FEDERAL UNIVERSITY OF UBERLÂNDIA

INSTITUTE OF BIOTECHNOLOGY
UNDERGRADUATE COURSE IN BIOTECHNOLOGY
DISCIPLINE: FERMENTATIVE PROCESS TECHNOLOGY

Teacher: Prof. Dr. Juliana de Souza Ferreira (FEQ/UFU)

PRACTICE 1. ALCOHOLIC FERMENTATION

✓ Determine the sugar content of sugary solutions

✓ Evaluate cell growth and determine the kinetic parameters (growth rate, time of
duplication, as well as yield–Y P/Sand in %, and Productivity (Prod) during the process
fermentative using Saccharomyces cerevisiae.

The media can be liquid and solid. Liquids are used when a large number is desired.
cells, being widely used to initiate fermentation or as an inoculum for large scales. As for
production of culture medium (liquid) for yeast propagation it is worth noting that yeast cells,
during the alcoholic fermentation process, they have nutritional needs as multiplication, the
cell growth and the efficiency of sugar conversion into alcohol directly influenced by the
nutrients present in the medium (AMORIM, 2005; RIBEIRO et al., 1987). As a nutritional source, studies
they show the need for nitrogen, since it is essential for growth and multiplication of
yeasts, the use of sugars in the generation of biomass and production of reserve carbohydrates, phosphorus
to integrate ATP molecules, sulfur as a constituent of amino acids, and the influence of ions
minerals (Zn, Co, Mn, Cu) in the enzymatic activity of cells (AMORIN, et al., 1996; BELLUCO, 2001).
According to Camili et al. (2006), the adequate concentration of nutrients has important relevance, and
they can negatively impact the fermentation process. The lack of nutrients can reduce the yield
alcoholic and the cellular viability of yeast.
To monitor the evolution of a fermentation process, it is necessary to determine the
concentration of sugars over time. One technique for such analysis uses refractometry (Index
of refraction or Brix). Refraction is the phenomenon by which, when a light ray passes obliquely
from one medium to another, of different density, it travels in a different direction. The use of such is emphasized

propriedade na identificação de compostos e na determinação de concentrações, em misturas binárias

homogeneous, of known compounds. Brix (symbol °Bx) is a numerical scale that measures the
amount of soluble solids in a sucrose solution. The Brix scale is used in the industry of
foods to measure the approximate amount of sugars in fruit juices, wines, and in the industry of
sugar. The Brix scale, created by Adolf F. Brix (1798 - 1870), was originally derived from the scale of
Balling, recalculating the reference temperature of 15.5 °C. The amount of soluble solid is the total of
all solids dissolved in water, starting with sugar, salt, proteins, acids, etc. The values of

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Measured reading is the sum of all of them. A solution of 25 °Bx has 25 grams of sucrose sugar per 100.
grams of liquid. Or 25 grams of sucrose sugar and 75 grams of water in 100 grams of the solution.
In general terms, fermentation involves the use of microorganisms in the transformation of
organic matter catalyzed by enzymes (MENEZES, 1980). Yeasts are the microorganisms most
important in the production of alcohol through fermentation. The genus Saccharomyces is one of the groups of
microorganisms most studied by the scientific community. This interest is a function of the wide application
these microorganisms in the biotechnology industry. This yeast has been reported as an agent of
transformation since 1800 (ANDRIETTA, STECKELBERG, ANDRIETTA, 2006).
Saccharomyces cerevisiae is a yeast widely used in fermentation processes.
alcoholic, showing the ability to convert the sugar present in the raw material (sucrose) into sugars.
simple and easily fermentable, such as glucose. The reaction of converting sugar (glucose) into ethanol is
represented in a generalized way by the expression below.

C6H12O6 2C2H5OH + 2CO2

Glucose 320C ethanol carbon dioxide

Through this expression, it can be observed that for every mole of sugar (glucose) consumed, two
moles of ethanol and carbon dioxide are produced. Under ideal conditions, the yield of product (ethanol)
Through the consumption of substrate (glucose) it is 0.511 g/g.
In alcoholic fermentation, substrates (sugars) are transformed into products (ethanol), and part
the energy (ATP) generated is used for the production of biomass (cells). Thus, the concentrations of
substrate (sugar), product (ethanol) and cells (yeast) vary with fermentation time. Hiss (2001)
define the variation of these concentrations as the chemical kinetics of the system.

3.1 Practice to be carried out at home by the students

✓ 2 tablespoons of sugar
✓ 1 tablespoon of wheat flour
✓ Water
✓ 2 saches (10 g) de Fermento biológico
✓ Spoons
✓ Stove
✓ 4 PET bottles of 500 ml
✓ 4 rubber balloons (party)
✓ Auxiliary Video 1 -[Link]
✓ Video auxiliary 2 - [Link]
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 3.2 Determination of sugar content (refractometer) (Material used for obtaining data in
laboratory
✓ Hygroscopic cotton;
✓ Samples of alcoholic fermentation;
✓ Banho termostático;
✓ ABBE type bench refractometer;
✓ Sucrose (or glucose);
✓ Vidraria diversa.

