Identification and effect of ozone and ultrasound pretreatments on Desmodesmus sp. and Tetradesmus obliquus proteins

1 Identification and effect of ozone and ultrasound pretreatments on Desmodemus sp. and Tetradesmus obliquus proteins R. M. González-Balderasab, S.B. Velásquez-Ortab, M. Felixc, C. Bengoecheac, I. Yañez Nogueza, M.T. Orta Ledesmaa,*. aInstituto de Ingeniería, Coordinación de Ingeniería Ambiental, Universidad Nacional Autónoma de México (UNAM), Instituto de Ingeniería UNAM, Circuito Escolar s/n, Ciudad Universitaria, Delegación Coyoacán, México, CDMX., C.P. 04510. bSchool of Chemical Engineering and Advanced Materials, Newcastle University, Newcastle upon Tyne NE1 7RU, UK. cDepartamento de Ingeniería Química, Universidad de Sevilla, Escuela Politécnica Superior, 41011, Sevilla, Spain. *Corresponding autor at: Coordinación de Ingeniería Ambiental UNAM, Circuito Escolar s/n, Ciudad Universitaria, Delegación Coyoacán, México, CDMX., C.P. 04510, Tel: +52- 555-623-3600 x 3672, Fax: +52-555-616-2164, E-mail address: [email protected]. 2 Abstract Either ozone or ultrasound pretreatment were applied to identify and evaluate their effect over the protein fraction of Desmodesmus sp. and Tetradesmus obliquus biomass cultivated in wastewater. Studies of SDS-PAGE, protein solubility, sulfhydryl and disulphide bond contents, and DSC were performed to evaluate the effect of each pretreatment on abundance, structure, and thermal properties of the protein concentrate obtained. Seven proteins were identified in Desmodesmus sp. biomass, while nine proteins were identified in Tetradesmus obliquus. The proteins identified may give a high add-value to microalgae biomass because they can find important applications in agricultural, food, and biomedical areas. On the other hand, the effect of pretreatment on these microalgal proteins mainly depends on protein structure. Desmodesmus sp. proteins showed a content of S-S groups an order of magnitude higher than Tetradesmus obliquus. This results in a higher glass transition temperature (Tg) for Desmodesmus sp. proteins (249 °C) than for Tetradesmus obliquus proteins (188 °C). On the contrary, the Tetradesmus obliquus native structure proteins tend towards cross-linking and protein-protein aggregation, as their primary structure has a high content of Methionine, Tyrosine, Tryptophan, Histidine, and Phenylalanine. Either ozone or ultrasound pretreatment, induces a Tg reduction in both Tetradesmus obliquus and Desmodesmus sp. proteins, however, this reduction is especially remarkable for Desmodesmus sp. proteins (drop in Tg of about 80 °C). The results showed that proteins in Desmodesmus sp. and Tetradesmus obliquus are highly resistant to pretreatment due to the nature of their biochemical structures which imply an excellent quality for protein valorisation, even after application of highly disruptive pretreatments. 3 Keywords: Ozonation; Ultrasonication; Proteins identification; Desmodesmus sp., Tetradesmus obliquus. Abbreviations DSC____Differential Scanning Calorimetry SDS-PAGE_____Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Tg____Glass Transition Temperature Td____Denaturation Temperature ΔHd____Denaturation Enthalpy Met____Methionine Tyr____Tyrosine Trp____Tryptophan His____Histidine Cys____Cysteine Phe____Phenylalanine PHA’s____Polyhydroxyalkanoates 4 1. Introduction Microalgae proteins are valuable due to can be applied as nutritional, functional, and therapeutic commodities [1]. Proteins are characterized by hydrodynamics properties like viscosity, coagulation, gelation, and bioactive properties [2, 3]. Proteins are also utilized because of their functional properties including solubility, wettability, encapsulation, and viscosity enhancement [2, 4]. Moreover, proteins can stabilize interfaces for example in foams or emulsions, and are surface-active [2, 5]. As one of the major components of the microalgae biomass (up to 50% [w/w]), proteins are expected to perform an important role in microalgae biorefinery since they have numerous biological properties [6, 7]. However, one of the major drawbacks that restrict the mass production of proteins, is that microalgae cells own a though cell wall that decreases the extraction of cell contents [7, 8]. Therefore, disruption methods that target cell walls are required to extend microalgae protein commercial applications [9]. For a given intracellular component such as proteins, an ideal disruption pretreatment have to selectively release the targeted component while using the lowest possible energy, minimizing the degradation of proteins and achieving maximum disruption