scispace - formally typeset

Journal ArticleDOI

Starch determination in Chlorella vulgaris—a comparison between acid and enzymatic methods

01 Jan 2012-Journal of Applied Phycology (Springer Netherlands)-Vol. 24, Iss: 5, pp 1203-1208

TL;DR: The enzymatic method (EM1) proved to be the most rapid and precise method for microalgal starch quantification and the evaluation of resistant starch by enzymes showed that no formation of this type of starch occurred in microalgae, meaning that this should not interfere with starch content determinations.

AbstractDifferent methods for estimating starch in Chlorella vulgaris were compared with the view of establishing a procedure suitable for rapid and accurate determination of starch content in this microalgal species. A close agreement was observed between methods that use perchloric acid and enzymatic methods that use α-amylase and amyloglucosidase to hydrolyze the starch of microalgae grown under different nitrogen culture conditions. Starch values obtained by these methods were significantly higher than those estimated by using hydrochloric acid as solubilizing and hydrolyzing agent. The enzymatic method (EM1) proved to be the most rapid and precise method for microalgal starch quantification. Furthermore, the evaluation of resistant starch by enzymatic methods assayed in nitrogen-sufficient and nitrogen-starved cells showed that no formation of this type of starch occurred in microalgae, meaning that this should not interfere with starch content determinations.

Topics: Resistant starch (65%), Starch (64%), Amylase (54%), Chlorella vulgaris (53%)

Summary (2 min read)

Introduction

  • The ongoing depletion of oil reserves coupled with economic growth and stability, and more significantly, the emerging concern about global warming arising from burning fossil fuels have become major drivers for the development of renewable and cost-effective energy sources (Stephens et al. 2010; Dragone et al. 2010).
  • Beyond that, some strains were also found to accumulate large amounts of starch under nitrogen starvation.
  • Therefore, an accurate and rapid method for the determination of starch is key to the commercial success of bioethanol production from microalgae.
  • Furthermore, enzyme digestion has been the preferred method of determining starch because, in theory, active, purified starch-degrading enzymes are specific for the hydrolysis of starch and yield highly accurate values (Rose et al. 1991).

Materials and methods

  • Cells in the late exponential growth phase were centrifuged at 8,750×g for 15 min, washed in distilled water and resuspended in nitrogen-sufficient and nitrogen-starved (standard medium without urea) culture media.
  • Cells were removed from the mortar and pestle using solvents (ethanol solution or acetone, depending on the method).
  • The different methods for determination of microalgal starch compared in this study are summarized in Table 1.
  • The removal of interfering substances is extreme-.

Step Method

  • Ly important since they are able to react colorimetrically, thus leading to the overestimation of starch values.
  • The solubilized starch solution was then reacted with a mixture of concentrated sulfuric acid and anthrone as described for AM1.
  • The microalgal biomass was extracted with acetone and boiling 80% (v/v) ethanol as described in the AM1 method aiming at the removal of interfering substances.
  • Samples were incubated at 50°C for 20 min and then cooled to room temperature.
  • The precision of each method was obtained by determining the percentage relative standard deviation (%RSD) according to Eq. 2. %RSD ¼ 100»s=CM ð2Þ where s = standard deviation and CM = mean starch concentration measured by a specific method.

Results and discussion

  • Comparison of starch content in C. vulgaris determined by acid and enzymatic methods Figure 1 shows the comparison of different methods for determining the starch content in microalgae cultivated under nitrogen sufficient conditions.
  • Perchloric acid has been considered to be the most efficient solvent for starch extraction from plant tissues (Ghiena et al. 1993).
  • The significant differences among the starch values obtained by both acid methods may be related to the difference in acid strengths between HClO4 and HCl, as explained above.
  • Additionally, both perchloric acid methods yielded similar precisions to that reported for the determination of starch in microalgae cultivated under nitrogen sufficient conditions (Table 2).
  • Resistant starch formation depends on several factors such as physical structure of starch, possible cross-linking/structural modification of starch and protein–starch/lipid–starch interactions (Tharanathan and Mahadevamma 2003).

Conclusions

  • The authors conclude that perchloric acid methods and enzymatic methods gave similar values for the concentrations of starch in C. vulgaris grown under different nitrogen conditions.
  • The enzymatic method EM1 that uses α-amylase and amyloglucosidase to hydrolyze the starch of microalgae proved to be the most rapid and precise procedure for starch determination in C. vulgaris.
  • The authors also acknowledge the financial support received through the projects INNOVALGAE (FCT PTDC/ AAC-AMB/108511/2008) and ALGANOL.

