3.2.

Pre-treatment
Pre-treatment's main objective is to break down lignin polymers that protect cellulose and
hemicellulose polymers present in the lignocellulosic material, and consequently increase the
accessibility of enzymes for the enzymatic hydrolysis of cellulose and hemicellulose.
In this work an acid pre-treatment of the spent coffee grounds (SCG) was applied. For this
purpose, it was weighed 10 g of SCG into conical flasks and added to each one of them, 200
mL of H2SO4 (Analar Normapur), HCl (commercial), HNO3 (Sigma-Aldrich), CH3COOH (Analar
Normapur), with various different concentrations.
Since acetic acid is a weak acid, compared to the other, an additional test was carried out with
higher concentration (7% v/v).
The reaction occurred in a thermostatic bath (Selecta, Unitronic OR-C) and the experimental
conditions used are described briefly in Table 3.1.
Table 3.1 - Operating conditions for the pre-treatment (10 g of SCG)

Acid
concentration
(% v/v)

Acid

1%
3%
5%

H2SO4
HCl
HNO3
CH3COOH

7%

CH3COOH

Acid
volume
(mL)

Temperature
(C)

Reaction
time
(min)

Stirring
speed
(rpm)

200

85

270

50

In order to analyse the efficiency of pre-treatment in the production of sugars, it was conducted
a test on SCG under the same operating conditions, but replacing the acid volume with
deionized water.
Two replicates of each test were performed. In Figure 3.7 it can be observed the appearance
of the samples before and after pre-treatment.
Before

After
Figure 3.7 - Appearance of the samples before and after pre-treatment

3.3. Enzymatic hydrolysis

To promote an efficient action of the enzyme used, pH of the pre-treatment assays was set to
5 (Figure 3.8).

Figure 3.8 - Adjustment of pH after pre-treatment

Then it was added the enzyme Viscozyme L (Novozymes), whose characteristics indicated by
the supplier are shown in Table 3.2, at a ratio of 100 L / g of SCG, and the flasks were placed
in a thermostatic bath (Selecta, Unitronic OR-C) for 120 minutes at a temperature of 50 C and
50 rpm (Figure 3.9), corresponding to the same conditions used by Silva (2011).

Figure 3.9 - Thermostatic bath used for enzymatic hydrolysis

Table 3.2 - Main features of Viscozyme L enzyme, according to its supplier, Novozymes

Physical state

Composition

Activity

Lquido

- glucanase
xylanase
cellulase
hemicellulase

100
FBG/g

Temperature
range (C)

40 - 55

pH

Group

4.0 6.0

-1-3 and -1-4

endoglucanases

FBG Fungal -glucanase

After hydrolysis, vacuum filtration (Neuberger) of the wort was performed and the samples
were frozen, in order to prevent sugar degradation (Figure 3.10).

Figure 3.10 - Vacuum filtration of samples after pre-treatment and enzymatic hydrolysis and respective
storage

3.4. Methods used for the quantification of sugars

For the determination of sugars, it was used different procedures that will be described.
3.4.1. 3,5-dinitrosalicilic acid method (DNS)
To determine the content of reducing sugars, it was used the methodology described by Miller
(1959) using the 3,5-dinitrosalicilic acid (DNS). This method is based on the reduction of DNS
(yellow color) to 3-amino,5-nitrosalicilic acid (orange color) and, simultaneously, the oxidation
of the free carbonyl group (C=O) that is present in the reducing sugars.
The concentration of reducing sugars was determined by absorbance reading ( = 540 nm)
using a UV-Vis spectrophotometer (Figure 3.11, Shimadzu, UV-160 A), using the previously
drawn calibration curve, using standards prepared with glucose at concentrations of 0.10 to
0.60 g/L (Annex A.2.1).

Figure 3.11 - UV-Vis spectrophotometer used in DNS method

3.4.2. Refractometry method in Brix scale

Refraction is generated by changing the direction of a beam of light, when it changes from one
medium to another, and can be measured in Brix scale.
The Brix corresponds to the numeric scale of refractive index and determines the amount of
soluble solids in a given sample, often being used to estimate the amount of total sugars
present in that sample.

The tests were performed using the direct method (NP EN 12143:1999), reading the Brix in
the Abbe refractometer (Atago, Figure 3.12) and, using a calibration curve prepared by reading
the Brix of standard solutions of glucose with concentrations of between 3.2 and 130 g/L, it
was determined the concentration of glucose in the samples.

Figure 3.12 - Apparatus used in the refractometer method

Once the temperature of the bath of the Abbe refractometer varied between readings, the
values measured were corrected to 20 C, corresponding to the reference temperature
(MAPA_Br, s/d).
3.4.3. Method of high performance liquid chromatography
High-performance liquid chromatography (HPLC) has as its basic principle the separation of
different chemical species through selective interaction between the molecules of the sample
(solute) and two phases - the mobile and stationary - and has been used as a method to
separate, identify, and quantify compounds.
Basically the equipment used to perform such separations consists of:

column - that contains the stationary phase, consisting of small particles of filling
material;

pump - that allows the movement of the mobile phase and sample through the column;

detector - that responds to the presence of a certain species, and indicates the retention
times of the molecules due to sample interactions between the stationary phase and
the mobile phase.

