Biorefinery-based approach to exploit mixed cultures of Lipomyces starkeyi and Chloroidium saccharophilum for Single Cell Oil production

: The mutualistic interactions between the oleaginous yeast Lipomyces starkeyi and the green microalga Chloroidium saccharophilum in mixed cultures were investigated to exploit possible synergistic effects. As a matter of facts, microalga could act as an oxygen generator for the yeast, while the yeast could provide carbon dioxide to microalga. A lignocellulosic hydrolysate from steam ex-ploded Arundo donax (Giant reed) was used as low cost feedstock. The overall lipid content and lipid productivity obtained in the mixed culture treating the hydrolysate of Arundo donax were equal to 0.081 g lipid .g biomass-1 and 37.2 mg lipid .L -1 .d -1 , respectively. They represented promising results if compared to the model systems where synthetic media were used. This study provided new input for the integration of Single Cell Oil (SCO) production with agro-industrial feedstock and the fatty acid distribution mainly consisting of stearic (C18:0) and oleic acid (C18:1) allows promising applications in biofuels, cosmetics, food additives and other products of industrial interest.


Introduction
A sustainable economic growth, devoted to the future generations, requires long-term available resources for industrial production, in terms of raw materials and energy. A biorefinery-based approach, aimed to the conversion of low cost feedstocks into marketable chemicals, fuels and products, has consequently to be preferred [1]. So far, several strategies have been explored to enhance the productivity and competitiveness of microbialbased processes and to improve simultaneously the biorefinery efficiency [2]. Particular attention has been devoted to the Single Cell Oil (SCO) production or microbial oil synthesis used as supplier of functional oils and for biodiesel [3] but the high fermentation costs make this production still undeveloped industrially. Therefore, the identification of substrates able to reduce the costs can be considered a solution that positively affects the related industrial implementation as well as the identification of strategies able to contribute positively in the same direction. Among them, co-culturing oleaginous yeasts and microalgae has been studied in the recent years for enhancing Single Cell Oil productivity by utilizing minimal resources in various fields such as wastewater treatment, biogas saccharophilum strains with a real system represented by Arundo donax hydrolysate previously subjected to a steam explosion pre-treatment. The incidence of inhibitors and volatile organic acids were also evaluated.

Strains
The oleaginous yeast Lipomyces starkeyi (DBVPG 6193) was supplied by the Dipartimento di Biologia Vegetale di Perugia, Italy. The strains were maintained at 5 °C on a YPD agar slants and then transferred in YPD medium for the seed culture contained (per liter): yeast extract 10 g, peptone 20 g, D-glucose 20 g and agar 20 g, when required. Both media were sterilized at 121 ˚C for 20 minutes before use.
The green microalga Chloroidium saccharophilum strain 042 was supplied by the ACUF microalgae collection (http://www.acuf.net) of the Department of Biology, at the University Federico II of Naples, Italy. Single colonies were picked up from the plates and suspended in BBM medium. Tubes and plates were grown at 25 °C under continuous light supply (100 μmol photons.m -2 .s -1 ).

Culture media and operating conditions
Lipomyces starkeyi and Chloroidium saccharophilum were grown in 500 mL Erlenmeyer flasks with an initial volume of 150 mL which contained (g/L): KH2PO4 (1.0), MgSO4·7H2O (0.5), (NH4)2SO4 (2.0), yeast extract (0.5), glucose (10.0) (YEG). The culture flasks were inoculated, separately and simultaneously, to achieve the initial cell density of about 3.0·10 6 Cells.mL -1 for both microorganisms. The pH was adjusted to 6-6.5 and prior to inoculation, the culture medium was sterilized at 121 °C for 20 min. As seed medium was also chosen (2 g.L -1 ) and glucose (10 g.L -1 ). The initial pH was adjusted to 6-6.5. The flasks were incubated at 25 °C and under continuous light intensity equal to 100 μmol photons.m -2 .s -1 . All chemicals were purchased from Sigma-Aldrich. Chloroidium saccharophilum and Lipomyces starkeyi were incubated into 500 mL Erlenmeyer flasks containing 150 mL of medium. The culture flasks were inoculated, separately and simultaneously, to achieve the initial cell density of about 3·10 6 Cells.mL -1 for both microorganisms.  (1) where t is the residence time in minutes, T is the temperature of pre-treatment, 100 is the reference temperature and 14.75 is the arbitrary constant.
