International Congress and Expo on Biofuels
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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
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