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The use of nanoparticles (NPs) is of increasing significance due to their large potential for various applications. Great attention should be paid on the possible impacts of nanoparticles on the environment as large amounts of them may reach the environment by accident or voluntarily. Marine algae are potential organisms for usage in nanopollution bioremediation in aquatic system, because of their ability to adapt to long exposure to NPs. Thus, it is of prime importance to study the possible interactions of different NPs with microalgae in assessing their potential environmental risks. Most studies on potential environmental effects of ZnO and TiO2 NPs have been performed independently and following the widely accepted, standardized test systems, which had been developed for the characterization of chemicals. In this study, we have examined the cumulative effect of ZnO and TiO2 NPs on Picochlorum sp. in addition to the individual effects of these NPs over 32 days. Our results indicate that the toxicity and availability of NPs to marine algae are reduced by their aggregation and sedimentation. NPs are found to have a negative effect on algal growth and chlorophyll a concentration during the early growth stages. In contrast, the case is reversed during the late growth stages. There is no significant difference between the effect of the NPs when they are used separately and when both ZnO and TiO2 are used together in the test (P > 0.05).

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni. We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis. Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge. IMPORTANCE Bacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.