2. Influence of wort composition on fermentation performance

#### 2.1. The chemical nature of sugarcane substrates and its by-products

In short, fermentation is initiated by the addition of the wort (also referred to as must), containing 18–25% (w/v) sugars, to a high-density yeast cell suspension, which represents 1/3 of the vat volume. Due to its large volumes, the feeding takes 4–6 h, and the fermentation is completed within 10 h. At the end of the fermentation, ethanol titres between 8 and 12% (v/v) are obtained, with a final cell density of 10–14% (w/v). Yeasts cells are then separated from the wine by centrifugation, which goes for distillation. The yeast slurry is diluted with equal volume of water and treated with sulfuric acid to reduce bacterial contamination, and reused in a subsequent fermentation cycle (Figure 1). This process configuration, using high cell densities and operating with cell recycling, is quite peculiar and allows two fermentation rounds per day during the harvest season that spans for almost 250 days. The reuse of cells reduces the need for yeast propagation, therefore diverting less sugar to yeast growth and

Figure 1. Simplified process flow diagram of the Brazilian ethanol production process (Courtesy of Jens C. F. Nielsen).

saving it to ethanol formation.

24 Fuel Ethanol Production from Sugarcane

As mentioned above, ethanol can be produced via direct fermentation of sugarcane juice, a mixture of juice and molasses, or molasses diluted in water [2, 3]. After shredding, cane is crushed in a milling tandem, constantly mixed with water, resulting in juices containing ca. 10– 15% sucrose content [3]. This process results in two types of juice: the primary – which leaves the first set of miller and is richer in nutrients – and the secondary – coming from the subsequent millers. The primary juice is commonly used for sugar production, whereas the secondary juice can either be used for sugar or ethanol production [3].

Sugarcane juice is passed through clarification (reducing impurities to less than 2%), decantation, and concentration (up to 18–25% total sugars) steps before sugar or ethanol production. These steps help reducing wild yeast and bacterial contaminations during the subsequent fermentation step, allowing for higher ethanol titre and yield [2].

During sugar production, the juice is clarified with lime, and concentrated through repetitive steps of evaporation and centrifugation. The concentrated juice later passes through a crystallization step, which catalyzes the formation of sucrose crystals that are later removed via centrifugation. The remaining sugar is left in the spent, dark and viscous liquid called molasses [4]. Molasses can be further recycled back into the sugar production process, resulting in several other types of molasses (i.e. A, B, C). The higher the number of recycles molasses is subjected to, the poorer its quality as a fermentation substrate [2].

Sugarcane juice and molasses are a complex mixture of carbohydrates, proteins, inorganic salts and organic acids [5], and wort prepared with either juice, molasses mixed with juice, or molasses diluted with water will have different nutrient composition, which will ultimately impact fermentation performance [1]. A comparison between sugarcane juice and molasses composition is shown below (Table 1).

Asparagine, glutamine and aspartic acid are the most abundant amino acids in sugarcanebased worts, while the disaccharide sucrose is by far the major sugar. Minor amounts of the monosaccharides, glucose and fructose, and the oligosaccharide, kestose, are also found [5]. Lipids are represented by a mixture of n-alkanes and ethyl and methyl esters of fatty acids (palmitate and oleate are the predominant), as well as of phytosterols (stigmasterol, β-sitosterol, and campesterol) [6, 7].

After fermentation cells are separated from the fermented wort (wine) by centrifugation, and this stream is sent for distillation. The distillation of ethanol generates a considerable amount of a wastewater stream named vinasse (stillage) [8].

Vinasse is currently applied in soil as a fertilizer (fertirrigation), due to its high potassium titre [4, 9]. However, this high organic matter load shows deleterious impacts on soil, water and groundwater [10]. A possible solution to this environmental issue is the anaerobic digestion of vinasse, reducing the organic matter of vinasse for later fertirrigation use, and generating a new stream of revenue (biogas) for the sugarcane ethanol plant [11].


Table 1. Composition of sugarcane juice and molasses.

Wort preparation influences final vinasse composition, which will have an impact on the performance of anaerobic digesters, and should be taken in consideration when designing such system. Vinasse can be defined as a mixture of various compounds, including organic acids, mainly fermentation-derived, such as succinic and malic acids, as well as lactic and acetic acids, derived from bacterial contamination. Glycerol is a substantial compound in vinasse, and non-distilled minor amounts of ethanol are also found [11, 12]. A general characterization of vinasse originated from different substrates is depicted in Table 2.

#### 2.2. Substrate related stresses on ethanol fermentation

Even being successfully used as substrate for ethanol production for decades, sugarcane based-worts present many challenging conditions for the fermenting yeast S. cerevisiae [1]. Besides nutrients, industrial worts used in the fermentation process, also carry inhibitors which can be both feedstock- or process-related [13]. During heating steps of juice pretreatment, some fermentation inhibitors are produced, from sugar degradation (e.g. furfural) and Maillard melanoidins [14].

