Actas de Ingeniería

Volumen 3, pp. 173-179, 2017

http://fundacioniai.org/actas

Determination of reducing sugars in extracts of Undaria pinnatifida (harvey) algae by UV-visible

spectrophotometry (DNS method)

Determinación de azúcares reductores en extractos de alga Undaria pinnatifida (harvey) por

espectrofotometría UV-visible (método DNS)

Marisa Garriga1, Melisa Almaraz2, Alicia Marchiaro3

1mgarriga@unpata.edu.ar, 2almarazmelis@hotmail.com, 3aliciam@unpata.edu.ar

Universidad Nacional de la Patagonia San Juan Bosco

Comodoro Rivadavia – Argentina

Artículo de Investigación

Abstract
Recent advances in the conversion of carbohydrates from algae biomass into liquid biofuels, such as bioethanol, have
demonstrated the potential of algae as a promising but relatively unexplored source of biofuels. Since the yield of conversion
to ethanol is functional to the saccharification process, it is of vital importance to have an appropriate analytical method for
the determination of reducing sugars obtained in said process. The aim of this work was to validate dinitrosalicylic acid
method (DNS) for the determination of reducing sugars in seaweed Undaria pinnatifida by ultraviolet spectrophotometry.
The validation plan included the evaluation of the following parameters: linearity, detection and quantification limits,
accuracy, precision, selectivity and robustness. Linearity were verified in a range of 0.28 to 1.00 g/L (r = 0.993; R 2 = 0.983;
CVs of the response factors lower than 5 %). Detection and quantification limits were 0.004 and 0.012 g/L, respectively. In
the accuracy study, over the range 0.30 to 0.90 g/L glucose concentration, high recoveries were reached (recovery average
97.741 %). Good results were obtained in the repeatability and precision studies (CV < 2 %). The selectivity study showed no
interferences regarding the determination of reducing sugars when formaldehyde was used instead of algae extract. Method
robustness was also verified, the analysis demonstrated that the method is robust to variations in the variables analyzed,
except to the pH changes. This study demonstrated the DNS method was selective, linear, accurate and robust, although
sensitive to the pH variations.
Keywords: validation; DNS method; reducing sugars; Undaria pinnatifida.

Resumen
Recent advances in the conversion of carbohydrates from algae biomass into liquid biofuels, such as bioethanol, have
demonstrated the potential of algae as a promising but relatively unexplored source of biofuels. Since the yield of
conversion to ethanol is functional to the saccharification process, it is of vital importance to have an appropriate
analytical method for the determination of reducing sugars obtained in said process. The aim of this work was to validate
dinitrosalicylic acid method (DNS) for the determination of reducing sugars in seaweed Undaria pinnatifida by ultraviolet
spectrophotometry. The validation plan included the evaluation of the following parameters: linearity, detection and
quantification limits, accuracy, precision, selectivity and robustness. Linearity were verified in a range of 0.28 to 1.00 g/L
(r = 0.993; R2 = 0.983; CVs of the response factors lower than 5%). Detection and quantification limits were 0.004 and
0.012 g/L, respectively. In the accuracy study, over the range 0.30 to 0.90 g/L glucose concentration, high recoveries were
reached (recovery average 97.741%). Good results were obtained in the repeatability and precision studies (CV < 2%).
The selectivity study showed no interferences regarding the determination of reducing sugars when formaldehyde was
used instead of algae extract. Method robustness was also verified, the analysis demonstrated that the method is robust
to variations in the variables analyzed, except to the pH changes.
Palabras clave: validación; Método DNS; reduciendo azúcares; Undaria pinnatifida.

