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The Brazilian technology of fuel ethanol fermentation – yeast inhibition
factors and new perspectives to improve the technology
Pedro de Oliva-Neto, Claudia Dorta, Ana Flavia Azevedo Carvalho, Valeria Marta Gomes de Lima,
Douglas Fernandes da Silva
Brazil is the second largest producer of ethanol in the world and the first in the technology of ethanol from sugar cane. The
Brazilian bioprocess of ethanol production is based on molasses or cane juice substrate. Currently, a fed-batch or
continuous or mixed process is operated with a stable cell recycle and high yeast concentration. The ethanol efficiency is
controlled by several industrial parameters of fermentation and depending on balance of these parameters and some
chemical and microbiological inhibitors. Sucrose and ethanol concentration, acid treatment of yeast cells, temperature and
pH, yeast cells flocculation, and some chemical inhibitors like organic acids and sulfite, beyond bacterial infection may
affect synergistically the viability of Saccharomyces cerevisiae and ethanol production. These parameters will be
extensively discussed in this chapter with focus on correct operation of the process to maximum ethanol production. The
inhibitory effects of these problems will be discussed in the metabolism of the yeast, making difficult the operational
procedures increasing the cost of the process and decreasing the ethanol efficiency. Other important aspects are screening
of yeast strains, type of bacterial and yeast contamination, antimicrobial compounds used to control microbial
contamination. New studies are been showed to control the yeast flocculation and bacterial infection to improve the
ethanol production.
Keywords: Fuel ethanol fermentation, chemical, bacterial infection, yeast metabolism inhibition.
1. The Brazilian process of fuel ethanol production
Brazil is the second largest producer of ethanol in the world and the first in the technology of ethanol from sugar cane.
The Brazilian production of sugar cane in the 2012/13 harvest was 589 million tons of cane, 38.3 million tons of sugar
(5% superior of the last harvest) and 23.64 billion liters de ethanol, (0.91% superior of the last harvest)1,2. In relation to
the area of sugar cane 8.5 million hectares1,2 was used in 2012/2013 harvest with expectation of growing. The FedBatch and Continuous process are currently used in Brazil. Driving the fed-batch process avoids the inhibitory effect of
sugar during the initial phase, resulting in higher ethanol concentration in the same period of time as compared to
conventional batch3, 4. The continuous process can be more advantageous than the fed-batch because it includes
optimization of process conditions for increased productivity, long period of continuous productivity, increased
volumetric productivity, cost reduction laboratory once reached the desired state and reduced cleaning time and
sanitizing the tanks5. The biggest drawback is that the continuous fermentations are more susceptible to bacterial
contamination for long periods of exposure and require a thorough knowledge to optimize the process conditions to
achieve the desired performance - especially when adding chemicals, changes in the rate of flow and mixing of nutrients
and changes in the estimated parameters6.
The Brazilian plants both Fed-Batch or Continuous process use the continuous yeast cells recycle, which is treated
with sulfuric acid before returning to the fermentation vats. The wort is thus added slowly over about 4 hours, and after
6-9 h arrives at the end of the fermentation. The treatment with sulfuric acid in S. cerevisiae is made to reduce yeast
flocculation and bacterial contamination. It is done after the centrifugation of yeast, lowering the pH of the cell
suspension diluted with water to a range from 2.0 to 3.0 by the addiction of sulfuric acid and water together and
maintained in constant stirring. The treatment time is variable, usually from 1.5 to 2 hours, but can reach up to 3 hours,
the higher this time is smaller cell viability of the yeast. Yeast cells younger and older are less resistant to treatment7.
The flowchart of the process of ethanol production (Figure 1) begins with washing of the cut cane after harvest.
Shredded cane is repeatedly mixed with water and crushed between rollers in the milling tandem; the collected juices
contain 10-15% sucrose. There are two types of cane juices: primary, leaving from the first set of mill and named
primary juice is richer and purer in sugar, and it is usually intended for the manufacture of sugar. From the others mill
sets was obtained the secondary juice, which can be used for both sugar production as alcohol. Both cane juice named
broth is normally preheated, sulfited with burning sulfur, and limed with CaO. These procedures lead to flocculation
process making flocs of no sugar products (paraffin, clay, dyes, protein, etc.) in flocculated broth. Phosphorylation is
another step for clarification of the cane juice for sugar production but not normally used for the broth used in ethanol
production. Then, the flocculated broth is heated to 105oC, and after decanted for 2 hs. The clarified broth is resulted for
this process and the liquid residue again will be heated and clarified by filtration system. This juice is named filtered
broth which usually returns to the beginning of the process of clarification.