Did you find this useful? Give us your feedback

...read more

Content maybe subject to copyright    Report

Starch determination in Chlorella vulgarisa comparison
between acid and enzymatic methods
Bruno Fernandes & Giuliano Dragone & Ana P. Abreu &
Pedro Geada & José Teixeira & António Vicente
Received: 12 September 2011 /Revised and accepted: 12 September 2011 /Published online: 4 December 2011
#
Springer Science+Business Media B.V. 2011
Abstract Different methods for estimating starch in Chlo-
rella vulgaris were compared with the view of establishing
a procedure suitable for rapid and accurate determination of
starch content in this microalgal species. A close agreement
was observed between methods that use perchloric acid and
enzymatic methods that use α-amylase and amyloglucosi-
dase to hydrolyze the starch of microalgae grow n under
different nitrogen culture conditions. Starch values obtained
by these methods were significantly higher than those
estimated by using hydrochloric acid as solubilizing and
hydrolyzing agent. The enzymatic method (EM1) proved to
be the most rapid and precise method for microalgal starch
quantification. Furthermore, the evaluation of resistant starch
by enzymatic methods assayed in nitrogen-sufficient and
nitrogen-starved cells showed that no formation of this type of
starch occurred in microalgae, meaning that this should not
interfere with starch content determinations.
Keywords Amylolytic enzymes
.
Biofuels
.
Chlorella
vulgaris
.
Microalgae
.
Perchloric acid
.
Starch hydrolysis
Introduction
The ongoing depletion of oil reserv es co upled with
economic growth and stability, and more significantly, the
emerging concern abou t gl obal wa rming arising from
burning fossil fuels have become major drivers for the
development of renewable and cost-effective energy sour-
ces (Stephens et al. 2010; Dragone et al. 2010). Among the
different potential sources of renewable energy, liquid
biofuels such as bioethanol (a petrol additive/substitute)
and biodiesel (a diesel alternative) are of most interest as
they are part of the few options to replace fossil fuels and
have the potential to limit greenhouse gas emissions (Chen
et al. 20 11; Nigam and Singh 2011). However, the
production of biofuels from terrestrial plants is controver-
sial mainly due to the impact on global food markets, on
food security (Brennan and Owende 2010), on arable land
usage, on potable water utilizat ion and on deforestation. In
contrast, microalgae have been considered as a promising
feedstock for biofuel production since they are able to
convert solar energy to chemical energy via CO
2
fixation
and do not compete for land with crops used for food
production (Ahmad et al. 2011). These photosynthe tic
microorganisms accumulate significant qu antities of lipids
and carbohydrates over short periods of time that can be
subsequently proces sed into biofuels (Brennan and Owende
2010; Spolaore et al. 2006; Chen et al. 2009).
Certain species of microalgae such as Chlorella vulgaris
have the ability to produce higher levels of starch than
lipids as reserve polymer (Dragone et al. 2011). Beyond
that, some strains were also found to accumulate large
amounts of starch under nitrogen starvation. These species
are suitable candidates for bioethanol production as starch
from microalgae can be extracted to produce fermentable
sugars (Mussatto et al. 2010).
Therefore, an accura te and rapid method for the
determination of starch is key to the commercial success
of bioethanol production from microalgae. On the other
hand, some difficulties are usually encountered in choosing
and implementing an appropriate methodology for micro-
algal starch quantification. The great number of different
B. Fernandes
:
G. Dragone (*)
:
A. P. Abreu
:
P. Geada
:
J. Teixeira
:
A. Vicente
IBBInstitute for Biotechnology and Bioengineering,
Centre of Biological Engineering, University of Minho,
Campus de Gualtar,
4710-057 Braga, Portugal
e-mail: gdragone@deb.uminho.pt
G. Dragone
e-mail: giulianodragone@hotmail.com
J Appl Phycol (2012) 24:12031208
DOI 10.1007/s10811-011-9761-5

