When designing a process for protein and lipid recovery, loss or degradation of either product should be minimized. Proteins are more vulnerable to temperature and shear induced degradation. Furthermore, when associated with lipids, they promote emulsion formation, impeding lipid separation from the aqueous media. A sequential extraction where most of the protein is released first is preferable as it can minimize protein degradation as well as prevent emulsion formation that could hinder lipid recovery. Thus, the first step was to solubilize and recover proteins.
Impact of temperature and time on cell disruption and protein and lipid recovery
Prior work by Soto-Sierra, Dixon (Soto-Sierra et al. 2017) demonstrated that treating C. reinhardtii with autolysin for 4 h at 25°C was an effective method for cell lysis and resulted in ~ 20% protein release. To evaluate and optimize protein recovery after the enzymatic treatment, biomass incubation with autolysin was performed at different temperatures (25, 35, and 50°C) and extended incubation times (8, 17, and 24 h) and total protein solubilized was compared among treatments. Biomass was incubated with either autolysin or control buffer at each temperature. At each time point, biomass was centrifuged, the supernatants were collected, and total soluble protein was calculated. Results (Fig. 2a) showed that at 24 h of autolysin treatment, protein solubilization for all temperatures was significantly higher when compared to the control. A significant increase in protein solubilization of approximately 10% was observed for the control treatment at 50°C when compared to 25°C. This indicated that there was protein being solubilized by the high temperature treatment (at 50°C) rather than by autolysin, as the treatment at 50°C solubilized a significantly lower amount of protein when compared to treatments at 25°C and 35°C (data not shown). Reduced protein extractability at 50°C could be attributed to a decrease of autolysin activity at the elevated treatment temperature (Wilken and Nikolov 2016).
Protein solubilization was significantly higher at 35°C when compared to autolysin treatments at 25°C and 50°C. On average, 50.1 ± 4.2% of the protein was solubilized after autolysin treatment at 35°C. Regarding incubation time, the amount of protein solubilized was approximately 15% higher for samples incubated 24 h when compared to 8 and 18 h of incubation (Fig. 2b). Based on these results, autolysin pretreatment for 24 h at 35°C was chosen as the optimum condition for cell wall disruption and protein release. In a large-scale setting, though, 24 h incubation might result in potential contamination, protein degradation by native proteases, and oxidation (Majumdar et al. 2018). Thus, further work to optimize extraction conditions regarding protein recovery, yield, and quality is necessary.
The remaining proteins, mostly photosynthetic (RuBisCO and LHC), were most likely still stored in the chloroplast along with lipids.
Effect of enhanced autolysin treatment on lipid recovery
In previous research, Soto-Sierra et al. (2017) reported that the autolysin treatment at 25°C achieved complete cell wall disruption while lipid bodies remained attached to cell remnants in the solid fraction (pellet). To determine the effect on lipid release of increasing temperature and incubation time, samples were treated with autolysin for 24 h at 35°C. TEM images of cell pellets were taken after autolysin treatment at 25°C and 35°C. Results (Fig. 3a) showed that for both temperature treatments, the majority of lipid bodies were still trapped in the solid fraction of the cell lysate. Presumably, lipid body surface proteins and phospholipids were associating with other proteins and polar biomolecules, preventing TAGs (triacylglycerols) from being released. Even though most of the lipid bodies were still contained in the solid fraction, TEM images also show an apparent reduction of lipid body size for the biomass treated at 35°C when compared to the treatment at 25°C.
To further explore these results, lipid release into the supernatants after 24 h of autolysin treatment at 35°C was quantified. Results showed that the prolonged incubation time of the microalgae cells with autolysin induced significant cell disruption (Fig. 3a), which resulted in the release of 40 ± 2 % and 43 ± 1% of total lipids at 25 and 35°C (Fig. 3b), respectively. Interestingly, Fig. 3a shows that while autolysin-treated cells were significantly disrupted, most lipid bodies (red triangles) were still attached to the cell remnants (Kirchhoff et al. 2008). We suspect that the autolysin treatment promoted the release of surface and other polar lipids while the internal and non-polar lipid bodies remained attached to the chloroplast membranes (Fan et al. 2011).
After autolysin treatment at 35°C, ~ 50 ± 4% of total proteins were extracted while ~ 57–60% of the lipids remained in the solid fraction. Future studies should focus on developing a processing strategy for recovering each product into a separate stream once they have been released. Alternatively, extraction conditions could also be optimized for maximizing the solubilization of protein, which is the most soluble product, first, while keeping the lipids retained in the cells for their subsequent extraction.