The clarified broth can be sent to the manufacture of sugar or ethanol. The residue from the manufacture of sugar is
molasses. Thus, the substrate for the production of ethanol (wort) can be made from the clarified broth, sugar cane
molasses or a mixture of both, adding water and minerals, if necessary. The wort is cooled to 30oC and prepared to
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contain 18-20oBx, which equates to approximately 13-15% of total sugars. The wort is then pumped into the
fermentation tanks to be mixed with the yeast and the fermentation process starts.
The feeding process of the wort, and withdrawal of the product is quite variable and depends on each plant. As
already mentioned it can be use Continuous or Fed-batch process for fuel ethanol production, but a mixture of these two
processes is also done. For example, the wort is initially fermented by the fed-batch process and ended by a Continuous
process (named Batcom). Alternatively, the Combat process starts by continuous fermentation system and ended by
Fed-batch.
The pH (3.8-4.5), Brix (18-20o), temperature (32-34oC) and concentration of yeast cells (10-14% wet mass) in the
vats are continuously monitored and controlled so that the ethanol content does not exceed 7.5% (w/v) and the residual
sugars do not exceed 0.1%, but these values depend on the process, substrate, etc. After fermentation, the broth is
centrifuged and the supernatant is sent to a special vat where hydro-alcoholic solution (called wine) will be stored to be
distilled and thus produce fuel ethanol. The aqueous residue of the distillery, called vinasse, is usually discarded or used
for ferti-irrigation of the plantation of sugarcane.
The heavy phase leaving the centrifuge is a yeast suspension (40-80% of wet weight), which is sent by gravity to
specific vats, where the yeast receive a hydration treatment, sulfuric acid and air, as mentioned before. After the acid
treatment, the yeast suspension is pumping to the fermentation vats. Excessive use of sulfuric acid to control of the
bacterial contaminant is harmful to yeast cells in a fermentation process. If this treatment is done in excess can has
abrasive effect on the wall of the yeast, weakening proton pump and nutrition of yeasts, which are essential for viability
and ethanol production8, 9, 10. Although the use of sulfuric acid is needed for bacterial decontamination and control of
yeast cells flocculation of the process, this practice could be not efficient. Some researchers believe in adjustment of the
processes by centrifugation, feed of wort, and nutrition, as the solution to a good fermentation. The acid in
excess causes many chemical reactions resulting in significant imbalance in the enzyme-substrate system in the
fermentation11, 12. Also, the acid treatment could not avoid totally the yeast flocculation13. Despite the effectiveness of
treatment on yeast deflocculation , this is not durable due to its dependence on pH. There is a pH increase when the
inoculum is mixed with wort in fermenting vat. Furthermore, the use of low pH (2.0-2.5) may affect the metabolism of
the yeast14, 15.
2. Inhibition of ethanolic fermentation
2.1 Contamination in the process of alcoholic fermentation
The consume of sucrose by microorganisms begins when the cut cane is still in the farm. The faster the cane is brought
to the mill to be processed, the more efficient the process of obtaining sugar and ethanol. According OLIVA-NETO11,
during the steps of the process from the harvest to the stage of sugar juice is fermented in the distillery, the microbial
flora undergoes a great selection due to the changes in pH, temperature, and inhibitors. Thus, during the fermentation,
the microflora is limited to a few microorganisms. As the pH becomes lower and there is an increase in the alcohol
content, making it difficult to adapt to most microorganisms. However, the process of fed-batch or continuous with cell
recycle used in alcohol production, favors the proliferation of some genera contaminants in fermentation vats17.
