**4. Culture parameters**

Most saccharolytic thermophiles use the Embden-Meyerhof-Parnas (EMP) pathway [5, 51] but do not use pyruvate decarboxylase for converting pyruvate to acetaldehyde as do yeasts. The theoretical yields of ethanol from 1 mol of hexose and pentose are 2.0 and 1.66 mol, respectively [5]. There are several routes from pyruvate to other end-products than ethanol. The following equations show the most common end-products from glucose with anaerobic bacteria:


including lactic acid, being common end-products. Examples of ethanol production from sugars by members of the genus include *Clostridium thermocellum* [32, 33] and *Clostridium* strain

The genus *Thermoanaerobacterium* is comprised of thermophilic anaerobes which fall within Cluster V of *Clostridia* [35]. Currently, the genus currently consists of nine species and *T. thermosulfurigenes* is the genus type species [36]. Species within *Thermoanaerobacterium* are usually amyloand xylanolytic with a *T*opt between 55 and 65°C and have been isolated from a diverse range of environments including geothermal features and from heat-treated canned foods [21, 37, 38]. They catabolize a broad range of hexoses, pentoses, and disaccharides to a mixture of ethanol, acetate,

from high initial substrate concentrations which considerably lower ethanol yields. Examples of ethanol production from sugars by members of the genus include *Thermoanaerobacterium saccharolyticum* with 1.18 mol ethanol/mol glucose [37] and *Thermoanaerobacterium* strain AK17 with 1.50

*Thermoanaerobacter* species have similar physiological characteristics as *Thermoanaerobacterium* species; all species within the genus are highly saccharolytic and produce end-products

species belong to the genus [24, 25]. The main difference between *Thermoanaerobacter* and

thiosulfate whereas *Thermoanaerobacterium* produces sulfur [37]. Additionally, the temperature optima for *Thermoanaerobacter* species (65–75°C) are higher as compared to *Thermoanaerobacterium* species (55–65°C). The type species, *Thermoanaerobacter ethanolicus* and several other species within the genus, have been extensively studied for ethanol production [40–43]. High ethanol yields have been observed by several members of the genus including *T. pseudoethanolicus*, *T. mathranii*, *T. pentosaceus*, *Thermoanaerobacter* strain AK5, and *Thermoanaerobacter* strain J1 [17, 38, 44–46]. The ethanol yields, however, vary extensively depending on culture conditions [17, 38]. Recently, *Thermoanaerobacter subterraneous* was moved to the genus *Caldanaerobacter* that currently comprises two species: *Caldanaerobacter subterraneous* (and its four subspecies) and *Caldanaerobacter uzonensis* [24, 25]. Other representative examples of thermophilic ethanologenic bacteria can be found within the genera of *Caldicellulosiruptor* [47], *Caloramator* [48], *Geobacillus* [49], *Caloramator boliviensis*, for example, produces 1.53 mol ethanol/mol xylose [50].

Most saccharolytic thermophiles use the Embden-Meyerhof-Parnas (EMP) pathway [5, 51] but do not use pyruvate decarboxylase for converting pyruvate to acetaldehyde as do yeasts. The theoretical yields of ethanol from 1 mol of hexose and pentose are 2.0 and 1.66 mol, respectively [5]. There are several routes from pyruvate to other end-products than ethanol. The following equations show the most common end-products from glucose with anaerobic

*Thermoanaerobacterium*, is that the majority of *Thermoanaerobacter* species produce H<sup>2</sup>

. One challenge for these organisms is achieving good ethanol yields

, and H<sup>2</sup>

. Nineteen species and five sub-

S from

AK1 with 1.5 mol ethanol/mol glucose [34].

and 1.33 mol ethanol/mol glucose and xylose, respectively [39].

which include ethanol, acetate, lactate, alanine, CO<sup>2</sup>

lactate, hydrogen, and CO<sup>2</sup>

104 Fuel Ethanol Production from Sugarcane

**4. Culture parameters**

bacteria:


Butyrate is not a commonly observed end-product with thermophilic anaerobes and alanine is not commonly assayed. The distribution of end products are known to be influenced by various factors which can be of direct relevance for the production of ethanol; these conditions include the substrate types and concentrations, the partial pressure of hydrogen, pH, and temperature. Some of these factors are discussed in detail below.

