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 contamination is a concurrent problem in industrial fermentations.

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

#### 3.1. Homo- and heterofermentative lactic acid bacteria

Sugarcane juice also presents potentially toxic metal ions on its composition. Walford (1996)

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

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)

Another important factor is the osmotic stress that is caused by elevated concentration of salts.

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

Component Concentration (% on total solids)

K2O 0.77–1.31 Na2O 0.01–0.04 CaO 0.24–0.48 MgO 0.1–0.39 Al2O3 0.005–0.17 Fe2O3 0.006–0.04 CuO 0.002–0.003 ZnO 0.003–0.012 MnO 0.007 CoO 0.00007 SO3 0.017–0.52 P2O5 0.14–0.4 Cl 0.16–0.27 SiO2 0.016–0.101

, and anions, like Cl and SO4

<sup>2</sup>, can all have severe effects

presented a compilation of mineral values from sugarcane juices (Table 3).

where added in the media at a concentration of 0.04 M [19].

, Na+

chelating property of molasses [2].

28 Fuel Ethanol Production from Sugarcane

Cations such as Ca2+, Mg2+, K+

Based on data compiled by [5].

Table 3. Inorganic composition of sugarcane juice.

on yeast growth and ethanol production [20].

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

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 have shown to include both types [31].

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 group that, differently from the homofermentative strains that cannot metabolize pentose sugars, they can ferment these sugars using an inducible phosphoketolase pathway, producing lactate and acetate [36].

availability and biological factors like yeast strain, population density and bacterial metabolism should all be considered when analyzing the impacts of contaminant species in industrial

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

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31

For instance, previous studies have demonstrated that, under certain fermentation conditions, competition towards nutrients is the most probable cause for yeast cells inhibition. S. cerevisiae in steady-state pure cultures withstood high concentrations of added lactic acid, without losing much of cell viability nor ethanol production/productivity, when compared to cocultivated S. cerevisiae and the facultative heterofermentative L. paracasei [26]. This shows that organic acid production by bacterial metabolism might not be, in some circumstances, directly

Co-cultivations experiments, in the context of ethanol production, are rather scarce in the literature. Very recently, a quite interesting investigation was performed using two common contaminant microorganisms in industrial ethanol plants, the heterofermentative bacterium L. fermentum, and the contaminating yeast, Dekkera bruxellensis [45]. The authors found that during co-cultivation experiments with S. cerevisiae strain PE-2 in the presence of both contaminants, the growth of the contaminating yeast was stimulated by the presence of the bacterium. This condition resulted in a more pronounced effect on the fermentation parameters than the effects observed in binary combinations of the fermenting yeast (PE-2) with each contaminant. Wild contaminants of S. cerevisiae were also evaluated in co-cultivations experiments [46]. They were found to be more detrimental to fermentative performance (resulting in lower ethanol production and higher residual sugars) when compared to co-cultivations with a

Bacteria showing different metabolic pathways may also impact differently yeast physiology and ethanol fermentation, on a context-dependent manner. Homofermentative bacteria (i.e. L. plantarum) was more inhibitory to yeast cells than heterofermentative bacteria (i.e. L. fermentum), when these strains where co-cultured with an industrial S. cerevisiae strain (CAT-1), under laboratory conditions (i.e. in equal cell concentrations). When the context of sugarcane ethanol fermentations is put in perspective (i.e. short fermentations catalyzed by high cell densities) L. fermentum were more deleterious, outcompeting yeast cells in the fermentation process. In these conditions, the fastidious metabolism of L. fermentum, and its faster uptake of fructose – a sugar typically not consumed as fast as glucose by S. cerevisiae – may have given L. fermentum a competitive advantage, when compared to L. plantarum, which had to compete with S. cerevisiae for the available glucose [27]. Therefore, it was concluded that under conditions similar to those used in the industrial production of fuel ethanol, heterofermentative strains have a more detri-

mental effect over yeast performance, in terms of ethanol yield and yeast viability.

Mannitol is a suitable indicator of sugarcane deterioration and bacterial contamination during industrial fuel ethanol fermentation [47, 48]. Glucose and fructose normally present a 1:1 ratio,

fermentations.

involved in yeast cells stress.

3.3. Co-cultivation studies

heterofermentative bacterium.

3.4. Monitoring bacterial contamination

#### 3.2. LAB effects on the fermenting yeast

Contaminating bacteria found in ethanol fermentations are often fastidious organisms which compete for nutrients against the fermenting yeast, negatively impacting its fermentation performance [25]. These nutrients are often several growth factors like nucleotides, amino acids and vitamins [25]. Due to the fast-growing nature of these bacterial strains, wort can become rapidly depleted for such nutrients [37]. This nutritional deficiency might result in lower ethanol titres, lower yeast crop viability and budding, longer fermentation periods and higher contents of residual sugars, a phenomenon called stuck or sluggish fermentation [27].

