Versatile method to obtain protein- and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae without mechanical pretreatment
Graphical abstract
Introduction
In the last few decades, interest in microalgae has increased notably due to their potential as a renewable source of bioproducts and bioenergy, as well as a biological alternative for the treatment of gaseous and liquid residual effluents in integrated processes [1]. In particular, microalgae are promising resources for protein and amino acids to satisfy the growing demand for food and feed [2], bioactive molecules [3], biofertilizers and biostimulants [4], culture media for microorganisms [5] and bulk chemicals [6]. Despite this interest, most of the applications of microalgae are currently limited to high-value bioproducts [7,8], mainly due to economic bottlenecks associated with some industrial process steps, such as harvesting, drying and cell disruption [9,10].
Several techniques have been tested for microalgal cell wall disruption, including mechanical, chemical, and/or enzymatic methods [11,12], and the choice of the method for a specific use will depend on the raw material resistance and the target biomolecules, among other economic, technical and environmental factors. In the extraction of proteins and amino acids, mild disruption methods are normally used in order to avoid damage to the molecules and to preserve their techno-functional properties, e.g., cooled bead milling, ultrasonication, pulsed electric field, enzymatic, etc. Nevertheless, these techniques are often not harsh enough to disrupt efficiently cell walls and result in low extraction yields [[13], [14], [15]], especially in recalcitrant species, which are more suitable for integrated depuration processes due to their natural resistance. Furthermore, direct extraction from fresh biomass, which avoids costly drying steps, is preferable but represents an additional difficulty for the recovery of the desired bioproducts in satisfactory yields, since cell walls maintain their rigid structure intact. Methods such as bead milling and high pressure homogenization are very effective for this purpose [16,17] but their high investment costs and energy requirements hamper their application in some cases [9,10]. By contrast, chemical and enzymatic methods require lower capital investment and operational energy consumption [11].
It has been demonstrated that highly alkaline treatments weaken the cell walls of microalgae and facilitate protein extraction [18,19], although when combined with heat these treatments can modify protein properties due to denaturation, racemization, and lysinoalanine formation [20]. Despite the possibility of structural modifications, the economic and energetic efficiency of this approach could justify its application to maximize nitrogen recovery from microalgae for use in numerous applications [21]. Furthermore, enzymatic methods that involve the use of proteases have proven to have a high efficiency in amino acid production from microalgae [6,22,23]. Nevertheless, the effectiveness of applying an alkaline treatment prior to enzymatic reaction requires further investigation [21].
In this study, we develop a versatile method to obtain proteinaceous material from microalgae after thoroughly evaluating the main variables. The method is composed of two sequential techniques, each of them characterized by high extraction yields when compared with other technological approaches [6,18,22]: (1) a first high alkaline reaction, as an initial harsh extraction, and (2) a second protease-assisted reaction, for an extensive extraction, in order to maximize the overall recovery of microalgal protein fraction. Such a comprehensive study of the different variables and the implementation of this new approach to fresh biomass from various recalcitrant species of microalgae has not been performed previously. In addition, our method is not only aimed at reaching a high overall extraction yield but also to produce different types of products with several potential uses. These include protein-enriched extracts that could be further fractionated for use as techno-functional compounds (emulsifiers and foaming agents), and/or hydrolyzed peptides and amino acids-enriched extracts, suitable for applications such as food and feed, culture media for microorganisms, bioactive peptides, bulk chemicals [12]. This approach could also be compatible with the co-extraction of other biocompounds (lipids, sugars, pigments, etc.), in biorefinery processes for the integral use of microalgal biomass. These characteristics make this an interesting alternative to existing technologies for the extraction of proteinaceous material from microalgae.
The aim of the study described here was to develop a sequential alkaline-enzymatic method to obtain protein- and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae without any supplementary pretreatment. The method was evaluated using a microalgal consortium previously isolated from a landfill leachate and the approach was also tested on other recognized recalcitrant species from different genera (C. vulgaris, N. gaditana and S. obliquus). The effect of the main variables – such as initial biomass concentration, the use of freeze-dried or fresh biomass, enzyme dosage, processing procedure (two-step and single-step, with and without pH control) and species of microalgae – were evaluated. Freeze-dried extracts obtained via different pathways of the method were characterized in total nitrogen, amino acid composition, and nitrogen-to-protein conversion factors. Additionally, a selected extract was characterized in terms of salt content, free mono- and di-saccharides, glycerol and organic acids.
Section snippets
Microalgae and culture
A non-axenic microalgae consortium was isolated from a landfill leachate by our research group in a previous study. A preliminary analysis of this consortium by optical microscopy, protein content and lipid capacity storage showed that could belong to Nannochloropsis sp. [24]. Chlorella vulgaris (SAG 211-12) and Scenedesmus obliquus (SAG 276-10) were acquired from the Algae Culture Collection (SAG), University of Göttingen (Germany). Nannochloropsis gaditana was obtained from the Marine Plant
Effect of the biomass concentration
The effect of the initial concentration of fresh biomass was evaluated in detail, through the two-step process without pH control (2Swo, Fig. 1), given its essential importance in the developed method, which was evidenced in our previous study of this approach [21]. The maximum total extraction yield of proteinaceous material from the two sequential extractions was achieved at 60 mg mL−1, with 81.2% of the total protein recovered (Fig. 2a). At biomass concentrations below 30 mg mL−1 the overall
Conclusions
A versatile and high yielding sequential alkaline-enzymatic method with several alternative pathways has been developed in this work to obtain protein- and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae species using a microalgal consortium. According to the results obtained, the pathways without pH control were recommended since those performed with pH control produce extracts with high salts content and very low of proteins. On the other hand, the pathways
CRediT authorship contribution statement
J.A. Callejo-López: Investigation, Methodology, Formal analysis, Visualization, Writing - original draft. M. Ramírez: Conceptualization, Methodology, Formal analysis, Supervision, Writing - review & editing. D. Cantero: Resources, Funding acquisition, Project administration, Writing - review & editing. J. Bolivar: Conceptualization, Methodology, Formal analysis, Supervision, Writing - review & editing.
Statement of informed consent, human/animal rights
No conflicts, informed consent, human or animal rights applicable.
Declaration of competing interest
There are no conflicts of interest to declare.
Acknowledgements
This work was financially supported by the Spanish Government (Ministerio de Economía y Competitividad) [grant number CTM2016-79089-R].
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