yields which reduce the use of high-cost reagents and the production of liquid wastes [1, 7, 9, 10]. In this regard, ozone and ultrasound pretreatment have showed high cell disruption and protein extraction efficiency [11]. Ozonation induces microalgal cell lysis through cell pre-oxidation which facilitates the extraction of intracellular material [12] whilst ultrasound pretreatment causes high cell disruption which induces the release of proteins to the aqueous phase [11]. Gonzalez-Balderas et al, [11] found that ozone may reach recovery yields of proteins up to 43% under an energy consumption of 30.64 kWh/kg dry biomass [11]. Whilst ultrasound pretreatment achieved a maximum protein recovery of 97 ± 0.4% [11]. Ultrasound has been 5 tested also for Nannochloropsis oculata achieving protein yields up to 75% [13]. Moreover, it was found that both pretreatments enhance protein recovery by increasing solvent efficiency from 3.7 g protein/L reagent using untreated biomass to up to 18.9 and 25.6 g protein/L reagent after ozone and ultrasound pretreatment, respectively [11]. Protein extraction time was also decreased by up to 87.5%. These results indicated significant intensification in the microalgae biorefinery pretreatment process as compared to other methods such as bead milling, pulsed electric field, and alkaline oxidation [14]. In addition, ozone and ultrasound pretreatments allow protein recovery from wet biomass which is an ideal solution to eliminate the costs and impacts that are associated with the dewatering and drying processes [15]. Sebii et al, [2] studied the effect of ultrasound pretreatment on the physicochemical, surface, and thermal properties of date palm pollen protein concentrate. They found that the thermal properties of proteins are markedly influenced by ultrasound pretreatment since the denaturation temperature and enthalpy associated decreased significantly. This approach is convenient in protein application as it seems that no chemical modifications are necessary to reach heat stability of a protein [2]. However, as microalgae proteins are sensitive to denaturation, in downstream processing is of vital importance to select a disruption method to rupture the cell wall and preserve protein functionality at the same time [12, 16]. The extent methods used for cell disruption may bestow distinct properties in bioactivity, digestibility, emulsification, and foaming of the final protein product [1]. Therefore, when developing a downstream process to produce microalgae-based protein products the end application should be considered [1]. To ensure the development of effective cell disruption methods for maximum recovery of microalgae proteins, it is necessary to study the pretreatment effect on both protein recovery and functional properties [16]. To the best of 6 our knowledge, there is no literature related to the Desmodesmus sp. and Tetradesmus obliquus protein identification or microalgae protein viability after ozone or ultrasound pretreatment. Based on these necessities, ozone or ultrasound pretreatment was applied in this study to Desmodesmus sp. and Tetradesmus obliquus biomass cultivated in wastewater to extract the microalgae proteins and study the effect of both pretreatments on them. The pretreatment effect was studied based on abundance, structure, and thermal properties of the protein concentrate obtained with a focus on protein valorisation. For this purpose, studies of SDSPAGE, protein solubility, sulfhydryl and disulphide bond contents, and DSC were performed. Additionally, protein identification was carried out with the purpose to give added-value to microalgae biomass. 2. Materials and Methods 2.1. Microalgae samples The microalgae used, Desmodesmus sp., were native microalgae isolated from the artificial lake ‘‘Lago Nabor Carrillo’’ located in Texcoco, Mexico. The microalgae, Tetradesmus obliquus (Turpin) Kützing (S), was provided by Instituto Politecnico Nacional, Mexico City, Mexico. Both microalgae biomass, Desmodesmus sp. and Tetradesmus obliquus, were grown using the method reported by González Balderas et al. [15]. For this purpose microalgae biomass was inoculated in 10 L reactors at room temperature (20 ± 3 °C). Municipal wastewater was used as culture medium (85 ± 16 mg/L of NH4+-N, 544 ± 5.5 mg/L of COD, 46 ± 4.5 mg/L of ortho-PO4-3, and 47 ± 1.5 mg/L of total phosphorus). Reactors were submitted under light/dark periods of 12:12 h, the light was provided by LED lamps (1620 lumen). After 27 days of cultivation, the biomass was harvested by 7 sedimentation, then oven-dried at 40 ℃ for 12 h and finally stored at 4 ℃ until