Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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 ©FORMATEX 2013 371 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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, Brettanomyces26,27,28,29,30 372 ©FORMATEX 2013 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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. ©FORMATEX 2013 373 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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 374 ©FORMATEX 2013 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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. ©FORMATEX 2013 375 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ 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. 376 ©FORMATEX 2013 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ [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. [11] OLIVA-NETO, P. Estudo de diferentes fatores que influenciam o crescimento da população bacteriana contaminante da fermentação alcoólica por leveduras. Tese (Doutorado)-Faculdade de Engenharia de Alimentos- Unicamp- Campinas, 1995, pgs.112. [12] OTENIO, M.H. Avaliação comparativa do efeito da retirada do tratamento ácido com ácido sulfúrico no fermento durante os reciclos, na Usina Bandeirantes, PR, na rotina industrial da Destilaria Anexa. Master thesis. Instituto de Biociências de Rio Claro, Universidade Estadual Paulista. Brazil, 1998, pgs. 69. [13] NUNES, M.A.; SOUTO, A; ALVES DINIS, N.C.; QUEIROZ, R.M. de; VIEIRA DE MEDEIROS, L.N.. BRITO DE OLIVEIRA, J.P. Processo de eliminação do ácido sulfúrico e outros bactericidas do sistema de fermentação alcoólica MelleBoinot. Álcool não corrosivo. Álc. Açúc., São Paulo, v.4, n.22, p39-50, 1991. [14] GUERRA, E.J.; ANGELIS, D.F. Floculação da levedura induzida por bactérias na fermentação etanólica: I. método de detecção preventiva e estudos para o controle. Stab: Açúc. Álc. Subp., Piracicaba, v. 16, n.6, p.25-27. 1998. [15] BOVI, R.; MARQUES, M.O. O tratamento ácido na fermentação alcoólica. Álcool e açúcar, v.3, n.9, p.10-13, 1983. [16] LUDWIG, K.M.; OLIVA-NETO, P.; ANGELIS, D.F. Quantificação da floculação de Saccharomyces cerevisiae por bactérias contaminantes da fermentação alcoólica. Ciênc. Tecnol. Aliment. Campinas, v.21, n.1, p.63-68. 2001. [17] OLIVA-NETO, P.; YOKOYA, F. Evaluation of bacterial contamination in fed-batch alcoolic fermentation process. W. J. Microbiol. Biotechnol., v.10, p.697-699, 1994. [18] NOBRE, T.P. Viabilidade celular de S. cerevisiae cultivada em associação com bactérias contaminantes da fermentação alcoólica. Master Thesis. Escola superior de Agricultura “Luiz de Queiroz“, Piracicaba., Universidade de São Paulo. Brazil, 2005, pgs.90. [19] OLIVA-NETO, P. Influência da contaminação por bactérias láticas na fermentação alcoólica pelo processo de batelada alimentada. Master thesis - Faculdade de Engenharia de Alimentos- Unicamp- Campinas, 1990, Brazil, pgs.190. [20] TILBURY, R.G. Occurrence and effects of acid bacteria in the sugar industry, 1975. In: ALVES, D.M.G., 1994; pgs.199. [21] PRIEST, F.G. Contamination, 