Origin 2022b

The carbon source consumption and production of organic acids were analyzed by HPLC using an Aminex HPX-87H column (300 cm × 7.8 mm; 9 µm particle size; 8% cross linkage) (Bio-Rad, USA) attached to 1260 Infinity (Agilent Technologies, USA) and using a system flow of 0.6 mL/min of a 5 mM sulfuric acid as the mobile phase at 60 °C. The refraction index detector was used for detection of sugars, and the UV detector at 210 nm for organic acids. Standard solutions with known concentration of glucose (1.0 to 5.0 g/L), oxalic acid (0.2 to 1.6 mM), citric acid (1.0 to 8.0 mM), malic acid (2.0 to 16 mM), formic acid (5.0 to 40 mM), and acetic acid (10 to 80 mM) were used for quantification of the components. For plasmid stability assessment, samples of the culture, collected right before induction and each hour after the induction, were diluted, plated on LB agar, and incubated for 24 h at 37 °C. Then, at least 100 colonies from each timepoint were simultaneously replicated in LB agar with and without kanamycin. After incubation at the same conditions, the percentage of antibiotic-resistant colonies in relation to the total colonies recovered without antibiotic was calculated and defined as the plasmid stability. The results obtained, together with the cell concentration measurement, were used to build the graph using the software Origin 2022b (OriginLab, USA).

The results were expressed as the mean ± standard deviation (SD). Statistical analysis was performed using Tukey's multiple comparison test (p < .05) to determine significant differences between two groups using Origin 2022b (OriginLab Corporation). With the same software, Spearman's list‐wise correlation analysis was performed to establish associations between the results.

Data were sorted and tabulated using Excel 2016 (Microsoft, Seattle, WA, USA), and graphs were generated using Origin 2022b (OriginLab, Northampton, MA, USA). All data were analyzed with IBM SPSS Statistics 19.0 (IBM Corp., Armonk, NY, USA). Tukey’s honestly significant difference (HSD) test was used to determine the significance of differences between means (P < 0.05).

Surface hardness was measured with a Zwick/Roell Indentec ZHµ hardness (Gravimeta, Oporto, Portugal) testing machine using a 300 gf load and a dwell time of 15 s. Analysis conditions were selected based on a recent study on the characterization and long-term stability of historical PMMA sheets [9 (link)]. Tests were performed on one sample out of the three exposed to each CO₂ trial. Hardness values and standard deviation were determined as the average of 10 independent readings (5 on each side) obtained at a distance > 5d from each other. Measurements were collected approximately 3 h after exposure to CO₂ and after 2 days and 1, 2, 4, and 35 weeks. Variations in hardness values for each sample over time were determined by comparison with a set of four control samples that had not been exposed to CO₂.
Hardness values for control and CO₂-exposed samples were compared using a one-way ANOVA statistical test. Where results were statistically different, a post hoc test (Tukey–Kramer multiple comparison) was performed. Results for these tests are reported above related graphs in lower-case letters. Where no statistical differences were revealed, the bars are labeled with the same letter(s). All statistical tests were conducted using the Origin 2022b software (OriginLab Corporation, Northampton, MA, USA).

Certified reference solutions were verified in addition to the biochar solution sample. Both internal (Y) and external standards were examined in every ICP-OES analysis. Standard solutions with R² > 0.995 were established before each study to calibrate the system. Three replicated measurements were made for every sample. ICP Expert Software v7.1.0.6821 (Agilent Technologies, Santa Clara, CA, USA) was used to analyze the ICP-OES data. All the containers utilized in the tests were washed with laboratory-grade detergent, steeped in a 10% nitric acid solution for the duration of the night, then flooded with deionized (DI) water before receiving a final rinse with DI water. Significant differences (p < 0.05) and the Tukey–Kramer honestly significant difference test were evaluated using the statistical tool JMP 15.2 (SAS Institute Inc., Cary, NC, USA). The Origin 2022b statistical program (OriginPro, Version 2022b, OriginLab Corporation, Northampton, MA, USA) was used to create data visualizations.