pretreatmet. To determine the approximate composition of the biomass cell counts were done using a Neubauer counting chamber and microscope (AX10 Lab A1, Zeizz, Jena, Deutschland). The Desmodesmus sp. dominant culture was composed of 90% Desmodesmus sp., 5% Cyanobacteria sp., and 5% Mychonastes sp. The Tetradesmus obliquus dominant culture was composed of 90% Tetradesmus obliquus, 5 % Cyanobacteria sp., and 5% Nannochloropsis sp. During cultivation, wastewater was used as control. All samples used for analyses were performed in triplicate. The chemical method reported by Safi et al. [17] was used to determine the content of proteins in the microalgae biomass. Desmodesmus sp. biomass was composed of 660 ± 56 mg/g of proteins, whilst Tetradesmus obliquus biomass was composed of 680 ± 12 mg/g of proteins. All experiments were done using the same batch of microalgal biomass to ensure consistency across experimentation. All analyses were performed in triplicate 2.2. Microalgae biomass pretreatment Before pretreatments, microalgae biomass was re-diluted in distilled water to form a suspension. 2.2.1 Ozone pretreatment Ozonation was conducted by applying the method reported by González-Balderas et al. [11]. For this purpose an ozone generator (Labo76, Emery Trailigaz, Wayne, NJ, US) with a production capacity of 19 g O3/h was used. For ozonation a 250 mL Erlenmeyer flask containing a 25 mL microalgae suspension was used, the ozone flow rate applied was 0.5 L/min. The Iodometric method was applied to determine the ozone concentration in the gas 8 phase [18]. Ozone dosage (OD) was calculated according to equation (1) after determining the ozone concentration in the gas phase. OD=𝐺𝐺𝐺𝐺𝐺𝐺 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝐺𝐺𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 �𝑚𝑚𝑚𝑚 𝐿𝐿 ��×𝑂𝑂𝑂𝑂𝑐𝑐𝑐𝑐𝑐𝑐 𝑓𝑓𝑓𝑓𝑐𝑐𝑓𝑓(𝐿𝐿𝑚𝑚𝑐𝑐𝑐𝑐 �)×𝑐𝑐𝑐𝑐𝑚𝑚𝑐𝑐(𝑚𝑚𝑐𝑐𝑐𝑐) 𝑆𝑆𝑆𝑆𝐺𝐺𝑆𝑆𝑐𝑐𝑐𝑐𝐺𝐺𝑐𝑐𝑐𝑐𝑐𝑐 𝑣𝑣𝑐𝑐𝑓𝑓𝑆𝑆𝑚𝑚𝑐𝑐 (𝐿𝐿) (1) Ozone flow (0.5 L/min) and suspension volume (0.025L) remained constant. 2.2.2. Ultrasound pretreatment Ultrasound pretreatment was conducted applying the method reported by González Balderas et al. [11]. For this purpose, a 100 W, 42 kHz (2510-MT, Branson, Hampton, NH, US) bath was used, experiments were carried out in 100 mL Erlenmeyer flasks. The pretreatment was carried out in flasks containing 25 mL of a microalgae suspension, immersed in the ultrasound bath, the temperature in the bath was controlled at 3 ± 1.5 °C using ice-water. To calculate the dissipated acoustic power applied to the microalgae solution during the study, the temperature increase during the first 30 s of the experiment was determined. Based on this, the 42-kHz and 100 W ultrasound bath supplied an intensity of 0.32 W/mL. To apply the desired energy to the microalgae suspension, ultrasonication time was calculated according to the following equations, using the output power (100 W) of the equipment and the concentration of biomass suspension. The relationship between the energy applied during pretreatment and the mass (kg) pretreated was calculated according to equation 2: 𝐸𝐸(𝑘𝑘𝑘𝑘ℎ)= �𝑘𝑘𝑘𝑘ℎ𝑘𝑘𝑘𝑘 𝑑𝑑𝑑𝑑𝑑𝑑 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 ��×(𝑘𝑘𝑘𝑘 𝑏𝑏𝑏𝑏𝑚𝑚𝑑𝑑𝑏𝑏𝑏𝑏𝑚𝑚𝑘𝑘𝑏𝑏𝑚𝑚 𝑝𝑝𝑑𝑑𝑚𝑚𝑝𝑝𝑑𝑑𝑚𝑚𝑏𝑏𝑝𝑝𝑚𝑚𝑑𝑑) (2) 9 Finally, the energy E(kWh) is equal to the power P in watts (W), times the time period t in hours (hr) divided by 1000, therefore: 𝑝𝑝(ℎ)= 𝐸𝐸(𝑘𝑘𝑘𝑘ℎ)×1000 𝑃𝑃(𝑓𝑓) (3) As a result we know the ultrasonication time needed to apply the desired energy to the microalgae biomass in suspension. Ultrasonication time during pretreatment can be found on supplementary material (Table 1). After either pretreatment, microalgae suspensions were lyophilized to preserve protein integrity and stored at 2 ℃ until used. According to previous works, where ultrasound and ozone pretreatments efficiency for lipids, carbohydrates, and proteins recovery was studied, the direct influence of pretreatment conditions applied on the protein recovery process after either pretreatment was diverse for both species of biomass (Desmodesmus sp. and Tetradesmus obliquus). Therefore, to study the effect of these pretreatments on the microalgae proteins recovered, the experimental parameters applied during pretreatment for each species of biomass were those that showed good protein recovery efficiency [11, 19]. Table 1 Experimental parameters applied during ozone and ultrasound pretreatment of Desmodesmus sp. and Tetradesmus obliquus biomass. 16 flow of the endothermic transition. Finally, the ΔHd was the area underneath the peak from the thermal curve. All analyses were performed in triplicate. 