**5. Production of ethanol from lignocellulose**

Production of bioethanol from lignocellulosic biomass by wild type thermophilic bacteria has been widely reported in the literature where the focus has been mostly on *Clostridium*  *thermocellum* and species within the genera *Thermoanaerobacterium* and *Thermoanaerobacter*. However, there is a large variation in the type and concentration of biomass used, fermentation processes (batch, semi-batch, continuous), pretreatment methods as well as whether pure or mixed cultures are used.

**4.2. Substrate loadings**

106 Fuel Ethanol Production from Sugarcane

**4.3. Ethanol tolerance**

ethanol yields dropped considerably.

**4.4. Other culture parameters**

In natural environments of thermophilic bacteria, the concentration of sugars is relatively low. It is thus not surprising that most thermophilic bacteria are inhibited at relatively low (often between 10 and 30 mM) initial substrate concentrations as compared to yeasts and *Z. mobilis* [4, 38, 39, 46]. This inhibition may be caused by accumulated hydrogen or by acid accumulation and pH drop, or it could also be an intriguing factor for thermophiles. *Thermoanaerobacter*, strain J1, has been shown to be very tolerant towards high sugar concentrations [17]. This high ethanol producing thermophile produces up to 1.7 mol ethanol/mol glucose at 100 mM initial glucose concentration. Recent work on *Thermoanaerobacter pentosaceus* showed a complete removal of xylose at 13.3 mM initial concentrations but only about 30% removal at 10 times higher concentrations [55]. Additionally, the ratio of ethanol to acetate and lactate decreased

One of the most important traits for good ethanol producers is their ethanol tolerance. For an economic ethanol recovery to occur, using classical downstream processes, the microorganism should grow and produce ethanol in the presence of at least 4% (v/v) ethanol [56]. It is well known that growth rates of many organisms decrease markedly with increasing ethanol concentrations because of leaky membranes resulting in loss of energy during cellular metabolism and finally cell lysis. Yeasts and *Z. mobilis* tolerate much higher ethanol concentrations as compared to thermophiles mainly due to their composition of fatty acid in their cell membrane.

Studies on ethanol tolerance of wild-type species of thermophiles show tolerance between 0.5 and 3.0% (v/v) [4, 46, 57, 58]. Substantial efforts to increase ethanol tolerance of wild type thermophiles, have been done. The highest ethanol tolerance observed for a thermophile has been with a mutant strain of *Thermoanaerobacter ethanolicus* (12.7% v/v) [28]. However, later studies with one of its mutant derivatives, JW200 Fe 4, showed much less tolerance [59]. *Thermoanaerobacter* BG1L1 showed 8.3% (v/v) tolerance in continuous culture studies [43] on xylose. Increased ethanol tolerance was also observed with *Thermoanaerobacter thermohydrosulfuricus* 39E by successively sub-culturing the strain to higher ethanol concentrations [57]. The resulting mutant strain 39EA tolerated 10.1% (v/v ethanol) at 45°C but only 2.6% (v/v) at 68°C. Additionally, the

Other environmental factors of importance for thermophilic bacteria is their pH and temperature growth optimum, their tolerance towards inhibitory compounds like furfuraldehyde and 5-hydroxymethyl-furfuraldehyde (5-HMF) and their need for trace elements and vitamins

Production of bioethanol from lignocellulosic biomass by wild type thermophilic bacteria has been widely reported in the literature where the focus has been mostly on *Clostridium* 

often originating from complex medium supplements like yeast extract.

**5. Production of ethanol from lignocellulose**

by a factor of more than six resulting in dramatic decrease in ethanol yields.

The theoretical maximum yield of ethanol obtained from glucose fermentation is 0.51 g ethanol/ g glucose (2 mol ethanol/mol glucose or 11.1 mM/g). Unsurprisingly, considering the complex structure of lignocellulosic biomass, ethanol yields are usually considerably lower from such substrates as seen in **Table 1**.


Cultivation were either in batch or continuous (con). Ethanol yields given in mM/g substrate degraded as well as substrate concentrations and incubation temperature are also shown. A—acid; Alk—alkaline; E—enzymatic; and WO wet oxidation.