starch methods reported in the literature complicates the
task of method evaluation and selection . Furthermore,
variations in the accuracy of starch determination methods
may confound the interpretation and compa rison of results
among different studies.
Methods for starch determination in microalgae can be
broadly grouped into acid hydrolysis or enzymatic proce-
dures. Exampl es of the former include hydrolysis with
perchloric acid (Chader et al. 2009) while the latter comprises
digestion with amylase and amyloglucosidase (Zemke-White
and Clements 1999). Both procedures hydrolyze the starch to
glucose, which is subsequently quantified colorimetrically.
However, acid-based procedures might be subject to error in
starch estimation due to the extraction of interfering
carbohydrates from other polymers. Furthermore, enzyme
digestion has been the preferred method of determining
starch because, in theory, active, purified starch-degrading
enzymes are specific for the hydrolysis of starch and yield
highly accurate values (Rose et al. 1991).
The objective of this study was to compare different
methodologies for the determination of starch in C. vulgaris,
and to establish a procedure suitable for rapid and accurate
routine measurement of starch content in microalgae.
Materials and methods
The freshwater microalga Chlor ella vulgaris P12 kindly
provided by the Algal Laboratory (CCALA), Institute of
Botany, Academy of Sciences of the Czech Republic was
precultivated in standard (nitrogen-sufficient) medium con-
taining (mg L
1
): 1100 (NH
2
)
2
CO, 237 KH
2
PO
4
, 204
MgSO
4
·7H
2
O, 40 FeNaC
10
H
12
O
8
N
2
,88CaCl
2
,0.83
H
3
BO
3
,0.95CuSO
4
·5H
2
O, 3.3 MnCl
2
·4H
2
O, 0.17
(NH
4
)
6
Mo
7
O
24
·4H
2
O, 2.7 ZnSO
4
·7H
2
O, 0.6 CoSO
4
·7H
2
O,
0.014 (NH
4
)VO
3
in distilled water (Fernandes et al. 2010).
Cells in the late exponential growth phase were centrifuged
at 8,750×g for 15 min, washed in distilled water and
resuspended in nitrogen-sufficient and nitrogen-starved (stan-
dard medium without urea) culture media. Photoautotrophic
cultivation of C. vulgaris was performed at 30°C in 1 L
photobioreactors containing 400 mL of medium with a surface
irradiance of 70 μmol photons m
2
s
1
provided by four
fluorescent lamps (Sylvania Standard F18W). All cultures
were agitated using air enriched with 2% (v/v) CO
2
at an
aeration rate of 0.833 vvm (volume of gas per volume of
culture suspension per minute).
Methods for starch determination
Microalgal cells at the beginning of the stationary growth
phase were harvested by centrifugation at 8,750×g for
15 min, washed in distilled water, lyophilized and disinte-
grated (10 mg) with a mortar and pestle prior to starch
analysis. The disintegration of the cells was performed for
5 min, monitored through microscopic observation. Cells
were remo ved from the mortar and pestle using solvents
(ethanol solution or acetone, depending on the method).
Starch content was expressed as % w/w (dry weight basis).
The different methods for determination of microalgal starch
compared in this study are summarized in Table 1.
Perchloric acid method (AM1)
In this method (Rose et al. 1991), the cells were first
extracted with acetone and subsequently extra cted with
boiling 80% (v/v) ethanol until the extract remained
colorless, in order to remove interfering substances (e.g.,
pigments, soluble sugars and lipids) and to gelatinize starch
granules. The removal of interfering substances is extreme-
1204 J Appl Phycol (2012) 24:12031208
Table 1 Comparison of reagents and estimated time employed in the determination of microalgal starch concentration by acid and enzymatic
methods
Step Method
AM1 AM2 AM3 EM1/EM2
Removal of interfering substances Acetone Acetone
80% ethanol 80% ethanol 80% ethanol 80% ethanol
8h 1h 2h 0.5h
Starch extraction and solubilization 35% HClO
4
30% HClO
4
1.1% HCl α-Amylase
Amyloglucosidase
0.5 h 1 h 0.5 h 1 h
Colorimetric determinationof glucose Anthrone Anthrone Anthrone Glucose oxidase + peroxidase
0.5 h 0.5 h 0.5 h 0.75
Total time 9.0 h 2.5 h 3.0 h 2.25 h
AM1 perchloric acid method (Rose et al. 1991), AM2 modified perchloric acid method (Brányiková et al. 2011), AM3 hydrochloric acid method
(Oren et al. 1988), EM1 enzymatic method (Megazyme 2009), EM2 enzymatic method for resistant starch (Megazyme 2009)