3.3 Alcoholic fermentation (Material used for data collection in the laboratory)
✓ Magnetic stirrer
✓ 70% alcohol;
✓ Analytical balance
✓ Centrifuge
✓ Spectrophotometer at 650 nm;
✓ Yeast Saccharomyces cerevisiae;
✓ Culture medium for yeast propagation
✓ Refractometer
✓ Glassware in general (Erlenmeyer, beaker, glass rod, etc).

4.1 Practice to be carried out at the residence by the students

Enumerate the PET bottles knowing what the content will be. Then, dissolve the yeast in 300
mL of warm water, with this solution (water + yeast) being equally distributed in 3
PET bottles. To one of them, add 1 spoon of sugar and to another add 1 spoon of wheat flour. In the
In the 4th flask, put 100 mL of water and add 1 tablespoon of sugar. Then, place a rubber balloon.
In each bottle. Observe the 4 bottles over time and wait until there is no longer any variation in volume.

4.2 Determination of sugar content (refractometer) (Conducted in the laboratory and made available the
results)
[Link]ção: Preparar as soluções de glicose, descritas na Tabela 1, para um volume total de 10 mL.
Attention to the importance of the number of decimal places considered. In reading the values
experimental, as in the angular coefficient of the line, verify that the respective range of variation is
about 0.01.

Table 1–Glucose solution for constructing the calibration curve (Barroso et al)
Solution 1 2 3 4 5 6 7 8 9 10 11 12
Glucose 0 15 30 45 60 75 90 100 218 320 436 544
(g/L)

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 n20D 1,3320 1,3355 1,3370 1,3395 1,3415 1,3440 1,3455 1,3465 1,3645 1,3785 1,3945 1,4085

[Link]

i. Connect the refractometer to a thermostatic bath at a temperature of 20°C.

ii. Open the prism and clean the surface using a paper towel moistened with distilled water;
iii. Add a few drops of distilled water onto the prism and close slowly to avoid any occurrence
formation of air bubbles;
iv. Turn the knob until the line of contact between the dark and light area seen through the eyepiece is at the
brand '0';
v. Open the prism by cleaning it with paper and using a pipette, place a few drops of
sample about the prism;
vi. Wait a few minutes for the liquid to reach thermal equilibrium with the prism;
vii. Slowly look through the ocular for the line of separation between the illuminated and dark regions, using for
this is the angle variation button;
viii. With the separation line very clear, the division between the two regions is positioned exactly in the center.
of the reticulum;

ix. The reading of the refractive index and °BRIX is carried out.

4.3 Fermentation (Conducted in the laboratory and results made available)

a. Microorganism: An industrial strain Y-904 of the yeast S. cerevisiae will be used in granular form.
produced by AB Brasil, in the concentration of inoculum (X0of 10 g/L. The hydration of the yeasts will be
carried out for up to two hours in water at room temperature, using 100 mL of water for each
10 g of cells.
[Link] medium: The culture medium for the yeasts will consist of glucose (150 g/L), KH2PO4(5
g/L), MgSO47H2O (1 g/L), NH4Cl (1.67 g/L), KCl (1 g/L) and yeast extract (6 g/L). Adjust the pH
for 4,5 with solution 1 M hydrochloric acid (HCl). The reagents to be used will all be of grade
analytical.
[Link] unit: The fermentation process will be conducted in a bench-top conical reactor.
(erlenmeyer), munidos de rolhas de borracha perfuradas, contendo um tubo de conexão acoplado a
centrifugation microtubes at each of their ends, with the outer microtube
filled with glycerol to allow for CO exhaustion2, avoiding air entry. It will be used
Erlenmeyer with a total volume of 250 mL and a usable volume of 150 mL, operated in batch mode. The volume of
inoculum will represent 10% of the usable volume and the culture medium 90%. After the inoculation of a

yeast suspension (X0= 10 g/L), this system will be initially weighed and kept under stirring in
magnetic stirrer at ~32 ºC. Every 30 minutes, the cell concentration, substrate, and will be evaluated
ethanol according to the procedures described below.
d. Determination of cell concentration: The cell concentration will be evaluated by turbidimetry. A
a aliquot of 1 mL of the sample (cell suspension) will be diluted 50x (use a volumetric flask) and its

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 absorbance measured at 650 nm. The cell concentration will be determined with the help of a curve
of calibration (cell concentration versus Absorbance) previously constructed (Annex 1).
e. Determination of glucose concentration: The determination of glucose will be carried out through the curve of
calibration (refractive index versus glucose concentration) established from the results
obtained from the experimental procedure described in item 4.2. 10 mL of the broth will be aliquoted
fermentation and centrifugation (5000 rpm for 15 min). After that, the refractive index of the supernatant
obtained from centrifugation will be determined and correlated with the concentration of glucose (g/L).
f. Determination of ethanol concentration: The concentration of ethanol formed will be determined by
gravimetry, evaluated from the volume of CO2released during the fermentation process with assistance
from an analytical balance. It is observed from the stoichiometry of the fermentation reaction (Eq. 1) that the

the amount of ethanol produced is directly related to the amount of CO2released. Soon,
evaluating the evolution of CO2(variation of reactor mass in grams) and dividing this value by the
volume of broth (in L) at a given moment (Vn) it is possible to estimate the value of the CO concentration2for
Eq. (2) is of ethanol Eq. (3).

