*3.4. Incubation Temperature*

Lastly, the final part of this investigation aimed at assessing the best incubation temperature for the extraction of metabolites from Blastocystis cultures. This part of the experiment used samples that had undergone extraction with methanol (extraction solvent) and bead bashing (lysis technique), chosen because they proved the most suitable methods, as described above.

Two sets of triplicates of metabolite extractions from *Blastocystis* cells were trialled under the following incubation temperatures: −20 ◦C or room temperature (RT). The efficacies of the incubation temperatures were compared using C μMA/B and NA/B between the two samples calculating the ratio of RT/−20 ◦C. Results of the extractions are summarised in Figure 3a,c as C μMRT/−20 ◦C for a selected set of molecules and NRT/−20 ◦C, respectively. The triplicates produced show that incubation at −20 ◦C vs. incubation at RT produced consistent results, with no outliers (Table S3, Supplementary Information). All but one of the result medians were within 0.1 of 1 (Table S3, Supplementary Information), meaning no significant results were produced. The number of molecules extracted also produced consistent results with a CV of 0.21, but there were no significant differences between the two temperatures. Therefore, neither temperature appeared to be the more efficacious for metabolite extraction.

In addition to investigating the effect of RT and −20 ◦C incubation temperatures, a 60 ◦C incubation was also trialled. Two sets of duplicates of metabolites extracted from Blastocystis cells were included, using −20 ◦C or 60 ◦C as the incubation temperatures. The efficacies of the incubation temperatures were compared using C μMA/B and NA/B between the two samples calculating the ratio of 60 ◦C/−20 ◦C. The results of the extractions are shown in Figure 3b,d as C μM 60 ◦C/−20 ◦C for a selected set of metabolites and N 60/−<sup>20</sup> ◦C, respectively. Duplicates were executed for this test and produced consistent results. The CVs all ranged between 0.01 and 0.29, suggesting that all results were reproducible (Table S4, Supplementary Information). There were no significant differences between the different extraction temperatures. Additionally, the number of metabolites extracted produced reproducible results, with a CV of 0.14 (Figure 3d).

Overall, it was determined that temperature was not an important factor in metabolite extraction here. This means that performing the experiment at RT would be sufficient to extract metabolites from Blastocystis.

The best extraction protocol (methanno/bead-bashing/RT) gave the 1D-1H-NMR spectrum shown in Figure 4, with Table S5, Supplementary Information containing the list of the most abundant molecules identified in this spectrum. Arabinitol and formate were the most abundant molecules. However, amino acids such as alanine and leucine were also be identified, along with molecules involved in Blastocystis energy metabolism such as acetate and succinate. Small sugars such as disaccharide trehalose and monosaccharide galactitol were identified, along with the lipid membrane component sn-Glycero-3-phosphocholine. Other molecules with biological roles such as betaine and malonate were also detected. Betaine has a role in regulating osmotic stress.

**Figure 3.** (**a**) Difference in concentrations between RT (5) and −20 ◦C (6) C μMRT/−20ºC incubation temperature for triplicates A–C. Numbers below 1 indicate an increased extraction for −20 ◦C incubation. (**b**) Difference in concentrations between 60 ◦C (7) and −20 ◦C (8) incubation temperatures for triplicates A–C. Number below 1 indicate an increased extraction for −20 ◦C incubation. (**c**) Difference in the number of different metabolites extracted between RT (5) and −20 ◦C (6) incubation temperatures. NRT/−20 ◦C for triplicates A–C. Numbers below 1 indicate an increased extraction for −20 ◦C incubation (**d**) Difference in the number of different metabolites extracted between 60 ◦C (7) and −20 ◦C (8) incubation temperatures N60 ◦C /−20 ◦C for triplicates A–C. Numbers below 1 indicate an increased extraction for −20 ◦C incubation. Numbers above the bars indicate measured ratios.

**Figure 4.** Final metabolite extraction protocol optimised by this study. Methanol is used as the extraction solvent, bead bashing as the lysis technique and incubation at RT.

#### **4. Discussion**

Herein, we have described an efficient protocol to extract metabolites from *Blastocystis* ST7 in culture, thus allowing an overview of its metabolome by 1H-NMR analysis to be established for the first time. The findings can be summarized as follows: (1) methanol is a more effective extraction solvent when compared against ethanol; (2) bead bashing is a more effective lysis method than sonication; (3) incubation temperature is not a significant factor in metabolite extraction of *Blastocystis*; thus, performing the extraction at room temperature (RT) is sufficient. These data were collated to produce a series of steps to form an effective protocol to perform metabolite extraction on *Blastocystis* (Figure 4).