2.5. Data analysis All samples used for analyses were performed in triplicate. Variability was expressed as standard deviations whose values were plotted in the parameters calculated. Average and standard deviations were obtained using Excel 2016 (Microsoft Corporation, Redmond, WA, USA). 3. Results and discussion 3.1 Protein identification In Desmodesmus sp. biomass, seven proteins were identified, among which the following may be highlighted: ATP synthase beta subunit, ribulose bisphosphate carboxylase long chain, photosystem II protein D2, and glyceraldehyde-3-phosphate dehydrogenase, (Table 2, Supplementary material). These proteins have important applications as biological markers, treatment for mycobacterial infections, and promoters in the production of isoprene, pinene, and polyhydroxyalkanoates (PHA’s), according to the US patent database and the NCBI [27]. In Tetradesmus obliquus biomass, nine proteins were identified, among these are ribulose bisphosphate carboxylase long chain, elongation factor Tu chloroplast, chlorophyll a/b chloroplast binding protein, and beta chloroplast subunit, (Table 2, Supplementary material). These proteins have applications as biological biomarkers for the detection of diseases and Hg soil contamination, precursors in the formation of biofilms and, as a treatment to mycobacterial infections, according to the US patent database and the NCBI [27]. It is important to highlight that studied microalgae biomass were cultivated in municipal 17 wastewater which may reduce protein application. However, the application of ultrasound or ozone pretreatments for cell disruption and protein extraction guarantees the destruction of potentially hazardous substances. Both pretreatment, ultrasound and ozone, have the capability to degrade micropollutants, destroy pathogenic microorganisms, and antibiotic resistance genes from wastewater. Therefore, both pretreatments may reduce considerably the concentration of residues and unwanted contaminants such as pathogens and pharmaceuticals contained in protein samples [28-30]. Results showed that protein fractions may give a high added-value to microalgae because they have important applications in agricultural, food, and biomedical areas. 3.2 Effect of pretreatment on microalgae proteins 3.2.1 Electrophoretic protein profile Fig. 1 and Table 3 supplementary material shows the effect of both pretreatments on the SDS-PAGE profile and densitometric analysis of proteins. In Desmodesmus sp. five sets of bands were identified, which are located at 54, 43, 39, 36, and 25 kD, respectively, Fig. 1a. Ozone pretreatment induced protein degradation since the relative concentration of proteins decreased, and degradation increased as contact time raised, Table 3 supplementary material. The relative concentration of proteins was reduced after 5 minutes of contact time for the bands at 50, 36.6, and 25 kD, these bands become practically imperceptible after 15 minutes of contact time. Also, the relative concentration of degraded protein increased considerably and only protein degradation adheres to the lane edges can be observed, Fig. 1a. According to the literature, the most sensitive amino acid to ozone is tryptophan which is degraded to formylkynurenine and, finally to kynurenine by hydrolysis [31]. Tyrosin is oxidized to an ortho-quinone and the ring is cleaved into aspartic acid by further oxidation. For the phenylalanine ring, the same fate as tyrosin can be expected although this reaction is 18 much slower in comparison to tyrosine. All amino acids and free amines (methilamine, dimethilamine, trimethilamine, butilamine) show great reactivite to ozone oxidation. However, the major aromatic amino acids, the sulphur-containing amino acids as well as the aliphatic amino acids arginine, lysine, proline, and histidine are especially sensitive to oxidation. The general amino acid reactivity towards ozone reported in literature is Tryptophan > Methionine >> Cystine >> Tyrosine >>> Phenylalanine [31, 32]. The same effect observed for ozone pretreatment was observed in DU4, which is the sample that was pretreated with ultrasound at the highest applied energy (200 kWh/kg). The densitometric analysis of these bands was omitted, as protein content is too low and cannot be estimated efficiently through densitometric analysis. In ultrasound pretreatment, energy applied induced reduction in the apparent concentration of proteins and an increase in degradation as energy applied raised, (Table 3, supplementary material). The most susceptible proteins to ultrasound pretreatment were at 39 and 36 kD since these were completely degraded as applied energy raised, Fig.1a. Under these same conditions (applied energy increased) proteins at 25 kD increased their apparent concentration, but this might be attributed to fragmentation of larger proteins. Biomass concentration also showed an important effect on proteins, DU1 and DU4 profiles indicated that an increase in biomass concentration raised protein degradation. This is because ultrasound pretreatment becomes more efficient as biomass concentration increases from 25 to 75 g/L, since as biomass concentration increases the chances of collision between cells rise, which enhances the shear between the cells, leading to an increase in cell disruption [11]. 