1981. In: ALVES, D.M.G. Fatores que afetam a formação de ácidos orgânicos bem como outros parâmetros da fermentação alcoólica. Tese (Doutorado). ESALQ. Piracicaba, 1994, pgs.199. [22] OLIVA-NETO, P.; YOKOYA, F. Effects of nutricional factors on growth of Lactobacillus fermentum mixed whith Saccharomyces cerevisiae in alcoholic fermentation. Rev. Microbiol, v.28, p.25-31, 1997 [23] HYNES, S.H.; KJARSGAARD, D.M.; THOMAS, K.C.; INGLEDEW, W.M. Use of virginiamycin to control the growth of lactic acid bacteria during alcohol fermentation. J. Ind. Microbiol. Biotech., v. 18, p. 284-291, 1997. [24] YOKOYA, F. Problemas com contaminantes na fermentação alcoólica. STAB. Açucar, Alcool, Subp. v.9, n.6, p.38-39, 1991. [25] ROSE, A.H. Industrial importance the Saccharomyces cerevisiae. In: SKINNER, F.A. et al. Biology and Activies of Yeast, Ed. Academic Press., 1980. [26] BEVAN, D.; BOND, J. Micro-organisms in field and mill - a preliminary survey. Proc. Ad. Soc. Sugar Cane Technol., 38 th Conference, p.137-143, 1971. [27] OLIVEIRA, M.C.F.L. Leveduras contaminantes da fermentação etanólica. Microbiologia da Fermentação Etanólica. Rio Claro. SP. Departamento de Bioquímica e Microbiologia Aplicada – IB UNESP, p. 92-104, 1987 [28] LIMA, U.A.; GOLDONI, J.S.; CEREDA, M.P.; SOUZA, L.G. Ocorrência de microrganismos em caldo bruto, caldo misto e água de embebição em uma usina de cana. Brasil Açucareiro, v.4, p.337-343, 1974. [29] TILBURY, R.H., Hollingsworth, B.S., Grahan, S. D., Pottage, P. Mill sanitation – a fresh approash to biocide evaluation. In Reis, F.S. ; Dick, J. (Eds) . Proceedings XVI Congress of Int. Soc. Sugar Cane Technology. 1977. [30] SILVA, R.B.O. Leveduras contaminantes na produção de etanol industrial por processo contínuo: Quantificação e identificação. Dissertação. Rio Claro – UNESP. 1994. 145 p. [31] CHAPMAN, C.; BARTLEY, W. The kinetics changes in yeast under conditions that cause the loss of mitochondria. Bioch. J., v.107, p. 455-465, 1968. [32] WEUSTHUIS, R. A.; VISSER, W.; PRONK, J.T.; SCHEFFERS, W. A.; van DIJKEN, J. Effects of oxygen limitation on sugar metabolism in yeasts: a continuous-culture study of the Kluyver effect. Microbiol., v. 140, p. 703-715, 1994. [33] BISSON, L.; BUTZKE, C.E. Diagnosis and rectification of stuck and sluggish fermentation. Am. J. Enol Vitic, v.51, p.168-177, 2000. [34] MALACRINÒ, P.; TOSI, E.; GARAMIA, G.; PRISCO, R.; ZAPPAROLI, G. The vinification of partially dried grapes: a comparative fermentation study on Saccharomyces cerevisiae strains under high stress. Lett. Appl. Microbiol., v. 40, p. 466472, 2005. [35] HOTTINGER, T.; SCHUTZ, P.;WIEMKEN, A. Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. J.Bacteriol., v.169, n.12, p.5518-5522, 1987. [36] PANEK, A.C.; MANSURE, J.J.A.; PASCHOALIN, M.F.; PANEK, A.D. Regulation of trehalose metabolism in Saccharomyces cerevisiae mutants during temperatures shifts. Bioch., v.72, p.77-79, 1990. [37] ALCARDE, A.R.; BASSO, L.C. Efeito da trealose na manutenção da viabilidade de células de leveduras desidratadas por liofilização. Scien. Agríc., v.54, n.3, p. 189-194, 