Metabolic changes and biochemical degradation during dark anoxic incubation of Nannochloropsis: implications for low-energy microalgal cell rupture

Abstract

Dark anoxic incubation has been identified as a low-cost method to facilitate the mechanical rupture of microalgae such as Nannochloropsis via autolysis-induced cell wall thinning. During this process, concentrated slurries of cells are incubated in the dark at an elevated temperature, to deprive them of light and oxygen. This work analyzed the integrity of proteins and lipids during dark anoxic incubation and investigated the cellular responses of Nannochloropsis through an in-depth proteomic analysis. Proteomic analysis identified enzymes associated with cellulose hydrolysis and glycolytic and fermentative pathways that are presumably activated to produce energy in the absence of light and oxygen. Progressive biochemical degradation was observed during 48 h of incubation, including the proteolysis and leakage of proteins, and the lipolysis and subsequent peroxidation of lipids. This provides further evidence of autolytic processes occurring during prolonged incubation, which can be attributed to uncontrolled action of intracellular proteases and lipases. Importantly, the resultant formation of peptides and free fatty acids will affect their use in food and fuel applications. It is therefore important to optimise the incubation time and parameters to achieve cell weakening while minimising the unnecessary degradation of biomacromolecules.

Keywords: Cell rupture; Dark anoxia; Lipolysis; Microalgae; Proteolysis.

Fig. 1

The effect of dark anoxic incubation on (a) the relative proteolysis of proteins as a % compared to day 0, b release of peptides and total protein (including peptides) from cells into the supernatant, and the (c) FFA content and (d) peroxide value (ΔPV) of the lipids. The data points and the error bars represent the average and standard deviation of duplicate measurements of triplicated experiments. Spearman correlation analysis returned (a) ρ = 0.822, p = 1E-3, (b) peptides: ρ = 0.982, p = 1E-5, and proteins: ρ = 0.928, p = 1E-5 (c) ρ = 0.928, p = 1E-5, (d) ρ = 0.928, p = 1E-5 confirming strong and statistically significant (p <  < 0.05) positive monotonic relationships with time for all aspects considered

Fig. 2

Functional groupings of proteins differentially expressed during dark anoxic incubation

Fig. 3

The effect of dark anoxic incubation time on the relative abundance of protein groups involved in the selected processes of interest. a Lipases, b cellulases, c proteases (sum of all proteases, peptidases and proteasome complex units), d redox enzymes (sum of all the enzymes/proteins having oxidoreducIe activity), (e) lipid peroxidation (sum of all lipoxygenases), f heat stress proteins (induced as a response to heat stress, with the function of these proteins mostly associated with protein folding and refolding), (g) glycolysis (proteins associated with the glycolytic pathway), h fermentation pathways (proteins identified to be involved in different fermentative pathways to maintain the redox balance of the cell), i respiration and energy generation (proteins involved in respiratory energy metabolism), j photosynthesis (proteins involved in photosynthesis including light harvesting complexes, photosystem I and II complexes and cytochrome proteins), k chlorophyll synthesis, l Calvin cycle (proteins involved in photosynthetic C fixation through the Calvin cycle). The proteins included in each of these groups and their associated data is included in supplementary information (Supplementary 2). Asterisk symbols represent statistically significant differences compared to the t = 0 time point

Fig. 4

Localisation of the main enzymes and intermediates identified in this study involved in ATP production through the glycolytic pathway. Green represents significantly upregulated enzymes, grey upregulated but not significantly, orange down-regulated but not significantly, and red significantly down regulated enzymes. Blue dashed line indicates the reactions in the lower glycolytic pathway and the rest belongs to the upper glycolytic pathway

Fig. 5

Identified enzymes and associated fermentative pathways in Nannochloropsis sp. under dark anoxia. Fermentative end products associated with the identified pathways are indicated by red boxes, with filled orange boxes representing end products that were detected during dark anoxic incubation of Nannochloropsis sp. in our previous study [8]. PPDK Pyruvate phosphate dikinase, PDH Pyruvate dehydrogenase, PYC Pyruvate carboxylase, ACS Acetate synthase, ACDC Acetyl CoA deacylase, ADH Aldehyde dehydrogenase, AcDH Alcohol Dehydrogenase, ALAT Alanine aminotransferase, CS Citrate synthase, ME malic enzyme, OGDH oxoglutarate dehydrogenase, SCL Succinyl CoA lyase, SDH Succinate dehydrogenase, FHy fumarate hydratase, MDH Malate dehydrogenase, AAT Aspartate aminotransferase, LDH Lactate dehydrogenase. Green: significantly increased. Grey: increased but not significantly. Orange: decreased but not significantly Black: not found

Fig. 6

Graphical summary of the proposed mechanistic responses of Nannochloropsis to dark anoxic incubation at 37 ℃. A The major catabolic processes occurring in cells that are deprived of light and oxygen. Red crosses and dashed arrows represent catabolic pathways that are not available under dark anoxic conditions. B Progressive physiological and biochemical effects of dark anoxic stress