The use of biofuels has grown in the last decades, due to environmental impacts of fossil fuels use. Elucidating structure, diversity, species interactions and assembly mechanisms of microbiomes is crucial for understanding the in uence of environmental disturbances. However, little is known about how contamination with biofuel/petrofuel blends alters the soil microbiome. This study aimed to characterize the soil microbiome of four long term eld experimental areas that received controlled releases of E10, E25 or B20 and were submitted to different bioremediation treatments, using 16S rRNA gene amplicon high throughput sequencing. Results indicated that the soil microbiome of biodieselareas is more diverse, resilient, and complex. It was also observed that in soils with low diversity and richness, the impact of bioremediation treatments on the microbiomes was higher. The network analysis showed that after applying the bioremediation treatment, the proportion of generalist taxa increased, suggesting a more connected and dynamic assembly. All abundant keystone taxa are well-known degraders, suggesting that the abundant species are core targets for biostimulation in soil remediation. Overall, these ndings extend our knowledge of the soil microbiome response triggered by pollution stress and bioremediation treatments, paving the way for future rationalized and e cient pollutant mitigation strategies.hypotheses that guided our study were: (i) The microbiome composition and dynamics vary according to the different fuel/biofuel blends; (ii) The bioremediation treatments in uence the microbial metabolism, resulting in shifts in microbial community structure and composition; (iii) The microbiome co-occurrence patterns and keystone species vary according to the different blends and bioremediation treatments, re ecting the level of complexity and strength of the interactions among microbial members depending on the substrate, nutrients and electron acceptor availability. It should be mentioned that our hypotheses did not consider environmental factors (e.g. soil type, chemistry, humidity, precipitation, temperature, water table, etc), because all four areas were under the same pedological and climatological conditions.
MATERIAL AND METHODS
Description of experimental areas and bioremediation treatmentsThe experimental site is located at the Ressacada Experimental Farm in Florianópolis, owned by the Federal University from Santa Catarina (UFSC), in the southeast of Santa Catarina Island, Brazil (Fig. S1a). The climate is mesothermic humid with an annual average precipitation of 1600 mm. The average groundwater temperature is 26°C in the summer and 22°C in the winter, and the water table varies from 0.7 to 2.0 m throughout the year. The subsurface soil consists of hydromorphic quartz sands with less than 5% clay. Soil organic carbon ranges between 0.16 and 0.68% (Ramos 2013, Ramos et al. 2010.Controlled releases of blends of biofuels/petrofuels were conducted in four experimental areas throughout time (2004)(2005)(2006)(2007)(...