Sequential Inoculation and Microbial Nutritional Strategies Influence the Multifunctionality of Cyanobacterium-Based Biofilms.

Multispecies biofilms combining Anabaena torulosa (An) with Trichoderma viride (Tr) along with either Providencia sp. (PW5) or Pseudomonas nitroreducens (B3) were optimized using eleven treatments with different inoculation strategies and evaluated from liquid to soil mesocosms scales. The optimal treatment, An-Tr (7 days after inoculation (DAI)) + PW5 (9 DAI), demonstrated significant increases in chlorophyll (45.2%), indole-3-acetic acid (66.9%) and total sugars (24.76%) over An alone, in liquid mesocosm, along with the highest polysaccharide content (6.56 mg g soil^-1) in soil-based mesocosms. Under controlled flask-conditions, An-Tr (7 DAI) + PW5 (9 DAI) and An-Tr (7 DAI) + B3 (9 DAI) exhibited higher nitrate reductase (30.4% and 25.1%) and glutamine synthetase (36.9% and 36.6%) activities compared to An alone, accompanied by elevated proteins. In soil-based micro- and mesocosms, An-Tr (7 DAI) + PW5 (9 DAI) demonstrated superior colonization potential reflected as enhanced enzyme activities, with one-fold increase in the activity of soil urease, β-glucosidase and soil chlorophyll against uninoculated soil. Phospholipid fatty acid (PLFA) analysis revealed distinct profiles, An-Tr (7 DAI) + B3 (9 DAI) exhibited higher 18:1 w9c and MUFA markers, with increased microbial activity and Gram-negative bacteria and anaerobic populations. In contrast, An-Tr (7 DAI) + PW5 (9 DAI) profiles demonstrated higher Gram-positive bacterial population, indicating enhanced resilience and nutrient cycling capacity. These collective findings underscore the promise of tailored inoculation strategies in optimizing biofilm composition for multifunctionality.