International Congress and Expo on Biofuels - 2015
Brazilian technology of fuel ethanol fermentation:new perspectives to improve the technology anddiversification. Dr. Pedro de Oliva Neto Lab. Industrial Biotechnology andUNESP Bioenergy Institute . Faculdade de Cincias e Letras Universidade Estadual Paulista UNESP Campus Assis So Paulo State - Brazil. Overview of Brazilians Fuel Ethanol Distilleries
- Some current numbers of Ethanol Industry:The estimatation for the 2014/15 crop: Cane Crop milh. Ton.(2%increase based on the last crop) Sugar cane milh. ton.(0.8%increase based on the last crop) Ethanol 29.2 billion liters (2.8% increase based on the last crop) - The characteristic parameters of fermentationare: Ethanol efficiency (conversion of sugar to ethanol) - 90% to 92% ; Ethanol titles for fermented must : GL; Fermentation times : 6 to 11 hours 8 hs in a Fed-Batch process (more predominant process) Yeast concentration in the fermented must: % v /v; Final volume of liquid residue after distillation: liters / liter of ethanol. Source: CONAB Brazil Flowchart of Brazilian Ethanol and sugar industry
Molasse (by-product of sugar manufacture) Hydro-alcoholic solution Fermentation vat (pH , 32-34oC)(Fed-Batch, Continuous, Conbat or Batcon process) Clarification (SO2and CaO addition) Filtration (Static or Rotary vacuum cane mud) FilteredBroth return to clarification e Phosphating Heat treatment and decanting (Decanter105oC/2 h) Clarified broth Sugar manufacture Cane washing Milling Alkaline water (pH 11) Pre-heating(70oC) Bagasse (Burning in the boiler -Energy, steam) Mud (fertilizer) Dilution water and/or Clarified sugarcane juice Preparation of Wort (18-22o Brix- 30oC.) YeastAcidification (pH 2.5) Fermented broth(Yeast 10-14%, ethanol % Residual sugar < 0.1%) Centrifugation Distillation Yeast cell suspension (40-80% wet mass) Acidification(H2SO4) of Yeast cells suspension -return to fermentation Oliva-Neto et al The Brazilian technology of fuel ethanol fermentation - yeast inhibition factors and new perspectives to improve the technology. In: A. Mndez-Vilas. (Org.). Materials and processes for energy: communicating current research and technological developments. 1 ed. Badajoz: Formatex, 2013, v. 1, p Important inhibitors of industrial fuel ethanol fermentation
Biological contaminants: Yeasts:Flocculant S. cerevisiae, Dekkera, Brettanomyces ,Candida, Hansenula, Kloeckera,Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces, Schwanniomyces, Torula, Torulopsis,Trichosporon, Cryptococcus . Problems:Decrease in ethanolicyield by sugar consume and yeast flocculation. Bacteria: The most important genus - Lactobacillus, Bacillus and Leuconostoc. The most importantspecies: Lactobacillus fermentum, L. plantarum Problems:Sugar consumption producing lactic acid which decrease the yeastviability. Increase of yeast flocculation causing the yeast settling at the bottom ofvats, and cell loss in centrifuges further contributing to the reduction in the ethanolyield. OLIVA-NETO, P.; YOKOYA, F. Evaluation of bacterial contamination in fed-batch alcoolic fermentationprocess. W. J. Microbiol. Biotechnol., v.10, p , 1994. OLIVA-NETO, P.; YOKOYA, F. Effects of nutricional factors on growth of Lactobacillus fermentum mixedwith Saccharomyces cerevisiae in alcoholic fermentation. Rev. Microbiol, v.28, p.25-31, 1997 YOKOYA, F. ; OLIVA-NETO, P. Characteristics of yeast flocculation by Lactobacillus fermentum. Rev.Microbiol. So Paulo. v. 22, p , 1991. Abiotic inhibitors of yeast fermentation
There important abiotic inhibitors are : a) The highest ethanol concentration used is 10% (v/v), The enzymes alcoholdehydrogenase and hexokinase are more sensitive to high concentrations of ethanol (Jones et al. 1976) b) pH and acidity - Acetic, formic and lactic acid have inhibitory effect by interfering inchemical maintenance functions of the cells, such as nutrient intake. Lactic acid shows inhibitory property in high concentrations (6-40 g/L) (Maiorella et al1987, Oliva-Neto & Yokoya, 1994) pH in the industrial fermentation should be maintained higher than 4.0. Lower pHacting in a synergistic effect with other inhibitors and they affect the proton pump andother cellular functions of S. cerevisiae. (Dorta et al. 2006) c) Sulphite Maximum level 50 100 mg/L to avoid inhibition on the metabolism ofsugar consumption. Sodium sulphite in the cane molasses to 700 mg/L, and in thewort up to300 mg SO2/L. Dissulphite reacts with acetaldehyde and blocks NAD+regeneration required for the glycolysis in yeast (Harada et al 1985, Alves, 1994). Sodium Sulphite MIC (Minimum Inhib.Concentr.) forS.cerevisiae is 5000 mg/L (Oliva- Neto & Yokoya, 2001) d) High temperature Maximum temperature possible to use in the fermentation is 34oC . Higher temperatures affect the cell membrane and yeast viability. DORTA et al.Synergism among lactic acid, sulfite, pH and ethanol in alcoholicfermentation ofS. cerevisiae (PE-2 and M-26). World Journal of Microbiology & Biotechnology, England, v. 22, p , Formulation of the fermentative media with stress factors: ethanol, lactic acid, sulphite and pH ______________________________________________________________________ sulfite (mg/L ) latic acid ethanol * pH toxicity MediumToC (NaHSO3) (g/L) (%) level ______________________________________________________________________ maximum low sulfite low lactic ac low ethanol normal pH control ____________________________________________________________________ *Sucrose atconcentrationof 16.37% or %(w/v) were used ascarbon CellsmorphologyA - pH 4.5 M 5B -pH 3.6 M source; Yeast budding and viabilitiy, residual protein and ethanolic yield in in medium after different fermentation conditions bySaccharomycescerevisiae PE-2( )andM-26 (). Flocs of S.cerevisiae and Lactobacillus