19 Fig. 1. SDS-PAGE profile of no pretreated ozone and ultrasound pretreated proteins of a) Desmodesmus sp. (D) and b) Tetradesmus obliquus (T). STD: standard, NP: no pretreatment, O3: ozone, U: ultrasound. A representative choice from a pool of results was make. The concurrence of protein aggregation can be concluded in Tetradesmus obliquus proteins after observing the SDS-PAGE profile and densitometric analysis, (Fig. 1b and Table 3, Supplementary material). The protein band at 150 kD for not pretreated, ozone, and ultrasound pretreated biomass confirmed it. Protein susceptibility to aggregation may be driven by the native structure, a degraded or altered structure, and a partially or fully deployed structure [33]. Moreover, the formation of non-covalent aggregates involves disruption of weak forces, such as hydrogen bonds, hydrophobic, electrostatic, and Van der Waals interactions, leading to protein denaturation and formation of new intermolecular bonds [34]. Covalent aggregates are formed by disulphide bonds through free thiol groups or formation of non-disulphide bonds such as dithyrosine formation [35]. In ozone pretreatment, oxidation process of certain amino acids such as Tyr, Cys, Trp, Met, His and Phe induce protein aggregation [36]. Table 2, supplementary material show that Tetradesmus obliquus proteins had a higher content (⁓5%) of those amino acids in comparison to Desmodesmus sp. proteins DO31 STD kD 250 100 150 75 50 37 25 20 DNP DO33 DU1 DU2 DU3 DU4 TO 3 1 STD kD 250 100 150 75 50 37 25 20 TNP TO 3 3 TU1 TU2 a) b) 20 (⁓2%). Furthermore, it has been described that ozone transform the thiol group of cysteine to disulphides by means of crosslinking [37, 38]. On the other hand, ultrasound produces cavitation, shear stress, turbulence, high pressure and generation of radicals in solution which may also induce aggregation [37]. The collapse of cavitation bubbles produces high temperatures within the bubbles, despite temperature control [39, 40]. High temperatures within the solution have a forthright effect on the conformation of polypeptide chains at scale of their secondary, tertiary, and quaternary structure, and can lead to aggregation through unfolding induced by temperature [33]. The heat produced during pretreatment with ultrasound induces changes in the SH groups and S–S bonds through oxidation of SH groups, which may also be due to water sonolysis [40]. Several studies have reported protein aggregation as a result of sonication. Stathpulos et al. [41] observed protein aggregation during ultrasound pretreatment and found that aggregates have a high content of β-sheet, also proteins that contained a meaningful native α-helical structure exhibited an increase in the βsheet structure in the aggregates [40, 41]. Gulserren et al. [42] found that after sonication, the number of reactive thiol groups in BSA decreased. They attributed the decrease in the number of thiol groups to protein aggregation [40, 42]. Densitometric analysis of SDS-PAGE showed that the relative concentration of aggregates decreased as contact time raised in ozone pretreatment. This may be due to the strong oxidizing effect of ozone, which in turn promotes protein degradation. This effect can be explained by ozone dosage. Ozone dosage applied during pretreatment increased considerably as ozone content time increased (eq.1), therefore it is probable that the highest ozone concentration achieved after 15 minutes contact time increased considerably protein degradation. On the contrary, in ultrasound pretreatment, the relative concentration of aggregates increased as energy applied raised. Moreover, 12 bands belonging to protein sets were identified, being the bands susceptible to both pretreatments 21 those found at 45.7, 39.6, 10.6, and 9.13 kD, since these four bands disappear when biomass is subjected to either of the pretreatments, Table 3 supplementary material. 