INTERNATIONAL CONGRESS AND EXPO
ON BIOFUELS - 2015
Brazilian technology of fuel ethanol fermentation:
new perspectives to improve the technology and
diversification.
Dr. Pedro de Oliva Neto
Lab. Industrial Biotechnology and UNESP Bioenergy Institute .
Faculdade de Ciências e Letras
Universidade Estadual Paulista – UNESP – Campus Assis
São Paulo State - Brazil.
Email: pedroolivaneto@gmail.com.br
OVERVIEW OF BRAZILIANS FUEL ETHANOL
DISTILLERIES

- Some current numbers of Ethanol Industry:
The estimatation for the 2014/15 crop:
-
Cane Crop – 653 milh. Ton. (2% increase based on the last crop)
-
Sugar cane - 35.7 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 fermentation are:
Ethanol efficiency (conversion of sugar to ethanol) - 90% to 92% ;

Ethanol titles for fermented must : 8.0-10º GL;

Fermentation times : 6 to 11 hours – 8 hs in a Fed-Batch process (more predominant process)

Yeast concentration in the fermented must: 12-13% v /v;

Final volume of liquid residue after distillation: 12-15 liters / liter of ethanol.

Source: CONAB
Brazil
FLOWCHART OF BRAZILIAN ETHANOL AND SUGAR INDUSTRY
Milling
Cane washing
Alkaline water (pH 11)
Filtration (Static or Rotary
vacuum cane mud) Filtered
Broth return to clarification e
Mud (fertilizer)
Pre-heating (70oC)
Bagasse (Burning in the
boiler - Energy, steam)
Heat treatment and decanting
(Decanter 105oC/2 h)
Clarification (SO2 and CaO
addition)
Phosphating
Sugar manufacture
Clarified broth
Molasse (by-product of
sugar manufacture)
Dilution water and/or Clarified sugarcane juice
Preparation of Wort
(18-22o Brix - 30oC.)
Yeast Acidification (pH 2.5)
Hydro-alcoholic solution
Distillation
Acidification (H2SO4) of
Yeast cells suspension return to fermentation
Centrifugation
Fermentation vat (pH 3.84.5, 32-34oC) (Fed-Batch,
Continuous, Conbat or
Batcon process)
Fermented broth (Yeast
10-14%, ethanol 7.5-10%
Residual sugar < 0.1%)
Yeast cell suspension (40-80% wet
mass)
Oliva-Neto et al 2013. The Brazilian technology of fuel ethanol fermentation - yeast inhibition factors and new perspectives
to improve the technology. In: A. Méndez-Vilas. (Org.). Materials and processes for energy: communicating current research
and technological developments. 1 ed. Badajoz: Formatex, 2013, v. 1, p. 371-379.









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 ethanolic yield by sugar consume and yeast flocculation.
Bacteria: The most important genus - Lactobacillus, Bacillus and Leuconostoc.
species: Lactobacillus fermentum, L. plantarum
The most important
Problems: Sugar consumption producing lactic acid which decrease the yeast
viability. Increase of yeast flocculation causing the yeast settling at the bottom of
vats, and cell loss in centrifuges further contributing to the reduction in the ethanol
yield.
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.
OLIVA-NETO, P.; YOKOYA, F. Effects of nutricional factors on growth of Lactobacillus fermentum mixed
with 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. São Paulo. v. 22, p. 21-27, 1991.
ABIOTIC INHIBITORS