The literature points as main contaminants through the fermentative Gram-positive compounds by Lactobacillus,
Bacillus and Leuconostoc, the latter being less common due to lower resistance to alcohol content11,18. OLIVA-NETO19
isolated as the main contaminant Lactobacillus fermentum from samples of "yeast suspension" in distilleries of state of
São Paulo (Brazil). Taxonomic analysis showed that L. fermentum was the predominant bacteria (62%), followed by L.
murinus (9%), L. vaccinostercus (9%), L. plantarum (2%) and Leuconostoc sp (2%). Lactobacillus are mainly
acidophilic species and tolerant to ethanol68, being demanding in nutritional terms, especially regarding the amino
acids22. According HYNES et al23 Lactic acid bacteria contamination is a major problem in industrial fermentation of
alcohol. The growth of these bacteria reduces the yield due to alcohol consumption of glucose that would be used in the
synthesis ethanol, and the competition of nutrients of the medium, and the toxic effect of lactic acid23,24. Moreover,
according LUDWIG et al16, these bacteria can induce the flocculation of yeast cells causing the yeast settling at the
bottom of vats, and cell loss in centrifuges further contributing to the reduction in the ethanol yield and cell
viability16,17,25.
Among the most important wild yeasts are undoubtedly other strains of the Saccharomyces cerevisiae. These quickly
dominate the industrial process early in the start of alcohol production. This is why the screening of yeast strains in
Brazil is made in their own factories, considering good fermentation and without flocculant characteristic, undesirable
in the process. But other genus have been found and which are detrimental to the process, competing for sugar and
decreasing the yield: Candida, Hansenula, Kloeckera, Kluyveromyces, Oidium, Pichia, Rhodotorula,
Schizosaccharomyces,
Schwanniomyces,
Torula,
Torulopsis,
Trichosporon,
Cryptococcus,
Dekkera,
Brettanomyces26,27,28,29,30
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Cane washing
Milling
Bagasse (Energy,
steam)
Alkaline water (pH 11)
Filtration (Static or Rotary
vacuum cane mud) Filtered
Broth return to clarification e
Pre-heating (70oC)
Heat treatment and decanting
(Decanter 105oC/2h)
Mud (fertilizer)
Sulphitation (SO2 addition
- 300-1400 ppm)
Phosphating (maintain in
300 ppm P2O5)
Sugar manufacture
Clarified broth
Molasse (by-product of
sugar manufacture)
Dilution water and/or Clarified broth
Wort (18-19o Brix 30oC.)
Yeast Acidification (pH 2.5)
Destillation
Hydro-alcoholic solution
Dilution and acidification
(H2SO4). Yeast cells suspension
return to fermentation
Centrifugation
Fermentation vat (pH
3.8-4.5, 32-34oC) (FedBatch, Continuous,
Conbat or Batcon
process)
Fermented broth
(Yeast 10-14%, ethanol
7.5% Residual sugar <
0.1%)
Yeast cell suspension (40-80% wet
mass)
Figure 1. The flowchart of the Brazilian process of fuel ethanol production.
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2.2 Metabolism of Saccharomyces cerevisiae inhibitory chemical agents
The understanding of inhibitory effect of various agents in the metabolism of S. cerevisiae during ethanolic
fermentation is extremely necessary to improve the technology of fuel ethanol. This micro-organism is heterotrophic
and has both metabolism, fermentation or cellular respiration. Also is necessary to understand how the inhibitory factors
affect the metabolism of S. cerevisiae synergistically since this is the industrial situation. Among the important factors
are: a) sucrose concentration influencing the osmotic pressure of the medium and the content of ethanol via
fermentation, b) pH and acidity - affecting respectively the proton pump and other cellular functions, such as nutrient
intake. c) sulfite - that may have an inhibitory effect on the metabolism of sugar consumption, and d) high temperature affecting the cell membrane.
CHAPMAN and second BARTLEY30, the yeast respiratory enzymes are inhibited from 2 g / L of glucose in the
medium, which makes the fermentation process the major route of sugar degradation, even under aerobic conditions.
Such inhibition is called Crabtree effect. High concentrations of sugars in the wort are responsible for the decrease or
stop of the fermentation due to the increase of osmotic pressure and high toxicity of ethanol in yeast cells31, 32, 33.
Trehalose is a storage carbohydrate and one important function is protection of yeast under stress such as high
temperature, toxicity of ethanol, cellular dehydration and increased osmotic pressure. This carbohydrate is accumulated
in the presence of oxygen at low concentrations of sugars, such as when there is exhaustion of glucose in the medium
during the diauxic growth34, 35, 36.