Lipid content on isolated chloroplasts
To develop a solvent-free extraction system, a secondary treatment that promoted lipid body release and oil demulsification was needed. First, we aimed to understand why the majority of the lipid bodies were not being released after the cells were disrupted. Based on research regarding lipid body accumulation of C. reinhardtii cells (Fan et al. 2011) and previous TEM images (Fig. 3a), lipids can be stored in the endoplasmic reticulum and/or inside the chloroplast. If stored in the chloroplast, the previously characterized (Moellering et al. 2009) LDSP could be associating with other polar biomolecules inside this organelle, preventing lipids from being extracted. Determining where lipid bodies are attached after the enzymatic treatment would provide additional information regarding which cell structures need to be cleaved to release the lipids. Thus, the next step was to confirm if the lipid bodies were enclosed in the remaining chloroplasts and chloroplasts remnants. To do so, chloroplast remnants and thylakoids were isolated after autolysin treatment based on a modified protocol of the one proposed by Mason et al. (2006) and lipid content in the intact chloroplast plus thylakoids fractions were calculated.
The increase in lipid content (DW) indicated that chloroplasts and the disrupted thylakoids concentrated most of the lipids trapped in the solid fraction. Results (Fig. 4) showed that lipid content of the chloroplasts fraction was almost 70% (g lipids/g total dry weight) while the lipid content of intact C. reinhardtii cells was 49%. This is a 1.42-fold lipid concentration when compared to whole cells. Furthermore, the gram dry basis sum of chloroplast lipids plus lipids released after autolysin treatment was approximately ~ 0.38 g lipids/g which is about 90% of the total lipid content in a C. reinhardtii cell after 48 h of nitrogen depletion (Soto-Sierra et al. 2017). These results indicated that the majority of the lipids in the remaining biomass after autolysin treatment were stored in the chloroplasts. Possibly, stacked membranes in the chloroplasts were trapping lipid bodies. Furthermore, the amphiphilic nature of the chloroplasts could be reducing the interfacial tension between the aqueous solution and the lipid bodies, contributing to the stabilization of dispersed droplets and avoiding their association. The attachment is possibly made between LDSP and proteins or other polar molecules in the chloroplast. Consequently, the next treatment to be designed should target not only the LDSP (Moellering et al. 2009), but also proteins and other molecules present in the chloroplast. Thus, the next step was to design an aqueous enzymatic treatment to disrupt chloroplast remnants and LDSP, so attached lipids could be released.
Effect of a secondary enzymatic treatment on lipid release
Thus far, autolysin treatment was able to permeabilize and disrupt the cells and release 50 ± 4% of the protein by extending incubation time. To release lipid bodies from internal compartments, in this case, the chloroplasts and LDSP need to be cleaved so the lipid bodies can be released from the disrupted chloroplasts remnants. To design an efficient AEE treatment, it is crucial to make sure that proteins are being cleaved by the protease chosen. Based on preliminary data (not shown), trypsin was selected as the best fit for cleaving LDSP and other chloroplast proteins. Trypsin was selected as it can approximately cleave the ~ 260 amino acid chain of the C. reinhardtii LDSP about 20 times based on the primary structure and cleavage specificity. Trypsin treatment could also promote the release of lipid bodies attached between thylakoids by disrupting membrane stacking as it was reported in chloroplasts of plants such as spinach (Jennings et al. 1981).
Biomass was treated with autolysin and lipids were recovered as specified in “Quantification of lipid release” section. If lipids were being released from the chloroplasts, the lipid recovery in the supernatant fraction was expected to increase. Results indicated a significant increase in lipid release for samples incubated with autolysin plus trypsin treatment. Figure 5a shows that more than 30% of lipids still trapped in the solid fraction (pellet) after autolysin treatment were released by trypsin treatment. After Nile Red staining, several lipid bodies were visible in the supernatants from trypsin-treated samples (Fig. 5b) which further confirmed lipid release.
With autolysin plus trypsin treatment, ~ 73 ± 7% of total lipids stored in C. reinhardtii cells were released from the solid fraction. Several authors have also reported an increase in lipid extractability after protease treatment of diverse biological substrates, such as microalgae (Wu et al. 2017), fish (Dumay et al. 2006; Kechaou et al. 2009), maize (Tester et al. 2007), and coconut (Patil and Benjakul 2019; Senphan and Benjakul 2017). The significant increase in lipid release can be attributed, among others, to the breakdown of particular protein structures (Gbogouri et al. 2006) keeping lipids attached to the cell remnants and inside of the lipid bodies.