1997. [38] SAMSOM, F.E.; KATZ, A.M.; HARRIS, D.L. Effects of acetate and short-chain fatty acids on metabolism. Arch. Biochem. Biophys., v.54, 406, 1955. [39] MAIORELLA, B.; BLANCH, H.W.; WILKE, C.R. By-product inhibition effects on ethanolic fermentation by Saccharomyces cerevisiae. Biotech. Bioeng., v.25, p. 103-121, 1983. [40] LAFON-LAFOURCADE, S.; GENEIX, C.; RIBEREAU-GAYON, P. Inhibition of alcoholic fermentation of grape must by fatty acids produced by yeasts and their elimination by yeasts ghosts. Appl. Env. Microbiol. v. 47, n.6, p. 1246-1249, 1984. [41] VIEGAS, C.A.; ROSA, M.F.; SÁ-CORREIA, I.; NOVAIS, J.M. Inhibition of yeast growth by octanoic and decanoic acids produced during ethanolic fermentation. Appl. Environ. Microb., v.55, n.1, p.21-28, 1989. ©FORMATEX 2013 377 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ [42] VIEGAS, A.C.; SÁ-CORREIA, I. Effects of low temperatures (9-33oC) and pH (3,3-5,7) in the loss of Saccharomyces cerevisiae viability by combining lethal concentrations of ethanol with octanoic and decanoic acids. Food Microbiol.,v.34, p.267-277, 1997. [43] HALM, M.; LILLIE, A.; SORENSEN, A. K.; JAKOBSEN, M. Microbiological and aromatic characteristics of fermented maize dough for ‘Kenkey’ production in Ghana. Int. J. Food Microbiol., v.19, p.135-143, 1993. [44] NOBRE, T.P. Viabilidade celular de S. cerevisiae cultivada em associação com bactérias contaminantes da fermentação alcoólica. Master thesis. Escola superior de Agricultura “Luiz de Queiroz“, Piracicaba., Universidade de São Paulo. Brazil, 2005, pgs.90. [45] DAESCHEL, M.A.; FLEMING, H.P.; MC FELTERS, R. F. Mixed culture fermentation of cucumber juice with Lactobacillus plantarum and yeasts. J. Food Scien., v.53, n.3, p. 863-864, 1988. [46] CASSIO, F; LEÃO, C.; van UDEN, N. Transport of lactate and other short-chain monocarbosylates in the yeast Saccharomyces. Appl. Environ. Microbiol., v.53, n.3, p. 509-513, 1987. [47] NARENDRANATH, N.V.; THOMAS, K.C; INGLEDEW, W.M. Effects of acetic acid and lactic acid on growth of Saccharomyces cerevisiae in minimal medium. J. Ind. Microbiol. Biotech., v.26, p.171-177, 2001. [48] HOLYOAK, C.D.; STRAFORD, M; McMULLIN, Z; COLE, M.B.; CRIMMINS, K.; BROEN, AJ.P.; COOTE, P. Activity of the plasma membrane H+- ATPase and optimal glycolytic flux are required for rapid adaptation and growth in the presence of the weak acid preservative sorbic acid. App. Environ. Microbiol., v. 62, p. 3158-3164, 1996. [49] BRUL, S.; COOTE, P. Presevative agents in food: mode of action and microbial resistence mechanisms. Int. Food Microbiol., v.174, p.125-128, 1999. [50] HALM, M.; HORNBAEK, T.; ARNEBORG, N.; SEFA-DEDEH, S.; JESPERSEN, L. Lactic acid tolerance determined by measurement of intracellular pH of single cells of Candida krusei and Saccharomyces cerevisiae isolated from fermented maize dough. Int. J. Food Microbiol., v.94, n.1, p.97-103, 2004. [51] GHOOSE, T.K.; TYAGI, R.D. Rapid ethanol fermentation of celulose hydrolysate: batch versus continous systems. Biotech. Bioeng., v.21, p. 1387-1400, 1979. [52] BEAVEN, M.J.; CHARPENTIER, C.; ROSE, A.H. Production and