The antagonistic effect of organic acids on the metabolism of the yeast is one of the important problems in industrial
alcoholic fermentation. According OLIVA-NETO10, the inhibitory effect of organic acids on yeast depends on: the acid
concentration, the strain of S. cerevisiae used in the fermentation process, the osmotic pressure of the medium and the
synergism with other inhibitors. There is a wide class of acids which cause damage to S. cerevisiae in ethanolic
fermentation, such as: acetic, propionic, butyric, isobutyric, valeric acid37, formic, lactic acid38 octanoic and decanoic39,
40, 41
. However, lactic acid stands out due to the contamination by lactic acid bacteria which are very frequent in
industrial fermentation processes18, 22, 42, 43. According MAIORELLA38, acetic, formic and lactic acid have inhibitory
effect by interfering in chemical maintenance functions of the cells. Lactic acid has a hydroxyl extra thus characterized
by a low solubility in lipids to the other two mentioned and its inhibitory property occurs in higher concentrations in the
range of 10-40 g/L. DAESHEL44 using species Saccharomyces cerevisiae and Saccharomyces rosei with Lactobacillus
plantarum as a contaminant during a fermentation of cabbage juice, established inhibitory concentration of lactic acid
for cell growth from 2g/L. OLIVA-NETO and YOKOYA16 found that after the 15th cycle of a fermentation process,
the efficiency alcoholic suffered a marked inhibition when the lactic acid exceeded 6 g/L and the number of
contaminating bacteria became greater than 1.2 x 109 / ml.
CASSIO et al.45 demonstrated the active process of proton-lactate (ratio 1:1) simultaneously, and lactate
accumulation within the cell of S. cerevisiae. According to these authors, the accumulation rate of this anion inside the
cell depends on the pH oscillation between inside and outside of the cell. Permeases were also cited as involved in the
active transport of lactate45, 46. In contrast, the undissociated form of lactic acid crosses the plasma membrane by passive
diffusion. Inside the cell, the undissociated form of lactic acid is ionized as the intracellular pH is around 6.0 to 7.0, and
the lactic acid pKa is 3.86, thereby causing acidification of the cytosol. With the accumulation of intracellular H +, the H
+
-ATPase intensifies its activity to expel the protons47. The increased activity of H +-ATPase activity as a function of
acidification inner result in a significant decrease of energy required for the yeast growth and other essential metabolic
functions48, and after certain time will not be possible to maintain intracellular pH leading to reduced growth and
ultimately cell death49.
Ethanol can become toxic to the yeast cell50, 51, 52. The tolerance to high concentrations of ethanol is strain dependent,
and for most tolerant strains, the maximum ethanol concentration that allows the yeast growth is 10% (w: v)53. The
enzymes alcohol dehydrogenase and hexokinase are more sensitive to high concentrations of ethanol 54, 55. MILLAR et
al56 found invertase, fructose-1, 6-bisphosphate aldolase and pyruvate decarboxylase the most sensitive enzymes.
According ZECH and GÖRISCH57, the Saccharomyces cerevisiae invertase undergoes inactivation up to 100% when
subjected to high ethanol concentrations (more than 8%) and salts concentrations present in the molasses. This enzyme
inactivation could be reversible if the inhibitors have a decreased concentration. The place of ethanol action that causes
inhibitory effect is on the phospholipid membranes, where binds to the hydrophobic interior of the membrane causing
stiffness and hence causes disruption of transport systems52, 58, 59. In addition, the selectivity ability of the plasma
membrane decreases, allowing the output of the cellular constituents and passive input of protons, thus reducing
membrane potential and ultimately interfering across all systems that require the proton-motive pump. This eventually
leads to uncontrolled cellular nutritional deficiencies, which enhances the inhibition by ethanol60, 61.