3.2.2 Protein solubility Enabling protein solubility is essential for functional properties of proteins, nonsoluble fractions do not take part in aqueous applications. Moreover, solubility is a practical measure of protein denaturation and aggregation since the solubility of proteins typically is reduced in these cases, giving a good index of its functionality [43]. Due to the different polarity of the amino acid groups, protein surfaces have net charges depending on the pH of the environment, except for when they are in the isoelectric point [44]. In this case, proteins show minimal solubility, and proteins tend to aggregate, favouring the formation of gels, but neither their solubilisation nor adsorption at interfaces, which is one of the most relevant functional properties of proteins [45]. At pH above the isoelectric point, proteins have a net negative charge, whereas, at lower pH than the isoelectric point, the proteins will have a net positive charge. In both cases, significant changes in the protein surface will result in intermolecular repulsion, and protein solubility increases [46]. Fig. 2a shows the effect of both pretreatments, ozone and ultrasound, on the solubility of Desmodesmus sp. proteins. Both pretreatments increase protein solubility by approximately 10% at acidic pH, compared to untreated proteins (from 2.6 ± 0.3% for DNP to 15.7 ± 6% and 11.05 ± 0.6% for DO33 and DU4 at pH 3, respectively). On the contrary, ozone pretreatment decreased protein solubility by 26% at alkaline pH (from 83 ± 1% to 57 ± 3.9% at pH 11), whilst ultrasound pretreatment decreased protein solubility by 20% and 40% (from 83 ± 1% to 41 ± 0.3% and 64 ± 1.6% at pH 11 for DU3 and DU4, respectively), Fig. 2a. The increased solubility of proteins at acidic pH values may be due to increased hydroxyl groups on the protein surface, 22 caused by radical reactions. Moreover, acidic pH below the isoelectric point has been related with an increase of protein functionality, due to protein opening, which can also involve an increase in protein solubility [47]. This agrees with the fact that proteins extracted after ozone pretreatment are the most soluble at acidic pH, where ozone can induce protein opening which consequently results in the loss of the native structure of the protein making it more flexible which exposed hydrophilic groups [48, 49]. This result may be confirmed by the effect of ozone pretreatment in disulphide and sulfhydryl groups (section 3.2.3). The low solubility of proteins at alkaline pH can be attributed to the formation of interpolymer complexes formed by strong electrostatic interactions. Despite ultrasound pretreatment decreased protein solubility in comparison to untreated biomass, during ultrasound pretreatment the increase in applied energy increased protein solubility. The increase in protein solubility as applied energy increase may be due to pretreatment with ultrasound could unfold proteins and break down the insoluble aggregates, which may result in the reduction of intermolecular interactions, leading to improved solubility [49]. Moreover, protein degradation increased as applied energy increased, the increased concentration of degraded protein also increased protein solubility. 23 Fig. 2. Solubility of no pretreated (NP) ozone (O3) and ultrasound (U) pretreated proteins of a) Desmodesmus sp. (D) and b) Tetradesmus obliquus (T) proteins. Data are shown as mean ± SD, derived from triplicate measurements. A representative choice from a pool of results was make. In Tetradesmus obliquus proteins, ozone pretreatments increased solubility over the entire pH range compared to untreated proteins, while ultrasound pretreatment only increased solubility at alkaline pH, Fig. 2b. Ozone pretreatments increased solubility by 10% at acidic pH (from 0.8 ± 0.2% for TNP to 9.2 ± 1% for TO33 at pH 3) and by 40% (from 47.9 ± 0.5% for TNP to 85.6 ±4% for TO33 at pH 11) at alkaline pH, while ultrasound pretreatment increased solubility by 20% (from 47.9 ± 0.5% for TNP to 68.1 ± 2.8% for TU4 at pH 11). As observed in the electrophoretic profile of Tetradesmus obliquus proteins, both aggregates and proteins, in general, are affected during ozone pretreatment. The increase in protein solubility in Tetradesmus obliquus may be also caused by conformational changes of proteins and the formation of soluble protein aggregates, from insoluble proteins during both pretreatments [50]. For all the samples, there is an increase in solubility at pH 8.9, particularly a) b) 2 4 6 8 10 12 0 20 40 60 80 Solubility (%) pH Sample Pretreatment Conditions DNP NA NA DO 3 3 Ozone 9 mg O 3 /L, 15 min DU3 Ultrasound 75 g/L, 50 kWh/kg DU4 Ultrasound 75 g/L, 125 kWh/kg 2 4 6 8 10 12 0 20 40 60 80 