tolerance of ethanol in regulation to phospholipid fatty-acyl composition in Saccharomyces cerevisiae NCYC 451. J. Gen. Microbiol., v.128, p.1447-1455, 1982. [53] LEÃO, C.; van UDEN, N. Effects of ethanol and other alkanois on the glucose transport system of Saccharomyces cerevisiae. Biotechnol. Bioeng., v. 24, p. 2601-2604, 1982. [54] JONES, R.P.; PAMMENT, N.; GREENFIELD, P.F. Alcohol fermentation by yeasts: The effect of environmental and other variables. Proc. Biochemist., v. 16, p.42-49, 1981. [55] CASEY, G.P.; INGLEDEW, W.M. Ethanol tolerance in yeasts. CRC Crit. Ver. Microb., v.13, p. 219-281, 1976. [56] SHARMA, S.; TAURO, P. Enzyme behavior during ethanol production by Saccharomyces cerevisiae. World J. Microb. Biotechnol., v.2, p. 112-115, 1987. [57] MILLAR, D.; GRAFFITHS-SMITH, U.; ALGAR, E.; SCOPES, R. Activity and stability of glycolitic enzymes in the presence of ethanol. Biotechnol. Lett., v.4, p. 601-605, 1982. [58] ZECH, M.; GÖRISCH, H. Invertase from Saccharomyces cerevisiae: reversible ativation by components of industrial molasses media. Enz. Microb. Technol, v.17, p. 41-46, 1995. [59] LOUREIRO-DIAS, M.C.; PEINADO, J.M. Effect of ethanol and other alkanols on the maltose transport system of Saccharomyces cerevisiae. Biotech. Lett., v.4, p.721-724, 1982. [60] INGRAM, L.O. Adaptation of membrane lipids to alcohols. J. Chem. Technol. Biotechnol., v. 35B, p. 235-238, 1985. [61] CASEY, G.P.; MAGNUS, C.A.; INGLEDEW, W.M. High-growth brewing: effect of nutrition on yeast composition, fermentation ability, and alcohol production. Appl. Env. Microbiol., v.48, n.3, p. 639-646, 1984. [62] DOMBEK, K.M.; INGRAM, L.O. Magnesium limitation and its role in apparent toxicity of ethanol during yeast fermentation. Appl. Env. Microbiol., v. 52, p. 471-481, 1986. [63] OLIVA-NETO, P.; YOKOYA, F. Susceptibility of Saccharomyces cerevisiae and lactic acid bacteria from the alcohol industry to several antimicrobial compounds. Braz. J. Microbiol., v.32, p. 10-14, 2001. [64] CARR, J.G.; PATRICIA, A.D.; SPARKS, A.H. The toxicity of sulphur dioxide towards certain lactic acid bacteria from fermented apple juice. J. Appl. Bacteriol., v.40, n.2, p. 201-212, 1976. [65] ANACLETO, J.; van UDEN, N. Kinectics and activation energetics of death in Saccharomyces cerevisiae induced by sulphor dioxide. Biotech. Bioeng., v.34, p. 2477-2486, 1982. [66] CARTWRIGHT, C.P.; ROSE, A.H.; CALDERBANK, J.; KEENAN, M.H.J. (1989) Solute Transport. In: The Yeasts. Ed. ROSE, A.H., Academic Press, London, v.3, p. 5-56. [67] HARADA, K.; HIGUCHI, R.; UTSUMI, I. Studies on sorbic acid, part 4. Inhibition of the respiration in yeast, 1968. In WARTH, A. D. Resistence of yeast species to benzoic and sobic acids and to sulfor dioxide. J. Food Protect., v. 48, n. 7, p. 564-569, 1985. [68] ALVES, D.M.G. Fatores que afetam a formação de ácidos orgânicos bem como outros parâmetros da fermentação alcoólica. Doctor thesis. ESALQ. Piracicaba, Brazil, 1994, pgs.199. [69] BASSO, L.C. 1991.In: ALVES, D.M.G. Fatores que afetam a formação de ácidos orgânicos bem como outros parâmetros da fermentação alcoólica. Doctor