S. cerevisiaecells flocculationby Lactobacillus fermentum Quantification of the yeast flocculation from induction by L. fermentum CCT 1396, after treatment with different concentrations ofproteases and carbohydrases The increase of yeast flocculation by the increase of L. fermentum Flocs of S.cerevisiae and Lactobacillus Source: Fermentec Ludwig et al Rev. Soc. Brasileira Cincia e Tecnologia de Alimentos , v.21, 1, p Industrial Yeast Deflocculation New perspectivies
Yeast cell deflocculation on the S. cerevisiae suspension from fuel ethanol distillery treated with soluble papain in 15 minutes of reaction. Effect of the soluble and immobilized papain in the suspension of flocculated yeast from fuel ethanol distillery Yeast cell deflocculation with the recycle of soluble papain by centrifugation of yeast suspension and enzyme recovery SILVA et al Enzyme Research , v. 2015, Article ID Development of new chemicalsfor control of microbial infection in fuel ethanol fermentation.
Minimum Inhibitory Concentration (MIC) for several chemicals against L.fermentum and S.cerevisiae, at 32oC - 24 h. ______________________________________________________________________________________________ Chemicals MIC (mg/l) Cultures _______________________________________________________________________________ S. cerevisiae S.cerevisiae L. fermentum L. fermentum 2 CCT FCLA M CCT CCT 1396 ______________________________________________________________________________________ TCC+CBe > TCC+CBe > > TCC+CBe > > TCC+CBa 5: > > TCC+CBa 2.5: > , ,12 TCC+CBa 1: > TCC+ CBa 2.5: > > HJ Kamoran > > HJ Kamoran > > _______________________________________________________________________________________ Symbols: TCC -3,4,4 trichlorocarbanilide,CBe -benzethonium chloride,CBa - benzalkonium chloride, CTA Cetyl trimethyl ammonium chloride.Hj Kamoran commercial product (antibioticMonensin) autoclavated product,culture in pH 6.0for L.fermentum,2 microfiltered product,culture in pH 6.0 for L.fermentum, 3 microfilteredproduct, culture in pH 4.0 for L.fermentum . Oliva-Neto et al Brazilian Archives of Biology and Technology. v. 57 (3), p , 2014. Diversification of subtrates for ethanol fermentation:
Search for more feedstocks and carbohydrases Amylasesproduction and Residues for starch industry Effect of culture time on enzymatic activity (dashed line) of amylase in R. oryzae (dark circle) and R. oligosporus (white circle) cultures and medium final pH (solid line). Conditions: 30C, pH 5.5, wheat flour type II as substrate Enzymatic reaction using enzyme produced from R. oligosporus (1.25 U/mL) in 5% and 10% (w/v) starch solution at 50C. Ethanolic efficiency from hydrolysis of cassava residue using R. oligosporus enzymes = 80% Freitas et al Chemical Papers,v. 68 (4) p Cellulases and Polygalacturonasesproduced in aCitrus residue (citrus pulp) culture medium in a cell recycle process A B B Poligalacturonaseactivity expressed (A) U/g and(B) U/ml in citric pulp culture of A. niger CCT and T. reesei QM 9414 with cell recycles of 72 h Cellulase activity expressed (A) U/g and(B) U/ml in citric pulp culture of A. niger CCT 3312 and T. reesei QM 9414 with cell recycles of 72 h Barbosa, M.F. Shynia, T. Y. Oliva-Neto, P.Adding value to the citrus pulp by enzymebiotechnology. Lambert Academic Press. Saarbruchen. Germany. 52 p. 2014 Production of xylo-oligosacharides from bagasse
XOS are Prebiotic: sugar oligomers that can not be digested by animals and humans.Advantages of prebiotics: - Increase the number of Probiotics in the gut.Decrease the number of harmful microbes.Improve the protection against osteoporosis, cardiovascular disease and colon cancer.- Prevent dental caries. - Low calories.Animal nutrition: decrease the number of diseases and reduce useof antibiotics. Effect of temperature and pH of reaction for xylanase activity of A. fumigatusM51 on xylan Carvalho et al Food Technology and Biotechnology, v. 53, p. 1. Carvalho et al Food Research International, v. 51, p Thank you! pedroolivaneto@gmail.com Collaborators:
Dr. Ana Flvia Azevedo Carvalho Postdocstudent CNPq Ms Bruna Escaramboni- PhD student CNPq Ms. Tania Sila Campioni PhD student CAPES Ms. Thas Yumi Shinya PhD student CAPES Fabiane Fernanda de Barros Correa Masters studentCNPq Douglas Fernandes da Silva Doctoral studentCNPq Louise Garbelotti Gonalves-Master student CAPES Franciane Figueiredo Master studentCAPES UNESP