Pretreated Arundo donax was mixed with distilled water (pH 5.2) to obtain a solution with 5% w/v solid content and was then treated with commercial enzymes purchased from Sigma-Aldrich consisting of cellulase from Trichoderma reesei ATCC 26921 (15 FPU/g of cellulose) and β-glucosidase from Aspergillus niger (30 CBU/g of cellulose). Cellulase activity was measured following the NREL filter paper assay [25] and reported in filterpaper units (FPU) per milliliter of solution. β-glucosidase activity was measured using the method described by Wood & Bhat [26] and reported in cellobiase units (CBU). Enzymatic hydrolysis was carried out at 160 rpm and 50 °C for 48 h (Minitron, Infors HT, Switzerland). The initial pH was adjusted to 6-6.5. At the hydrolysate of Arundo donax (ADH), 8 mL of phosphate buffer (0.2 M) were added, then it was inoculated with Lipomyces starkeyi and Chloroidium saccharophilum alone and mixed to achieve the initial cell density of 3.0·10 6 Cells.mL -1 for both microorganisms.

Analytical methods
Measurements of pH were made by an inoLab® Multi 740 Multimeters pH-meter (WTW).
The biomass concentration was monitored with a Shimadzu UV6100 spectrophotometer (Japan) and by measuring turbidity of liquid samples at 600 and 680 nm. Microbial biomass (g.L -1 ) was determined by filtering 2-3 mL of culture over pre-weight PES filters (0.45 μm; Sartorius Biolab, Germany). The retained biomass on filters was washed, dried at 105 °C for 24 hours and then stored in a desiccator before being weighed. The individual cell counts of yeast and microalga were determined with a haemocytometer, using the microscope, due to the different appearance of L. starkeyi and C. saccharophilum being different under the microscope. After centrifugation and filtration with 0.2 μm cut-off filters, the liquid samples were analysed for residual substrate content (glucose) and soluble fermentation products (VFA, alcohols). Glucose and VFAs were analyzed by HPLC (LC2010, Shimadzu, Japan), equipped with a refractive index detector (RID-20A, Shimadzu, Japan).
The total concentration of phenolic compounds was determined using Folin-Ciocalteu assay [27]. A simple method based on UV spectra was followed for the estimation of total furans (furfural and hydroxymethylfurfural) in the hydrolysates [28]. Lipids were extracted following a method adapted from Bligh and Dyer [29]. The samples were stirred in a CHCl3/CH3OH mixture (2:1 w/v) over 24 hours, and the oleaginous biomass was filtered off and washed with additional CHCl3. This procedure was repeated three times.
The solvent was then removed by evaporation under N2 stream. The total lipid concentration was estimated by gravimetric method. To calculate the lipid concentration of the cells, they were dried to a constant weight in the oven at 80 °C. The lipids extracted were subjected to transesterification reaction in a stirred container at 60 °C for 10 min, using NaOH (1% w/v) as catalysts and using methanol as reagent. The samples were dried by N2 stream and subsequently 1 mL of heptane was added for the analysis. The content of fatty acid methyl esters was determined by gas chromatography. The GC (GC-MS 2010, Shimadzu, Japan) was equipped with a flame ionization detector and an Omegawax 250 (Supelco) column (30 m x 0.25 mm I.D., 0.25 μm). Helium was used as carrier gas (flow rate: 30 mL/min). The FAME samples were initially dissolved in 1 mL of heptane and 1 μL of this solution was loaded onto the column. The temperature of the column was kept at 50 °C for 2 min, then heated to 220 °C at a rate of 4 °C/min, and finally kept constant for 2 minutes. Methyl decanoate was used as internal standard. The peak of each methyl ester was identified by comparing the retention time with the peak of the pure standard compound.