Concomitantly to nutritional competition, these contaminants also deviate carbon to their catabolic pathways and produces organic acids and polyalcohols [27]. These weak organic acids also have a deleterious effect on yeast metabolism. In low pH conditions, usually found in fermentations (i.e. 6.5–4.5) they are found mostly in their protonated form, due to their high pKa values (3.86 for lactic acid; 4.75 for acetic acid) [27]. In this form, these uncharged molecules can permeate the plasma membrane and, when encounters the cytosolic pH (7.0), rapidly dissociates releasing its proton and acidifies the intracellular pH (pHi) [38].

This acidification of the pHi affects many cellular processes, by influencing the ionization states of acidic and basic side chains of amino acids in important enzymes, changing their tertiary structure and activity [39]. Among the several vital functions that are inhibited by pHi acidification is the glycolysis [40], thus inhibiting the cell's ability to recover ATP.

Saccharomyces responds, partially, to this acidification via the plasma membrane H+ -ATPase pump, Pma1p. It pumps out H<sup>+</sup> using ATP hydrolysis, at a 1:1 ratio [41]. This response mechanism comes with a cost: almost 20% of all ATP formed during normal conditions is drained by this process40; at starvation conditions, when exposed to weak acid stress, the amount of consumed ATP can go as high as 60% [42].

Other responses are also triggered, when yeast cells are exposed to weak acid stress. The plasma membrane ATP binding cassette (ABC) transporter Pdr12p is induced when cells face this stressful condition [43], and it is believed to play an important role on yeast cells adaptation to grow in the presence of weak organic acids by pumping out H+ ions, under the expense of either ATP or proton gradient [42].

When protons and anions are pumped out of cells, they re-associate. Once protonated, these molecules can permeate again to the interior of yeast cells, forcing them to pump out these ions repeatedly. This process is defined as a futile cycle36 and is a major energetic drain in industrial processes. These energetic drains increase cells maintenance coefficient (m) and reduce their fitness, inhibiting their growth and reducing their viability [44].

Despite these universal cellular responses, yeast might be more or less susceptible to a given contaminant, depending on its metabolism and also on the physiological context these organisms are exposed to. Growth conditions such as temperature, pH, sugar content, nutrients availability and biological factors like yeast strain, population density and bacterial metabolism should all be considered when analyzing the impacts of contaminant species in industrial fermentations.

For instance, previous studies have demonstrated that, under certain fermentation conditions, competition towards nutrients is the most probable cause for yeast cells inhibition. S. cerevisiae in steady-state pure cultures withstood high concentrations of added lactic acid, without losing much of cell viability nor ethanol production/productivity, when compared to cocultivated S. cerevisiae and the facultative heterofermentative L. paracasei [26]. This shows that organic acid production by bacterial metabolism might not be, in some circumstances, directly involved in yeast cells stress.

#### 3.3. Co-cultivation studies

group that, differently from the homofermentative strains that cannot metabolize pentose sugars, they can ferment these sugars using an inducible phosphoketolase pathway, producing

Contaminating bacteria found in ethanol fermentations are often fastidious organisms which compete for nutrients against the fermenting yeast, negatively impacting its fermentation performance [25]. These nutrients are often several growth factors like nucleotides, amino acids and vitamins [25]. Due to the fast-growing nature of these bacterial strains, wort can become rapidly depleted for such nutrients [37]. This nutritional deficiency might result in lower ethanol titres, lower yeast crop viability and budding, longer fermentation periods and higher contents of residual sugars, a phenomenon called stuck or sluggish fermentation [27]. Concomitantly to nutritional competition, these contaminants also deviate carbon to their catabolic pathways and produces organic acids and polyalcohols [27]. These weak organic acids also have a deleterious effect on yeast metabolism. In low pH conditions, usually found in fermentations (i.e. 6.5–4.5) they are found mostly in their protonated form, due to their high pKa values (3.86 for lactic acid; 4.75 for acetic acid) [27]. In this form, these uncharged molecules can permeate the plasma membrane and, when encounters the cytosolic pH (7.0),

rapidly dissociates releasing its proton and acidifies the intracellular pH (pHi) [38].

acidification is the glycolysis [40], thus inhibiting the cell's ability to recover ATP.

amount of consumed ATP can go as high as 60% [42].

their fitness, inhibiting their growth and reducing their viability [44].

of either ATP or proton gradient [42].

