Elsevier

Algal Research

Volume 50, September 2020, 101961
Algal Research

Effect of milling and enzymatic hydrolysis in the production of glucose from starch-rich Chlorella sorokiniana biomass

https://doi.org/10.1016/j.algal.2020.101961Get rights and content

Highlights

  • Starch accumulation in C. sorokiniana reached 26% (m/m) upon nitrogen starvation.

  • Cellulases were not effective for the disruption of the microalga cell wall.

  • Milling of freeze-dried cells allowed high hydrolysis yields with fungal amylases.

  • Starch gelatinization was not required for high hydrolysis yields of milled cells.

Abstract

Starch-accumulating microalgae are a potential source of glucose, a valuable industrial feedstock. However, the recalcitrance of their cell wall hinders the enzymatic hydrolysis of the intracellular starch. This work studied starch accumulation, cell wall disruption, and enzymatic hydrolysis of the green microalga Chlorella sorokiniana. Starch accumulation reached 26% of the microalgal biomass in response to nitrogen starvation. The use of cellulases for cell wall disruption was not successful; however, disruption of the dry biomass using a vibratory mill was very effective for the hydrolysis of the intracellular starch by amylases. In reaction mixtures with a solids loading of 5% (m/m), the starch of milled cells was hydrolyzed by fungal amylases, without a prior gelatinization step, to a final respective glucose yield and concentration of 99% and 16 g.L−1 within 5 h. The increase of the solids loading to 25% (m/m) resulted in a glucose yield and concentration of 75% and 70 g.L−1, respectively, within the same reaction time.

Introduction

Microalgae have been proposed as feedstock to produce energy, materials for industrial applications, as well as high-value products based on their general chemical composition, which includes proteins, lipids, and carbohydrates [1]. Although much attention has been given to biodiesel production from microalgae [2], recent studies have also focused on the carbohydrate fraction of these microorganisms, such as starch, that can be enzymatically hydrolyzed to glucose [3,4]. This sugar has several applications in biochemical and chemical processes and can be used as a precursor of platform molecules to produce energy and chemicals [5].

Microalgae starch accumulation is triggered under nutritional stress, such as nitrogen or sulfur limitation [6,7]. However, as nutrient deficiency may have a negative impact on cell growth, a careful evaluation of the cultivation conditions must be conducted to obtain starch-rich cells.

The processing of starch-rich microalgal cells to obtain glucose syrups is hindered by the presence of a rigid cell wall, which prevents access to the intracellular starch. Even though several methods, based on either chemical or physical treatments, have been proposed for the rupture of this structure, the cell wall rupturing remains an open issue [8,9]. This is one of the most energy-intensive steps on the downstream processing of microalgae for the recovery of intracellular products and its effectiveness is highly dependent on the targeted molecule and the studied species [10,11].

Enzymatic hydrolysis has been frequently studied to obtain glucose from microalgae of the genus Chlorella as it would allow both the rupture of the cell wall and the production of glucose in a single step [12]. Cellulases are commonly proposed enzymes for such a process as cellulose has been thought to be one of the main components of the Chlorella cell wall [13]. However, both the effectiveness of this enzyme for rupturing the Chlorella cell wall [14,15] and the very presence of cellulose in this structure are controversial, with some studies indicating the complete absence of this molecule in the cell wall of Chlorella [16,17].

Once the cell wall is ruptured, amylases are commonly used to hydrolyze the intracellular starch and produce a glucose syrup [18,19]. In the plant starch industry, this hydrolysis is usually preceded by a gelatinization step for the disruption of the structure of the starch granule [20]. However, no studies so far have addressed how the pretreatment step necessary for cell wall rupture of microalgae may affect the structure of the intracellular starch and how this would reflect in the need for a gelatinization step in the microalgal processing into glucose.

The aim of the present work was, therefore, to efficiently trigger starch accumulation during the cultivation of the microalga Chlorella sorokiniana and to process the starch-rich biomass to obtain glucose syrups. The algae biomass was directly submitted to enzymatic hydrolysis with cellulases and amylases to assess the effectiveness of cellulases to disrupt the cell wall. Alternatively, freeze-dried algae biomass was submitted to physical treatment by milling before hydrolysis with amylases, without a gelatinization step. The effect of high solids loadings in the hydrolysis yields was evaluated.

Section snippets

Cultivation and storage conditions

The cultivation of Chlorella sorokiniana (UTEX 1663), a green spherical microalga, in Bold's Basal Medium (BBM) and the cell growth measurements by cell counting and dry weight were done as previously reported [21]. Cells were harvested by centrifugation at 8000g for 5 min and freeze-dried at a pressure up to 1.65 mbar for 24 h (Christ Delta 1-24 LSC, Christ, Osterode am Harz, Germany) before being stored in a freezer at −4 °C. For comparison, freshly collected cells, characterized in terms of

Cultivation of C. sorokiniana for starch accumulation

The green microalga Chlorella sorokiniana was grown in a one-stage batch process and its cell and dry weight concentrations and glucose content were assessed during 25 days.

As shown in Fig. 1a, the cell concentration increased up to the 10th day of cultivation and cells entered the stationary phase from this time onwards. However, the dry weight and the glucose content of the algal biomass showed an increasing trend up to the 20th day of cultivation; the glucose content increased over five

Conclusion

Nitrogen starvation was able to trigger the accumulation of starch by the microalga Chlorella sorokiniana in a one-stage batch process. Cellulases were not able to disrupt the cell wall of the microalgal starch-rich cells, resulting in low hydrolysis yields even after gelatinized algae biomass samples were used as substrate. However, milling of the freeze-dried cells resulted in a microalgal biomass highly susceptible to the enzymatic hydrolysis with fungal amylases without the need of a prior

CRediT authorship contribution statement

Marcella Fernandes de Souza:Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft.Marcoaurelio Almenara Rodrigues:Formal analysis, Writing - review & editing.Suely Pereira Freitas:Formal analysis, Writing - review & editing.Elba Pinto da Silva Bon:Formal analysis, Writing - review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was financed by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) project “Microalgae: production, characterization, and fractioning to obtain biofuels and bioproducts with bioactive potential” [Grant Number 4074812013].

No conflicts, informed consent, or human or animal rights are applicable to this study.

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