© 2017. IAI All rights reserved

173
1. Introduction
As global energy demand continues rising and fossil
resources are depleted, biomass is emerging as one of the
most important energy sources in the near future. The
biomass provides a number of local environmental gains
[1-3]. The thermochemical transformation of biomass
generates gases, liquids and solid fuels. Among all the
procedures used to transform biomass into useful
product, liquefaction, pyrolysis and gasification are the
most appropriate methods [4]. Biomass is a renewable
energy resource providing transportation fuels such as
biothanol or biodiesel [5, 6]. The benefits of biofuels over
traditional fuels include greater energy security, reduced
environmental impact, foreign exchange savings, and
socioeconomic issues [7, 8].
Bioethanol has been considered an important
substitute for replacing liquid fuels of fossil origin [9];
additionally, it finds application in diesel engines, as
additive of the gasolines and in fuel cells. The
technologies available for the production of first
generation bioethanol, based on biomass used for food
production, are accessible and widespread but the
associated cost of raw materials is high: food price
increase or shortages of them, reduction of agricultural
biodiversity, erosion and soil contamination with
fertilizers and pesticides [10-15]. The alternatives for the
production of second generation bioethanol,
lignocellulosic biomass, are not currently profitable
options due of degrading the lignin associated with
cellulose; despite they present advantages as the low cost
of raw materials, minimal land use change, and
avoidance of the competition between food and fuel.
Given this situation, algae are presented as an alternative
source of biomass for the production of third generation
fuels.
They constitute a renewable and abundant resource,
can produce carbohydrates, lipids and proteins in a short
period of time, which can be transformed into biofuels;
they lack lignocellulosic material, which facilitates the
chemical and enzymatic pre-treatment to degrade these
materials to fermentable sugars and their processing
generates value-added co-products; have a higher rate of
carbon dioxide fixation compared to terrestrial biomass,
they may have greater potential for carbon dioxide
remediation [16, 17].
Recent advances in the conversion of carbohydrates
from algae biomass into liquid biofuels, such as
bioethanol, have demonstrated the potential of algae as a
promising but relatively unexplored source of biofuels
[18]. The carbohydrate contents of seaweed are in a
range of 30-70%. It depends on the species and culture
conditions [19].
In the region of the Gulf San Jorge, Patagonia
Argentina there are about 178 species of green, brown
and red algae [20]; in particular, the kelp Undaria
pinnatifida (Phaeophyceae) is an invasive brown
seaweed, native to northeastern Asia, which was
introduced in Argentina through international ships in
the Golfo Nuevo region, and from there has been
progressively extended along the Patagonian coast [21,
22] negatively impacting the biodiversity of marine
species. Through a manipulative experiment involving
Undaria removal in 2001, was found that its presence is
associated with a dramatic decrease in species richness
and diversity of native seaweeds in Golfo Nuevo. Future
prospects are worrisome, as, in addition to the negative
impact from a biodiversity viewpoint, native commercial
macroalgae and invertebrates might also be posh [23].
Carbohydrates in brown seaweed consist of alginate,
laminaran, fucoidan and mannitol [24]. Carbohydrate
content in U. pinnatifida is 52% carbohydrate including
crude fiber as cellulose on dry solid basis [19].
The critical step in the conversion of this
carbohydrates to ethanol involves the degradation of the
polysaccharides to fermentable sugars in a process called
saccharification. The goals of the research have focused
on developing pre-treatments of the biomass in order to
make this saccharification easier and thus achieve a
higher yield in the process. The main objective of pre-
treatment is to decrease the degree of crystallinity of the
polysaccharide mesh that forms the cell wall and thus
make them more susceptible to saccharification [25].
One study found that seaweed carbohydrates from all
three classes of macroalgae (brown, red, and green) can
be effectively hydrolyzed to monosaccharides by dilute
H2SO4 treatment at high temperature [26]. Since the yield
of conversion to ethanol is functional to the
saccharification process, it is of vital importance to have
an appropriate analytical method for the determination
of reducing sugars obtained in said process. One of the
most widespread analytical techniques for the
quantification of reducing sugars is that developed by
Sumner [27-29] with various co-worker and later
modified by Miller [30].
With the passage of time and the dizzying
development of technology, after almost 50 years, this
technique has undergone some modifications, although
in essence it remains the same. When applying old
methodologies, it is advisable to carry out a retrospective
validation of the method in order to demonstrate its
suitability in the application in which it is intended to be
used.
ISO 9000 [31] defines validation as the "confirmation,
through the provision of objective evidence, that the
requirements for a specific intended use or application
have been fulfilled". The validation of an analytical
method is a fundamental step to ensure that the method
meets the requirements for the specific intended use or
application and that the results delivered by that method
are reliable. When the validation of a method is
performed, it is sought to be able to determine on a
statistical basis, that the method is suitable for the
intended purposes.
In this sense, it is important for the process of
validation to be assigned to a responsible person to
