Biodiesel is a renewable fuel that can be mixed with toluene and be accidentally released into anoxic ecosystems and impact soil microbial communities. Therefore, the aim of the present work was to examine, under nitrate-reduction conditions, the biodegradation of toluene in the presence of two different types of biodiesel (sunflower and rapeseed), and their impact on the bacterial community structure.

Sediment samples were spiked individually with toluene, biodiesel, and their blends in laboratory-designed microcosms. Sunflower oil biodiesel was produced in the laboratory, while rapeseed oil biodiesel was a commercial product. Degradation of biodiesels and blends was monitored by directly measuring the substrate or indirectly by determining nitrate removal during the course of the experiment. Denitrification rates were estimated with the acetylene inhibition technique. Denaturing gradient gel electrophoresis was used to assess impacts on the bacterial community structure exposed to biodiesels, blends, and toluene.

The results of this study showed that toluene and biodiesel were completely degraded within 10 days. Biodiesel significantly affected the bacterial community structure at a similar magnitude, independently of its origin. Additionally, toluene impacted the bacterial community and denitrification process to a lower extent than biodiesel and a clear decrease in the relative bacterial richness and diversity was shown in samples with biodiesel and blends. To the best of our knowledge, this is one of the first reports describing degradation of biodiesel alone and blends under nitrate-reducing conditions, and also the effects of these compounds on the denitrification process. In addition, due to the recently discovered “oxygenic denitrification” process, the acetylene inhibition technique and nitrous oxide quantification may not be the most adequate tool to estimate denitrification rates. Further detailed studies are advised to understand whether the identified bacterial community shift impacts ecosystem functions.

Our results help to understand the biodegradation of toluene, biodiesel, and their blends in sediments under nitrate-reducing conditions and might be important in implementing bioremediation strategies in anoxic environments.