This acidification of the pHi affects many cellular processes, by influencing the ionization states of acidic and basic side chains of amino acids in important enzymes, changing their tertiary structure and activity [39]. Among the several vital functions that are inhibited by pHi

Saccharomyces responds, partially, to this acidification via the plasma membrane H+ -ATPase pump, Pma1p. It pumps out H<sup>+</sup> using ATP hydrolysis, at a 1:1 ratio [41]. This response mechanism comes with a cost: almost 20% of all ATP formed during normal conditions is drained by this process40; at starvation conditions, when exposed to weak acid stress, the

Other responses are also triggered, when yeast cells are exposed to weak acid stress. The plasma membrane ATP binding cassette (ABC) transporter Pdr12p is induced when cells face this stressful condition [43], and it is believed to play an important role on yeast cells adaptation to grow in the presence of weak organic acids by pumping out H+ ions, under the expense

When protons and anions are pumped out of cells, they re-associate. Once protonated, these molecules can permeate again to the interior of yeast cells, forcing them to pump out these ions repeatedly. This process is defined as a futile cycle36 and is a major energetic drain in industrial processes. These energetic drains increase cells maintenance coefficient (m) and reduce

Despite these universal cellular responses, yeast might be more or less susceptible to a given contaminant, depending on its metabolism and also on the physiological context these organisms are exposed to. Growth conditions such as temperature, pH, sugar content, nutrients

lactate and acetate [36].

30 Fuel Ethanol Production from Sugarcane

3.2. LAB effects on the fermenting yeast

Co-cultivations experiments, in the context of ethanol production, are rather scarce in the literature. Very recently, a quite interesting investigation was performed using two common contaminant microorganisms in industrial ethanol plants, the heterofermentative bacterium L. fermentum, and the contaminating yeast, Dekkera bruxellensis [45]. The authors found that during co-cultivation experiments with S. cerevisiae strain PE-2 in the presence of both contaminants, the growth of the contaminating yeast was stimulated by the presence of the bacterium. This condition resulted in a more pronounced effect on the fermentation parameters than the effects observed in binary combinations of the fermenting yeast (PE-2) with each contaminant.

Wild contaminants of S. cerevisiae were also evaluated in co-cultivations experiments [46]. They were found to be more detrimental to fermentative performance (resulting in lower ethanol production and higher residual sugars) when compared to co-cultivations with a heterofermentative bacterium.

Bacteria showing different metabolic pathways may also impact differently yeast physiology and ethanol fermentation, on a context-dependent manner. Homofermentative bacteria (i.e. L. plantarum) was more inhibitory to yeast cells than heterofermentative bacteria (i.e. L. fermentum), when these strains where co-cultured with an industrial S. cerevisiae strain (CAT-1), under laboratory conditions (i.e. in equal cell concentrations). When the context of sugarcane ethanol fermentations is put in perspective (i.e. short fermentations catalyzed by high cell densities) L. fermentum were more deleterious, outcompeting yeast cells in the fermentation process. In these conditions, the fastidious metabolism of L. fermentum, and its faster uptake of fructose – a sugar typically not consumed as fast as glucose by S. cerevisiae – may have given L. fermentum a competitive advantage, when compared to L. plantarum, which had to compete with S. cerevisiae for the available glucose [27]. Therefore, it was concluded that under conditions similar to those used in the industrial production of fuel ethanol, heterofermentative strains have a more detrimental effect over yeast performance, in terms of ethanol yield and yeast viability.

#### 3.4. Monitoring bacterial contamination

Mannitol is a suitable indicator of sugarcane deterioration and bacterial contamination during industrial fuel ethanol fermentation [47, 48]. Glucose and fructose normally present a 1:1 ratio, since sucrose is the prevalent sugar in this feedstock. Therefore, mannitol titres can be used to predict sucrose losses due to bacterial contamination as well as dextran synthesis, which results in problems such as viscosity, evaporation, crystallization and, to a lesser extent, poor filterability in sugarcane factories [49].

References

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Another interesting indicator of bacterial contamination is lactic acid. This organic acid is considered by many a suitable indicator of bacterial contamination during industrial ethanol fermentation. However, because D- and L-lactic acids is formed by industrial lactobacilli isolates [50], as a result of varying proportions of racemases [51, 52], conflicting results are expected depending on the technique employed to quantify this by-product. This is because most commercial enzymatic kits usually employed in the routine analysis of lactic acid in ethanol plants, normally detect only the L-form.