perform this task. So that the validation is done in a
methodical, orderly, traceable and reliable way to
establish the scope of validation, it is essential:
 Know the method to be validated and its
applicability, i.e. the analyte, its concentration and
the matrix (or matrices) in which it is desired to
use.
174
  Identify factors of influences that could change
these parameters or performance characteristics.
 Determine the limitations of the method.
Since the dinitrosalicylic acid method (DNS) has been
used since the beginning for the determination of
reducing sugars in foods, its validation is necessary in
order to establish if this method is suitable for the matrix
in which it is intended to apply: extracts of the seaweed
Undaria pinnatifida. In this work, the dinitrosalicylic acid
technique was validated for the determination of
reducing sugars obtained in the extracts of U. pinnatifida
algae by acid hydrolysis for later application in the
production of bioethanol.
2. Materials and Methods
 Chemicals: Sodium potassium tartrate and 3,5-
dinitrosalicylic acid, Sigma-Aldrich S.p.a.; Sodium
3, 5-dinitrosalicylic D-Glucose 3-amino-5-nitrosalicylic
acid (yellow) acid (red-brown)
Figure 1. Conversion of reducing sugars by DNS
The reagent is a solution formed by the following
compounds: 3, 5-Dinitrosalicylic acid (2-hydroxy-3,5-
dinitrobenzoic acid), which acts as an oxidant, Rochelle
salt (sodium-potassium tartrate), which prevents the
dissolution of oxygen in the reagent and sodium
hydroxide to provide the medium required for the redox
reaction to occur.
 Preparation of DNS Reactive
1. Solution A: Dissolve 1.00 g of DNS in 20 mL of
NaOH 2 M.
2. Solution B: Dissolve 30 g of sodium and
potassium tartrate tetrahydrate in 50 mL of
distilled water.
3. Stir until complete dissolution.
4. Solution A onto B.
5. Heat and mix to homogenize.
6. Complete the volume to 100 mL with distilled
water.
7. Store in amber bottle at 4 °C.
To carry out the validation of the DNS method, the
same was applied to standard glucose solutions
and U. pinnatifida seaweed extracts. Glucose
solutions of different concentrations were
obtained by dilutions from a stock solution of 1
g/L. The algae extracts were obtained by
hydrolysis of 1 g dry weight of U. pinnatifida
seaweed in 50 mL of 0.25 N sulfuric acid in a
thermostatted bath at 50 °C for 1 hour. For both
glucose solutions and algae extracts, the
procedure was the same:
hydroxide and Sulfuric acid 98%, Biopack of
analytical reagent grade.
 Apparatus: balance Mettler Toledo model ME,
precision 0,0001 g; spectrophotometer UV-
Visible-Lambda 465, Perkin Elmer.
The DNS method is a colorimetric technique that
consists of a redox reaction between the 3,5-
dinitrosalicyclic acid and the reducing sugars present in
the sample. The reducing power of these sugars comes
from their carbonyl group, which can be oxidized to the
carboxyl group by mild oxidizing agents, while the DNS
(yellow) is reduced to 3-amino-5-nitrosalicylic acid (red-
brown) which can be quantified by spectrophotometry at
540 nm, wavelength of maximum absorbance [30]. The
intensity of the color is proportional to the concentration
of sugars. The reaction is carried out in an alkaline
medium. The Figure 1 shows the oxidation-reduction
reaction mentioned.
D-Gluconic acid
1. In tubes of 10 mL, is placed 1 mL of algae
extract and 1 mL of DNS reagent.
2. The pH was brought to pH 10.
3. The tubes are taken to a bath
thermostatized at 100 °C, 5 minutes.
4. It is cooled to room temperature.
5. The volume is completed with 8 mL of
distilled water.
6. It is homogenized and read at 540 nm in a
spectrophotometer.
The reading is compared against a reagent blank
solution, in which the algae extract is replaced by
distilled water and steps 2 to 6 are repeated.
2.1 Validation of the analytical methodology
In order to determine the optimal operating
wavelength, the absorption spectrum of a glucose
solution of medium concentration was performed for
wavelengths from 520 to 625 nm. The figure 2 shows the
absorption spectrum obtained.
Figure 2. Absorption spectrum of glucose
175
 The maximum absorption wavelength corresponded
to 540 nm, coincident with Miller [30]. For the validation
of the analytical methodology, the following parameters
were studied: linearity, limit of detection, limit of
quantification, accuracy, precision, selectivity and
robustness. For linearity, the absorbance standard curve
was constructed as a function of concentration, with
glucose solutions of concentrations less than 1 g/L, and
the correlation coefficient was calculated. Subsequently,
the regression line was found and the validity of the
linear model was verified through a hypothesis testing on
the slope of the line and the y-intercept.
Detection and quantification limits were calculated as
3 and 10 times the standard deviation, respectively, of 9
reagents blank solution, according to Owen [32]. The
accuracy of measurement expresses the proximity of a
single result to a reference value [33]. The accuracy of the
method was determined by the percentage of recovery,
using the method of Addition of Standard Solution in
triplicate: on an algae extract, whose content of reducing
sugars was previously determined, solutions of glucose
concentrations of 0.30, 0.60 and 0.90 g/L were added in
equal amounts. The DNS method was applied to the algae
extracts enriched and the percentage of recovery was
calculated according to equation (1).
𝑀𝑎 −𝑀𝑠
%𝑅 = (1)
𝑎
Where:
% R: Percentage of recovery.
Ma : Initial concentration of algae extract with
addition of standard.
Ms : Initial concentration of algae extract without
addition of standard.
a : Concentration of the standard solution added.
ISO 5725 uses term “precision” to refer to the
closeness of agreement between test results [33]. The
precision was determined by repeatability tests (the
same analyst performed 5 replicates of the same algae