Thesis. ESALQ. Piracicaba, 1994, pgs.199. [70] BRECHOT, P.; CROSON, M. ; MATSURA, S. Fermentation and respiration of yeasts in presence sulphur dioxide. Ant. van Leeuw., v.35, p. 21-22, 1969. [71] GIBBONS, W.R; WESTBY, C.A. Effects of sodium meta bisulfite on diffusion fermentation of fodder beets fuel ethanol production. Biotechn. and Bioeng., v.30, p. 906-916, 1987. [72] MAIER, K.; HINZE, H.; LEUSCHEL, L. Mechanism of sulfite action on the energy metabolism of Saccharomyces cerevisiae. Bioch. Bioph. Acta, v. 848, p. 120-130, 1986. [73] ALVES da SILVA, E. F. Fermentação etanólica: influência do ácido sulfúrico sobre a viabilidade da levedura de processo e bactérias e leveduras contaminantes. Master thesis. Instituto de Biociências, Unesp, Rio Claro, 1993, pgs. 134. 378 ©FORMATEX 2013 Materials and processes for energy: communicating current research and technological developments (A. Méndez-Vilas, Ed.) ____________________________________________________________________________________________________ [74] KANDLER, O.; WEISS, N. Regular nonsporing Gram positive Rods. In: SNEATH et al. Bergeys Manual of Systematic Bacteriology. Baltimore. Willians e Wilkens, v.2, p. 1208-1234, 1986. [75] SOUZA, M.A.A.; TRÓPIA, M.J.; BRANDÃO, R.L. New aspects of glucose activation of the H+_ ATPase un the yeast Saccharomyces cerevisiae. Microbiol., v.147, p. 2849- 2855, 2001. [76] BECHER DOS PASSOS, J.B.; VANHALEWYN, M.; BRANDÃO, R.L.; CASTRO, I.M.; NICOLI, J.R.; THEVELEIN, J. Glucose-induced activation of plasma membrane H+-ATPase in mutants of the yeast Saccharomyces cerevisiae affected in cAMP metabolism, cAMP-dependent protein phosphorilation and initiation of glycolysis. Biochim. Biophys. Acta, v.1136, p.57-67.1992. [77] CONWAY, E.J.; BRADY, T.G. Biological production of acid an alkali, 1950. In: ALVES, D.M.G., 1994. [78] COOTE, N.; KIRSOP, B.H. Factors responsible for the decrease in pH during beer fermentations,.J. Inst. Brew., v.82, p.149156, 1976. [79] BLANPAIN, J.P.; RONJAT, M.; SUPPLY, P.; DUFOUR, J.P.; GOFFEAU, A.; DUPONT, Y. The yeast plasma membrane H+_ATPase. J. Biolog. Chem., v.267, n.6, p. 3735-3740, 1992. [80] ERASO, P; GANCEDO, C. Activation of yeast plasma membrane ATPase by acid pH during growth. FEBS Lett., v. 224, n.1, p.187-192, 1987. [81] GAO, C.; FLEET, G.H. The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloekera apiculata. J. App. Bacteriol., v. 65, p. 405-409, 1988. [82] VIEGAS, A. C.; SÁ-CORREIA, I. Activation of plasma membrane ATPase of Saccahromyces cerevisiae by octanoic acid. J.Gen. Microbiol., v.137, p.645-651. 1991. [83] ALEXANDRE, H.; MATHIEU, B; CHARPENTIER, C. Alteration in membrane fluidity and lipid composition, and modulation of H+-ATPase activity in Saccharomyces cerevisiae caused by decanoic acid. Microbiol., v. 142, p. 469-475, 1996. [84] CARMELO, V.; SANTOS, H.; SÁ-CORREIA, I. Effect of extracellular acidification on the activity of plasma membrane ATPase and on the cytosolic and vacuolar pH of Saccharomyces cerevisiae. Bioch. Biophy. Acta, v.1325, p. 63-70, 1997. [85] VIEGAS, C.A; ALMEIDA, P.F.; CAVACO, M.; SÁ-CORREIA, I. The