Supplementary file 1_Effect of tequila vinasse irrigation on soil physicochemical properties, heavy metals, and bacterial communities in soils cultivated with Agave tequilana.docx

Tequila production yields 10–12 liters of tequila vinasses (TV) per liter of product, posing significant waste management challenges. Land application offers a cost-effective disposal method with potential fertilization benefits, though its impact on soil microbial ecology remains uncertain. In this one-year field case study in Amatitán, Jalisco, Mexico, seven TV treatments were tested: raw TV at 1, 3, 5, and 10 L/m²/month; neutralized TV at 10 L/m²/month; and raw or neutralized TV at 30 L/m² every three months. These were compared with three controls (fertilizer, no input, water). Physicochemical properties were measured quarterly, and bacterial communities analyzed via 16S rRNA sequencing. Results showed that TV irrigation increases soil organic matter, potassium, and heavy metal levels without exceeding regulatory safety limits. However, chromium concentrations were higher than initial conditions, indicating potential risk of accumulation. Soil pH rose from 6.4 to 6.5 across all treatments. Phosphorus peaked at 1,231 mg/kg in August with neutralized TV, then returned to baseline by December 2022. Bacterial communities were mainly composed of Actinomycetota (36–47%) and Pseudomonadota (21–27%). Low TV doses increased beneficial bacteria such as Conexibacter, Bradyrhizobium, and Burkholderia-Caballeronia-Paraburkholderia, while high raw TV favored stress-tolerant genera like Desulfitobacterium and Kocuria. Redundancy analysis showed that Kocuria was positively correlated with As, Cd and Pb, while Amphiplicatus, Nitrospira, Chryseolinea, and Actinocorallia were positively correlated with Cu, Cr, Ni and Zn. Neutralized TV supported nitrogen-fixing bacteria such as Micromonospora and Frankia. Moderate TV doses improved soil fertility while maintaining bacterial diversity. However, high doses caused shifts in bacterial communities, highlighting the need for dose optimization in sustainable TV management.