extract on the same day and on the same equipment) on
three algae extracts of known concentrations. For each
case the coefficient of variation was calculated.
The selectivity study was tested using formaldehyde
as an interfering, instead of the carbonyl group present
in the reducing sugars. The effect of the interfering in
concentrations of 4, 2 and 1% on a solution of glucose of
concentration 0.50 g/L, and on an extract of algae of
concentration 0.28 g/L, were analyzed. "The robustness
of an analytical procedure is a measure of its capacity to
remain unaffected by small, but deliberate variations in
method parameters and provides an indication of its
reliability during normal usage" [34].
For the study of robustness of the methodology, the
recommendations of Youden and Steiner [35] adopted
later by Quatrochi and Plackett and Burman [36, 37], and
published by Vander [38] were taken as references.
According to Vander [38], for their statistical validity the
study requires that the experimental design includes at
least seven real factors, which are evaluated by means of
the realization of eight independent experiments. For
this, each variable is studied through a high and a low
value.
The factors chosen for this test, were: reaction time,
reagent volume (DNS), pH, cell volume, volume of algae
extracts, operator, reaction water bath cooling; and for
each one of them was established its minimum and
maximum value. For each factor the difference between
the average absorbance of the upper level of the effect (X)
and the average absorbance of the lower level of the
effect (x) was calculated. The greater the difference, X-x,
the more influence this variable will have on the
analytical method. The acceptance criterion was
established as a function of the standard deviation of the
method (S); if (X - x) < √2 𝑆, the method was considered
not to be sensitive to the variable observed [36].
3. Results and discussion
 Linearity. The method demonstrated linearity in a
range of 0.28 to 1.00 g/L. For the realization of the
calibration curve, it worked with concentration
values lower than 1 g/L. With the absorbance
values obtained, the correlation coefficient (r =
0.993) was found to be statistically significant
when performing a hypothesis testing with a
significance level of 5%. Figure 3 shows the
calibration curve for the determination of
reducing sugars in extracts of U. pinnatifida
seaweed by UV-Visible spectrophotometry.
Figure 3. Calibration curve for the determination of
reducing sugars in extracts of Undaria pinnatifida algae by UV-
Visible spectrophotometry
Subsequently, the regression line between the
absorbance values (y) and the concentrations (x)
was found by minimizing the sum of the squares
of the residuals so as to find the slope (m) and the
y-intercept (b). The values obtained were m =
1.592 and b=-0.077 with a coefficient of
determination (R2) of 0.983, which indicates that
98.32% of the variation in absorbance can be
explained by variation in concentration. In this
way the calibration curve is represented by
y=1.592 x-0.077.
In order to verify the validity of the linear model
in the range of concentrations studied, in addition
to finding the coefficient of determination, a
hypothesis testing was performed on the slope of
the regression line and the y-intercept, it was
verified that the coefficient of variation (CV) of the
response factors (quotient between ycalculated and
x) are less than 5%. It worked with the statistical
variable Student´s t and a significance level of 5%,
where tc = t8, 0.025 = 2.751. The calculated values
were tb = -2.039 and tm = 23.04, for the y-intercept
and the slope respectively; and the CV = 4.04.
From them it is concluded that there is linearity
176
 and that there is not sufficient evidence to suppose LOD are indicative of the absence of analyte in
that the y-intercept is different from zero; detectable amounts. The limit of quantification
The standard errors (s) are sb = 0.038 and sm = (LOQ) is strictly the lowest analyte concentration
0.069 and considering a 95% confidence level that can be quantified with an acceptable level of
results that the slope and the y-intercept true accuracy and precision [40]. The limit of
would be found in the interval (1.433, 1.752) and quantification can be calculated from equation (3).
(-0.165, 0.010) respectively; of second interval it Limit of quantification, LOQ = 10 * S = 0.012 (3)
can be deduced that the y-intercept is close  Accuracy. Table 1 shows the results of the
enough to zero. These acceptance criteria coincide accuracy test. The mean recovery rate for the
with [39], who verifies the linearity, through the accuracy test resulted to be 97.741%, see Table 1.
study of the same parameters. According to A.O.A.C. [41], for a concentration
 Limits. The limit of detection (LOD) is the above 100 mg/kg (algae extract concentration =
minimum concentration of analyte that can be 280 mg/kg) the acceptable recovery value is 90 to
detected and identified, but not necessarily 107% whereby the value obtained in the
quantified with a certain degree of certainty [40]. experiment would be acceptable for the level
The limit of detection can be calculated from the measured in the matrix analyzed. Standard
equation (2). deviations below 1 can be observed at three levels
Limit of detection, LOD = 3 * S = 0.004 (2) of concentrations, in addition to percentages of
recovery close to 100%, both results coincide with
Values above the LOD can be attributed to the Herrera [42].
presence of the analyte and the values below of
Table 1. Recovery rate and coefficient of variation for glucose samples (G) + algae extract (H)
Sample Glucose g/L G + H g/L Average Recovery% Stand. Dev. C.V.%
0.574
1 0.300 0.569 0.571 97.222 0.003 0.003
0.572
0.879
2 0.600 0.868 0.870 98.333 0.008 0.893
0.864
1.166
3 0.900 1.157 1.159 97.667 0.007 0.574
1.153
AlgaeExtract 0.280
Recovery Average% 97.741 0.490

