an increased rate of sugar consumption, reduced contamina­tion due to the presence of ethanol and a reduced number of reactors. However, SSF is constrained by different temperature optima for each process (the cellulase optimum is usu­ally 40-50 °C, whereas the fermentation temperature usually cannot exceed 35 °C for most ethanol producers) and carbon source limitation in the early stages of the process. Several modifications of SSF to ease the problems and increase productivity have been published. These include the use of thermotolerant ethanol producers [100, 101], application of a pre­saccharification step [102] or the use of recombinant strains consuming both hexose and pentose sugars (a simultaneous saccharification and co-fermentation process (SSCF)) [103] in batch or fed-batch mode [104]. Consolidated bioprocessing (CBP), which combines cellulase production, cellulose hydrolysis and fermentation into a single step have been investigated as a way of reducing the cost of cellulolytic enzymes, increasing volumetric productivity and reducing capital investment [105]. Some biofuel companies (e. g. Mascoma and Qteros) have been founded based on this concept [105]. CBP microorganisms should combine high cellulase production and secretory capability, the ability to utilize a broad range of sugars, tolerance to high concentrations of salts, solvents and inhibitors, high ethanol productivity and yield, have a known genomic DNA sequence and developed recombinant technologies and ideally be usable as feed protein after fermentation [105]. There is a lack of native organ­isms that combine the ability to produce cellulolytic enzymes and be homoethanolic with high titres and yields. Although some thermophilic anaerobic bacteria e. g. Clostridium ther — mocellum, are high cellulase producers and utilize both pentose and hexose sugars, they have a low tolerance to ethanol ~30 g/l [106] and an insufficient yield ~0.2 g/g [107]. There­fore recombinant strains have been prepared by engineering cellulolytic microorganisms (e. g. C. thermocellum, C. phytofermentans, C. cellulolyticum, T. reesei or F. oxysporum) to produce ethanol. Knockout mutants of Thermoanaerobacterium saccharolyticum that lack lactic and ace­tic acid production exhibited an ethanol yield from xylose of 0.46 g/g [108], while recombi­nant Geobacillus thermoglucosidasius produced 0.42-0.47 g/g of ethanol from hexoses [65]. Another attempt, to create a recombinant cellulose-utilizing microorganism using non-cellu­lolytic strains with high ethanol production have not been very successful; although some recombinant ethanologenic strains secreting some active cellulases have been prepared [106, 109, 110], their requirement for a nutrient rich medium and often sensitivity to end-product inhibition hamper their use [105].