#### **4.1. Partial pressure of hydrogen**

Early observations of the influence of hydrogen concentrations on the end-product formation of *Thermoanaerobacter ethanolicus* were reported in 1981 [15]. Higher partial pressure of hydrogen (*p*H<sup>2</sup> ) leads to increased ethanol production and less acetate production from glucose fermentations [15, 38, 46]. Strict anaerobes produce H<sup>2</sup> either via pyruvate ferredoxin oxidorecutase or NAD(P)-dependent oxidoreductase [52]. It has been well established that the high concentrations affects mesophilic bacteria more severely than thermophiles because the NADH ferredoxin oxidoreductase (FNOR) that converts NADH to Fdred is more strongly inhibited. The reduction potential is −400 mV for the Fdred/Fdox couple but −320 mV for the NADH/NAD<sup>+</sup> couple [52, 53]. Therefore, at low temperatures, elevated hydrogen concentrations inhibit H<sup>2</sup> evolution at much lower concentrations as compared to at high temperatures. Microorganisms respond to this by directing their reducing equivalents to other more favorable electron acceptors and consequently produce reduced products such as ethanol and lactate. In nature, hydrogen accumulation usually does not occur because of hydrogen-utilizing organism such as methanogens and sulfate-reducers, allowing for a complete catabolism of glucose to end-products. However, batch fermentation with monocultures allows hydrogen to accumulate leading to a change in end production profile in some *Thermoanaerobacter* species [15, 38, 41]. For instance, during degradation of glucose and xylose, the major end-product for *Thermoanaerobacter brockii* was ethanol [54]. Under hydrogen scavenging conditions, however, the flow of electrons from glucose degradation was directed away from ethanol but towards acetate with extra ATP produced. Several experiments using different liquid-to-gas ratios have revealed that changes in end-product formation occur during hydrogen accumulation among species of *Clostridium, Thermoanaerobacter,* and *Caloramator*. Hydrogen accumulation in these cultures can either change the carbon flow to more reduced end-products or inhibit substrate degradation. The inhibition observed can be either direct, inhibiting the hydrogenases, or indirect by productions of acids, lowering the pH in a closed system, and thus stopping further degradation of the substrates.

#### **4.2. Substrate loadings**

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

*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

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

> **Pretreatment**

**Ethanol yields (mM/g)**

Batch None 7.20–8.00 60 [61]

Progress in Second Generation Ethanol Production with Thermophilic Bacteria

Batch None 6.10–8.00 60 [61]

Batch None 3.26 60 [62]

Con Alk 1.40 70 [55]

Batch WO/E 2.61 70 [63]

Batch None 4.81 65 [64]

Batch WO/E 8.50–9.20 70 [42]

Batch WO/E 8.50–9.20 70 [65]

Batch E 3.30–4.50 70 [66]

Batch WO 6.30 60 [67]

Grass (2.5 g/L) Batch A/Alk/E 5.5 60 [39]

**Temp (°C)**

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

**References**

107

**mode**

*Clostridium thermocellum* Avicel (2.5 g/L) Batch A 5.00 60 [60]

*Clostridium* strain AK1 Hemp (5.0 g/L) Batch A/Alk 3.5 50 [34]

*Thermoanaerobacter* strain AK5 Grass (4.5 g/L) Batch A/E 4.31 65 [38] *Thermoanaerobacter* strain J1 Hemp (4.5 g/L) Batch A/E 4.3 65 [17]

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

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—

or mixed cultures are used.

from such substrates as seen in **Table 1**.

*Clostridium thermocellum* Whatman

*Clostridium thermocellum* Paddy straw

*Thermoanaerobacter pentosaceus* Rapeseed

*Thermoanaerobacter mathranii* Wheat straw

*Thermoanaerobacter ethanolicus* Beet molasses

*Thermoanaerobacter* BG1L1 Corn stover,

*Thermoanaerobacter* BG1L1 Wheat straw

*T. ethanolicus* Wood HL

*T. saccharoylticum* Xylan

*Thermoanaerobacterium* strain

AK17

wet oxidation.

*Clostridium* strain DBT-IOC-C19

**Organisms Substrate Fermentation** 

paper (8.0 g/L)

straw (5 0 g/L)

(6.7 g/L)

(30.0 g/L)

wheat straw (25.0–150.0 g/L)

(30.0–120.0 g/L)

(8.0 g/L)

(10.0 g/L)

(8.0 g/L)

Avicel (10.0 g/L)

#### **4.3. Ethanol tolerance**

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 ethanol yields dropped considerably.

#### **4.4. Other culture parameters**

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 often originating from complex medium supplements like yeast extract.