 Selectivity. Analytical selectivity refers to "the components of similar behavior" [43]. Tests were
extent to which a method can be used to performed in duplicate and the results are shown
determine certain analyses in mixtures or in Table 2.
matrices without interference from other
Table 2. Mean absorbance of pure interferent, glucose + interferent, Seaweed extract + interferent
Average Absorbance Increment%*
Formaldehyde% V Formaldehyde Glucose + interferent AlgaeExtract + interferent Glucose Algae Extract
1 0.012 0.296 1.459 2.957 1.531
2 0.014 0.303 1.503 5.391 4.593
4 0.0153 0.313 1.548 8.870 7.724
Glucose 0.2875
AlgaeExtract 1.437
*Percent increase in absorbance value of the solution with interferant relative to pure solution

Brown algae contain alginic acid with carbonyl
groups in their structure (as in DNS acid), these
units are bound so that the carboxyl group is free
while the aldehyde groups are linked by a
glycosidic bond, it is to be expected then, that due
to steric matters, the carbonyl groups present in
the algae are not reduced in the presence of
reducing sugars and the carbonyl groups in the
DNS do so, which would demonstrate the
selectivity of the method. The selectivity study
was tested using formaldehyde as an interferent,
instead of the carbonyl group present in the
reducing sugars.
The maximum influence was observed for a
concentration of 4% formaldehyde on the glucose
solution. While for the case under study, U.
pinnatifida extract, the maximum increase was
only 7.724% for the most concentrated solution in
formaldehyde: 4%. The method, therefore, proved
to be selective with respect to the reducing sugars
present in U. pinnatifida seaweed extracts.
 Precision. Table 3 shows the results of the
repeatability test. In terms of repeatability, the
results do not show significant differences in the
samples analyzed the same day, by the same
analyst, in the same laboratory, with the same
equipment and the same reagents; testing the
precision of the method. The CV for the three
concentrations analyzed were lower than the
acceptance limit commonly required for this type
of analysis: 2% [44, 45], which ensures the
repeatability of the results when the method is
applied.
177
 Table 3. Repeatability test results (n = 5)
Concent. Absorbance Average Stand. Dev. C.V.%
1.362
1.333
0.900 1.335 1.342 0.014 1.058
1.328
1.351
0.618
0.621
0.560 0.618 0.621 0.006 0.886
0.631
0.619
0.186
0.184
0.180 0.183 0.184 0.002 0.822
0.183
0.186