The inhibitory effect of sulfite is another industrial problem since it is used in the sugar factory and it is present in the
molasses and sugarcane juice. The sodium sulfide in the cane molasses is present in the range of 200 to 700 mg/L62, and
up to 300 mg SO2 / L in the wort, especially when it involves the presence of juice sulfide from plant of sugar. Sulfur
dioxide is a very reactive substance and its inhibitory action is directly related to the pH, as characterized by two
dissociation constants. In the lower pH values coexist bisulfite (HSO3-) and sulfur dioxide (SO2) with pK1 = 1.77,
whereas on pH 5.0 to 9.0, there is a mixed composition of bisulfite and sulfite (SO3-2), and the pK2 = 6.9. Once the pH
of the fermentation is acid, the sulfite is the most toxic form (SO2 and HSO3-)63, 64, 65.Bisulfite can form sulfonic acid by
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reaction with carbonyl groups of aldehydes, organic acids, and other66. According to CARR et al.64, compounds such as
isulfite are bactericidal. HARADA et al.66 observed that disulfide reacts with acetaldehyde and blocks NAD+
regeneration required for the glycolysis in yeast. According ALVES67 and BASSO68 found that the addition of 100 mg
SO2/L in the form of NaHSO3, 40% of this substance reacted with components of the sugar cane must, and only 45% of
the theoretical acetaldehyde could be detected. In this experiment there was a decrease in the efficiency of fermentation
and cell growth. BRÉCHOT et al.69 reported an inhibition of 30 to 40% of the fermentation and between 40 and 80% in
the cell respiration by adding sulfite in the wort. Higher concentrations of metabisulphite in the production of fuel
ethanol from sugar beet were responsible for reduction in productivity and decrease of alcohol on cell viability70.
OLIVA-NETO and YOKOYA62 concluded that the CMI (Minimum Inhibitory Concentration) for sodium sulfite in pH
4.5 was in the range of 10-40 mg/L for lactic acid bacteria, and for S. cerevisiae 5000 mg/L, in the same conditions.
Levels of yeasts ribonucleosides phosphates undergo a drastic decrease in the presence of 2mM SO2 pH 3.6. The
ATPase activity is increased with 1 mM SO2 71.
An important factor for producing ethanol is the pH of the must, which influence on yeast growth, ethanol
fermentation rate as well as for the control of bacterial contamination72. The bacteria are less resistant to low pH and has
slower growth than yeasts in such situation. While for lactic acid bacteria the ideal pH is 6.073, S. cerevisiae presents a
good ethanol yield at pH above 4.0. According to SOUZA et al.74, the enzyme H+ -ATPase from plasma membrane of
S. cerevisiae controls an important physiological process. Through the proton pump, such enzyme regulates intracellular
pH and promotes the driving force for the nutrition. A striking feature of this enzyme is the fact that it is activated in the
presence of glucose that causes internal acidification increasing the level of its activity in yeast cells74,75. The yeast to
prevent the decrease of pHi (inner pH in the cell) releases H+ to the external environment through the activation of
H+-ATPase, besides absorbing K+ and basic amino acids, organic acids and excrete carbon dioxide release76,77. The
H+-ATPase plasma shows conformational changes as a function of H+ 78 and at pH 4.0 increase in three times its
activity, affinity to ATP folding without however causing changes in optimum pH (6.0)79. When the pH decreases from
6 to 3, there is an increased sensitivity of yeast to ethanol80 dissipating the proton motive force of the membrane. When
the cell metabolism was damage, the H+-ATPase presumably help activating proton motive force across the plasma
membrane with the consumption of ATP. The intracellular acidification occurs in the presence of stressors affecting the
organization of the plasma membrane 81,82,83,84,85.
Ethanol86, octanoic acid, decanoic41, succinic acid83, cinnamic acid87, low pH79, lack of nitrogen source88 and supraoptimal temperatures89 stimulate in vivo the activity of H+-ATPase of yeast. According to some researchers, the
activation of this enzyme could not be attributed to its synthesis but the post-translational modifications of this protein,
since the total number of enzyme decreases in conditions of stress and its activity is increased83, 89. This ATPase
activation may be caused, at least in part, by the change in plasma membrane lipid moiety that modifies the arrangement
of their enzymes contributing to the greater contact with their substrate83, 89 .There is a correlation between the glucose
phosphorylation and the activation of H+- ATPase, but the mechanism of activation of this enzyme during stress
situations is not yet fully understood90.