H+-ATPase in the plasma membrane of Saccharomyces cerevisiae is activated growth latency in octanoic acid supplemented médium acompanying the decrease in intracelular pH and viability. Appl. Environ. Microbiol., v. 64, p. 779-783, 1998. [86] FERNANDES, AR.; PEIXOTO, F.P; SÁ-CORREIA, I. Activation of H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress. Arch. Microbiol., v.171, p.6-12, 1998. [87] ROSA, M.F.; SÁ-CORREIA, I. In vivo activation by ethanol of plasma membrane ATPase of Saccharomyces cerevisiae. Appl. Environ. Microbiol, v.57, p. 830-835, 1991. [88] CHAMBEL, A.; VIEGAS; C.A.; SÁ-CORREIA, I. Effect of cinnamic acid on the growth and on plasma membrane H+- ATPase activity of Saccharomyces cerevisiae. Internat. J. Food Microb., v. 50, p. 173-179, 1999. [89] BENITO, B.; PORTILLO, F.; LAGUNAS, R. In vivo activation of the plasma membrane ATPase during nitrogen starvation. Identification of the regulatory domain that controls activation. FEBS Lett., v.300, p. 271-274, 1992. [90] VIEGAS, C.A; SEBASTIÃO, P.; NUNES, A.G.; SÁ-CORREIA, I. Activation of plasma membrane H+-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supra-optimal temperatures. Appl. Environ. Microbiol., v.61, n.5, p.1904-1909, 1995. [91] CHANG, A; SLAYMAN, C.W. Maturation of the yeast plasma membrane [H+] ATPase involves phosphorylation during intracelular transport. J. Cell Biol., v.115, p.289-295, 1991. [92] DORTA, C.; OLIVA-NETO, P; ABREU-NETO, M.S.; NICOLAU-JUNIOR, N; NAGASHIMA, A.I. Synergism among lactic acid, sulfite, pH and ethanol in alcoholic fermentation of Saccharomyces cerevisiae (PE-2 and M-26). World J. Microbiol. Biotechnol., v.22, p.177-182, 2006. [93] YOKOYA, F. ; OLIVA-NETO, P. Characteristics of yeast flocculation by Lactobacillus fermentum. Rev. Microbiol.São Paulo. v. 22, p. 21-27, 1991. [94] SANTOS, M.T.; YOKOYA, F. Characteristics of yeast cell flocculation by Lactobacillus fermentum. J. Ferment. Bioeng., v.75, n.2, p.151-154, 1993. [95] STRATFORD, M.; KEENAN, M.H.J. Yeast flocculation: quantification. Yeast, v.4, p.107-115, 1988. [96] MENEGHINI, S.P., REISI, F.C, ALMEIDA, P.G, CECCATO-ANTONINI, S.R. Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation. Brazilian Journal of Microbiology. v.39, p. 337-343, 2008. [97] ROSALES, S.Y.R. Contaminantes bacterianos da fermentação etanólica: isolamento em meios diferenciais, identificação e avaliação de desinfetantes. Rio Claro/SP: UNESP, 1989, 200p. Doctor Thesis - Instituto de Biociências, Universidade Estadual Paulista, Brazil, 1989. [98] OLIVEIRA, M.C.; FURLETTI, M.E.; ANGELIS, D.F. Novos biocidas propostos a indústria alcooleira (I) Análise do desempenho do Busan 881. V Simpósio Nacional de fermentação Universidade Federal de Viçosa - MG. Brazil, 1982. [99] OLIVA-NETO, P.; YOKOYA, F. Effect of 3,4,4'-triclorocarbanilide on growth of lactic acid bacteria contaminants in alcoholic fermentation. Biores. Technol., v.63, p.17-21, 1998. [100] OLIVA-NETO, P.; FERREIRA M. A.; YOKOYA, F. Screening for yeast with antibacterial properties from an ethanol distillery. Biores. Technol. v. 92, p. 1-6. 2004. ©FORMATEX 2013 379