 Robustness. Table 4 summarizes the factors that absorbance obtained in the 8 trials. Table 5
were taken into account for the robustness presents the values of the differences, in absolute
analysis and the maximum and minimum values value, for each factor.
of each. S = 0.031; Standard deviation of the mean
Table 4. Average Absorbance for the Robustness Test of Youden and Steiner
Condition variable Tests
Reference* Max. Min. 1 2 3 4 5 6 7 8
1 10 5 10 10 10 10 5 5 5 5
2 2.5 1.5 2.5 2.5 1.5 1.5 2.5 2.5 1.5 1.5
3 10 5 10 5 10 5 10 5 10 5
4 15 10 25 25 10 10 10 10 25 25
5 2.5 1.5 2.5 1.5 2.5 1.5 1.5 2.5 1.5 2.5
6 M o M o o M M o o M
7 Yes No Yes No No Yes No Yes Yes No
Average Absorbance 0.016 0.001 0.002 0.056 0.089 0.002 0.028 0.023
*1. Reaction time; 2. DNS volume (mL); 3. pH; 4. Cell Volume; 5. Volume of Algae extract; 6. operator; 7. Cooling in water bath of the reaction

Table 5. Influence of variation of some parameters on the response

Reference High value Low value Average X Average x Dif.
1 10 5 0.019 0.035 0.017
2 2 1 0.027 0.027 0.000
3 10 5 0.034 0.081 0.047
4 25 10 0.068 0.037 0.031
5 2 1 0.011 0.043 0.033
6 M o 0.046 0.008 0.038
7 Si No 0.025 0.029 0.003

Those parameters, which presented a value for the
difference, (X - x) greater than √2 𝑆, according to
equation (4), were considered critical [36].
√2 𝑆 = 1.414 x 0.031 = 0.044
It was concluded that the method is solid
compared to the small variations made in the
considered variables, except for the pH, a
parameter in which special attention must be paid
during the tests for the determination of reducing
sugars. The variation generated by the pH is also
detected by Herrera [42] in the validation of a
spectrophotometric method.
4. Conclusions
The proposed analytical methodology for the
determination of reducing sugars in U. pinnatifida
seaweed extracts by UV-Visible spectrophotometry,
fulfilled the requirements to consider it validated,
proving to be a specific, linear, accurate, precise and
robust method against possible variations in the
conditions of the method except pH.
In this way the DNS method can be reliably used on
(4) the matrix in which it is intended to apply: extracts of
algae U. pinnatifida for the production of bioethanol. It
should be noted that one of the methods of
saccharification is acid hydrolysis, which is why the
extract obtained will have an acidic pH. When applying
the DNS method, we must ensure an alkaline pH, but
given that the robustness analysis proved to be sensitive
to changes in pH, the same pH should be ensured for all
experiments, so that the data are comparable.
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