Parameter analysis
The specific growth rate (μx) and Yield factor (Yx/s) were calculated according to Equations 2, 3 and 4: where X and X0 are the concentrations of microbial biomass at time t and at initial time, respectively. μx is the specific growth rate of microbial biomass. S0 and S are the values of substrate concentrations at initial time and during the cultivation time, respectively. Lipid content, lipid yield (Ylipid) and lipid productivity were calculated according to Equations 5, 6 and 7, respectively: 3. Results and discussion 3.1. Effects of synthetic media on C. saccharophilum and L. starkeyi growth performances in mono and mixed culture A preliminary experimental campaign was conducted in order to evaluate the behaviour of the two pure strains selected for this study, L. starkeyi and C. saccharophilum, in two different synthetic media, BBM+G and YEG, at a C/N ratio equal to 11, that was not very different from the Redfield one equal to about 7 [30], and thus avoiding initial N limitations. Tests were performed by inoculating each batch with single or mixed cultures. In this latter case, the inoculum ratio between the yeast and the microalga was 1:1. Anyway, no evidences were observed in terms of microbial biomass and cell proliferation, even changing this ratio, as already demonstrated in a previous study [31]. In addition, an increase in the microalga:yeast ratio would promote algal metabolic activity by inducing an increase in pH, that could inhibit yeast growth. On the contrary, microalgae growth would be inhibited, due to excessive nutrient consumption caused by a high yeast:microalga ratio [32]. In BBM+G, the growth of C. saccharophilum showed an initial trend correlated to the glucose consumption (Fig. 1a). This could be justified by an initial photosynthetic activity lower than the metabolism of organic carbon, that could produce an endogenic source of CO2 [31] reducing CO2-limiting effect, explaining the increase in microbial biomass once the predominantly heterotrophic metabolism has stopped in the latter part of the cultivation time (Fig. 1a). In the meantime, the reduced cell proliferation -from 2.7·10 7 to 3.6·10 7 cells.mL -1 -would presuppose an increase in size more than in number, due to a phase of lipid accumulation. This hypothesis seemed to be confirmed by the trend of microbial biomass, which increased from 0.93 to 2.3 g.L -1 . The biomass growth rate (μx), the related value of biomass productivity and the yield factor (Yx/s), equal to 0.381 d -1 , 328.6 mg.L -1 .d -1 , 0.471 g.g -1 respectively (Tab. 1), are comparable with the values obtained from Herrera-Valencia et al. and Tan and Johns [19,20], taking into account the different growth conditions. On the other hand, the single cultures of L. starkeyi did not show any growth ( Fig. 1b), in spite of the presence of yeast extract and glucose. Probably, the initial glucose consumption led to the production of ethanol and acetic acid due to a reduced activity of TCA-cycle, that, in turn, moved the culture to anaerobiosis, due to oxygen limitation in the medium, essential for glucose transport. The batch culture of L. starkeyi was exposed to transient fermentation inhibition (Custers effect), and the absence of an alternative oxygen-independent transport mechanism for glucose absorption, led to the lack of microbial growth [33]. Fig 1c shows the performances of C. saccharophilum and L. starkeyi grown in mixed cultures. From the trends associated to the cell proliferation it seemed evident the presence of a synergistic effect, as evidenced by the comparison of glucose consumption rate values. The microalga was able to provide the oxygen necessary for L. starkeyi to metabolize the glucose, which was partially assimilated by C. saccharophilum and this effect led to an increase of glucose consumption rate from 1.47 g.L -1 .d -1 for C. saccharophilum alone to 2.15 g.L -1 .d -1 for mixed culture. The final constant values in terms of cell concentration (Fig. 1c) are justified by the metabolic shift due to an imbalance in the C/N ratio, which induced lipid accumulation phase rather than cell duplication. Since the microbial biomass productivity is a result of gravimetric methodology, the relative value increased from 328.6 mg.L -1 .d -1 for C. saccharophilum alone, to 366.2 mg.L -1 .d -1 for mixed culture (Tab. 1). An increase was also observed in terms of specific growth rate, but not in terms of yield factor (Yx/s), which is likely affected by endogenous metabolism and a faster glucose consumption. In mixotrophic conditions, the microalga should be less dependent on the yeast for CO2, whereas the latter still benefits from the alga for O2 production, that is partially consumed by respiration of the alga, negatively affecting yeast growth. However, this aspect did not affect the metabolic activity of the yeast, which, on the contrary, was favored by the presence of alga (Fig. 1b, 1c). On the other hand, if comparing the max values of cells.mL -1 for C. saccarophilum alone and in mixed culture (3.6·10 7 and 5.8·10 7 cells.mL -1 respectively), it emerges that the trend is similar; therefore, there was no real competition for N or P sources nor an effective reduced production of O2 by the microalga, necessary to metabolize glucose, which would otherwise have been the reason for a reduced growth of yeast as well. Moreover, the yeast proliferation has undoubtedly induced a progressive photo-inhibition effect which could be another reason for a slowdown of microalgal growth in the final part of the test. The latter effect was associated to a limiting O2 concentration which in turn did not allow the yeast to grow further and to produce CO2 available to the microalga. Tests were carried out in an attempt to verify the behavior