ly important since they are able to react colorimetrically,
thus leading to the overestimation of starch values.
Subsequently, the microalgal starch was extracted and
solubilized with 35% (v/v) perchloric acid. The solubilized
starch solution was then reacted with a mixture of concentrated
sulfuric acid and anthrone (2 g anthrone in 1 L of 72% (v/v)
H
2
SO
4
) to quantify glucose spectrophotometrically at 625 nm.
Modified perchloric acid method (AM2)
In this method (Brányiková et al. 2011), the removal of
interfering substances using acetone was avoided, and the
duration of this and subsequent (starch extraction and
solubilization) steps was shortened, when compared with
AM1 (Table 1). Pigments were extracted three times using
80% ethanol for 15 min at 68°C. For total hydrolysis of
starch, 30% perchloric acid was added to the sediment,
stirred for 15 min at 25°C and centrifuged. This procedure
was repeated three times. The solubilized starch solution was
then reacted with a mixture of concentrated sulfuric acid and
anthrone as described for AM1.
Hydrochloric acid method (AM3)
The microalgal biomass was extracted with acetone and
boiling 80% (v/v) ethanol as described in the AM1 method
aiming at the removal of interfering substances. Subse-
quently, starch granules were hydrol yzed with 1.1% hydro-
chloric acid at 100°C for 30 min (Oren et al. 1988).
Glucose was determined colorimetrically by the anthrone
reaction after starch hydrolysis.
Enzymatic method (EM1)
The starch content of C. vulgaris was assayed by enzymatic
degradation of the starch to glucose with α-amylase and
amyloglucosidase, using the total starch assay procedure from
Megazyme (Megazyme 2009) accepted by AOACAssocia-
tion of Analytical Communities (Official Method 996.11) and
AACCAmerican Association of Cereal Chemists (Method
76.13). Lyophilized microalgal biomass, previously disinte-
grated, was resuspended in 80% (v/v) ethanol and incubated in
a water bath at 8085°C for 5 min, in order to extract interfering
compounds. Thermostable α-amylase (3,000 U mL
1
)in
MOPS buffer (50 mM, pH 7.0) containing 5 mM CaCl
2
,
was added to each sample. The samples were incubated in a
boiling water bath for 6 min, with mixing at 2 min intervals,
andthenplacedinablockheaterat50°Candallowedto
equilibrate for 5 min. Amyloglucosidase (3,300 U mL
1
)in
sodium acetate buffer (200 mM, pH 4.5) plus sodium azide
(0.02% w/v) was subsequently added to each sample. After
that, samples were incubated at 50°C for 30 min, and
centrifuged for 10 min at 4,500 × g to separate any remaining
insoluble material. Aliquots of the supernatant were assayed
for glucose. Each aliquot was added to 3.0 mL of GOPOD
reagent in distilled water. This reagent contained, according
to the manufacturers spec ifications: glucose oxidase
(>12,000 U); peroxidase (>650 U) and 4-aminoantipyrine
(80 mg). Samples were incubated at 50°C for 20 min and
then cooled to room temperature. The absorbance of samples
and the
D-glucose control were measured at 510 nm in a
spectrophotometer against a reagent blank solution consist-
ing of 0.1 mL of water and 3.0 mL of GOPOD reagent.
Enzymatic method for resistant starch (EM2)
The enzymatic method for resistant starch proposed by
Megazyme (Megazyme 2009) was based on the EM1 method
detailed above, except that after extraction of interfering
compounds with hot ethanol, the microalgal biomass was
predissolved with 2 M KOH in an ice/water bath, followed by
neutralization with sodium acetate buffer and further hydro-
lysis with α-amylase and amyloglucos idas e.
Statistical design and analysis
Each of the five methods was tested in triplicate on the
microalgal biomass. The replicates were performed using cells
from two cultivations, one under nitroge n sufficient and another
under nitrogen starved conditions. The accuracy of each
method was evaluated as the percentage relative error (%Er)
between their mean results and those obtained by the official
method EM1 according to the equation given below (Eq. 1).
%Er ¼ 100
»
C
M
C
OF
ðÞ=C
OF
ð1Þ
where C
M
= mean starch concentration measured by a specific
method and C
OF
= mean starch content obtained by the
official enzymatic method EM1. The precision of each
method was obtained by determining the percentage relative
standard deviation (%RSD) according to Eq. 2.
%RSD ¼ 100
»
s=C
M
ð2Þ
where s = standard deviation and C
M
= mean starch
concentration measured by a specific method. Results were
analyzed by the Experimental Design Module of the Statistica
8.0 software (Statsoft, USA).
Results and discussion
Comparison of starch content in C. vulgaris determined by
acid and enzymatic methods
Figure 1 shows the comparison of different methods for
determining the starch content in microalgae cultivated
J Appl Phycol (2012) 24:12031208 1205