OF YEAST FERMENTATION
There important abiotic inhibitors are :
a) The highest ethanol concentration used is 10% (v/v), The enzymes alcohol
dehydrogenase 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 in
chemical maintenance functions of the cells, such as nutrient intake.
Lactic acid shows inhibitory property in high concentrations (6-40 g/L) (Maiorella et al
1987, Oliva-Neto & Yokoya, 1994)
pH in the industrial fermentation should be maintained higher than 4.0. Lower pH
acting in a synergistic effect with other inhibitors and they affect the proton pump and
other cellular functions of S. cerevisiae. (Dorta et al. 2006)
c) Sulphite – Maximum level – 50 – 100 mg/L to avoid inhibition on the metabolism of
sugar consumption. Sodium sulphite in the cane molasses - 200 to 700 mg/L, and in the
wort up to 300 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.) for S.cerevisiae is 5000 mg/L (OlivaNeto & 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 alcoholic fermentation of
S. cerevisiae (PE-2 and M-26). World Journal of Microbiology & Biotechnology, England, v. 22, p. 177-182, 2006
Formulation of the fermentative media with stress factors: ethanol, lactic acid, sulphite and pH
______________________________________________________________________
sulfite (mg/L )
latic acid
ethanol *
pH
toxicity
Medium ToC
(NaHSO3)
(g/L)
(%)
level
______________________________________________________________________
1
32
200
6.0
9.5
3.6
maximum
2
32
50
6.0
9.5
3.6
low sulfite
3
32
200
2.0
9.5
3.6
low lactic ac.
4
32
200
6.0
7.5
3.6
low ethanol
5
32
200
6.0
9.5
4.5
normal pH
6
32
0
0.0
7.5
4.5
control
____________________________________________________________________
* Sucrose at concentration of 16.37% or 20.65% (w/v) were used as carbon
source;
Cells morphology A - pH 4.5 – M 5 B - pH 3.6 –M1
Yeast budding and viabilitiy, residual protein and ethanolic yield in in medium after different fermentation conditions by
Saccharomyces cerevisiae PE-2 ( ) and M-26 ( ).
35
70
30
60
Total turbidity average (600nm)
Total turbidity average (600nm)
S. cerevisiae cells flocculation by Lactobacillus fermentum
25
20
15
10
50
40
30
20
10
5
0
0
2
2
2,5
2,7
3
3,5
4
2,5
2,7
3
4,5
3,5
4
4,5
5
pH
pH
0
0
175 min.
1360 min.
175 min.
pH determination of 4650 UE Protease Novozyme 642 in deflocculation of
Saccharomyces cerevisiae flocculated by Lactobacillus fermentum CCT 1396.
pH determination of Carbohydrase SP 299 in deflocculation of Saccharomyces
cerevisiae flocculated by Lactobacillus fermentum CCT 1396.
Quantification of the yeast flocculation from induction by L. fermentum CCT 1396,
after treatment with different concentrations of proteases and carbohydrases
Flocs of S.cerevisiae and Lactobacillus
Source: Fermentec
Ludwig et al 2001 Rev. Soc. Brasileira Ciência e Tecnologia de
Alimentos , v. 21, 1, p. 63-68.
5,5
The increase of yeast
flocculation by the increase
of L. fermentum
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 2015 Enzyme Research , v. 2015, Article ID 573721
Development of new chemicals for 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 1
S.cerevisiae 2
L. fermentum1
L. fermentum 2
CCT 2652
FCLA M26
CCT 0559
CCT 1396
______________________________________________________________________________________
TCC+CBe1
>12.5
12.5
6.25
6.25
TCC+CBe2
>12.5
>12.5
3.12
3.12
TCC+CBe3
>12.5
>12.5
6.25
6.25
TCC+CBa 5:12
>12.5
>12.5
6.25
6.25
TCC+CBa 2.5:12
>12.5
12.5
3,12
3,12
TCC+CBa 1:12
>12.5
12.5
1.56
1.56
TCC+ CBa 2.5:11
>12.5
>12.5
12.5
12.5
HJ Kamoran1
>0.312
>0.312
0.156
0.078
HJ Kamoran2
>0.625
>0.625
0.312
0.156
_______________________________________________________________________________________
Symbols: TCC - 3,4,4’ trichlorocarbanilide, CBe - benzethonium chloride, CBa - benzalkonium chloride, CTA – Cetyl
trimethyl ammonium chloride. Hj Kamoran – commercial product (antibiotic Monensin). 1 - autoclavated product,
culture in pH 6.0 for L.fermentum, 2 – microfiltered product, culture in pH 6.0 for L.fermentum, 3 – microfiltered
product, culture in pH 4.0 for L.fermentum .
-
Oliva-Neto et al. 2014. Brazilian Archives of Biology and Technology. v. 57 (3), p. 441-447, 2014.
Diversification of subtrates for ethanol fermentation:
Search for more feedstocks and carbohydrases
Amylases production 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: 30◦C, 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 50°C.
Ethanolic efficiency from hydrolysis of cassava
residue using R. oligosporus enzymes = 80%
Freitas et al 2014 Chemical Papers, v. 68 (4) p. 442-450.
Cellulases and Polygalacturonases produced in a Citrus residue (citrus
pulp) culture medium in a cell recycle process
A
FPU /g Substrate
8
6
4
2
0
1
2
3
4
Cycle
B
B
FPU / mL)
0.80
0.60
0.40
0.20
0.00
1
2
3
4
Cycle
A. niger
Poligalacturonase 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
T. reesei
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 enzyme
biotechnology. 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 use of antibiotics.
Effect of temperature and pH of reaction for xylanase activity of A.
fumigatus M51 on xylan
Carvalho et al 2015. Food Technology and
Biotechnology, v. 53, p. 1.
Carvalho et al 2013 Food Research
International, v. 51, p. 75-85.
Collaborators:
Dr. Ana Flávia Azevedo Carvalho – Postdoc student CNPq
Ms Bruna Escaramboni - PhD student CNPq
Ms. Tania Sila Campioni – PhD student CAPES
Ms. Thaís Yumi Shinya – PhD student CAPES
Fabiane Fernanda de Barros Correa – Masters student CNPq
Douglas Fernandes da Silva – Doctoral student CNPq
Louise Garbelotti Gonçalves - Master student CAPES
Franciane Figueiredo – Master student CAPES
UNESP