**3. Ethanol producing thermophilic anaerobes**

efficiency must increase by 20% while greenhouse gases must decrease by 20% by 2020 [1]. This has led to a dramatic increase in the production of bioethanol from 48 billion liters in 2007 to 2.6 billion liters in 2017 [2]. Both the United States and Brazil are by far the largest producers of bioethanol although the vast majority of ethanol produced is from first generation biomasses such as sucrose-rich sugarcane and easily fermentable starch-rich crops such as corn. However, there is a growing concern over the use of these feedstocks because they are food and feed related and thus in a direct competition with food production [3–5]. In addition, increased concern has been regarding the negative impact on agricultural areas used for the

Production of bioethanol by second-generation non-food (lignocellulosic) biomass, such as agricultural residues, addresses some of the above mentioned environmental concerns although poses several challenges as a raw material for bioprocessing. Second generation biomass requires extensive and costly chemical or physical pretreatment in addition to enzymatic treatment processes which negatively impacts its industrial feasibility. Lignocellulosic biomass is often difficult to degrade due to the lignin sheath and the highly crystalline nature of cellulose [6]. In addition to cellulose, lignocellulose is also composed of lignin and hemicelluloses of which the latter contains a plethora of monosaccharides with various connectivities and varying degrees of branching. Therefore, processing lignocellulosic biomass and subsequent fermentation of the liberated sugars to ethanol has proven to be a major complication

To address the challenges posed by lignocellulosic biomass, fermentative organisms that can meet these process needs will help improve the feasibility of bioethanol production from lignocellulosic biomass. At present, the majority of bioethanol is produced using well-established mesophilic organisms despite some of the inherent advantages to the use of thermophilic microorganisms such as higher operating temperatures and utilizing a non-glucose hexoses and pentoses such as xylose and arabinose. This work focuses on the physiology of ethanol-producing thermophiles with an emphasis on their salient features relevant to the utilization of lignocellulosic biomass as well as the use of genetic engineering to improve their

For the fermentative production of ethanol from biomass to be commercially successful, several key processes and organisms need to be considered [3, 4, 7–9]. These process requirements needed to simultaneously consider two viewpoints: the physiological properties of the ethanologen used and process requirements. Concerning organism requirements, an ideal strains should be homoethanologenic, with high productivity (> 1/g/L/h), have broad substrate spectra and high tolerance of ethanol, inhibitory compounds and high initial substrate concentrations. Other key factors include high cellulolytic activity, simple nutritional needs, low biomass production and ease of genetic manipulation. Ideally, a single organism that

**2. Selected aspects of ethanol production from lignocellulosic** 

production of this biomass.

102 Fuel Ethanol Production from Sugarcane

for large-scale production [3–5].

potential for bioethanol utilization.

**biomass**

While more than 300 species of thermophilic anaerobic bacteria have described as of 2008 from a wide range of environs with new species being continuously discovered. Thermophilic anaerobes have been isolated from a diverse range of environments [13] including deep-sea vents [14], geothermal areas [15–17], compost piles [18], municipal solid waste or sewage sludge [19], oil wells [20], and canned goods [21]. Most thermophilic microorganisms are either obligatory or facultative anaerobic, likely due to the limited availability of oxygen and highly reducing nature of geothermal features [22]. The majority of the those that are highly ethanologenic that have been described in the literature are often strict anaerobes within the genera of *Clostridium*, *Caloramator*, *Caldanaerobacter*, *Thermoanaerobacter*, or *Thermoanaerobacterium* [3, 23].

The highly polyphyletic genus *Clostridium* within the class *Clostridia* (family *Clostridiaceae*, order *Clostridiales*) currently has greater than 200 species with standing in nomenclature although only about 15 are strains within the genus are thermophilic [24, 25] usually with temperature optima for growth between 45 and 65°C although several strains reportedly grow at temperatures as high at 75°C. All species within the genus are strictly anaerobic and can typically be isolated from a broad range of nutrient-rich environments [26]. Many members within the genus can hydrolyze cellulose and produce ethanol, making them target of extensive research on biofuel production from complex [27, 28].

*C. thermocellum* is a thermophilic species that degrades crystalline cellulose using a cellulosome which is comprised of a complex arrangement of glycohyldrolases attached to a scaffold-like matrix [6]. Several other members of *Clostridium* have glycohydrolases including *C. acetobutylicum* [29, 30] and *C. cellulovorans* [31]. Ethanol yields by *Clostridium* species are often moderate and vary depending on environmental conditions with other organic acids, 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 AK1 with 1.5 mol ethanol/mol glucose [34].

**1.** 1 Glucose 2 Ethanol +2 CO2

**4.1. Partial pressure of hydrogen**

further degradation of the substrates.

**3.** 1 Glucose 2 Acetate +2 CO2 + 4H<sup>2</sup>

**4.** 1 Glucose 1 Butyrate +2 CO2 + 2 H<sup>2</sup>

<sup>+</sup> 2 Alanine

temperature. Some of these factors are discussed in detail below.

cose fermentations [15, 38, 46]. Strict anaerobes produce H<sup>2</sup>

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

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

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

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

) leads to increased ethanol production and less acetate production from glu-

Progress in Second Generation Ethanol Production with Thermophilic Bacteria

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

105

couple [52, 53]. Therefore, at low temperatures, elevated hydrogen concentra-

evolution at much lower concentrations as compared to at high temperatures.

either via pyruvate ferredoxin

**2.** 1 Glucose 2 Lactate

**5.** 1 Glucose +2 NH4

hydrogen (*p*H<sup>2</sup>

NADH/NAD<sup>+</sup>

tions inhibit H<sup>2</sup>

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, lactate, hydrogen, and CO<sup>2</sup> . One challenge for these organisms is achieving good ethanol yields 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 and 1.33 mol ethanol/mol glucose and xylose, respectively [39].

*Thermoanaerobacter* species have similar physiological characteristics as *Thermoanaerobacterium* species; all species within the genus are highly saccharolytic and produce end-products which include ethanol, acetate, lactate, alanine, CO<sup>2</sup> , and H<sup>2</sup> . Nineteen species and five subspecies belong to the genus [24, 25]. The main difference between *Thermoanaerobacter* and *Thermoanaerobacterium*, is that the majority of *Thermoanaerobacter* species produce H<sup>2</sup> S from 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].