of L. starkeyi alone in a more suitable medium, classified as YEG since it was found to be unable to grow in a BBM+G medium although enriched with yeast extract and glucose because of a Cluster effect above discussed. The individual cultures of C. saccharophilum ( Fig. 2a) and L. starkeyi (Fig. 2b) show similar trends, confirmed also by the corresponding values of specific growth rate (μx) equal to 0.94 d -1 and 0.8 d -1 , respectively, and by the maximum value of cell concentration (cells.mL -1 ) which for the alga was equal to 3.3·10 7 and for the yeast to 3.1·10 7 . Mixotrophic activity of C. saccharophilum seemed to be confirmed by glucose consumption profile (Fig. 2a). Instead, L. starkeyi alone showed an initial lag phase (Fig. 2b). Therefore, although initially there were no limiting conditions with regard to oxygen concentration, the yeast needed an evident acclimation phase in terms of microbial biomass rather than the profile associated to the number of cells (cells.mL -1 ), that increase with linear trend from the beginning (Fig. 2b). Once the oxygen became limiting, i.e. after the third day, as observed also by the absence of further substrate consumption able to affect C/N ratio and consequently cell duplication, L. starkeyi showed a shift in metabolic activity directed on lipid accumulation. The effect of this metabolic turnover could be explained by the significant increase in microbial biomass, related to the occurrence of lipid bodies within the cell. Concerning C. saccharophilum alone, an initial phase of mixotrophic metabolism was followed by an autotrophic final phase, visible from the interruption of glucose assimilation. Also for the alga, therefore, during the final phase of the test a lipid accumulation seems to be present, but it was related to a significant increase in the cell concentration due to the autotrophic metabolism. When C. saccharophilum and L. starkeyi were grown in mixed culture of YEG medium (Fig. 2c), a synergistic effect was observed, confirming the results obtained with the enriched BBM+G medium. Therefore, the mixotrophic metabolism of the microalga and the yeast fermentation were not in competition for the organic C source, nor for N and P, but rather they benefited from each other's metabolic activity, i.e. the consequent presence of a O2-CO2 virtuous loop. This seems evident looking at the initial glucose consumption rate in case of L. starkeyi alone (Fig. 2b), and in L. starkeyi and C. saccharophilum mixed culture (Fig. 2c). The presence of O2 overproduction by the microalgal metabolism supported the fermentation and what already observed in a previous study [31] and according to what already highlighted, for example by Ling et al. [34] and Li et al. [32]. An imbalance in the microalga:yeast inoculum ratio generally motivated by their different growth rates, besides not inducing a net increase in terms of lipid productivity, can sometimes lead to an accentuation for N and P competition, and consequently to an imbalance in pH. Fig. 2c also shows that once the carbon source was depleted, the consequence was, also in this case, the stopping of cell proliferation as shown by the unmodified cell concentration. to maximize the concentration in fermentable sugars. The steam explosion represents a pre-treatment able to facilitate the access of the crystalline structure of lignocellulosic biomass to hydrolytic enzymes and to limit the negative effects just discussed. Enzymatic hydrolysis, on the other hand, has been chosen for its operating conditions, which remain less impacting compared to other hydrolytic processes, further reducing the risk to increase the production and the release of inhibitory compounds [35]. As observed in Fig.   3a, C. saccharophilum alone did not present any form of microbial growth that could be associated to the presence of inhibitory compounds. The choice of C. saccharophilum was made also in view of its high tolerance to the presence, for example, of phenols, recognizing to these latter compounds also a positive effect on the regulation of enzymatic activity, cell membrane structure and macromolecule synthesis [36,37,38]. Furans could be also a reason of inhibition since they are able to cause long lag-phase [39]. Therefore, the permanence of this regime led to the exhaustion of CO2 and subsequently of O2 source, making the culture substantially anaerobic and therefore unable to grow up. Further aspect preliminarily taken into account was on the possible inhibition due to the turbidity of the culture medium that already in other studies led to adopt a dilution factor to promote photosynthetic activity [40] involving, at the same time, a loss in organic substrates concentration. On the other hand, the inhibition could be attributed to acetate potentially present in the dissociated form at neutral pH, as in our case [41]. Russel [42] attributed the inhibitory effects of weak acids to two mechanisms: uncoupling and intracellular anion Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 30 December 2020 accumulation. For all these reasons, i.e. associated to the possible occurrence of limiting conditions in terms of CO2 or O2, and to the potential inhibiting aspects such as turbidity or to high concentration in acetate, the study of mixed culture was found to be fundamental and potentially able to answer the doubts resulting from the single culture of C. saccharophilum. Another not secondary aspect that could have been the cause of algae inhibition is C/N ratio. In fact, the use of low cost feedstock, which is motivated by the need to reduce the costs of the growth medium and at the same time by the positive effect on the increase of lipid accumulation, presents, however, a high C/N ratio that exceeds the optimal values for the algae.
