Starch determination in Chlorella vulgaris—a comparison between acid and enzymatic methods
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.
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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)....
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Cites methods from "Starch determination in Chlorella v..."
...Starch determination methods As reported by Fernandes et al. (2012), there are different approaches for the determination of starch in microalgae: i) perchloric acid method - in this method, first, interfering compounds (pigments, soluble sugars, and lipids) are extracted with acetone or ethanol,…...
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...For all these methods, it is first necessary to break microalgae cells either by using chemical or physical methods. i) chemical methods: these can be used as in the cases of starch quantification methods previously mentioned (see Section 5.1) (Fernandes et al., 2012; Margarites and Costa, 2014)....
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References
53 citations
"Starch determination in Chlorella v..." refers background in this paper
...According to Raessler et al. (2010), the accurate determination of starch is dependent on both its complete extraction from the sample and its complete hydrolysis into glucose....
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29 citations
26 citations
"Starch determination in Chlorella v..." refers background in this paper
...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)....
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...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)....
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25 citations
12 citations
"Starch determination in Chlorella v..." refers background in this paper
...It has been reported (Esposito et al. 2006) that the protein content in green microalgae is about threefold higher in nitrogensufficient cells with respect to nitrogen-starved cells....
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