The effect of synergism between lactic acid, sulfite, pH and ethanol as inhibitors of alcoholic fermentation of sugar
cane fermented broth was carried out with two strains of S. cerevisiae for industrial use in Brazilian distilleries (Pe-2
and M-26). The strains were subjected to inhibitors concentration used in industrial conditions including adding of : 200
mg/L of NaHSO3, 6 g/L lactic acid, 7.5% or 9.5% ethanol and pH 3.6 or 4.5. Among these factors, the low pH (3.6)
followed by ethanol 9.5% were major stressors for the yeast during fermentation. The pH 4.5 probably protected the
proton motive force even in the presence of all other stress factors, as demonstrated by the largest size, a greater number
of more oval shape and yeast without the presence of yeast ghost (A) in relation yeast inhibited closer and fewer and
presence of yeast ghost 91.
3. The control of microbial contamination
The control of microbial contaminants in the fermentation is extremely necessary and avoid over-acidity and excessive
consumption of sulfuric acid because the contaminants are primarily responsible for the yeast flocculation 92, 93, yeast94,
polymers95. Cell flocculation is responsible for recycling more bacterial contaminants, difficulty of the action of
antimicrobial agents, and spending on more antifoams and sulfuric acid. However this control is not easily conducted in
distilleries, which makes the common high level of microbial contamination. Among the difficulties are the short supply
of products that selectively act efficiently only in contaminants being harmless to the growth of S. cerevisiae. Another
difficulty is directly associated with the inability to work in aseptic conditions, yet it is done sterilization of the wort.
The microbial infections are controlled using antimicrobial chemicals in milling and fermentation. The biocides
quaternary ammonium and organosulfur are currently used in the mill. For the fermentation are currently used
antibiotics monesin (Kamoran) and virginiamycin (innocuous to yeast) and the biocide chlorine dioxide that affects
S.cerevisiae at dosages higher than 50 mg/L, but this dosage partially inhibit Lactobacillus fermentum96. Unfortunately,
monensin has been detected in powder yeast exported as a byproduct by ethanol distilleries, which is limiting the use of
this product and demanding that other alternatives are created.
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Minimum Inhibitory Concentration (MIC) of penicillin V acid against Leuconostoc mesenteroides and Lactobacillus
fermentum, both isolated from ethanol distilleries, were 0.1 to 0.2 mg/L. Cefamandole presented a MIC of 0.26 to
0.36 mg/L for L. fermentum, but it was less effective for L. mesenteroides (0.36 to 1.45 mg/L). Clindamycin was most
effective antibiotic for L. fermentum (0.05-0.10 mg/L), but less effective against L. mesenteroides with CMI of 0.05 to
0.40 mg/L 92.
In a study of biocides92 for industrial use were determined MICs for S. cerevisiae, L. fementum and L. mesenteroides,
and that stood out for being effective against bacteria, without which affected the yeast were methyldithiocarbamate that
was effective against L. fermentum (MIC = 2.5 mg/L) or less against S. cerevisiae (MIC = 5.0mg / L), and
formaldehyde (MIC for L. fermentum and L. mesenteroides from 11.5 to 23 mg/L) and 46.3 mg/L for S. cerevisiae.
Thiocyanate (MIC = 1.5-5.0 mg/L), methyldithiocarbamate (2.5 mg/L),bromine-phenate (MIC 11.5 to 23 mg/L) were
effective against bacteria and yeasts, which limits the possibility for use only in the mill. ROSALES97 demonstrated that
the use of quaternary ammonium has a positive effect in controlling bacterial fermentation and further decrease in the
counts detected from Bacillus subtilis, through the combination of penicillin and quaternary ammonium. OLIVEIRA et
al 98 studied the performance of biocide brand busan 881 (Buckmann lab.) and this product was more effective when
placed in the initial phase of the process, getting higher in alcohol and lower acidity compared to control.