Before arriving at the mixed culture analysis, an additional control culture was monitored considering L. starkeyi alone as inoculum. Fig. 3b and 4b show the relative trends in terms of metabolites (substrates and products) and microbial biomass. The lag phase associated to the first two days seemed caused by the presence of furans, which, although not in high concentration, represented a compound able to delay the growth phase. The hypothesis that the yeast inhibition is affected by the C/N ratio has been ruled out, given its ability to tolerate values of even equal to 100 [4]. The lag phase was therefore followed by a growth phase, more visible in terms of microbial biomass than in terms of cells.mL -1 , which was the result of the simultaneous assimilation of glucose, xylose and acetate. This aspect confirmed what has already been observed by Anschau et al. [43], Gong et al. [44] and Yang et al. [45] related to the potential use of this microorganism in presence of more complex feedstocks where co-metabolism is of great interest to solve inhibition problems due to volatile organic acids which are formed during the pretreatments and it should be valuable towards conversion of acetate lignocellulosic biomass materials into Single Cell Oils.
Moreover, acetate assimilation by L. starkeyi represents a positive aspect with respect to pH adjustment, but it leads to an additional demand for dissolved oxygen.
Figs. 3b and 4b show how the consumption patterns of the substrates, i.e. mainly glucose, xylose and acetate, followed a diauxic mechanism. Once the dissolved oxygen was depleted, the respiratory metabolism became fermentative. The concentration and productivity of microbial biomass (Tab. 1) were 3.7 g.L -1 and 411 mg.L -1 .d -1 , respectively. These values can be considered in line with those already obtained by Pirozzi et al. [46] taking into account that Arundo donax was submitted to different pre-treatment processes, but different by one order of magnitude when compared to hydrolysates of wheat corn, corn bran and corn stover [15,47,48]. observed in single culture. The max value in terms of cells.mL -1 for L. starkeyi and C. saccharophilum in mixed culture was 1.1·10 8 and 2.1·10 7 , respectively. These values were understandably lower than those found for mixed culture in YEG medium, respectively equal to 5.6·10 8 and 6.7·10 7 cells.mL -1 , because of faster glucose consumption and absence of inhibition phenomena.
What seemed evident, confirming what has already been observed in previous studies [31,32,49,50,51], was the presence of a synergistic effect that in our case was attributed to different processes. During the lag phase, L. starkeyi showed a latent growth phase (Fig.   3c) that induced a progressive endogenous source of CO2 able to avoid limiting conditions for the microalga growth. As observed in the single culture (Fig. 3a), the microalga didn't grow neither according to a heterotrophic metabolism, nor mixotrophic because of the various forms of inhibition just discussed. This progressive accumulation of CO2, not to the point of representing a form of inhibition for the yeast, could be the cause of a partial acidification of the culture medium, but both C. saccharophilum and L. starkeyi tolerate acid pH. This acidification was therefore counteracted by acetate consumption after the fourth day, since a pH increase is typically observed when a microorganism grows in a salt of an organic acid. The progressive consumption of the organic substrate, and thus the decrease in the C/N ratio that was crucial for the inhibition of single microalgae growth, together with the pH control due to acetate consumption to balance the potential acidification due to yeast respiration, were the positive aspects that favoured the symbiotic growth of C.