under nitrogen sufficient conditions. It can be observed that
there was no statistically significant difference (p<0.05)
between the starch content of C. vulgaris determined either
by perchloric acid methods (AM1 and AM2) and enzymatic
methods (EM1 and EM2).
On the other hand, the hydrochloric acid method (AM3)
provided nearly 20% lower estimat es of starch content than
those obtained by perchloric acid methods (AM1 and
AM2). These results may be attributed to the higher
efficiency of starch extraction and hydrolysis with HClO
4
in comparison with HCl. According to Raessler et al.
(2010), the accurate determin ation of starch is dependent on
both its complete ext raction from the sample and its
complete hydrolysis into glucose. In green algae, starch is
synthesized and stored within the chloroplast, which
considerably limits the accessibility of the solvent during
extraction. Consequently, extraction of starch generally
needs rather harsh conditions to allow thorough access of
the solvent. A previous study (Fontana et al. 2001) reported
that the higher the acid strength, the higher the yield of
glucose released from starch by acid hydrolysis. As a result,
the higher starch values obtained by the perchloric acid
method can be related to the greater acid strength of HClO
4
compared to that of HCl. In this sense, perchloric acid has
been considered to be the most efficient solvent for starch
extraction from plant tissues (Ghiena et al. 1993).
Starch content of C. vulgaris estimated by the hydro-
chloric acid method was also significantly lower than those
obtained by both perchloric acid methods at the end of
microalgae cultivation under nitrogen starvation conditions
(Fig. 2). As can be seen in Fig. 2, the specific starch
concentration in nitrogen-depleted microalgae reached 30%
as determined by the hydrochloric acid method, while the
estimation of the microalgal starch content attained 33.5%
and 35% according to AM1 and AM2 methods, respec -
tively, which use HClO
4
as a solubilizing and hydrolyzing
agent. The significant differences among the starch values
obtained by both acid methods may be related to the
difference in acid strengths between HClO
4
and HCl, as
explained above.
Figure 2 also shows that enzymatic methods yielded
significantly higher microalgal starch concentrations than
that obtained by the hydrochloric acid method. On the other
hand, the starch values estimated by using amylolytic
enzymes did not differ significantly (at a 95% confidence
level) from those resul ting from perchloric acid methods.
Table 2 summarizes the precision and accuracy of the
tested methods for the determination of starch in microalgae
cultivated under nitrogen sufficient conditions. According
to this table, the accuracy of AM2 method was similar to
that of the perchloric acid method AM1 for the estimation
of the microalgal starch content. Moreover, it can be
observed in Table 2 that precisions (10.8%) achieved by
both perchloric acid methods were comparable to that
obtained by the enzymatic method EM1 (9.2%).
Although the perchloric acid method AM2 provided a
higher relative error (lower accuracy) than method AM1
AM1 AM2 AM3 EM1 EM2
0
10
20
30
40
b
a
a
a
Starch (%)
Anal
y
tical Method
a
Fig. 2 Starch concentration (mean of three replicates±standard
deviation) in C. vulgaris grown under nitrogen starvation conditions.
Columns with the same letters are not significantly different (p<0.05)
according to the Tukey's test for mean comparisons. (AM1 perchloric
acid method (Rose et al. 1991), AM2 modified perchloric acid method
(Brányiková et al. 2011), AM3 hydrochloric acid method (Oren et al.
1988), EM1 enzymatic method (Megazyme 2009), EM2 enzymatic
method for resistant starch (Megazyme 2009))
AM1 AM2 AM3 EM1 EM2
0
1
2
3
4
ab
b
ab
a
Starch (%)
Anal
y
tical Method
a
Fig. 1 Starch concentration (mean of three replicates±standard
deviation) in C. vulgaris grown under nitrogen sufficient conditions.
Columns with the same letters are not significantly different (p<0.05)
according to the Tukeys test for mean comparisons. (AM1 perchloric
acid method (Rose et al. 1991), AM2 modified perchloric acid method
(Brányiková et al. 2011), AM3 hydrochloric acid method (Oren et al.
1988), EM1 enzymatic method (Megazyme 2009), EM2 enzymatic
method for resistant starch (Megazyme 2009))
1206 J Appl Phycol (2012) 24:12031208

(3.4 and 0.9, respectively) for determining starch concen-
tration in nitrogen-starved cultures of C. vulgaris (Table 3),
starch values estimated by both perchloric acid methods did
not differ significantly from that obtained by the enzymatic
method EM1 (Fig. 2). Additionally, both perchloric acid
methods yielded sim ilar precisions to that reported for the
determination of starch in microalgae cultivated under
nitrogen sufficient conditions (Table 2).
By comparing Tables 2 and 3, it can also be noticed that
methods AM1 and AM2 provided a more accurate value of
the starch content in nit rogen-starv ed ce lls than that
estimated in cells grown under nitrogen-sufficient condi-
tions. This result may be due to the lower concentration of
interfering compounds (e.g., proteins) present in microalgae
grown under nitrogen starvation conditions. It has been
reported (Esposito et al. 2006) that the protein content in
green microalgae is about threefold higher in nitrogen-
sufficient cells with respect to nitrogen-starved cells.
According to Chow and Landhäusser (2004), water-
soluble compounds such as proteins react with the
concentrated sulfuric acid in the glucose assay and,
therefore, significantly interfere with the absorbance read-
ing. Tables 2 and 3 also show that the enzymatic method
EM1 had the best precision in starch quantification, while
method AM3 yielded the highest percentage relative
standard deviation.
Evaluation of resistant starch in C. vulgaris
Many of the properties of starches including gelatinisation
characteristics, solubility, and the formation of resistant
starch determine their suitability for particular applications
(Maršálková et al. 2010). By defin ition, resistant starch is
the portion of starch that resists hydrolysis by amylolytic
enzymes in the small intestine. It is a linear molecule of α-
1,4-D-glucan, essentially derived fr om the retrog raded
amylose fraction, and has a relatively low molecular weight
(1.2×10
5
Da) (Fuentes-Zaragoza et al. 2010). Resistant
starch formation depends on several factors such as
physical structure of starch, possible cross-linking/structural
modification of starch and proteinstarch/lipidstarch inter-
actions (Tharanathan and Mahadevamma 2003).
In our study, formation of resistant starch in C. vulgaris
was evaluated by comparing starch contents determined
either by the enzymatic method EM1 for nonresistant starch
and the enzymatic method for samples containing resistant
starch (EM2). According to Figs. 1 and 2, no statistically
significant differences were found between the starch
values determined by both enzymatic methods at the end
of the cultivation period under nitrogen sufficient and
nitrogen starvation conditions, respectively. These findings
led us to hypothesize that formation of resistant starch did
not occur under either growing condition.
Conclusions
We conclude that perchloric acid methods and enzymatic
methods gave similar values for the concentrations of starch
in C. vulgaris grown under different nitrogen conditions.
On the other hand, hydrochloric acid method resulted in
significantly lower estimates of star ch in microalgae. The
enzymatic method EM1 that uses α-amylase and amylo-
glucosidase to hydrolyze the starch of microalgae proved to
be the m ost rapid and precise procedure for starch
determination in C. vulgaris.
By comparing the enzymatic method for nonresistant
starch and the enzymatic method for samples containing
resistant starch, we concluded that no resistant starch was
formed in microalgae cultivated neither under nitrogen
sufficient nor nitrogen star vation conditions.
Acknowledgement This research work was supported by the grants
SFRH/BD/44724/2008 (Bruno Fernandes) from Fundação para a
CiênciaeaTecnologia(Portugal)andSFRH/BPD/44935/2008
(Giuliano Dragone). The authors also acknowledge the financial
support received through the projects INNOVALGAE (FCT PTDC/
AAC-AMB/108511/2008) and ALGANOL.
Table 2 Comparison of accuracy and precision of methods for
estimating starch content in nitrogen-sufficient microalgae
Method Accuracy (%) Precision (%)
AM1 17.8 10.8
AM2 15.1 10.8
AM3 11.7 40.3
EM1 9.2
EM2 1.1 9.3
AM1 perchloric acid method (Rose et al. 1991), AM2 modified
perchloric acid method (Brányiková et al. 2011), AM3 hydrochloric
acid method (Oren et al. 1988), EM1 enzymatic method (Megazyme
2009), EM2 enzymatic method for resistant starch (Megazyme 2009)
Table 3 Comparison of accuracy and precision of methods for
estimating starch content in nitrogen-starved microalgae
Method Accuracy (%) Precision (%)
AM1 0.9 11.8
AM2 3.4 11.4
AM3 11.2 7.7
EM1 1.5
EM2 0.1 1.7
AM1 perchloric acid method (Rose et al. 1991), AM2 modified
perchloric acid method (Brányiková et al. 2011), AM3 hydrochloric
acid method (Oren et al. 1988), EM1 enzymatic method (Megazyme
2009), EM2 enzymatic method for resistant starch (Megazyme 2009)
J Appl Phycol (2012) 24:12031208 1207