Furfural, for instance, has been shown to reduce the specific growth rate, the biomass yield on ATP, and both ethanol yield and productivity [15], and is lethal to cells in concentrations above 84 nmol/gDW [16]. During fermentation, furfural is reduced to furfuryl by NADH-dependent alcohol dehydrogenase (ADH) [17, 18], in a NAD+ regenerative manner, resulting in lower glycerol formation, and higher ethanol titres, when furfural concentration is kept under 29 nmol/l [16].

Tauer et al. (2004) have investigated the effect of Maillard derived products during fermentation of different beverages (i.e. Tequila, Mezcal, whiskey and beer). In their study they observed a reduction in the formation of ethanol of up to 80%. Also, it was observed that the inhibition of these Maillard products is pH dependent, showing little inhibition at pH 4, and increasing at higher pH values.

Wort

composition

 Parameters

CODa

> Sugarcane juice

Molasses

Mix Based on data compiled by [11].

aChemical oxygen demand.

bBiochemical

cTotal solids. dVolatile solids. Table 2. Vinasse

composition

 from different

sugarcane-based

 worts.

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 oxygen demand.

 27.7

69

37.8

 5.8

 15.7

 4.4

 0.5

 0.03

 0.04

 2.3

 0.9

 1.4

 0.3

 105

 52.5

 8.4

 4.7

 0.04

 1.1

 0.5

 1.1

 0.1

 24.7

 28.6

 13.8

 0.9

 0.13

 0.21

 4.1

 2.3

 1.9

 0.6

 82

 0.2

 1.4

 1.9

 2.4

 0.6

 7.3

 14.7

 4.9

 0.5

 0.11

 0.1

 1.5

 0.7

 0.4

 0.2

 22.5

 1.5

 17.9

60

 1.8

 0.1

 0.4

 1.0

 0.8

 0.2

BODb

N

P

K

SO4

2

Ca

Mg

TSc

VSd

 (in g/L)


Wort preparation influences final vinasse composition, which will have an impact on the performance of anaerobic digesters, and should be taken in consideration when designing such system. Vinasse can be defined as a mixture of various compounds, including organic acids, mainly fermentation-derived, such as succinic and malic acids, as well as lactic and acetic acids, derived from bacterial contamination. Glycerol is a substantial compound in vinasse, and non-distilled minor amounts of ethanol are also found [11, 12]. A general charac-

Composition Sugarcane juice (g/L) Sugarcane molasses (g/kg)

Total solids 140–190 735–875 Total sugars 105–175 447–587 Sucrose 98–167 157–469 Reducing sugars 6–11 97–399 Nitrogen 0.08–0.3 0.25–1.5 Phosphorous 0.02–0.1 0.3–0.7 Potassium 0.7–1.5 19–54 Calcium 0.1–0.5 6–12 Magnesium 0.1–0.5 4–11

Even being successfully used as substrate for ethanol production for decades, sugarcane based-worts present many challenging conditions for the fermenting yeast S. cerevisiae [1]. Besides nutrients, industrial worts used in the fermentation process, also carry inhibitors which can be both feedstock- or process-related [13]. During heating steps of juice pretreatment, some fermentation inhibitors are produced, from sugar degradation (e.g. furfural)

Furfural, for instance, has been shown to reduce the specific growth rate, the biomass yield on ATP, and both ethanol yield and productivity [15], and is lethal to cells in concentrations above 84 nmol/gDW [16]. During fermentation, furfural is reduced to furfuryl by NADH-dependent alcohol dehydrogenase (ADH) [17, 18], in a NAD+ regenerative manner, resulting in lower glycerol formation, and higher ethanol titres, when furfural concentration is kept under

Tauer et al. (2004) have investigated the effect of Maillard derived products during fermentation of different beverages (i.e. Tequila, Mezcal, whiskey and beer). In their study they observed a reduction in the formation of ethanol of up to 80%. Also, it was observed that the inhibition of these Maillard products is pH dependent, showing little inhibition at pH 4, and

terization of vinasse originated from different substrates is depicted in Table 2.

2.2. Substrate related stresses on ethanol fermentation

and Maillard melanoidins [14].

Based on data compiled by [2].

26 Fuel Ethanol Production from Sugarcane

Table 1. Composition of sugarcane juice and molasses.

increasing at higher pH values.

29 nmol/l [16].

Table 2. Vinasse composition from different sugarcane-based worts.

Sugarcane juice also presents potentially toxic metal ions on its composition. Walford (1996) presented a compilation of mineral values from sugarcane juices (Table 3).