The chromatographic separation is achieved due to the varying degrees of affinity of

substances present in the sample, to the mobile and stationary phases. The compound with
higher affinity for the stationary phase is the last to elute, unlike the one that has the lower
affinity, which is the first to elute, therefore, it allows one to identify different retention times for
each compound (Chust, 1990).

In this method a HPLC (Gilson, Figure 3.13) was used. It was composed by an injection valve
(Gilson, 231XL) equipped with a 20 L sample loop and a pump (Gilson 307). The column
used was a Knauer Eurospher II 100, NH2, 5 m, 250 x 4.6 mm, with pre column. It was used
at room temperature and the detector was of type Evaporative Light Scattering (ELSD)
(Polymer Laboratories, PLEMD 960), operated with an air flow rate of 7.2 L/min, at 70 C, with
attenuation of 1 and a time constant of 1 s. The resulting chromatogram of samples was traced
by an integrator (Shimadzu, C-R3A Chromatopac) with slope of 7500 and attenuation of 6.
Column

Detector

Eluent

Injection
valve

Pump
Integrator

Figure 3.1 - HPLC used in the determination of sugars

The flow rate of the mobile phase (eluent), consisting of Acetonitrile and ultrapure water with
80:20 (v/v) ratio, was 0.60 mL/min, with a column pressure of 28 bar, that increased to 32 bar
along the carrying out of the measurements. Compressed air was used in order to vaporize
the eluent in the detector.
Prior to manual injection, each sample was filtered with a 0.2 m pore syringe nylon filter. The
total time of each analysis was 40 to 45 minutes.
Initially it was prepared, from standard solutions, the calibration curves of glucose, arabinose,
and xylose. From the retention time of each one of the standards, it was possible to identify, in
the chromatogram of the real sample, the compounds of interest and then to quantify it by
using the previously prepared calibration curve.

A.2. Quantification of sugars

A.2.1. DNS method
The reducing sugars, as previously described, were determined using the DNS method, which
is described below.
Material:

Spectrophotometer UV-Vis (Shimadzu, UV-160 A), = 540 nm;

Vortex (2 Genie, Scientific Industries);

Cuvettes PMMA;

Heating plate (Selecta);

Screw vial;

Micropipette;

Gobelets;

Graduated pipette 10 mL.

Reagents/Solutions:

3,5-dinitrosalicilic acid solution and Rochelle salt (DNS);

Deionized water;

Solution obtained after pre-treatment and enzymatic hydrolysis of SCG.

Procedure:

Defrost the samples and mix using a vortex;

If necessary, dilute the samples with deionized water;

Put in a screw test tube 1 mL of sample and add 1 mL of DNS solution;

Stir in vortex for approximately 10 seconds;

Put the mixture in a boiling water bath for 5 minutes;

Add 10 mL of deionized water, after fast cooling of the tubes;

Stir again in the vortex;

Read absorbance at 540 nm and using the calibration curve previously prepared,
determine the concentration of total reducing sugars.

Figure A.6 - Evolution of color along the boiling time, due to reduction of DNS

A.2.2. Refractometry method

Total sugars concentration was determined by refractometry using the procedure described
below.
Material:

Abbe refractometer (Atago);

Pasteur pipettes;

Cotton.

Reagents/Solutions:

Solution obtained after pre-treatment and enzymatic hydrolysis of SCG;

Ethyl alcohol (commercial grade).

Procedure:

Defrost the samples and mix using vortex;

Connect the thermostatic bath control equipment. Register the temperature between
the performed readings;

Turn on the refractometer and check whether the lighting is operating;

Carefully clean with cotton wool, soaked in ethyl alcohol, the surface of the two prisms
and until it evaporates;

With the aid of a Pasteur pipette pour a drop of sample on the primary prism surface,
in order to form a thin film. The amount used should be enough to allow to distinguish
through the eyepiece, two hemispheres in the field of light - a light and a dark one;

Use the knobs in order to adjust the line that separates the hemispheres, with the cross
of the screen itself and the sharpness of the image reproduced;

Read the refractive index in Brix. Adjust the readings by the effects of the acidity and
to the temperature of 20 C and determine the concentration of the sugars present in
the sample with the aid of a previously prepared calibration curve.

A.2.3. Method of high performance liquid chromatography (HPLC)

The concentration of xylose, arabinose and glucose was determined by HPLC. Corresponding
procedure is described below.
Material:

HPLC and integrator;

Syringe;

Nylon filters.

Reagents/Solutions:

Solution obtained after pre-treatment and enzymatic hydrolysis of SCG;

Ultrapure water;

Acetonitrile.

Procedure:

Defrost the samples and mix using a vortex;

If necessary, dilute the samples with deionized water;

Turn on the equipment and follow the instructions for use, programming to the desired
operating conditions;

Use a syringe to collect a certain volume of sample and pass the sample through a
nylon filter;

After filtering, inject the sample manually in the equipment and wait for the
chromatogram result;

From the chromatogram obtained and using the retention time, identify the compounds
and calculate the corresponding concentration, using the calibration curve, previously
prepared.

From: Gomes, DSJ. 2016. Estudo do Pr-Tratamento cido e Hidrlise Enzimtica da

Borra de Caf Visando a Produo de Bioetanol. Dissertao do Mestrado em
Engenharia Qumica Tecnologias de Proteo Ambiental. DEQ/ISEP. November 2016.