Citations
More filters

Journal ArticleDOI
TL;DR: This comprehensive review article spots the light on one of the most interesting microalga Chlorella vulgaris and assembles the history and a thorough description of its ultrastructure and composition according to growth conditions.
Abstract: Economic and technical problems related to the reduction of petroleum resources require the valorisation of renewable raw material Recently, microalgae emerged as promising alternative feedstock that represents an enormous biodiversity with multiple benefits exceeding the potential of conventional agricultural feedstock Thus, this comprehensive review article spots the light on one of the most interesting microalga Chlorella vulgaris It assembles the history and a thorough description of its ultrastructure and composition according to growth conditions The harvesting techniques are presented in relation to the novel algo-refinery concept, with their technological advancements and potential applications in the market

479 citations


Journal ArticleDOI
TL;DR: Mixotrophic cultivation of C. vulgaris using the main dairy industry by-product could be considered a feasible alternative to reduce the costs of microalgal biomass production, since it does not require the addition of expensive carbohydrates to the culture medium.
Abstract: Growth parameters and biochemical composition of the green microalga Chlorella vulgaris cultivated under different mixotrophic conditions were determined and compared to those obtained from a photoautotrophic control culture. Mixotrophic microalgae showed higher specific growth rate, final biomass concentration and productivities of lipids, starch and proteins than microalgae cultivated under photoautotrophic conditions. Moreover, supplementation of the inorganic culture medium with hydrolyzed cheese whey powder solution led to a significant improvement in microalgal biomass production and carbohydrate utilization when compared with the culture enriched with a mixture of pure glucose and galactose, due to the presence of growth promoting nutrients in cheese whey. Mixotrophic cultivation of C. vulgaris using the main dairy industry by-product could be considered a feasible alternative to reduce the costs of microalgal biomass production, since it does not require the addition of expensive carbohydrates to the culture medium.

269 citations


Journal ArticleDOI
TL;DR: It is demonstrated that optimization of microalgal cultivation conditions can be considered a useful strategy for maximizing CO2 bio-mitigation by C. vulgaris.
Abstract: Biofixation of CO2 by microalgae has been recognized as an attractive approach to CO2 mitigation. The main objective of this work was to maximize the rate of CO2 fixation (RCO2 ) by the green microalga Chlorella vulgaris P12 cultivated photoautotrophically in bubble column photobioreactors under different CO2 concentrations (ranging from 2% to 10%) and aeration rates (ranging from 0.1 to 0.7 vvm). Results showed that the maximum RCO2 (2.22 g L � 1 d � 1 ) was obtained by using 6.5% CO2 and 0.5 vvm after 7 days of cultivation at 30 C. Although final biomass concentration and maximum biomass productivity of microalgae were affected by the different cultivation conditions, no significant differences were obtained in the biochemical composition of microalgal cells for the evaluated levels of aeration and CO2. The present study demonstrated that optimization of microalgal cultivation conditions can be considered a useful strategy for maximizing CO2 bio-mitigation by C. vulgaris.