**Table 1.** Examples of ethanol production from lignocellulosic biomass by thermophilic bacteria.

Early experiments on ethanol production from lignocellulose included as the ethanol-producing organisms *Thermoanaerobacter ethanolicus* and *Clostridium thermocellum* with hemicellulose from birch- and beechwood as a feedstock [66]. *Clostridium thermocellum* produced between 7.2 mM ethanol /g and 8.0 mM/g from avicel and Whatman paper, respectively. Studies of ethanol production from paddy straw, sorghum stover and corn stubs, pretreated with alkali showed similar results [68]. However, these results were obtained with relatively low substrate loadings (8.0 g/L) but later studies showed that increased substrate loadings lowered the ethanol yields considerable [69]. The highest ethanol yields reported from lignocellulose are by *Thermoanaerobacter* BG1L1 grown on corn stover and wheat straw [42, 43] that were pretreated with acid or wet oxidation, or 9.2 mM/g for biomass hydrolysates. *Thermoanaerobacterium* strain AK17 showed ethanol yields of 2.0 (paper) mM/g, 2.9 (grass) mM/g and 5.8 (cellulose) mM/g biomass [23]. Optimization experiments showed an increase in ethanol yields on grass and cellulose up to 4.0 and 8.6 mM·g−1, respectively. The main culture factors increasing ethanol yields was obtained by lowering of the substrate concentration from 7.5 to 2.5 g/L [39]. Recent investigations on two *Thermoanaerobacter* strains, AK5 and J1, showed promising results from various types of hydrolysates made from chemically and enzymatically pretreated lignocellulosic biomass [17, 38] (**Table 1**).

to higher substrate concentrations and demonstrated higher ethanol tolerance and substrate utilization [72]. Thus, evolutionary adaptation, may still be used for evolving of wild type strains and further improving GM strains to meet requirements for tolerance to high ethanol titers, improve substrate utilization, and potentially resistance to inhibitory compounds generated

Progress in Second Generation Ethanol Production with Thermophilic Bacteria

http://dx.doi.org/10.5772/intechopen.78020

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Despite other promising features, one of the main drawback of most wild type thermophiles is their production of mixed end-products resulting in lower ethanol yields and the fact that highly ethanologenic organisms are not natively cellulolytic and *vice versa*. Two main strategies have been used to metabolically engineer thermophilic organisms for consolidated bioprocessing (CBP). The first strategy is to increase the ethanol yields of cellulase-producing organisms while the other is to express enzymes for biomass deconstruction in highly ethanologenic microorganisms [73, 74]. The first approach involves increasing ethanol yields by redirecting the flow of carbon and electrons which involves eliminating other potential fermentation products. Obvious targets include knocking out acetate and lactate pathways. The second approach involves addition of cellulolytic genes to the genome of a good ethanol

The first thermophilic bacterium to be genetically modified to increase ethanol yields was *Thermoanaerobacterium saccharolyticum* in 2004 [75]. Since then, several other ethanologenic thermophiles have been genetically modified to increase ethanol titers and minimize the for-

Deletion of genes involved to the production of various end-products to increase ethanol production capacity is the most obvious way to increase ethanol titers. This has been done by knocking out lactate dehydrogenase in *Thermoanaerobacterium saccharolyticum* [73, 82], *Thermoanaerobacter mathranii* [79], *Clostridium thermocellum* [83] and *Geobacillus thermogluco-*

Wild type *Clostridium thermocellum* produces a mixture of ethanol, acetate, lactate, hydrogen, and carbon dioxide [84] from cellulose and other substrates. The first successful transformation of the species was performed in 2006 [85], later on leading to the development of genetic systems to knock out the *pta* gene and thus acetate formation [85]. However, growth of the resultant strain was abnormal although cellulase active remained intact. Later work on *C. thermocellum* showed improved ethanol yields in an adapted strain (Δ*hpt*, Δ*ldh*, Δ*pta*) lacking acetate and lactate pathways and was successfully used in co-culture with *Thermoanaerobacterium* 

Early work on *Thermoanaerobacterium saccharolyticum* were performed by using electroporation and shuttle vectors [86], but later on this strain has been further modified by inserting a cellobiohydrolase gene from *Clostridium thermocellum* into its genome [77]. Also a *ldh* gene knock out was done using *Thermoanaerobacterium saccharolyticum* [75] and then a double knock out of both *ldh* and *ak* [73]. The knocking out of acetate production led to less available energy,

during biomass pretreatment such as 5-HMF and fufuraldehyde.

**6.2. Genetic engineering**

producing bacterium.

*sidasius* [78].

*saccharolyticum* [85].

mation of other end-products (**Table 2**).