saccharophilum in mixed culture. While in terms of the final microbial biomass, the differences were not evident when passing from 4.72 to 4.14 g.L -1 for mixed cultures in YEG medium and in Arundo donax hydrolysate (Fig 2c and 3c), different was what evaluated in terms of microbial biomass productivity measured in the same time-range, and equal respectively to 611.9 and 365.5 mg.L -1 .d -1 , paying in the latter case the incidence of inhibiting phenomena that slowed down the growth phase. The microbial biomass growth rate (μx) and the yield factor (Yx/s) in mixed culture were respectively equal to 0.791 d -1 and 0.539 g.g -1 (Tab. 1). Once this synergistic effect stopped as a consequence of limiting conditions achievement in terms of O2, yeast growth rate changed, switching to a fermentative metabolism and consequently to ethanol accumulation, negatively influencing also the alga, whose growth also suffered a slowdown (Fig. 3c).
Studies about the tolerance of single microbe species to inhibitory compounds are quite limited. Several attempts have been made to promote the microbial growth by reducing toxic compounds before fermentation process with certain detoxification steps. However, the detoxification which directly leads to high cost will decrease the economy of the whole process [52]. The fact that depletions of hydrolysis degradation products were enhanced by the mixed culture mode should be regarded positively because the overall process of microbial lipid production from lignocellulosic hydrolysate can be simplified by omitting the need of detoxification step [49].

Lipid production and fatty acid distribution
Different parameters were evaluated and reported in Tab. 1, in an attempt to compare the incidence of the culture conditions adopted onto the lipid content, lipid yield and lipid productivity. The lipid content reached a maximum value of 0.081 glipid.gbiomass -1 for mixed culture in Arundo donax hydrolysate, which was higher when compared to the mixed culture in BBM+G or YEG where the same value was equal about to 0.063-0.064 glipid.gbiomass -1 .
Therefore, these values were significantly lower to those obtained by Liu et al. [49], where the maximum lipid content was 0.53 glipid.gbiomass -1 for Chlorella pyrenoidosa and Rhodotorula glutinis consortium. The explanation could be found in the differences in substrate concentration, which in the latter case, amounted from 30 to 60 g.L -1 promoting consequently an imbalance in the C/N ratio that could have favoured microbial oil accumulation phase mainly by the yeast, whose metabolic activity is positively conditioned by the increase of this ratio [4].  [55]. The degree of unsaturation in the fatty acids affects the oxidative stability of the biodiesel with SFAs being the most stable followed by MUFAs compared to the least stable PUFAs, respectively [56]. Additionally, C16:0 and C18:1 are used as food additives and cosmetics, indicating as Single Cell Oils from mixed culture using low cost lignocellulosic feedstock as Arundo donax could have several potential applications.

Conclusions
Lignocellulosic extracts have the potential to provide a complex substrate of fermentable sugars and volatile organic acids, mainly acetate, at low cost. In this study, it was shown that C. saccharophilum and L. starkeyi were able to grow according to a synergistic effect on complex substrate such as Arundo donax hydrolysate, while showing that this synergistic effect allowed to overcome the problems associated to inhibitory phenomena due to lignin or sugar degradation products and a non-optimal C/N ratio. The reason was mainly attributed to the virtuous exchange of O2 and CO2, but also to phenomena of pH regulation. The promising results in terms of microbial growth and lipid accumulation were correlated with those of cultures in less complex synthetic media. The mixed cultures, in all cases, proved to be the most performing. The operational and economic impacts associated with the introduction of a detoxification phase in an attempt to overcome the inhibition effects of the above mentioned products remain to be clarified and deepened. Single Cell Oil production from lignocellulosic biomass offers a new direction for bio-refinery approach, and it will have a great future if the above mentioned problems will be properly handled. In fact, Single Cell Oils represent intermediates for biodiesel production, polymers, biosurfactants and the control of unsaturation degree in their chain, for example by hydrogenation, could be critical to ensure selectivity and stability. The exploitation of Single Cell Oils (SCOs) related to the possibility of ensuring mono or polyunsaturation could represent an alternative to the emergence of diseases such as atherosclerosis. Therefore, a decisive step in the direction of developing an economically sustainable method for the recovery of high purity SCOs is still awaited.