Solubility (%) pH Sample Pretreatment Conditions TNP NA NA TO33 Ozone 9 mg O3/L, 15 min TU4 Ultrasound 25 g/L, 200 kWh/kg 24 in the ultrasound pretreated proteins (Fig. 2b). This effect may be due to the interaction of the buffer (Tris base) with the reagent used for protein solubility [51]. Moreover, Tris base buffer is characterized for breaking electrostatic interactions (salt bridges) in the proteins, although the untreated biomass protein already shows this effect [52]. Solubility increase becomes more evident when pretreatment is applied and can be attributed to an increase in the number of ions in proteins, as a consequence of radical reactions that can be carried out during both pretreatments. 3.2.3 Disulphide and sulfhydryl groups Fig. 3 shows that either ozone or ultrasound pretreatment of biomass induced a decrease in S-S groups, whereas the content of SH groups increased for both proteins, Desmodemus spp. and Tetradesmus obliquus. The increase in SH groups can be attributed to the breakage of S-S bonds. However, the content of SH in Tetradesmus obliquus proteins is ~50% higher in comparison to Desmodesmus sp. proteins, Fig. 3a. These results agreed with protein solubility since Tetradesmus obliquus proteins were more soluble after either pretreatment than Tetradesmus obliquus no pretreated proteins or Desmodemus spp. proteins. The influence of SH groups in protein solubility has been previously observed by other authors, attributing this effect to protein polarity [53]. Moreover, this difference may be also related to the presence of some amino acids such as methionine and cysteine, being Tetradesmus obliquus proteins which have a higher content of these amino acids, this information was confirmed with the relative concentration of such amino acids in Desmodemsus sp. and Tetradesmus obliquus proteins, (Table 4, supplementary material). The relative concentration of Tyr, Cys, Trp, Met, His, and Phe is 204% higher in Tetradesmus obliquus proteins than in Desmodesmus sp. proteins. On the other hand, Desmodemus sp. proteins showed a higher content of S-S groups in one order of magnitude than Tetradesmus 25 obliquus proteins (1734 ± 56 µmol/g soluble protein for Desmodesmus sp. and 154 ± 4.7 µmol/g soluble protein for Tetradesmus obliquus), Fig. 3b. Although in microalgae proteins are mostly intracellular material, highly glycosylated proteins are important components of microalgae cell walls [54]. Microalgae cell wall proteins improve tensile strength, help maintaining wall structure, and promote plaque formation and defence responses [55]. They are also key constituents responsible for the rigidity of the cell wall, as a consequence of cross-linking with each other and other cell wall constituents such as signalling molecules, polysaccharides, and possibly receptors on plasma membranes [56]. Therefore, the high content of S-S groups on Desmodemus spp. proteins can be associated with the rigidity of their cell wall. Desmodemus species have four sporopollenin wall layers that form crosslinks among straight-chain aliphatic compounds, phenolic and aromatic groups while Tetradesmus species show a distinctive trilaminar structure [57] composed of algaenans in the trilaminar layer and cellulose in the internal wall layers [58]. Moreover, the amount of SH groups (7.8 ± 0.5 µmol/g soluble protein) in Desmodemus spp. proteins is residual as compared to the total sulphide content (3476.4 ± 111.8 µmol/g soluble protein), which points out the high occurrence of disulphide bonding. This information is consequent with the low protein solubility show in the previous experiments and suggested that Desmodemus spp. proteins were more resistant to pretreatment than Tetradesmus obliquus proteins. 32 including nutritional value, biological activity, and tech functionality [61]. Also, proteins may be applied as biofertilizers and biostimulants [62], culture media for microorganisms, [63] and bulk chemicals [64]. Because of this, it is important to found a pretreatment method that not only increases protein extraction efficiency but also improves protein functionality and avoids damage to the properties of the molecules [65]. In this manuscript cell disruption pretreatments (ozone and ultrasound) that increase protein extraction and functionality were presented. According to the results found and presented previously both pretreatments increase microalgae protein functionality to be implemented in the food, cosmetic, and pharmaceutical industry, since protein solubility increases at certain pretreatment parameters [66]. Protein solubility is of particular importance because it impacts