Protein release after secondary extraction process
Once lipid release was achieved, protein solubilization after the secondary extraction process was monitored. First, the protein release of autolysin-treated cells after incubation in buffer vs. buffer plus trypsin was compared (Fig. 6). Then, the molecular weight (MW) protein profiles (Fig. 7) were analyzed to identify proteins that remain in the cell debris/solids, solubilized, or degraded during trypsin treatment.
Results showed that the trypsin treatment caused further solubilization of 14 ± 1% of the protein, bringing the cumulative protein release after autolysin plus trypsin treatment to ~ 64 ± 6% (Figs. 2 and 6). Even though only a small amount of the protein stored in the chloroplast was solubilized by trypsin, the specific digestion was enough to release lipids stored between the thylakoid membranes. The pellet after autolysin plus trypsin treatment (Fig. 7b, lane 6, LHC arrow) showed a decrease in band intensity of a complex of proteins of MW ~ 17 to 30 kDa, which could potentially correspond to the LHC. Solubilization of these proteins was possibly induced by the trypsin digestion. After trypsin, a slight decrease in proteins of ~ 35 kDa, ~ 45 kDa and ~ 98 kDa is also apparent (Fig. 7b, lanes 6 & 7). Moreover, the gel shows that after autolysin treatment (Fig. 7b, lane 3), high molecular weight proteins (between 98 and 198 kDa) are completely solubilized. These proteins can potentially be the glycosylated cell wall proteins, which are characterized by a high molecular weight (Mathieu-Rivet et al. 2013), and were being solubilized early on after autolysin treatment. Proteins that have not yet been solubilized can be recovered from the solid fraction using a mechanical or chemical, treatment. One advantage of preserving the proteins in the solid fraction is that it allows for the selective recovery of lipids from the liquid phase while keeping most of the proteins in the solid fraction (pellet). The separation caused by the density difference between both products, could potentially decrease steps and energy involved in the extraction process allowing for recovery of each product at higher purities.
Effect of trypsin treatment on cell structure and bioproduct release
To better understand why trypsin treatment was promoting lipid release while keeping proteins in the solid fraction, the effect of autolysin plus trypsin and autolysin treatment only on lipid release was analyzed and compared by TEM imaging. Results showed that the autolysin treatment caused the disruption of the cell wall and chloroplast envelopes (Fig. 8a, A, B). Nevertheless, numerous lipid bodies were still attached to the internal portion of the thylakoid membranes (Fig. 8a, D).
For the autolysin plus trypsin-treated samples, Fig. 8b, G and H, shows an apparent decrease in membrane stacking and relaxation of thylakoid (T) membranes when compared to samples only treated with autolysin (Fig. 8a, D). This effect was previously reported (Jennings et al. 1981) when treating spinach chloroplasts with trypsin. According to Grebanier (1979), the main effect of trypsin on chloroplast membranes is to digest a small fragment from the light-harvesting protein complex. Possibly, the relaxation of the thylakoid membranes accompanied by the disruption of lipid body proteins induced the release of lipid bodies. A reduction in the amount of lipids still attached to the pellet together with the presence of empty lipid and starch bodies (Fig. 8b, G & H) after trypsin treatment confirms the abovementioned effects.
Interestingly, the autolysin plus trypsin-treated samples (Fig. 8b, E & F) showed large amounts of free starch granules in some of the TEM sections. Insoluble starch appeared to be released from the chloroplasts and sedimented at the bottom of the pellets. This is most likely due to the higher density of the starch granules (~ 1.5 g/cm3) when compared to the thylakoid fragments (~ 1 g/m3) and lipid bodies (~ 0.9 g/m3). If starch is one of the products to be recovered, the difference in density when compared to other cell components will allow this product to accumulate at the bottom of the solid phase, facilitating its recovery and further purification.
Certainly, the AEE treatment designed not only facilitates lipid and protein extraction, but also propitiates starch recovery. Further research should aim to optimize the fractionation and extraction of these three products after the enzymatic treatment.
With the primary and secondary enzymatic treatments developed, intact cells with intact cell walls were transformed into highly disrupted cells, and finally, into partially fractionated bioproducts (Fig. 9a–c).