The compound 3,4,4' trichlorocarbanilide (TCC) combined with sodium dodecyl sulfate (DDS) in a 1:4 ratio (m / m)
in aqueous solutions is one of the few biocides that selectively inhibits Lactobacillus fermentum and Leuconostoc
mesenteroides (CMI <0.125 -1.0 mg/L) without inhibiting S. cerevisiae, in view of the CMI for yeast was much greater
(16 mg/L). 1.8 g/L of TCC was immobilized in sodium alginate, and applied in fermentation with high rate of L.
fermentum as a contaminant. There bacterial inhibition, control of acidity, increased the viability of the yeast to 20.8%
increase in the efficiency of alcoholic fermentation during 8 cycles). These same authors conducted another experiment
with 0.075 g / L of TCC in alginate and 1.67 mg/L DDS in the wort and it inhibited L. fermentum inoculated at the
beginning of the process, and remained stable for 24 recycles of fermentation with the same pellet of TCC99. Some
studies were conducted in order to obtain strains of yeast that naturally inhibit the lactic acid bacteria. The comparative
study between the strain S. cerevisiae M26, isolated from ethanol distilleries in screening of strains able to inhibit
L. fermentum 100 produced higher acidity than the Pe-2, with higher production of succinic acid, an important inhibitor
of lactic acid bacteria91.
4. Conclusion
The Brazilian distilleries of fuel ethanol use the continuous yeast cells recycle, and the yeast cells needs to operate with
high viability to maintain high ethanol efficiency. One aspect of the innovation in ethanol technology corresponds the
more restricted use of sulfuric acid with innovation of new technologies, sulfite control, and especially greater control
of bacteria because they are responsible for significant production of organic acids in the process, especially lactic acid,
which is seen as extremely damaging to the metabolism of the yeast and ethanol production. In this sense, the
appropriate pH of wort is important to protect the yeast metabolism, more control of some chemicals used in sugar
factory like sulfite, and new antimicrobial chemicals are necessary to control the bacterial contamination. The
knowledge of synergistic effect of several chemical and biological inhibitors and the correct use of industrial parameters
to avoid yeast inhibition are fundamental to reach high ethanol efficiency and low cost.
5. References
[1] Mapa 2013 – Ministério da Agricultura, Pecuária e Abastecimento. Brazilian government department of Agriculture, Livestock
and Supply. http://www.agricultura.gov.br/vegetal/safras-estoques. Access in the site: Access in: April 23, 2013.
[2] Conab 2013 - Brazilian National Company of Supply.
http://www.conab.gov.br/OlalaCMS/uploads/arquivos/13_04_09_10_30_34_boletim_cana_portugues_abril_2013_4o_lev.pdf.
Access in the site: Access in: April 23, 2013.
[3] WINKLER, M.A. (1991): in Genetically-Engineered Proteins and Enzymes from Yeast: Production Control. Ed. Wiseman, A.,
Ellis Horwood, pp. 96-146.
[4] WINKLER, M.A. (1995): in HandBook of Enzyme Biotechnology. Ed. Wiseman, A. Ellis Horwood, p. 9-30.
[5] ABUD, C.L. Avaliação de uma população de células de Saccharomyces cerevisiae submetida a processos fermentativos em
condições de temperaturas elevadas. Dissertação (Mestrado). I.Q. UNESP-Araraquara, 1997.
[6] CYSEWSKI, G.R. e WILKIE, C.W. Process design and economic studies of fermentation methods for the production of ethanol.
Biotechnol. and Bioeng. v. 20, p. 1421-1430, 1978.
[7] INGLEDEW, W.M. Continuous fermentation in the fuel alcohol industry: how does the technology affect yeast. 2003. In:
BAYROCK, D.P. E INGLEDEW, W.M. Ethanol production in multistage continuous, single stage continuous, Lactobacilluscontaminated continuous, and batch fermentations. W. J. Microbiol. Biotechnol., v.21, p. 83-88, 2005.
[8] BOVI, R.; MARQUES, M.O. O tratamento ácido na fermentação alcoólica. Álc. e Açúc., v.3, n.9, p.10-13, 1982.
[9] PATERSON, M.; BORBA, J.M.M.; MELO, F.A.D.; MORAES, J.I. Avaliação do desempenho da fermentação etanólica em
diferentes situações do processo industrial. Brás. Açuc., v.106, n.516, p.27-32, 1988.
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[10] RODINE, M. A. T. 1985. Isolamento, caracterização e identificação de bactérias contaminantes de dornas de fermentação nas
destilarias de etanol. Dissertação de Mestrado, ESALQ-USP, Piracicaba, SP, pgs. 92.
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fermentação alcoólica por leveduras. Tese (Doutorado)-Faculdade de Engenharia de Alimentos- Unicamp- Campinas, 1995,
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