other techno-functional properties, for example, solubility affects the proteins emulsifying ability, poor protein solubility may induce poor stability in emulsions [67]. Gel formation also requires high protein solubility, to form three-dimensional protein networks for mobility and interaction with other proteins [68]. Both pretreatmentr increased microalgae protein applicability in bioplastics production. Hence protein denaturation induced during pretreatment increased their thermoplastic properties [2]. Finally, the pretreatments applied for cell disruption may increase the potential application of microalgae proteins as biofertilizers, culture media for microorganisms, and bulk chemicals, since the decrease of molecular weight of proteins may improve their assimilation by plant and microorganisms. Future studies can be focused to study the effect of cell disruption to harvest therapeutic proteins. Moreover, as microalgal proteins showed a degree of resistance to mechanical degradation, other cell disruption treatments can be studied for recalcitrant species. 33 Conclusions Seven proteins were identified in Desmodesmus sp. biomass while nine proteins were identified in Tetradesmus obliquus. Proteins identified (i.e. ATP synthase beta subunit, ribulose bisphosphate carboxylase long chain, photosystem II protein D2, and glyceraldehyde-3-phosphate dehydrogenase) may give a high added-value to microalgae biomass because they have important applications in agricultural, food, and biomedical areas such as the production of bioplastics, the stabilization of dispersed systems or the manufacture of compatible biomaterials. The pretreatments carried out (ozone and ultrasound) had different effects on functional properties of Desmodesmus sp. and Tetradesmus obliquus microalgae proteins, which depend on protein structure. The solubility of Desmodesmus sp. proteins increased below the isoelectric point, whereas it was reduced above the isoelectric point regardless of the pretreatment applied. However, the solubility of Tetradesmus obliquus increased within the overall pH interval analysed when it was pretreated with ozone, whereas it increased only at alkali pH when it was pretreated with ultrasound. Thus, Desmodesmus sp. proteins showed a content of S-S groups one order of magnitude higher than Tetradesmus obliquus which induces a Tg 60 °C higher for Desmodesmus sp. proteins than for Tetradesmus obliquus proteins. On the contrary, the native structure of Tetradesmus obliquus proteins constitutes a tendency toward cross-linking and protein-protein aggregation, due to proteins primary structure has a high content of Met, Tyr, Trp, His and Phe. This tendency to aggregates formation induces a Tg reduction in only 10 °C after pretreatment or even an increase of 3 °C, while the Tg of Desmodesmus sp. proteins decreased by up to 80 °C after either ozone or ultrasound pretreatment. The results showed that proteins in Desmodesmus sp. and Tetradesmus obliquus are highly resistant due to the nature of their biochemical structures 34 which imply an excellent quality for protein valorisation because considerably strong pretreatments were applied. 35 Acknowledgements The authors recognize Dr. Jorge Herrera Diaz from USAI in the Facultad de Química, UNAM, for his valuable help in the proteomic analysis; thanks to Dr. Ignacio Monje-Ramirez and Prof. Rob Dixon for their valuable help in laboratory processes. The experimental work was carried out in the Laboratorio de Ingeniería Ambiental, Instituto de Ingeniería, UNAM and Electrochem Fabrication Laboratory, Newcastle University. Funding: This work was supported by the Fondo Sectorial CONACYT-SENER-Sustentabilidad Energética, project 220704 and by Consejo Nacional de Ciencia y Tecnología (CONACYT grant 385966/253075). This research has been also funded by FEDER/Ministerio de Ciencia e Innovación - Agencia Estatal de Investigación through the RTI2018-097 100-B-C21 project. 36 References [1] L. Soto-Sierra, P. Stoykova, Z.L. Nikolov, Extraction and fractionation of microalgae-based protein products, Algal Res. 36 (2018) 175-192, https://doi.org/10.1016/j.algal.2018.10.023 [2] H. Sebii, S. Karra, B. Bchir, A.M. Ghribi, A.M. Ghribi, S. Danthine, C. Blecker, H. Attia, S. Besbes, Effect of sonication on Physico-chemicla, surface and thermal properties of date palm pollen protein concentrate, Food Sci. and Technol. 106 (2019) 128-126, https://doi.org/10.1016/j.lwt.2019.02.041 [3] R. Y. Yada, Proteins in food processing, Woodhead Publishing, Cambridge, 2017. [4] J. O'Sullivan, B. Murray, C. Flynn, I. 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