International Congress and Expo on  - PowerPoint Presentation

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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 NetoLab. 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 Oliva-Neto et al 2013. The Brazilian technology of fuel e

thanol 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.

Molasse

(by-product of sugar manufacture)

Hydro-alcoholic solution  Fermentation vat (pH 3.8-4.5, 32-34oC) (Fed-Batch, Continuous, Conbat or Batcon process)

Clarification (

SO2 and CaO addition)

Filtration

(Static or Rotary vacuum cane mud) Filtered Broth return to clarification e

Phosphating

Heat

treatment and decanting

(Decanter 105

o

C/2 h)

Clarified broth

Sugar manufacture

Cane washing

Milling

Alkaline water (pH 11)

Pre-heating (70

o

C)

Bagasse (Burning in the boiler - Energy, steam)

Mud

(fertilizer)

Dilution water and/or Clarified sugarcane juice

Preparation of

Wort

(

18-22

o

Brix - 30

o

C.)

Yeast

Acidification

(pH 2.5)

Fermented

broth

(Yeast 10-14%, ethanol

7.5-10%

Residual sugar < 0.1%)

Centrifugation

Distillation

Yeast

cell suspension (40-80% wet mass)

Acidification

(H

2

SO

4

) of

Yeast cells suspension

-return

to fermentation

Important inhibitors of industrial fuel ethanol

fermentationBiological 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. The most important species: Lactobacillus fermentum, L. plantarum 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,

1997YOKOYA

, 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 (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 alcoholic fermentation of S. cerevisiae (PE-2 and M-26). World Journal of Microbiology & Biotechnology, England, v. 22, p. 177-182, 2006Formulation of the

fermentative media with stress factors: ethanol, lactic acid, sulphite

and

pH

______________________________________________________________________ sulfite (mg/L ) latic acid ethanol * pH toxicityMedium 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 Cells morphology A - pH 4.5 – M 5

B - pH 3.6 –M1 source;

Yeast budding and viabilitiy, residual protein and

ethanolic

yield in in medium after different fermentation conditions by Saccharomyces

cerevisiae

PE-2 ( ) and M-26 ( ).

S. cerevisiae

cells flocculation by Lactobacillus fermentum

Quantification of the yeast flocculation from induction by

L.

fermentum

CCT 1396, after treatment with different concentrations of proteases and carbohydrases

The

increase of yeast flocculation

by

the

increase

of

L.

fermentum

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.

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 recoverySILVA et al 2015 Enzyme Research , v. 2015, Article ID 573721

Development of new chemicals for

control of microbial infection in fuel ethanol fermentation.Oliva-Neto et al. 2014. Brazilian Archives of Biology and Technology. v. 57 (3), p. 441-447, 2014.

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+CBe

1

>12.5 12.5 6.25 6.25

TCC+CBe

2

>12.5 >12.5 3.12 3.12

TCC+CBe

3

>12.5 >12.5 6.25 6.25

TCC+CBa

5:1

2

>12.5 >12.5 6.25 6.25

TCC+CBa

2.5:1

2

>12.5 12.5 3,12 3,12

TCC+CBa

1:1

2

>12.5 12.5 1.56 1.56

TCC+

CBa

2.5:1

1

>12.5 >12.5 12.5 12.5

HJ Kamoran

1

>0.312 >0.312 0.156 0.078

HJ Kamoran

2

>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 .

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 substrateEthanolic efficiency from

hydrolysis of cassava residue using

R.

oligosporus

enzymes = 80% Diversification of subtrates for ethanol fermentation: Search for more feedstocks and carbohydrases Enzymatic reaction using enzyme produced from R. oligosporus (1.25 U/mL) in 5% and 10% (w/v) starch solution at 50°C.

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

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. 2014BA

B

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 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

- 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. XOS are Prebiotic: sugar oligomers that can not be digested by animals and humans. Advantages of prebiotics:Production of xylo-oligosacharides from bagasse

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.

Thank you!

pedroolivaneto@gmail.com

UNESP

Collaborators

:Dr. Ana Flávia Azevedo Carvalho – Postdoc student CNPqMs Bruna Escaramboni - PhD student CNPqMs. Tania Sila Campioni – PhD student CAPESMs. 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