182 citations


Cites methods from "Starch determination in Chlorella v..."

  • ...Starch content of C. vulgaris was determined by enzymatic hydrolysis of the microalgal starch to glucose with a-amylase and amyloglucosidase, as previously described by Fernandes et al. (2012)....

    [...]


Journal ArticleDOI
TL;DR: The present work is a review of the biotechnological and biochemical potential of microalgae in food, wherein the main functional constituent compounds are in the biomass.
Abstract: Microalgae contain several bioactive compounds that can supplement the nutritional and energy needs of the population. The biochemical composition of microalgae can be manipulated by altering the culture conditions and environmental stress to induce the microorganisms to produce high concentrations of a biocompound of interest. In addition, microalgae do not require arable land and can be grown in regions where land use change is not a concern. Thus, the present work is a review of the biotechnological and biochemical potential of microalgae in food, wherein the main functional constituent compounds are in the biomass. The biological effects of these bioactive substances and possible applications for food products are also discussed.

138 citations


Journal ArticleDOI
TL;DR: After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.
Abstract: Photosynthetic carbon partitioning into starch and neutral lipids, as well as the influence of nutrient depletion and replenishment on growth, pigments and storage compounds, were studied in the microalga, Parachlorella kessleri. Starch was utilized as a primary carbon and energy storage compound, but nutrient depletion drove the microalgae to channel fixed carbon into lipids as secondary storage compounds. Nutrient depletion inhibited both cellular division and growth and caused degradation of chlorophyll. Starch content decreased from an initial value of 25, to around 10% of dry weight (DW), while storage lipids increased from almost 0 to about 29% of DW. After transfer of cells into replenished mineral medium, growth, reproductive processes and chlorophyll content recovered within 2 days, while the content of both starch and lipids decreased markedly to 3 or less % of DW; this suggested that they were being used as a source of energy and carbon.

105 citations


Cites background from "Starch determination in Chlorella v..."

  • ...These can be commercially processed into biofuels, particularly biodiesel (Yang et al., 2011) and bioethanol (Fernandes et al., 2012)....

    [...]


References
More filters

Journal ArticleDOI
Abstract: Sustainability is a key principle in natural resource management, and it involves operational efficiency, minimisation of environmental impact and socio-economic considerations; all of which are interdependent. It has become increasingly obvious that continued reliance on fossil fuel energy resources is unsustainable, owing to both depleting world reserves and the green house gas emissions associated with their use. Therefore, there are vigorous research initiatives aimed at developing alternative renewable and potentially carbon neutral solid, liquid and gaseous biofuels as alternative energy resources. However, alternate energy resources akin to first generation biofuels derived from terrestrial crops such as sugarcane, sugar beet, maize and rapeseed place an enormous strain on world food markets, contribute to water shortages and precipitate the destruction of the world's forests. Second generation biofuels derived from lignocellulosic agriculture and forest residues and from non-food crop feedstocks address some of the above problems; however there is concern over competing land use or required land use changes. Therefore, based on current knowledge and technology projections, third generation biofuels specifically derived from microalgae are considered to be a technically viable alternative energy resource that is devoid of the major drawbacks associated with first and second generation biofuels. Microalgae are photosynthetic microorganisms with simple growing requirements (light, sugars, CO 2 , N, P, and K) that can produce lipids, proteins and carbohydrates in large amounts over short periods of time. These products can be processed into both biofuels and valuable co-products. This study reviewed the technologies underpinning microalgae-to-biofuels systems, focusing on the biomass production, harvesting, conversion technologies, and the extraction of useful co-products. It also reviewed the synergistic coupling of microalgae propagation with carbon sequestration and wastewater treatment potential for mitigation of environmental impacts associated with energy conversion and utilisation. It was found that, whereas there are outstanding issues related to photosynthetic efficiencies and biomass output, microalgae-derived biofuels could progressively substitute a significant proportion of the fossil fuels required to meet the growing energy demand.

3,930 citations


"Starch determination in Chlorella v..." refers background in this paper

  • ...These photosynthetic microorganisms accumulate significant quantities of lipids and carbohydrates over short periods of time that can be subsequently processed into biofuels (Brennan and Owende 2010; Spolaore et al. 2006; Chen et al. 2009)....

    [...]

  • ...However, the production of biofuels from terrestrial plants is controversial mainly due to the impact on global food markets, on food security (Brennan and Owende 2010), on arable land usage, on potable water utilization and on deforestation....

    [...]