PCO2(g/L)=change in mass / Vn (2)

Pethanol(g/L) = 92 * PCO2/88 or 46 * PCO2/44 (3)

[Link] of conversion factors from substrate to product (YP/SThe conversion factor of substrate into
the product will be calculated using Equation 4. In which Pf is the final concentration of ethanol (g/L) and P0é
initial concentration of ethanol (g / L). Sfit is the concentration of final substrate (gsubstrate/L), S0 is the
initial substrate concentration (gsubstrate/L).

(4)
h. Determination of the maximum specific growth rate (μ)maxThe maximum specific rate of
growth (µmax) will be obtained by linear regression from Equation 5 in which X0and X represents the
cell concentration at the initial time and at time t, respectively. The generation time (tg) will be
calculated by Equation 6 (Bailey and Ollis, 1986).

(5)

ln ( 2 )
t g=
max
(6)

[Link] of volumetric productivity (Productivity): The productivity of ethanol will be calculated

through Equation 7. In which PfIt is the final concentration of ethanol (g/L) and P0is initial concentration of
ethanol (g / L). Vmeio is the useful volume, that is, of the fermentation medium (L), t is the time of

fermentation (h).
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 (7)

a) Make records (photo/video) of the rehearsal done at home. Analyze qualitatively the results obtained.
and to discuss the observed phenomena.
b) Build calibration curves for determining sugar content using the refractometer from
data provided by the teacher (Table 1) (Quantitative part).
c) Build the curve showing the variation in the concentration of glucose, ethanol, and cells over time
and analyze regarding the phases of cell growth and the classification of the product according to Garden and
Ludecking-Piret equation.
d) Determine the maximum specific growth rate (µ)max) and the cell doubling time (tg),
as well as the biomass to ethanol conversion factor (YP/S), yield (%) and productivity.
e) Evaluate the total ethanol production, and compare it with literature data

6. Bibliography
AMORIM, H. V. Ethanol production in a petroleum dependent world: the Brazilian Experience. Sugar Journal, v. 67, p.
November 14, 2005.

AMORIM, H.V., BASSO, L.C., ALVES, D.M.G. Processos de produção de álcool–Controle e monitoramento,
Fermentec Publishing/FEALQ/ESALQ- USP, Piracicaba, São Paulo, 103 p, 1996.
ANDRIETTA, S. R.; STECKELBERG, C.; ANDRIETTA, M. G. S. Bioethanol–Brazil, 30 years at the forefront.
Multiscience, University of Campinas, 2006.
BAILEY J. E.; OLLIS D. F. Bichemical Engineering Fundamentals. Second edition. New York, 1986.
BARROSO, M., PINEIRO, M., SILVA SERRA, E., SEIXAS DE MELO, J. Refractometry in the determination of the content of
sugar and aspartame in commercial beverages. Department of Chemistry at the University of Coimbra, Rua Larga,
3049-535 Coimbra, Portugal, p.61-64.
BELLUCO, A. E. S. Physiological and compositional changes in Saccharomyces cerevisiae under non-...
proliferating. Thesis (Doctorate, Luiz de Queiroz College of Agriculture), University of São Paulo,
Piracicaba, Brazil, 2001.
BORZANI, W., SCHMIDEL, W., LIMA, U.A., Aquarone, E. Industrial Biotechnology - Fundamentals. Vol. 01. Publisher
Edgard Blücher Ltda. 2001.
CAMILI, E. A.; CABELLO, C.; CARDOSO, M. G. Production of ethanol treated with flotation process. 2nd ed.
Lavras: UFLA, 2006.
HISS, H. Kinetics of fermentation processes In: SCHMIDELL, W.; LIMA, U.A.; AQUARONE, E.; BORZANI, W.R
(Coord.) Industrial Biotechnology. São Paulo: Edgard Blücherp, 93-122, 2001.
MENEZES, T. J. B. Etanol, o combustível do Brasil. São Paulo: Editora Agronômica Ceres Ltda., p. 141-178, 1980.
Refractometry, Institute of Chemistry–UFG.
RIBEIRO, E. J., LOPES, J.J.C., FERRARI, S.E. Continuous nitrogen supplementation in the process of
alcoholic fermentation, Brazil Sugar Industry, v.105, n.1, p. 26-30, 1987.

ANNEX 1: Calibration curve for the determination of cell concentration.

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