(900–1800 ppm/Brix) acids. Other acids found in lower concentrations are succinic, oxalic, tartaric and glycolic acids [5]. During fermentation, lactic and acetic acids are also formed via

Clash of Kingdoms: How Do Bacterial Contaminants Thrive in and Interact with Yeasts during Ethanol Production?

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Undissociated organic acids are lipid permeable and thus enter yeast cells. Due to the higher intracellular pH as compared to the environment, dissociation into the corresponding anion, leads to intracellular acidification and ATP expenditure, a mechanism known as weak acid uncoupling [22], which is described in more details in the following sections. Moreover, the anion accumulated

inside the cells may reach toxic concentrations that impair essential metabolic functions.

nation is a concurrent problem in industrial fermentations.

yield, yeast cell flocculation, and low yeast viability [24–27].

plants found that Lactobacillus was the most abundant genus [28].

3.1. Homo- and heterofermentative lactic acid bacteria

have shown to include both types [31].

3. The interplay between yeast and bacteria in the fermentation process

Fuel ethanol fermentation performed in Brazil is one of the largest industrial biotechnological processes in the world, with the most favorable energy balance as compared to other similar processes for ethanol production [13, 23]. However, in view of the nature of the process and the large volumes processed, aseptic conditions are never achieved. Therefore, bacterial contami-

This is regarded as a major drawback that deviates sugars away from ethanol formation and lead to detrimental effects upon yeast fermentative performance, such as reduced ethanol

Bacterial contaminants found present in the fermentation step of ethanol production comprise mainly lactic acid bacteria (LAB) [28], probably because of their higher tolerance towards acidic pH and ethanol titres when compared to other microorganisms [29, 30]. Studies that investigated the identity of these contaminants during yeast fermentation in Brazilian ethanol

Contaminating lactic acid bacteria are traditionally classified in two major metabolic subgroups according to the pathway used to metabolize hexose sugars: homo- and heterofermentative [29]. In general, bacteria isolates from industrial fermented sugarcane substrates

Homofermentative bacteria catabolize hexoses via the so-called Embden-Meyerhof-Parnas (EMP) pathway, in which 1 mol of hexose results in the formation of 2 mol of lactic acid and 2 mol of ATP. In comparison, in heterofermentative bacteria another pathway is active, 6 phosphogluconate/phosphoketolase (6-PG/PK) pathway [29], and hexoses are converted to equimolar amounts of lactic acid, ethanol or acetate, and carbon dioxide, yielding 1 mol of ATP per mol of hexose fermented [32, 33]. With the conversion of acetyl phosphate to acetate instead of ethanol, an additional ATP can be produced. Then, regeneration of surplus NAD+ must be achieved by an alternative electron acceptor. Under aerobic conditions, oxygen may serve as the electron acceptor [34], but under anaerobic or even oxygen-limited conditions, fructose is reduced to mannitol, serving as an electron sink [35]. There is a third classification

contaminating bacteria.

Aluminum shows high toxicity under acidic conditions (being present in Al3+ form) and is particularly deleterious to yeast cells, reducing cell viability, trehalose content, and ethanol yield [1]. Its deleterious effects can be alleviated by magnesium concentration in the broth, or completely abolished by mixing sugarcane juice with molasses. This might be related to some chelating property of molasses [2].

Other metal ions also play an important role in yeast fermentation inhibition. Sugarcane molasses showed inhibitory effect towards invertase activity of a laboratory S. cerevisiae strain. This inhibition could be further replicated in laboratory media when copper ions (CuCl2) where added in the media at a concentration of 0.04 M [19].

Another important factor is the osmotic stress that is caused by elevated concentration of salts. Cations such as Ca2+, Mg2+, K+ , Na+ , and anions, like Cl and SO4 <sup>2</sup>, can all have severe effects on yeast growth and ethanol production [20].

Sugarcane juice and molasses have a complex composition of organic acids. Even though they do not respond to a major fraction of the composition of these raw materials, they are responsible for their pH values (ca. 5–6) and their buffering capacity [5]. The composition, and concentration, of such acids depend on several factors, such as the maturity stage and variety of the plant, weather, soil and health state [21]. The most common organic acids found in sugarcane are trans-aconitic (5000–8000 ppm/Brix), malic (1200–1800 ppm/Brix) and citric


Table 3. Inorganic composition of sugarcane juice.

(900–1800 ppm/Brix) acids. Other acids found in lower concentrations are succinic, oxalic, tartaric and glycolic acids [5]. During fermentation, lactic and acetic acids are also formed via contaminating bacteria.

Undissociated organic acids are lipid permeable and thus enter yeast cells. Due to the higher intracellular pH as compared to the environment, dissociation into the corresponding anion, leads to intracellular acidification and ATP expenditure, a mechanism known as weak acid uncoupling [22], which is described in more details in the following sections. Moreover, the anion accumulated inside the cells may reach toxic concentrations that impair essential metabolic functions.
