Extractive disruption process integration using ultrasonication and an aqueous two-phase system for protein recovery from Chlorella sorokiniana

Abstract

Microalgae emerge as the most promising protein sources for aquaculture industry. However, the commercial proteins production at low cost remains a challenge. The process of harnessing microalgal proteins involves several steps such as cell disruption, isolation and extraction. The discrete processes are generally complicated, time-consuming and costly. To date, the notion of integrating microalgal cell disruption and proteins recovery process into one step is yet to explore. Hence, this study aimed to investigate the feasibility of applying methanol/potassium ATPS in the integrated process for proteins recovery from Chlorella sorokiniana. Parameters such as salt types, salt concentrations, methanol concentrations, NaCl addition were optimized. The possibility of upscaling and the effectiveness of recycling the phase components were also studied. The results showed that ATPS formed by 30% (w/w) K₃PO₄ and 20% (w/w) methanol with 3% (w/w) NaCl addition was optimum for proteins recovery. In this system, the partition coefficient and yield were 7.28 and 84.23%, respectively. There were no significant differences in the partition coefficient and yield when the integrated process was upscaled to 100-fold. The recovered phase components can still be recycled effectively at fifth cycle. In conclusions, this method is simple, rapid, environmental friendly and could be implemented at large scale.

Keywords: Aqueous two‐phase system; Cell disruption; Microalgae; Process integration; Proteins recovery.

Figure 1

Phase diagrams of (A) methanol/dipotassium phosphate ATPS with approximate pH range 10.4–10.8; and (B) methanol/tripotassium phosphate ATPS with approximate pH range 12.8–13.6.

Figure 2

Effect of (A) dipotassium phosphate concentration; and (B) tripotassium phosphate concentration on partitioning and yield of proteins. The optimization of the system was performed by varying the potassium salt concentration. Above dipotassium phosphate concentration of 37% (w/w) and tripotassium phosphate concentration of 35% (w/w), respectively, white insoluble complex was formed upon the addition of BCA working reagent into the sample and blocked absorbance measurement. Partition coefficient and yield were calculated using Eqs. (1), and (2), (3), respectively. The results were expressed as the means of triplicate readings (mean ± SD).

Figure 3

Effect of methanol concentration on partitioning and yield of proteins. The optimization of the methanol/tripotassium phosphate system was performed by varying the methanol concentration from 20–40% (w/w), while the concentration of K₃PO₄ was maintained at 30% (w/w). Above methanol concentration of 40% (w/w), white insoluble complex was formed upon the addition of BCA working reagent into the sample and blocked absorbance measurement. Partition coefficient and yield were calculated using Eqs. (1), and (2), (3), respectively. The results were expressed as the means of triplicate readings (mean ± SD).

Figure 4

Effect of sodium chloride (NaCl) concentration on partitioning and yield of proteins. The optimization of the methanol/tripotassium phosphate system was performed by varying the NaCl concentration from 0–5% (w/w). However, precipitation at the bottom phase was observed due to salt saturation when the concentration of NaCl was added above 3.5% (w/w). Partition coefficient and yield were calculated using Eqs. (1), and (2), (3), respectively. The results were expressed as the means of triplicate readings (mean ± SD).

Figure 5

Light microscopy image of (A) untreated C. sorokiniana (control) (1000x); and (B) C. sorokiniana after treated with integrated process (1000x).

Figure 6

(A) Partitioning efficiency and yield of proteins using recycled phase components were investigated. The recycling processes were repeated for up to 5 times. Partition coefficient and yield were calculated using Eqs. (1), and (2), (3), respectively. (B) The influence of number of recycling cycle on the concentration of proteins in the top phase. The protein concentrations were estimated by BCA protein assay. The results were expressed as the means of triplicate readings (mean ± SD).