Journal ArticleDOI
TL;DR: The first use of microalgae by humans dates back 2000 years to the Chinese, who used Nostoc to survive during famine, while future research should focus on the improvement of production systems and the genetic modification of strains.
Abstract: The first use of microalgae by humans dates back 2000 years to the Chinese, who used Nostoc to survive during famine. However, microalgal biotechnology only really began to develop in the middle of the last century. Nowadays, there are numerous commercial applications of microalgae. For example, (i) microalgae can be used to enhance the nutritional value of food and animal feed owing to their chemical composition, (ii) they play a crucial role in aquaculture and (iii) they can be incorporated into cosmetics. Moreover, they are cultivated as a source of highly valuable molecules. For example, polyunsaturated fatty acid oils are added to infant formulas and nutritional supplements and pigments are important as natural dyes. Stable isotope biochemicals help in structural determination and metabolic studies. Future research should focus on the improvement of production systems and the genetic modification of strains. Microalgal products would in that way become even more diversified and economically competitive.

3,400 citations


"Starch determination in Chlorella v..." refers background in this paper

  • ...These photosynthetic microorganisms accumulate significant quantities of lipids and carbohydrates over short periods of time that can be subsequently processed into biofuels (Brennan and Owende 2010; Spolaore et al. 2006; Chen et al. 2009)....

    [...]


Journal ArticleDOI
Abstract: This article is an up-to-date review of the literature available on the subject of liquid biofuels. In search of a suitable fuel alternative to fast depleting fossil fuel and oil reserves and in serious consideration of the environmental issues associated with the extensive use of fuels based on petrochemicals, research work is in progress worldwide. Researchers have been re-directing their interests in biomass based fuels, which currently seem to be the only logical alternative for sustainable development in the context of economical and environmental considerations. Renewable bioresources are available globally in the form of residual agricultural biomass and wastes, which can be transformed into liquid biofuels. However, the process of conversion, or chemical transformation, could be very expensive and not worth-while to use for an economical large-scale commercial supply of biofuels. Hence, there is still need for much research to be done for an effective, economical and efficient conversion process. Therefore, this article is written as a broad overview of the subject, and includes information based on the research conducted globally by scientists according to their local socio-cultural and economic situations.

1,760 citations


"Starch determination in Chlorella v..." refers background in this paper

  • ...Among the different potential sources of renewable energy, liquid biofuels such as bioethanol (a petrol additive/substitute) and biodiesel (a diesel alternative) are of most interest as they are part of the few options to replace fossil fuels and have the potential to limit greenhouse gas emissions (Chen et al. 2011; Nigam and Singh 2011)....

    [...]

  • ...…energy, liquid biofuels such as bioethanol (a petrol additive/substitute) and biodiesel (a diesel alternative) are of most interest as they are part of the few options to replace fossil fuels and have the potential to limit greenhouse gas emissions (Chen et al. 2011; Nigam and Singh 2011)....

    [...]


Journal ArticleDOI
TL;DR: This review presents recent advances in microAlgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production and aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.
Abstract: Microalgae have the ability to mitigate CO(2) emission and produce oil with a high productivity, thereby having the potential for applications in producing the third-generation of biofuels. The key technologies for producing microalgal biofuels include identification of preferable culture conditions for high oil productivity, development of effective and economical microalgae cultivation systems, as well as separation and harvesting of microalgal biomass and oil. This review presents recent advances in microalgal cultivation, photobioreactor design, and harvesting technologies with a focus on microalgal oil (mainly triglycerides) production. The effects of different microalgal metabolisms (i.e., phototrophic, heterotrophic, mixotrophic, and photoheterotrophic growth), cultivation systems (emphasizing the effect of light sources), and biomass harvesting methods (chemical/physical methods) on microalgal biomass and oil production are compared and critically discussed. This review aims to provide useful information to help future development of efficient and commercially viable technology for microalgae-based biodiesel production.

1,473 citations


Journal ArticleDOI
Abstract: Of the three generations of biodiesel feedstocks described in this paper, food crops, non-food crops and microalgae-derived biodiesel, it was found that the third generation, microalgae, is the only source that can be sustainably developed in the future. Microalgae can be converted directly into energy, such as biodiesel, and therefore appear to be a promising source of renewable energy. This paper presents a comparison between the use of microalgae and palm oil as biodiesel feedstocks. It was found that microalgae are the more sustainable source of biodiesel in terms of food security and environmental impact compared to palm oil. The inefficiency and unsustainability of the use of food crops as a biodiesel source have increased interest in the development of microalgae species to be used as a renewable energy source. In this paper, the main advantages of using microalgae for biodiesel production are described in comparison with other available feedstocks, primarily palm oil.

830 citations


"Starch determination in Chlorella v..." refers background in this paper

  • ...In contrast, microalgae have been considered as a promising feedstock for biofuel production since they are able to convert solar energy to chemical energy via CO2 fixation and do not compete for land with crops used for food production (Ahmad et al. 2011)....

    [...]


Frequently Asked Questions (1)
Q1. What are the contributions mentioned in the paper "Starch determination in chlorella vulgaris—a comparison between acid and enzymatic methods" ?

In this paper, Dragone et al. compared different methods for estimating starch in Chlorella vulgaris and established a procedure suitable for rapid and accurate determination of starch content in this microalgal species.